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Actions of benzophenanthridine alkaloids and various synthetic compounds on the cannabinoid-1 (CB1) receptor pathway of mouse brain with particular reference to the
effects on [3H]CP55940 and [3H]SR141716A binding, interference with basal and
CP55940-stimulated [35S]GTPγS binding, and modification of WIN55212-2-dependent
inhibition of L-glutamate release from synaptosomes
by Amey S. Dhopeshwarkar
MSc., University of Abertay Dundee, 2007 B.Pharm., University of Pune, 2004
THESIS SUBMITTED IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
in the
Department of Biological Sciences
Faculty of Science
Amey S. Dhopeshwarkar 2012
SIMON FRASER UNIVERSITY Summer 2012
All rights reserved. However, in accordance with the Copyright Act of Canada, this work may
be reproduced, without authorization, under the conditions for “Fair Dealing.” Therefore, limited reproduction of this work for the
purposes of private study, research, criticism, review and news reporting is likely to be in accordance with the law, particularly if cited appropriately.
ii
Approval
Name: Amey S. Dhopeshwarkar Degree: Doctor of Philosophy (Biological Sciences) Title of Thesis: Actions of benzophenanthridine alkaloids and various
synthetic compounds on the cannabinoid-1 (CB1) receptor pathway of mouse brain with particular reference to the effects on [3H]CP55940 and [3H]SR141716A binding, interference with basal and CP55940-stimulated [35S]GTPγS binding, and modification of WIN55212-2-dependent inhibition of L-glutamate release from synaptosomes.
Examining Committee: Chair: Dr Julian Christians, Associate Professor
Dr Russell A. Nicholson Senior Supervisor Associate Professor
Dr Christopher Kennedy Supervisor Professor
Dr Francis C.P. Law Supervisor Professor
Dr Gordon Rintoul Internal Examiner Associate Professor Department of Biological Sciences, SFU
Dr Andrew Gifford External Examiner Scientist, Medical Department Brookhaven National Laboratory
Date Defended/Approved: August 15, 2012
iii
Partial Copyright Licence
Ethics Statement
The author, whose name appears on the title page of this work, has obtained, for the research described in this work, either:
a. human research ethics approval from the Simon Fraser University Office of Research Ethics,
or
b. advance approval of the animal care protocol from the University Animal Care Committee of Simon Fraser University;
or has conducted the research
c. as a co-investigator, collaborator or research assistant in a research project approved in advance,
or
d. as a member of a course approved in advance for minimal risk human research, by the Office of Research Ethics.
A copy of the approval letter has been filed at the Theses Office of the University Library at the time of submission of this thesis or project.
The original application for approval and letter of approval are filed with the relevant offices. Inquiries may be directed to those authorities.
Simon Fraser University Library Burnaby, British Columbia, Canada
update Spring 2010
iii
Abstract
Benzophenanthridine alkaloids (chelerythrine and sanguinarine) inhibited the binding of
[3H]CP55940 and [3H]SR141716A to mouse brain membranes (IC50s approx. 1-2 µM).
Piperonyl butoxide and (S)-methoprene were more potent inhibitors of [3H]CP55940
binding (IC50s: 8.2 µM and 16.4 µM respectively) than of [3H]SR141716A binding (IC50s:
21 µM and 63 µM respectively). Binding experiments demonstrated selectivity towards
the brain CB1 versus spleen CB2 receptor.
Benzophenanthridines reduced the Kd of [3H]CP55940 binding to brain membranes
whereas (S)-methoprene and piperonyl butoxide lowered Bmax. These study
compounds reduced the association of [3H]CP55940 and [3H]SR141716A, however
benzophenanthridines were consistently more effective.
In the presence of a saturating concentration of SR141716A, (S)-methoprene and
piperonyl butoxide increased dissociation of [3H]SR141716A above that observed with
SR141716A alone. All compounds activated [3H]SR141716A dissociation when assayed
alone, but (S)-methoprene was the least effective. In separate studies, phthalate
diesters reduced the Bmax of [3H]SR141716A without affecting Kd, and increased
[3H]SR141716A dissociation above a saturating concentration of AM251.
Benzophenanthridines antagonized CP55940-stimulated and basal binding of
[35S]GTPγS to the G-protein of mouse brain, whereas piperonyl butoxide and (S)-
methoprene inhibited CP55940-stimulated [35S]GTPγS binding only. Inhibition of
CP55940-stimulated binding of [35S]GTPγS was also demonstrated with phthalates.
4-Aminopyridine- (4-AP-) induced release of L-glutamate from mouse brain
synaptosomes was partially inhibited by WIN55212-2. The inhibitory effect of
WIN55212-2 was completely neutralized by AM251, (S)-methoprene, piperonyl butoxide
and phthalate diesters, whereas in the presence of WIN55212-2, the
benzophenanthridines enhanced 4-AP-induced L-glutamate release above that caused
by 4-AP alone.
iv
The [3H]CP55940 and [3H]SR141716A binding data suggest that the study compounds
modify radioligand binding allosterically. The [35S]GTPγS binding results suggest that
chelerythrine and sanguinarine are inverse agonists of G-protein-coupled CB1 receptors,
while piperonyl butoxide, (S)-methoprene and phthalate diesters are neutral lower
potency antagonists. Modulation 4-AP-evoked L-glutamate release from synaptosomes
by the study compounds with WIN-55212-2 present strongly supports this latter profiling.
Although these compounds exhibit lower potencies versus many conventional CB1
receptor inhibitors, further studies are warranted, given their potential to 1) modify CB1
receptor-dependent behavioral/physiological outcomes in the whole animal, and 2) serve
as starting structures for synthesis of novel/more potent G-protein-coupled CB1 receptor
blocking drugs.
Keywords: Benzophenanthridines; (S)-methoprene; piperonyl butoxide; [3H]CP55940; [35S]GTPγS; L-glutamate; synaptosomes;cannabinoid-1 (CB1) receptor
v
Dedication
To My Beloved Mom and Dad
vi
Acknowledgements
I wish to express my deepest gratitude and appreciation to my senior supervisor, Dr
Russell A. Nichoson for his guidance, patience and indefatigable support throughout my
graduate research career. I remember the days when Dr Nicholson spared his time
even on weekends and holidays to discuss my research and his invaluable suggestions
and encouragements have always made me feel confident about my research work.
Thorough discussion sessions with him about project and related scientific issues and
perspectives have enriched my knowledge in this field. Without Dr Nicholson’s support
and effort, I would not have completed my PhD research in time. I believe that I was
lucky to have such a knowledgeable senior supervisor and I am fortunate to be his last
graduate student.
I am very much thankful to Dr Chris Kennedy and Dr Francis C.P. Law for serving as my
committe members and their valuable time and inputs during my PhD. They have
always been supportive during my studies at SFU.
I am also thankful to Mr Saurabh Jain and Ms Kathleen M. Bisset for their help and
advice during my research.
Finally, I would like to thank my family for their love, support encouragement and always
believing in me.
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Table of Contents
Approval .......................................................................................................................... ii Abstract .......................................................................................................................... iii Dedication ....................................................................................................................... v Acknowledgements ........................................................................................................ vi Table of Contents .......................................................................................................... vii List of Tables ................................................................................................................. xii List of Figures................................................................................................................xiv Glossary ........................................................................................................................xxi
1. Introduction .......................................................................................................... 1 1.1. Historical significance of cannabis use and cannabinoids ....................................... 1
1.1.1. The early Chinese/Indian era ...................................................................... 1 1.1.2. The period encompassing the early Christian era through to the 18th
century ........................................................................................................ 2 1.1.3. The Western medicine era of the 19th and 20th centuries ............................. 2
1.2. Cannabinoids ......................................................................................................... 5 1.2.1. G protein-coupled receptors (GPCRs) and their activation cycle ................. 7 1.2.2. The [35S]GTPγS binding assay .................................................................... 8
1.3. Other cannabinoid receptors .................................................................................. 8 1.4. Cannabinoid-1 Receptors (CB1-Rs) ........................................................................ 9
1.4.1. The structure and activation of CB1-Rs ....................................................... 9 1.4.2. The distribution of CB1-Rs in mammalian brain ......................................... 17
1.5. CB1-R-mediated intracellular signaling pathways .................................................. 18 1.5.1. Inhibition of cyclic AMP (cAMP) ................................................................ 18 1.5.2. Stimulation of cAMP production ................................................................ 20 1.5.3. CB1-Rs and the modulation of Ca2+ fluxes and phospholipases C
and A ........................................................................................................ 21 1.5.4. CB1-R-dependent regulation of ion channels ............................................. 21 1.5.5. Involvement of CB1-Rs in the suppression of neurotransmitter
release ...................................................................................................... 22 1.6. Homodimerization and heterodimerization of CB1-Rs ........................................... 24 1.7. Constitutive activity of CB1-Rs .............................................................................. 25 1.8. The biochemistry of endocannabinoids ................................................................. 25
1.8.1. Anandamide biosynthesis ......................................................................... 28 1.8.2. 2-Arachidonoyl glycerol (2-AG) biosynthesis ............................................. 30
1.9. Degradation pathways for endocannabinoids ....................................................... 32 1.10. Transport of endocannabinoids ............................................................................ 33 1.11. Endocannabinoid-mediated short term depression (DSI and DSE) ....................... 35 1.12. Endocannabinoids as synaptic circuit breakers and retrograde messengers ........ 35 1.13. Mechanisms of endocannabinoid mediated short term depression (eCB-
STD) ..................................................................................................................... 38 1.13.1. CaER ........................................................................................................ 38 1.13.2. Basal RER ................................................................................................ 38 1.13.3. Ca2+-assisted RER .................................................................................... 39
1.14. Termination of eCB-STD ...................................................................................... 39 1.15. Endocannabinoid-mediated long term depression (eCB-LTD) .............................. 41
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1.16. Other important aspects of endocannabinoid signaling ......................................... 41 1.16.1. Regulation of excitability ........................................................................... 41 1.16.2. Basal activity of endocannabinoid signaling .............................................. 42 1.16.3. Plasticity of endocannabinoid signaling ..................................................... 42
1.17. Subcellular distribution of various signaling molecules involved in regulation of the endocannabinoid system ............................................................................ 42 1.17.1. Gq Protein α subunit .................................................................................. 42 1.17.2. Phospholipase Cβ (PLCβ) ......................................................................... 43 1.17.3. Diacylglycerol lipase (DAGL) ..................................................................... 43 1.17.4. N-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D
(NAPE-PLD) ............................................................................................. 43 1.17.5. Monoacylglycerol lipase (MAGL) ............................................................... 44 1.17.6. Fatty acid amide hydrolase (FAAH) ........................................................... 44
1.18. Physiological roles of the endocannabinoid system .............................................. 44 1.18.1. Learning and Memory ............................................................................... 44 1.18.2. Anxiety ...................................................................................................... 45 1.18.3. Depression ................................................................................................ 45 1.18.4. Addiction ................................................................................................... 46 1.18.5. Appetite ..................................................................................................... 46 1.18.6. Pain .......................................................................................................... 46
1.19. Classification of ligands that bind to cannabinoid receptors .................................. 47 1.19.1. Cannabinoid receptor agonists .................................................................. 47
1.19.1.1. Classical cannabinoids ............................................................... 47 1.19.1.2. Non-classical cannabinoids ........................................................ 47 1.19.1.3. Aminoalkylindoles ....................................................................... 47 1.19.1.4. Eicosanoids/Endocannabinoids .................................................. 48
1.19.2. Cannabinoid receptor antagonists/ Inverse agonists ................................. 48 1.19.2.1. Diarylpyrazoles ........................................................................... 48 1.19.2.2. Other inverse agonists primarily active at CB1-Rs ....................... 48
1.20. Cannabinoid receptor 2 (CB2-R) ........................................................................... 53 1.20.1. CB2-R receptor signaling ........................................................................... 53
1.20.1.1. Adenylyl cyclase regulation......................................................... 53 1.20.1.2. Mitogen-activated protein kinase regulation ................................ 53
1.20.2. Therapeutic aspects of CB2-R modulators ................................................. 54 1.21. Brief overview of the test chemicals used in my research ..................................... 55
1.21.1. Benzophenanthridine alkaloids ................................................................. 55 1.21.2. Piperonyl butoxide (PBO) .......................................................................... 56 1.21.3. Methoprene ............................................................................................... 57 1.21.4. Phthalate esters ........................................................................................ 58 1.21.5. Tributyl tin (TBT) compounds .................................................................... 59
1.22. Rationale behind my research and the general approach ..................................... 62 1.22.1. Summary of objectives .............................................................................. 62
2. The actions of benzophenanthridine alkaloids, piperonyl butoxide and (S)-methoprene at the G-protein coupled cannabinoid CB1 receptor in vitro. .................................................................................................................... 64
2.1. Abstract ................................................................................................................ 64 2.2. Introduction .......................................................................................................... 65 2.3. Materials and Methods ......................................................................................... 67
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2.3.1. Radioligands, drugs and study compounds ............................................... 67 2.3.2. Animals ..................................................................................................... 67 2.3.3. Determination of the effects of study compounds on the binding of
[3H]CP55940 to CB1 receptors in mouse brain membranes....................... 67 2.3.4. Determination of the effects of study compounds on basal and
CP55940-stimulated [35S]GTPγS binding to mouse brain membranes ............................................................................................... 69
2.3.5. Data analysis ............................................................................................ 70 2.4. Results ................................................................................................................. 70 2.5. Discussion ............................................................................................................ 71 2.6. Figures and Tables ............................................................................................... 75
3. The G protein-coupled cannabinoid-1 (CB1) receptor of mammalian brain: Inhibition by phthalate esters in vitro. ................................................... 88
3.1. Abstract ................................................................................................................ 88 3.2. Introduction .......................................................................................................... 89 3.3. Materials and methods ......................................................................................... 91
3.3.1. Animals ..................................................................................................... 91 3.3.2. Investigation of the effects of phthalate esters on the binding of
[3H]CP55940 and [3H]SR141716A to CB1 receptors of mouse brain. ......... 92 3.3.3. Investigation of phthalate interference with CB1 receptor agonist-
stimulated [35S]GTPγS binding to the Gα-protein. ...................................... 93 3.3.4. Data analysis ............................................................................................ 95
3.4. Results ................................................................................................................. 95 3.4.1. Effects of phthalate esters on binding of [3H]CP55940 to CB1
receptors. .................................................................................................. 95 3.4.2. Effects of selected phthalate esters on binding of [3H]SR141716A to
CB1 receptors. ........................................................................................... 95 3.4.3. Influence of selected phthalates on the saturation binding of
[3H]SR141716A to CB1 receptors .............................................................. 96 3.4.4. Effects of selected phthalates on [3H]SR141716A kinetics ........................ 96 3.4.5. Effects of phthalates on CB1 receptor agonist-stimulated [35S]GTPγS
binding to the Gα-protein .......................................................................... 96 3.5. Discussion ............................................................................................................ 97 3.6. Note in added proof ............................................................................................ 100
3.6.1. Background ............................................................................................. 100 3.6.2. Experimental approach ........................................................................... 101 3.6.3. Results .................................................................................................... 101 3.6.4. Conclusion .............................................................................................. 101
3.7. Figures and Tables ............................................................................................. 102
4. Benzophenanthridine alkaloid, piperonyl butoxide and (S)-methoprene action at the cannabinoid-1 receptor (CB1-R) pathway of mouse brain: interference with [3H]CP55940 and [3H]SR141716A binding and modification of WIN55212-2-dependent inhibition of synaptosomal L-glutamate release. ............................................................................................ 115
4.1. Abstract .............................................................................................................. 115 4.2. Introduction ........................................................................................................ 116
x
4.3. Materials and Methods ....................................................................................... 118 4.3.1. Chemicals and supplies .......................................................................... 118 4.3.2. Animals ................................................................................................... 119 4.3.3. Isolation of membranes from mouse brain for binding studies ................. 119 4.3.4. Effects of benzophenanthridines, (S)-methoprene and piperonyl
butoxide on equilibrium binding of [3H]CP55940 and [3H]SR141716 to brain CB1 receptors ............................................................................. 120
4.3.5. Effect of benzophenanthridines, (S)-methoprene and piperonyl butoxide on the association and dissociation kinetics of [3H]CP55940 and [3H]SR141716A .......................................................... 121
4.3.6. Interaction of benzophenanthridines, methoprene and piperonyl butoxide with CB2 receptors in mouse spleen ......................................... 121
4.3.7. Preparation of synaptosomes from mouse whole brain ........................... 122 4.3.8. Release of L-Glutamate from synaptosomes........................................... 123 4.3.9. Analysis of radioligand binding data and glutamate release data ............ 124
4.4. Results ............................................................................................................... 124 4.4.1. Effects of benzophenanthridines, piperonyl butoxide and (S)-
methoprene on binding of [3H]SR141716A to CB1 receptors ................... 124 4.4.2. Influence of study compounds on the saturation binding of
[3H]SR141716A to CB1 receptors of mouse brain .................................... 125 4.4.3. Effects of sanguinarine, chelerythrine, piperonyl butoxide, and (S)-
methoprene on the kinetics of CB1 receptor-selective radioligand binding .................................................................................................... 125
4.4.4. Effects of study compounds on mouse spleen CB2 receptors as assessed by inhibition of [3H]CP55940 binding ....................................... 126
4.4.5. Effects of study compounds on WIN55212-2-dependent inhibition of 4-aminopyridine- (4-AP-) evoked release of L-glutamate from mouse brain synaptosomes ................................................................................ 126
4.5. Discussion .......................................................................................................... 127 4.6. Figures and Table .............................................................................................. 132
5. Effects of organotins on the CB1 receptor pathway of mouse brain in vitro. .................................................................................................................. 150
5.1. Introduction ........................................................................................................ 150 5.2. Materials and methods ....................................................................................... 151 5.3. Results ............................................................................................................... 152
5.3.1. Displacement of [3H]CP55940 binding to mammalian CB1 receptors by organotin compounds ......................................................................... 152
5.3.2. Basal and CP55940-stimulated [35S]GTPγS binding to the Gα subunit as influenced by tributyltin compounds ....................................... 152
5.3.3. Modulation by tributyltin acetate and phenylethynyl tributyltin of WIN55212-2-dependent inhibition of 4-aminopyridine-evoked release of L-glutamate from mouse brain synaptosomes ........................ 153
5.4. Discussion .......................................................................................................... 153 5.5. Figures and Table .............................................................................................. 156
xi
6. Conclusion and future prospects .................................................................... 162
References ................................................................................................................. 164
xii
List of Tables
Table 2.1 Inhibition of specific [3H]CP55940 binding to mouse brain membranes by isoquinoline type compounds and PMSF. Isoquinolines were present in the assay at 30 µM and PMSF was present at 0.5 mM. Data represent mean ± S.E.M. of 3 independent experiments. ...................................................................... 84
Table 2.2 Inhibition of 100 nM CP55940-stimulated and basal [35S]GTPγS binding to mouse brain membranes by AM251. Data represent mean ± S.E.M. of 3 independent experiments. ND = not determined. Results provided by Mr Saurabh Jain. ................................ 85
Table 2.3 Lack of effect of isoquinoline type compounds on CP55940-stimulated and basal [35S]GTPγS binding to mouse brain membranes. Study compounds were present in the assay at 40 µM. Data represent mean ± S.E.M. of 3 independent experiments. ........ 86
Table 2.4 Lack of effect of piperonyl butoxide and (S)-methoprene on the basal binding of [35S]GTPγS to mouse brain membranes. Values represent mean ± S.E.M. of 3 independent experiments. ....................... 87
Table 3.1 Inability of PMSF to influence the inhibitory effects of n-butylbenzylphthalate (nBBP) and di-n-butylphthalate (DnBP) on [3H]CP55940 binding to mouse brain membranes. Phthalate esters were present in the assay at 20 µM and PMSF was used at 50 µM. Each value represents the mean ± S.E.M. of 3-6 independent experiments. .................................................................... 113
Table 3.2 Inhibitory effects of n-butylbenzylphthalate (nBBP), di-n-butylphthalate (DnBP), diethylhexylphthalate (DEHP), mono-isohexylphthalate (MiHP) and mono-n-butyl phthalate (MnBP) on the specific binding of [3H]SR141716A to mouse brain membranes. Diesters were present at concentrations producing 50% inhibition of [3H]CP55940 binding. Each value represents the mean ± S.E.M. of 3 independent experiments. ..................................... 114
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Table 4.1 Inhibitory effects of chelerythrine, sanguinarine, piperonyl butoxide and (S)-methoprene on spleen CB2 receptors as determined with [3H]CP55940. Each study compound was added at a concentration that achieved an IC50 for [3H]CP55940 binding to brain CB1 receptors (Dhopeshwarkar et al. 2011). All values represent mean percentage inhibition ± S.E.M. of at least 3 independent experiments. Parallel experiments with [3H]CP55940 corroborated our previously published IC50s at brain CB1 receptors (2.2 µM chelerythrine gave 49.03 ± 0.94 % inhibition, 1.2 µM sanguinarine gave 51.33 ± 0.49 % inhibition, 8.2 µM piperonyl butoxide gave 47.50 ± 1.17 % inhibition and 16.4 µM methoprene gave 50.22 ± 1.10 % inhibition). ...................................... 149
Table 5.1 Inhibitory effects of tributyl and triphenyltins on the binding of [3H]CP55940 to CB1 receptors in mouse brain. All values are as IC50s (with 95% confidence intervals in brackets) calculated from curves based on at least 3 independent experiments except for triphenyltin chloride where the IC50 was estimated from 2 independent experiments). ................................................................... 161
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List of Figures
Figure 1.1 The spread of the use of cannabis across the globe (Adapted from Zuardi, 2006). ........................................................................................... 3
Figure 1-2 Structure of two important phytocannabinoids. Structures redrawn using ChemDraw Ultra 11.0 from structures reported in Pertwee et al. (2010). ................................................................................................. 6
Figure 1.3 Two dimensional representation of the CB1-R (Adapted from Shim et al., 2011). ........................................................................................... 12
Figure 1.4 Diagramatic representation of the C terminal domain of the CB1-R (Adapted from Stadel et al., 2011) .......................................................... 13
Figure 1.5 Structures of prominent endocannbinoids (All structures redrawn using ChemDraw Ultra 11.0 from Kano et al., 2009). ............................. 27
Figure 1.6 Transacylation-phosphodiesterase pathway for biosynthesis of anandamide (Adapted from Cadas et al., 1997). .................................... 29
Figure 1.7 Metabolic pathways for biosynthesis of 2-AG (Adapted from Kano et al., 2009). ........................................................................................... 31
Figure 1.8 Blockade of DSI by CB1-R antagonists. .................................................. 37
Figure 1.9 The pathway involved in the termination of endocannabinoid-mediated short term depression (eCB-STD) (Adapted from Kano et al., 2009). ........................................................................................... 40
Figure 1.10 Structures of ∆9-THC, ∆8-THC, HU210, DALN, CP47497, CP55244, CP55940, WIN55212-2, JWH015 and L-768242. All structures redrawn using ChemDraw 11.0 ultra from Howlett et al. (2002). ................................................................................................... 49
Figure 1.11 Structures of anandamide, 2-AG ether and 2-AG. All structures redrawn using ChemDraw Ultra 11.0 from Howlett et al. (2002). ............ 50
Figure 1.12 Structures of SR141716A, AM251, AM281, LY320135 and AM630. All structures redrawn using ChemDraw Ultra 11.0 from Howlett et al. (2002). .............................................................................. 51
Figure 1.13 Structures of (S)-methoprene, piperonyl butoxide, sanguinarine, chelerythrine, nBBP and DnBP. Structures redrawn using ChemDraw 11.0 from Dhopeshwarkar et al. (2011) and Bisset et al., (2011). .............................................................................................. 52
Figure 1.14 Structures of selected phthalate esters and tributyl tin compounds. All structures redrawn using ChemDraw Ultra 11.0. ............................... 61
xv
Figure 2.1 The structures of sanguinarine, berberine, papavarine and possible comparison of conformations of piperonyl butoxide and (S)-methoprene with anandamide and 2-arachidonoyl glycrol. Also possible comparison of sanguinarine and (S)-methoprene with ∆9-tetrahydrocannabinol and ∆9-tetrahydrocannabivarin. ............................ 76
Figure 2.2 Concentration-dependent inhibition of [3H]CP55940 binding to mouse brain CB1 receptors by sanguinarine and chelerythrine. Values represent mean ± S.E.M. of at least 3 independent experiments each performed in duplicate. Ki values were 0.38 µM (sanguinarine) and 0.57 µM (chelerythrine). ........................................... 77
Figure 2.3a Inhibition of A) CP55940-stimulated and B) basal binding of [35S]GTPγS to mouse brain membranes by chelerythrine. Values represent mean ± S.E.M. of 3 independent experiments each performed in triplicate. ............................................................................ 78
Figure 2.3b Inhibition of A) CP55940-stimulated and B) basal binding of [35S]GTPγS to mouse brain membranes by chelerythrine. Values represent mean ± S.E.M. of 3 independent experiments each performed in triplicate.Basal binding data provided by Mr Saurabh Jain. ....................................................................................................... 79
Figure 2.4a Inhibition of A) CP55940-stimulated and B) basal binding of [35S]GTPγS to mouse brain membranes by sanguinarine. Values represent mean ± S.E.M. of 3 independent experiments each performed in triplicate. ............................................................................ 80
Figure 2.4b Inhibition of A) CP55940-stimulated and B) basal binding of [35S]GTPγS to mouse brain membranes by sanguinarine. Values represent mean ± S.E.M. of 3 independent experiments each performed in triplicate. Basal binding data provided by Mr Saurabh Jain. ......................................................................................... 81
Figure 2.5a A) Concentration-dependent inhibition of [3H]CP55940 binding to mouse brain CB1 receptors by (S)-methoprene and piperonyl butoxide. Ki values were 2.13 µM (methoprene) and 4.25 µM (piperonyl butoxide). B) Inhibition of CP55940-stimulated binding of [35S]GTPγS to mouse brain membranes by (S)-methoprene and piperonyl butoxide. Values represent mean ± S.E.M. of 3 independent experiments each performed in triplicate. .......................... 82
Figure 2.5b A) Concentration-dependent inhibition of [3H]CP55940 binding to mouse brain CB1 receptors by (S)-methoprene and piperonyl butoxide. Ki values were 2.13 µM (methoprene) and 4.25 µM (piperonyl butoxide). B) Inhibition of CP55940-stimulated binding of [35S]GTPγS to mouse brain membranes by (S)-methoprene and piperonyl butoxide. Values represent mean ± S.E.M. of 3 independent experiments each performed in triplicate. .......................... 83
xvi
Figure 3.1 (a-f) The structures of phthalate diesters: n-butylbenzylphthalate (nBBP); di-n-hexylphthalate (DnHP); di-n-butylphthalate (DnBP); di-ethylhexylphthalate (DEHP); di-isooctylphthalate (DiOP) and di-n-octylphthalate (DnOP).(g-i) The structures of phthalate monoesters: mono-2-ethylhexyl-phthalate (M2EHP), mono-isohexyl-phthalate (MiHP) and mono-n-butyl-phthalate (MnBP). All structures have been redrawn from Bissett et al. (2011) using IsisDraw. .............................................................................................. 102
Figure 3.2 Inhibitory effects of phthalate esters (DnBP, nBBP, DnOP, MiHP and MnBP) on the binding of [3H]CP55940 to mouse brain CB1 receptors in vitro. Each point represents the mean ± SEM of 3 independent experiments. Results provided by Ms Kathleen M. Bisset. .................................................................................................. 103
Figure 3.3 Inhibitory effects of phthalate esters (DEHP, DnHP, DiOP and M2EHP) on the binding of [3H]CP55940 to mouse brain CB1 receptors in vitro. Each point represents the mean ± SEM of 3 independent experiments. Results provided by Ms Kathleen M. Bisset. .................................................................................................. 104
Figure 3.4 The effect of nBBP and DnBP (both at 35 µM) on the equilibrium binding of of [3H]SR141716A to CB1 receptors of mouse whole brain. Kd and Bmax values are displayed for each treatment and 95% confidence intervals were as follows: control (Kd 0.628 to 0.859. Bmax 0.303 to 0.343), nBBP (Kd 0.761 to 1.333. Bmax 0.176 to 0.229) and DnBP (Kd 0.624 to 0.846. Bmax 0.120 to 0.136). R2 values were 0.9877 (control), 0.9756 (nBBP) and 0.9887 (DnBP). Data points represent the means ± SEMs of 3 independent experiments (most SEM bars are obscured by data symbols). ............. 105
Figure 3.5a Influence of nBBP (35 µM) and DnBP (50 µM) on the time course of association of [3H]SR141716A with CB1 receptors of mouse brain. In a) membranes received the standard 15 min preincubation with phthalate esters prior to [3H]SR141716A addition. In b) the phthalate ester and [3H]SR141716A were applied simultaneously. Data points represent the means ± SEMs of 3 independent experiments (most SEM bars are obscured by data symbols). ...................................................................................... 106
Figure 3.5b Influence of nBBP (35 µM) and DnBP (50 µM) on the time course of association of [3H]SR141716A with CB1 receptors of mouse brain. In a) membranes received the standard 15 min preincubation with phthalate esters prior to [3H]SR141716A addition. In b) the phthalate ester and [3H]SR141716A were applied simultaneously. Data points represent the means ± SEMs of 3 independent experiments (most SEM bars are obscured by data symbols). ...................................................................................... 107
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Figure 3.6 Dissociation of the [3H]SR141716A:CB1 receptor complex (initiated by challenge with 5 µM AM251) in the absence (control) or in the presence of 35 µM nBBP or 50 µM DnBP. Data represent mean ± SEM of at least 3 independent experiments, each performed in triplicate. .......................................................................... 108
Figure 3.7 Inhibition of CP55940-stimulated binding of [35S]GTPγS to mouse whole brain membranes by phthalate esters. Phthalate esters were assayed at 75 µM throughout. Each column represents the mean, and error bar the ± SEM of 7 independent experiments. ............ 109
Figure 3.8 Relationship between the ability of study compounds to inhibit the binding of [3H]CP55940 and CP55940-stimulated binding of [35S]GTPγS in mouse whole brain membrane fractions. All assays were performed 75 µM; r2 = 0.7844. ..................................................... 110
Figure 3.9 With WIN55212-2 present, BBP (at 30 µM but not 5 µM) enhances 4-AP-evoked L-glutamate release above the level produced by 4-AP alone. ............................................................................................. 111
Figure 3.10 With WIN55212-2 present, MnBP (both at 30 µM and 5 µM) does not enhance 4-AP-evoked L-glutamate release above the level produced by 4-AP alone. ...................................................................... 112
Figure 4.1 Concentration dependency of inhibition by chelerythrine (open circles), sanguinarine (solid circles), piperonyl butoxide (solid triangles) and (S)-methoprene (squares) on [3H]SR141716A binding to mouse brain CB1 receptors. IC50 and 95% confidence interval values are provided in Section 4.4.1. ....................................... 132
Figure 4.2 Effect of chelerythrine (1 µM; open circles), sanguinarine (1 µM; solid circles), piperonyl butoxide (30 µM; solid triangles) and (S)-methoprene (60 µM; squares) on equilibrium binding of [3H]SR141716A to mouse brain CB1 receptors. Control data points are identified by the diamond symbols. Kd values (as nM): control 0.51 ± 0.04; chelerythrine 0.47 ± 0.08; sanguinarine 0.46 ± 0.04; (S)-methoprene 1.5 ± 0.6 and piperonyl butoxide 2.5 ± 1.1. Bmax values (as pmol [3H]SR141716A/mg protein): control 0.79 ± 0.02; chelerythrine 0.32 ± 0.02; sanguinarine 0.50 ± 0.01; (S)-methoprene 0.44 ± 0.08 and piperonyl butoxide 0.56 ± 0.13. ............... 133
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Figure 4.3 Effect of chelerythrine (2.5 µM; open circles), sanguinarine (1.5 µM; solid circles), piperonyl butoxide (10 µM; solid triangles) and (S)-methoprene (20 µM; squares) on equilibrium binding of [3H]CP55940 to mouse brain CB1 receptors. Control data points are identified by the diamond symbols. Kd values (as nM): control 0.36 ± 0.07; chelerythrine 2.32 ± 0.43; sanguinarine 2.28 ± 0.77; (S)-methoprene 1.37 ± 0.25 and piperonyl butoxide 0.34 ± 0.19. Bmax values (as pmol [3H]SR141716A/mg protein): control 0.6 ± 0.03; chelerythrine 0.65 ± 0.06; sanguinarine 0.63 ± 0.11; (S)-methoprene 0.25 ± 0.02 and piperonyl butoxide 0.35 ± 0.05. ............... 134
Figure 4.4a Influence of study compounds on the time course of association of [3H]SR141716A and [3H]CP55940 with CB1 receptors of mouse brain. In a) membranes received a standard 15 min preincubation with sanguinarine (2.5 µM), chelerythrine (2.5 µM), piperonyl butoxide (30 µM) and (S)-methoprene (30 µM) prior to [3H]SR141716A addition. .................................................................... 135
Figure 4.4b Influence of study compounds on the time course of association of [3H]SR141716A and [3H]CP55940 with CB1 receptors of mouse brain.The same study compound concentrations were applied simultaneously with [3H]SR141716A.. .................................................. 136
Figure 4.4c The effects of benzophenanthridines (5 µM), piperonyl butoxide (20 µM) and (S)-methoprene (20 µM) on the association of [3H]CP55940 under preincubation conditions are shown Symbols: diamonds = control; solid circles = sanguinarine; open circles = chelerythrine; triangles = piperonyl butoxide and squares = (S)-methoprene.Data points represent the means ± SEMs of 3 independent experiments (a number of SEM bars are obscured by data symbols) ....................................................................................... 137
Figure 4.5a The influence of study compounds on the dissociation of CB1 receptor-selective radioligands. Figure 4.5a shows the effects of piperonyl butoxide (30 µM) and (S)-methoprene (60 µM) on the dissociation of [3H]SR141716A when initiated by challenge with a saturating concentration (5 µM) of SR141716A. ................................... 138
Figure 4.5b The influence of study compounds on the dissociation of CB1 receptor-selective radioligands. Figure 4 5b, defines the effects of sanguinarine (5 µM), chelerythrine (5 µM), piperonyl butoxide (30 µM) and (S)-methoprene (60 µM) when added alone on the dissociation of [3H]SR141716A from the [3H]SR141716A:CB1 receptor complex .................................................................................. 139
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Figure 4.5c The influence of study compounds on the dissociation of CB1 receptor-selective radioligands. In Figure 4 5c, the effects of sanguinarine (5 µM), chelerythrine (5 µM), piperonyl butoxide (20 µM) and (S)-methoprene (60 µM) on the dissociation of [3H]CP55940 when initiated by application of a saturating concentration (5 µM) of CP55940 are given. Symbols: diamonds = control; solid circles = sanguinarine; open circles = chelerythrine; triangles = piperonyl butoxide and squares = (S)-methoprene. Data represent mean ± SEM of at least 3 independent experiments, each performed in triplicate......................... 140
Figure 4.6 Relationship between concentration of (S)-methoprene and inhibition at CB2 receptors of mouse spleen based on interference with [3H]CP55940 binding. .................................................................... 141
Figure 4.7a Inhibition of 50 µM veratridine-evoked release of L-glutamate from mouse brain synaptosomes by 5 µM tetrodotoxin (TTX) ...................... 142
Figure 4.7b Failure of 5 µM TTX to modify 3 mM 4-AP-evoked release of L-glutamate from synaptosomes. ............................................................ 143
Figure 4.8 Partial inhibition of 4-AP-evoked release of L-glutamate from synaptosomes by the CB1-R agonist WIN55212-2, and full relief of WIN55212-2-dependent inhibition by the CB1-R antagonist AM251. ................................................................................................ 144
Figure 4.9 With WIN55212-2 present, sanguinarine (at 2 µM but not 0.25 µM) enhances 4-AP-evoked L-glutamate release above the level produced by 4-AP alone. ...................................................................... 145
Figure 4.10 With WIN55212-2 present, chelerythrine (at 2 µM but not 0.25 µM) enhances 4-AP-evoked L-glutamate release above the level produced by 4-AP alone. ...................................................................... 146
Figure 4.11 With WIN55212-2 present, (S)-methoprene (at 25 µM but not 5 µM) enhances 4-AP-evoked L-glutamate release above the level produced by 4-AP alone. ...................................................................... 147
Figure 4.12 With WIN55212-2 present, piperonyl butoxide (at 25 µM but not 5 µM) enhances 4-AP-evoked L-glutamate release above the level produced by 4-AP alone. ...................................................................... 148
Figure 5.1 Structures of tributyl and triphenyltin compounds examined in the present investigation. Structures were constructed using Isis Draw. ................................................................................................... 156
xx
Figure 5.2 Concentration-dependent inhibition of specific [3H]CP55940 binding to mouse brain CB1 receptors by tributyltin benzoate, tributyltin acetate and phenylethynyl tributyltin. Each data point represents the mean ± S.E.M. inhibition of specific [3H]CP55940 binding for at least three independent assays, each performed in triplicate. Experiments conducted by Mr. Saurabh Jain. This figure was originally published in the M.Sc. thesis of Mr. Saurabh Jain (Simon Fraser University, 2011). .......................................................... 157
Figure 5.3 Concentration-dependent inhibition of CP55940 (100 nM)-stimulated [35S]GTPγS binding by tributyltin benzoate and phenylethynyl tributyltin. Each data point represents the mean ± S.E.M. percentage inhibition of CP55940 stimulated [35S]GTPγS binding determined by three independent assays each performed in triplicate. These experiments were conducted by Mr Saurabh Jain and this figure was originally published in the M.Sc. thesis of Mr. Saurabh Jain (Simon Fraser University, 2011). .............................. 158
Figure 5.4 Modulation of WIN55212-2-dependent inhibition of 4-aminopyridine (4-AP-)-evoked release of L-glutamate from mouse brain synaptosomes by tributyltin acetate (TBT acetate). Typical release profiles are displayed with mean % changes (± SEM) to 4-AP-evoked and control release in the adjacent table. ........................... 159
Figure 5.5 Modulation of WIN55212-2-dependent inhibition of 4-aminopyridine (4-AP-)-evoked release of L-glutamate from mouse brain synaptosomes by phenylethynyl tributyltin (TBPE tin). Typical release profiles are displayed with mean % changes (± SEM) to 4-AP-evoked and control release in the adjacent table. .......... 160
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Glossary
2-AG 2-Arachidonyl glycerol
2-AGE 2-Arachidonyl glycerol ether
4-AP 4-Aminopyridine
AEA Anandamide
Bmax Maximum concentration of binding sites
BSA Bovine serum albumin
CBD Cannabidiol
CB1-R Cannabinoid receptor-1
CB2-R Cannabinoid receptor-2
CHEL Chelerythrine
DAGL Diacylglycerol lipase
DSE Depolarization-induced suppression of excitation
DSI Depolarization-induced suppression of inhibition
DMSO Dimethylsulfoxide
EDTA Ethylenediamine tetraacetic acid
EGTA Ethylene glycol-bis(2-aminoethyl)-N,N,N’,N’-tetraacetic acid
EPSCs Excitatory post synaptic currents
eCB-STD Endocannabinoid mediated short term depression
eCB-LTD Endocannabinoid mediated long term depression
FAAH Fatty acid amide hydrolase
GPCR G-protein coupled receptor
GTP Guanosine-5’-triphosphate
GDP Guanosine-5’-diphosphate
GABA γ-Aminobutyric acid
IC50 Concentration effective in producing 50% inhibition
IPSCs Inhibitory post synaptic currents
KCl Potassium chloride
Kd Dissociation constant
Lys Lysine
L-GLU L-glutamic acid
MAGL Monoacylglycerol lipase
xxii
MnBP Mono-n-butyl phthalate
METHO Methoprene
NADA N-Arachidonyl dopamine
NAPE N-Arachidonyl-phosphatidylethanolamine
nBBP n-Butylbenzylphthalate
PLD Phospholipase D
PI Phosphatidyl inositol
PLA1 Phospholipase A1
PMSF Phenylmethane sulfonyl fluoride
PBO Piperonyl butoxide
SANG Sanguinarine
TTX Tetrodotoxin
TBT Tributyltin
∆9 -THC ∆9- Tetrahydrocannabinol
∆8-THC ∆8- Tetrahydrocannabinol
VGSCs Voltage-gated sodium channels
VTD Veratridine
1
1. Introduction
1.1. Historical significance of cannabis use and cannabinoids
Cannabis sativa and its preparations have been used throughout the millennia for
recreational and various therapeutic purposes (Hollister, 2001). Cannabis sativa is one
of the oldest cultivated plants in the history of humankind dating back at least 10,000
years (Jiang, 2006).
