BMC Pharmacology BioMed CentralBioMed Central Page 1 of 13 (page number not for citation purposes) BMC Pharmacology Research article Open Access Differential modulation of microglia

BioMed CentralBMC Pharmacology

Open AcceResearch articleDifferential modulation of microglia superoxide anion and thromboxane B2 generation by the marine manzaminesAlejandro MS Mayer1 Mary L Hall2 Sean M Lynch3 Sarath P Gunasekera4 Susan H Sennett5 and Shirley A Pomponi6

Address 1Department of Pharmacology Chicago College of Osteopathic Medicine Midwestern University 555 31st Street Downers Grove Illinois 60515 USA 2Department of Pharmacology Chicago College of Osteopathic Medicine Midwestern University 555 31st Street Downers Grove Illinois 60515 USA 3Department of Biochemistry Chicago College of Osteopathic Medicine Midwestern University 555 31st Street Downers Grove Illinois 60515 USA 4Division of Biomedical Marine Research Harbor Branch Oceanographic Institution Inc 5600 US 1 North Fort Pierce Florida 34946 USA 5Division of Biomedical Marine Research Harbor Branch Oceanographic Institution Inc 5600 US 1 North Fort Pierce Florida 34946 USA and 6Division of Biomedical Marine Research Harbor Branch Oceanographic Institution Inc 5600 US 1 North Fort Pierce Florida 34946 USA

Email Alejandro MS Mayer - amayermidwesternedu Mary L Hall - mhallxmidwesternedu Sean M Lynch - slynchmidwesternedu Sarath P Gunasekera - sgunasekerhboiedu Susan H Sennett - sennetthboiedu Shirley A Pomponi - pomponihboiedu

Corresponding author

AbstractBackground Thromboxane B2 (TXB2) and superoxide anion (O2

-) are neuroinflammatorymediators that appear to be involved in the pathogenesis of several neurodegenerative diseasesBecause activated-microglia are the main source of TXB2 and O2

- in these disorders modulation oftheir synthesis has been hypothesized as a potential therapeutic approach for neuroinflammatorydisorders Marine natural products have become a source of novel agents that modulateeicosanoids and O2

- generation from activated murine and human leukocytes With the exceptionof manzamine C all other manzamines tested are characterized by a complex pentacyclic diaminelinked to C-1 of the β-carboline moiety These marine-derived alkaloids have been reported topossess a diverse range of bioactivities including anticancer immunostimulatory insecticidalantibacterial antimalarial and antituberculosis activities The purpose of this investigation was toconduct a structure-activity relationship study with manzamines (MZ) A B C D E and F onagonist-stimulated release of TXB2 and O2

- from E coli LPS-activated rat neonatal microglia in vitro

Results The manzamines differentially attenuated PMA (phorbol 12-myristate 13-acetate)-stimulated TXB2 generation in the following order of decreasing potency MZA (IC50 lt0016 microM)gtMZD (IC50 = 023 microM) gtMZB (IC50 = 16 microM) gtMZC (IC50 = 298 microM) gtMZE and F (IC50 gt10 microM)In contrast there was less effect on OPZ (opsonized zymosan)-stimulated TXB2 generation MZB(IC50 = 144 microM) gtMZA (IC50 = 316 microM) gtMZC (IC50 = 334 microM) gtMZD MZE and MZF (IC50 gt10microM) Similarly PMA-stimulated O2

- generation was affected differentially as follows MZD (apparentIC50lt01 microM) gtMZA (IC50 = 01 microM) gtMZB (IC50 = 316 microM) gtMZC (IC50 = 343 microM) gtMZE andMZF (IC50 gt10 microM) In contrast OPZ-stimulated O2

- generation was minimally affected MZB (IC50= 417 microM) gtMZC (IC50 = 93 microM) gtMZA MZD MZE and MZF (IC50 gt 10 microM) From thestructure-activity relationship perspective contributing factors to the observed differentialbioactivity on TXB2 and O2

- generation are the solubility or ionic forms of MZA and D as well aschanges such as saturation or oxidation of the β carboline or 8-membered amine ring In contrast

Published 11 March 2005

BMC Pharmacology 2005 56 doi1011861471-2210-5-6

Received 25 January 2005Accepted 11 March 2005

This article is available from httpwwwbiomedcentralcom1471-221056

copy 2005 Mayer et al licensee BioMed Central Ltd This is an Open Access article distributed under the terms of the Creative Commons Attribution License (httpcreativecommonsorglicensesby20) which permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

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BMC Pharmacology 2005 56 httpwwwbiomedcentralcom1471-221056

the fused 13-membered macrocyclic and isoquinoline ring system and any substitutions in theserings would not appear to be factors contributing to bioactivity

Conclusion To our knowledge this is the first experimental study that demonstrates that MZAat in vitro concentrations that are non toxic to E coli LPS-activated rat neonatal microglia potentlymodulates PMA-stimulated TXB2 and O2

- generation MZA may thus be a lead candidate for thedevelopment of novel therapeutic agents for the modulation of TXB2 and O2

- release inneuroinflammatory diseases Marine natural products provide a novel and rich source of chemicaldiversity that can contribute to the design and development of new and potentially useful anti-inflammatory agents to treat neurodegenerative diseases

BackgroundThe hallmark of brain inflammation is the activation ofglia particularly microglia the resident immune cells ofthe brain [1] Microglia activation in brain pathologies ascaused by infectious diseases inflammation traumabrain tumors ischemia and AIDS may result in neuronalinjury and ultimately neurodegeneration [1] Similar toother tissue macrophages when microglia become acti-vated they release potentially neurotoxic mediators [2]followed by sublethal and lethal injury to the central nerv-ous system The two different phenotypic forms of micro-glia namely the activated but nonphagocytic microgliafound in inflammatory pathologies and the reactive orphagocytic microglia present in trauma infection andneuronal degeneration appear to have the capacity toexpress cell-surface receptors and release mediators ofinflammation such as cytokines coagulation factorscomplement factors proteases nitric oxide eicosanoidsand reactive oxygen species [2]

Over the last three decades the marine environment hasbeen demonstrated to be a source of novel therapeuticagents many of which have anti-inflammatory properties[3] We have previously shown that selected marine natu-ral products modulate eicosanoids [45] and O2

- genera-tion from activated rat [6] and human neutrophils [7] aswell as liver [8] and alveolar macrophages [9] Based onthese observations we hypothesized that selected marinenatural products might potentially attenuate activatedbrain microglia [2] Since the discovery by Sakai and Higathat the marine sponge-derived manzamine A (MZA) hadpotent antitumor activity [10] there has been a sustainedinterest in the chemistry [11] as well as the pharmacologyof the manzamines a class of β-carboline marine-derivedalkaloids More than 40 manzamine-type alkaloids havebeen isolated from 9 different genera of marine spongesfrom the Indian and Pacific Oceans and in addition to theantitumor activity [10] manzamines have been shown tobe immunostimulatory [12] insecticidal [13] antibacte-rial[13] antimalarial [14] antiparasitic [15] antiviral[16] and to possess antituberculosis activity [17]

In preliminary communications we have reported thatMZA isolated from the Okinawan marine sponge Hali-clona sp potently inhibited TXB2 and O2

- generation byactivated rat neonatal microglia while showing very lowconcomitant toxicity [18-20] We now extend these previ-ous communications by reporting the results of a struc-ture-activity relationship study with manzamines A B CD E and F on agonist-stimulated release of O2

