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Journal of Pharmacology And Experimental Therapeutics Fast Forward
First published on April 4, 2006; DOI: 10.1124/jpet.105.100495

0022-3565/06/3181-255-261$20.00
JPET 318:255-261, 2006
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CARDIOVASCULAR

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, Brody School of Medicine, East Carolina University, Greenville, North Carolina

Received December 22, 2005; accepted March 31, 2006.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
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) (2.5 mg/kg) or 1 h after i.p. 4) dipyridamole (5 mg/kg) or 5) an 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. Because clonidine is a mixed I1/{alpha}2 agonist, we also investigated whether adenosine signaling is linked to the I1 or the {alpha}2A receptor by administering the selective I1 (rilmenidine, 25 µg) or {alpha}2A [{alpha}-methylnorepinephrine ({alpha}-MNE), 4 µg] agonist 30 min after central adenosine receptor blockade (8-SPT; 10 µg i.c.) or artificial cerebrospinal fluid. The hypotensive response elicited by rilmenidine or {alpha}-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 [5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-{epsilon}]-1,2,4-triazolo[1,5-c]-pyrimidine (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.

The mechanism of the centrally mediated hypotension elicited by clonidine and similar drugs is not fully understood. Reported studies have identified the brainstem and, in particular, the rostral ventrolateral medulla (RVLM), as the major neuroanatomical substrate for clonidine (Tingley and Arneric, 1990Go; Ernsberger and Haxhiu, 1997Go). Suppression of the activity of sympathetic neurons in the RVLM, assessed electrochemically (Mao et al., 2003Go) and electrophysiologically (Wang et al., 2004bGo), 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., 2003Go, 2004aGo).

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., 1995Go; Estato et al., 2004Go), it only manifests in anesthetized, but not conscious, normotensive rats (El-Mas et al., 1994Go; Yang et al., 2005Go). Reported studies including ours have shown that partial or complete baroreceptor denervation unmasks the hypotensive effect of clonidine in conscious normotensive rats (Abdel-Rahman, 1992Go; El-Mas and Abdel-Rahman, 1997Go; Medvedev et al., 1998Go). Up-regulation of the I1- and {alpha}2-adrenergic receptors in the brainstem of aortic barodenervated rats has been reported in previous studies from our laboratory (El-Mas and Abdel-Rahman, 1995Go, 1997Go). 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 to the spontaneously hypertensive rat because both exhibit baroreflex dysfunction (Judy and Farrell, 1979Go; Abdel-Rahman, 1992Go; El-Mas and Abdel-Rahman, 1995Go) and enhanced hypotensive responsiveness to clonidine when used in the conscious state compared with intact normotensive rats (Abdel-Rahman, 1992Go; El-Mas and Abdel-Rahman, 1995Go). Second, although not investigated in aortic barodenervated (ABD) rats, the centrally mediated hypotensive response elicited by adenosine is substantially enhanced in spontaneously hypertensive rats compared with Wistar-Kyoto rats (Abdel-Rahman and Tao, 1996Go). Third, clonidine and adenosine seem to trigger a similar signaling pathway. For example, nitric oxide is implicated in the hypotensive effects of clonidine and moxonidine (Soares de Moura et al., 2000Go; Moreira et al., 2004Go) as well as in the hypotension elicited by adenosine injection into the nucleus tractus solitarii (Lo et al., 1998Go). Notably, clonidine increases the release of glutamate, which subsequently enhances the release of neuronal adenosine in the brainstem (Tingley and Arneric, 1990Go; Iliff et al., 2003Go; Paes-de-Carvalho et al., 2005Go). Finally, activation of adenosine A2A and A2B receptors in the RVLM leads to a reduction in blood pressure (Scislo et al., 2001Go; Harden et al., 2002Go).

