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