- and TXB2from LPS-activated rat neonatal microglia

ResultsEffect of manzamine A on LPS-activated neonatal brain microglia TXB2 O2

- and LDH releaseAs shown in Fig 1 MZA has a pentacyclic diamine groupattached to the β-carboline moiety and it was tested as itshydrochloride salt As is shown in Fig 2A MZA potentlyinhibited PMA-stimulated TXB2 generation (IC50 = 0016microM) with a maximum 955 inhibition observed at 10microM (MZA vs vehicle respectively 783 plusmn 45 vs 1413 plusmn439 pg of TXB2 per 200000 microglia per 70 min P lt001 n = 4) Furthermore as depicted in Fig 3A MZAinhibited PMA-stimulated O2

- generation with an appar-ent IC50 = 01 microM (MZA vs vehicle respectively 75 plusmn 18vs138 plusmn 19 nmol of O2

- per 200000 microglia per 70min P lt 001 n = 4) Significantly increasing MZA con-centrations to 10 microM resulted in O2

- inhibition of 559 plusmn61 P lt 001 n = 4

In contrast as shown in Fig 2A the effect of MZA on OPZ-stimulated TXB2 generation was weaker (apparent IC50 =316 microM) with a maximum 58 inhibition at 10 microM(MZA vs vehicle respectively 1927 plusmn 474 vs 4504 plusmn 308pg of TXB2 per 200000 microglia per 70 min P lt 005 n= 2) Similarly as depicted in Fig 3A MZA did not appearto affect OPZ-stimulated O2

- generation even at 10 microM(MZA vs vehicle respectively 95 plusmn 1 vs 94 plusmn 07 O2

nmol per 200000 microglia per 70 min P gt 005 n = 2)

As shown in Fig 2A and 3A the cytotoxicity of MZA toneonatal brain microglia measured as LDH release wasnot significantly different from controls even at 10 microM(MZA vs vehicle respectively 213 plusmn 7 vs 139 plusmn 37 of total LDH released by 01 Triton X-100 treated-

The chemical structures of manzamines A B C D E and FFigure 1The chemical structures of manzamines A B C D E and F Manzamines are indole-derived alkaloids isolated from the marine sponges Haliclona sp [10] Amphimedon sp [66] and Xestospongia sp [1067] Molecular weights are respectively = 5852 5508 3475 5913 5647 5808

Manzamine A HCl Manzamine B Manzamine C

Manzamine D HCl Manzamine E Manzamine F

Differential effects of manzamines A B C D E and F on PMA and OPZ-stimulated TXB2 generation by LPS-activated rat neo-natal microgliaFigure 2Differential effects of manzamines A B C D E and F on PMA and OPZ-stimulated TXB2 generation by LPS-activated rat neonatal microglia Rat neonatal microglia (200000 cellswell) were activated with LPS (03 ngmL) for 17 hours Manzamines were added 15 min before stimulation with either PMA (1 microM) or OPZ (05 mgmL) After 70 min agonist-triggered TXB2 was measured as described in Materials and Methods LDH release indicator of cytotoxicity was determined as described in Materials and Methods Data are expressed as percentage of control TXB2 release triggered by either PMA (MZA 1423 plusmn 439 pg TXB2 70 min MZB C D E F 3279 plusmn 281 pg TXB2 70 min) or OPZ (4504 plusmn 308 pg TXB2 70 min) Data show mean plusmn SEM of indicated number (n) of experiments P lt 005 P lt 001

Manzamine A

0 01 1 100

LDH (n=6)

PMA (n=4)

[ M][ M]

OPZ (n=2)

se Total LD

elease

Manzamine B

0 01 1 100

LDH (n=5)

PMA (n=3)

[ M][ M]

OPZ (n=2)

se Total LD

elease

Manzamine C

0 01 1 100

LDH (n=5)

OPZ (n=2)

[ M][ M]

PMA (n=-3)

se Total LD

elease

Manzamine D

0 01 1 100

LDH (n=5)

OPZ(n=2)

[ M][ M]

PMA (n=3)

se Total LD

elease

Manzamine E

0 01 1 100

LDH (n=5)

PMA (n=3)

[ M][ M]

OPZ (n=2)

se Total LD

elease

Manzamine F

0 01 1 100

LDH (n=5)

OPZ(n=2)

[ M][ M]

PMA (n=3)

se Total LD

elease

Differential effects of manzamines A B C D E and F on PMA and OPZ-stimulated O2- generation by LPS-activated rat neona-tal microgliaFigure 3

Differential effects of manzamines A B C D E and F on PMA and OPZ-stimulated O2- generation by LPS-

activated rat neonatal microglia Rat neonatal microglia (200000 cellswell) were activated with LPS (03 ngmL) for 17 hours Manzamines were added 15 min before stimulation with either PMA (1 microM) or OPZ (05 mgmL) After 70 min agonist-triggered O2

- release was determined as described in Materials and Methods LDH release indicator of cytotoxicity was meas-ured as described in Materials and Methods Data are expressed as percentage of control O2

- release triggered by PMA (MZA 138 plusmn 19 nmoles O2

-70 min MZB C D E F 108 plusmn 06 nmoles O2-70 min) or OPZ (94 plusmn 07 nmoles O2

- 70 min) Data show mean plusmn SEM of indicated number (n) of experiments P lt 005 P lt 001

Manzamine A

0 01 1 100

LDH (n=6)

PMA (n=4)

[ M][ M]

OPZ (n=2)

2- Rel

Total LD

elease

Manzamine B

0 01 1 100

LDH (n=5)

PMA (n=3)

[ M][ M]

OPZ (n=2)

2- Rel

Total LD

elease

Manzamine C

0 01 1 100

LDH (n=5)

OPZ(n=2)

[ M][ M]

PMA (n=3)

2- Rel

Total LD

elease

Manzamine D

0 01 1 100

LDH (n=5)

PMA (n=3)

[ M][ M]

OPZ (n=2)

2- Rel

Total LD

elease

Manzamine E

0 01 1 100

LDH (n=5)

PMA (n=3)

[ M][ M]

OPZ (n=2)

2- Rel

Total LD

elease

Manzamine F

0 01 1 100

LDH (n=5)

PMA (n=3)

[ M][ M]

OPZ (n=2)

2- Rel

Total LD

elease

microglia n = 6 P = 01735) This data suggests that theeffect of MZA on both PMA-stimulated TXB2 and O2

- maybe of a pharmacological nature

Effect of manzamine B on LPS-activated neonatal brain microglia TXB2 O2

- and LDH releaseMZB differs from MZA in having a tetracyclic diaminecomplex and an epoxide ring system (Fig 1) As shown inFig 2B MZB which was tested as a free base was lesspotent than MZA in affecting PMA-stimulated TXB2 gener-ation (IC50 = 16 microM) MZB 10 microM reduced TXB2 release to155 of control (MZB vs vehicle respectively 5463 plusmn281 vs 3279 plusmn 281 pg of TXB2 per 200000 microglia per70 min P lt 001 n = 3) Similarly as depicted in Fig 3BMZB was less potent than MZA in affecting PMA-stimu-lated O2

- generation (IC50 = 316 microM) MZB 10 microMreduced O2

- release to 20 of control (MZB vs vehiclerespectively 214 plusmn 214 vs 108 plusmn 06 nmol of O2

- per200000 microglia per 70 min P lt 001 n = 3)

As shown in Fig 2B MZB affected OPZ-stimulated TXB2(IC50 = 144 microM) more than MZA MZB 10 microM reducedTXB2 generation to 256 of control (MZB vs vehiclerespectively 1160 plusmn 207 vs 4504 plusmn 308 pg of TXB2 per200000 microglia per 70 min P lt 005 n = 2) Further-more as shown in Fig 3B MZB reduced OPZ-stimulatedO2

- generation (IC50 = 417 microM) more than MZA MZB 10microM reduced O2

- generation to 165 of control (MZB vsvehicle respectively 15 plusmn 07 vs 94 plusmn 07 O2

- nmol per200000 microglia per 70 min P lt 001 n = 2)