In the present study, we tested the hypothesis that central adenosine signaling plays a key role in mediating the hypotensive effects of clonidine. Conscious ABD rats used in these studies received clonidine in the absence and presence of pharmacological interventions that inhibited or enhanced central adenosine signaling. Because the findings with clonidine (mixed I1/{alpha}2A agonist) supported the tested hypothesis, we investigated whether adenosine signaling is linked to the I1 or the {alpha}2A receptor by investigating the effect of central adenosine receptor blockade on the hypotension elicited by selective I1 (rilmenidine) or {alpha}2A-adrenergic receptor ({alpha}-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.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
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 ± 1°C, 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 postoperative 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, 1995Go, 1997Go). In brief, 5 days before the experiment was started, 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 mater covering the foramen magnum was exposed. A hole was drilled 1 to 1.5 mm distal to the caudal edge of the occipital bone (Joh et al., 2005Go). A steel cannula (23 gauge; 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 postmortem 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 i.v. injections, respectively. The catheters were advanced 5 cm into the femoral vessels and secured with sutures.

Barodenervation was accomplished by bilateral transaction of the aortic depressor nerves after a midline incision in the cervical region as in our previous studies (Abdel-Rahman, 1992Go; El-Mas and Abdel-Rahman, 1995Go, 1997Go). 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 i.m. injection of 60,000 units of penicillin G benzathine and penicillin G procaine in aqueous suspension (Durapen) and a s.c. injection of buprenorphine 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 Instruments, Quincy, MA). Heart rate was computed from blood pressure waveforms by a Grass tachograph and was 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 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 water-soluble [8-(p-sulfophenyl)theophylline (8-SPT) 2.5 mg/kg] nonselective adenosine receptor blockers, or 3) an equal volume of saline. Groups 4 and 5 received the same dose of clonidine 1 h after dipyridamole (5 mg/kg i.p.) or an equal volume of sesame oil. Changes in BP and HR were followed for an additional 45 min after clonidine or vehicle injection.

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, 1995Go). The injector was connected via a PE-10 catheter 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 a 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, 2002Go). 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 to 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 I1- or {alpha}2A-evoked-hypotension, additional groups of ABD rats received the selective I1 agonist rilmenidine (25 µg i.c.) or the {alpha}2 agonist {alpha}-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 an equal volume of DMSO (n = 4) 30 min after adenosine A2A receptor blockade (SCH58261; 150 µg i.c.).

Drugs
Clonidine hydrochloride, dipyridamole, 8-SPT, theophylline, (-)-{alpha}-MNE, DMSO, pentobarbital sodium, and SCH58261 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 + 1/3 (systolic pressure - diastolic pressure). Mean arterial pressure and heart rate are expressed as mean ± S.E. change from their respective baselines. 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 pretreatment to post-treatment values in each group. The pretreatment to post-treatment comparisons were made through the use of contrasts that compared the pretreatment to post-treatment values in each group. The contrasts essentially averaged the pretreatment 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.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Baseline MAP and HR were not significantly different in all groups of rats used in the study (Table 1). Furthermore, 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 {alpha}-MNE, the baseline MAP and HR values were not significantly different from the corresponding baseline values of the other groups shown in Table 1.


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  		TABLE 1 Baseline MAP and HR values obtained 30 min after stabilization and before the following pretreatments Data are presented as means ± S.E.M.

 

Figure 1
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  		Fig. 1. Time course changes in MAP and HR after i.v. clonidine (10 µg/kg) administration in conscious ABD rats. 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 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 means ± S.E.M. The number of rats in each group is shown in parentheses. *, P < 0.05, comparing the effects of theophylline to saline pretreatment and dipyridamole to sesame oil on clonidine-evoked responses. The repeated measures ANOVA error term showed that post-treatment was significantly different from pretreatment in the groups that received clonidine after saline or 8-SPT but not after theophylline.

 

Effect of Systemic Adenosine Receptor or Adenosine Uptake Blockade on Clonidine-Evoked Hypotension. Changes in MAP and HR evoked by systemic clonidine after pretreatment with 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 to 3 min in control (saline-pretreated) animals, reaching a maximum (-23 ± 2.2) at approximately 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 nonselective 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. 1, A and B). 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). The repeated measures ANOVA error term showed that posttreatment was significantly (P < 0.05) different from pretreatment in the groups that received clonidine after saline or 8-SPT but not after theophylline.

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 (Fig. 2, C and D). Pretreatment with 8-SPT (10 µg i.c.) virtually abolished the hypotensive and bradycardic responses elicited by i.c. clonidine (0.6 µg) (Fig. 2, A and B). Figure 2, C and D, shows 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) mm Hg in the absence and presence of dipyridamole, respectively. The repeated measures ANOVA error term showed that post-treatment was significantly (P < 0.05) different from pretreatment in the groups that received clonidine after aCSF, DMSO, and dipyridamole but not after 8-SPT.


