CENTRAL ADENOSINE SIGNALING PLAYS A KEY ROLE IN …jpet.aspetjournals.org › content › jpet › early › 2006 › 04 › 04 › jpet.105.… · JPET # 100495 4 Introduction The

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CENTRAL ADENOSINE SIGNALING PLAYS A KEY ROLE IN CENTRALLY

MEDIATED HYPOTENSION IN CONSCIOUS AORTIC-BARODENERVATED RATS

NOHA NASSAR and ABDEL A. ABDEL-RAHMAN

Department of Pharmacology and Toxicology (NN and ARA)

Brody School of Medicine

East Carolina University

Greenville, NC 27834

JPET Fast Forward. Published on April 4, 2006 as DOI:10.1124/jpet.105.100495

Copyright 2006 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on April 4, 2006 as DOI: 10.1124/jpet.105.100495

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Running title: Adenosine & Clonidine hypotension

Correspondence to: Abdel A. Abdel-Rahman, Ph.D. Department of Pharmacology and Toxicology,

School of Medicine, East Carolina University, Greenville, NC 27834.

Tel: +1- (252) 744-3470; Fax: +1- (252) 744-3203; E-mail: [email protected]

Number of text pages: 28

Number of tables: 1

Number of figures: 4

Number of reference: 42

Number of words

Abstract: 230

Introduction: 592

Discussion: 1315

List of non-standard abbreviations

ABD: aortic barodenervated rat

α-MNE: alpha methylnorepinephrine

8-SPT: 8-(p-sulfophenyl) theophylline

RVLM: rostral ventrolateral medulla

NTS: nucleus tractus solitarius

α2A-AR: alpha 2A adrenergic receptors

SHR: spontaneously hypertensive rat

SCH58261: 5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3- ]-1,2,4-triazolo[1,5-c]pyrimidine


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Abstract

We tested the hypothesis that clonidine-evoked hypotension is dependent on central adenosinergic

pathways. Five groups of male conscious aortic baroreceptor denervated (ABD) rats received

clonidine (10 µg/kg; i.v.) 30 min after i.v. : (1) saline, (2) theophylline (10 mg/kg), or (3) 8-(p-

sulfophenyl) theophylline (8-SPT; 2.5mg/kg); or 1 hr after i.p. (4) dipyridamole (5 mg/kg) or (5)

equal volume of sesame oil. Blockade of central (theophylline) but not peripheral (8-SPT) adenosine

receptors abolished clonidine hypotension. In contrast, dipyridamole substantially enhanced the

bradycardic response to clonidine. In additional groups, intracisternal (i.c.) dipyridamole (150 µg)

and 8-SPT (10 µg), enhanced and abolished, respectively, clonidine (0.6 µg, i.c.) evoked

hypotension. Since clonidine is a mixed I1/α2 agonist, we also investigated whether adenosine

signaling is linked to the I1 or the α2A receptor by administering the selective I1 (rilmenidine, 25 µg)

or α2A (α-methylnorepinephrine; α-MNE, 4 µg) agonist 30 min after central adenosine receptor

blockade (8-SPT; 10 µg, i.c.) or aCSF. The hypotensive response elicited by rilmenidine or α-MNE

was abolished in 8-SPT pretreated rats. To delineate the role of the adenosine A2A receptor in

clonidine-evoked hypotension, i.c. clonidine (0.6 µg) was administered 30 min after central

adenosine receptor A2A blockade (SCH58261; 150 µg, i.c.). The latter virtually abolished the

hypotensive and bradycardic responses elicited by clonidine. In conclusion, central adenosine A2A

signaling plays a key role in clonidine-evoked hypotension in conscious aortic barodenervated rats.


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Introduction

The mechanism of the centrally mediated hypotension elicited by clonidine and similar drugs

is not fully understood. Reported studies have identified the brainstem, and particularly the rostral

ventrolateral medulla (RVLM), as the major neuroanatomical substrate for clonidine (Tingley and

Arneric, 1990; Ernsberger and Haxhiu, 1997). Suppression of the activity of sympathetic neurons in

the RVLM, assessed electrochemically (Mao et al., 2003), and electrophysiologically (Wang et al.,

2004b) plays a crucial role in the hypotensive response elicited by clonidine. The dependence of

clonidine-evoked hypotension on central neurotransmitters/neuromodulators that directly or

indirectly influence the activity of sympathetic neurons in the RVLM has enhanced our knowledge

of the pharmacology of this class of drugs as well as central control of blood pressure (Wang et al.,

2003; Wang et al., 2004a).