As shown in Fig 2B and 3B in contrast to MZA MZB wascytotoxic to neonatal brain microglia at concentrationsabove 1 microM In fact considerable LDH release wasobserved at 10 microM (883 plusmn 12 of total LDH released by01 Triton X-100 treated-microglia n = 5 P lt 001)Taken together these data suggest that the reduction ofboth O2

- and TXB2 generation resulted from both pharma-cological and toxic effects of MZB on LPS-activated micro-glia cells

Effect of manzamine C on LPS-activated neonatal brain microglia TXB2 O2

- and LDH releaseMZC differs from MZA in having a monocyclic amine ringattached to the β-carboline moiety (Fig 1) As shown inFig 2C MZC which was tested as a free base was lesspotent than MZA in affecting PMA-stimulated TXB2 gener-ation (IC50 = 298 microM) MZC 10 microM reduced TXB2releaseto 196 of control (MZC vs vehicle respectively 677 plusmn293 vs 3279 plusmn 281 pg of TXB2 per 200000 microglia per70 min P lt 001 n = 3) Similarly as depicted in Fig 3CMZC was less potent than MZA in affecting PMA-stimu-lated O2

- generation (apparent IC50 = 343 microM) MZC 10microM reduced O2

- release to 361 of control (MZC vs

vehicle respectively 37 plusmn 22 vs108 plusmn 06 nmol of O2- per

200000 microglia per 70 min P lt 005 n = 3)

As shown in Fig 2C MZC affected OPZ-stimulated TXB2(IC50 = 334 microM) similar to MZA MZC 10 microM reducedTXB2 generation to 298 of control (MZC vs vehiclerespectively 1345 plusmn 160 vs 4504 plusmn 308 pg of TXB2 per200000 microglia per 70 min P lt 005 n = 2) Further-more as depicted in Fig 3C MZC reduced OPZ-stimu-lated O2

- generation (apparent IC50 = 93 microM) higher thanMZA MZC 10 microM reduced O2

- generation to 416 ofcontrol (MZC vs vehicle respectively 39 plusmn 03 vs 94 plusmn07 O2

- nmol per 200000 microglia per 70 min P lt 005n = 2)

As shown in Fig 2C and 3C and in contrast to MZA MZCwas cytotoxic to neonatal brain microglia though not asmuch as MZB Substantial LDH release was observed at 10microM (598 plusmn 11 of total LDH released by 01 Triton X-100 treated-microglia n = 5 P lt 005) In summary sim-ilar to MZB the data suggests that the reduction of bothO2

- and TXB2 generation resulted from both pharmacolog-ical and toxic effects of MZC on LPS-activated microgliacells

Effect of manzamine D on LPS-activated neonatal brain microglia TXB2 O2

- and LDH releaseMZD differs from MZA in having a tetrahydrocarbolinegroup in the molecule (Fig 1) As shown in Fig 2D MZDthat was tested as its hydrochloride salt strongly affectedPMA-stimulated TXB2 generation (IC50 = 023 microM) MZD10 microM reduced TXB2 release to 188 of control (MZD vsvehicle respectively 611 plusmn 342 vs 3279 plusmn 281 pg of TXB2per 200000 microglia per 70 min P lt 001 n = 3) Fur-thermore as shown in Fig 3D MZD also strongly affectedPMA-stimulated O2

- generation MZD 01 microM reduced O2-

release to 14 of control (MZD vs vehicle respectively07 plusmn 07 vs 108 plusmn 06 nmol of O2

- per 200000 microgliaper 70 min P lt 001 n = 3)

In contrast as shown in Fig 2D the effect of MZD onOPZ-stimulated TXB2 generation was limited MZD 10microM reduced TXB2 generation to 799 of control (MZDvs vehicle respectively 3613 plusmn 469 vs 4504 plusmn 308 pg ofTXB2 per 200000 microglia per 70 min P gt 005 n = 2)Furthermore as shown in Fig 3D MZD did not affectOPZ-stimulated O2

- generation even at 10 microM

As shown in Fig 2D and 3D and in contrast to MZB andC MZD was very cytotoxic to microglia when PMA wasused as an agonist to trigger O2

- and TXB2 release MZD at01 microM caused 615 plusmn 13 of total LDH released by 01 Triton X-100 treated-microglia (n = 5 P lt 005) In con-trast to the limited effect of MZD on OPZ-stimulatedmicroglia the data suggests that the reduction of PMA-

stimulated O2- and TXB2 generation resulted from both

pharmacological and toxic effects of MZD on LPS-acti-vated microglia cells

Effect of manzamine E on LPS-activated neonatal brain microglia TXB2 O2

- and LDH releaseAs shown in Fig 1 MZE differs from MZA in having a sat-urated ketone functionality in the eight-membered aminering As depicted in Fig 2E MZE inhibited PMA-stimu-lated TXB2 generation with a maximum 494 inhibitionobserved at 10 microM (MZE vs vehicle respectively 1614 plusmn628 vs 3279 plusmn 281 pg of TXB2 per 200000 microglia per70 min P gt 005 n = 3) Furthermore as shown in Fig 3EMZE inhibited PMA-stimulated O2

- generation with amaximum 263 inhibition observed at 10 microM (MZE vsvehicle respectively 85 plusmn 21 vs108 plusmn 06 nmol of O2

- per200000 microglia per 70 min P gt 005 n = 3)

As shown in Fig 2E MZE had limited effect on OPZ-stim-ulated TXB2 generation with a maximum 431 inhibi-tion at 10 microM (MZE vs vehicle respectively 2594 plusmn 646vs 4504 plusmn 308 pg of TXB2 per 200000 microglia per 70min P gt 005 n = 2) Similarly as depicted in Fig 3EMZE did not affect OPZ-stimulated O2

- generation even at10 microM

As shown in Fig 2E and 3E cytotoxicity of MZE to micro-glia measured as LDH release was low even at 10 microM(MZE vs vehicle respectively 20 plusmn 67 vs 198 plusmn 77 of total LDH released by 01 Triton X-100 treated-microglia P gt 005 n = 5)

Effect of manzamine F on LPS-activated neonatal brain microglia TXB2 O2

- and LDH releaseMZF differs from MZA in having a saturated ketone func-tionality in the eight-membered amine ring and hydroxy-lation at the C-8 position of the β-carboline ring system(Fig 1) As shown in Fig 2F MZF did not inhibit PMA-stimulated TXB2 generation In the presence of 10 microM MZTXB2 release was 1041 plusmn 247 of control TXB2 genera-tion (MZF vs vehicle respectively 3202 plusmn 1139 vs 3279plusmn 281 pg of TXB2 per 200000 microglia per 70 min P gt005 n = 3) Similarly as shown in Fig 3F MZF did notinhibit PMA-stimulated O2

- release In the presence of 10microM MZF O2

- release was 113 plusmn 145 of control O2- gen-

eration (MZF vs vehicle respectively 128 plusmn 16 vs 108 plusmn06 nmol of O2

- per 200000 microglia per 70 min P gt005 n = 3)

As shown in Fig 2F MZF effect on OPZ-stimulated TXB2generation was weak with a non-statistically significant262 inhibition at 10 microM (MZF vs vehicle respectively3317 plusmn 121 vs 4504 plusmn 308 pg of TXB2 per 200000 micro-glia per 70 min P gt 005 n = 2) Similarly as depicted inFig 3F MZF was minimally effective in inhibiting OPZ-

stimulated O2- generation only a non-statistically signifi-

cant 157 inhibition observed at 10 microM

As shown in Fig 2F and 3F cytotoxicity of MZF to neona-tal brain microglia measured as LDH release was low evenat 10 microM (MZF vs vehicle respectively 19 plusmn 72 vs 198plusmn 77 of total LDH released by 01 Triton X-100treated-microglia n = 5)

Effect of manzamine A B C D E and F on hypoxanthine-xanthine oxidase generated O2