The history of cannabis use can be broadly classified into three eras, the early
Chinese/Indian era, the early Christian era through to the 18th century, and the era of
Western medicine of the 19th and 20th centuries (Zuardi, 2006).
1.1.1. The early Chinese/Indian era
The earliest references to the use of different parts of the cannabis plant were
documented in the Han dynasty in China (Zuardi, 2006). Fibers obtained from the stem
were used for preparing ropes, strings and paper, while fruits were used as food by the
ancient Chinese (Li, 1973).
The world’s oldest pharmacopoeia, Pen-ts’ao ching documented the use of
cannabis as a medicine for the treatment of rheumatic pain, constipation and disorders
of the female reproductive system (Zuardi, 2006). Other evidence for the use of
cannabis in ancient China was reported by Jiang et al. (2006), where a clay bowl
containing cannabis was discovered in the 2500 old Yanghai tombs of Northwestern
China. It is believed that stores such as this were probably used for medicinal purposes
and psychomanupulation (Russo et al., 2008).
The ancient Indian culture (around 1000 years B.C.) regularly employed
cannabis for medicinal and religious reasons (Zuardi, 2006). In ancient Indian medicine,
2
the plant was used for various purposes including induction of analgesia and hypnotic
states and reducing the occurence of epileptic seizures. It was also used as an
antiparasitic, an antispasmodic, an antibiotic, an expectorant and an aphrodisiac (Zuardi,
2006).
1.1.2. The period encompassing the early Christian era through to the 18th century
During this period, the use of cannabis gained increasing acceptance throughout
the Middle East and Africa. Around 1000 A.D., Arabic medical compendiums described
the use of cannabis as a plant beneficial in the treatment of diuretic disorders and
gastrointestinal problems including flatulence (Zuardi, 2006). In the 16th century,
cannabis was introduced to South America through the arrival of African slaves, while
Arab traders introduced cannabis to the European sub-continent firstly in Spain and then
to various Mediterranean countries including Italy (Zuardi, 2006).
1.1.3. The Western medicine era of the 19th and 20th centuries
Pioneering scientific studies and several books published by the Irish physicist
William B. O’Shaughnessy and the French psychiatrist Jacques-Joseph Moreau
facilitated the rapid introduction of cannabis to Western medicine. In their books, they
documented a range of therapeutic uses as well as psychomimetic and experimental
manipulations based on the use of cannabis and hashish (cannabis resin) (Di Marzo,
2006).
Western medicine readily accepted many of their proposed uses of cannabis
since during this period there were very few realistic therapeutic options available for the
treatment of disorders such as rheumatism, muscular spasms, pain and convulsive
states (Zuardi, 2006). Figure 1.1 summarizes the global spread of cannabis use from
South East Asia through Africa and South America to Europe and the USA (as published
by Zuardi (2006)), while Table 1.1 details the main landmarks in cannabinoid research
up until the late 1990s (Novarro and Fonseca, 1998).
3
Figure 1.1 The spread of the use of cannabis across the globe (Adapted from Zuardi, 2006).
4
Table 1.1 Major advances in the use of cannabis and research on the cannabinoid system of mammalian brain (Adapted from Navarro and Fonseca, 1998).
Event Date
Medical, ceremonial and recreational uses of Cannabis 3000 B.C. onwards
Isolation of psychoactive cannabinoids 1964
Discovery of synthetic cannabinoids 1980 onwards
Discovery of the cannabinoid-1 receptor ( CB1-R) in mammalian brain
1988
Mapping of the CB1-R in mammalian brain 1990
Cloning of the CB1-R 1990
Neuropharmacology of the CB1-R 1988-1995
Discovery and isolation of a natural cannabinoid anandamide in brain
1992-1995
Synthesis of diarylpyrazole CB1 receptor antagonists, (e.g. SR141716A and AM251)
1994
Isolation and identification of 2-arachidonyl glycerol (2-AG) as another important endocannabinoid
1995-1997
Functional neuroanatomy of CB1 receptors 1996-1997
Delineation of anandamide biosynthesis and its mechanism of uptake
1997
5
1.2. Cannabinoids
Gaoni and Mechoulam (1964) identified ∆9-tetrahydrocannabinol (∆9-THC)
(Figure 1.2) as the main psychoactive component of Cannabis sativa, a discovery that
eventually led to the synthesis of various analogs of ∆9-THC (Howlett et al., 2002).
Compounds that mimic the actions of the cannabis derivative ∆9-THC are defined
as cannabinoids (Howlett et al., 2002). A critical advance in cannabinoid research
occured with the discovery of specific membrane receptors to which ∆9-THC actively
binds in brain tissue (Devane et al., 1988). Matsuda et al. (1990) cloned and
characterized the first cannabinoid-1 receptor (CB1-R) while a cannabinoid-2 receptor
(CB2-R) was identified by Munro et al. in 1993.
Before the discovery of these receptors, the psychoactive actions of ∆9-THC and
related cannabinoids were assumed to arise from their ability to 1) dissolve in lipids
(Seeman et al., 1972), 2) modify the fluidity of synaptic plasma membranes (Hillard et
al., 1985) and 3) intercalate with lipids and other components of neuronal plasma
membranes (Pertwee, 1988).
Both CB1-Rs and CB2-Rs belong to the rhodopsin-like subfamily of receptors
which are G protein-coupled receptors (GPCRs) with seven transmembrane spanning
domains (TMH1-7). CB1-Rs and CB2-Rs were found to be sensitive to inhibition by
Pertussis toxin treatment, indicating that the response to cannabinoid drugs was
mediated through the Gi/o family of G proteins (Howlett et al., 1986).
Moreover, both CB1-Rs and CB2-Rs are found to have varied tissue distributions
in vertebrates. CB1-Rs are densely located in many regions of the central nervous
system with much lower levels in kidney, testis, uterus, heart and vascular tissue. On
the other hand, CB2-Rs are abundantly expressed in tissues of the immune system,
including spleen, tonsils and haematopoietic cells, but are found at much lower levels in
central nervous system (CNS) (Kano et al., 2009; Brown, 2007).
6
∆9-Tetrahydrocannabinol (∆9-THC)
∆8-Tetrahydrocannabinol (∆8-THC)
Figure 1-2 Structure of two important phytocannabinoids. Structures redrawn using ChemDraw Ultra 11.0 from structures reported in Pertwee et al. (2010).
7
G protein-coupled CB1-Rs and G protein-coupled CB2-Rs are differentiated on
the basis of predicted amino acid sequence, signaling mechanisms, affinity towards
specific agonists and antagonists and tissue distribution. They each share 48% amino
acid sequence homology and both have their G proteins coupled to adenylyl cyclase and
mitogen-activated protein kinase (MAPK) (Howlett et al., 2002). The CB1-R is larger than
the CB2-R with 13 more amino acid residues on the C terminal, an extra 72 amino acid
residues on the N terminal and 15 additional residues on the third extracellular loop
(Childers, 2006).
These G protein-coupled cannabinoid receptors are activated by certain
cannabis-derived compounds as well as endogenous lipid molecules termed
endocannabinoids. The endocannabinoids, their receptors and associated biochemical
machinery (including precursors, critical biosynthetic enzymes, degradative enzymes,
mediators and transporters) collectively constitute the endocannabinoid system (ECS).
The ECS represents a highly conserved system within all vertebrate phyla as well as
some invertebrates, with subtle structural differences in the structure of receptors,
enzymes and other components, thus underscoring the importance of the ECS for
survival of many life forms (De Petrocellis et al., 2004).
1.2.1. G protein-coupled receptors (GPCRs) and their activation cycle
GPCRs are seven transmembrane spanning receptors and are coupled to
specific heterotrimeric guanine nucleotide-binding proteins (G proteins) (Drake et al.,
2006). G proteins transduce an extracellular signal to an intracellular effector (Drake et
al., 2006). These receptors represent an attractive target for drug discovery and it has
been estimated that nearly half of the drugs marketed today target GPCRs (Kroeze et
al., 2003).
G proteins are made up of a monomer (Gα subunit) and dimer (Gβ and Gγ
subunit). In their inactive state, the Gα subunit is bound to guanosine diphosphate (GDP)
and exists as Gα(GDP)βγ (Harrison and Traynor, 2003). When activated, the Gα subunit
exchanges GDP for guanosine-5’-triphosphate (GTP) and this binary complex (Gα-GTP)
then detaches from the Gβγ subunit to act on different effectors (Griffin et al., 1998;
8
Harrison and Traynor, 2003). Inactivation of GPCRs occurs by the intrinsic GTPase
activity of the Gγ subunit which hydrolyses GTP to GDP. Finally, Gα-GDP and Gβγ
subunits recombine to form the inactive Gα(GDP)βγ.
1.2.2. The [35S]GTPγS binding assay
The [35S]GTPγS binding assay is a functional assay that can be employed to
measure the extent of G protein activation following the binding of ligand(s) to the GPCR
(Breivogel and Childers, 2000; Breivogel et al., 2001; Harrison and Traynor, 2003). It is
an excellent assay to measure the primary functional event that immediately follows the
activation of the GPCR by its ligand (Harrison and Traynor, 2003).
This assay is characterized by the replacement of endogenous GTP by
[35S]GTPγS which binds to Gα subunit to form the Gα[35S]GTPγS complex. The γ-
thiophosphate bond on [35S]GTPγS is highly resistant to GTPase-mediated hydrolysis
and therefore inactivation of the GPCR cycle is blocked and the extent of activation can
be conveniently quantified by measuring the [35S]-label bound (Griffin et al., 1998;
Harrison and Traynor, 2003).
1.3. Other cannabinoid receptors
Besides CB1-Rs and CB2-Rs, other two GPCRs, GPR55 and GPR119 (Lambert
and Muccioli, 2007) have been proposed as novel cannabinoid receptors based on their
affinity towards endocannabinoids. There is much ongoing debate in the scientific
community regarding classification of these receptors as genuinely cannabinoid
selective (Okunu and Yokomizo, 2011). Oka et al., (2007) reported the activation of
GPR55 by lysophosphatidylinositol derivatives but not cannabinoids. In marked
contrast, Lauckner et al., (2008) found that AEA and ∆9-THC increased intracellular
calcium in a cell line expressing GPR55. Nevertheless, this review will be focused
mainly on CB1-Rs and CB2-Rs. Phylogenetic analysis by Brown (2007) revealed that
CB1-Rs and CB2-Rs belong to family of lipid receptors (formerly endothelial
differentiation gene receptors (EDG)) which are activated by acylethanolamide
9
analogues typified by 2-arachidonylglycerol (2-AG) and anandamide
(arachidonylethanolamide, AEA).
Sharir et al., (2010) described the work of researchers at AstraZeneca who found
that nanomolar concentrations of cannabinoid agonists stimulate [35S]GTPγS binding
and this response was antagonized by cannabidiol, a natural product cannabinoid
receptor antagonist. The ion channel, transient receptor potential vanilloid 1 (TRPV1)
share several similarities with cannabinoid receptors in terms of intracellular signaling,
shared ligand and tissue distribution, their role in pathophysiological conditions and
binding of the endocannabinoid/endovanilloid anandamide (AEA) (Starowicz et al.,
2007).
1.4. Cannabinoid-1 Receptors (CB1-Rs)
1.4.1. The structure and activation of CB1-Rs
The first cloning and expression of a 473 amino acid CB1-R from rat brain was
achieved by Matsuda et al. (1990) (Figure 1.3). The human homolog of 472 amino acids
was reported by Gerard et al. (1990) and a 473 amino acid sequence from mouse brain
was identified by Chakrabarti et al. (1995). Significantly these three CB1-Rs exhibit 97-
99% amino acid sequence homology (Kano et al., 2009). Like any GPCR, the CB1-R is
an integral membrane protein consisting of seven hydrophobic transmembrane helices
(7TMH) linked by three extracellular (E1-3) and three intracellular loops (I1-3) which are
flanked by an N-terminal on the periplasmic domain and a C terminal on the cytoplasmic
domain (Montero et al., 2005). Between the cytoplasmic extension of TMH7 and the
proximal C terminus lies another helix designated helix 8, which runs parallel to the
cytosolic membrane surface (Patny et al., 2006). The cytoplasmic regions are
responsible for G protein binding, desensitization and cellular signal trafficking. Binding
of an agonist triggers activation of the heterotrimeric G proteins by exchanging GTP for
GDP on the α subunit. This leads to the dissociation of G proteins from receptors and
cleavage of α and β/γ subunits which in turn modulate downstream effectors (Stadel et
al., 2011). According to the two state model proposed for GPCRs, CB1-Rs exist in the
active (R) state and the inactive (R*) state (Samama et al., 1993; Gullapalli, 2010).
10
These states are in equilibrium in the absence of ligand and, following ligand binding, the
equilibrium can shift to either state. Thus, an agonist will actively bind to the active state
of this receptor while an inverse agonist will bind to the inactive state. A classical
antagonist will be overall neutral having an equal affinity towards both R and R* the state
of the receptors. These properties can be conveniently studied in CB1-Rs due to the
availability of selectively acting agonists (e.g. ∆9-THC, CP55940 and WIN55212-2),
antagonists (e.g. cannabidiol and AM251) and the inverse agonist (SR141716A)
(Gullapalli, 2010; Gatley et al., 1997; Herkenham et al., 1990; Pertwee, 2006; Rinaldi-
Carmona et al., 1994). Studies by various research groups have greatly improved our
understanding the role of CB1 transmembrane helices (TMH), extracellular loops (ECs)
in particular the E2 loop and the carboxyl terminus in cannabinoid binding and receptor
activation (Shim et al., 2011a; Shim et al., 2011b, McAllister, 2003; Ahn et al., 2009;
Stadel et al., 2011). However, despite much effort, little information is available on the
way in which ligands orient and dock at their respective active sites within the CB1-R
binding pocket. Chin et al. (1998) and Song and Bonner (1996) showed that the
hydrogen bonding interaction between residue K192 on the TMH and the phenolic
hydroxyl group of CP55940 and the carboxamide oxygen of the inverse agonist
SR141716A (Hurst et al., 2002, 2006) were critical for ligand binding. Moreover, residue
S383, which has been proposed to induce a bend in the TMH7, again appears essential
for agonist (CP55940) binding (Kapur et al., 2007). In addition, C386 (on TMH7) has
also been implicated as a critical amino acid for SR141716A binding (Fay et al., 2005).
McAllister et al., (2003) demonstrated that aromaticity at TMH5 (imparted by
residues such as F201, W280 and W357) was important for accomodating the agonist
CP55940 and inverse agonist SR141716A within the binding pocket of the CB1-R. By
employing molecular modeling, McAllister and associates also proposed that the binding
pocket for various ligands was primarily located in the hydrophobic transmembrane helix
bundle of the receptor. In related studies, Shi and Javitch (2004) found that the
extracellular loops also play a vital role regards ligand recognition, ligand sensitivity and
access of the ligand to the binding pocket of dopamine receptors (which are also a
GPCRs). This observation also proved relevant for CB1-Rs, since Ahn and coworkers
(2009) showed that the second extracellular loop (E2) was important for ligand binding
and receptor localization. They were also able to demonstrate that alanine (Ala)
11
mutations in the C terminal residues on E2 led to reduced agonist binding but had no
effect on the binding of inverse agonists. In addition, Bertalovitz et al. (2010) reported
that point mutations on the C terminal region of E2 can lead to loss of agonist and
antagonist binding capacity of CB1-Rs but have no effect on inverse agonist binding.
Shim et al. (2011) further elucidated the structure of the E2 loop by using a
combination of simulated annealing and molecular dynamics simulations. They studied
the molecular structure of E2 in two forms, disulphide (E2disulphide) and dithiol (E2dithiol). It
was found that that E2disulphide helical segment has a amphipathic alignment (at the
membrane:water interface) which stabilizes the receptor and imparts greater flexibility
compared to E2dithio. This further led to E2/TMH coupling and rearrangement of the TMH.
These coupling/interactions and TMH rearrangement are important for receptor
activation. However, the extent of this coupling was distinct in both the forms of E2.
Since in CB1 E2disulphide E2 offers more flexibility, the C terminal region of E2 inserts into
the extracellular H3/H5 region causing H5 to move away from H3 and H6 to move into
H3 at the extracellular region, leading to efficient coupling of E2 to TMH. However, in
CB1 E2dithiol, E2 has reduced flexibility leading to less insertion of E2 C terminal residues
and thus weak coupling of E2/TMH. This work confirmed the importance of the E2 C
terminal for receptor activation and also served as supporting evidence of the original
findings by Ahn et al. (2009).
12
Figure 1.3 Two dimensional representation of the CB1-R (Adapted from Shim et al., 2011).
13
Figure 1.4 Diagramatic representation of the C terminal domain of the CB1-R (Adapted from Stadel et al., 2011)
14
The C terminal is responsible for interaction with G proteins, CRIP 1a, protein
kinases, arrestins and with itself or other receptors to form a dimer (Ahn et al., 2009)
(Figure 1.4). Several characteristic features of the C terminal sequence such as
transmembrane interaction sites, palmitoylation sites, phosphorylation sites and the PDZ
binding domain make it a potential candidate for roles in biogenesis, receptor localization
and activity (Ahn et al., 2009).
The CB1-R carboxy terminus has 73 amino acid residues (R400-L472) and differs
from CB2-R by being 14 residues longer. Despite rough similiarities in length, the sharing
of a few ligands and participation in similar signal pathways, there is no significant
homology between the C termini of these receptors (Bramblett et al., 1995; Xie and
Chen, 2005; Choi et al., 2005). Hydropathy plot analyses conducted by Kyte and Dolittel
(1982) predicted the C terminus of the CB1-R to be less hydrophobic than the equivalent
region of CB2-R, but there remained difficulties in purifying the full length CB1 terminus
because of its high flexibility and relatively unstructured nature. Not until 2009 was this
problem overcome when successful purification of a peptide corresponding to the full
length CB1-R carboxy terminus was achieved. NMR analysis using doubly tagged (15N
and 13C) full length C terminus in dodecylphosphocholine revealed the presence of two
amphipathic α helical domains (Ahn et al., 2009). Importantly, Ahn and associates were
also successful in identifying the specific amino acids of these helical domains (S401-
F412 and A440-M461, respectively) thus suggesting an amphipathic role for each. They
also reported that the hydrophobic face of each helix was intimately associated with the
membrane surface, thus stabilizing the helical domains for binding with other proteins
involved in receptor function, while the polar face of the helices was able to project into
the cytosol (Ahn et al., 2009). Besides its role in receptor function, the C terminus has
been found to be a requirement for receptor exit from endoplasmic reticulum (Tai et al.,
1999; Bermak et al., 2001; Duvernay et al., 2004, Robert et al., 2005).
The cytoplasmic extension of TMH7 is characterized by the presence of a highly
conserved motif within the rhodopsin class A GPCRs, NPXXY and is termed Helix 8
(Patny et al., 2006; Tiburu et al., 20011). This amphipathic helical domain is an integral
part of the intracellular GPCR binding connection to G proteins (Tiburu et al., 2011;
Rosenbaum et al., 2009, Fritze et al., 2003) and is reported to play a crucial role both in
15
ligand recognition and signal transduction (Tiburu et al., 2011). The distal region on the
C-terminus carries another helical domain, Helix 9. However, Helix 9 has only recently
been identified within the CB1-R and very little work has so far been done in defining the
precise structure and role of this helix (Ahn et al., 2010).
TMH7/H8 region has always been an area of interest in probing various structural
determinants involved in activation of the GPCR cycle. Tiburu et al. (2011), using solid
state nuclear magnetic resonance (NMR) and site directed spin labeling-electron
paramagnetic resonance (SDSL/EPR) demonstrated short range electrostatic
interactions between TMH/H8 (with its conserved motif and proline kink) and the
phospolipid bilayer/membrane microenvironment. By employing local helix distortion
studies, they postulated that the conserved but flexible NPXXY motif likely plays a vital
role in ligand binding and signaling events involving TMH7/H8 (Tiburu et al., 2011;
Tiburu et al., 2009; Tyukhtenko, 2009; Hall et al., 2009). Furthermore, experiments
conducted by Tiburu et al. (2011) demonstrated dynamic functional interactions between
TMH7/H8 and the membrane phospholipid environment which modifies the membrane
bilayer structure at discrete loci. These findings hinted at a potential mechanochemical
role of the phospholipid bilayer in mediating CB1/GPCR signal transmission and hence
signal transduction.
Helix 8 is also reported to have a contributory role towards receptor signaling by
interaction with intracellular loops (ICs). This helix interacts with distal part of the C
terminus, offering it rigidity and potential to interact with the third intracellular loop (IC3).
This interaction is essential for proper receptor signaling (Ahn et al., 2010). Moreover,
Swift et al., (2006) identified various noncovalent interactions between helix 8, TMH 7
and IC1, which again were important for receptor signaling. Several studies in various
laboratories indicated a possible role of Helix 8 in receptor biosynthesis, folding and
trafficking (Oksche et al., 1998; Duvernay et al., 2004; Thielen et al., 2005).
Studies by Ahn et al., (2010) helped to understand the importance of this helical
domain (H8) for ligand binding and activation. As mentioned earlier, this helix is
amphipathic with leucines and/or phenylalanines and basic residues common to both
hydrophobic and hydrophilic faces. Ahn and coworkers employed point mutations in
several key residues on both faces. The first mutant involved substitution of the
16
hydrophobic groups, Leu404, Phe408 and Phe412 with alanine to reduce hydrophobicity
while the second involved replacement of the basic residues, Lys402, Arg405 and
Arg409 with glutamine to remove positive charge. The first mutant yielded low Bmax
values (based on saturation binding isotherms), minimal Emax (from [35S]GTPγS binding
studies) and defective localization when compared to the wild type CB1-R. Intriguingly,
the second mutant was virtually identical when compared with the wild type with respect
to the same parameters as the first mutant. Circular dichroism spectroscopy further
revealed that intact hydrophobic residues were indeed vital for maintenance of the helix
while positively charged residues could be easily replaced by less polar or neutral
residues without affecting the structure/function of the helix. These data supported the
importance of the formation of this helical domain for receptor localization and hence
ligand binding and activation. Moreover, various other groups have independently
reported that a defective H8 leads to impaired receptor localization and β arrestin
translocation to the plasma membrane (Suvorova et al., 2009; Yasuda et al., 2009; Ahn
et al., 2010).
Apart from this, the L404F point mutation on H8 domain displayed faster agonist
induced internalization when compared to the wild type, thus underlying its importance in
CB1-R trafficking (Anavi-Goffer et al., 2007).
Helix 9 (H9) consists of charged residues which have been implicated in the
formation of contacts with the cytoplasmic helical extension of TMH5, TMH6 and IC3.
Schertler (2008) suggested the probable role of hydrophobic residues on H9 as a point
of contact with the G protein (Gq).
Besides, extracellular loops, TMH/H8 and H9, distal C terminus ((∆418-472) of
the CB1-R have also been implicated in receptor localization, receptor stability, G protein
binding, desensitization, intracellular sorting during internalization and cellular trafficking
of the receptor (Stadel et al., 2011; Ahn et al., 2009).
Truncation of the CB1 distal C terminal domain led to changes in the magnitude
and kinetics of Ca2+ current inhibition via G protein coupling, suggesting the role of this
domain in cellular signal regulation (Nia and Lewis, 2001). Chillakuri et al. (2007)
demonstrated that after deletion of this domain of the CB1-R expressed in insect cells,
17
Sf9 resulted in a two-fold increase in receptor production and increased basal activity as
compared to wild type. Moreover, residues 418-439 on this domain were found to be
important for receptor desensitization but not internalization, while internalization was
affected when residues within the 460-473 were phosphorylated (Hsieh et al., 1999; Jin
et al., 1999).
Despite the accumulation of much data concerning the role of various domains of
theCB1GPCR, much more research is needed to completely understand the subtle
complexities of this fundamental signaling unit.
1.4.2. The distribution of CB1-Rs in mammalian brain
Understanding the distribution pattern of cannabinoid receptors in brain was
made possible by employing ligand binding studies with the highly specific synthetic
agonist [3H]CP55940 (Herkenham et al., 1991; Herkenham et al., 1990; Mailleux and
Vanderhaeghen, 1992).
The binding of [3H]CP55940 was found to be widely distributed with intensities
dependent on the area of brain concerned, with the general pattern of binding conserved
across mammalian species. The inner most layers of the olfactory bulb, the
hippocampus (in particular the dentate molecular layer and the CA3 region), the lateral
striatum, globus pallidus, entopenduncular nucleus, substantia nigra, pars reticularis and
the cerebellar molecular layer displayed the highest levels of binding of [3H]CP55940,
while moderate levels were found in the cerebral cortex, septum amygdala,
hypothalamus, lateral subnucleus of interpeduncular nucleus, parabrachial nucleus,
nucleus of the solitary tract and the spinal dorsal horn. Low ligand binding was noted
particularly in the thalamus, and various other nuclei in the brain stem and the spinal
ventral horn (Kano et al., 2009; Herkenham, 1990).
The telencephalic and cerebellar regions, where high intensity binding of
[3H]CP55940 occurs, control motor and cognitive function which can explain the
profound effect of cannabinoids on motor and cognitive responses, while lower brain
stem which controls cardiovascular and respiratory functions exhibited low density
binding. This latter distribution is compatible with the fact that high doses of
cannabinoids are not fatal (Herkenham et al., 1991; Mailleux and Vanderhaeghen,
18
1992). Similarly, moderate binding in spinal dorsal horn is consistent with the analgesic
action of intrathecally administered cannabinoid. Moreover, the anti-anorexic and anti-
emetic actions of cannabinoid agonists are likely achieved by moderate binding in the
ventromedial hypothalamic nucleus (located in the hypothalamus and amygdala) which
forms a major part of the satiety center (Kano et al., 2009).
The CB1-R expression pattern has been further investigated by histochemical
analysis. Depending on the region of brain, two distinct forms or labelings of CB1-R
mRNA expression have been reported, specifically, uniform labeling and non-uniform
labeling (Mailleux and Vanderhaeghen, 1992; Matsuda et al., 1993). Uniform labeling
reflecting a large number of cells expressing high level of CB1-R mRNA was detected in
the striatum, thalamus, hypothalamus, cerebellum and lower brain stem. In contrast,
non-uniform labeling resulting from a low number of cells expressing high levels of CB1-
R mRNA was found in the cerebral cortex, amygdala and hippocampus (Kano et al.,
2009). Furthermore, CB1-R expression is invariably greater at inhibitory synapses than
excitatory synapses; however the density of receptors at inhibitory synapse varies
considerably according to brain region. CB1-R density as assessed by immunogold
labeling was 30 times higher on inhibitory synapses than excitatory ones for
hippocampal CA1 pyramidal cells, 6 times for Purkinje cells and nearly 4 times higher for
striatal medium spiny neurons (Kawamura et al., 2006; Uchigashima et al., 2007).