It is imperative to note, however, that the expression of the hypotensive effect of clonidine is

dependent on the preparation used. Whereas clonidine-evoked hypotension manifests in

hypertensive rats, conscious or anesthetized (Jastrzebski et al., 1995; Estato et al., 2004), it only

manifests in anesthetized, but not conscious, normotensive rats (El-Mas et al., 1994; Yang et al.,

2005). Reported studies including ours have shown that partial or complete baroreceptor denervation

unmasks the hypotensive effect of clonidine in conscious normotensive rats (Abdel-Rahman, 1992;

El-Mas and Abdel-Rahman, 1997; Medvedev et al., 1998). Upregulation of the I1- and α2-adrenergic

receptors in the brainstem of aortic barodenervated rats has been reported in previous studies from

our laboratory (El-Mas and Abdel-Rahman, 1995; El-Mas and Abdel-Rahman, 1997). However, the

mechanisms that underlie the enhancement of clonidine evoked hypotension in this model are not

fully understood. We focused on central adenosine signaling as a key contributor to this

phenomenon for the following reasons. First, the aortic barodenervated rat bears some resemblance


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to the SHR because both exhibit baroreflex dysfunction (Judy and Farrell, 1979; Abdel-Rahman,

1992; El-Mas and Abdel-Rahman, 1995), and enhanced hypotensive responsiveness to clonidine

when used in the conscious state compared with intact normotensive rats (Abdel-Rahman, 1992; El-

Mas and Abdel-Rahman, 1995). Second, although not investigated in ABD rats, centrally mediated

hypotensive response elicited by adenosine is substantially enhanced in the SHR compared with

WKY rats (Abdel-Rahman and Tao, 1996). Third, clonidine and adenosine seem to trigger a similar

signaling pathway. For example, nitric oxide (NO) is implicated in the hypotensive effects of

clonidine and moxonidine (Soares de Moura et al., 2000; Moreira et al., 2004) as well as in the

hypotension elicited by adenosine injection into the NTS (Lo et al., 1998). Notably, clonidine

increases the release of glutamate, which subsequently enhances the release of neuronal adenosine in

the brainstem (Tingley and Arneric, 1990; Iliff et al., 2003; Paes-de-Carvalho et al., 2005). Finally,

activation of adenosine A2A and A2B receptors, in the RVLM, leads to a reduction in blood pressure

(Scislo et al., 2001; Harden et al., 2002).

In the present study, we tested the hypothesis that the central adenosine signaling plays a key

role in mediating the hypotensive effects of clonidine. Conscious aortic barodenervated (ABD) rats

used in these studies, received clonidine in the absence and presence of pharmacological

interventions that inhibited or enhanced central adenosine signaling. Since the findings with

clonidine (mixed I1/α2A agonist) supported the tested hypothesis, we investigated whether adenosine

signaling is linked to the I1 or the α2A receptor by investigating the effect of central adenosine

receptor blockade on the hypotension elicited by selective I1 (rilmenidine) or α2A AR (α-MNE)

agonist. Finally, we sought direct evidence for the involvement of the adenosine A2A receptor by

investigating the effect of the selective A2A antagonist SCH58261 on clonidine-evoked hypotension.


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Methods

Animals: A total of 97 male Sprague-Dawley rats (12-13 weeks) obtained from Harlan Farms

(Indianapolis, IN) weighing 300 ± 20 g were used in the present study. All rats were housed in a

room with controlled environment at a constant temperature of 23 ± 1ºC, humidity of 50 ± 10%, and

a 12 h light/dark cycle. Food (Prolab RMH300, Granville Milling, Creedmoor, NC) and water were

available ad libitum. Surgical procedures and post-operative care were performed in accordance

with, and approved by, the Institutional Animal Care and Use Guidelines.