In order to determine a potential scavenging effect ofMZA B C D E and F on O2

- a standard hypoxanthine-xanthine oxidase system was used as a cell-free source ofO2

- [21] As shown in Fig 4 O2- generation by incubation

of purified xanthine oxidase with hypoxanthine was abol-ished by superoxide dismutase Furthermore DMSO thevehicle used to prepare the manzamines did not affect O2

formation (control vs DMSO respectively 123 plusmn 23 vs12 plusmn 11 nmoles30 min n = 2 P gt 005) Similarly MZAB and E did not significantly affect O2

- generation (MZAB E vs control respectively 153 plusmn 44 153 plusmn 24 163 plusmn22 vs 12 plusmn 11 nmoles30 min n = 2 P gt 005) In con-trast MZC D and F appeared to enhance O2

- formation(MZC D F vs control respectively 207 plusmn 31 222 plusmn 2201 plusmn 36 vs 12 plusmn 11 nmoles30 min n = 2 P lt 005(MZC F) P lt 001 MZD) Thus we conclude that the inhi-bition of either PMA or OZ stimulated-O2

- release fromLPS-activated microglia by the manzamines was not theresult of a direct O2

- scavenging effect

DiscussionThe important role of neuroinflammation and glial acti-vation in the pathogenesis of brain disorders has progres-sively been established [1222] Because in vitro LPS-activated microglia appear to mimic the functions ofactivated microglia found in neuroinflammatory condi-tions in vivo [223] we used LPS-activated rat microglia asa relevant in vitro paradigm to search for marine naturalproducts that may modulate the enhanced release of TXB2and O2

- from activated microglia [24] Using this in vitromodel we have previously communicated that MZA a sec-ondary metabolite isolated from the Okinawan marinesponge Haliclona sp[10] inhibited TXB2 and O2

- genera-tion by microglia [18] The current study extends our ini-tial observations and reports a structure-activityrelationship study with manzamines A B C D E and Fon both PMA and OPZ-stimulated release of TXB2 and O2

from LPS-activated rat neonatal microglia

Members of the eicosanoid family (ie prostaglandinsleukotrienes and thromboxanes) are important mediatorsof inflammation that would appear to be play a causativerole in the pathogenesis of several CNS disorders [25-27]Increased levels of eicosanoids have been observed in

neurodegenerative disorders such as amyotrophic lateralsclerosis [28] multiple sclerosis [29] ischemia and sei-zures [30] prion diseases [31] human immunodeficiencyvirus-associated dementia [32] and Alzheimers disease[33] Following the seminal observation that microgliarelease eicosanoids [34] numerous studies have increas-ingly supported the notion that activated brain macro-phages may be the main source of both prostaglandins[3335-39] and thromboxanes [23373940] in theseneurodegenerative diseases Thus modulation of micro-glia enhanced prostanoid synthesis has been investigatedas a potential drug therapeutic approach for interventionin neuroinflammatory disorders of the CNS [364142]One possible approach to diminish enhanced eicosanoidproduction has been to search for inhibitors of signaltransduction pathways involved in eicosanoid synthesisin activated microglia [42] In our study we used PMAand OPZ agonists known to activate p4442 and p38mitogen-activated protein kinases in microglia [43] totarget distinct signal transduction pathways that causeTXB2 release in rat neonatal microglia activated by an invitro exposure to 03 ngmL of LPS for 17 hours [23] Asshown in Fig 2 the 6 marine-derived manzamine analogsattenuated PMA-stimulated TXB2 generation differentiallyand in the following order of decreasing potencyMZAgtMZDgtMZB gtMZCgtMZE and F In contrast the

manzamine analogs inhibited OPZ-stimulated TXB2 gen-eration with reduced potency MZBgtMZAgtMZCgtMZDMZE and MZF Thus with the exception of MZB and MZCwhich modulated both PMA and OPZ-stimulated TXB2release with similar potency MZA MZD MZE and MZFinhibited TXB2 release with lower potency when OPZ wasused as an agonist

It is interesting to compare our differential results with theMZA B C D E and F with those reported for other agentsthat have been shown to modulate microglia eicosanoidrelease by targeting the cyclooxygenase I and II enzymeswhich are expressed in activated rat and human microglia[3544] PGE2 and TXB2 synthesis that occurs concomi-tantly with LPS-induced activation of rat [384546] andhuman microglia [35] has been shown to be attenuatedby nonsteroidal anti-inflammatory drugs (NSAIDs) withdiffering activities towards the two isoforms of COXThus LPS-induced microglia PGE2 synthesis was reducedby COX-1 inhibitors acetylsalicylic acid (aspirin) (IC50 =312ndash10 microM) [3846] flurbiprofen (apparent IC50 = 100nM) [45] and indomethacin (apparent IC50 = 1 nM) [38]and the COX-2 inhibitor NS-398 (apparent IC50 = 1ndash5nM) [38] Even though NSAIDs have been reported toattenuate neurotoxicity in vitro [47] and neuroinflamma-tion in animal models [48-50] an important caveat is thefact that determining the best NSAIDs for clinical neuro-degenerative disease management appears to remain amatter of considerable debate in view of their well knownadverse effects [51-53] Thus although the molecularmechanism by which the manzamines inhibit TXB2release in LPS-activated cells remains currently undeter-mined MZA inhibited PMA-stimulated eicosanoid gener-ation in vitro with potency similar to that of the COX-1inhibitor indomethacin [38] potency that was higherthan that of other NSAIDs that have been reported tomodulate enhanced eicosanoid release in both activatedrat and human microglia [35384546]

The involvement of reactive oxygen species (ROS) hasbeen documented in CNS pathologies such as Parkin-sons disease Alzheimers disease Huntingtons diseaseDowns syndrome cerebral ischemia and reperfusionamyotrophic lateral sclerosis multiple sclerosis and men-ingitis [54] Prolonged exposure to ROS may potentiallydamage neurons particularly their synapses [55] as wellas oligodendrocytes the myelin producing cell of the CNS[56] by overriding normal CNS antioxidant defensemechanisms eg superoxide dismutase catalase glutath-ione-S-transferase glutathione peroxidase permanentlyaffecting cellular function [57] Thus the mechanism ofROS generation by CNS leukocytes ie infiltrating neu-trophils and monocytes as well as resident microglia pro-duction of O2

- hydrogen peroxide and nitric oxide inCNS has received considerable attention since the mid-

Differential effect of manzamine A B C D E and F and superoxide dismutase on hypoxanthine-xanthine oxidase sys-tem-generated O2-Figure 4

Differential effect of manzamine A B C D E and F and superoxide dismutase on hypoxanthine-xanthine oxidase system-generated O2

- Cell-free source of O2-

was generated by a standard hypoxanthine-oxidase system as described in Materials and Methods Data are expressed as O2

- released (nmoles O2-30 min) and correspond to the

mean plusmn SD of 2 representative experiments in duplicate P lt 005 P lt 001 versus control

Positiv

1980s [2] In fact during the past 18 years numerousresearch groups have shown that O2

- may be generated bymicroglia isolated from rat mice hamsters dogs swineand humans when stimulated with a variety of agonistssuch as phorbol ester opsonized zymosan calcium iono-phore antiviral antibodies antibody-coated red bloodcells and myelin (reviewed in [2]) We and others havehypothesized that rather than scavenge ROS with antioxi-dants the modulation of the signal transduction mecha-nism leading to microglia ROS generation might beputatively a better therapeutic strategy to turn off orreduce ROS generation that could lead to neuronal injury[25859] As depicted in Fig 3 the manzamine analogsattenuated PMA-stimulated O2

- generation in the follow-ing order of decreasing potencyMZDgtMZAgtMZBgtMZCgtMZE and MZF In contrast andsimilarly to their weaker effect on OPZ-stimulated TXB2generation all the manzamine analogs modulated OPZ-stimulated O2