1.5. CB1-R-mediated intracellular signaling pathways
1.5.1. Inhibition of cyclic AMP (cAMP) Activation of the CB1-R following agonist binding triggers a cascade of multiple
signal transduction pathways, a finding supported by [35S]GTPγS assays and studies
examining Pertussis toxin sensitivity of cannabinoid-dependent effects (Pertwee, 1997).
The first characterized pathway for agonist-stimulated CB1-R activation was the
inhibition of adenylyl cyclase and this effect was completely blocked by Pertussis toxin
indicating that it was mediated through Gi/o proteins (Howlett, 1985; Howlett and Fleming,
1984; Howlett et al., 1986). Further evidence towards the role of CB1-R and Gi/o proteins
was supported by work of Derkinderen and co-workers (1996 and 2001) who reported
19
that cannabinoid receptor stimulation led to Tyr-phosphorylation of the focal adhesion
kinase in hippocampal slices and this effect was readily blocked by both SR141716A
and Pertussis toxin.
In their activated states, Gi/o proteins were found to regulate adenylyl cyclase
isoforms 1,3,5,6 or 8 since co-expression of these isoforms resulted in CB1-R-mediated
inhibition of cAMP accumulation (Rhee et al., 1998).
Depending upon the precise signal transduction pathway, different subtypes of
Gi/o were found to be activated by the same ligand, agonist, antagonist or inverse agonist
indicating a CB1-R-biased signaling mechanism (Kenakin, 2007; Houstan and Howlett,
1998; Glass and Northup, 1999; Mukhopadhyay et al., 2002).
In N18TG2 cells, WIN55212-2 displayed agonist behavior for Gi1, Gi2, and Gi3
while desacetyllevonantradol (a CB1-R agonist) was an agonist at Gi1, Gi2 and an inverse
agonist at Gi3. Methanandamide (an agonist and stable analog of anandamide) behaved
as an inverse agonist at Gi1 and Gi2, but it displayed full agonist activity at Gi3.
Interestingly, SR141716A acted as an inverse agonist at all three G protein subtypes
(Mukhopadhyay and Howlett, 2005; Turu and Hunyady, 2010). Furthermore, Houston
and Howlett (1998) suggested the existence of different affinity states for the CB1-R
representing different conformations. This proposal was supported by Georgieva and co-
workers (2008) where, using plasmon-waveguide resonance spectroscopy they found
that the CB1-R assumed distinctly different conformations when bound by CP55940 or
WIN55212-2.
Deadwyler et al. (1995) and Hampson et al. (1995) reported that in hippocampal
cells, agonist-mediated stimulation of the CB1-R decreased intracellular cAMP, lowered
net dephosphorylation of ion channels, activated A-type potassium currents and in turn
led to hyperpolarization of the membrane. The cyclic AMP-dependent protein kinase
(PKA) pathway as regulated by CB1-Rs is intimately involved in synaptic plasticity and
neuronal remodeling (Howlett, 2005).
20
1.5.2. Stimulation of cAMP production
Intriguingly and in contrast to the above studies, CB1-R-mediated increases in
cAMP concentrations have also been observed in response to treatment with
cannabinoids (Howlett, 2005). Maneuf and Brotchie (1997) found an increase in basal
cAMP production in globus pallidus slice preparations treated with cannabinoid agonists
such as CP55940 and WIN55212-2.
Surprisingly, the order of potency of these agonists was similar in mediating this
effect through the CB1-R, and SR141716A was a competitive inhibitor for both inhibitory
and stimulatory components of this mechanism (Bonhaus et al., 1998).
Three mechanisms have been put forward by different research groups to
explain this observation. The first mechanism suggested the possibility of endogenous
synthesis of an adenylyl cyclase activator possibly CB-R-mediated synthesis of
prostaglandins (Burstein et al., 1986, 1994). Here prostaglandins might operate as
cellular stimulators for cannabinoid-mediated cAMP production (Hillard and Bloom,
1983).
The second possibility relates to potential differences in the type of adenylyl
cyclase isoforms expressed by the target cells and the capacity of expressed isoform to
respond to Gi/o-mediated regulation (Howlett, 2005). Agonist stimulation of recombinant
cannabinoid receptors co-expressing adenylyl cyclase isoforms of the 5/6 family or the
1/3/8 family displayed inhibition of adenylyl cyclase as a result of inhibition by Gi (α-
subunit), while those expressing isoforms of the 2/4/7 family led to stimulation of
adenylyl cyclase as a result of the increased Gs response by the Gi (βγ subunit) released
following cannabinoid receptor stimulation (Rhee et al., 1998).
The third mechanism could involve direct interaction between CB1-Rs and Gs. It
was reported that neurons and CHO cells expressing recombinant CB1-Rs pre-treated
with Pertussis toxin, supported cannabinoid agonist-mediated stimulation of cAMP
(Glass and Felder, 1997; Felder et al., 1998; Bonhaus et al., 1998)
21
1.5.3. CB1-Rs and the modulation of Ca2+ fluxes and phospholipases C and A
Sugiura and co-workers (1996, 1997, 1999) using a fura-2 fluorescence assay,
reported cannabinoid and endocannabinoid-mediated increases in intracellular free
calcium in N18TG2 neuroblastoma and NG108-15 neuroblastoma-glioma hybrid cells.
This response was due to a CB1-R and Gi/o interaction since it was blocked by both
SR141716A and Pertussis toxin. Other data also support the ability of cannabinoid
receptors to signal through an inositol 1,4,5-triphosphate (IP3)-Ca2+ mobilization pathway
(Howlett, 2005). Ca2+ mobilization in the N18TG2 neuroblastoma cell line could be
blocked by a phospholipase C (PLC) inhibitor, suggesting that PLCβ could be the
effector (Sugiura et al., 1996, 1997). Netzeband and co-workers (1999) found that
activation of CB1-Rs caused an increase in Ca2+ levels in response to depolarization via
glutamate receptor activation or high K+ which primarily resulted in mobilization of Ca2+
from caffeine-sensitive and IP3 receptor-sensitive stores. This signal was reduced by
SR141716A, Pertussis toxin and a PLC inhibitor, again suggesting that this Ca2+
mobilization originated from a CB1-R dependent PLC mechanism (Netzeband et al.,
1999).
Cannabinoid receptors have also been implicated in the regulation of
phospholipase A2 activity. Cannabinoid pre-treatment of several cultured cell types has
been shown to release arachidonic acid, and this effect is believed to be mediated by
phospholipase (Burstein 1991; Burstein et al., 1994; Shivachar et al., 1996).
1.5.4. CB1-R-dependent regulation of ion channels
Agonist-mediated stimulation of CB1-Rs results in activation of G protein-coupled
inwardly-rectifying potassium channels (GIRKs) (Henry and Chavkin 1995; Mackie et al.,
1995) and inhibition of L-type (Gebremedhin et al., 1999), N-type (Mackie and Hill, 1992)
and P/Q -type (Mackie et al., 1995) calcium channels. When treated with anandamide or
WIN55212-2, AtT-20 pituitary tumor cells expressing CB1-Rs, were found to activate
inwardly rectifying potassium currents (Kir) and this response was Pertussis toxin-
sensitive, indicating the involvement of Gi/o proteins (Mackie et al., 1995; Henry and
Chavkin, 1995; McAllister et al., 1999). McAllister and co-workers (1999) also
demonstrated the activation of GIRKs in the Xenopus oocyte system.
22
The agonist stimulation of CB1-Rs expressed in N18 neuroblastoma and NG108-
15 neuroblastoma-glioma hybrid cells resulted in inhibition of N-type voltage gated Ca2+
channels (Mackie and Hill, 1992; Mackie et al., 1993; Howlett, 2005). Fura-2
fluorescence studies also confirmed that anandamide and 2-AG inhibited the
depolarization-induced intracellular free Ca2+ increase in NG108-15 cells (Sugiura et al.,
1997). When treated with WIN55212-2 and anandamide, AtT-20 pituitary cells
expressing recombinant CB1-Rs, were found to inhibit Q-type Ca2+ channels (Mackie et
al., 1995). Pertussis toxin sensitivity suggested the mediation of this response through
Gi/o proteins. Hampson and coworkers (1998), using the fura 2 assay, showed that P/Q-
type Ca2+ fluxes are also inhibited by anandamide and this inhibition was suppressed by
SR141716A and Pertussis toxin, again suggesting the involvement of CB1-R and Gi/o
proteins. Similar results were reported for inhibition of L-type Ca2+ currents in cat brain
arterial smooth muscle cells expressing CB1-Rs by WIN55212-2 and anandamide and
these effects were inhibited by SR141716A and Pertussis toxin.
1.5.5. Involvement of CB1-Rs in the suppression of neurotransmitter release
Gill et al. (1970) first reported the CB1-R-mediated inhibition of transmitter
release in guinea pig ileum by investigating electrically-evoked twitch response
recordings. Since then, numerous studies using electrophysiological and biochemical
techniques have been carried which demonstrate CB1-R-mediated suppression of
transmitter release (Schlicker and Kathmann, 2001).
CB1-Rs control the release of several neurotransmitters in mammalian brain
including glutamate (Levenes et al., 1998), GABA (Szabo et al., 1998), glycine (Jennings
et al., 2001), acetylcholine (Gifford et al., 1996), norephinephrine (Ishac et al., 1996),
dopamine (Cadogan et al., 1997), serotonin (Nakazi et al., 2000) and cholecystokinin
(CCK) (Beinfeld and Connolly, 2001).
Shen and co-workers (1996) using cultured hippocampal neurons were the first
to gain evidence for the suppression of glutamate release by a cannabinoid agonist.
They found that WIN55212-2 decreased excitatory postsynaptic currents (EPSCs) thus
indicating a reduction in neurotransmitter release. Importantly, this suppression of
EPSCs was reversed by SR141716A demonstrating involvement of CB1-Rs. Schlicker
23
and Kathamann (2001) detected a similar kind of CB1-R-mediated decrease in
neurotransmitter release in the cerebellum, the cortex and the striatum. Wang (2003)
using rat brain synaptosomes demonstrated a CB1-R agonist-mediated decrease in 4-
AP-evoked L-glutamate release in response to low micromolar concentrations of
WIN55212-2. This decrease was concentration-dependent and was reversed by the
CB1-R antagonist AM281. Similar results were obtained by Godino et al. (2007), where
they showed a moderate (approx. 30%) decrease in KCl-evoked L-glutamate release by
low micromolar concentrations of WIN55212-2, this effect again being reversed by low
micromolar concentrations of AM281.
Interestingly, similar effects were noted on GABA release by CB1-R agonists in
the striatum, substantia nigra, pars reticularis, hippocampus, nucleus accumbens and
cerebellum (Kano et al., 2009). In these neurons, GABAergic inhibitory postsynaptic
currents (IPSCs) were effectively suppressed by WIN55212-2, but the postsynaptic
response to muscimol (a GABAA receptor agonist) was unaffected, clearly pointing to a
presynaptic locus of action. This effect was also blocked by SR141716A, strongly
supporting the role of CB1-Rs (Chan and Yung, 1998).
Several possible mechanisms have been proposed by various research groups
regarding the molecular targets involved in CB1-R-mediated suppression of transmitter
release (Schlicker and Kathmann, 2001).
The first mechanism advanced involved the possible involvement of voltage-
dependent Ca2+ channels. It was found that cadmium, a non-selective blocker of Ca2+
channnels, readily reduced or blocked the WIN55212-2-mediated inhibitory effects on
spontaneous EPSCs in rat striatal slices and spontaneous GABAergic IPSCs in rat
hippocampal slices (Huang et al., 2001; Hoffman and Lupica, 2000).
Besides regulation via voltage-gated Ca2+ channels, evidence also exists for
involvement of sites downstream of the Ca2+ channels. Takahashi (2000) conducted
electrophysiological experiments and reported that Ca2+-independent miniature EPSCs
and miniature IPSCs were reduced by a cannabinoid receptor agonist indicating a direct
influence on release machinery. Another possible mechanism is through involvement of
K+ channels, including GIRKs and KA channels (Pertwee, 1997; Ameri, 1999), since the
24
inhibitory effects of WIN55212-2 on evoked EPSCs in the mouse nucleus accumbens
were reduced by BaCl2 (a KA channel blocker) or 4-aminopyridine (4-AP) (a GIRK
channel blocker) and completely blocked when BaCl2 and 4AP were co-administered
(Robbe et al., 2001).
However, the complexity of the target systems impacted by cannabinoids
increases further when sodium channel involvement is considered. Nicholson et al.
(2003), Liao et al. (2004) and Duan et al. (2008) in our laboratory showed that
endocannabinoid anandamide and synthetic cannabinoid agonists WIN55212-2,
CP55940 and AM404 along with the cannabinoid antagonists AM251 inhibit sodium
channels at low micromolar concentrations. Hence more studies are needed to fully
elucidate and understand this complex phenomenon of suppression of neurotransmitter
release by cannabinoid and cannabimimetic drugs.
1.6. Homodimerization and heterodimerization of CB1-Rs
Another intriguing aspect of the CB1-R is its capacity to undergo dimerization in
the presence of other receptors (Turu and Hunyady, 2010). Wager-Miller et al. (2002)
were first to show homodimerization of the CB1-R where they showed a high molecular
weight band of a dimerized CB1-R using Western blotting. Resonance energy transfer
studies have also revealed the heterodimerization of CB1-Rs with D2 and A2A receptors
(Carriba et al., 2008) and also with opiate receptors (Rios et al., 2006) in transfected cell
lines. Kearn et al. (2005) reported an interesting interaction between heterodimerized
CB1 and D2 receptors. They noted that independent activation of both receptors resulted
in decreased forskolin-induced adenylyl cyclase activity. However, when co-expressed,
activation of the CB1-R reversed the inhibition caused by D2 stimulation and in turn led
to elevated cAMP levels (Kearn et al., 2005).
Furthermore, there are also reports of coupled trafficking of CB1-Rs to and from
the plasma membrane as a consequence of dimerization (Ellis et al., 2006). Orexin-1
receptor distribution in cells was found to be dramatically altered (from plasma
membrane to intracellular vesicles) following the coexpression of CB1-Rs in same
system (Ellis et al., 2006)
25
To summarize, dimerization is an emerging and interesting area for further
exploration of CB1-R activity in relation to regulation of endocannabinoid system.
1.7. Constitutive activity of CB1-Rs
Like several other GPCRs, CB1-Rs have been found to exhibit constitutive
activity wherein they show high basal activity in absence of agonist binding, both in
expression systems and in native tissues (Gifford and Ashby, 1996; Turu and Hunyady,
2010; Bouaboula et al., 1997; Rinaldi-Carmona, 1998; Pertwee, 2005). Moreover, the
CB1-R inverse agonist, SR141716A has been found to act on certain other receptors
(Savinainen et al., 2003; Lauckner et al., 2008). This further complicates the
interpretation of data. Hence the introduction of a neutral antagonist can help study the
constitutive property of CB1-Rs (Turu and Hunyady, 2010). Neutral antagonists can be
defined as agents or compounds that do not change the basal activity of CB1-Rs (Turu
and Hunyady, 2010). This definition remains valid if there is an assumption of the
existence of CB1 constitutive activity however, if this activity was caused due to
endocannabinoids innately present in tissues, then these neutral antagonists can be
referred as partial agonists. Hence, more studies are warranted in this area of
cannabinoid research to fully elucidate the constitutive versus basal activity of the CB1-
Rs (Turu and Hunyady, 2010).
1.8. The biochemistry of endocannabinoids
The first endocannabinoid was isolated from porcine brain and was named
anandamide (arachidonyl ethanolamide, from the Sanskrit word ‘anand’ means bliss or
happiness) (Figure 1-5) (Devane et al., 1992).
Sugiura and co-workers (2002) found that anandamide was a partial agonist at
cannabinoid receptors and a full agonist at transient receptor potential vanilloid receptors
1 (TRPV1) (Kano et al., 2009). Another breakthrough was the discovery of another major
endocannabinoid, 2-arachidonyl glycerol (2-AG) which was isolated from canine gut and
rat brain (Mechoulam, 1995; Sugiura et al., 1995). 2-AG is more abundant in the brain
26
and acts as a full agonist at cannabinoid receptors and hence can be termed as true
natural endocannabinoid (Sugiura et al., 2006).
Other endocannabinoids so far identified are dihomo-γ-linolenoyl ethanolamide
(Hanus et al., 2001), decosatetraenoyl ethanolamide (Hanus et al., 2001), 2-AG ether
(noladin ether) (Hanus et al., 2001), O-arachidonylethanolamine (virodhamine) (Porter et
al., 2002) and N-arachidonoyldopamine (Huang et al., 2002). See Figure 1-5 for
structures.
Dihomo-γ-linolenoyl ethanolamide and decosatetraenoyl ethanolamide belong to
a broad family of N-acylethanolamides like anandamide. They are present in mammalian
brain and bind to CB1-Rs with medium affinity (Felder et al., 1993; Hanus et al., 1993)
and with lower affinity to CB2-Rs (Felder et al., 1995) respectively.
Noladin ether was isolated from pig brain and was found to bind CB1-Rs with
medium affinity, although this compound has lower affinity for CB2-Rs (Hanus et al.,
2001). Virodhamine (from Sanskrit word, ‘Virodh’ meaning opposition or antagonism)
was isolated from rat brain (Porter et al. (2002) and acts as an antagonist or a partial
agonist at the CB1-R but as a full agonist at CB2-Rs. N-Arachidonoyldopamine, first
identified in bovine and rat nervous tissue, displays agonist actions on both the
endocannabinoid and endovanilloid system.
27
Anandamide 2-Arachidonoyl glycerol ether 2-Arachidonoyl glycerol
Virodhamine Dihomo-γ-linolenoyl ethanolamide
Docosatetraenoyl ethanolamide N-Arachidonoyl dopamine
Figure 1.5 Structures of prominent endocannbinoids (All structures redrawn using ChemDraw Ultra 11.0 from Kano et al., 2009).
28
Among all of these endocannabinoids, anandamide and 2-AG have been widely
accepted as the major endocannabinoids acting on CB1-Rs. Hence, although several
reviews are available (Basavarajappa, 2007; Bisogno et al., 2005; Okamoto et al., 2007;
Sugiura et al., 2006; Vandevoorde and Lambert, 2007; Kano et al., 2009) that explore
the metabolic pathways for formation and degradation of a whole range of potential
endocannabinoids, only anandamide and 2-AG will be discussed further.
1.8.1. Anandamide biosynthesis
Di Marzo and co-workers (1994) were the first to show the Ca2+ ionophore
ionomycin or high K+ depolarization causes anandamide production in rat striatal or
cortical neurons. The extracellular Ca2+ chelator EGTA effectively abolished anandamide
production indicating a role of Ca2+ entry in promoting this mechanism. Interestingly,
Stella and Piomelli (2001) noted that carbachol-evoked generation of anandamide was
not blocked by EGTA (an extracellular Ca2+ chelator) but was effectively blocked by
BAPTA-AM (a membrane permeable Ca2+ chelator). Besides these biochemical
experiments, electrical stimulation has also been shown to raise anandamide levels in
rat hypothalamic slices as measured by mass spectrometric analysis (Di et al., 2005).
The classical ‘transacylation-phosphodiesterase pathway’ has been suggested
for biosynthesis of anandamide (Figure 1.6).
29
Figure 1.6 Transacylation-phosphodiesterase pathway for biosynthesis of anandamide (Adapted from Cadas et al., 1997).
30
This is a two step enzyme-catalyzed reaction where the initial step involves
transfer of an arachidonate group from the sn-1 position of the phospholipids to the
primary group of phosphatidylethanolamine (PE) in the presence of the enzyme N-
acyltransferase (NAT), yielding N-arachidonyl PE or NAPE (Figure 1.6). The second
step involves the catalytic hydrolysis of NAPE to anandamide and phosphatidic acid in
the presence of N-acylphosphatidylethanolamine-hydrolysing phospholipase D (NAPE-
PLD) enzyme. The rate limiting step in anandamide synthesis is the NAT activity which
is regulated by Ca2+ (Kano et al., 2009). The anandamide formed is then presumed to
diffuse from the membrane into the surrounding medium (Kreitzer and Regehr, 2002).
1.8.2. 2-Arachidonoyl glycerol (2-AG) biosynthesis
Unlike anandamide, 2-AG displays full agonism at the CB1-R. Electrical
stimulation (Stella et al., 1997) in hippocampus as well as ionomycin treatment in
N18TG2 neuroblastoma cells (Bisogno et al., 1997) leads to increased levels of 2-AG.
Several biochemical pathways have been suggested for biosynthesis of 2-AG
(Figure 1-7), however the more important pathway involves phospholipase C (PLC) and
diacylglycerol lipase (DAGL) (Kano et al., 2009). The first step involves formation of
diacylglycerol (DAG) (which has an arachidonic acid moiety) by enzymatic hydrolysis of
arachidonic acid containing membrane phospholipids (like phosphatidylinositol) by PLC.
In the second step, DAGL acts on DAG to generate 2-AG (Kano et al., 2009). Other
biosynthetic pathways of possible relevance include those reactions leading to the
production of lysophospholipid from membrane phospholipid initially mediated by
phospholipase A1 (PLA1) and final formation of 2-AG by the action of Lyso-PLC on PLA1
(Sugiura et al., 1995; Tsutsumi et al., 1994; Ueda et al., 1993). Nakane et al. (2002)
suggested that 2-AG could also be formed by the action of a phosphatase on 2-
arachidonoyl lysophosphatidic acid (2-arachidonoyl LPA), while the generation of 2-
arachidonoyl phosphatidic acid from 1-acyl-2-arachidonoylglycerol has been suggested
by Bisogno et al. (1999) and Carrier et al. (2004) (Figure 1.7).
31
Figure 1.7 Metabolic pathways for biosynthesis of 2-AG (Adapted from Kano et al., 2009).
32
1.9. Degradation pathways for endocannabinoids
After endocannbinoids are released they can be degraded by two main
mechanisms i.e. hydrolysis and oxidation (Vandevoorde and Lambert, 2007). The
hydrolytic pathway involves the breakdown of anandamide by the action of fatty acid
amide hydrolase (FAAH) and 2-AG by monoacylglycerol lipase (MAGL) while the
oxidative pathway includes oxidation of the arachidonic acid moiety of endocannabinoids
by cyclooxygenase (COX) and lipoxygenase (LOX).
FAAH was first identified, purified and cloned from rat liver by Cravatt et al.
(1996). Formerly named as ‘anandamide amidohydrolase’, FAAH was identified in the
brain and many other organs. FAAH was reported to sensitive to the serine protease
inhibitor phenylmethylsulfonyl fluoride (PMSF), and was also found to act on other fatty
acid amides but with anandamide as the preferred substrate (Cravatt et al., 1996;
McFarland and Baker, 2004). In vitro studies have revealed that FAAH has the ability to
hydrolyse the ester linkage of 2-AG; however, this activity was minimal in vivo (Cravatt et
al, 1996). However, the importance of FAAH in anandamide breakdown was underlined
further when FAAH knockout mice were reported to be more responsive towards
exogenously administered anandamide (Cravett et al, 2001).
Tornqvist and Belfrage (1976) were the first to describe MAGL but this enzyme
was not cloned until 1997 by Karlsson and co-workers (1997) who cloned it from a
mouse adipocyte cDNA library. In vivo, MAGL is found to be the main enzyme that
catalyzes the hydrolysis of 2-AG (Dinh et al., 2002; Dinh et al., 2004; Vandevoorde and
Lambert, 2007). MAGL has 303 amino acids and is present in various organs including
brain. This enzyme accounts for about 85% of 2-AG degradation, while the remainder is
attributed to two less characterized enzymes namely, ABHD6 and ABHD12 (Blankman
et al., 2007).
Among the three known forms of COX enzymes in mammalian tissues, COX-2
accepts anandamide as a substrate generating prostaglandin-ethanolamides
(Vandevoorde and Lambert, 2007). Anandamide and 2-AG also apparently serve as a
33
substrates for LOX, however much less work has been done in this area (Chen et al.,
1994).
1.10. Transport of endocannabinoids
A two step process mediates the extracellular removal of endocannabinoids, the
first being transport into the cells followed by enzymatic hydrolysis (McFarland and
Baker, 2004; Kano et al., 2009).
Anandamide (AEA) transport has been widely studied and compared to 2 AG.
AEA uptake has been reported in cortical neurons (Fegley et al., 2004), striatal neurons,
astrocytes (Di Marzo et al., 1994; Beltramo et al., 1997) and cerebellar granule cells
(Hillard et al., 1997). Various aspects of AEA transport have been noted by research
groups. It has been reported that AEA transport is (i) temperature sensitive (ii) inhibited
by certain fatty acid amide derivatives (iii) relatively fast (t1/2 approx. 2.5 minutes) (iv)
controlled by other signal transduction pathways and (v) saturable at 37oC (Di Marzo et
al., 1994; Beltramo et al., 1997; Hillard et al., 1997; Maccarrone et al., 1998; Maccarrone
et al., 2000; Rakshan et al., 2000).
Several mechanisms have been advanced to explain the cellular uptake of AEA
including a protein carrier-mediated process, facilitated diffusion regulated by FAAH,
AEA sequestration by cellular machinery and endocytotic uptake of AEA (McFarland and
Baker, 2004).
Accumulation of a substrate on the cis side of the membrane leads to carrier
protein accumulation on the trans side of the membrane thus causing movement of an
extracellular substrate into intracellular compartment against a concentration gradient, a
phenomenon termed as ‘trans flux coupling’ (McFarland and Baker, 2004). ‘Trans flux
coupling for AEA was observed in cerebellar granule neurons (Hillard and Jarrahian,
2000). The intracellular presence (or expression) of FAAH positively modulated the
cellular uptake of AEA (McFarland and Baker, 2004). Neuroblastoma or glioma cells
expressing FAAH when treated with the FAAH inhibitor,
methylarachidonylflurophosphonate (MAFP) display close to a 50% reduction in AEA
accumulation (Deutsch et al., 2001). Furthermore, Day et al., (2001) showed a 2-fold
34
increase in the uptake of AEA following FAAH transfection of HeLa cells (which are
otherwise devoid of FAAH activity). Glaser et al. (2003) suggested FAAH-dependent
facilitated diffusion as a mechanism for cellular uptake of AEA. Kinetic analysis by
Glaser et al. (2003) demonstrated that AM404 (an AEA transport inhibitor and FAAH
inhibitor, Hillard and Jarrahian, 2000), when added at the 5 min time point, significantly
decreased AEA cellular accumulation, while no such effect was reported at earlier time
points (25 sec and 45 sec). This observation was consistent in both neuroblatoma cells
(with FAAH activity) and astrocytoma cells (without FAAH activity) (Glaser et al. 2003).
Hence, these researchers concluded that AEA accumulation was saturable at 5 min time
and this coincided with the inhibition of downstream components of AEA uptake
especially FAAH (Glaser et al., 2003).
Besides the above mentioned mechanisms, Hillard and Jarrahian (2003)
hypothesized that AEA could be taken up through sequestration by cellular components.
They found that radiolabeled AEA reached intracellular concentrations that were higher
than those in the extracellular media (Hillard and Jarrahian, 2003). They suggested the
existence of two distinct intracellular pools of AEA, one was free AEA and the other was
AEA sequestered by a cellular component. This sequestration of AEA is saturable and
may involve certain membranous compartments that serve as reservoir for this lipophilic
molecule (Hillard and Jarrahian, 2003). Since the AEA that is sequestered or bound is
not available for free diffusion across plasma membrane, it generates a positive inward
concentration gradient of AEA that is maintained by FAAH (Hillard and Jarrahian, 2003;
McFarland and Lambert, 2004).
The final model or mechanism proposed for AEA degradation is through the
caveolae-related endocytotic process (McFarland et al., 2003; McFarland et al., 2004).
McFarland (2004) showed that by inhibiting the caveolae-related endocytosis process
(by treating cells with N-ethylmaleimide and tyrosine kinase inhibitor, genistein), there
was marked reduction of cellular accumulation of AEA (McFarland et al., 2004)
As mentioned earlier, few studies have been directed towards understanding the
cellular uptake and degradation of 2-AG (Kano et al., 2009). However, some reviews
suggest the existence of a very similar mechanism for 2-AG transport as for AEA
(Beltramo and Piomelli, 2000; Bisogno et al., 2001; Piomelli et al., 1999).
35
1.11. Endocannabinoid-mediated short term depression (DSI and DSE)
In 2001, four research groups demonstrated independently that
endocannabinoids mediate synaptic plasticity through retrograde signaling in the CNS
and play a vital role towards short term and long term synaptic tonicity (Wilson et al.,
2001; Kreitzer et al., 2001; Ohno-Shosaku et al., 2001; Maejima et al., 2001).
Endocannabinoids are biosynthesized de novo and then released into the synaptic cleft
either tonically under basal conditions or in an activity-dependent manner (Kano et al.,
2009, Katona and Freund, 2008). The endocannbinoids that are released then travel
retrogradely (unlike usual anterograde transmission) and activate presynaptically-located
CB1-Rs (Katona and Freund, 2008), thus causing suppression of transmitter release
either transiently (endocannbinoid mediated short term depression, eCB-STD) or over
extended duration (eCB-long term depression, eCB-LTD) (Kano et al., 2009).
1.12. Endocannabinoids as synaptic circuit breakers and retrograde messengers
Pitler and Alger (1994) reported that following a train of action potentials from the
post synaptic cell, the spontaneous inhibitory postsynaptic potentials (IPSPs) from CA1
pyramidal cells of hippocampal slices were suppressed, thus indicating a reduction of
GABA release from presynaptic nerve endings: a novel phenomenon termed
‘depolarization-induced suppression of inhibition’ (DSI).
It was found that depolarization of the post synaptic neuron triggers Ca2+ entry
through voltage-dependent Ca2+ channels which then elevates Ca2+ levels in post
synaptic neurons. Support for this mechanism was clear since cerebellar DSI is
inhibited by chelation of extracellular Ca2+ or by adding Cd2+ to the bath solution (Llano
et al., 1991). Moreover, DSI was enhanced by the L-type Ca2+ channel activator, BAY K
8644 while blocked by calcium chelators like BAPTA and EGTA (Pitler and Alger, 1992;
Vincent and Marty, 1993). An ultimate presynaptic locus for DSI expression was
36
confirmed by Llano et al. (1991) when they found that DSI correlated directly with the
decrease in frequency of IPSCs (IPSCs).
Almost the same time, Ohno-Shosaku et al. (2001) using rat hippocampal
cultures and Wilson and Nicoll (2001) using rat hippocampal slices, reported that DSI
was completely abolished by the CB1 receptor antagonists SR141716A, AM251 and
AM281, while a metabotropic glutamate receptor antagonist failed to do this, indicating
that the retrograde signaling mechanism was mediated through CB1-Rs.
Meanwhile, in cerebellar Purkinje cells, Kreitzer and Regehr (2001) reported a
phenomenon similar to DSI where they observed that following postsynaptic
depolarization, the excitatory transmission was transiently suppressed, an effect termed
as ‘depolarization-induced suppression of excitation’ or DSE. The presynaptic Ca2+
currents generated in response to stimulation of the excitatory climbing fibers were found
to be inhibited or suppressed during DSE, thus providing good evidence for a
presynaptic locus of this effect (Kreitzer and Regehr, 2001). Moreover, DSE was
abolished by treatment with the calcium ion chelator BAPTA and occluded by the CB1-R
antagonist, AM251. This effect was not blocked by mGlu, adenosine and GABA
receptor antagonists suggesting that like DSI, DSE is also mediated through the
endocannabinoid system (Kreitzer and Regehr, 2001).
37
Figure 1.8 Blockade of depolarization-induced suppression of inhibition (DSI) CB1-R antagonists by AM251 and SR141716A in rat hippocampal neurons (Kano et al., 2009).
A: Examples of inhibitory postsynaptic currents (IPSCs) (right panel) and
control results showing that DSI can be evoked repetitively without change in its
magnitude (left panel)
B: Example of IPSCs, control and after treatment with AM281 (left panel).
Average time courses for DSI before and after treatment with AM281 (right panel)
C: Example of IPSCs, control and after treatment with SR141716a (left
panel). Average time courses for DSI before and after treatment with SR141716A (right
panel)
38
1.13. Mechanisms of endocannabinoid mediated short term depression (eCB-STD)
Unlike classical neurotransmitters, endocannabinoids are de novo synthesized
on demand and released and not stored in vesicles. Precisely what initiates the
endocannabinoid production and hence eCB-STD in neurons has been the subject of
much scientific debate (Kano et al., 2009). Two different pathways have been put forth
i.e. a PLCβ-independent pathway triggered by large rise in intracellular Ca2+
concentration alone (CaER), while the other is a PLCβ-dependent pathway that is
activated by stimulation of basal (receptor-driven) endocannabinoid release, (basal
RER) or elevated calcium-driven endocannabinoid release (Ca2+-assisted RER).
1.13.1. CaER
In the hippocampus, influx of Ca2+ ions into the postsynaptic cell triggers DSI
thus indicating vital role of Ca2+ elevation in initiating DSI and hence eCB-STD (Wilson
and Nicoll, 2001). The main sources for postsynaptic Ca2+ elevation are through voltage
gated Ca2+ channels (Ohno-shosaku et al., 2007; Pitler and Alger, 1992) or through
release from an intracellular Ca2+ reservoir (Isokawa and Alger, 2006).
According to this model, micromolar concentrations of Ca2+ trigger the activation
of voltage-dependent calcium channels which then produce 2-AG, likely through a
DAGL-mediated pathway and a PLCβ-independent pathway (Kano et al., 2009). 2-AG
released then initiates DSI/DSE by activating CB1-Rs.