Aortic barodenervation, intracisternal cannulation and intravascular catheterization: These

surgical procedures were performed as in our previous studies (El-Mas and Abdel-Rahman, 1995;

El-Mas and Abdel-Rahman, 1997). Briefly, 5 days before starting the experiment, a stainless steel

guide cannula was implanted into the cisterna magna under pentobarbital sodium anesthesia

(60 mg/kg, i.p.). The head was placed in a David Kopf stereotaxic frame. The dura matter covering

the foramen magnum was exposed. A hole was drilled 1-1.5 mm distal to the caudal edge of the

occipital bone (Joh et al., 2005). A steel cannula (23G; Small Parts, Miami, FL) was passed between

the occipital bone and the cerebellum so that its tip protruded into the cisterna magna. The cannula

was secured in place with small metal screws and dental acrylic cement (Durelon; Thompson Dental

Supply, Raleigh, NC). The guide cannula was considered patent when spontaneous outflow of

cerebrospinal fluid was observed and by gross post-mortem histological verification after injection of

5 µl of fast green dye (EM Science, Cherry Hill, NJ). Catheters (polyethylene 50) were placed in the

abdominal aorta and vena cava via the femoral artery and vein for measurement of blood pressure

and intravenous injections, respectively. The catheters were advanced 5 cm into the femoral vessels

and secured with sutures.


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Barodenervation was accomplished by bilateral transaction of the aortic depressor nerves

following a midline incision in the cervical region as in our previous studies (Abdel-Rahman, 1992;

El-Mas and Abdel-Rahman, 1995; El-Mas and Abdel-Rahman, 1997). Finally, the catheters were

tunneled subcutaneously and exteriorized at the back of the neck between the scapulae. The catheters

were flushed with heparin in saline (200 U/ml) and plugged by stainless steel pins. Incisions were

closed by surgical staples and swabbed with povidone-iodine solution. Each rat received an

intramuscular injection of 60,000 U of penicillin G benzathine and penicillin G procaine in aqueous

suspension (Durapen) and a subcutaneous injection of buprnorphine hydrochloride (Buprenex, 30

µg/kg) and was housed in a separate cage. The arterial catheter was connected to a Gould-Statham

pressure transducer (Oxnard, CA) and BP was displayed on a polygraph (model 7D; Grass

Instrument Co., Quincy, MA). Heart rate was computed from blood pressure waveforms by a Grass

tachograph and is displayed on another channel of the polygraph.

Protocols and Experimental Groups

Effect of systemic adenosine receptor or uptake blockade on clonidine evoked hypotension. A

total of 32 animals were used in this part of the study. On the day of the experiment, the arterial

catheter was connected to a pressure transducer for measurement of blood pressure (BP) and heart

rate (HR). A period of 30 min was allowed at the beginning of the experiment for stabilization of BP

and HR at baseline. Five groups (n=5-13) of conscious ABD rats received clonidine (10 µg/kg, i.v.)

30 min after systemic (i.v.): (1) lipid soluble (theophylline, 10 mg/kg), (2) or the water soluble [8-(p-

sulfophenyl) theophylline, 8-SPT, 2.5 mg/kg) nonselective adenosine receptor blocker or (3) equal

volume of saline. Groups 4 and 5 received the same dose of clonidine 1 hr after dipyridamole (5

mg/kg, i.p.) or equal volume of sesame oil. Changes in BP, HR were followed for additional 45 min

after clonidine or vehicle injection.


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Effect of central adenosine receptor or uptake blockade on centrally mediated hypotension. A

total of 56 animals were used in this part of the study. On the day of the experiment, the arterial

catheter was connected to a pressure transducer for measurement of BP and HR as mentioned above.

Intracisternal (i.c.) injections were made in unrestrained rats through a 30 gauge stainless steel

injector, which extended 2.0 mm beyond the tip of the previously implanted guide cannula so that

injections were made into the cisterna magnum as in our reported studies (El-Mas and Abdel-

Rahman, 1995). The injector was connected via PE 10 to a Hamilton microsyringe. A period of at

least 30 min was allowed at the beginning of the experiment for stabilization of blood pressure and

heart rate. To avoid potential problems as a result of multiple drug injections and removal of the

injector, drugs or vehicles were separated with small air bubble. Each injected volume did not

exceed 4 µl delivered by hand over a period of 1 min as in reported studies (Zhang and Abdel-