- generation with lower potencyMZBgtMZCgtMZA MZD MZE and MZF Interestingly asshown in Fig 4 the effect of MZD A B and C on PMA-stimulated O2

- formation was not the result of any detect-able scavenging O2

- because none of the manzaminesinhibited a standard hypoxanthine-xanthine oxidase sys-tem that was used as a cell-free source of O2

- Thusalthough the exact mechanism by which the manzaminesmodulate O2

- release by microglia remains currentlyundetermined we have demonstrated that these com-pounds clearly modulate the signal transduction pathwaythat PMA triggers in microglia and ultimately leads to O2

generation

It is interesting to compare our differential results with theMZD A B and C with those reported for other agents thatmodulate PMA-stimulated O2

- generation in microgliaInterestingly MZD A B and C demonstrated higherpotency than three clinically available agents shown toinhibit PMA-stimulated O2

- generation propentofylline aselective phosphodiesterase inhibitor (IC50 gt100 microM)[60] cabergoline a potent and selective agonist of D2-dopamine receptors (IC50 gt100 microM) [59] and nicergo-line an ergoline derivative used for cerebrovascular dis-eases (IC50 = 10ndash15 microM) [58] It is noteworthy that theseagents have been proposed to confer protective effectsagainst neurodegenerative diseases which may involve O2

release by activated rat microglia

In order to determine if the effect of the manzamines onPMA or OPZ-triggered TXB2 and O2

- generation was eitherpharmacological or toxic we investigated LDH releasefrom LPS-activated rat neonatal microglia LDH has exten-sively been used as a marker for cell cytotoxicity [61] Theresults from our investigation appear to demonstrate thatthe manzamine analogs clearly differed in their effect onLDH release from LPS-primed microglia MZA MZE and

MZF generated less than 50 of maximal LDH release at10 microM and thus were the least toxic analogs MZB andMZC induced greater than 50 of LDH release at 10 microMwhile MZD showed greater than 50 of LDH release at01 microM when PMA rather than OPZ was used as anagonist thus its toxicity contrasted with the other analogstested Thus even though MZD inhibited PMA-stimulatedO2

- generation with slightly higher potency than MZAbecause MZD caused concomitant LDH release at lowconcentrations (01 microM) the nature of the inhibitoryeffect on PMA-triggered O2

- and TXB2 release either toxicor pharmacological remains currently unresolved Insummary the in vitro studies described herein suggest thatMZA is the most potent manzamine analog of the seriesinvestigated because both PMA-stimulated O2

- and TXB2were potently inhibited with the lowest concomitantrelease of LDH

It is of interest to consider the results of our structure-activity relationship (SAR) study with the manzaminesalkaloids characterized by a complex heterocyclic ring sys-tem attached to the C-1 of the β-carboline moiety Fromthe SAR perspective the potent effect of MZA and Dhydrochloride salts on PMA-stimulated O2

- and TXB2 sug-gests that the solubility or ionic forms are contributingfactors to their bioactivity Furthermore the fused 13-membered macrocyclic and octahydroisoquinoline ringsystem and any substitutions in these rings would appearto be less important for their in vitro activity Finallychanges such as saturation or oxidation of the β carbolineor the 8-membered amine ring tended to decrease bioac-tivity in both O2

- and TXB2 assays

Taken together our current data demonstrates that themost potent and least toxic manzamine analog namelyMZA was less effective in attenuating O2

- and TXB2 fromLPS-activated microglia when the triggering agonist wasOPZ rather than PMA Similar differential effects betweenPMA and OPZ-triggered signaling have been observedwith other natural products [62] Furthermore the currentdata suggest the following on the as yet undefined mech-anism of action of MZA Firstly that the MZA moleculartarget plays a critical role in O2

- and TXB2 generation initi-ated by PMA upon binding to PKC [6364] and activationof the p4442 mitogen-activated protein kinase signalingpathway [43] Secondly that the MZA molecular targetprobably plays a less critical role in O2

- and TXB2 releaseelicited by OPZ a ligand of the microglial cell surfacecomplement receptor 3 shown to activate the p38mitogen-activated protein kinase signaling pathway [43]Studies to determine which element is targeted by MZA inthe p38 andor p4442 mitogen-activated protein kinasepathways in LPS-activated rat microglia are currentlyunderway in our laboratory

ConclusionOur present results provide the first experimental evi-dence to support the hypothesis that the marine-derivedβ-carboline alkaloid manzamines differentially modulateboth O2

- and TXB2 generated by E coli LPS-activated ratneonatal microglia Additional conclusions are the fol-lowing Firstly SAR studies demonstrated that at in vitroconcentrations that were non-toxic to E coli LPS-activatedrat neonatal microglia MZA was the most potent inhibi-tor of O2

- and TXB2 Secondly although the mechanism bywhich MZA inhibited PMA-stimulated TXB2 generation invitro is as yet unclear its potency was similar to the COX-1 inhibitor indomethacin [38] and thus higher than otherNSAIDs reported to modulate enhanced eicosanoidrelease in both activated rat and human microglia[35384546] Thirdly although the mechanism by whichMZA inhibited PMA-stimulated O2

-generation in vitroremains undetermined MZA was more potent than pro-pentofylline a selective phosphodiesterase inhibitorcabergoline a potent and selective agonist of D2-dopamine receptors and nicergoline an ergoline deriva-tive used for cerebrovascular diseases compounds whichhave been proposed to confer protective effects againstneurodegenerative diseases by affecting O2

- release by acti-vated rat microglia Fourthly SAR studies which demon-strated that the ionic forms are a contributing factor to thebioactivity of the complex manzamine heterocyclic ringsystem attached to a β-carboline moiety may explain thepotent effect of MZA and D hydrochloride salts on PMA-stimulated O2

- and TXB2 Interestingly the fused 13-mem-bered macrocyclic amine and octahydroisoquinoline ringsystem as well as substitutions in these rings appeared tobe a non-factor for the in vitro activity of the manzaminesFinally the reported pharmacokinetic properties and thelack of significant in vivo toxicity [14] of MZA a β-carbo-line alkaloid whose complete synthesis has been reported[11] would suggest that MZA is a prime candidate for fur-ther investigation of its potential utility as a pharmacoph-ore from which new and novel therapeutic agents forneuroinflammatory diseases might be developed

MethodsReagentsLPS B (Escherichia coli 026B6) was obtained from DifcoLaboratories (Detroit MI) Wright Giemsa stain (modi-fied) ferricytochrome c type III (from horse heart) (FCC)superoxide dismutase (from bovine liver) phorbol 12-myristate 13-acetate (PMA) zymosan and dimethyl sul-phoxide (DMSO) were obtained from Sigma ChemicalCo (St Louis MO) PMA was maintained at -80degC as a 10mM stock solution in DMSO Opsonized zymosan (OPZ)was maintained at -20degC in a stock solution of 15 mgmlin PBS and prepared as described [65] Dulbeccos modi-fied Eagle medium (DMEM) with high glucose (4500mgl) Hanks balanced salt solution (HBSS) penicillin

(P) streptomycin (S) trypsin (025)-EDTA (1 mM) andtrypan blue were purchased from GIBCO-BRL (GrandIsland NY) certified heat-inactivated fetal bovine serum(FBS) was obtained from Hyclone (Logan UT) a LPSstock of 1 mgml was prepared in a 09 sodium chloridenonpyrogenic solution from Baxter Healthcare Corp(Toronto ONT Canada) and then diluted with DMEMplus 10 FBS plus P and S to the appropriate concentra-tion used in our experiments Both the LPS stock solution[10 ngml] and dilutions were stored at -80degC thawedprior to each experiment and discarded after use