1.13.2. Basal RER
The G-protein coupled receptors (Gq/11) mGlu1/5, M1/M3 muscarinic receptors,
glucocorticoid receptors, oxytocin receptors and orexin receptors were found to induce
eCB-STD when strongly activated (Hashimotodani et al., 2007; Hashimotodani et al.,
2007; Maejima et al., 2005). The most likely pathway for induction of eCB-STD by this
pathway is thought to involve stimulation of postsynaptic G-protein-coupled receptors
39
and DAG generation (through a PLCβ-dependent pathway), which then generates 2-AG
by the action of DAGL. The 2-AG then mediates induction of DSI or DSE (Kano et al.,
2009).
1.13.3. Ca2+-assisted RER
This model proposed to understand eCB-STD is a combination of the above two
models except here a weak stimulation of postsynaptic Gq/11 receptors cause a small rise
in Ca2+ to submicromolar concentrations which in turn activates PLCβ and thus 2-AG
and DSI/DSE (Kano et al., 2009; Hashimotodani et al., 2007).
1.14. Termination of eCB-STD
The 2-AG biosynthesized by the above mechanisms may then be partially
degraded by the COX-2 enzyme which is located in the postsynaptic cell and the
remaining 2-AG then diffuses rapidly into extracellular space by lateral diffusion
ultimately binding and activating presynaptic CB1-Rs. Presynaptically-located MAGL can
then degrade the remaining 2-AG thus terminating eCB-STD signalling (Kano et al.,
2009).
40
Figure 1.9 The pathway involved in the termination of endocannabinoid-mediated short term depression (eCB-STD) (Adapted from Kano et al., 2009).
41
1.15. Endocannabinoid-mediated long term depression (eCB-LTD)
As the name indicates, eCB-LTD induces a prolonged suppression of
neurotransmitter release in the brain and this effect is mediated through presynaptically
located CB1-Rs. However, the precise location of the initiation of eCB-LTD in specific
regions of brain also plays an important role as a deciding factor for involvement of CB1-
Rs and/or presynaptic components (Kano et al., 2009). It was found that application of
the CB1-R agonist (CP55940) at both excitatory synapses in the nucleus accumbens
(Robbe et al., 2002) and inhibitory synapses in the hippocampus (Chevaleyre et al.,
2007) induces LTD, indicating that CB1-Rs alone are sufficient to initiate the LTD
response in these regions of brain. However, at excitatory synapses in the dorsal
striatum, LTD cannot be evoked by CB1-R activation alone (Ronesi et al., 2004) since it
requires simultaneous activation of CB1-Rs and low frequency presynaptic activity
(Singla et al., 2007).
Studies have also been directed towards seeking an understanding of the way in
which a relatively short activation of CB1-Rs triggers LTD. It was reported that
presynaptic inhibition of the cAMP/PKA cascade and P/Q type voltage-gated calcium
channels was required for induction of LTD in the nucleus accumbens (Mato et al.,
2008), while presynaptic cAMP/PKA signalling and RIM1α were needed for expression
of LTD in the hippocampus and amygdala (Chavaleyre et al., 2007).
1.16. Other important aspects of endocannabinoid signaling
1.16.1. Regulation of excitability
Endocannabinoids have been found to control neuronal firing in several brain
regions (Kano et al., 2009). Kreitzer et al. (2002) showed in that in rat cellebellar cells,
interneuronal excitability was reduced in a CB1-R-dependent manner when Purkinje cells
were depolarized. They suggested that depolarization triggers generation and release of
endocannabinoids that then bind to CB1-Rs causing opening of K+ channels and hence
hyperpolarization (Kreitzer et al., 2002; Kano et al., 2009).
42
1.16.2. Basal activity of endocannabinoid signaling
CB1-Rs expressed exogenously using recombinant expression systems have
been found to support constitutive activity, but the case for such activity in native
membranes is weak (Howlett, 2004). SR141716A has shown to behave as an inverse
agonist in many native brain membrane preparations by inhibiting basal G protein
binding. This effect of SR141716A however, was at micromolar concentrations, in
contrast to the competitive antagonist behaviour in electrophysiological systems which
can occur at nanomolar concentrations (Sim-Selley et al., 2001). This again opens the
door to the debate as to whether CB1-Rs are constitutively active or this inverse agonist
activity of SR141716A (in the micromolar range) can be interpreted as its action on the
constitutively active adenosine receptor (Savinainen et al., 2003).
Moreover, CB1-Rs have been found to display basal activity and this basal effect
can be attributed to the tonically-released endocannabinoids (Kano et al., 2009;
Hoffman, 2003).
1.16.3. Plasticity of endocannabinoid signaling
Endocannabinoid system is tonically regulated and can be up or down regulated
in different situations (Kano et al., 2009). Chen et al., (2007) showed that DSI can be
potentiated by tetanic stimulation in hippocampal slice preparations and this was
mediated through CB1-Rs. Similarly, chronic exposure of nucleus accumbens to ∆9-THC
or WIN55212-2 reduced the sensitivity of CB1-R and abolished eCB-LTD (Hoffman et al.,
2003).
1.17. Subcellular distribution of various signaling molecules involved in regulation of the endocannabinoid system
1.17.1. Gq Protein α subunit
So far four isoforms of Gq protein α-subunits have been identified by
immunochemical studies. These include Gαq, Gα11, Gα14 and Gα15/16. Among these,
Gαq and Gα11 are the main isoforms found mostly in brain (Tanaka et al., 2000) and they
43
also represnt the major ones that are attached to the extrasynaptic membrane
containing mGluR1 in Purkinje cells and mGluR5 in hippocampal pyramidal cells
(Tanaka et al., 2000).
1.17.2. Phospholipase Cβ (PLCβ)
All four isoforms (1-4) of PLCβ are abundantly expressed in the brain largely in a
nonoverlapping expression manner (Kano et al., 2009). PLCβ1 is mainly expressed in
the telencephalon, PLCβ2 in white matter, PLCβ3 in the caudal cerebellum, PLCβ4 in
the raustral cerebellum, thalamus and brain stem (Ross et al., 1989; Roustan, 1995;
Tanaka and Kondo, 1994; Watanabe et al., 1998).
1.17.3. Diacylglycerol lipase (DAGL)
DAGLα is widely distributed in various regions of brain with its highest density
found in cerebellar Purkinje cells, pyramidal cells in hippocampus and medium spiny
neurons in the striatum (Kano et al., 2009). However the distribution of DAGLα varies
from region to region. For example, DAGLα is at the highest amount in the spine neck
(as compared to spine head) in Purkinje cells (Yoshida et al., 2006), while in
somatodendritic membranes, DAGLα levels were highest in spine followed by the
dendritic shaft with lower amounts in the soma (Katona et al., 2006; Yoshida et al.,
2006).
1.17.4. N-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D (NAPE-PLD)
NAPE-PLD is reported to be widely expressed in presynatic regions with the
highest levels in granule cells of the dentate gyrus and medium to low levels in CA3
pyramidal cells of the hippocampus, olfactory bulb, piriform cortex and thalamic nuclei
(Kano et al., 2009). Interestingly, the presence of NAPE-PLD in the axonal terminal may
indicate a presynaptic locus for anandamide synthesis which in turn may be functioning
as an anterograde messenger (Kano et al., 2009).
44
1.17.5. Monoacylglycerol lipase (MAGL)
Dinh et al. (2002) described the MAGL mRNA expression patterns in various
brain regions. They found the presence of this enzyme in the synapse-rich neuropil
region of the hippocampus, the amygdala and the cerebral cortex, where this enzymes is
mostly distributed as a seat for axon terminals (Dinh et al., 2002). Interestingly, MAGL
activity in brain was also found to be responsible for determining basal endocannabinoid
tonicity and retrograde signaling since presynaptically-located MAGL was reported to
breakdown the 2-AG released from the postsynaptic cell, thus restricting the
accumulation of 2-AG in and around the synaptic cleft (Hashimotodani et al., 2007).
1.17.6. Fatty acid amide hydrolase (FAAH)
FAAH presence is complementary to the expression of MAGL and CB1-Rs, for
example, FAAH is absent in the globus pallidus where CB1-Rs are abundantly expressed
while it is present at high density in somatodendritic elements of principal neurons but
mostly absent in interneurons (where CB1 and MAGL are widely expressed) (Kano et al.,
2009).
1.18. Physiological roles of the endocannabinoid system
Behavioural studies have helped enormously in exploring the physiological role
of the endocannabinoid system in a variety of brain functions like learning and memory,
depression, addiction, appetite and feeding behaviour, pain, as well as neuroprotection.
1.18.1. Learning and Memory
It has been now well established that phytocannabinoids and synthetic
cannabinoids cause memory and learning impairment in humans and laboratory animals
(Davies et al., 2002; Kano et al., 2002). In laboratory animals, Morris water maze tests
have been employed to investigate the effects of cannabinoid agonists. Mice (and rats)
failed to perform well in this test following systemic administration of CB1-R agonists
(Verval et al., 2001). Moreover, CB1 knockout mice and wild type mice treated with
SR141716A (CB1-R antagonist) exhibited similar performance in the fixed hidden
45
platform water maze task. Interestingly, when the same task was repeated with change
in location of the hidden platform there was a marked difference in the behaviour of both
animals with wild-type returning to the new location and CB1 knock out returning to the
old location of the hidden platform, thus indicating impairment of the extinction process
(Varvel and Lichtman, 2002).
1.18.2. Anxiety
There is increasing evidence suggesting a relationship between anxiety and
endocannabinoid tone. The effects of cannabinoid agonists on anxiety in laboratory
animals can be studied by employing different tests like elevated plus-maze, the light-
dark crossing test, the vocalization test and the social interaction test. The results are
quite complex to interpret although generally, cannabinoid agonists at low doses were
anxiolytic but at high doses were anxiogenic (Viveros et al., 2005). A cross talk
mechanism was suggested by Berrendero and Maldonaldo (2002) between the
cannabinoid and opioid systems since in the light-dark crossing test, the anxiolytic
effects of ∆9-THC were completely blocked by the opoid antagonist, naltrindole.
Moreover, in the plus-maze test, the anxiogenic effect of CP55940 was almost
completely blocked by an opioid antagonist, nor-binaltorphimine (Marin et al., 2003).
1.18.3. Depression
The endocannabinoid system has also been implicated in depressive episodes
(Kano et al., 2009). Hill and Gorzalka (2005) reported an antidepressant effect in
rodents following activation of the CB1-R. They found that in the rat forced swim test
(FST), HU210 (a CB1-R agonist) (5-25µg/kg i.p.) and AM404 (an anandamide transport
inhibitor) (5 mg/kg, i.p.) had antidepressant effects similar to the well-know
antidepressant drug, desipramine and this antidepressant effect was reversed by AM251
(a CB1-R antagonist) (Hill and Gorzalka, 2005).
Moreover recently, the CB1-R antagonist SR141716A (Rimonabant) was
withdrawn from European markets after reports of suicidal tendencies in some patients
employing this drug to treat obesity (Christensen et al., 2007).
46
1.18.4. Addiction
The endocannabinoid system has been known for its drug seeking and drug
reward effects and its likely involvement in addiction (De Vries and Schoffelmeer, 2005;
Fattore et al, 2007; Maldonado et al, 2006). Castane et al. (2002) showed that there
was a significant rewarding effect in wild type mice administered with nicotine (0.5 mg/kg
sc) but not in CB1 knockout mice. Furthermore, this effect was reduced by
administration of SR141716A (Le Foll and Goldberg, 2004).
However, the exact mechanism by which the endocannabinoid system
contributes to drug seeking and addiction behaviour is still rather obscure (Kano et al.,
2009).
1.18.5. Appetite
Food intake, appetite and feeding behaviour are precisely regulated through
involvement of the endocannabinoid system, and the mechanisms involved are subject
to ongoing investigation (Kano et al., 2009). Pagotto et al. (2006) showed that CB1-R
agonists increase food intake in a dose- dependent manner in laboratory animals while
CB1-R antagonists lead to decreased food intake in wild type mice but not in CB1
knockout mice (Di Marzo et al., 2001).
Rodent models and clinical studies strongly support the notion that CB1-Rs can
be targeted for the treatment of appetite disorders and obesity (Kano et al., 2009). ∆9-
THC has been employed effectively as an appetite stimulant in patients with HIV-
induced wasting syndrome and cancer while, as mentioned earlier, rimonabant
(SR141716A) was briefly used in European markets for the treatment of obesity
(Christensen et al., 2007; Kano et al., 2009).
1.18.6. Pain
The endocannabinoid system is intimately involved in pain modulation, and the
antinociceptive effects of cannbinoids have been reported to be at par with the opiates
(Hohmann and Suplita, 2006). Evidence for this came from the studies by Calignano et
al. (1998) who showed that SR141716A induced hyperalgesia in the Formalin test and
47
the hot plate test (Richardson et al., 1998). Also, analgesia induced by electrical
stimulation of periaqueductal gray matter was blocked by SR141716A (Walker et al.,
1999), thus indicating the role of the endocannabinoid system in pain modulation.
1.19. Classification of ligands that bind to cannabinoid receptors
1.19.1. Cannabinoid receptor agonists
1.19.1.1. Classical cannabinoids
This group encompasses derivatives of ABC-tricyclic benzopyran compounds
obtained from the Cannabis plant or synthetic analogs. The prototypical example of this
class is ∆9-THC (Figure 1.10). Others includes ∆8-THC, (6aR,10aR)- 9-(hydroxymethyl)-
6,6-dimethyl-3-(2-methyloctan-2-yl)-6a,7,10,10a-tetrahydrobenzo[c]chromen-1-ol (HU-
210) and desacetyl-L-nantradol (DALN). Here, ∆9-THC and ∆8-THC are
phytocannabinoids while HU-210 and DALN are synthetic (Howlett et al., 2002) (Figure
1.10).
1.19.1.2. Non-classical cannabinoids
Much of the success in developing the non-classical cannabinoids can be
attributed to Pfizer researchers who synthesized new compounds lacking the
dihydropyran ring of ∆9-THC. CP47497, CP55244 and CP55940 are the examples of
this group (Melvin et al., 1993; Devane et al., 1988) (Figure 1.10).
1.19.1.3. Aminoalkylindoles
This group of compounds were developed by Sterling Winthrop researchers and
are structurally related analogs of pravadoline, structurally unrelated to ∆9-THC (unlike
other cannabimimetics) and typified by R-(+)-WIN55212 (Bell et al., 1991; Pacheco et
al., 1991; Howlett et al., 2002) (Figure 1.10).
Other examples of this group include JWH-015 and L-768242 (Figure 1.10).
48
1.19.1.4. Eicosanoids/Endocannabinoids
These compounds which belong to 20:4, n-6 series of fatty acid amides, are
mostly endogenous cannabinoid agonists that are de novo synthesized and released by
mammalian brain (Howlett et al., 2002). Examples of eicosanoids/endocannabinoids
include anandamide (AEA), 2-arachidonyl glycerol (2-AG),
docosatetraenoylethanolamide and 2-AG ether (noladin ether) (Figure 1.11).
1.19.2. Cannabinoid receptor antagonists/ Inverse agonists
1.19.2.1. Diarylpyrazoles
The first cannabinoid receptor antagonist namely, SR141716A (CB1-R-selective)
and SR144528 (CB2-R-selective) were synthesized at Sanofi (Rinaldi-Carmona et al.,
1994 and 1998). Initially identified as cannabinoid antagonists, they were later found in
some preparations to exert effects opposite to cannabinoid agonists and hence were
classified as inverse agonists (Pertwee, 1999). Two other analogs in this diarylpyrazole
series include AM251 and AM281 (Figure 1.12).
1.19.2.2. Other inverse agonists primarily active at CB1-Rs
The Eli Lilly compound, LY320135 (a substituted benzofuran) has a higher
affinity for CB1-Rs then CB2-Rs and displays an inverse agonist profile similar to
SR141716A (Howlett et al., 2002). Similarly, another aminoalkylindole, 6-
iodopravadoline (AM630) was reported to be an inverse agonist at CB2-Rs (Howlett et
al., 2002) (Figure 1-12).
Dhopeshwarkar et al. (2011) found that (S)-methoprene and piperonyl butoxide
were antagonistic at CB1-R while sanguinarine and chelerythrine displayed inverse
agonist-like profiles. Bisset et al. (2011) reported that certain phthalate diesters (nBBP,
DnBP) were antagonist at CB1-Rs. However, all these compounds require low to
moderate micromolar concentrations for activity at CB1-Rs (for structures of these
compounds see Figure 1.13).
49
∆9-THC ∆8-THC HU210
DALN CP47497 CP55244
CP55940 WIN55212-2
JWH015 L-768242
Figure 1.10 Structures of ∆9-THC, ∆8-THC, HU210, DALN, CP47497, CP55244, CP55940, WIN55212-2, JWH015 and L-768242. All structures redrawn using ChemDraw 11.0 ultra from Howlett et al. (2002).
50
Anandamide 2-Arachidonyl glycerol ether (noladin ether)
2-Arachidonyl glycerol (2-AG)
Figure 1.11 Structures of anandamide, 2-AG ether and 2-AG. All structures redrawn using ChemDraw Ultra 11.0 from Howlett et al. (2002).
51
SR141716A AM251 AM281
LY320135 AM630
Figure 1.12 Structures of SR141716A, AM251, AM281, LY320135 and AM630. All structures redrawn using ChemDraw Ultra 11.0 from Howlett et al. (2002).
52
Methoprene Piperonyl butoxide
Sanguinarine Chelerythrine
nBBP DnBP
Figure 1.13 Structures of (S)-methoprene, piperonyl butoxide, sanguinarine, chelerythrine, nBBP and DnBP. Structures redrawn using ChemDraw 11.0 from Dhopeshwarkar et al., (2011) and Bisset et al., (2011).
53
1.20. Cannabinoid receptor 2 (CB2-R)
1.20.1. CB2-R receptor signaling
Like CB1-Rs, CB2-Rs couple to Gi/o proteins and modulate adenylyl cyclase and
MAPK activity. However, unlike CB1-Rs, CB2-Rs do not couple to the Gs subunit of G
protein, and hence cannot modulate ion channel activity (Demuth and Molleman, 2006;
Felder et al., 1995; Kobayashi et al., 2001).
1.20.1.1. Adenylyl cyclase regulation
CB2-Rs are negatively coupled to adenylate cyclase thus they decrease cAMP
production. Slipetz et al. (1995) reported that in cell lines transfected with CB2-Rs,
forskolin-stimulated cAMP production was inhibited by CB2-R agonists and this effect
was concentration-dependent. Similar results were obtained by Felder et al. (1995) with
∆9THC and anandamide. Another line of evidence for coupling of CB2-R to Gi/o proteins
came from experiments where following pretreatment of CB2 transfected CHO cells with
Pertussis toxin was found to attenuate the inhibition of cAMP production (Pertwee,
1997).
1.20.1.2. Mitogen-activated protein kinase regulation
In common with CB1-Rs, CB2-Rs have been implicated in the regulation of MAP
kinase (Pertwee, 1997). CP55940 and WIN55212-2 were found to activate MAP kinase
in CHO cells transfected with CB2-Rs in a concentration-dependent manner (Bouaboula
et al., 1996). This effect of CP55940 was blocked by pretreatment of cells with Pertussis
toxin, indicating the involvement of Gi/o G proteins (Bouaboula et al., 1996). Bouaboula
and co-workers (1996) also found that MAP kinase regulation was mediated through
protein kinase C, since inhibitors of this enzyme decreased CB2-R-mediated activation of
MAP kinase. Interestingly, CHO cells stably transfected with the CB1-R showed
activation of MAP kinase but this activation of unaffected by treatment with a protein
kinase C inhibitor, indicating a distinct difference in the signal transduction mechanism of
each receptor (Bouaboula et al., 1996).
54
1.20.2. Therapeutic aspects of CB2-R modulators
Studies have indicated a potential role of CB2-R agonists in the treatment of
chronic pain (Mackie, 2006). Like CB1 receptor agonists, CB2-Rs agonists (like AM1241,
HU308, JWH133) can be effectively employed for neuropathic and inflammatory pain
treatment with the advantage that they are devoid of the major psychoactive side effects
that often accompany therapy with CB1-R agonists (Mackie, 2006). Moreover, analgesia
produced by CB2-R agonists was found to be sensitive to naloxone (an opioid
antagonist), implicating the opioid pathway and offering a novel indirect mechanism for
how these agonists might relieve neuropathic pain (Ibrahim et al., 2003; Elmes et al.,
2004).
Interestingly, CB2-Rs have also been implicated in bone development and bone
mass density increases in mouse models. CB2-R knockout mice displayed decreased
bone mass compared to normal mice thus hinting towards the possible role of CB2-R
agonists in treatment of bone disorders and osteoporosis in humans (Bab, 2005). Bab
(2005) also found that HU308, a CB2 receptor agonist, reduced bone mass loss in
ovariectomized mice.
Another interesting role of CB2 receptor agonists is their potential therapeutic
utility for treatment of atherosclerotic lesions. Steffens et al. (2005) showed that
treatment of mice on an atherogenic diet with a low dose of ∆9-THC, decreased the
progression of atherosclerotic lesions and this effect was abolished by SR144258, a
CB2-R antagonist. Benito et al. (2003) also predicted the possible role of impaired CB2-
R signaling in plaque formation in Alzheimer’s disease, as well as in the chronic
inflammatory response seen during retroviral encephalitis (Benito et al., 2003; Benito et
al., 2005).
CB2-R agonists may represent promising candidates for treatment of the various
diseases/disorders as mentioned above. However, more studies are clearly warranted
as the exact physiological role of CB2-Rs, their signaling transducers and their agonists
remain to be defined (Mackie, 2006). Preclinical studies and animal models have shown
promise in the development of the treatment of a variety of CB2-R mediated
diseases/disorders. However, detailed clinical trials are needed to understand the exact
role and the therapeutic potential of the CB2-R agonists in human subjects.
55
1.21. Brief overview of the test chemicals used in my research
1.21.1. Benzophenanthridine alkaloids
Alkaloids are a group of nitrogen-containing biological compounds which are
mostly generated as metabolic by-products by higher plants (Maiti and Kumar, 2007).
Their spectrum of activity ranges from anti-inflammatory, antitumor and anti-microbial to
analgesic and spasmolytic (Jursky and Baliova, 2011). Benzophenanthridine alkaloids
(as exemplified by sanguinarine and chelerythrine; Figure 1-13) are class of substances
that can be isolated from plants of the Papaveraceae, Fumariaceae, Rutaceae,
Meliaceae and Caprifoliaceae families. These plants are also known for antifungal,
nematocidal and various other beneficial properties (Simanek et al., 2003).
Benzophenanthridine also show an array of biological and physiological activities when
introduced into mammalian cell preparations; they therefore represent an area of
immense interest (Simanek et al., 2003).
In Russia, Sanguinaria® and Sanguinatrine® containing extract from S.
canadensis and M. cordata respectively is used in toothpastes and mouthwashes
primarily as antiplaque agents and also in feed additives (Simanek et al., 2003).
However, toxic effects attributed to benzophenanthridines are known (Das and Khanna,
1997). Das and Khanna (1997) found that the consumption of edible oil containing
Argemone mexicana seed oil (which contains about 0.5% benzophenanthridines) was
associated with a dropsy-like syndrome. Moreover, long term use of dental products
containing benzophenanthridines have been associated with increased frequency of
leukoplakia of the maxillary vestibule (Eversole et al., 2000).
At the molecular level, benzophenanthridine react with nucleophilic and anionic
moieties in various amino acids that form the backbones of peptides and proteins
(Schmeller et al., 1997). Benzophenanthridines also inhibit protein kinase C (Wang et
al, 1997) and are capable of intercalating with DNA and complexing with genetic material
(Maiti et al., 1982).
Sanguinarine and chelerythrine can exist in the iminium (charged) or
alkanolamine (neutral, pseudobase or hydroxide adduct) forms at pH 1.0-6.0 and 8.5-
56
11.0 respectively, with equilibrium between these two forms at pKa 7.4 (Maiti and
Kumar, 2007).
The biological activity of benzophenanthridines can in part be attributed to their
capacity to maintain equilibrium between both forms at physiological pH (Dvorak and
Simenak, 2007). Benzophenanthridines penetrate the cell membrane in the
alkanolamine form since this form imparts the lipophilicity necessary for efficient
penetration into membranes and the cell (Dvorak and Simenak, 2007; Simenak et al.,
2003). Once inside the cell benzophenanthridines convert to the iminium form (Simenak
et al., 2003). Both form can exert biological effects, and this would depend on how
readily the iminium or pseudobase can access and bind to their respective molecular
targets. Another important consideration of benzophenanthridine activity is the iminium
bond. Since this bond is prone to nucleophilic attack benzophenanthridines readily bind
to SH-groups on proteins (Simenak et al., 2003). Stiborova et al. (2002) also reported
the formation of DNA adducts following activation of sanguinarine and chelerythrine by
cytochrome P450 of human liver microsomes. Moreover, Dvorak and Simenak (2007)
reported the metabolism of sanguinarine by cytosolic and microsomal reductases in rats
and guinea pig intestine. The product (dihydrosanguinarine) can then be metabolized to
different conjugates by sulfation, glucuronidation, O-demethylation and N-demethylation
(Dvorak and Simenak, 2007).
1.21.2. Piperonyl butoxide (PBO)
Piperonyl butoxide (PBO) is a methylenedioxyphenyl compound which is
synthesized from natural saffrole derivatives found in many plant tissues (Wang et al.,
2012, Ugolini et al., 2005). PBO is commercially available as a potent synergist of
pyrethrin-, pyrethroid- and carbamate-based insecticides (Ugolini et al., 2005) (Figure 1-
13). Here synergists can be defined as agents that increase the insecticidal activity of
the insecticidal compound but virtually lack any insecticidal activity on their own
(Franklin, 1976). The popularity of PBO as a synergist for insecticidal compounds is due
to its broad spectrum of synergistic action and very low acute mammalian toxicity (acute
oral LD50 = 10 g/kg in the rat) (Franklin, 1976). PBO was also found to be safe on
chronic exposure at low doses (Franklin, 1976). However, chronic exposures at very
high doses have been reported to be hepatotoxic and to cause kidney damage (Franklin,
57
1976). Franklin (1976) demonstrated that the methylenedioxy moiety was very important
for synergistic activity and metabolic enzymes were capable of cleaving the methylene
carbon.
The synergistic mechanism of PBO action was first studied by Sun and Johnson
(1960) and this was later confirmed by Casida in 1970. PBO was found to inhibit the
cytochrome P450 enzymes responsible for oxidation of insecticides and xenobiotics.
Thus inhibition of cytochrome P450 spared insecticides from metabolic breakdown
therefore allowing them to exert their toxic actions on insects. Binding conformation
studies performed by Keseru et al. (1999) showed that PBO reduced the conformational
mobility of cytochrome P450 and also created a steric hindrance in the channel
responsible for substrate access ito the enzyme.
Schleier III and coworkers (2007) found that PBO undergoes exponential decay
in water over a 36 hour time period indicating a relatively short t1/2 of PBO in the
environment.
1.21.3. Methoprene
Methoprene is a non-cyclic, amber colored synthetic terpenoid commonly used
as a biochemical pesticide belonging to a potent class of insect growth regulators which
mimics the insect juvenile growth hormone (Monteiro et al., 2005; Wilson, 2004; Siddall,
1976)(Figure 1-13). Juvenile growth hormone is important for insect development,
reproduction, behaviour, pheromone production and adult diapauses (Wilson, 2004).
Methoprene, when applied to insects, mimics the action of this hormone (juvenile
hormone III) thus causing disruption of development which leads to death (Wilson,
2004). Another advantage of methoprene as an insect control chemical is its remarkable
insect specificity. Thus methoprene is extremely active against dipteran insects but
practically inactive against lepidopterans (Staal, 1975; Wilson, 2004).
Methoprene is practically non-toxic to mammals with an oral LD50 of > 34,600
mg/kg in rats (Hawkins et al., 1977). However, some toxic effects have been reported in
non-target aquatic insects and some fish at high concentrations (Miura and Takahashi,
1973, Breaud et al., 1977, Quistad et al., 1976). Using the model microorganism
Bacillus stearothermophilus, Monteiro et al. (2005) suggested that the toxic actions of
58
methoprene on non-target organisms may be due to membrane interactions and
disturbance of cell bioenergetics.
Methoprene is rapidly metabolised by mammals (Hawkins et al., 1977). Hawkins
et al. (1977) showed that following oral administration of methoprene to rats, some
methoprene was metabolised by gut flora. Methoprene was also extensively distributed
throughout rat tissues with the highest concentration in the adrenal cortex (Hawkins et
al., 1977). In the environment, methoprene is rapidly degraded by sunlight and it is also
rapidly broken down in soil (t1/2 approx. 10 days in soil; Siddall, 1976; Hawkins et al.,
1977).
1.21.4. Phthalate esters
Phthalate esters are an important class of industrial chemicals that are widely
used as additives in plastic products (Xu et al., 2010, Staples et al., 2011) (Figure 1-13).
Phthalates having higher molecular weights like di-2-ethylhexyl phthalate (DEHP) are
employed as plasticizers to soften polyvinylchloride products, while phthalates with lower
molecular weights like di-n-butyl phthalate (DBP) and butyl benzyl phthalate (BBP) are
primarily used to hold color and scents in various consumer products (Cao, 2010).
Phthalates have been widely reported environmental contaminants (Rudel et al., 2003;
Bornehag et al., 2004; Bornehag et al., 2005) including some foods (Cao, 2010). This
ubiquitous contamination by phthalates is a cause of increasing concern since
toxicological studies have indicated a number of adverse effects displayed by these
compounds. DEHP was found to be a liver carcinogen in rodents (Cao, 2010) while
DBP, BBP and other phthalates were reported to have teratogenic effects in mice and
rats (Blount et al., 2000). Bornehag and coworkers (2004) also showed that there was a
relationship between phthalates in house dust and asthma and allergy in children.
Another important source of human exposure to phthalate is through food (Cao, 2010).
Indeed, several reports have been published to date showing the migration of phthalates
from plastic containers into cooking oil, mineral water (Xu et al., 2010), soft drinks
(Bosnir et al., 2007) and infant foods packed in recycled paperboard (Gratner et al.,
2009).
59
In humans, phthalates tend not to accumulate in the body (Schmid and Schlatter,
1985) because they are quickly metabolized to their respective monoesters which then
undergo glucuronidation and excretion in urine and faeces (Blount et al., 2000).
Phthalates are rapidly photodegraded in air (half life ~1 day), but in water the t1/2 was
found to be slightly longer (Staples et al., 1997). Staples and coworkers (1997) also
showed that phthalates were hydrolysed to form their respective alcohols and phthalic
acid and these intermediates are then further degraded aerobically or anaerobically.
Factors affecting the rate of phthalate degradation were availability of oxygen,
temperature and nutrient content (Staples et al., 1997).
1.21.5. Tributyl tin (TBT) compounds
TBT compounds are organic derivatives of tin (Sn4+) which are widely used as
biocides or as stabilizers in industrial or agricultural sectors (Kannan et al., 1999, Okoro
et al., 2011) (Figure 1-14). Since the 1970s, TBTs have been employed as paint
additives to reduce bio-fouling on ship hulls, marine docks and fishing nets (Dubey and
Roy, 2002). However, a decade later, France became the first country to ban TBT
application as anti-fouling agent in boats less than 25 m long since researchers in
France and UK reported adverse effects of TBTs on nontarget aquatic organisms
(Dubey and Roy, 2002). TBTs have been found to weaken oyster and mussel shells
and also affect the growth and development of aquatic snails (Dubey and Roy, 2002).
The lipophilic nature of these compounds leads to easy entry into cells. TBTs
then often interfere with energy transduction and this can represent a major mechanism
leading to toxicity and death (Cooney and Wuertz, 1989; Cooney, 1995; Dubey and Roy,
2002). However, TBT resistant (or tolerant) bacteria have also been reported (Dubey
and Roy, 2002). These include Alteromonas sp M-1, E.coli, Pseudomonas fluorescens,
Pseudomonas aeruginosa, B. Subtilis and S.aureus. The TBT resistance (tolerance)
can be explained by the inherent capacity of these microorganisms to a) perform
dealkylation of TBTs generating less toxic compounds b) effectively pump TBTs out of
the cell (via Pgp efflux proteins) c) carry out metabolic utilization of TBTs as source of
carbon or d) complex TBTs to metallothionein-like proteins (Fukagawa et al., 1994; Blair
et al., 1982).
60
In humans, the greatest chance of exposure to TBTs is either in drinking TBT-
contaminated water and beverages or eating contaminated aquatic organisms (Antizar-
Ladislao, 2008). TBTs are potentially dangerous for pregnant humans since Nakanishi
(2007) showed that these compounds stimulate human placental oestrogen biosynthesis
and human chronic gonadotropin production in vitro. TBTs have also been showed to
cause cell necrosis or apoptosis in healthy mammalian cells (Nakanishi, 2007; Saitoh et
al., 2001).
The degradation of TBTs in soil, fresh water and marine and estuarine
environments is mostly mediated through biotic processes (Barug, 1981; Dowson et al.,
1996). The t1/2 of most TBTs in soil is >89 days (Wuertz et al., 1991), but in fresh water
it is much less (9 days; Seligman et al., 1986), while in sediments it can be up to 2.1
years (Sarradin et al., 1995). Abiotic degradation processes like breaking of the Sn-C
bond as a result of UV and gamma irradiation and also chemical and thermal cleavage
are also thought to occur (Sheldon, 1975).