Rahman, 2002). Blood pressure and heart rate were recorded continuously. Four groups (n=4-6)

received clonidine (0.6 µg, i.c.) 30 min after i.c. injection of: (1) aCSF, (2) 8-SPT (10 µg), (3)

dipyridamole (150 µg) or (4) DMSO. Groups 5-8 served as controls as follows: group 5 received

only aCSF; group 6 received 8-SPT followed by aCSF; Group 7 received only DMSO and group 8

received dipyridamole followed by aCSF. To delineate whether adenosine mediates the I1 or the α2A

evoked-hypotension, additional groups of ABD rats received the selective I1 agonist rilmenidine (25

µg, i.c.) or α2 agonist α-methylnorepinephrine (α-MNE, 4 µg, i.c.) 30 min after central (i.c.)

adenosine receptor blockade (8-SPT, 10 µg) or aCSF (n=6-8 each).

Effect of central adenosine A2A receptor blockade on clonidine-evoked hypotension:

Conscious ABD rats received i.c. clonidine (0.6 µg, n=5) or equal volume of DMSO (n=4) 30 min

following adenosine A2A receptor blockade (SCH58261; 150 µg, i.c.).


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Drugs. Clonidine hydrochloride, dipyridamole, 8-(p-Sulfophenyl)-theophylline (8-SPT),

theophylline, (-)-α-methylnorepinephrine (α-MNE), dimethysulfoxide (DMSO), pentobarbital

sodium, and SCH58261 [5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3- ]-1,2,4-triazolo[1,5-

c]pyrimidine] were purchased from Sigma-Aldrich (St. Louis, MO). Rilmenidine was generously

provided by Technologie Servier (Neuilly Sur Seine, France). aCSF had the following composition:

123 mM NaCl, 0.86 mM CaCl2, 3 mM KCl, 0.89 mM MgCl2, 25 mM NaHCO3, 0.5 mM NaH2PO4,

and 0.25 mM Na2HPO4, pH 7.4.

Statistical analysis. Mean arterial pressure (MAP) was calculated as: diastolic pressure + one third

(systolic pressure - diastolic pressure). Mean arterial pressure and heart rate are expressed as mean ±

SE change from their respective baseline. The time-course data were analyzed by repeated measures

ANOVA using SPSS 13.0 statistical package for windows, for differences in time and treatment

trends followed by a one way ANOVA to assess individual differences at different time points

among different groups. Tukey’s (equal variance) and Games Howell (unequal variance) tests were

used for post hoc analysis. Contrasts based on the t-test and the ANOVA error terms were used to

compare pre-treatment-to-post-treatment values in each group. The pre- to post- treatment

comparisons were made through the use of contrasts that compared the pre- to post-treatment values

in each group. The contrasts essentially averaged the pre-treatment values and compared them to the

averaged post-treatment values using the repeated measures ANOVA error term. These contrasts

examined whether there were drug-evoked changes from baseline. A value of P<

0.05 was considered significant.


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Results

Baseline MAP and HR, were not significantly different in all groups of rats employed in the study

(Table1). Further, none of the pretreatments caused any significant change in the blood pressure or

heart rate at the time of clonidine administration except for a slight increase in MAP and HR caused

by theophylline (Fig. 1A). In the groups that received aCSF or the 8-SPT pretreatment followed by

rilmenidine or α-MNE, the baseline MAP and HR values were not significantly different from the

corresponding baseline values of the other groups shown in Table 1.

Effect of systemic adenosine receptor or adenosine uptake blockade on clonidine evoked

hypotension. Changes in MAP and HR evoked by systemic clonidine following pretreatment with

theophylline, 8-(p-sulfophenyl) theophylline (8-SPT) or saline in freely moving conscious ABD rats

are shown in Fig.1. Clonidine (10 µg/kg, i.v.) lowered MAP within 2-3 min in control (saline

pretreated) animals reaching a maximum (-23±2.2) at approx 15 min (Fig. 1A). The hypotension was

accompanied by bradycardia reaching a maximum (-48±13 beats/min) at 15 min (Fig. 1B). Blockade

of adenosine receptors by the non-selective lipid soluble adenosine blocker theophylline (10 mg/kg,

i.v.), which caused a slight increase in BP and HR (Fig. 1A), abolished clonidine evoked

hypotension (Fig. 1A). On the other hand, 8-SPT (2.5 mg/kg, i.v.) had no significant effect on

clonidine evoked reductions in blood pressure or heart rate (Fig. 1A, 1B). Conversely, pretreatment

with dipyridamole, an adenosine uptake blocker, in sesame oil (5 mg/kg, i.p.), substantially

enhanced the reduction in heart rate elicited by clonidine (Fig. 1D). Compared with saline, the

vehicle, sesame oil, had no effect on clonidine evoked hemodynamic responses (data not shown).