Figure 2
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  		Fig. 2. Time course changes in MAP and HR after i.c. clonidine (0.6 µg) administration in conscious ABD rats. A and B, arrow marks clonidine or its vehicle, aCSF, administration 30 min after i.c. aCSF or 8-SPT (10 µg). C and D, arrow marks clonidine or its vehicle, aCSF, administration 30 min after i.c. dipyridamole (150 µg) or its vehicle DMSO. Values are means ± S.E.M. The number of rats in each group is shown in parentheses. *, P < 0.05, comparing the effects of 8-SPT to aCSF and dipyridamole to DMSO pretreatment on clonidine-evoked responses. The repeated measures ANOVA error term showed that post-treatment was significantly different from pretreatment in the groups that received clonidine after aCSF, DMSO, and dipyridamole but not after 8-SPT.

 
Effect of Intracisternal 8-(p-Sulfophenyl)theophylline Pretreatment on Rilmenidine- or {alpha}-MNE-Evoked Hypotension. Figure 3 shows the changes in blood pressure and heart rate in conscious ABD rats after i.c. administration of the selective {alpha}2 agonist {alpha}-MNE (4 µg) (Fig. 3, A and B) or the I1 agonist rilmenidine (25 µg) (Fig. 3, C and 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 mm Hg) or {alpha}-MNE (-16 ± 3 mm Hg) in aCSF-pretreated animals was virtually abolished in 8-SPT-pretreated rats. The repeated measures ANOVA error term showed that post-treatment was significantly different from pretreatment in the group that received {alpha}-MNE or rilmenidine after aCSF but not after 8-SPT.


Figure 3
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  		Fig. 3. Time course changes in MAP and HR after i.c. administration of {alpha}-MNE (4 µg) (A and B) or rilmenidine (25 µg) (C and D) in conscious ABD rats. The arrow marks rilmenidine or {alpha}-MNE administration 30 min after i.c. aCSF or 8-SPT (10 µg). Values are means ± S.E.M. The number of rats in each group is shown in parentheses *, P < 0.05, comparing the effects of 8-SPT to aCSF pretreatment on {alpha}-MNE- and rilmenidine-evoked responses. The repeated measures ANOVA error term showed that posttreatment was significantly different from pretreatment in the group that received {alpha}-MNE (left panels) or rilmenidine (right panels) after aCSF but not 8-SPT.

 

Effect of Intracisternal SCH58261 Pretreatment on Clonidine-Evoked Hypotension. Figure 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 (replotted from Fig. 2), pretreatment with SCH58261 virtually abolished the hypotensive response elicited by clonidine (Fig. 4A). Administration of the adenosine A2A antagonist SCH58261 alone (n = 4) had no effect on blood pressure or heart rate (Fig. 4). The repeated measures ANOVA error term showed that post-treatment was significantly different from pretreatment in the group that received clonidine after DMSO but not after SCH58261.