61
R1 = R2 = -(CH2)3-CH3, DnBP MnBP
R1 = R2 = -(CH2)5-CH3, DnHP
R1 = -CH2-C6H6, R2= -(CH2)3-CH3, nBP
R= -H, TBT hydride Triphenyl tin chloride
R= -Br, TBT bromide
R= -OCH3, TBT methoxide
R= -OCOOCH3, TBT acetate
R= -C6H6, Tributyl phenyl tin
Figure 1.14 Structures of selected phthalate esters and tributyl tin compounds. All structures redrawn using ChemDraw Ultra 11.0.
62
1.22. Rationale behind my research and the general approach
The pioneering work by Quistad et al., (2002, 2006) at the University of
California, Berkeley has established that various organophosphorus pesticides inhibit the
binding of the radioligand [3H]CP55940 to brain CB1-Rs. However, we know very little of
the extent to which environmental chemicals might interfere with CB1-Rs in the brain.
Both synthetic and natural product chemicals represent a significant component of the
human environment and show an extraordinary diversity in structure. Given the power
of the endocannabinoid system as a fundamental regulator of synaptic strength in many
neuronal networks, even a relatively low level of interference by a moderately potent
xenobiotic would be anticipated to cause some physiological, behavioral or
psychological alterations. Such subtle outcomes, especially if they are psychological or
behavioral, could possibly be missed in the standard toxicological evaluation of
environmental chemicals. Another potential consequence of examining a diverse group
of environmental chemicals at CB1-Rs is that one might obtain a new perspective on a
chemical structure or molecular feature capable of offering a useful way forward in the
design of novel agonists, antagonists or inverse agonists. Such compounds may have
therapeutic value.
Initial exploratory experiments in our laboratory identified a number of chemicals
capable of interfering with the binding of [3H]CP55940 to receptors of mouse brain.
Included in this group were two benzophenanthridine alkaloids (sanguinarine and
chelerythrine), (S)-methoprene and piperonyl butoxide.
The objectives of each phase of my research are detailed below and elaboration
of each objective is provided in the Introductory sections of Chapters 2, 3, 4 and 5.
1.22.1. Summary of objectives
1) To confirm that benzophenanthridine alkaloids (sanguinarine and
chelerythrine), (S)-methoprene and piperonyl butoxide interfere with the binding of
63
[3H]CP55940 to CB1 receptors in mouse brain and establish concentration:inhibition
relationships to determine inhibitory potencies based on IC50 estimates.
2) To classify study compounds as agonists, antagonists or inverse agonists at
CB1-Rs of mouse brain. This phase of the pharmacological profiling utilized the
[35S]GTPγS binding assay, since it allows any modification by study compounds to the
primary functional response of the G-protein to be investigated. These experiments
required effects on basal [35S]GTPγS binding and CB1-R agonist-stimuated [35S]GTPγS
binding to be defined.
3) To explore in greater depth the mechanisms by which study compounds
inhibit the binding of [3H]CP55940 and [3H]SR141716A (radioligands specific for the
brain CB1-R binding pocket) using saturation binding and kinetic approaches. This
phase of the study was extended to phthalates.
4) To investigate the ability of benzophenanthridine alkaloids, (S)-methoprene
and piperonyl butoxide to interfere with the binding of [3H]CP55940 to CB2 receptors in
mouse spleen and explore concentration:inhibition relationships to a level sufficient to
draw conclusions on CB1-R vs CB2-R selectivities.
5) To pharmacologically classify study compounds based on the ultimate
functional presynaptic outcome of drugs interfering with CB1-Rs, that is the release of
neurotransmitter from the nerve ending. For this I employed a continuous fluorometric L-
glutamate release assay. Prior to profiling the study compounds, it was necessary to
verify the inhibitory effect of a standard cannabinoid agonist (WIN55212-2) and
demonstrate the neutralization of WIN55212-2's inhibitory effect by a diarylpyrazole
(AM251), along the lines reported by (Wang et al., 2003). This study compound testing
phase of the investigation was later extended to selected phthalate esters and
tributyltins.
Note: Other researchers in my laboratory were involved in pursuing some of the
above objectives. Appropriate acknowledgements to their work are given, both in the
Results sections and in Figure legends.
64
2. The actions of benzophenanthridine alkaloids, piperonyl butoxide and (S)-methoprene at the G-protein coupled cannabinoid CB1 receptor in vitro.
2.1. Abstract
This investigation focused primarily on the interaction of two
benzophenanthridine alkaloids (chelerythrine and sanguinarine), piperonyl butoxide and
(S)-methoprene with G-protein-coupled cannabinoid CB1 receptors of mouse brain in
vitro.
Chelerythrine and sanguinarine inhibited the binding of the CB1 receptor agonist
[3H]CP55940 to mouse whole brain membranes at low micromolar concentrations (IC50s:
chelerythrine 2.20 µM; sanguinarine 1.10 µM). The structurally related isoquinoline
alkaloids (berberine and papaverine) and the phthalide isoquinoline ((-)-β-hydrastine)
were either inactive or considerably below IC50 at 30 µM. Chelerythrine and sanguinarine
antagonized CP-55940-stimulated binding of [35S]GTPγS to the G-protein (IC50s:
chelerythrine 2.09 µM; sanguinarine 1.22 µM). In contrast to AM251, both compounds
strongly inhibited basal binding of [35S]GTPγS (IC50s: chelerythrine 10.06 µM;
sanguinarine 5.19 µM).
Piperonyl butoxide and S-methoprene inhibited the binding of [3H]CP55940
(IC50s: piperonyl butoxide 8.2 µM; methoprene 16.4 µM), and also inhibited agonist-
stimulated (but not basal) binding of [35S]GTPγS to brain membranes (IC50s: piperonyl
butoxide 22.5 µM; (S)-methoprene 19.31 µM). PMSF did not modify the inhibitory effect
of (S)-methoprene on [3H]CP55940 binding.
Our data suggest that chelerythrine and sanguinarine are effacacious
antagonists of G-protein-coupled CB1 receptors. They exhibit lower potencies compared
65
to many conventional CB1 receptor blockers but act differently to AM251. Reverse
modulation of CB1 receptor agonist binding resulting from benzophenanthridines
engaging with the G-protein component may explain this difference. Piperonyl butoxide
and (S)-methoprene are effacacious, low potency, neutral antagonists of CB1 receptors.
Certain of the study compounds may represent useful starting structures for
development of novel/more potent G-protein-coupled CB1 receptor blocking drugs.
Acknowledgement: This chapter adheres closely to our paper published by the
European Journal of Pharmacology as: Dhopeshwarkar A.S., Jain S., Liao C., Ghose
S.K., Bisset K.M., & Nicholson R.A. (2011). The actions of benzophenanthridine
alkaloids, piperonyl butoxide and (S)-methoprene at the G-protein coupled cannabinoid
CB1 receptor in vitro. European Journal of Pharmacology, 654(1), 26-32.
2.2. Introduction
Cannabinoid CB1 receptors are widely distributed in mammalian brain and occur
at high density in the cerebral cortex, hippocampus, cerebellum and basal ganglia
(Herkenham et al., 1991; Tsou et al., 1998). CB1 receptors are predominantly
presynaptic and interface directly with G-proteins in the neuronal membrane, forming the
initial presynaptic element of a negative feedback mechanism regulating transmitter
exocytosis (Howlett et al., 1986; Katona et al., 1999; Kawamura et al., 2006). During
heightened synaptic activity, postsynaptic neurons generate endocannabinoids which
translocate retrogradely to activate presynaptic CB1 receptors. Activation of the coupled
G-protein leads to inhibition of voltage-sensitive Ca++ channels (Mackie and Hille, 1992;
Twichell et al., 1997, Kushmerick et al., 2004), negative modulation of adenylate cyclase
(Howlett and Fleming, 1984; Howlett, 1985) and activation of K+ currents (Deadwyler et
al., 1993; Mackie et al., 1995). The net effect is a downward adjustment of transmitter
release from the nerve ending (Chevaleyre et al., 2006; Kreitzer and Regehr, 2001;
Wilson and Nicoll, 2001; Howlett, et al., 2002).
66
In addition to endocannabinoids, various other natural and synthetic compounds
including ∆9-tetrahydrocannabinol, CP55940 and WIN55212-2 exert agonist effects at
brain cannabinoid receptors (Devane et al., 1988; Compton et al., 1992). Selective CB1
receptor antagonists such as the diarylpyrazoles AM251 and SR141716A, and the
phytocannabinoid, ∆9-tetrahydrocannabivarin have also been discovered (Rinaldi-
Carmona et al., 1994; Lan et al., 1999; Thomas et al., 2005). These CB1 receptor
modulators exert potent effects in vitro, acting in the nanomolar range. Despite
unfavorable psychiatric side effects associated with the first group of CB1
antagonists/inverse agonists developed to treat obesity, compounds with this
pharmacological profile remain of substantial interest (Szabo et al., 2009; Wu et al.,
2009; Riedel et al., 2009).
Chelerythrine and sanguinarine are quaternary benzophenanthridine alkaloids of
plant origin. We considered that the pseudobase forms of chelerythrine and
sanguinarine might engage CB1 receptors in a similar way to ∆9-tetrahydrocannabinol or
∆9-tetrahydrocannabivarin (see Fig. 2.1) based on preliminary findings that both natural
products displace the binding of [3H]CP55940 to mouse brain membranes. In other
exploratory experiments, binding inhibition was noted for two synthetic chemicals used in
insect pest management: piperonyl butoxide, which we hypothesized may adopt an
endocannabinoid-like conformation; and (S)-methoprene, which may represent a highly
flexible analog of ∆9-tetrahydrocannabinol or ∆9-tetrahydrocannabivarin or perhaps
mimic 2-AG (see Figure 2.1).
The aim of the present work was to investigate the in vitro effects of these study
compounds on the G-protein coupled CB1 receptor in mouse brain in more depth.
Interactions with this signaling complex were evaluated on the basis of ability to 1)
displace the binding of [3H]CP55940, a radioligand that binds to a region of the CB1
receptor shared with the recognition sites for endocannabinoids, classical cannabinoids,
aminoalkylindoles and diarylpyrazoles (Devane et al., 1988; Song and Bonner, 1996;
McAllister et al., 2003) and 2) modify the binding of [35S]GTPγS to brain G-proteins in the
presence and absence of agonist, an assay which determines functional coupling of the
CB1 receptor to its G-protein (Selley et al., 1996; Petitet et al., 1997).
67
2.3. Materials and Methods
2.3.1. Radioligands, drugs and study compounds
[3H]CP-55940[(1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-
hydroxy-propyl)cyclohexan-1-ol; side chain-2,3,4-[3H]; sp. act. 139.6 and 174.6 Ci/
mmol) and guanosine 5'-O-(γ35S]thio)-triphosphate ([35S]GTPγS; sp. act. 1250 Ci/ mmol)
were obtained from Perkin Elmer Life and Analytical Sciences, Canada. Chelerythrine,
berberine, sanguinarine, (as chloride or hydrochloride salts), papaverine, (-)-b-
hydrastine, CP55940, N-piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-
1H-pyrazole-3-carboxamide (AM251), 2,3-dihydro-5-methyl-3-[(4-
morpholinyl)methyl]pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl](1- naphthyl)methanone
(WIN55,212-2), phenylmethanesulfonylfluoride (PMSF) and piperonyl butoxide were
purchased from Sigma-Aldrich, Canada. (S)-Methoprene (98.5% purity) was kindly
supplied by Doug Vangundy, Director of Speciality Product Development, Wellmark
International (Dallas, Texas).
2.3.2. Animals
Male CD1 mice (20-25g) obtained from Charles River Laboratories, (Saint-
Constant, Quebec, Canada) were used for all experiments. Animals were maintained on
a 12 h light:dark cycle with food and water provided ad libitum. All procedures using
mice adhered to the Canadian Council on Animal Care standards regarding the use of
animals in research and had approval of the Simon Fraser University Animal Care
Committee.
2.3.3. Determination of the effects of study compounds on the binding of [3H]CP55940 to CB1 receptors in mouse brain membranes.
We evaluated several published procedures for the measurement of specific
binding of [3H]CP55,940 to CB1 receptors. The method described by Quistad et al.,
(2002) was adopted with minor modifications for the present investigation. Mice were
euthanized by rapid cervical dislocation and all isolation procedures were carried out at
0-4 oC. Mouse whole brains were homogenized (10 up/down strokes) in ice-cold buffer
68
(Trisma base (100 mM), EDTA (1 mM) adjusted to pH 9 with HCl; 1 brain/10 ml buffer)
using a motor driven homogenizer (pestle rotation approx. 1500 rpm). Homogenates
were centrifuged in a Beckman J2HS centrifuge at 900 x g for 10 min in a JA20 rotor.
The supernatant containing the neuronal membranes was centrifuged at 11,500 x g for
20 min. Pellets were thoroughly resuspended to a protein concentration of close to 6.5
mg/ml in storage buffer (Trisma base (50 mM), EDTA (1 mM) and MgCl2.6H2O (3 mM),
adjusted to pH 7.4 with HCl) and stored in aliquots at -80 oC. When required for
experiments, membranes were thawed on ice, taken up in a 5 ml syringe and thoroughly
resuspended by moving the suspension out and in (6 times) through an 18g needle (with
its square cut tip held close to the base of the tube) and then vortexed. For assay,
compounds (in DMSO; 5 µl) were added to borosilicate glass culture tubes (13 x 100
mm; Kimble-Chase; no siliconization), followed by binding buffer (500 µl; Trisma base
(50 mM), EDTA (1 mM), MgCl2.6H2O (3 mM), BSA (fatty acid free; 3 mg/ml) adjusted to
pH 7.4 with HCl). Membranes (154.3 + 3.5 µg protein) were then added to each tube
and the mixture vortexed and incubated for 15 minutes at room temperature. Following
addition of [3H]CP-55940 (added in 10 µl DMSO; final radioligand concentration 1.0 nM),
the tube contents were thoroughly mixed and incubations run for 90 min at 30 oC with
gentle shaking. Binding reactions were stopped by adding ice-cold wash buffer (0.9%
NaCl containing 2 mg/ml BSA; 1 ml) and membranes were collected by rapid vacuum
filtration on pre-soaked Whatman GF/C filters. Membranes trapped on the filter were
immediately washed (3 x 4 ml) with ice-cold wash buffer. Filters were thoroughly air
dried before adding scintillant (4 ml; BCS, Amersham Bioscience UK) and radioactivity
was quantitated using liquid scintillation counting. Non-specific binding, measured in the
presence of unlabeled CP55,940 or WIN55,212-2 (both at 10 µM), was subtracted from
total binding to yield the specific binding signal which averaged 80.9 + 4.7 % and 80.7 =
3.1% respectively. In each experiment, binding in the absence and presence of
unlabeled CP55,940 or WIN55212-2 was performed in triplicate and test compounds
were assayed in duplicate. A minimum of three experiments were conducted for every
treatment. All protein measurements were carried out as described by Peterson (1977).
69
2.3.4. Determination of the effects of study compounds on basal and CP55940-stimulated [35S]GTPγS binding to mouse brain membranes
The procedure for isolating brain membranes and measuring the effects of study
compounds on basal and agonist-stimulated [35S]GTPγS binding was adapted from that
of Breivogel et al., (2000). The isolation of brain membranes was carried out at 0-4 oC.
Immediately following the cervical dislocation procedure, whole brains were removed
from two mice and homogenized (Polytron Kinematica GmBH; speed setting 6 for 15
seconds) in isolation buffer (Trisma base (50 mM), MgCl2.6H20 (3 mM), EGTA (0.2 mM),
NaCl (100 mM) with pH adjusted to 7.4 with HCl). The homogenate was centrifuged in a
Beckman J2HS centrifuge (JA20 rotor) at 24,000 x g for 25 min, and the resulting pellet
was then resuspended in isolation buffer and re-centrifuged The final membrane pellet
was thoroughly homogenized in isolation buffer, the protein concentration adjusted to 7
mg/ml and aliquots transfered to the -80 oC freezer. After removal from storage at -80 oC, brain membranes were thawed on ice and thoroughly dispersed as described in
Section 2.3. [35S]GTPγS binding experiments were performed as follows. The test
compound (in DMSO; 5 µl) or DMSO control, as appropriate, was placed in the tube first
followed by assay buffer (500 µl; isolation buffer (pH 7.4) containing, bovine serum
albumin (fatty-acid free; 1 mg/ml), guanosine diphosphate (GDP; 100 µM), dithiothreitol
(20 µM), [35S]GTPγS (0.14 nM final concentration) and adenosine deaminase (0.004
units/ml). The brain membranes (70.1 + 4.2 µg protein) were then added and after
thorough vortexing, a 15 min preincubation at room temperature was carried out.
Following this, CP55940 (100 nM final concentration; in 5 µl DMSO) or DMSO control
was added (total assay volume = 535 µl), the samples were mixed thoroughly and the
incubation continued at 30 0C for 90 min with gentle shaking. Where effects on basal
binding were investigated, no agonist addition was made after the preincubation. The
incubation was terminated by the addition of ice-cold wash buffer (2 ml; Trisma base:
HCl; pH 7.4) and rapid filtration under vacuum through pre-soaked Whatman GF/B
filters. This was quickly followed by three 4 ml washes of the membranes on the filter.
Membrane-bound 35S was quantitated as described in Section 2.3. Assay tubes
(borosilicate glass culture tubes; 13 x 100 mm) were siliconized with Sigmacote (Sigma-
Aldrich Canada) 24h prior to assay. All assays were conducted in triplicate. Basal
binding, defined as the binding occuring in the absence of agonist (CP55940) minus
70
non-specific binding (measured with 100 µM unlabelled GTPγS present) averaged 95.4
+ 1.4%. 100 nM CP-55940 increased the basal binding of [35S]GTPγS by 65.6 + 2.8%.
2.3.5. Data analysis
Results are given as the mean ± S.E.M. Curve fitting by non-linear regression
analysis and estimation of IC50 (concentration of study compound producing 50%
inhibition) was carried out using Prism 4 (GraphPad Software Inc., San Diego, CA). Ki
values for study compounds were determined using the Cheng-Prusoff equation with
0.35 nM as the Kd for [3H]CP55940.
2.4. Results
Fig. 2.2 shows the concentration-dependent inhibition of [3H]CP55940 binding to
mouse brain CB1 receptors by benzophenanthridines under equilibrium conditions.
Sanguinarine and chelerythrine exhibited inhibitory potencies as estimated from IC50s of
1.10 µM (95% CI = 0.62-1.93 µM) and 2.20 µM (95% CI = 1.55-3.13 µM) respectively.
At higher concentrations both approached full inhibition of [3H] radioligand binding, with
sanguinarine slightly more effacacious than chelerythrine.
The isoquinoline alkaloids berberine and papaverine, as well as the phthalide
isoquinoline (-)-β-hydrastine individually achieved no greater than 17.6 % inhibition of
[3H] radioligand binding at 30 µM (Table 1). PMSF (0.5 mM) also had no effect on
[3H]CP55940 binding (Table 2.1).
Chelerythrine and sanguinarine inhibited both agonist- (CP55940-) stimulated
and basal binding of [35S]GTPγS binding to mouse brain membranes (Figs. 2.3 and 2.4).
In the agonist-stimulated [35S]GTPγS binding assays (Figs. 2.3a and 2.4a), chelerythrine
produced an additional 54.92 + 2.54% and 67.52 + 3.40% encroachment into the basal
signal at 20 µM and 50 µM respectively and sanguinarine caused an additional 50.12 +
2.84%, 68.28 + 0.56% and 74.76 + 1.05% encroachment into the basal signal at 4 µM,
10 µM and 50 µM respectively. The IC50s for agonist stimulation were: chelerythrine 2.09
µM (95% CI = 1.73-2.44 µM) and sanguinarine 1.22 µM (95% CI = 1.05-1.50 µM). The
IC50s for basal binding were: chelerythrine 10.06 µM (95% CI = 7.18-15.54 µM) and
71
sanguinarine 5.19 µM (95% CI = 4.59-5.89 µM). Under identical conditions, AM251
inhibited 100 nM CP55940-stimulated [35S]GTPγS binding by 59.59% and 98.93% at
0.01 µM and 1 µM respectively (Table 2.2), confirming that the G-protein under
investigation is coupled to CB1 receptors. However, at 20 µM AM251 produced minimal
(circa 10%) inhibition of basal [35S]GTPγS binding (Table 2.2). No significant inhibitory
effects of berberine, papaverine, (-)-β-hydrastine (all at 40 µM) on CP55940-stimulated
or basal [35S]GTPγS binding were detected (Table 2.3).
The effects of piperonyl butoxide and (S)-methoprene on [3H]CP55940 binding
and agonist-stimulated [35S]GTPγS binding are shown in Fig. 5a and 5b. Piperonyl
butoxide and (S)-methoprene inhibited [3H]CP55940 binding to CB1 receptors [IC50s:
piperonyl butoxide 8.2 µM (95% CI = 7.08-9.32 µM) and (S)-methoprene 16.4 µM (95%
CI = 13.7-19.06 µM)]. In parallel experiments, 4 µM (S)-methoprene inhibited [3H]CP-
55940 binding by 27.32 + 4.11% and 28.16 + 4.17% in the absence and presence of 50
µM PMSF respectively, showing that esterases were not limiting the inhibitory potency of
this compound. Both compounds also blocked CP55940-stimulated binding of
[35S]GTPγS to the G-protein [IC50s: piperonyl butoxide 22.5 µM (95% CI = 18.98-36.02
µM) and (S)-methoprene 19.31 µM (95% CI = 17.01-21.61 µM)]. No effects of piperonyl
butoxide or (S)-methoprene on basal binding of [35S]GTPγS were observed (Table 2.4).
2.5. Discussion
The results of this investigation demonstrate that benzophenanthridine alkaloids,
piperonyl butoxide and (S)-methoprene inhibit the G-protein-coupled CB1 receptor of
mammalian brain, and suggest a clear functional difference between the actions of the
natural product alkaloids and the synthetic compounds at this complex.
The IC50s of sanguinarine and chelerythrine in the [3H]CP55940 binding assay lie
in the 1-2 µM range, which places them very similar in potency to cannabidiol,
virodhamine, various ∆8-tetrahydrocannabinol derivatives and certain bicyclic resorcinols
(Devane et al., 1988; Compton et al., 1993; Steffens et al., 2005; Wiley et al., 2002),
however, these benzophenanthridines are considerably less potent than ∆9-
tetrahydrocannabinol and ∆9-tetrahydrocannabivarin, which inhibit the binding of
72
[3H]CP55940 at low nanomolar concentrations (Devane et al., 1988; Thomas et al.,
2005).
Antagonist-like actions for sanguinarine and chelerythrine at the G-protein-
coupled CB1 were strongly indicated since both alkaloids produce inhibition of CP55940-
stimulated [35S]GTPγS binding at IC50s identical to those for displacement of [3H]CP-
55940 binding to mouse brain membranes. The effect of these benzophenanthridines in
the agonist-stimulated [35S]GTPγS assay is qualitatively similar to that of the CB1
receptor-selective antagonist AM251. However, at greater than maximum effect
concentrations, and in marked contrast to AM251, sanguinarine and chelerythrine
showed considerable encroachment into the basal component of [35S]GTPγS binding
with CP55940 present. Again unlike AM251, chelerythrine and sanguinarine strongly
inhibited basal [35S]GTPγS binding. On the basis of these G-protein modulatory profiles,
chelerythrine and sanguinarine may be more reasonably classified as inverse agonists
at the G-protein-coupled CB1 receptor. The slightly higher inhibitory potency of
sanguinarine compared to chelerythrine in the [3H]CP55940 and agonist stimulated
[35S]GTPγS binding assays was likely due to the methylenedioxy moiety in place of the
two methoxy groups on ring A. We cannot say whether the cationic (quaternary
ammonium) form or the hydroxide adduct (pseudobase; which is formed at physiological
pH; Slalinova et al., 2001) binds to the G-protein-coupled CB1 receptor. However, the
existence of an equilibrium state raises the possibility that that lower concentrations of
an active species may cause the effects described here. The failure of berberine (which
can also form the pseudobase), papaverine and (-)-β-hydrastine to interact with the G-
protein-coupled CB1 receptor in these assays indicates that aromatic ring stacking and
position of the nitrogen atom are important for inhibitory activity.
Our original hypothesis that the benzophenanthridines may bind in a similar way
to phytocannabinoids at the CB1 receptor seems improbable. However, since it is known
that 1) GTP and non-hydrolysable GTP analogs including GTPγS and guanyl-5’-yl
imidodiphosphate allosterically dissociate [3H]CP55940 from the CB1 receptor (Devane
et al., 1988; Houston and Howlett, 1993) and that 2) low micromolar concentrations of
sanguinarine and chelerythrine inhibit the binding of a fluorescent GTP probe to the GTP
binding protein Rac1b in a competitive fashion (Beausoleil et al., 2009), we think it most
73
likely that chelerythrine and sanguinarine exert retrograde allosteric inhibition of agonist
binding to the CB1 receptor by targeting the guanine nucleotide recognition site on the
associated G-protein. In theory, a drug that acts selectively in this fashion could offer an
alternative mechanism to the diarylpyrazoles for downregulating endocannabinoid-
mediated signaling in the CNS, and therefore may have potential in weight reduction and
the treatment of various metabolic disorders in humans. It is also possible that the level
of inhibition of endocannabinoid activation of CB1 receptors in vivo may be more readily
managed with moderate potency drugs that are selective for the G-protein component of
this complex, potentially reducing psychiatric side effects. Some evidence exists for
selectivity of benzophenanthridines, since it is known that chelerythrine and
sanguinarine bind more avidly to certain GTP binding proteins than members of the
berberine series (Beausoleil et al., 2009). In addition, our data on basal and agonist-
stimulated [35S]GTPγS binding, which reflect respectively the sum of binding to all GTP
binding sites in the membrane fraction versus only those specifically activated by
CP55940, indicate that the latter response is 4-5 fold more sensitive to blockade by
chelerythrine and sanguinarine. Benzophenanthridines may therefore offer a novel area
of chemistry for development of drugs that negatively regulate the endocannabinoid
system, but achieving selectivity at CB1 over CB2 receptors through this mechanism may
be difficult. It is important to note that chelerythrine was reported originally as a potent
and specific protein kinase C inhibitor (Herbert et al., 1990), although considerable doubt
now exists (Lee et al. 1998), and studies by Garcia et al. (1998) found that stimulation of
protein kinase C with phorbol 12-myristate 13-acetate phosphorylates the CB1 receptor
which in turn suppresses both cannabinoid-induced activation of an inwardly rectifying
K+ current and depression of P/Q type Ca++ channel activity. If, in our experiments, the
benzophenanthridines were inhibiting protein kinase C through this particular
mechanism, we would predict they would not block (or possibly facilitate) CP-55940-
mediated activation of the G-protein. Consistent with the results presented here,
chelerythrine has been reported to inhibit desacetyllevonantradol-dependent activation
of the G-protein-coupled CB1 receptor in N18TG2 neuroblastoma cells, however, this
observation was interpreted as supporting other direct evidence for modulation of a
downstream protein kinase C by the CB1 receptor (Rubovitch et al., 2004).
74
We also considered the possibility that our study compounds may act on CB1
receptors indirectly by increasing the levels of anandamide and/or 2-AG through
inhibition of endocannabinoid degrading enzymes (eg FAAH or MAGL). Such a
mechanism is involved in the action of organophosphorus compounds (Nomura et al.,
2008). However, since these endocannabinoids are CB1 receptor agonists, our study
compounds would be expected to significantly increase the basal [35S]GTPγS binding
signal (as we show with CP55940). This is clearly not the case since the
benzophenanthridines strongly inhibit, and methoprene and piperonyl butoxide fail to
influence basal [35S]GTPγS binding. Furthermore, 0.5 mM PMSF (a concentration which
would be expected to fully inhibit FAAH and provide about 50% inhibition of MAGL) has
no influence on the binding of [3H]CP55940 to CB1 receptors in our in vitro system,
offering another line of evidence that our study compounds could not act by this
mechanism.
Piperonyl butoxide is widely used as a synergist for insecticides (Jones, 1998),
while methoprene exerts growth regulatory effects in insects rather than direct toxicity
(Henrick, 2007). Both exhibit low mammalian toxicity [acute oral LD50s in the rat: 7,500-
10,000 mg/kg (piperonyl butoxide) and >34,600 mg/kg (methoprene); Siddall, 1976;
Hawkins et al., 1977]. Our results indicate that in contrast to the benzophenanthridines,
piperonyl butoxide and (S)-methoprene act as neutral antagonists of the CB1 receptor,
giving some support to our hypothesis that they act as structural mimics of
phytocannabinoids or endocannabinoids. However, based on displacement of
[3H]CP55940 binding, their potencies are approximately 4-10 fold lower than the CB1
receptor inhibitor virodhamine (Steffens et al., 2005) and 2-hydroxyphenyl
arachidonamide (Edgemont et al., 1995), 12-24 fold lower than LH-21 (Chen et al.,
2008) and 130-260-fold lower than ∆9-tetrahydrocannabivarin (Thomas et al., 2005).
While a variety of hydroxylated analogs of anandamide have been evaluated at CB1
receptors (Van der Stelt et al., 2002), we are not aware of attempts to optimize
alkylalkoxymethylenedioxyphenyl systems, or methoprene. Such compounds may offer
alternative starting templates for optimization of novel CB1 receptor modulators.
75
2.6. Figures and Tables
N
O
O
O
O
C
H
3
C
H
3
N
C
H
3
O
O
O
O
C
H
3
C
H
3
(a) Sanguinarine (b) Berberine (c) Chelerythrine
N
O
O
C
H
3
O
O
C
H
3
C
H
3
C
H
3
N
O
O
O
C
H
3
O
O
C
H
3
O
H
H
C
H
3
O
H
N
H
O
(d) Papavarine (e) (-)(1R,9S)-β-Hydrastine (f) Anandamide
O
O
O
O
O
O
O
O
H
O
H
(g) Piperonyl butoxide (h) 2-Arachidonoyl glycerol
O
O
O
C
H
3
H
(i) (S)- Methoprene
Figure 2.1 The structures of sanguinarine, berberine, papavarine and possible comparison of conformations of piperonyl butoxide and (S)-methoprene with anandamide and 2-arachidonoyl glycrol. Also possible comparison of sanguinarine and (S)-methoprene with ∆9-tetrahydrocannabinol and ∆9-tetrahydrocannabivarin (continued on page 76).
76
N
C
H
3
O
H
O
O
O
O
O
O
H
(j) Sanguinarine(pseudobase form) (k) ∆9-Tetrahydrocannabinol
O
O
O
C
H
3
H
O
O
H
(l) (S)-methoprene(m) ∆9- Tetrahydrocannabivarin
Figure 2.1 (continued) The structures of sanguinarine, berberine, papavarine and possible comparison of conformations of piperonyl butoxide and (S)-methoprene with anandamide and 2-arachidonoyl glycrol. Also possible comparison of sanguinarine and (S)-methoprene with ∆9-tetrahydrocannabinol and ∆9-tetrahydrocannabivarin.
(a) - (e): The structures of sanguinarine, chelerythrine, berberine, papaverine and (-) (1R,9S)-β-hydrastine. (f) - (i): Comparisons of possible conformations of piperonyl butoxide and (S)-methoprene with anandamide and 2-arachidonoyl glycerol respectively. (j - m): Comparison of the pseudobase form of sanguinarine and a possible conformation of (S)-methoprene with ∆9-tetrahydrocannabinol and ∆9-tetrahydrocannabivarin.
All structures redrawn using IsisDraw from Dhopeshwarkar et al. (2011).
77
Figure 2.2 Concentration-dependent inhibition of [3H]CP55940 binding to mouse brain CB1 receptors by sanguinarine and chelerythrine. Values represent mean + S.E.M. of at least 3 independent experiments each performed in duplicate. Ki values were 0.38 µM (sanguinarine) and 0.57 µM (chelerythrine).
78
Figure 2.3a Inhibition of A) CP55940-stimulated and B) basal binding of [35S]GTPγS to mouse brain membranes by chelerythrine. Values represent mean + S.E.M. of 3 independent experiments each performed in triplicate.
79
Figure 2.3b Inhibition of A) CP55940-stimulated and B) basal binding of [35S]GTPγS to mouse brain membranes by chelerythrine. Values represent mean + S.E.M. of 3 independent experiments each performed in triplicate.Basal binding data provided by Mr Saurabh Jain.
80
Figure 2.4a Inhibition of A) CP55940-stimulated and B) basal binding of [35S]GTPγS to mouse brain membranes by sanguinarine. Values represent mean + S.E.M. of 3 independent experiments each performed in triplicate.
81
Figure 2.4b Inhibition of A) CP55940-stimulated and B) basal binding of [35S]GTPγS to mouse brain membranes by sanguinarine. Values represent mean + S.E.M. of 3 independent experiments each performed in triplicate. Basal binding data provided by Mr Saurabh Jain.
82
Figure 2.5a A) Concentration-dependent inhibition of [3H]CP55940 binding to mouse brain CB1 receptors by (S)-methoprene and piperonyl butoxide. Ki values were 2.13 µM (methoprene) and 4.25 µM (piperonyl butoxide). B) Inhibition of CP55940-stimulated binding of [35S]GTPγS to mouse brain membranes by (S)-methoprene and piperonyl butoxide. Values represent mean + S.E.M. of 3 independent experiments each performed in triplicate.
83
Figure 2.5b A) Concentration-dependent inhibition of [3H]CP55940 binding to mouse brain CB1 receptors by (S)-methoprene and piperonyl butoxide. Ki values were 2.13 µM (methoprene) and 4.25 µM (piperonyl butoxide). B) Inhibition of CP55940-stimulated binding of [35S]GTPγS to mouse brain membranes by (S)-methoprene and piperonyl butoxide. Values represent mean + S.E.M. of 3 independent experiments each performed in triplicate.