Repeated measures ANOVA error term showed that post-treatment was significantly (p<0.05)

different from pretreatment in the groups that received clonidine following saline or 8-SPT but not

following theophylline.


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Effect of intracisternal 8 (p-sulfophenyl) theophylline pretreatment or dipyridamole

pretreatment on clonidine mediated hypotension. In the control groups that received only aCSF

or DMSO, no change occurred in blood pressure or heart rate over the observation period. Therefore,

the data from both vehicle groups were pooled for comparison with other drug pretreatments (Figs.

2C& D). Pretreatment with 8-SPT (10 µg, i.c.) virtually abolished the hypotensive and bradycardic

responses elicited by i.c. clonidine (0.6 µg; Figs. 2A, B). Figs. 2C, D show the effect of intracisternal

pretreatment with dipyridamole (150 µg) on clonidine evoked reductions in MAP and HR.

Compared with the control (vehicle), dipyridamole significantly (p<0.05) enhanced the magnitude

and the duration of clonidine (0.6 µg; i.c.) evoked hypotension. Maximal reductions in MAP caused

by i.c. clonidine were (-10.3±3) and (-19.3±1.8) mmHg in the absence and presence of dipyridamole,

respectively. Repeated measures ANOVA error term showed that post-treatment was significantly

(p<0.05) different from pretreatment in the groups that received clonidine following aCSF, DMSO

and dipyridamole but not following 8-SPT.

Effect of intracisternal 8 (p-sulfophenyl) theophylline pretreatment on rilmenidine or α-MNE

evoked hypotension. Fig. 3 shows the changes in blood pressure and heart rate in conscious ABD

rats following i.c. administration of the selective α2 agonist α-methylnorepinephrine (α-MNE, 4 µg

Figs. 3A, B) or I1 agonist rilmenidine (25 µg, Figs. 3C, D) 30 min after central (i.c.) adenosine

receptor blockade (8-SPT, 10 µg) or aCSF (n=6-8). The hypotensive response elicited by rilmenidine

(-24 ±5 mmHg) or α-MNE (-16±3 mmHg) in aCSF pretreated animals was virtually abolished in 8-

SPT pretreated rats. Repeated measures ANOVA error term showed that post-treatment was

significantly different from pretreatment in the group that received α-MNE or rilmenidine following

aCSF but not 8-SPT.


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Effect of intracisternal SCH58261 pretreatment on clonidine evoked hypotension

Fig. 4 depicts the effect of central adenosine A2A receptor blockade (SCH58261; 150 µg, i.c) on the

blood pressure and heart rate responses elicited by clonidine (0.6 µg; i.c.; n=5) in conscious ABD

rats. Compared with DMSO (re-plotted from Fig. 2), pretreatment with SCH58261 virtually

abolished the hypotensive response elicited by clonidine (Figs. 4A). Administration of the adenosine

A2A antagonist SCH58261 alone (n=4) had no effect on blood pressure or heart rate (Fig. 4).

Repeated measures ANOVA error term showed that post-treatment was significantly different from

pretreatment in the group that received clonidine following DMSO but not SCH58261.


