INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

FOR MANY YEARS IT WAS generally acknowledged that two adenosine-receptor subtypes (A₁ and A₂) were present in the mammalian myocardium. In 1992, however, Zhou et al. (31) cloned a new adenosine-receptor subtype, which they termed the A₃ receptor, that was resistant to blockade by alkylxanthine-type adenosine-receptor antagonists. Subsequently, sheep and human adenosine A₃ receptors were cloned (14, 22). Concurrent with these molecular studies, Fozard and Carruthers (6) reported that the hemodynamic effects of the adenosine analog N⁶-[2-(4-aminophenyl)ethyl]adenosine (APNEA) in the rat were not blocked by the adenosine- receptor blocker 8-(p-sulfophenyl)-theophylline (8-SPT).

All three of the initial cloning studies indicated only very weak expression of the adenosine A₃ receptor in rat, sheep, and human hearts compared with other tissues (14, 22, 31). To date there have been no studies on cardiac adenosine A₃-receptor density or on the physiological role that this receptor plays in normal mammalian ventricular myocardium. There have, however, been several studies in rabbit myocardium (3, 10, 27) and rabbit (1) and chick ventricular myocytes (25) indicating that the adenosine A₃ receptor may play a role in the cardioprotective effects of adenosine. These studies have used the relatively new, selective adenosine A₃ agonists N⁶-(3-iodobenzyl)-9-[5-(methylcarbamoyl)--D-ribofuranosyl]adenine (IB-MECA) and 2-chloro-N⁶-(3-iodobenzyl)-9-[5-(methylcarbamoyl)--D-ribofuranosyl]adenine (Cl-IB-MECA). It has been reported that IB-MECA has a very high affinity for both cloned rat (1 nM, Ref. 7) and rabbit A₃ receptors (2 nM, Ref. 9), and Cl-IB-MECA has an even higher affinity (~0.3 nM) for the rat A₃ receptor (13).

Although these agonists are relatively selective for A₃ receptors at low doses, these agents are typically used at higher doses in intact cells, in which their functional selectivity for specific adenosine-receptor subtypes has yet to be determined. For example, doses of IB-MECA (30 nM) used in myocardial ischemia studies in the rabbit bind to cloned rabbit adenosine A₁ receptors (9). In addition to species differences in cloned adenosine A₃-receptor sensitivity to blockade by xanthine antagonists, there also appear to be species differences in the functional role of A₃ receptors. In the rat, adenosine-induced mast cell degranulation appears to be mediated by the A₃ receptor (6, 8, 20, 29), whereas in the dog, mast cell degranulation is thought to be the result of A_2b-receptor activation (2). In terms of effects in the cardiovascular system, administration of APNEA, IB-MECA, and Cl-IB-MECA each increases plasma histamine levels and decreases systemic blood pressure (6, 8, 20, 29); however, IB-MECA had no such effect (3) in the only study to date in rabbit. There have been no studies on the effects of these adenosine A₃-receptor agonists in intact animals larger than the rabbit. Although isolated rabbit heart studies report no effects of IB-MECA on ventricular function or coronary flow in the normal heart (10, 27), there have been no studies with these agents in the rat heart. Therefore, the purpose of this study was to compare the hemodynamic effects of IB-MECA and Cl-IB-MECA in nonischemic, isolated perfused rat and rabbit hearts and in an in situ porcine preparation.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

All animals in this study received humane care according to the guidelines set forth by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and also by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication No. 86-23, Revised 1996). In addition, animals were used in accordance with the guidelines of the University of Kentucky Institutional Animal Care and Use Committee.

Isolated perfused heart preparation. Experiments were conducted on male Sprague-Dawley rats (300-350 g) and New Zealand White rabbits of either sex (2-2.5 kg). Rats and rabbits were anesthetized with pentobarbital sodium (70 mg/kg ip), and they were then administered heparin (500 U/kg iv). The hearts were rapidly excised and immediately placed into ice-cold Krebs-Henseleit buffer to produce cardiac arrest. After cannulation of the aorta, hearts were perfused at a constant pressure of 70 mmHg with Krebs-Henseleit buffer composed of (in mM) 118 NaCl, 4.7 KCl, 1.2 MgSO₄, 1.2 KH₂PO₄, 1.5 CaCl₂, 25.0 NaHCO₃, and 11.0 glucose, 10 µM EDTA, and 1 µM ascorbic acid. The perfusate was maintained at 37°C in a constant temperature reservoir and was bubbled with 95% O₂-5% CO₂ resulting in pH 7.35-7.40, PCO₂ 36-40 mmHg, and PO₂ 560-620 mmHg. Myocardial temperature was maintained at 37°C by partially submersing the heart into a water-jacketed chamber filled with Krebs buffer draining from the pulmonary artery.