84
Table 2.1 Inhibition of specific [3H]CP55940 binding to mouse brain membranes by isoquinoline type compounds and PMSF. Isoquinolines were present in the assay at 30 µM and PMSF was present at 0.5 mM. Data represent mean + S.E.M. of 3 independent experiments.
Compound Inhibition (%)
Berberine 12.05 + 2.2
1R, 9S-(-)-β-Hydrastine 4.09 + 1.64
Papaverine 17.56 + 0.5
PMSF 1.27 + 3.12
85
Table 2.2 Inhibition of 100 nM CP55940-stimulated and basal [35S]GTPγS binding to mouse brain membranes by AM251. Data represent mean + S.E.M. of 3 independent experiments. ND = not determined. Results provided by Mr Saurabh Jain.
AM251
(µM)
Inhibition of CP55940-stimulated
[35S]GTPγS binding (%)
Inhibition of
basal [35S]GTPγS
binding (%)
0.010 59.59 + 2.66 ND
1.0 98.93 + 1.62 ND
10.0 100 7.61 + 6.32
20.0 ND 10.01 + 1.37
9.21 + 0.76% encroachment of AM251 on the basal component of [35S]GTPγS
binding was observed in experiments involving 10 µM CP55940 agonist, as observed by
others with the closely related analog SR141716A (Selley et al., 1996; Petitet et al.,
1997).
86
Table 2.3 Lack of effect of isoquinoline type compounds on CP55940-stimulated and basal [35S]GTPγS binding to mouse brain membranes. Study compounds were present in the assay at 40 µM. Data represent mean + S.E.M. of 3 independent experiments.
Compound
Inhibition of CP55940-
stimulated [35S]GTPγS
binding (%)
Effect on basal
[35S]GTPγS binding
(+ = % increase; - = % decrease)
Berberine 2.82 + 0.89 a 1.98 + 0.35
1R, 9S-(-)β-
Hydrastine
2.23 + 2.23 - 0.50 + 5.77
Papaverine 5.79 + 1.77 3.90 + 2.32
a represents a % increase in CP-55940-induced stimulation
87
Table 2.4 Lack of effect of piperonyl butoxide and (S)-methoprene on the basal binding of [35S]GTPγS to mouse brain membranes. Values represent mean + S.E.M. of 3 independent experiments.
Compound
Concentration
(% increase)
Piperonyl butoxide 20 µM 8.93 + 6.20
30 µM 7.21 + 1.61
40 µM 1.47 + 0.47
(S)-Methoprene 25 µM 4.33 + 6.32
40 µM 2.80 + 0.80
50 µM 0.03 + 5.59
88
3. The G protein-coupled cannabinoid-1 (CB1) receptor of mammalian brain: Inhibition by phthalate esters in vitro.
3.1. Abstract
This research examines the in vitro interaction of phthalate diesters and
monoesters with the G protein-coupled cannabinoid 1 (CB1) receptor, a presynaptic
complex involved in the regulation of synaptic activity in mammalian brain. The diesters,
n-butylbenzylphthalate (nBBP), di-n-hexylphthalate (DnHP), di-n-butylphthalate (DnBP),
di-2-ethylhexylphthalate (DEHP), di-isooctylphthalate (DiOP) and di-n-octylphthalate
(DnOP) inhibited the specific binding of the CB1 receptor agonist [3H]CP55940 to mouse
whole brain membranes at micromolar concentrations (IC50s: nBBP 27.4 µM; DnHP 33.9
µM; DnBP 45.9 µM; DEHP 47.4 µM; DiOP 55.4 µM; DnOP 75.2 µM). DnHP, DnBP and
nBBP achieved full (or close to full) blockade of [3H]CP55940 binding, whereas DEHP,
DiOP and DnOP produced partial (55-70%) inhibition. Binding experiments with
phenylmethane-sulfonylfluoride (PMSF) indicated that the ester linkages of nBBP and
DnBP remain intact during assay. The monoesters mono-2-ethylhexylphthalate
(M2EHP) and mono-isohexylphthalate (MiHP) failed to reach IC50 at 150 µM and mono-
n-butylphthalate (MnBP) was inactive. Inhibitory potencies in the [3H]CP55940 binding
assay were positively correlated with inhibition of CB1 receptor agonist-stimulated
binding of [35S]GTPγS to the G protein, demonstrating that phthalates cause functional
impairment of this complex. DnBP, nBBP and DEHP also inhibited binding of
[3H]SR141716A, whereas inhibition with MiHP was comparatively weak and MnBP had
no effect. Equilibrium binding experiments with [3H]SR141716A showed that phthalates
reduce the Bmax of radioligand without changing its Kd. DnBP and nBBP also rapidly
enhanced the dissociation of [3H]SR141716A. Our data are consistent with an allosteric
mechanism for inhibition, with phthalates acting as relatively low affinity antagonists of
CB1 receptors and cannabinoid agonist-dependent activation of the G-protein. Further
89
studies are warranted, since some phthalate esters may have potential to modify CB1
receptor-dependent behavioral and physiological outcomes in the whole animal.
Acknowledgement: This chapter adheres closely to our paper published by
Neurochemistry International as: Bisset, K.M., Dhopeshwarkar, A.S., Liao C., Nicholson
R.A. (2011). The G protein-coupled cannabinoid-1 (CB1) receptor of mammalian brain:
Inhibition by phthalate esters in vitro. Neurochemistry International, 59(1), 706-713.
3.2. Introduction
CB1 receptors together with their associated G-proteins form an integral part of
the endocannabinoid system which regulates the activity at many synapses in the brain
via a negative feedback mechanism. When synaptic activity intensifies, postsynaptic
neurons generate endocannabinoids which translocate retrogradely and bind to
presynaptic CB1 receptors. Activation of the G-protein then causes inhibition of voltage-
gated Ca++ channels (Mackie and Hille, 1992; Twichell et al., 1997, Kushmerick et al.,
2004) and activation of inwardly rectifying K+ channels (Deadwyler et al., 1993; Mackie
et al., 1995). In addition, the lowering of cAMP levels through negative modulation of
adenylate cyclase by the G-protein (Howlett and Fleming, 1984; Howlett, 1985; Bidault-
Russell et al., 1990) can reduce protein kinase A activity (Kim et al., 2006) which favors
activation of A-type K+ channels (Hoffman and Johnson 1998). Stimulation of
presynaptic CB1 receptors therefore downregulates Ca++-dependent release of
neurotransmitters (Chevaleyre et al., 2006; Kreitzer and Regehr, 2001; Wilson and
Nicoll, 2001) and changes in K+ conductances reduce presynaptic excitability (Mackie et
al., 1995; Hoffman et al., 1997; Zona et al., 2002).
In addition to endocannabinoids, several classes of synthetic CB1 receptor
activators have been identified (Devane et al., 1988; Compton et al., 1993; Felder et al.,
1995) and correlations between their binding affinities at CB1 receptors and in vivo
outcomes such as antinociception, hypothermia, catelepsy and psychoactivity have been
90
demonstrated (Compton et al., 1993). Moreover, various behavioral responses of
classical CB1 receptor agonists are typically blocked by antagonists of CB1 receptors
such as SR141716A (Compton et al., 1996; Rinaldi-Carmona et al., 1995).
Investigations involving phytocannabinoids of Cannabis sativa, various synthetic ligands
and endogenous cannabinoids of the CNS have radically improved our understanding of
the neurobiology and pharmacology of the endocannabinoid system to the extent that
novel therapeutic applications can now be pursued (Pertwee, 1997; Di Marzo, 2009;
Muccioli, 2007; Thakur et al., 2009).
By contrast, considerably less emphasis has been placed on synthetic
environmental chemicals and their potential to modify CB1 receptor function in
mammalian brain. However, it is known that binding of [3H]CP55940 to CB1 receptors is
strongly inhibited by certain organophosphorus and organosulfur compounds
incorporating longer chain alkyl moieties (Segall et al., 2003), and also by various
organophophorus pesticides (Quistad et al., 2002). We have demonstrated that at
micromolar concentrations, methoprene and piperonyl butoxide (chemicals of low acute
toxicity used in pest management) can inhibit CP55940 action at CB1 receptors in vitro,
possibly by adopting endocannabinoid-like conformations or by methoprene acting
alternatively as a flexible analog of ∆9-tetrahydrocannabinol (Dhopeshwarkar et al.,
2011).
During exploratory investigations we found that certain phthalate esters reduce
the binding of [3H]CP55940. Phthalate esters are found in a wide range of consumer and
industrial products (Schettler, 2006). They are utilized as formulation components in
paints, adhesives and medicines as well as in personal care and pest management
products. As phthalate esters remain relatively mobile within polymeric matrices, they
are frequently added to plastics to enhance the flexibility, elasticity and self-lubricating
properties of the end products. Significant quantities of phthalate esters are incorporated
into plastics used to manufacture toys, food wrappings and medical devices (Heudorf et
al., 2007). Human exposures to phthalate esters can occur following dermal, oral and
vapor phase contact and exposures resulting from medical procedures and PVC
manufacturing can be significant for some phthalates (Kavlock et al., 2002a and 2002b)
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The acute toxicities of phthalate esters in rats and mice are generally quite low
(Calley et al., 1966; Heudorf et al., 2007). However, following chronic administration to
rodents, several phthalate esters have been identified as reproductive and
developmental toxicants (Huendorf et al., 2007) and evidence of hepatic and renal
impairment has also been demonstrated for example with DEHP in some rodent models
(Kavlock, et al., 2002a). Kim et al. (2009) reported a positive association between
phthalate ester exposure (measured as urinary phthalate metabolites) and symptoms of
attention-deficit/hyperactivity disorder in school-age children. A number of phthalate
esters influence brain function following intraperitoneal injection as evidenced by their
ability to modify hexobarbital sleep time in rodents (Calley et al., 1966). Additionally, the
cholinergic system of vertebrate brain is sensitive to phthalate esters. Various diesters
(and to a lesser extent monoesters) inhibit nicotinic acetylcholine receptor-mediated
calcium signaling in human neuroblastoma cells (Liu et al., 2009; Lu et al., 2004) and
fish exposed to dietary dibutyl- and diethylhexyl- phthalates show reduced brain
acetylcholinesterase activity (Jee et al., 2009).
In the present investigation we examined a group of phthalate diesters and
monoesters for their ability to influence the binding of [3H]CP55940 and [3H]SR141716A.
These radioligands and also the endocannabinoid anandamide bind to closely
associated loci within the CB1 receptor binding pocket (McAllister et al., 2003). We then
carried out experiments with [35S]GTPγS to evaluate potential interference of phthalates
with the functional coupling of the CB1 receptor to its G-protein (Selley et al., 1996;
Petitet et al., 1997).
3.3. Materials and methods
3.3.1. Animals
This research was carried out on male mice (weight approx. 25 g; CD1 strain)
that were purchased from Charles River Laboratories (Saint-Constant, Quebec,
Canada). Animals had continuous access to water and food (rodent Lab Diet 5001).
Mice were subjected to a 12 h light/12 h dark cycle and were maintained at 21 + 2 oC
and 50 + 10 % relative humidity. All experimental procedures using mice followed
92
guidelines developed by the Canadian Council on Animal Care and had prior approval of
the Animal Care Committee at Simon Fraser University.
3.3.2. Investigation of the effects of phthalate esters on the binding of [3H]CP55940 and [3H]SR141716A to CB1 receptors of mouse brain.
We used the method of Quistad et al., (2002) to investigate the effects of
phthalates on [3H]CP55940 binding. Membrane isolation procedures were carried out at
0-4 oC. Mouse whole brains were homogenized (10 up/down strokes) in ice-cold buffer
(Trisma base (100 mM), EDTA (1 mM) adjusted to pH 9 with HCl; 1 brain/10 ml buffer)
using a motor driven homogenizer (pestle rotation rate approx. 1500 rpm). The
homogenate was centrifuged at 900 x g (Beckman J2HS; JA20 rotor) for 10 min. The
supernatant was centrifuged at 11,500 x g for 25 min. After thorough resuspension of the
membrane pellet in a small volume of ice-cold buffer [Trisma base (50 mM), EDTA (1
mM) and MgCl2.6H2O (3 mM); adjusted to pH 7.4 with HCl], the protein concentration
was diluted to approx. 6.5 mg/ml and the binding preparation then stored in aliquots at -
80 oC. Just prior to experiments, the membrane suspension was thawed on ice, taken up
in a 5 ml syringe and slowly forced into a tube through an 18g needle (with its square cut
tip held close to the base of the tube). The membranes were moved in and out of the
syringe 5-6 times then thoroughly vortexed, a procedure that helped reduce variability
between replicates. Phthalate esters (see Figure 3.1 for structures), dissolved in 5 µl
DMSO (or 5 µl DMSO as the solvent control) were added to borosilicate glass culture
tubes (13 x 100 mm; without siliconization) and this was followed by addition of binding
buffer (500 µl; Trisma base (50 mM), EDTA (1 mM), MgCl2.6H2O (3 mM), BSA (fatty acid
free; 3 mg/ml) adjusted to pH 7.4 with HCl). Brain membranes (170.67 + 0.84) µg
protein) were then placed in each tube and the suspension was then thoroughly
vortexed and pre-incubated at room temperature for 15 minutes. Additions of [3H]CP-
55940 (side chain-2,3,4-[3H]; sp. act. 174.6 Ci/ mmol; Perkin Elmer Life and Analytical
Sciences, Canada) to each tube were made in 10 µl DMSO (final radioligand
concentration 1.0 nM; final DMSO concentration 2.8%), and after careful mixing,
incubations were run for 1.5 h at 30 oC with gentle shaking. Incubations were stopped by
the addition of 1 ml of ice-cold wash buffer (0.9% NaCl containing 2 mg/ml BSA) and
membranes were quickly harvested by vacuum filtration on pre-soaked Whatman GF/C
93
filters. Membranes trapped on filters were immediately washed (3 x 4 ml) with ice-cold
wash buffer. Filters were completely dried (in a fume hood). Scintillation cocktail (BCS,
Amersham Bioscience UK) was then added and radioactivity was measured using liquid
scintillation counting. Non-specific binding, measured in the presence of unlabeled
WIN55,212-2 (10 µM), was subtracted from total binding to calculate the specific binding
signal (76.8 + 1.1% of total binding). This is very similar to the 80% obtained by Quistad
et al. (2002) using 1 µM WIN55212-2. Under the present assay conditions the IC50 for
WIN55212-2 was 6 nM and maximum displacement of [3H]CP55940 by WIN55212-2
was achieved at both 1 and 10 µM.
Selected phthalate esters were also evaluated in binding assays using the CB1
receptor antagonist [3H]SR141716A (sp. act. 56 Ci/ mmol; Perkin Elmer Life and
Analytical Sciences, Canada). For competitive displacement assays an identical
experimental procedure to that described above was used. [3H]SR141716A was
present at 1.2 nM and AM251 (at 2 µM) was introduced to estimate the specific binding
signal, which averaged (71.0 + 0.7%). For association experiments, membranes were
either preincubated with the phthalate ester for 15 min. before [3H]SR141716A addition
or received simultaneous application of phthalate and radioligand. Dissociations were
initiated on CB1 receptors equilibrated with [3H]SR141716A using either a saturating
concentration of AM251 or this concentration of AM251 plus the phthalate ester.
In each experiment with [3H]CP55940 or [3H]SR141716A, binding in the
absence and presence of unlabeled WIN55212-2 or AM251 was performed in triplicate
and test compounds were assayed in duplicate. A minimum of three independent
experiments were performed for every treatment. Protein measurements were
conducted according to Peterson (1977).
3.3.3. Investigation of phthalate interference with CB1 receptor agonist-stimulated [35S]GTPγS binding to the Gα-protein.
The method we used to isolate the mouse whole brain membrane fraction and
determine the effects of phthalates on agonist-stimulated [35S]GTPγS binding generally
followed the procedure published by Breivogel et al. (2000). Whole brains were quickly
removed from two mice and homogenized for 15 sec in 10 ml of ice-cold isolation buffer
94
(Trisma base (50 mM), MgCl2.6H20 (3 mM), EGTA (0.2 mM), NaCl (100 mM) with pH
adjusted to 7.4) using a tissue fragmenter (Polytron Kinematica GmBH; setting 6). The
suspension was centrifuged in a Beckman J2HS centrifuge (24,000 x g for 25 min at 2 oC) and the pellet was then resuspended in fresh ice-cold isolation buffer and re-
centrifuged. The washed membrane pellet was thoroughly dispersed in isolation buffer,
then protein concentration was adjusted to approx. 7 mg/ml before aliquots were
transferred to a -80 oC freezer. Prior to experimentation, the membrane fractions were
thawed on ice and completely dispersed as described in the previous section. This
procedure helped improve the reproducibility between replicates without obvious loss in
agonist-stimulated [35S]radioligand binding. Binding experiments were performed using
guanosine 5'-O-(g-γ35S]thio)-triphosphate ([35S]GTPγS) of sp. act. 1250 Ci/mmol
purchased from Perkin Elmer Life and Analytical Sciences, Canada.
The phthalate esters (dissolved in DMSO; 5 µl) or DMSO control (as required)
were placed in borosilicate glass tubes (13 x 100 mm; siliconized 24 h prior to assay with
Sigmacote [Sigma-Aldrich Canada]) and then 500 µl of isolation buffer (pH 7.4) was
added which contained fatty-acid free bovine serum albumin (1 mg/ml), guanosine
diphosphate (GDP; 100 µM), dithiothreitol (20 µM), [35S]GTPγS (0.14 nM final
concentration) and adenosine deaminase (0.004 units/ml). The membrane fraction (70.1
+ 4.2 µg protein/assay) was then added and after thorough vortexing, initial 15 min
incubation was carried out at room temperature. The CB1 receptor agonist CP55940
(100 nM final concentration; in 5 µl DMSO) or DMSO solvent control were then added
and, after thorough mixing, incubations were continued for 1.5 h at 30 0C with gentle
shaking. The final concentration of DMSO was 1.9%. Incubations were terminated by
introduction of 2 ml of ice-cold wash buffer (50 mM Trisma base:HCl; pH 7.4) which was
immediately followed by rapid vacuum filtration through pre-soaked Whatman GF/B
filters. Membranes trapped on filters were subjected to three 4 ml washes with the same
buffer. Membrane-bound 35S was measured using liquid scintillation counting. All assays
were conducted in triplicate. Specific binding of [35S]GTPγS (90.7 + 1.4%) was
calculated by subtracting [35S]GTPγS bound in the presence of 100 µM unlabelled
GTPγS from total binding. 100 nM CP55940 stimulated the basal specific [35S]GTPγS
binding signal by 57.7 + 0.6% and the effect of phthalates on this signal was
investigated.
95
3.3.4. Data analysis
Values are given as mean ± S.E.M. All values of IC50 (concentration of phthalate
ester producing 50% inhibition) were estimated from the concentration:response
relationships defined by non-linear regression analysis using Prism 4 (GraphPad
Software Inc., San Diego, CA, USA). Other non-linear and linear regression analyses
were likewise carried out with Prism 4.
3.4. Results
3.4.1. Effects of phthalate esters on binding of [3H]CP55940 to CB1 receptors.
The effects of the di- and mono-esters on the binding of [3H]CP55940 to CB1
receptors in mouse whole brain membranes are shown in Figures 3.2 and 3.3. Apart
from MnBP, all compounds produced concentration-dependent inhibition of [3H]CP55940
binding. Within the diester series, nBBP and DnHP were the most potent as indicated by
IC50s of 27.4 µM (95% CI = 20.7-36.5 µM) and 33.9 µM (95% CI = 26.5-38.5 µM)
respectively. DnBP, DEHP and DiOP were of intermediate potency (IC50s of 45.9 µM
(95% CI = 35.9-58.6 µM), 47.4 µM (95% CI = 41.5-54.1 µM) and 55.4 µM (95% CI =
45.8-67.0 µM) respectively), while DnOP was the weakest (IC50: 75.2 µM (95% CI =
65.9-87.2 µM). Based on the level of inhibition at 150 µM (the maximum concentration
employed), BBP, DnHP and DnBP were the most effacacious (85-100% inhibition),
followed by DEHP and DiOP (60-70% inhibition), while DnOP displayed lower efficacy
(50-60% inhibition). At 150 µM MiHP and M2EHP achieved less than 50% inhibition of
[3H]CP55940 binding and MnBP was inactive. In a separate series of experiments,
PMSF failed to modify the inhibitory effects of nBBP and DnBP on [3H]CP55940 binding
(Table 3.1).
3.4.2. Effects of selected phthalate esters on binding of [3H]SR141716A to CB1 receptors.
Table 3.2 shows the inhibitory effects of selected di- and mono-esters on the
specific binding of [3H]SR141716A to CB1 receptors of mouse brain. In the case of the
diesters nBBP, DBP and DEHP, the extent of inhibition of [3H]SR141716A binding at
96
concentrations that achieve 50% inhibition of [3H]CP55940 binding resulted in inhibitory
effects that were approximately 35-45% higher (nBBP, DnBP) and 25% lower (DEHP).
The monoester MnBP (100 µM) had no effect on specific binding of [3H]SR141716A
while MiHP produced circa. 33% inhibition.
3.4.3. Influence of selected phthalates on the saturation binding of [3H]SR141716A to CB1 receptors
The control saturation binding curve was constructed by measuring the specific
binding of [3H]SR141716A to CB1 receptors at equilibrium over a range of radioligand
concentrations (0.032 to 2.8 nM). Experiments were concurrently performed in the
presence of nBBP or DnBP (Figure 3.4). Analyses revealed that phthalates have
negligible effect on the Kd of radioligand binding but reduce the Bmax by 37% (nBBP) and
60% (DnBP).
3.4.4. Effects of selected phthalates on [3H]SR141716A kinetics
The association time course of [3H]SR141716A between 0 and 3 min in the
absence of test compounds (Figures 3.5a and 3.5b) aligns with data published by
Rinaldi-Carmona et al., 1996 using synaptosomes. nBBP (35 µM) and DnBP (50 µM)
reduce the ability of [3H]SR141716A to equilibrate with CB1 receptors both when applied
in advance of the [3H]SR141716A (Figure 3.5a) and to a lesser extent when introduced
simultaneously with radioligand (Figure 3.5b). When combined with a saturating
concentration of AM 251, nBBP (35 µM) and DnBP (50 µM) increased the dissociation of
the [3H]SR141716A: CB1 receptor complex to levels much greater than that produced by
a saturating concentration of AM251 alone (Figure 3.6).
3.4.5. Effects of phthalates on CB1 receptor agonist-stimulated [35S]GTPγS binding to the Gα-protein
Both diesters and monoesters also had the capacity to inhibit CB1 receptor
agonist-activated binding of [35S]GTPγS to the Gα-protein and in agreement with
[3H]CP55940 binding data, the diesters were consistently more active (Figure 3.7).
Inhibitory effects of the study compounds on [3H]CP55940 binding and CP55940-
97
stimulated binding of [35S]GTPγS to the Ga-protein were closely associated (r2 = 0.7844;
Figure 3.8).
3.5. Discussion
The present investigation demonstrates that certain phthalate esters interfere
with the binding of [3H]CP55940 and [3H]SR141716A to CB1 receptors of mouse brain at
micromolar concentrations in vitro. Since we found that CB1 receptor agonist-stimulated
[35S]GTPγS binding is also decreased by phthalate esters, these compounds appear to
inhibit activation of the associated G-protein receptors by operating as low affinity CB1
receptor antagonists.
In the [3H]CP55940 binding assay, the IC50 values of the phthalate esters nBBP,
DnHP, DnBP and DEHP lie in the 27-47 µM range, which put them at similar potency to
cis-9,10-octadecenyl-a-methylethanolamide an analog of the sleep-inducing lipid cis-
oleamide (Boring et al., 1996), of higher potency than thujone, cis-oleamide and cis-
9,10-octadecenylethanolamide (Boring et al, 1996; Meschler and Howlett, 1999), but of
lower potency compared to the antagonists sanguinarine, chelerythrine, piperonyl
butoxide and (S)-methoprene (Dhopeshwarkar et al., 2011). In distinct contrast to
thujone and cis-oleamide and its analogs (Boring et al., 1996; Meschler and Howlett,
1999), the phthalate esters were able to inhibit CB1 receptor agonist activation of the G-
protein. It must be emphasized that nBBP, DnHP, DnBP and DEHP are obviously much
weaker as inhibitors of [3H]CP55940 binding, [3H]SR141716A binding and CB1 receptor
agonist-stimulated [35S]GTPγS binding when compared to the antagonists SR141716A
and AM251 which exert their effects in the low nanomolar range (Rinaldi-Carmona et al.,
1995; Rinaldi-Carmona et al., 1996; Gatley et al., 1997).
Within the group of phthalate esters examined in the present investigation, a
broad range of CB1 receptor inhibitory effects were demonstrated. Our data indicate that
the diesters are more potent inhibitors of [3H]CP55940 binding than the monoesters. A
similar differential was also noted by Liu et al., (2009) in studies on the inhibitory effects
of phthalates on Ca++ transients triggered by nAChR activation in human neuroblastoma
cells. In contrast, the potency of the monoester MEHP as an inhibitor of follicle -
98
stimulating hormone binding to Sertoli cells was reported to be at least three orders of
magnitude higher than for the diester DEHP; the latter phthalate showing no activity at
100 µM (Grasso et al., 1993).
Our results suggest that the overall relationship between phthalate diester
structure and inhibitory effects on [3H]CP55940 binding to CB1 receptors is complex.
nBBP and DnHP showed the highest inhibitory potencies (IC50s = 27.4 and 33.9 µM
respectively) combined with robust efficacy (85% or greater inhibition at maximum
concentration). Reducing the length of each n-hexyl group of DnHP by 2 carbons (i.e.
giving DnBP), reduces inhibitory potency but efficacy is retained at circa. 85%. By
contrast, DEHP, which can be considered as a bis 2-ethyl analog of DnHP or a bis 2-
propyl analog of DnBP, demonstrates reduced potency and efficacy against DnHP, and
similar potency with reduced efficacy compared to DnBP. The phthalate diesters with the
longer alkyl substituents (DiOP and DnOP) exhibited lower inhibitory potencies (IC50s
55.4 and 75.2) and efficacies were also comparatively low (55-65%). The critical nature
of the diester configuration for inhibition of [3H]CP55940 binding is emphasized by
comparison of nBBP and DnBP (which are amongst the most effective compounds
studied) with MnBP (a phthalate devoid of activity).
For the experiments with PMSF, we reasoned that using phthalates of
intermediate (DnBP) and higher (nBBP) potency at < IC50 would offer a sensitive basis
for assessment. Moreover, DnBP and nBBP (study compounds with alkyl and aryl
substituents respectively) might be expected to show different susceptibilities to
breakdown by serine hydrolases. Nonetheless, based on our experiments, there was no
evidence that serine hydrolases limit the inhibitory effect of either of these analogs in the
[3H]CP-55940 binding assay.
The equilibrium binding and dissociation data using [3H]SR141716A provide a
useful insight into the mechanism by which nBBP and DnBP inhibit radioligand binding.
The saturation isotherms demonstrate that these phthalates act by eliminating binding
sites for radioligand (i.e. Bmax is reduced), without affecting the affinity of radioligand for
the remaining sites (i.e. Kd is unchanged). Moreover, the dissociation experiments
strongly suggest that these compounds act allosterically with respect to the
[3H]SR141716A binding site, since under our assay conditions any access by phthalate
99
diesters to the radioligand binding site is completely prevented by the saturating levels of
AM251. The dissociation data also argue against an irreversible or tight binding of
phthalate esters to the [3H]SR141716A recognition site, another potential explanation of
the reduced Bmax and unchanged Kd. The time courses for dissociation of
[3H]SR141716A in the presence of nBBP and DnBP indicate that the binding of
phthalates to this allosteric binding site and subsequent negative modulation of
radioligand binding occurs very rapidly. Rapid engagement of phthalates with a site
coupled allosterically to the [3H]SR141716A binding site is also consistent with the
reduced levels of nBBP and DnBP binding in the association experiments. However, the
association profiles in the presence of nBBP and DnBP are likely markedly influenced by
the effect of these compounds on availability of receptors (Bmax) that can bind
[3H]SR141716A. Overall, our results indicate that a critical mechanism underlying
inhibition of [3H]SR141716A to CB1 receptors involves phthalates engaging with a site
that is distinct from but negatively coupled to the radioligand recognition site. The
proposed binding region for phthalate esters on the CB1 receptor may represent a novel
target that could be exploited therapeutically by phthalate ester analogs or other drugs to
produce downregulation of endocannabinoid action in the brain.
Unlike phthalate diesters, MEHP and other monoesters inhibit the binding of
follicle stimulating hormone (FSH) to G-protein coupled FSH receptors, an action that
may involve direct engagement of MEHP with the G-protein (Grasso et al., 1993). The
allosteric inhibition of [3H]SR141716A binding to the CB1 receptor by phthalate diesters
could also arise from a direct interaction with its G-protein as we have postulated for
chelerythrine and sanguinarine (Dhopeshwarkar et al. 2011). However, in contrast to the
findings of Grasso et al., (1993), we found that monoesters are, at best, exceptionally
weak inhibitors of CB1 receptor radioligand binding. Therefore, negative allosteric
coupling between a phthalate diester recognition site on the CB1 receptor and the
radioligand binding site is likely a more productive area for future exploration.
Phthalate diesters have potential to access the brain, since a number of these
compounds interfere with barbiturate-induced sleep duration following systemic
administration (Calley et al., 1966) and phthalate ester exposure in school children has
been associated with a behavioral (attention-deficit/hyperactivity) disorder (Kim et al.
2009). The presynaptic CB1 receptor plays a fundamental role at many synapses in
100
mammalian brain and activation of this complex by endocannabinoids promotes a
variety of physiological and behavioral responses. Moreover, downregulation of CB1
receptors and other components of the endocannabinoid system in human epilepsy is
associated with increased excitability in neuronal networks and has been linked to
reduced seizure thresholds (Ludanyi et al., 2008). A critical question is whether brain
CB1 receptors are exposed to phthalate diesters in vivo at concentrations that are
sufficient to interfere with the activation of this signaling pathway by endocannabinoids.
Phthalate esters undergo extensive ester cleavage in the gastrointestinal tract and
hydrolysis would be expected to limit the ability of diesters to reach the brain particularly
after acute oral exposure. However, individuals receiving higher exposure to phthalate
esters on a continuous basis (perhaps as a result of occupational exposure) or hospital
patients exposed to phthalates released from medical devices may be more likely to
accumulate these chemicals in the brain. In the present investigation, threshold inhibitory
effects of DEHP, DnOP, DiOP and nBBP on [3H]CP55940 binding are evident between 1
and 10 µM. Even concentrations within this range in brain may be sufficient to
antagonize endocannabinoid-mediated signaling at CB1 receptors to an extent that
causes low level synaptic perturbations and subtle pathophysiological and affective
responses.
Finally, it must be stressed that further studies aimed at determining 1) phthalate
ester levels in brain following short term systemic and chronic exposures and 2) the
ability of these compounds to modify critical effects of cannabinoid agonists in intact
animals are essential to improve our understanding of the potential phthalate diesters
might have in modulating the endocannabinoid system in vivo.
3.6. Note in added proof
3.6.1. Background
Subsequent to our Neurochemistry International publication appearing, I
established the L-glutamate release assay originally described by Nicholls et al. (1987)
in our laboratory. This assay uses purified synaptosomes and has been utilized by
Wang (2003) to study inhibition of 4-aminopyridine- (4-AP-) evoked release of L-
101
glutamate by the CB1-R agonist WIN55212-2. Since inhibition by WIN55212-2 is
blocked by a diarylpyrazole CB1-R antagonist (Wang, 2003), this system provides a
rigorous functional test for compounds that might antagonize CB1-Rs in the brain.
3.6.2. Experimental approach
We selected two phthalates for investigation, BBP and MnBP. The former was
one of the more potent diesters, both for inhibition of [3H]CP55940 binding and inhibition
of CP55940-stimulated [35S]GTPγS binding. The latter, a monoester, gave no inhibition
of [3H]CP55940 binding and at best produced marginal (<20%) inhibition of [35S]GTPγS
binding. The methods for the isolation of synaptosomes and the fluorimetric
measurement of L-glutamate release are described in detail in Chapter 4.
3.6.3. Results
Consistent with the findings of Wang (2003), WIN55212-2 inhibited of 4-AP-
evoked release of L-glutamate and this effect was fully blocked by AM251. In agreement
with our [3H]CP55940 binding results and as predicted by the [35S]GTPγS binding data,
BBP at 30 µM (but not 5 µM) fully reversed the inhibition by WIN55212-2 (Figure 3.9).
Again in agreement with the [3H]CP55940 binding experiments and the [35S]GTPγS
assays, MnBP failed to modify the inhibitory effect of WIN55212-2 (Figure 3.10).
3.6.4. Conclusion
The L-glutamate assay results strongly supports the idea that 1) phthalate
diesters (as exemplified by BBP) exert antagonist actions at presynaptic CB1-Rs at low
to moderate micromolar concentrations in vitro, and 2) monoesters (as exemplified by
MnBP) are inactive.
102
3.7. Figures and Tables
O
O
O
O
O
O
O
O
O
O
O
O
(a) nBBP (b) DnHP (c) DnBP
O
O
O
O
O
O
O
O
O
O
O
O
(d) DEHP (e) DiOP (f) DnOP
O
O
O
O
H
O
O
O
O
H
O
O
O
O
H
(g) M2EHP (h) MiHP (i) MnBP
Figure 3.1 (a-f) The structures of phthalate diesters: n-butylbenzylphthalate (nBBP); di-n-hexylphthalate (DnHP); di-n-butylphthalate (DnBP); di-ethylhexylphthalate (DEHP); di-isooctylphthalate (DiOP) and di-n-octylphthalate (DnOP).(g-i) The structures of phthalate monoesters: mono-2-ethylhexyl-phthalate (M2EHP), mono-isohexyl-phthalate (MiHP) and mono-n-butyl-phthalate (MnBP). All structures have been redrawn from Bissett et al. (2011) using IsisDraw.