Figure 4
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  		Fig. 4. Time course changes in MAP (A) and HR (B) after i.c. clonidine (0.6 µg) administration in conscious ABD rats after SCH58261 (150 µg i.c.). The arrow marks administration of clonidine or its vehicle aCSF 30 min after i.c. DMSO (replotted from Fig. 2) or SCH58261. Values are means ± S.E.M. The number of rats in each group is shown in parentheses. *, P < 0.05, significantly different compared with DMSO pretreatment. The repeated measures ANOVA error term showed that posttreatment was significantly different from pretreatment in the group that received clonidine after DMSO but not SCH58261; the latter had no effect on the measured variables.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
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., 1994Go; Yang et al., 2005Go). 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., 1994Go). The most important findings of the present study, undertaken in conscious ABD rats to circumvent the potential confounding effects of anesthetics, are the following. 1) Systemic pretreatment with theophylline, a lipid-soluble nonselective adenosine receptor antagonist, abolished the hypotension elicited by clonidine. 2) Systemic pretreatment with the water-soluble analog 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, 1993Go); 3) Dipyridamole, an adenosine uptake blocker, enhanced the hemodynamic effects of clonidine, particularly after their central (i.c.) administration. 4) Central blockade of adenosine receptors by i.c. 8-SPT virtually abolished the hemodynamic responses elicited by i.c. administration of the mixed I1/{alpha}2A agonist clonidine, the selective I1 agonist Rilmenidine, and the selective {alpha}2A agonist {alpha}-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 after systemic administration of both 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, 2000Go) and theophylline gains access to the CNS to block central adenosine receptor (Abdel-Rahman and Tao, 1996Go). 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, 2001Go). By contrast, clonidine lowers blood pressure and heart rate by activating central G{alpha}i-coupled {alpha}2A receptors, causing a reduction in cAMP (Piascik et al., 1996Go). Although we used a dose (10 mg/kg) of theophylline that is known to block central adenosine receptors (Tao and Abdel-Rahman, 1993Go) and is much less than the reported doses (25-80 mg/kg) that inhibit PDE (Abdollahi and Simaiee, 2003Go), 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., 1995Go) attenuated the hypotensive effect of clonidine after 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, 1993Go; Abdel-Rahman and Tao, 1996Go), 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. Furthermore, central administration of SCH58261, a selective A2A receptor blocker (Ongini et al., 1999Go; Beukers et al., 2000Go), virtually abolished the clonidine-evoked hypotension. Together, the findings suggest the dependence of clonidine-evoked hypotension on central adenosine A2A receptors.

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., 2003Go), 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., 2001Go). 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., 2003Go). 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 imidazoline-binding sites, it is still considered a mixed I1/{alpha}2A agonist (Bousquet et al., 2003Go; Estato et al., 2004Go). Therefore, it was difficult to ascertain the receptor, I1 or {alpha}2A, whose activation triggers central adenosine signaling. 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 {alpha}2A ({alpha}-MNE) receptors. Interestingly, 8-SPT pretreatment attenuated the hypotensive response elicited by both drugs. It is imperative to note that although {alpha}-MNE is considered a "pure" {alpha}2A receptor agonist (Szabo, 2002Go), the selective I1 agonist rilmenidine also exhibits {alpha}2A agonist activity (Szabo et al., 1999Go; Estato et al., 2004Go). Together, these findings raise the interesting possibility that the {alpha}2A receptor activation triggers central adenosine signaling. However, an alternative explanation is that I1 activation by rilmenidine might depend on a downstream {alpha}2A receptor activation as proposed by Head (1999Go). The findings suggest that the adenosine system plays a critical role in mediating centrally evoked hypotension. However, because both theophylline and 8-SPT are nonselective 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 to hypotension (Scislo et al., 2001Go). SCH58621 was preferred over ZM241385 to block the A2A receptor because it is devoid of A2B antagonist activity (Ongini et al., 1999Go; Beukers et al., 2000Go). 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., 2003Go; Catelli et al., 2003Go). 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/{alpha}2A agonist clonidine as well as the selective I1 and {alpha}2A agonists rilmenidine and {alpha}-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 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 nondenervated intact rats the adenosine A1 receptor, which mediates pressor response (Scislo and O'Leary, 2002Go), might serve to counteract (mask) clonidine-evoked hypotension.


   Acknowledgements
 
We thank Dr. Kevin O'Brien for valuable assistance with the statistical analyses and Kui Sun for excellent technical assistance.


   Footnotes
 
This study was supported by National Institutes of Health Grant 2R01 AA07839.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.105.100495.

ABBREVIATIONS: RVLM, rostral ventrolateral medulla; ABD, aortic barodenervated rat; {alpha}-MNE, {alpha}-methylnorepinephrine; SCH58261, 5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-{epsilon}]-1,2,4-triazolo[1,5-c]pyrimidine; BP, blood pressure; HR, heart rate; 8-SPT, 8-(p-sulfophenyl)theophylline; i.c., intracisternal; aCSF, artificial cerebrospinal fluid; DMSO, dimethyl sulfoxide; MAP, mean arterial pressure; CNS, central nervous system; PDE, phosphodiesterase inhibitor; ZM241385, 4-{2-[7-amino-2-(2-furyl)[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-ylamino]ethyl}phenol.

Address correspondence to: Dr. Abdel A. Abdel-Rahman, Department of Pharmacology and Toxicology, School of Medicine, East Carolina University, Greenville, NC 27834. E-mail: abdelrahmana{at}ecu.edu


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
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