LPS containmentTo inactivate extraneous LPS all glassware and metal spat-ulas were baked for 4 hours at 180degC Sterile and LPS-free75- and 162-cm2 vented cell culture flasks 24-well flat-bottom culture clusters 96-well cell culture clusters anddisposable serological pipettes were purchased from Cos-tar Corporation (Cambridge MA) while polystyrene cellculture dishes (60 times 15 mm) were obtained from CorningGlass Works (Corning NY) Sterile and LPS-free Eppen-dorf Biopur pipette tips were purchased from BrinkmannInstruments Inc (Westbury NY)

Manzamines A B C D E and FManzamine A (MZA) was isolated from a marine spongespecies of the genus Haliclona collected off ManzamoOkinawa in waters at a depth of 30 m in April 1985 [10]Manzamine B (MZB) manzamine C (MZC) and man-zamine D (MZD) were isolated from a sponge of thegenus Amphimedon collected by SCUBA off ManzamoOkinawa [66] Manzamine E (MZE) and manzamine F(MZF) were isolated from a sponge species of the genusXestospongia collected off the coast of Miyako IslandOkinawa in June 1986 [67] All manzamines were dis-solved in DMSO to prepare a 10 mM stock and stored at -80degC prior to use in the experiments

Isolation and culture of rat neonatal microgliaAll experiments were performed with adherence to theNational Institutes of Health guidelines on the use ofexperimental animals and with protocols approved byMidwestern Universitys Research and Animal Care Com-mittee To isolate rat neonatal microglia cerebral corticesof 1ndash2 day-old Sprague-Dawley rats purchased from Har-lan (Indianapolis IN) were surgically removed and placedin cold DMEM + 10 FBS + 120 Uml P and 12 microgml Sthe meninges carefully removed and brain tissue mincedand dissociated with trypsin-EDTA at 36degC for 3ndash5 minThe mixed astroglial cell suspension was plated in either75- or 162-cm2 vented cell culture flasks with DMEMmedium supplemented with 10 FBS + 120 Uml P + 12microgml S and grown in a humidified 5 CO2 incubator at36degC for 12ndash14 days On day 14 and every 3ndash4 days there-after microglia were detached using an orbital shaker

(150 rpm 05 hours 36degC 5 CO2) centrifuged (400 timesg 25 min 4degC) and microglia number and viabilityassessed by trypan blue exclusion Microglia were charac-terized as described earlier [23] Depending on the partic-ular experimental protocol (see below) microgliaaveraging greater than 95 viability were plated in 24-well cell culture clusters with DMEM supplemented with10 FBS + 120 Uml P + 12 microgml S and placed in ahumidified 5 CO2 incubator at 36degC 18ndash24 hours priorto the experiments

Experimental protocol to study the effect of manzamines A ndash F on microglia release of TXB2 and O2

To study the effects of manzamines A B C D E and F onthe generation of TXB2 and O2

- rat neonatal microglia (2times 105 cells24-well cell culture clusters) were treated to thefollowing protocol Seventeen hours prior to the experi-ments microglia cells were treated with LPS (03 ngml)in a final volume of 1 ml of DMEM supplemented with10 FBS + 120 Uml P + 12 microgml Thereafter the mediawas removed and replaced with 1 ml warm HBSS one ofthe manzamines (01ndash10 microM final concentration) or vehi-cle (DMSO) was added and the microglia incubated forfifteen minutes in a humidified 5 CO2 incubator at359degC After the fifteen minute preincubation periodwith either manzamines or vehicle PMA (1 microM) or OPZ(05 mgmL) was added and microglia incubated for 70minutes in a humidified 5 CO2 incubator at 359degC inthe presence of the manzamines or vehicle The final con-centration of DMSO did not affect microglia viability orLDH release O2

- TXB2 and lactate dehydrogenase (LDH)release were assayed as described below

Assay for TXB2Following the incubation of LPS-activated microglia withHBSS manzamines or vehicle as explained above PMA-(1 microM) or OPZ- (05 mgmL)triggered TXB2 generation inthe culture supernatants was measured using immu-noassays (Cayman Chemical Ann Arbor MI) as indicatedby the manufacturers protocol Results were expressed aspgmL produced after 70 min of PMA or OPZ stimulation

Assay for O2-

O2- generation was determined by the SOD-inhibitable

reduction of FCC [23] Briefly PMA (1 microM) or OPZ (05mgmL)- triggered O2

- release from LPS-activated micro-glia was measured in the presence of FCC (50 microM) andHBSS with or without SOD (700 Units) which inhibitedgt95 of FCC reduction during the 70 min incubationdescribed above All experimental treatments were run intriplicate and in a final volume of 1 ml Changes in FCCabsorbance were measured at 550 nm using a BeckmanDU-650 spectrophotometer Differences in the amount ofreduced FCC in the presence and absence of SOD wereused to determine O2

- generation and expressed in nmol

by employing the molecular extinction coefficient of 210times 103 M-1 cm-1

Experimental protocol to study the effect of manzamines on superoxide anion by the hypoxanthine-xanthine oxidase systemA standard hypoxanthine-xanthine oxidase system wasused as a cell-free source of O2

- O2- was generated by incu-

bation of purified xanthine oxidase (002 Unitsml) withhypoxanthine (15 mM) at 37degC [21] O2

- formation wasassessed spectrophotometrically as the increase in absorb-ance at 550 nm associated with the SOD (30 UmL)-inhibitable reduction FCC (50 microM) as described above forrat microglia O2

- generation and expressed in nmoles30minutes

Lactate dehydrogenase assayLactate dehydrogenase (LDH) release from microglia wasdetermined spectrophotometrically as described else-where [9] Microglia LDH release was expressed as a per-centage of total LDH Total LDH resulted from 01Triton X-100-lysed microglia cells (intracellular LDH)plus LDH released to the extracellular medium

Statistical analysis of the dataData were analyzed with the Prismreg software package pur-chased from GraphPad (San Diego CA) One-way analy-sis of variance followed by Dunnetts test was performedon all sets of data Manzamine-treated groups were com-pared with the vehicle-treated group shown as 0 or con-trol in the corresponding figures Differences wereconsidered statistically significant at p lt 005 and reportedin each figure legend

AbbreviationsCNS central nervous system DMEM Dulbeccos modi-fied Eagle medium DMSO dimethyl sulphoxide FBSfetal bovine serum certified FCC ferricytochrome c typeIII HBSS Hanks balanced salt solution LPS lipopolysac-charide MZA manzamine A MZB manzamine B MZCmanzamine C MZD manzamine D MZE manzamine EMZF manzamine F O2

- superoxide anion OPZopsonized zymosan P penicillin PBS phosphate buff-ered saline PMA phorbol-12-myristate-13-acetate Sstreptomycin SOD superoxide dismutase TXB2 throm-boxane B2

Authors contributionsAMS M designed and conducted the experimentsdescribed and prepared the manuscript draft

MLH performed the statistical analysis of the data andprepared Fig 2 3 and 4

S M L helped conduct the xanthine oxidase studies withManzamines A B C D E and F

SPG supplied pure Manzamines A B C D E and Ffrom the Division of Biomedical Marine Research deposi-tory prepared Fig 1 and contributed to various sectionsof the manuscript

SAP provided financial support and contributed to var-ious sections of the manuscript

SHS contributed to various sections of the manuscript

All authors read and approved the manuscript

AcknowledgementsThis publication was made possible by grant number 1 R15 ES12654-01 (to AMSM) from the National Institute of Environmental Health Sciences NIH Its contents are solely the responsibility of the authors and do not necessarily represent the official view of the NIEHS NIH Additional sup-port from Midwestern University and Harbor Branch Oceanographic Insti-tution is gratefully acknowledged The authors wish to specially thank Katie Koning for excellent technical assistance