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Discussion

The present study demonstrates, for the first time, a key role for central adenosine signaling

in the hypotensive effect of centrally acting hypotensive agents in conscious ABD rats. The use of

the ABD rat circumvented the need for anesthesia; the latter unmasks the hypotensive response

elicited by centrally acting drugs in normotensive rats but may confound data interpretation (El-Mas

et al., 1994; Yang et al., 2005). Sham operated normotensive rats were not included in the present

study because centrally acting drugs produce little or no reduction in blood pressure in these animals

in the conscious state (El-Mas et al., 1994). The most important findings of the present study,

undertaken in conscious ABD rats to circumvent the potential confounding effects of anesthetics,

are: (i) systemic pretreatment with theophylline, a lipid soluble nonselective adenosine receptor

antagonist, abolished the hypotension elicited by clonidine; (ii) systemic pretreatment with the water

soluble analogue 8-p-sulphophenyl-theophylline (8-SPT) failed to attenuate clonidine evoked

hypotension. It is noteworthy that the systemic dose of 8-SPT was equieffective to that of

theophylline based on previous work from our laboratory (Tao and Abdel-Rahman, 1993), (iii)

dipyridamole, an adenosine uptake blocker, enhanced the hemodynamic effects of clonidine,

particularly following their central (i.c.) administration, and (iv) central blockade of adenosine

receptors by i.c. 8-SPT virtually abolished the hemodynamic responses elicited by i.c. administration

of the mixed I1/α2A agonist clonidine, the selective I1 agonist rilmenidine and the selective α2A

agonist α-MNE. Finally selective blockade of the central adenosine A2A receptor virtually abolished

clonidine-evoked hypotension. Together, these findings suggest the dependence of centrally evoked

hypotension on central adenosine A2A receptor signaling in conscious aortic barodenervated rats.

Results of the present study showed, for the first time, that in the ABD rat theophylline

virtually abolished the hypotensive effect of clonidine following systemic administration of both


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drugs. This finding inferred that the interaction between the two drugs occurred within the CNS

because clonidine lowers blood pressure via a central mechanism of action (van Zwieten, 2000) and

theophylline gains access to the CNS to block central adenosine receptor (Abdel-Rahman and Tao,

1996). It is also plausible to consider the possibility that opposite effects of theophylline and

clonidine on central cAMP contributed to the attenuation of the hypotensive effect of clonidine

caused by theophylline for the following reasons. Theophylline, being a PDE inhibitor, is expected

to increase cAMP (Somerville, 2001). By contrast, clonidine lowers blood pressure and heart rate by

activating central Gαi coupled α2A receptor causing a reduction in cAMP (Piascik et al., 1996).

Although we used a dose (10 mg/kg) of theophylline known to block central adenosine receptors

(Tao and Abdel-Rahman, 1993) and is much less than the reported doses (25-80 mg/kg) that inhibit

PDE (Abdollahi and Simaiee, 2003), the possibility still exists that PDE inhibition contributed to

theophylline action in the present study; no reported study has ruled out a PDE inhibitory action for

theophylline at the 10 mg/kg dose level. It is imperative to note, however, that 8-SPT, which blocks

adenosine receptors and is devoid of PDE inhibitory activity (Rabe et al., 1995) attenuated the

hypotensive effect of clonidine following central administration of both drugs. Intracisternal

administration of 8-SPT along with clonidine was adopted in the present study to confirm the

involvement of central adenosine signaling in the hypotensive response elicited by clonidine. The

inability of systemic 8-SPT in a dose that blocks peripheral (Tao and Abdel-Rahman, 1993; Abdel-

Rahman and Tao, 1996), but not central, adenosine receptors to influence clonidine-evoked

hypotension bolsters the conclusion that central but not peripheral adenosine receptors are implicated

in clonidine-evoked hypotension. Further, central administration of SCH58261, a selective A2A

receptor blocker (Ongini et al., 1999; Beukers et al., 2000), virtually abolished the clonidine-evoked


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hypotension. Together, the findings suggest the dependence of clonidine evoked hypotension on

central adenosine A2A receptor.

Based on the findings with adenosine receptor antagonists, we reasoned that increased

availability of adenosine at its receptors would enhance the hypotensive effect of clonidine.