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Figure 3.2 Inhibitory effects of phthalate esters (DnBP, nBBP, DnOP, MiHP and MnBP) on the binding of [3H]CP55940 to mouse brain CB1 receptors in vitro. Each point represents the mean + SEM of 3 independent experiments. Results provided by Ms Kathleen M. Bisset.
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Figure 3.3 Inhibitory effects of phthalate esters (DEHP, DnHP, DiOP and M2EHP) on the binding of [3H]CP55940 to mouse brain CB1 receptors in vitro. Each point represents the mean + SEM of 3 independent experiments. Results provided by Ms Kathleen M. Bisset.
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Figure 3.4 The effect of nBBP and DnBP (both at 35 µM) on the equilibrium binding of of [3H]SR141716A to CB1 receptors of mouse whole brain. Kd and Bmax values are displayed for each treatment and 95% confidence intervals were as follows: control (Kd 0.628 to 0.859. Bmax 0.303 to 0.343), nBBP (Kd 0.761 to 1.333. Bmax 0.176 to 0.229) and DnBP (Kd 0.624 to 0.846. Bmax 0.120 to 0.136). R2 values were 0.9877 (control), 0.9756 (nBBP) and 0.9887 (DnBP). Data points represent the means + SEMs of 3 independent experiments (most SEM bars are obscured by data symbols).
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Figure 3.5a Influence of nBBP (35 µM) and DnBP (50 µM) on the time course of association of [3H]SR141716A with CB1 receptors of mouse brain. (a) membranes received the standard 15 min preincubation with phthalate esters prior to [3H]SR141716A addition.
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Figure 3.5b Influence of nBBP (35 µM) and DnBP (50 µM) on the time course of association of [3H]SR141716A with CB1 receptors of mouse brain. (b) The phthalate ester and [3H]SR141716A were applied simultaneously. Data points represent the means + SEMs of 3 independent experiments (most SEM bars are obscured by data symbols).
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Figure 3.6 Dissociation of the [3H]SR141716A:CB1 receptor complex (initiated by challenge with 5 µM AM251) in the absence (control) or in the presence of 35 µM nBBP or 50 µM DnBP. Data represent mean + SEM of at least 3 independent experiments, each performed in triplicate.
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Figure 3.7 Inhibition of CP55940-stimulated binding of [35S]GTPγS to mouse whole brain membranes by phthalate esters. Phthalate esters were assayed at 75 µM throughout. Each column represents the mean, and error bar the SEM of 7 independent experiments.
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Figure 3.8 Relationship between the ability of study compounds to inhibit the binding of [3H]CP55940 and CP55940-stimulated binding of [35S]GTPγS in mouse whole brain membrane fractions. All assays were performed 75 µM; r2 = 0.7844.
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Figure 3.9 With WIN55212-2 present, BBP (at 30 µM but not 5 µM) enhances 4-AP-evoked L-glutamate release above the level produced by 4-AP alone.
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Figure 3.10 With WIN55212-2 present, MnBP (both at 30 µM and 5 µM) does not enhance 4-AP-evoked L-glutamate release above the level produced by 4-AP alone.
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Table 3.1 Inability of PMSF to influence the inhibitory effects of n-butylbenzylphthalate (nBBP) and di-n-butylphthalate (DnBP) on [3H]CP55940 binding to mouse brain membranes. Phthalate esters were present in the assay at 20 µM and PMSF was used at 50 µM. Each value represents the mean + S.E.M. of 3-6 independent experiments.
Treatment Inhibition (%)
PMSF -2.82 + 3.18
nBBP 28.25 + 2.11
nBBP + PMSF 27.01 + 4.55
DnBP 20.50 + 1.40
DnBP + PMSF 22.61 + 3.31
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Table 3.2 Inhibitory effects of n-butylbenzylphthalate (nBBP), di-n-butylphthalate (DnBP), diethylhexylphthalate (DEHP), mono-isohexylphthalate (MiHP) and mono-n-butyl phthalate (MnBP) on the specific binding of [3H]SR141716A to mouse brain membranes. Diesters were present at concentrations producing 50% inhibition of [3H]CP55940 binding. Each value represents the mean + S.E.M. of 3 independent experiments.
Treatment Inhibition of specific binding (%)
nBBP (27 µM) 67.82 + 1.71
DnBP (46 µM) 72.30 + 3.23
DEHP (47 µM) 37.42 + 3.48
MiHP (100 µM) 33.23 + 4.15
MnBP (100 µM) 0
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4. Benzophenanthridine alkaloid, piperonyl butoxide and (S)-methoprene action at the cannabinoid-1 receptor (CB1-R) pathway of mouse brain: interference with [3H]CP55940 and [3H]SR141716A binding and modification of WIN55212-2-dependent inhibition of synaptosomal L-glutamate release.
4.1. Abstract
Benzophenanthridine alkaloids (chelerythrine and sanguinarine) inhibited binding
of [3H]SR141716A to mouse brain membranes (IC50s: <1 µM). Piperonyl butoxide and
(S)-methoprene were less potent (IC50s: 21 and 63 µM respectively).
Benzophenanthridines and piperonyl butoxide were more selective towards brain CB1-
Rs versus spleen CB2-Rs.
All compounds reduced Bmax of [3H]SR141716A binding to CB1-Rs, but only
methoprene and piperonyl butoxide increased Kd (3-5-fold). Benzophenanthridines
increased the Kd of [3H]CP55940 binding (6-fold), but did not alter Bmax. (S)-methoprene
increased the Kd of [3H]CP55940 binding (by almost 4-fold) and reduced Bmax by 60%.
Piperonyl butoxide lowered the Bmax of [3H]CP55940 binding by 50%, but did not
influence Kd.
All compounds reduced [3H]SR141716A and [3H]CP55940 association with CB1-
Rs. Combined with a saturating concentration of SR141716A, only piperonyl butoxide
and (S)-methoprene increased dissociation of [3H]SR141716A above that of SR141716A
alone. Only piperonyl butoxide increased dissociation of [3H]CP55940 to a level greater
than CP55940 alone. Binding results indicate predominantly allosteric components to
the study compounds action.
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4-Aminopyridine- (4-AP-) evoked release of L-glutamate from synaptosomes was
partially inhibited by WIN55212-2, an effect completely neutralized by AM251, (S)-
methoprene and piperonyl butoxide. With WIN55212-2 present, benzophenanthridines
enhanced 4-AP-evoked L-glutamate release above 4-AP alone. Modulatory patterns of
L-glutamate release (with WIN-55212-2 present) align with previous antagonist/inverse
agonist profiling based on [35S]GTPγS binding. Although these compounds exhibit lower
potencies compared to many classical CB1 receptor inhibitors, they may modify CB1-R-
dependent behavioral/physiological outcomes in the whole animal and could offer
templates for synthesis of novel and more potent CB1-R blocking drugs.
Note: The research described in this chapter will be submitted shortly for
publication in an appropriate neurochemical/neuropharmacological journal. The
submission will adhere closely to the format laid out here.
4.2. Introduction
Cannabinoid-1 receptors (CB1-Rs) are present in numerous regions of
mammalian brain and are particularly abundant within the cerebral cortex, hippocampus,
cerebellum and basal ganglia (Herkenham et al., 1991; Tsou et al., 1998). CB1-Rs
couple to G-proteins in the plasma membrane of nerve terminals and together they
constitute the primary presynaptic element of the endocannabinoid signaling pathway
that regulates transmitter release through negative feedback (Howlett et al., 1986;
Katona et al., 1999; Kawamura et al., 2006). Endocannabinoids, generated in
postsynaptic neuronal cell bodies when synaptic activity intensifies, migrate retrogradely
and bind to presynaptic CB1-Rs. G-protein activation leads to inhibition of voltage-
sensitive Ca++ channels (Mackie and Hille, 1992; Twichell et al., 1997, Kushmerick et al.,
2004; Guo and Ikeda, 2004), negative modulation of adenylate cyclase (Howlett and
Fleming, 1984; Howlett, 1985) and activation of K+ currents (Deadwyler et al., 1993;
Mackie et al., 1995, Childers and Deadwyler, 1996; Guo and Ikeda, 2004). Since these
various signaling mechanisms reduce the ability of action potentials impinging on the
nerve ending to depolarize and activate calcium entry, transmitter release is adjusted
downwards, thus completing the negative feedback loop (Chevaleyre et al., 2006;
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Kreitzer and Regehr, 2001; Wilson and Nicoll, 2001; Howlett, et al., 2002; Freund et al.,
2003).
Certain plant natural products and synthetic drugs mimic endocannabinoid
activation of this signaling pathway by exerting potent (nanomolar) agonist actions at
CB1-Rs. Prominent xenocannabinoid agonists include ∆9-tetrahydrocannabinol, the main
psychoactive principle of Cannabis sativa (Razdan, 1986), CP55940 (Johnson and
Melvin, 1986) and the aminoalkylindole WIN55212-2 (Compton et al., 1992). Selective
high potency CB1-R antagonists, notably the phytocannabinoid ∆9-
tetrahydrocannabivarin and the diarylpyrazole antagonist/inverse agonists AM251 and
SR141716A, have also been reported (Rinaldi-Carmona et al., 1994; Lan et al., 1999;
Thomas et al., 2005).
There is considerable interest in possible therapeutic applications of CB1-R
modulators. Agonists and allosteric activators of agonist action have been considered in
the relief of pain, muscle spasms, anxiety states and depressive illness, and they can
also block emesis, improve sleep and stimulate appetite (Van Sickle et al., 2001;
Iversen, 2003, Ligresti et al., 2009; Bradshaw and Walker, 2005; Di Marzo, 2009). On
the other hand, CB1-R antagonists/inverse agonists such as the diarylpyrazole
rimonabant (SR141716A) have shown effectiveness in reducing body weight through
suppression of appetite (Colombo et al., 1998), but rimonabant use in human medicine
was curtailed due to adverse psychiatric side effects. Nevertheless, discovery of a CB1-
R inhibitor divorced of such unfavourable symptoms clearly remains of considerable
interest (Szabo et al., 2009; Wu et al., 2009; Riedel et al., 2009).
Research in our laboratory has focused on other natural products and synthetic
environmental chemicals capable of interacting with the endocannabinoid system.
Specifically we have demonstrated that at very low to moderate micromolar
concentrations, the benzophenanthridine alkaloids (sanguinarine and chelerythrine), the
pesticides (piperonyl butoxide and (S)-methoprene), certain phthalate dialkyl ester
plasticizers, as well as the more acutely toxic tributyltin derivatives (tributyltin acetate
and tributylethynyl tin) inhibit both the binding of [3H]CP55940 to CB1-Rs, as well as CB1-
R agonist-dependent activation of the G-protein (Dhopeshwarkar et al., 2011; Bisset et
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al., 2011; Jain S., M.Sc. Thesis, Simon Fraser University, 2011; Dhopeshwarkar A.S.,
Ph.D. Thesis, Simon Fraser University, 2012, Chapter 5).
Endocannabinoids, the high potency synthetic agonists (e.g. CP55940 and
WIN55212-2) and dihydropyrazole antagonists/inverse agonists (e.g. AM251 and
SR141716A) engage with a discrete binding pocket on the CB1-R and available
evidence suggests individual binding domains are distinct or may have tendency to
partially overlap (Shim, 2010; Kapur et al., 2007). The purpose of this phase of our
research program was to examine the effects of sanguinarine, chelerythrine, piperonyl
butoxide and (S)-methoprene in greater detail with regards their ability to interfere with
the equilibrium binding and kinetic properties of radioligands that engage with the
binding pocket of CB1-Rs (specifically [3H]55940 and [3H]SR141716A). Moreover, since
we predicted that, based on their abilities to modulate basal and CB1-R-agonist-
(CP55940-) stimulated binding of [35S]GTPγS to the G protein, the benzophenanthridines
were likely inverse agonists and piperonyl butoxide and (S)-methoprene were neutral
antagonists of CB1-Rs (Dhopeshwarkar et al., 2011), it was critical to investigate whether
modification of neurotransmitter release (i.e. the ultimate consequence of presynaptic
CB1-R engagement by these compounds) was consistent with this functional profiling.
4.3. Materials and Methods
4.3.1. Chemicals and supplies
2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl)-cyclohexyl]-5-(2-methyloctan-2-yl)-
phenol (CP-55940),5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-(piperidin-1-yl)-
1H-pyrazole-3-carboxamide (AM251), (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinyl-
methyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone mesylate
(WIN55212-2), dimethylsulfoxide (DMSO), sanguinarine, chelerythrine, piperonyl
butoxide, Ethylene diamine tetraacetic acid (EDTA), Ethylene glycol-bis(2-
aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), 4-aminopyridine (4-AP), veratridine
(VTD), tetrodotoxin (TTX), acetone, Percoll®, glutamate dehydrogenase (Type II from
bovine liver), β-nicotinamide adenine dinucleotide phosphate (sodium salt hydrate;
NADP+), bovine serum albumin (BSA; fatty acid free) and all other chemicals required for
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assay buffers and salines, were purchased from Sigma Aldrich, Canada. 5-(4-
chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-
carboxamide (SR141716A) was obtained from Cayman Chemical and (S)-methoprene
(98.5% purity) was supplied by Doug Vangundy, Director of Speciality Product
Development, Wellmark International (Dallas, Texas). Radioligands, [3H]CP55940 (side
chain-2,3,4-[3H]; specific activities 139.6 and 174.6 Ci/mmol) and [3H]SR141716A
(piperidine ring 3,4-[3H]; specific activity 56 Ci/mmol) were purchased from Perkin Elmer
Life and Analytical Sciences, Canada.
4.3.2. Animals
All experiments described in this report were performed with male CD1 mice (20-
25 g) purchased from Charles River Laboratories (Saint-Constant, Quebec). Animal
orders were placed by Animal Care Services of Simon Fraser University, Burnaby,
Canada. Upon receipt, mice were housed under standardized environmental conditions
(21 0C; 55 % relative humidity; 12 hour light/dark cycle) and allowed unlimited access to
food and water. Our animal housing, handling and experimental procedures conformed
to Canadian Council on Animal Care guidelines and were formally approved by the
Simon Fraser University Animal Care Committee prior to embarking on this investigation.
4.3.3. Isolation of membranes from mouse brain for binding studies
The isolation of brain membranes were carried out at 0-4 oC according to a
method published previously (Dhopeshwarkar et al., 2011). Mouse whole brains were
homogenized (10 pestle excursions; pestle rotation approx. 1500 rpm) in ice-cold buffer
[Trisma base (100 mM), EDTA (1 mM), adjusted to pH 9 with HCl; 1 brain per 10 ml
buffer]. The homogenate was centrifuged for 10 min at 900 x g in a JA20 rotor of a
Beckman J2HS centrifuge. Centrifugation of the supernatant at 11,500 x g for 25 min
produced a membrane pellet which was resuspended in ice-cold buffer [Trisma base (50
mM), EDTA (1 mM) and MgCl2.6H2O (3 mM); adjusted to pH 7.4 with HCl] at a protein
concentration of approx. 6.5 mg/ml. The membrane preparation was then frozen in
aliquots at -80 oC. Just prior to assay, the preparation was thawed on ice and carefully
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dispersed by slowly moving the membrane suspension in and out of a syringe fitted with
a 18 gauge needle (6 times) followed by vortexing.
4.3.4. Effects of benzophenanthridines, (S)-methoprene and piperonyl butoxide on equilibrium binding of [3H]CP55940 and [3H]SR141716 to brain CB1 receptors
The saturation binding method of Steffens et al. (2004) was adopted (with
modifications as detailed below), and effects on radioligand saturation binding
characteristics were investigated by assaying the study compound (at ≥ IC50) with
different concentrations of [3H]CP55940 or [3H]SR141716A (0.032-3.5 nM). To achieve
these concentrations of [3H]CP55940 and [3H]SR141716A, radioligand specific activity
was reduced by addition of the required quantity of unlabelled CP55940 and
SR141716A. For assay, compounds formulated in DMSO (5 µl) or DMSO control (5 µl),
as appropriate, were rapidly injected into borosilicate glass tubes (13 x 100 mm; Kimble-
Chase) containing binding buffer [500 µl; Trisma base (50 mM), EDTA (1 mM),
MgCl2.6H2O (3 mM), BSA (fatty acid free; 3 mg/ml) adjusted to pH 7.4 with HCl]. After
addition of brain membranes (227.09 + 0.66 µg protein), the mixture was gently vortexed
and the incubation continued for 15 minutes at room temperature. [3H]CP55940 or
[3H]SR141716A was then added at the required concentrations and incubations were
run for 90 min to ensure equilibration of radioligand. Incubations were concluded by
addition of 4 ml ice-cold wash buffer (0.9% NaCl containing 2 mg/ml BSA (fatty acid
free)) and membranes were harvested on Whatman GF/C filters (presoaked with wash
buffer) using a (Hoefer FH 225V) vacuum filtration system attached to a vacuum pump
(Hoefer FH 225V). Membranes collected on filters were washed (3 x 4 ml) with ice-cold
wash buffer. Each filter was removed from its filtration well and placed in a scintillation
vial and allowed to dry completely. After drying, 4 ml of scintillation cocktail (BCS,
Amersham Biosciences, UK) was added and radioactivity measured using liquid
scintillation counting. Specific binding was calculated by subtracting non-specific binding
(binding in presence of 10 µM CP55940 or 10 µM SR141716A) from total binding and
this was determined for each concentration of [3H]CP55940/CP55940 or
[3H]SR141716A/SR141716A in the absence and presence of study compound. Values
were used to construct equilibrium binding isotherms which allowed calculation of the Kd
(equilibrium dissociation constant) and Bmax (number of receptors available for
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radioligand binding). At least three experiments were conducted for each treatment and
protein measurement was done as described by Peterson (1977).
4.3.5. Effect of benzophenanthridines, (S)-methoprene and piperonyl butoxide on the association and dissociation kinetics of [3H]CP55940 and [3H]SR141716A
Association studies were initiated by addition of study compound in DMSO (5 µl)
or DMSO control (5 µl), as appropriate, to 500 µl assay buffer in borosilicate glass tubes.
Membranes (approx. 230 µg protein) were then added and the system allowed to
incubate for 15 min at room temperature. Meanwhile, the Hoefer FH 225V filtration
apparatus was prepared by inserting pre-soaked Whatman GF/C filters and allowing the
vacuum to draw down a few minutes before the end of the preincubation. [3H]CP55940
or [3H]SR141716A (1 nM final concentration) was added at t = 15 min, and the brain
membrane suspensions filtered at various time points between 0 and 180 secs. After
three 4 ml washes, filters were dried and radioactivity associated with the membranes
quantified. The time course of radioligand association was also tracked after each study
compound (or DMSO control) was added a few seconds before the radioligand (defined
as co-treatment situation).
Dissociation studies were conducted by equilibrating brain membranes (approx.
230 µg protein) with [3H]CP55940 or [3H]SR141716A (1 nM final concentration) for 90
minutes at 37 oC with gentle shaking. At equilibrium, either a saturating (5 µM)
concentration of (CP55940) or AM251 (added in 10 µl DMSO), or study compound at ≥
IC50 (added in 5 µl DMSO) plus 5 µM (CP55940) or AM251 (added in 5 µl DMSO) was
added, Dissociation of radioligand was monitored over 300 seconds.
4.3.6. Interaction of benzophenanthridines, methoprene and piperonyl butoxide with CB2 receptors in mouse spleen
After evaluation of several methods, the method of Hillard et al. (1999) was
adopted for this investigation. Two mice were euthanized by rapid cervical dislocation
and the spleens were rapidly removed and homogenized in 10 ml TME buffer (Tris-HCl
(50 mM), EDTA (1 mM) and MgCl2.6H2O (3 mM), titrated to pH 7.4 with HCl) using a
pre-chilled motor driven homogenizer (10 strokes up and down, pestle rotation 1500
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rpm). The homogenate was then centrifuged at 500 x g for 10 minutes in a Beckman
J2HS centrifuge using JA-20 rotor. The pellet was discarded and the supernatant
centrifuged at 17,500 x g for 30 minutes. The fresh spleen membranes were
immediately used for experimentation. For assay, 500 µl of binding buffer (Tris-HCl (50
mM), EDTA (1 mM) and MgCl2. 6H2O (3 mM) and BSA (fatty acid free, 3 mg/ml), pH 7.4
with HCl) was transferred to borosilicate glass tubes (13 x 100 mm; Kimble-Chase).
Study compounds (in 5 µl DMSO) or DMSO controls, as appropriate, were then
introduced followed by spleen membranes (200.20 ± 2.07 µg of protein per tube). The
mixture was gently vortexed and incubated for 15 min at room temperature, whereupon
[3H]CP55940 (1 nM final concentration; added in 10 µl in DMSO) was injected, the tube
contents thoroughly mixed, and a 90 minute incubation at 30 oC with gentle shaking
carried out. The binding reactions were terminated by addition of ice-cold wash buffer
(0.9% NaCl containing 2 mg/ml BSA; 1 ml) and membranes were collected on pre-
soaked Whatman GF/C filter papers using vacuum filtration (Hoefer FH 225V).
Harvested membranes were then washed with three washes of 4 ml ice-cold wash
buffer. Scintillant (4 ml, BCS, Amersham Bioscience UK) was added after thorough
drying of the filter and radioactivity measured using liquid scintillation counting. Non-
specific binding (measured in the presence of 10 µM CP55940) was subtracted from
total binding to yield specific binding to CB2 receptors and this averaged 75.01 ± 1.4%.
All assays were performed in triplicate and a minimum of three experiments were
conducted for every treatment.
4.3.7. Preparation of synaptosomes from mouse whole brain
Synaptosomes (pinched-off nerve endings) were prepared and isolated from the
whole brains of male CD1 mice using the discontinuous isoosmotic gradient technique of
Dunkley et al. (2008). On the day of experiment, Percoll gradients were prepared in two
12 ml polycarbonate centrifuge tubes by carefully layering 2 ml of each Percoll gradient
solution, starting with 23% at the bottom followed by 15%, 10% and finally 3% at the top.
A flow rate of less than 2 ml/min was maintained while layering so that Schlieren lines
remained clearly visible at the interface between Percoll layers. Gradients were then
transferred onto ice and stored in the cold room until needed. All centrifuge tubes,
glassware and buffers were pre-chilled on ice before start of experiment and all
processing steps were performed at 0-40C. Two mice were sacrificed by cervical
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dislocation and whole brains removed within 15 seconds. The brains were then
thoroughly rinsed with 5 ml of ice-cold homogenizing buffer [sucrose (0.32 M), EDTA (1
mM) and Trisma base (5 mM); with pH adjusted to 7.4 using HCl]. Next, brain tissue
was transferred to a specially constructed glass/plexiglass tissue homogenizer
containing 15 ml of homogenization buffer and homogenized (7 strokes up and down;
pestle rotation 700 rpm). The homogenate was immediately centrifuged in a Beckman
J2HS centrifuge using JA-20 rotor for 10 minutes (1000 x g). The supernatant obtained
was centrifuged at 15000 x g for 30 minutes and the pellet was then gently homogenized
(2 ml glass homogenizer; Uniform, England). The homogenate was then diluted to 4 ml
with homogenizing buffer and then 2 ml carefully layered on top of the 3% Percoll layer
in each polycarbonate tube. Tube contents were then centrifuged at 31,000 x g (at 4 0C)
for 5 minutes (excluding acceleration and deceleration time). Tubes were carefully then
transferred to ice and synaptosome-rich fractions at the 10-15% and 15-23% interfaces
were pooled in a chilled 100 ml beaker. Synaptosomal fractions were then slowly diluted
with 80 ml assay buffer [NaCl (130 mM), KCl (4.5 mM), NaHCO3 (5 mM), MgCl2.6H2O (1
mM), Na2HPO4 (1.2 mM), HEPES (10 mM), glucose (10 mM), BSA (1 mg/ml) with pH
adjusted to 7.4 with NaOH]. This dilution was performed over a 30 minute period
whereupon the suspension was centrifuged at 20,000 x g (at 4 0C) for 30 minutes. The
pellets containing purified synaptosomes were gently resuspended in 1 ml assay buffer
and 100 µl aliquots of pure synaptosomes [0.767 ± 0.11 µg of protein per aliquot as
determined by Peterson (1977)] were apportioned to 9 snap top vials which were held on
ice in the cold room until required.
4.3.8. Release of L-Glutamate from synaptosomes
The enzyme-linked fluorescence technique originally described by Nicholls et al.
(1987) was adopted with minor modifications to measure the release of endogenous
glutamate from mouse brain synaptosomes. Briefly, 100 µl synaptosomes were added
to 2 ml of ice-cold assay buffer and the suspension incubated in shaking water bath at
37 0C for 15 mins. The suspension was then transferred to stirred quartz cuvette
thermostated at 37 0C in a Perkin Elmer LS 50 spectrophotometer. NADP+ (1 mM),
glutamic dehydrogenase (100 units) and CaCl2 (as appropriate; 1.3 mM) were added
followed by the cannabinoid agonist (WIN55212-2) and/or antagonist (AM251) along
with study compounds, inhibitors, EGTA or solvent controls, as appropriate, and the
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system allowed to incubate for another 5 minutes. Fluorescence recording was started
and 4-AP (3 mM) was added at 100 secs to activate glutamate release from
synaptosomes and the experiment was terminated at 500 secs. The excitation
wavelength was set at 360 nm (slit width 5) and the emission was sampled at 460 nm
(slit width 5). Fluorescence output was measured at 1 sec intervals. The amount of
glutamate released was monitored as an increase in fluorescence due to NADPH
forming from NADP+ as a result of the oxidative deamination of released glutamate by
glutamate dehydrogenase. Standard glutamate was added to allow quantitation of the
released glutamate as nmol glutamate/mg synaptosomal protein.
All compound additions were done with microsyringes (Hamilton, USA).
4.3.9. Analysis of radioligand binding data and glutamate release data
Curve fitting and calculation of binding parameters was performed using Prism,
GraphPad Software Inc., San Diego, CA, USA). In the glutamate release assays,
fluorescence changes were measured with the vertical analysis tool of the Perkin Elmer
LS-50 software and the percentage change to the 4-AP-induced fluorescence signal
above the control (assay buffer added at 100 secs instead of 4-AP) was calculated.
Time resolved fluorescence data were also transferred to GraphPad Prism (5.0) and full
fluorescence traces constructed. Percentage changes induced by standard
pharmacological agents and study compounds were calculated as mean ± S.E.M. based
on 3-4 independent experiments.
4.4. Results
4.4.1. Effects of benzophenanthridines, piperonyl butoxide and (S)-methoprene on binding of [3H]SR141716A to CB1 receptors
Figure 4.1 shows the inhibitory effects of benzophenanthridines, piperonyl
butoxide and (S)-methoprene on specific binding of [3H]SR141716A to CB1 receptors in
the mouse brain membrane preparation. The benzophenanthridines, sanguinarine and
chelerythrine were the most potent inhibitors based on IC50 values of 732 nM (95%
confidence interval (CI) = 364-1470 nM) and 911 nM (95% CI = 713-1164 nM)
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respectively. Piperonyl butoxide and methoprene had weaker effects on [3H]SR141716A
binding, achieving IC50s of 21.1 µM (95% CI 17.4-25.7) and 62.8 µM (95% CI 48.4-81.5)
respectively. At maximum effect concentrations, the benzophenanthridines produced
>90% inhibition, in contrast to piperonyl butoxide (70-80%) and methoprene (approx.
50%).
4.4.2. Influence of study compounds on the saturation binding of [3H]SR141716A to CB1 receptors of mouse brain
The control saturation binding curve was constructed by measuring the specific
binding of [3H]SR141716A to CB1 receptors at equilibrium over a range of radioligand
concentrations (0.032 to 2.8 nM). Control experiments were always performed
concurrently with benzophenanthridines, piperonyl butoxide or (S)-methoprene assayed
at ≥ IC50 (Figure 4.2). All study compounds reduced the apparent Bmax, but only
methoprene and piperonyl butoxide increased the Kd of [3H]SR141716A binding (by
approx. 3- and 5-fold respectively). Analogous equilibrium binding experiments were
conducted with [3H]CP55940 (Figure 4.3) and differences between benzopenanthridines
and synthetic compounds were again indicated. The benzophenanthridines increased
the Kd of [3H]CP55940 binding by approximately 6-fold, but did not alter Bmax. In
contrast, methoprene increased the Kd of [3H]CP55940 binding by almost 4-fold and
reduced Bmax by approx. 60%. Piperonyl butoxide reduced the Bmax of [3H]CP55940
binding by close to 50%, but had no influence on the Kd.
4.4.3. Effects of sanguinarine, chelerythrine, piperonyl butoxide, and (S)-methoprene on the kinetics of CB1 receptor-selective radioligand binding
The effects of sanguinarine, chelerythrine, piperonyl butoxide, and (S)-
methoprene on the association of [3H]SR141716A with CB1 receptors over the initial
phase of the binding reaction are displayed in Figure 4.4. All compounds reduced the
ability of [3H]SR141716A to progressively bind to CB1 receptors, when applied both
before radioligand (Figure 4.4a) and together with radioligand (Figure 4.4b). In similar
experiments, all study compounds reduced the association of [3H]CP55940 (Figure
4.4c). When added together with a saturating concentration of SR141716A, piperonyl
butoxide (30 µM) and (S)-methoprene (60 µM) increased the dissociation of the
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[3H]SR141716A:CB1 receptor complex to a rate greater that induced by the saturating (5
µM) concentration of SR141716A alone (Figure 4.5a). Sanguinarine and chelerythrine
(at 5 µM) failed to increase SR141716A-induced dissociation under these conditions
(data not shown). However, when applied alone, all study compounds initiated
dissociation of [3H]SR141716A, (S)-methoprene being the least effective (Figure 4.5b).
In the presence of a saturating (5 µM) concentration of CP55940, only piperonyl
butoxide accelerated the dissociation of [3H]CP55940 to a level greater than that induced
by CP-55940 alone (Figure 4.5c).
4.4.4. Effects of study compounds on mouse spleen CB2 receptors as assessed by inhibition of [3H]CP55940 binding
After verifying the IC50s of sanguinarine, chelerythrine, piperonyl butoxide and
(S)-methoprene that we reported previously for [3H]CP55940 binding to mouse brain CB1
receptors (Dhopeshwarkar et al. 2011), we employed identical concentrations to
investigate the inhibitory actions of study compounds on [3H]CP55940 binding to CB2
receptors of mouse spleen. In contrast to (S)-methoprene, which showed 51% inhibition
of [3H]CP55940 binding to CB2 receptors, the three other compounds were very much
weaker, achieving 4% inhibition (chelerythrine), 14% inhibition (sanguinarine) and 21%
inhibition (piperonyl butoxide), (Table 4.1). Since (S)-methoprene was of very similar
inhibitory potency at brain CB1 as spleen CB2 receptors, the relationship between
concentration and inhibition of [3H]CP55940 binding to spleen CB2 receptors was
explored in more detail. The results demonstrated (S)-methoprene to be a partial
inhibitor, unable to produce greater than 50% inhibition of [3H]CP55940 binding (Figure
4.6).
4.4.5. Effects of study compounds on WIN55212-2-dependent inhibition of 4-aminopyridine- (4-AP-) evoked release of L-glutamate from mouse brain synaptosomes
In marked contrast to 50 µM veratridine-evoked release of L-glutamate from
synaptosomes, the release of L-glutamate induced by 3 mM 4-AP was completely
insensitive to inhibition by 5 µM tetrodotoxin (TTX; Figure 4.7). In these preliminary
experiments, we also verified that 4-AP-evoked release of L-glutamate is partially (28.4 +
1.59 %) inhibited by the CB1-R agonist WIN55212-2 (8 µM), that this inhibition by
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WIN55212-2 is completely relieved by the CB1-R antagonist AM251 (8 µM), and that
preincubation with AM251 alone has no influence on the amount of neurotransmitter
released by 4-AP (Figure 4.8). All study compounds were then tested at a low and high
concentration for their ability to modify WIN55212-2-dependent inhibition of L-glutamate
release from mouse brain synaptosomes. Sanguinarine and chelerythrine (both at 0.25
µM) and (S)-methoprene at 5 µM failed to affect inhibition by WIN55212-2 of 4-AP-
evoked release, however a weak (approx. 5%) reduction in inhibition by WIN55212-2
was observed with piperonyl butoxide at 5 µM (Figures 4.9-4.12). In the presence of
WIN55212-2, sanguinarine and chelerythrine (at 2 µM) enhanced the release of L-
glutamate over and above that of 4-AP alone (Figures 4.9 and 4.10), whereas (S)-
methoprene (25 µM) and piperonyl butoxide (25 µM) fully suppressed WIN55212-2-
dependent inhibition of L-glutamate release without any tendency for a
benzophenanthridine-like overshoot (Figures 4.11 and 4.12). Like AM251, chelerythrine
(2 µM), sanguinarine (2 µM), (S)-methoprene (25 µM) and piperonyl butoxide (2 µM), did
not enhance the baseline release of L-glutamate from mouse brain synaptosomes
(Figures 4.9-4.12).
4.5. Discussion
In an earlier investigation the benzophenanthridine alkaloids (chelerythrine and
sanguinarine) and the pesticide formulation components ((S)-methoprene and piperonyl
butoxide) were found to inhibit the binding of [3H]CP55940 to CB1-Rs of mouse brain and
inhibit CB1-R agonist-dependent activation of [35S]GTPγS binding to the G protein Gα
subunit (Dhopeshwarkar et al., 2011). The present research provides insight into how
these compounds inhibit the binding of [3H]CP55940 and [3H]SR141716A to CB1-Rs of
mouse brain and explores putative antagonist-like actions further by investigating the
ability of study compounds to modify evoked (CB1-R-sensitive) release of L-glutamate
from mouse brain synaptosomes.