References1 Kreutzberg GW Microglia a sensor for pathological events in

the CNS Trends Neurosci 1996 19312-3182 Mayer AMS Therapeutic implications of microglia activation

by lipopolysaccharide and reactive oxygen species genera-tion in septic shock and central nervous system pathologiesa review Medicina (B Aires ) 1998 58377-385

3 Mayer AMS Hamann MT Marine pharmacology in 2000 marinecompounds with antibacterial anticoagulant antifungalanti-inflammatory antimalarial antiplatelet antituberculo-sis and antiviral activities affecting the cardiovascularimmune and nervous systems and other miscellaneousmechanisms of action Mar Biotechnol (NY) 2004 637-52

4 Mayer AMS Jacobson PB Fenical W Jacobs RS Glaser KB Pharma-cological characterization of the pseudopterosins novelanti- inflammatory natural products isolated from the Car-ibbean soft coral Pseudopterogorgia elisabethae Life Sci1998 62L401-L407

5 Mayer AMS Glaser KB Jacobs RS Regulation of eicosanoid bio-synthesis in vitro and in vivo by the marine natural productmanoalide a potent inactivator of venom phospholipases JPharmacol Exp Ther 1988 244871-878

6 Mayer AMS Spitzer JA Modulation of superoxide anion gener-ation by manoalide arachidonic acid and staurosporine inliver infiltrated neutrophils in a rat model of endotoxemia JPharmacol Exp Ther 1993 267400-409

7 Mayer AMS Brenic S Glaser KB Pharmacological targeting ofsignaling pathways in protein kinase C- stimulated superox-ide generation in neutrophil-like HL-60 cells effect of phor-bol ester arachidonic acid and inhibitors of kinase(s)phosphatase(s) and phospholipase A2 J Pharmacol Exp Ther1996 279633-644

8 Mayer AMS Spitzer JA Modulation of superoxide generation inin vivo lipopolysaccharide- primed Kupffer cells by stau-rosporine okadaic acid manoalide arachidonic acid genis-tein and sodium orthovanadate J Pharmacol Exp Ther 1994268238-247

9 Mayer AMS Brenic S Stocker R Glaser KB Modulation of super-oxide generation in in vivo lipopolysaccharide- primed ratalveolar macrophages by arachidonic acid and inhibitors ofprotein kinase C phospholipase A2 protein serine-threo-nine phosphatase(s) protein tyrosine kinase(s) andphosphatase(s) J Pharmacol Exp Ther 1995 274427-436

10 Sakai R Higa T Manzamine A a novel antitumor alkaloid froma sponge J Am Chem Soc 1986 1086404-6405

11 Hu JF Hamann MT Hill R Kelly M The manzamine alkaloidsAlkaloids Chem Biol 2003 60207-285

12 Ang KK Holmes MJ Kara UA Immune-mediated parasite clear-ance in mice infected with Plasmodium berghei followingtreatment with manzamine A Parasitol Res 2001 87715-721

13 Edrada RA Proksch P Wray V Witte L Muller WE van Soest RWFour new bioactive manzamine-type alkaloids from the Phil-ippine marine sponge Xestospongia ashmorica J Nat Prod1996 591056-1060

14 Ang KKH Holmes MJ Higa T Hamann MT Kara UAK In vivo anti-malarial activity of the beta-carboline alkaloid manzamineA Antimicrobial Agents amp Chemotherapy 2000 441645-1649

15 Rao KV Kasanah N Wahyuono S Tekwani BL Schinazi RF HamannMT Three new manzamine alkaloids from a common Indo-nesian sponge and their activity against infectious and tropi-cal parasitic diseases J Nat Prod 2004 671314-1318

16 Yousaf M Hammond NL Peng J Wahyuono S McIntosh KA Char-man WN Mayer AM Hamann MT New manzamine alkaloidsfrom an Indo-Pacific sponge Pharmacokinetics oral availa-bility and the significant activity of several manzaminesagainst HIV-I AIDS opportunistic infections and inflamma-tory diseases J Med Chem 2004 473512-3517

17 El Sayed KA Kelly M Kara UA Ang KK Katsuyama I Dunbar DCKhan AA Hamann MT New manzamine alkaloids with potentactivity against infectious diseases J Am Chem Soc 20011231804-1808

18 Mayer AMS Gunasekera SP Pomponi SA Sennett SH Inhibition ofLPS-primed rat brain microglia superoxide and thrombox-ane B2 generation by the marine Manzamines Society Neuro-science Abstracts 2000 261346

19 Mayer AMS Gunasekera SP Pomponi SA Sennett SH Anti-inflam-matory uses of Manzamines U S Patent 2002 6387916

21 Lynch SM Frei B Mechanisms of copper- and iron-dependentoxidative modification of human low density lipoprotein JLipid Res 1993 341745-1753

22 Streit WJ Mrak RE Griffin WS Microglia and neuroinflamma-tion a pathological perspective J Neuroinflammation 2004 114[httpPM15285801]

23 Mayer AMS Oh S Ramsey KH Jacobson PB Glaser KB Romanic AMEscherichia Coli Lipopolysaccharide potentiation and inhibi-tion of rat neonatal microglia superoxide anion generationcorrelation with prior lactic dehydrogenase nitric oxidetumor necrosis factor-a thromboxane B2 and metallopro-tease release SHOCK 1999 11180-186

24 Mayer AMS Oh S Presto E Glaser KB Jacobson PB LPS-primedrat brain microglia a convenient in vitro model to search foranti-inflammatory marine natural products SHOCK 19977(Suppl 2)49

25 Eikelenboom P Bate C van Gool WA Hoozemans JJ Rozemuller JMVeerhuis R Williams A Neuroinflammation in Alzheimers dis-ease and prion disease GLIA 2002 40232-239

26 Moore AH OBanion MK Neuroinflammation and anti-inflam-matory therapy for Alzheimers disease Adv Drug Deliv Rev2002 541627-1656

27 Minghetti L Cyclooxygenase-2 (COX-2) in inflammatory anddegenerative brain diseases J Neuropathol Exp Neurol 200463901-910

28 McGeer PL McGeer EG Inflammatory processes in amyo-trophic lateral sclerosis Muscle Nerve 2002 26459-470

29 Fretland DJ Potential role of prostaglandins and leukotrienesin multiple sclerosis and experimental allergicencephalomyelitis Prostaglandins Leukot Essent Fatty Acids 199245249-257

30 Farooqui AA Horrocks LA Excitatory amino acid receptorsneural membrane phospholipid metabolism and neurologi-cal disorders Brain Res Brain Res Rev 1991 16171-191

31 Williams AE Van Dam AM Man AHWK Berkenbosch F Eikelen-boom P Fraser H Cytokines prostaglandins and lipocortin-1are present in the brains of scrapie-infected mice Brain Res1994 654200-206

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32 Griffin DE Wesselingh SL McArthur JC Elevated central nervoussystem prostaglandins in human immunodeficiency virus-associated dementia Ann Neurol 1994 35592-597

33 Hoozemans JJ Rozemuller AJ Janssen I De Groot CJ Veerhuis REikelenboom P Cyclooxygenase expression in microglia andneurons in Alzheimers disease and control brain Acta Neu-ropathol (Berl) 2001 1012-8

34 Gebicke-Haerter PJ Bauer J Schobert A Northoff H Lipopolysac-charide-free conditions in primary astrocyte cultures allowgrowth and isolation of microglial cells J Neurosci 19899183-194

35 Hoozemans JJ Veerhuis R Janssen I van Elk EJ Rozemuller AJ Eike-lenboom P The role of cyclo-oxygenase 1 and 2 activity inprostaglandin E(2) secretion by cultured human adult micro-glia implications for Alzheimers disease Brain Res 2002951218-226