Dipyridamole, used in a dose that inhibits adenosine uptake in vivo (da Silva Torres et al., 2003), did

not produce the expected enhancement of clonidine evoked hypotension. It was noticeable; however,

that the duration of the hypotensive action of clonidine was somewhat longer in dipyridamole treated

rats. More importantly, the bradycardic effect of clonidine was substantially enhanced by

dipyridamole. The latter, administered systemically, may have synergistically enhanced the

bradycardic effect of clonidine by increasing the availability of adenosine at the receptor site in the

heart, which is expected to reduce myocardial function (Usta et al., 2001). It is likely that these

synergistically enhanced cardiac depressant effects in dipyridamole-clonidine treated rats

contributed to the noticeable motor incoordination (data not shown). Accordingly, in a second set of

experiments, we circumvented the confounding peripheral effects of dipyridamole by directly

injecting the drug into the cisterna magna. As there are no reports on the use of i.c. dipyridamole, the

dose chosen was based on the rule that the dose administered by this route constitutes 1/10 to 1/20

the systemic dose (Catelli et al., 2003). We report, for the first time, that i.c. pretreatment with

dipyridamole significantly enhanced clonidine-evoked hypotension in ABD rats without the

confounding peripheral effects. This finding bolsters the conclusion that central adenosine receptor

signaling contributes, at least partly, to the hemodynamic effects of clonidine.

Although, clonidine is classified by Bousquet as an agonist at the imidazoline binding sites,

it is still considered a mixed I1/α2A agonist (Bousquet et al., 2003; Estato et al., 2004). Therefore, it

was difficult to ascertain the receptor, I1 or α2A, whose activation triggers central adenosine signaling.


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To address this issue, we investigated the effect of central adenosine receptor blockade (i.c. 8-SPT)

on the hypotensive response elicited by the selective activation of central I1 (rilmenidine) or α2A (α-

MNE) receptor. Interestingly, 8-SPT pretreatment attenuated the hypotensive response elicited by

both drugs. It is imperative to note that although α-MNE is considered “pure” α2A receptor agonist

(Szabo, 2002), the selective I1 agonist rilmenidine also exhibits α2A agonist activity (Szabo et al.,

1999; Estato et al., 2004). Together, these findings raise the interesting possibility that the α2A

receptor activation triggers central adenosine signaling. However, an alternative explanation is that I1

activation by rilmenidine might depend on a downstream α2A receptor activation as proposed by

Head (Head, 1999). The findings suggest that the adenosine system plays a critical role in mediating

centrally-evoked hypotension. However, because both theophylline and 8-SPT are non-selective

adenosine receptor blockers, we were unable to ascertain the adenosine receptor subtype implicated

in the mediation of clonidine-evoked hypotension. We reasoned that the A2A receptor would be a

viable candidate because its activation within the brainstem leads hypotension (Scislo et al., 2001).

SCH58621 was preferred over ZM241385 to block the A2A receptor because it is devoid of A2B

antagonist activity (Ongini et al., 1999; Beukers et al., 2000). As there are no reports on the use of

i.c. SCH58261, we adopted the criterion that the i.c. dose is equivalent to 1/10 to 1/20 the systemic

dose (Carta et al., 2003; Catelli et al., 2003). We report, for the first time, that i.c. pretreatment with

SCH58261 virtually abolished clonidine-evoked hypotension; a finding that implicates the A2A

receptor as a mediator of clonidine-evoked hypotension in the ABD rat.

We present evidence that central adenosine signaling is implicated in the hypotension evoked

by the mixed I1/α2A clonidine as well as the selective I1 and α2A agonists rilmenidine and α-MNE,

respectively, in conscious ABD rats. This new evidence is based on the use of pharmacological

interventions that enhanced or reduced adenosine signaling in the CNS. The findings with the


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selective A2A antagonist suggest the involvement of central A2A adenosine receptors in mediating the

hypotensive action of clonidine. However, the present findings pertain to the aortic barodenervated

rat and the possibility cannot be ruled out that in non-denervated intact rats the adenosine A1

receptor, which mediates pressor response (Scislo and O'Leary, 2002), might serve to counteract

(mask) clonidine evoked hypotension.


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Acknowledgement:

The authors thank Dr Kevin O’Brien for his valuable assistance with the statistical analyses and Ms.

Kui Sun for her excellent technical assistance.


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Footnotes:

This study was supported by NIH grant 2R01 AA07839

Reprint requests to: Abdel A. Abdel-Rahman, PhD, Department of Pharmacology and Toxicology,

School of Medicine, East Carolina University, Greenville, NC 27858.