Present evidence indicates that [3H]CP55940 and [3H]SR141716A bind to
specific regions within the CB1 receptor binding pocket (Shim, 2010). Our initial
experiments of this investigation established IC50s for study compounds in the
[3H]SR141716A binding assay. When compared to [3H]CP55940 values
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(Dhopeshwarkar et al., 2011), the inhibitory potencies for [3H]SR141716A binding were 1
to 2-fold higher for benzophenanthridines and 2.5-fold and 3.8-fold lower for piperonyl
butoxide and (S)-methoprene respectively, indicating different capacities to impact on
radioligand-specific binding loci within the CB1-R binding pocket. Our saturation binding
and kinetic data for these radioligands support this and other mechanistic differences
between study compounds in their actions.
The saturation binding constants obtained for [3H]SR141716A agree closely with
those published by Rinaldi-Carmona et al. (1996) using a fraction from rat brain. We
found that chelerythrine and sanguinarine decrease the total number of binding sites
(Bmax) available to [3H]SR141716A without affecting the affinity (Kd). This may be
explained by the benzophenanthridines binding to an allosteric site and triggering a
profound conformational modification to the [3H]SR141716A recognition site such that
radioligand cannot bind. However our results do not exclude benzophenanthridines
binding irreversibly (or in a very slowly reversible manner) to the orthosteric site. At
similar concentrations to those used for the benzophenanthridines in this study,
Beausoleil et al., (2009) found that sanguinarine and chelerythrine competitively inhibit
the binding of a GTP fluoroprobe to Rac1b (a GTP binding protein), and this led us to
propose that benzophenanthridines may inhibit [3H]CP55940 binding to the CB1-R by
targeting the guanine nucleotide recognition site on its associated G-protein
(Dhopeshwarkar et al., 2011). However, while it is well recognized that GTP and its
GTP analogs allosterically dissociate [3H]CP55940 from the CB1 receptor (Devane et al.,
1988; Houston and Howlett, 1993), the binding of [3H]SR141716A is known to be
unaffected by 300 µM GTPγS (Rinaldi-Carmona et al., 1996). Nevertheless, our
saturation binding assays with [3H]CP55940 indicated that the benzophenanthridines
reduce radioligand binding affinity without changing Bmax, lending support to an apparent
competitive mechanism for inhibition of [3H]CP55940 binding by benzophenanthridine
alkaloids, with potential for allosteric involvement. The kinetic results support the idea
that benzophenanthridine-dependent changes to the equilibrium binding constants of
[3H]CP55940 and [3H]SR141716A can arise both through a slowing of the rate of
association and by increasing the rate of dissociation of these radioligands.
It is improbable that (S)-methoprene and piperonyl butoxide engage with the
[3H]SR141716A recognition site itself because the pattern of inhibition by these
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compounds is mixed, with both compounds affecting Bmax and Kd of [3H]SR141716A
binding. The possibility of an allosteric mechanism with respect to [3H]SR141716A
binding is supported because (S)-methoprene and piperonyl butoxide increase the
dissociation of equilibrated radioligand from its recognition site, irrespective of whether a
saturating concentration of unlabeled SR1417126A is present or not.
Our findings regarding the mechanism of interference with the binding of
[3H]CP55940 by (S)-methoprene are not so clear. Whereas a reduction in the initial rate
of formation of the radioligand:recognition site complex by (S)-methoprene, combined
with its failure to influence the dissociation of [3H]CP55940 lends strong support to a
simple competition, this compound clearly increases Kd and reduces Bmax in saturation
binding assays, an outcome in apparent conflict with such a mechanism. We have
hypothesized that (S)-methoprene may represent a flexible analog of ∆9-
tetrahydrocannabinol (Dhopeshwarkar et al., 2011), a phytocannabinoid that binds to the
same site region of the CB1-R binding pocket as CP55940 (Gatley et al., 1997; Thomas
et al., 2005). A model that involves binding of (S)-methoprene to the [3H]CP55940
binding region in two conformations, one that allows radioligand to bind but with reduced
affinity, and one that blocks access to [3H]CP55940, could reconcile these observations.
The manner in which (S)-methoprene and piperonyl butoxide interfere with
[3H]CP55940 binding are evidently different since the ability of piperonyl butoxide when
combined with a maximum effect concentration of unlabeled CP55940 to accelerate
dissociation of radioligand over and above the rate observed with a maximum effect
concentration of CP55940 alone suggests a prominent allosteric component. We
conclude that the reduction in the Bmax of [3H]CP55940 binding observed with piperonyl
butoxide in saturation binding experiments is most likely allosterically-mediated and
therefore not inconsistent with our premise (Dhopeshwarkar et al., 2011) that piperonyl
butoxide could bind to the endocannabinoid receptor within the CB1-R binding pocket.
The L-glutamate release experiments reported in this investigation provide
strong support to the pharmacological profiling we originally proposed for
benzophenanthridines, (S)-methoprene and piperonyl butoxide that was based on
inhibition of CB1-R agonist-activated [35S]GTPγS binding (Dhopeshwarkar et al., 2011).
In preliminary experiments, we confirmed, as found originally by Wang (2003), that 4-
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AP-dependent release of L-glutamate is inhibited by WIN55212-2, and that the inhibition
is relieved by a diarylpyrazole (AM251 in present studies). The lower level of inhibition
(approx. 28%) by WIN55212-2 observed in the present investigation aligns more closely
with the level of inhibition by WIN55212-2 of KCl-evoked L-glutamate release from
synaptosomes (Godino et al., 2007). We also found that this WIN55212-2-sensitive
component of release required extrasynaptosomal Ca++ (data not shown) and that 3 mM
4-AP-induced release of L-glutamate from synaptosomes was unaffected by
tetrodotoxin. This latter result confirmed a lack of participation of voltage-sensitive
sodium channels in the 4-AP response, a critical prerequisite because CB1-R drugs,
including WIN55212-2 and diarylpyrazoles inhibit voltage-sensitive sodium channels at
low micromolar concentrations (Nicholson et al. 2003; Kim et al., 2005; Liao et al., 2004),
and very similar concentrations of these drugs are necessary to reveal CB1-R-dependent
effects on L-glutamate release from synaptosomes.
A salient finding of the present study is that concentrations of sanguinarine,
chelerythrine, (S)-methoprene and piperonyl butoxide that have no effect on basal
release of L-glutamate are able to neutralize (reverse) the inhibitory effect of WIN55212-
2 on 4-AP-evoked release of this neurotransmitter. In parallel experiments, the classical
CB1-R antagonist AM251 displayed an identical profile in agreement with other studies
using the diarylpyrazole AM281 (Wang 2003; Godino et al., 2007). The effects of the
study compounds on WIN55212-2-dependent inhibition of 4-AP-evoked release are
concentration-dependent. Moreover, the concentrations of sanguinarine, chelerythrine,
(S)-methoprene and piperonyl butoxide that we show are capable of neutralizing the
inhibitory effects of WIN55212-2 on L-glutamate release are very similar to those needed
to produce significant inhibition of radioligand ([3H]CP55940 and [3H]SR141716A)
binding and inhibition of CB1-R agonist-stimulated [35S]GTPγS binding to the G protein.
In marked contrast to the neutral antagonist actions of (S)-methoprene and
piperonyl butoxide in the L-glutamate release assay, an inverse agonist-like action was
indicated for sanguinarine and chelerythrine since at 2 µM (and in the presence of 4-AP),
these alkaloids promote release of neurotransmitter that is greater than that achieved by
4-AP alone. Our results show that this latter phenomenon is exclusively dependent on
WIN 55212-2 being present, so for benzophenanthridines to act in this way in vivo, a
significant level of endocannabinoid tone would be necessary.
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In conclusion, we demonstrate that binding sites for [3H]CP55940 and
[3H]SR141716A within the CB1-R binding pocket are differentially influenced in the very
low micromolar range by benzophenanthridine alkaloids (chelerythrine and
sanguinarine), and in the low to moderate micromolar range by piperonyl butoxide and
(S)-methoprene. Certain structural features of these study compounds or their binding
sites may be useful points of consideration for discovery of more potent G-protein-
coupled CB1 receptor blocking drugs that might be capable of downregulating the central
effects of endocannabinoid agonists.
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4.6. Figures and Table
Figure 4.1 Concentration dependency of inhibition by chelerythrine (open circles), sanguinarine (solid circles), piperonyl butoxide (solid triangles) and (S)-methoprene (squares) on [3H]SR141716A binding to mouse brain CB1 receptors. IC50 and 95% confidence interval values are provided in Section 4.4.1.
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Figure 4.2 Effect of chelerythrine (1 µM; open circles), sanguinarine (1 µM; solid circles), piperonyl butoxide (30 µM; solid triangles) and (S)-methoprene (60 µM; squares) on equilibrium binding of [3H]SR141716A to mouse brain CB1 receptors. Control data points are identified by the diamond symbols. Kd values (as nM): control 0.51 + 0.04; chelerythrine 0.47 + 0.08; sanguinarine 0.46 + 0.04; (S)-methoprene 1.5 + 0.6 and piperonyl butoxide 2.5 + 1.1. Bmax values (as pmol [3H]SR141716A/mg protein): control 0.79 + 0.02; chelerythrine 0.32 + 0.02; sanguinarine 0.50 + 0.01; (S)-methoprene 0.44 + 0.08 and piperonyl butoxide 0.56 + 0.13.
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Figure 4.3 Effect of chelerythrine (2.5 µM; open circles), sanguinarine (1.5 µM; solid circles), piperonyl butoxide (10 µM; solid triangles) and (S)-methoprene (20 µM; squares) on equilibrium binding of [3H]CP55940 to mouse brain CB1 receptors. Control data points are identified by the diamond symbols. Kd values (as nM): control 0.36 + 0.07; chelerythrine 2.32 + 0.43; sanguinarine 2.28 + 0.77; (S)-methoprene 1.37 + 0.25 and piperonyl butoxide 0.34 + 0.19. Bmax values (as pmol [3H]SR141716A/mg protein): control 0.6 + 0.03; chelerythrine 0.65 + 0.06; sanguinarine 0.63 + 0.11; (S)-methoprene 0.25 + 0.02 and piperonyl butoxide 0.35 + 0.05.
135
Figure 4.4a Influence of study compounds on the time course of association of [3H]SR141716A and [3H]CP55940 with CB1 receptors of mouse brain. In a) membranes received a standard 15 min preincubation with sanguinarine (2.5 µM), chelerythrine (2.5 µM), piperonyl butoxide (30 µM) and (S)-methoprene (30 µM) prior to [3H]SR141716A addition. Symbols: diamonds = control; solid circles = sanguinarine; open circles = chelerythrine; triangles = piperonyl butoxide and squares = (S)-methoprene. Data points represent the means + SEMs of 3 independent experiments (a number of SEM bars are obscured by data symbols).
136
Figure 4.4b Influence of study compounds on the time course of association of [3H]SR141716A and [3H]CP55940 with CB1 receptors of mouse brain. b) The same study compound concentrations were applied simultaneously with [3H]SR141716A. Symbols: diamonds = control; solid circles = sanguinarine; open circles = chelerythrine; triangles = piperonyl butoxide and squares = (S)-methoprene.Data points represent the means + SEMs of 3 independent experiments (a number of SEM bars are obscured by data symbols).
137
Figure 4.4c Influence of study compounds on the time course of association of [3H]SR141716A and [3H]CP55940 with CB1 receptors of mouse brain. c) The effects of benzophenanthridines (5 µM), piperonyl butoxide (20 µM) and (S)-methoprene (20 µM) on the association of [3H]CP55940 under preincubation conditions are shown Symbols: diamonds = control; solid circles = sanguinarine; open circles = chelerythrine; triangles = piperonyl butoxide and squares = (S)-methoprene. Data points represent the means + SEMs of 3 independent experiments (a number of SEM bars are obscured by data symbols).
138
Figure 4.5a The influence of study compounds on the dissociation of CB1 receptor-selective radioligands. Figure 4.5a shows the effects of piperonyl butoxide (30 µM) and (S)-methoprene (60 µM) on the dissociation of [3H]SR141716A when initiated by challenge with a saturating concentration (5 µM) of SR141716A. Symbols: diamonds = control; triangles = piperonyl butoxide and squares = (S)-methoprene. Data represent mean + SEM of at least 3 independent experiments, each performed in triplicate.
139
Figure 4.5b The influence of study compounds on the dissociation of CB1 receptor-selective radioligands. Figure 4 5b, defines the effects of sanguinarine (5 µM), chelerythrine (5 µM), piperonyl butoxide (30 µM) and (S)-methoprene (60 µM) when added alone on the dissociation of [3H]SR141716A from the [3H]SR141716A:CB1 receptor complex. Symbols: diamonds = control; solid circles = sanguinarine; open circles = chelerythrine; triangles = piperonyl butoxide and squares = (S)-methoprene. Data represent mean + SEM of at least 3 independent experiments, each performed in triplicate.
140
Figure 4.5c The influence of study compounds on the dissociation of CB1 receptor-selective radioligands. Figure 4 5c, the effects of sanguinarine (5 µM), chelerythrine (5 µM), piperonyl butoxide (20 µM) and (S)-methoprene (60 µM) on the dissociation of [3H]CP55940 when initiated by application of a saturating concentration (5 µM) of CP55940 are given. Symbols: diamonds = control; solid circles = sanguinarine; open circles = chelerythrine; triangles = piperonyl butoxide and squares = (S)-methoprene. Data represent mean + SEM of at least 3 independent experiments, each performed in triplicate.
141
Figure 4.6 Relationship between concentration of (S)-methoprene and inhibition at CB2 receptors of mouse spleen based on interference with [3H]CP55940 binding.
142
Figure 4.7a Inhibition of 50 µM veratridine-evoked release of L-glutamate from mouse brain synaptosomes by 5 µM tetrodotoxin (TTX)
143
Figure 4.7b Failure of 5 µM TTX to modify 3 mM 4-AP-evoked release of L-glutamate from synaptosomes.
144
Figure 4.8 Partial inhibition of 4-AP-evoked release of L-glutamate from synaptosomes by the CB1-R agonist WIN55212-2, and full relief of WIN55212-2-dependent inhibition by the CB1-R antagonist AM251.
145
Figure 4.9 With WIN55212-2 present, sanguinarine (at 2 µM but not 0.25 µM) enhances 4-AP-evoked L-glutamate release above the level produced by 4-AP alone.
146
Figure 4.10 With WIN55212-2 present, chelerythrine (at 2 µM but not 0.25 µM) enhances 4-AP-evoked L-glutamate release above the level produced by 4-AP alone.
147
Figure 4.11 With WIN55212-2 present, (S)-methoprene (at 25 µM but not 5 µM) enhances 4-AP-evoked L-glutamate release above the level produced by 4-AP alone.
148
Figure 4.12 With WIN55212-2 present, piperonyl butoxide (at 25 µM but not 5 µM) enhances 4-AP-evoked L-glutamate release above the level produced by 4-AP alone.
149
Table 4.1 Inhibitory effects of chelerythrine, sanguinarine, piperonyl butoxide and (S)-methoprene on spleen CB2 receptors as determined with [3H]CP55940. Each study compound was added at a concentration that achieved an IC50 for [3H]CP55940 binding to brain CB1 receptors (Dhopeshwarkar et al. 2011). All values represent mean percentage inhibition + S.E.M. of at least 3 independent experiments. Parallel experiments with [3H]CP55940 corroborated our previously published IC50s at brain CB1 receptors (2.2 µM chelerythrine gave 49.03 + 0.94 % inhibition, 1.2 µM sanguinarine gave 51.33 + 0.49 % inhibition, 8.2 µM piperonyl butoxide gave 47.50 + 1.17 % inhibition and 16.4 µM methoprene gave 50.22 + 1.10 % inhibition).
Compound Inhibition of [3H]CP55940 binding to CB2 receptors of mouse spleen
Chelerythrine (2.2 µM) 4.14 + 0.14
Sanguinarine (1.2 µM 14.34 + 0.23
Piperonyl butoxide (8.2 µM) 20.86 + 0.23
(S)-Methoprene (16.4 µM) 50.98 + 0.21
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5. Effects of organotins on the CB1 receptor pathway of mouse brain in vitro.
Note: The research described in this chapter will be submitted shortly for
publication in an appropriate neurochemical/neuropharmacological journal. The
submission will adhere closely to the format laid out here.
5.1. Introduction
This Chapter constitutes a report on the modulatory effects of eight tributyltins
and two triphenyltin compounds at G protein-coupled cannabinoid-1 receptors (CB1-Rs)
of mouse brain in vitro. Organotin compounds have been exploited extensively for their
ability to preserve lumber and prevent biofouling on marine structures. Concerns over
toxicity to many marine and terrestrial species has led to severe restrictions on organotin
use, however they continue to be used on large ocean-going cargo ships because of the
huge reductions in fuel costs (and CO2 output) that can be achieved by minimizing
biofouling and the associated frictional drag on hulls. Tributyltin derivatives have also
seen significant uses as chemical intermediates and also as catalysts for various
chemical reactions.
The nervous system is known to be a highly sensitive target of tributyltins and a
number of mechanisms appear to be involved in their toxic actions. For example, in vivo
administration of tributyltin chloride to pregnant mice elevates dopamine concentrations
in the striatum and 5-hydroxytryptamine (5-HT) concentrations in the medulla oblongata
of F1 offspring, while dams exhibit widespread decreases in brain 5-HT levels (Tsunoda
et al., 2006). Tributyltins have been reported to lower the binding of [3H]MK801 to NMDA
receptors in the cerebral cortex of mice both in vitro and in vivo (Konno et al., 2001).
151
Tributlytins modulate glutathione levels in the striatum, hippocampus and cortex (Fortier
et al., 2010). Measurements of extracellular glutamate concentrations indicate that
tributyltins cause significant release of L-glutamate from neurons, leading to
excitotoxicity (Nakatsu et al., 2006).
The potential of tributyltins to interfere with the binding of [3H]CP55940 was
demonstrated originally as a result of experiments conducted by Dr. Chengyong Liao in
our laboratory. Mr. Sudip Ghose and Mr. Saurabh Jain of our laboratory went on to test
other organotin compounds in the [3H]CP55940 binding assay. Selected tributyltin
compounds were subsequently assayed for their capacity to modulate basal and
CP55940-stimulated binding of [35S]GTPγS to the G protein by Mr. Saurabh Jain (M.Sc.
Thesis Simon Fraser University, 2011) These assays suggested that some tributyltins
could possibly act as inverse agonists of CB1-Rs at low micromolar concentrations. My
task was to examine selected tributyltins for their ability to modify WIN55212-2-
dependent inhibition of evoked transmitter (L-glutamate) release from synaptosomes,
since we hypothesized that the inhibitory effect of WIN55212-2 should be neutralized
and transmitter release may possibly undergo further enhancement.
5.2. Materials and methods
All organotin compounds were purchased from Sigma-Aldrich. Methodological
details of the binding assays for [3H]CP55940 and [35S]GTPγS have been adequately
described in Chapter 2, Section 2.3.3 and 2.3.4 and Chapter 3, Section 3.3.2 and 3.3.3
of this thesis. The isolation of synaptosomes from mouse brain and the assay of L-
glutamate release was described in sufficient detail in the previous Chapter 4 (Section
4.3.7 and 4.3.8).
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5.3. Results
5.3.1. Displacement of [3H]CP55940 binding to mammalian CB1 receptors by organotin compounds
Eight tributyltins and 2 triphenyltins were tested for their ability to interfere with
[3H]CP55940 binding to mammalian CB1 receptors. The two triphenyltins and all
tributyltins except two (tributyltin hydride and tributylphenyltin; both inactive) achieved
IC50s at low micromolar concentrations (Table 5.1). All except the two inactive
compounds gave well-defined sigmoidal inhibition curves. Apart from phenylethynyl
tributyltin (which had the highest IC50 value of the group of active compounds and
produced about 80% inhibition at its maximum effect concentration), all other active
compounds achieved full (93-100%) inhibition. Concentration:inhibition curves are
displayed for tributyltin benzoate, tributyltin acetate and phenylethynyl tributyltin (Figure
5.1).
5.3.2. Basal and CP55940-stimulated [35S]GTPγS binding to the Gα subunit as influenced by tributyltin compounds
The inhibitory effects of tributyltin benzoate and phenylethynyl tributyltin on
[35S]GTPγS binding to the Ga subunit are shown in Figure 5.3. Tributyltin benzoate
reached IC50 at 1.43 µM (95% CI = 1.35 - 1.53 µM) and produced complete inhibition of
agonist-stimulated [35S]GTPγS binding around 2.5 µM. In addition tributyltin benzoate
encroached 51.03 ± 3.24% into basal binding at 5 µM with agonist present.
Phenylethynyl tributyltin achieved IC50 at 1.87 µM (95% CI = 1.71 - 2.02 µM) and
reached complete inhibition of agonist-stimulated [35S]GTPγS binding around 4 µM with
48.98 ± 14.64% and 68.53 ± 8.81% encroachment into the basal binding signal at 10 µM
and 20 µM respectively. Tributyltin benzoate (on its own) reduced basal [35S]GTPγS
binding by 22.12 ± 3.60% (at 2.5 µM) and 50.87 ± 1.87% (at 5 µM). Phenylethynyl
tributyltin (on its own) also reduced basal binding signal by 63.06 ± 3.62% at 20 µM.
153
5.3.3. Modulation by tributyltin acetate and phenylethynyl tributyltin of WIN55212-2-dependent inhibition of 4-aminopyridine-evoked release of L-glutamate from mouse brain synaptosomes
The capacity of tributyltin acetate and phenylethynyl tributyltin to modify CB1-R
agonist- (WIN55212-2-) dependent inhibition of 4-AP-evoked release of L-glutamate
from synaptosomes was explored to test the hypothesis that tributyltins negatively
modulate CB1-Rs by acting as inverse agonists. The fluorescence traces of L-glutamate
release from synaptosomes under these circumstances are displayed in Figures 5.4 and
5.5. Tributyltin acetate and phenylethynyl tributyltin (at 3 µM) fully relieved the inhibition
of 4-AP-evoked release caused by 8 µM WIN55212-2. This effect was identical to that of
the classical diarylpyrazole inverse agonist/antagonist AM251 at 8 µM. No relief of
WIN55212-2-dependent inhibition was observed at 0.5 µM with either compound. In
contrast to phenylethynyl tributyltin, tributyltin acetate (at 3 µM) actually enhanced the
release of L-glutamate over and above that observed with 4-AP alone. Little effect (2-8%
change) of the organotin compounds alone on background release of L-glutamate from
mouse brain synaptosomes was detected (Figures 5.4 and 5.5).
5.4. Discussion
This investigation demonstrates that a number of tin-containing compounds have
the capacity to inhibit the binding of [3H]CP55940 to CB1-Rs in mouse brain at very low
micromolar concentrations. However, the relationships between structural features and
inhibitory potencies are complex. Compounds containing a wide variety of substituents
attached to the central tin atom of the tributyltin system (including benzoate, acetate,
methoxide, hydroxide, bromide and trifluoromethane sulfonate), as well as the hydroxide
derivative of triphenyltin, all achieved IC50 between 2.0 and 3.3 µM. The chloride
derivative of triphenyltin and the phenylethynyl derivative of tributyltin were less potent
(IC50s approx. 5 and 15 µM respectively). Intriguingly, the highly bulky three phenyl
substituent system (in triphenyltin hydroxide and triphenyltin chloride) fails to reduce
inhibitory potency greatly. Furthermore, in the tributyltin series, if a single phenyl ring is
attached directly to the central tin atom (e.g. tributylphenyl tin), inhibitory activity is
eliminated. However, if an ethynyl or carboxylate spacer is inserted between the tin atom
154
and the phenyl ring (as in phenylethynyl tributyltin or tributyltin benzoate), the ability to
inhibit the binding of [3H]CP55940 to CB1-Rs dramatically recovers. In addition, within
the subtituted tributyltin series, the hydride is inactive; nevertheless, replacement of the
hydrogen with bromine produces the most potent organotin inhibitor we have found to
date.
The results of the [35S]GTPγS binding experiments (conducted by Mr. Saurabh
Jain) provided the first indication that tributyltins are able to interfere functionally with
CB1-R agonist-dependent activation of the CB1-R:G protein complex. For these
experiments, we focused specifically on tributyltin benzoate and phenylethynyl tributyltin.
Since the results demonstrated that both compounds inhibit basal binding of [35S]GTPγS,
CP55940-stimulated binding of [35S]GTPγS to the Gα subunit and they also inhibit the
basal [35S]GTPγS signal with CP55940 present, we infer that they likely act as inverse
agonists of CB1-Rs.
The possibility that tributyltins negatively modulate CB1-Rs by acting as inverse
agonists, was pursued further by investigating the ability of two analogs (tributyltin
acetate and phenylethynyl tributyltin) to modulate CB1-R agonist- (WIN55212-2-)
dependent inhibition of the release L-glutamate from synaptosomes following challenge
with 4-aminopyridine. This assay is well suited for such studies because it has been
shown by Wang (2003) and Godino et al. (2007) that inhibition of evoked L-glutamate
release from synaptosomes by WIN55212-2 is blocked by AM281, a classical
diarylpyrazole inverse agonist. Tributyltin acetate and phenylethynyl tributyltin fully
suppressed the inhibition of 4-AP-evoked release caused by WIN55212-2. However, in
contrast to phenylethynyl tributyltin, tributyltin acetate actually enhanced release over
and above that observed with 4-AP alone. Tributyltin acetate and phenylethynyl
tributyltin are certainly functional inhibitors of CB1-Rs as assessed by their modulatory
effects on presynaptic release of L-glutamate. Moreover, they closely mimic the standard
antagonist/inverse agonist AM281 in this assay. The profile of tributyltin acetate is
particularly consistent with an inverse agonist action at brain CB1-Rs. Given the findings
of this study, the ability of tributyltins to cause significant release of L-glutamate from
neurons and excitotoxicity (Nakatsu et al., 2006) may be caused in part by blockade of
endocannabinoid-dependent inhibition of L-glutamate release.
155
Although organotin compounds can be quite toxic to mammals, there may be
potential to develop from these structures a new class of drug that acts at CB1 receptors.
A practical approach may be to develop hybrid molecules that link the tin-containing
center with various pharmacophoric moieties present in the many classical CB1-R active
drugs. These hybrids may or may not be problematic from the toxicological standpoint.
Additionally, replacement of the central tetravalent tin atom with silicon or carbon could
generate useful structure activity data that could assist in drug design especially if tin-
containing prototypes turned out to be too toxic.
156
5.5. Figures and Table
Sn
O
O
Sn
O
Sn
O
S
O
C
F
3
O
TBT acetate TBT methoxide Tributylstannyl trifluoromethane sulphonate
Sn
Br
Sn
Sn
TBT bromide Tributylphenyl tin Tributyl(phenyl ethynyl)tin
Sn
O
O
Sn
O
H
Sn
Cl
TBT benzoate Triphenyl tin hydroxide Triphenyl tin chloride
Sn
H
TBT hydride
Figure 5.1 Structures of tributyl and triphenyltin compounds examined in the present investigation. Structures were constructed using Isis Draw.
157
Figure 5.2 Concentration-dependent inhibition of specific [3H]CP55940 binding to mouse brain CB1 receptors by tributyltin benzoate, tributyltin acetate and phenylethynyl tributyltin. Each data point represents the mean ± S.E.M. inhibition of specific [3H]CP55940 binding for at least three independent assays, each performed in triplicate. Experiments conducted by Mr. Saurabh Jain. This figure was originally published in the M.Sc. thesis of Mr. Saurabh Jain (Simon Fraser University, 2011).
158
Figure 5.3 Concentration-dependent inhibition of CP55940 (100 nM)-stimulated [35S]GTPγS binding by tributyltin benzoate and phenylethynyl tributyltin. Each data point represents the mean ± S.E.M. percentage inhibition of CP55940 stimulated [35S]GTPγS binding determined by three independent assays each performed in triplicate. These experiments were conducted by Mr Saurabh Jain and this figure was originally published in the M.Sc. thesis of Mr. Saurabh Jain (Simon Fraser University, 2011).
159
Figure 5.4 Modulation of WIN55212-2-dependent inhibition of 4-aminopyridine (4-AP-)-evoked release of L-glutamate from mouse brain synaptosomes by tributyltin acetate (TBT acetate). Typical release profiles are displayed with mean % changes (+ SEM) to 4-AP-evoked and control release in the adjacent table.
160
Figure 5.5 Modulation of WIN55212-2-dependent inhibition of 4-aminopyridine (4-AP-)-evoked release of L-glutamate from mouse brain synaptosomes by phenylethynyl tributyltin (TBPE tin). Typical release profiles are displayed with mean % changes (+ SEM) to 4-AP-evoked and control release in the adjacent table.
161
Table 5.1 Inhibitory effects of tributyl and triphenyltins on the binding of [3H]CP55940 to CB1 receptors in mouse brain. All values are as IC50s (with 95% confidence intervals in brackets) calculated from curves based on at least 3 independent experiments except for triphenyltin chloride where the IC50 was estimated from 2 independent experiments).
Organotin IC50 with 95% confidence interval (µM)
TBT benzoate 2.6 (1.7 - 3.9)
TBT acetate 2.7 (2.3 - 3.3)
TBT methoxide 3.3 (2.7-3.8)
TBT bromide 2.0 (1.6 - 2.5)
Tributylstannyl-TMS 2.9(2.3- 3.6)
Triphenyltin hydroxide 2.6 (1.5-4.5)
Triphenyltin chloride 5.1
Tributylphenylethynyl tin 14.8 (9.8 - 22.2)
TBT hydride >100
Tributylphenyltin >100
The above IC50 values were calculated from concentration:inhibition experiments carried out by Dr. Chengyong Liao, Mr. Sudip Ghose and Mr. Saurabh Jain. TBT = tributyltin, TMS = trifluoromethane sulfonate
162
6. Conclusion and future prospects
The conclusions of my Ph.D. research have already been discussed in detail
elsewhere (see Chapter 2, Section 2.5; Chapter 3, Section 3.5; Chapter 4, Section 4.5;
Chapter 5, Section 5.4).
In summary, I conclude that sanguinarine, chelerythrine, (S)-methoprene and
piperonyl butoxide exert inhibitory actions at CB1-Rs in mouse brain in vitro. These
compounds act at very low to moderate micromolar concentrations with an inhibitory
potency ranking (estimated from [3H]CP55940, [3H]SR141716A and [35S]GTPγS binding
data) of sanguinarine ~ chelerythrine > piperonyl butoxide > methoprene.
Based on my saturation binding and kinetic experiments I infer that these
compounds inhibit via predominently allosteric mechanisms with respect to [3H]CP55940
and [3H]SR141716A binding.
My experiments with mouse brain synaptosomes demonstrate that WIN-55212-2-
dependent inhibition of 4-AP-evoked L-glutamate release is blocked by sanguinarine,
chelerythrine, (S)-methoprene and piperonyl butoxide. The actions of (S)-methoprene
and piperonyl butoxide are indistinguishable from AM251 (a classical diarylpyrazole CB1-
R antagonist), demonstrating an antagonist action at CB1-Rs. In addition to blocking the
inhibitory effect of WIN55212-2 on evoked release of L-glutamate, sanguinarine and
chelerythrine (with WIN55212-2 present) increase the release of neurotransmitter to a
level greater than that caused by WIN55212-2 alone. This suggests an inverse agonist-
like action of the benzophenanthridines. The L-glutamate release results therefore align
with our previous profiling of these compounds in the [35S]GTPγS binding assay.
Building on the findings of Dr. Chengyong Liao, Ms. Kathleen M. Bisset and Mr.
Saurabh Jain in my laboratory, I further explored the pharmacological actions of
phthalate esters and tributyltins at brain CB1-Rs. Based on [35S]GTPγS binding and L-
163
glutamate release results I conclude that these common environmental pollutants are
antagonists of CB1-R function.
The environmental chemicals highlighted in this thesis represent a broad range
of structural classes. It is interesting that when their potential to modify functional
outcomes of the CB1-R signaling pathway was investigated (i.e. using [35S]GTPγS
binding and L-glutamate release) only inhibitory (antagonist or inverse agonist-like)
actions were revealed. From these observations I infer that environmental chemicals
possessing the structural features of a CB1-R agonist might be more rarely encountered.
It would be logically predicted that if the study compounds were able to enter the
brain and engage with CB1-Rs, they should reduce the effectiveness of
endocannabinoids (e.g. anandamide and 2-AG) in activating CB1-Rs. I recommend that
future studies examine the ability of these compounds to inhibit both [3H]anandamide
binding to CB1-Rs and anandamide-induced suppression of L-glutamate release from
synaptosomes. Another very important line of future research would be to examine how
in-vivo administration of study compounds might modify the classical behavioral
manifestations of CB1-R agonists. Should any compound show CB1-R antagonist or
inverse agonist-like effects in vivo, careful consideration should be given to its potential
as a prototype for rational design of more effective analogs. As mentioned in the
introduction, CB1-R antagonists are effective in body weight reduction. Despite of the
issue with Rimonabant (SR141716A) and the USFDA's recent approval of Lorcaserin (a
weight reducing 5-HT2C agonist), there is a huge demand for drugs with this property.
Certain study compounds (e.g. (S)-methoprene and piperonyl butoxide) would represent
very low acute toxicity starting points and I would also suggest that improvements in
potency to the level of SR141716A may not be needed as some CNS drugs are effective
and well tolerated at low micromolar concentrations.
164
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