36 Minghetti L Polazzi E Nicolini A Creminon C Levi G Interferon-gamma and nitric oxide down-regulate lipopolysaccharide-induced prostanoid production in cultured rat microglialcells by inhibiting cyclooxygenase-2 expression J Neurochem1996 661963-1970

37 Minghetti L Levi G Induction of prostanoid biosynthesis bybacterial lipopolysaccharide and isoproterenol in rat micro-glial cultures J Neurochem 1995 652690-2698

38 Greco A Ajmone-Cat MA Nicolini A Sciulli MG Minghetti L Para-cetamol effectively reduces prostaglandin E2 synthesis inbrain macrophages by inhibiting enzymatic activity ofcyclooxygenase but not phospholipase and prostaglandin Esynthase J Neurosci Res 2003 71844-852

39 Slepko N Minghetti L Polazzi E Nicolini A Levi G Reorientationof prostanoid production accompanies activation of adultmicroglial cells in culture J Neurosci Res 1997 49292-300

40 Giulian D Corpuz M Richmond B Wendt E Hall ER Activatedmicroglia are the principal glial source of thromboxane inthe central nervous system NEUROCHEM INT 1996 2965-76

41 Aldskogius H Regulation of microglia - potential new drug tar-gets in the CNS Expert Opin Ther Targets 2001 5655-668

42 Minghetti L Levi G Microglia as effector cells in brain damageand repair focus on prostanoids and nitric oxide ProgNeurobiol 1998 5499-125

43 Koistinaho M Koistinaho J Role of p38 and p4442 mitogen-acti-vated protein kinases in microglia GLIA 2002 40175-183

44 Minghetti L Polazzi E Nicolini A Creminon C Levi G Up-regula-tion of cyclooxygenase-2 expression in cultured microglia byprostaglandin E2 cyclic AMP and non-steroidal anti-inflam-matory drugs Eur J Neurosci 1997 9934-940

45 Ajmone-Cat MA Nicolini A Minghetti L Differential effects of thenonsteroidal antiinflammatory drug flurbiprofen and itsnitric oxide-releasing derivative nitroflurbiprofen on pros-taglandin E(2) interleukin-1beta and nitric oxide synthesisby activated microglia J Neurosci Res 2001 66715-722

46 Fiebich BL Lieb K Hull M Aicher B van Ryn J Pairet M EngelhardtG Effects of caffeine and paracetamol alone or in combina-tion with acetylsalicylic acid on prostaglandin E(2) synthesisin rat microglial cells NEUROPHARMACOLOGY 2000392205-2213

47 Klegeris A Walker DG McGeer PL Toxicity of human THP-1monocytic cells towards neuron-like cells is reduced by non-steroidal anti-inflammatory drugs (NSAIDs) NEUROPHARMA-COLOGY 1999 381017-1025

48 Pompl PN Ho L Bianchi M McManus T Qin W Pasinetti GM Atherapeutic role for cyclooxygenase-2 inhibitors in a trans-genic mouse model of amyotrophic lateral sclerosis FASEB J2003 17725-727

49 Mohanakumar KP Muralikrishnan D Thomas B Neuroprotectionby sodium salicylate against 1-methyl-4-phenyl-123 6-tet-rahydropyridine-induced neurotoxicity Brain Res 2000864281-290

50 Lim GP Yang F Chu T Chen P Beech W Teter B Tran T Ubeda OAshe KH Frautschy SA Cole GM Ibuprofen suppresses plaquepathology and inflammation in a mouse model for Alzhe-imers disease J Neurosci 2000 205709-5714

51 McGeer PL Schulzer M McGeer EG Arthritis and anti-inflam-matory agents as possible protective factors for Alzheimersdisease a review of 17 epidemiologic studies NEUROLOGY1996 47425-432

52 Pasinetti GM Cyclooxygenase as a target for the antiamy-loidogenic activities of nonsteroidal anti-inflammatory drugsin Alzheimers disease Neurosignals 2002 11293-297

53 Hoozemans JJ Veerhuis R Rozemuller AJ Eikelenboom P Non-ster-oidal anti-inflammatory drugs and cyclooxygenase in Alzhe-imers disease Curr Drug Targets 2003 4461-468

54 Halliwell B Role of free radicals in the neurodegenerative dis-eases therapeutic implications for antioxidant treatmentDrugs Aging 2001 18685-716

55 Colton CA Fagni L Gilbert D The action of hydrogen peroxideon paired pulse and long-term potentiation in thehippocampus Free Radic Biol Med 1989 73-8

56 Griot C Vandevelde M Richard A Peterhans E Stocker R Selectivedegeneration of oligodendrocytes mediated by reactive oxy-gen species Free Radic Res Commun 1990 11181-193

57 Fridovich I Superoxide dismutases Adv Enzymol Relat Areas MolBiol 1986 5861-97

58 Yoshida T Tanaka M Okamoto K Inhibitory effect of nicergolineon superoxide generation by activated rat microglias meas-ured using a simple chemiluminescence method Neurosci Lett2001 2975-8

59 Yoshida T Tanaka M Suzuki Y Sohmiya M Okamoto K Antioxi-dant properties of cabergoline inhibition of brain auto-oxi-dation and superoxide anion production of microglial cells inrats Neurosci Lett 2002 3301-4

60 Si Q Nakamura Y Ogata T Kataoka K Schubert P Differentialregulation of microglial activation by propentofylline viacAMP signaling Brain Res 1998 81297-104

61 Morgenstern S Flor R Kessler G Klein B Automated determina-tion of NAD-coupled enzymes Determination of lacticdehydrogenase Anal Biochem 1966 13149-161

62 Nunez CV Zacheu FM Pinto E Roque NF Colepicolo P BrigagaoMR Sesquiterpene lactone from Wunderlichia crulsianainhibits the respiratory burst of leukocytes triggered by dis-tinct biochemical pathways Life Sci 2003 732161-2169

63 Giulian D Baker TJ Characterization of ameboid microglia iso-lated from developing mammalian brain J Neurosci 198662163-2178

64 Colton CA Gilbert DL Production of superoxide anions by aCNS macrophage the microglia FEBS Lett 1987 223284-288

65 Johnston RBJ Measurement of O2- secreted by monocytesand macrophages Methods Enzymol 1984 105365-369

66 Sakai R Kohmoto S Higa T Jefford CW Bernardinelli G Man-zamine B and C two novel alkaloids from the sponge Hali-clona sp Tetrahedron Lett 1987 285493-5496

67 Ichiba T Sakai R Kohmoto SSG New manzamine alkaloids froma sponge of the genus Xestospongia Tetrahedron Lett 1988293083-3086

Abstract

Background

Results

Conclusion

Background

Results

Effect of manzamine A on LPS-activated neonatal brain microglia TXB2 O2- and LDH release

Effect of manzamine B on LPS-activated neonatal brain microglia TXB2 O2- and LDH release

Effect of manzamine C on LPS-activated neonatal brain microglia TXB2 O2- and LDH release

Effect of manzamine D on LPS-activated neonatal brain microglia TXB2 O2- and LDH release

Effect of manzamine E on LPS-activated neonatal brain microglia TXB2 O2- and LDH release

Effect of manzamine F on LPS-activated neonatal brain microglia TXB2 O2- and LDH release

Effect of manzamine A B C D E and F on hypoxanthine- xanthine oxidase generated O2-

Discussion

Conclusion

Methods

Reagents

LPS containment

Manzamines A B C D E and F

Isolation and culture of rat neonatal microglia

Experimental protocol to study the effect of manzamines A - F on microglia release of TXB2 and O2-

Assay for TXB2

Assay for O2-

Experimental protocol to study the effect of manzamines on superoxide anion by the hypoxanthine-xanthine oxidase system

Lactate dehydrogenase assay

Statistical analysis of the data

Abbreviations

Authors contributions

Acknowledgements

References