E-mail: [email protected]


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Figure Legends

Fig. 1. Time course changes in mean arterial pressure (MAP) and heart rate (HR) following i.v.

clonidine (10 µg/kg) administration in conscious aortic barodenervated (ABD) rats. Panels A and B:

arrow marks clonidine administration 30 min after i.v.: saline, theophylline (10 mg/kg), or 8-SPT

(2.5 mg/kg). In panels C and D, dipyridamole (5 mg/kg) or its vehicle, sesame oil, was injected i.p.

60 min before clonidine; the data over the 30 min that preceded clonidine administration are shown

in the figure. Values are mean ± SEM. Number of rats in each group is shown in parenthesis.

*P<0.05 comparing the effects of theophylline to saline pretreatment and dipyridamole to sesame oil

on clonidine-evoked responses. Repeated measures ANOVA error term showed that post-treatment

was significantly different from pretreatment in the groups that received clonidine following saline

or 8-SPT but not following theophylline.

Fig. 2. Time course changes in mean arterial pressure (MAP) and heart rate (HR) following

intracisternal (i.c.) clonidine (0.6 µg) administration in conscious aortic barodenervated (ABD) rats.

Panels A and B: arrow marks clonidine or its vehicle, aCSF, administration 30 min following i.c.

aCSF or 8-SPT (10 µg). Panels C and D: arrow marks clonidine or its vehicle, aCSF, administration

30 min following i.c. dipyridamole (150 µg) or its vehicle DMSO. Values are mean ± SEM. Number

of rats in each group is shown in parenthesis. * P<0.05 comparing the effects of 8-SPT to aCSF and

dipyridamole to DMSO pretreatment on clonidine-evoked responses. Repeated measures ANOVA

error term showed that post-treatment was significantly different from pretreatment in the groups

that received clonidine following aCSF, DMSO and dipyridamole but not following 8-SPT.


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Fig. 3. Time course changes in mean arterial pressure (MAP) and heart rate (HR) following

intracisternal administration of α-methylnorepinephrine (α-MNE, 4 µg; panels A and B) or

rilmenidine (25 µg; panels C and D) in conscious aortic barodenervated (ABD) rats. Arrow marks

rilmenidine or α-MNE administration 30 min following i.c.: aCSF or 8-SPT (10 µg). Values are

mean ± SEM. Number of rats in each group is shown in parenthesis *P<0.05 comparing the effects

of 8-SPT to aCSF pretreatment on α-MNE and rilmenidine evoked responses. Repeated measures

ANOVA error term showed that post-treatment was significantly different from pretreatment in the

group that received α-MNE (left panels) or rilmenidine (right panels) following aCSF but not 8-SPT.

Fig. 4. Time course changes in mean arterial pressure (MAP, A) and heart rate (HR, B) following

intracisternal (i.c.) clonidine (0.6 µg) administration in conscious aortic barodenervated (ABD) rats

following SCH58261 (150 µg, i.c.). Arrow marks clonidine, or its vehicle aCSF, administration 30

min following i.c. DMSO (re-plotted from figure 2) or SCH58261. Values are mean ± SEM.

Number of rats in each group is shown in parenthesis. *P<0.05 significantly different compared to

DMSO pretreatment. Repeated measures ANOVA error term showed that post-treatment was

significantly different from pretreatment in the group that received clonidine following DMSO but

not SCH58261; the latter had no effect on the measured variables.


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Table 1. Baseline mean arterial pressure (MAP, mm Hg) and heart rate (HR, beats/min) values

obtained 30 min after stabilization and before the following pretreatments

Data presented as mean ± SEM *and # SCH58261 pretreatment before clonidine and aCSF, respectively.

Treatment

MAP

HR

Systemic (i.v.) Saline (n=13)

110.0±3.0

402±10

Theophylline (n=6)

112.5±9.0

428± 22

8SPT (n=8)

110.4±4.0

394±12

Dipyridamole (n=5)

103.3 ±5.0

402±14

Central (intracisternal aCSF (n=4)

109.2±5.0

395±16

DMSO (n=5) 102.3±3.0

390±10

Dipyridamole (n=5)

110.0 ±3.0 410±10

8-SPT (n=4) 107.1±6.0

395±21

SCH58261 (n-5)*

114.3±9.5 430±12

SCH58261 (n=4) #

101.0 ±3.0 344±26


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