In situ pig preparation. Domestic pigs of either sex weighing 22-27 kg were premedicated with ketamine (30 mg/kg im) and anesthetized with pentobarbital sodium (20 mg/kg iv). Anesthesia was maintained with pentobarbital sodium (1.5-2 mg/kg iv) every 15 min. A tracheostomy was performed, and the animals were mechanically ventilated with a mixture of room air and 100% O₂. Tidal volume, respiratory rate, and percent oxygen in the inspired air were adjusted to maintain normal arterial blood gas and pH values. Core body temperature was monitored with an esophageal temperature probe and maintained at 37.0-37.5°C with a heating pad. The left femoral artery was cannulated to obtain samples for arterial blood gas analysis and for monitoring blood pressure. The left femoral vein was cannulated for the infusion of fluids and anesthesia. Lactated Ringer solution was administered via an ear vein or femoral vein at 5-7 ml · kg¹ · min¹ after an initial bolus of 300-400 ml.

Ventricular function. Regional ventricular function was assessed by myocardial segment shortening. Piezoelectric segment shortening crystals (Crystal Biotech, Houston, TX) were placed in the LAD perfused bed to measure regional segment shortening via sonomicrometry. Crystals were placed in the midmyocardium (~4-6 mm from the epicardium) between 5 and 15 mm apart and aligned such that the intercrystal axis was parallel to the direction of myocardial fiber shortening. End diastole was defined as the onset of +dP/dt, and end systole was defined as 20 ms before peak dP/dt. Systolic shortening was defined as end-diastolic length (EDL)  end-systolic length (ESL), and percent segment shortening (%SS) was calculated as (EDL  ESL/EDL) × 100. All hemodynamic and sonomicrometric signals were fed through a 32-bit analog-to-digital converter directly into an online data acquisition computer with customized software (Coyote Bay Instruments, Manchester, NH). These signals were continuously displayed on a computer monitor.

Isolated heart protocols. Rabbit and rat hearts (n  5 except where noted) were allowed a 20-min equilibration period after placement of the left ventricular balloon before any intervention. Separate groups of isolated rabbit hearts were submitted to 2-min infusions of 50 nM IB-MECA and 50 nM Cl-IB-MECA. Isolated perfused rat hearts were studied in the following groups: 1) multiple IB-MECA infusions; 2) multiple infusions of the adenosine A_2a-receptor agonist 2-[p-(2-carboxyethyl)-phenylethylamino]-5'-N-ethylcarboxyamidoadenosine (CGS-21680); 3) IB-MECA + the adenosine A₃-receptor antagonist 3-ethyl-5-benzyl-2-methyl-6-phenyl-4-phenylethynyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (MRS-1191); 4) CGS-21680 + MRS-1191 (n = 3); 5) IB-MECA + the selective adenosine A_2a-receptor antagonist 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1, 5-c]pyrimidine (Sch-58261); and 6) CGS-21680 + Sch-58261.

In situ protocols. Animals were allowed a period of 30 min for recovery and stabilization after all instrumentation was completed before the experimental protocol was initiated. Six pigs received IB-MECA treatment. The first pig was administered two doses of IB-MECA (12.5 µg/kg). The first treatment was a slow infusion (over 5 min) into the left atrium followed by a 20-min recovery period before a second treatment was administered intravenously. The subsequent five pigs were given two intravenous boluses (5 µg/kg). Three additional pigs were treated with Cl-IB-MECA; one pig was treated with 10 µg/kg iv, and the other two pigs were treated with 25 µg/kg iv.

Drugs and chemicals. IB-MECA, 8-(3-chlorostyryl)caffeine (CSC), MRS-1191, and CGS-21680 were obtained from Research Biochemicals International (RBI, Natick, MA). Sch-58261 was a gift from Schering-Plough Research Institute (Milan, Italy). Cl-IB-MECA was provided by RBI as part of the Chemical Synthesis Program of the National Institute of Mental Health (contract N0IMH-30003). With the exception of CGS-21680 that was dissolved in distilled water (with heating), all drugs were solubilized in DMSO. IB-MECA, Cl-IB-MECA, CGS-21680, MRS-1191, and Sch-58261 were all made as 1 mM stock solutions. The concentration of the vehicle (DMSO) that the hearts were exposed to (0.01%) had no effect on coronary flow or ventricular function.

Data analysis. Results are means ± SE. The effects of adenosine agonists and antagonists on coronary flow are expressed as absolute values and as a percentage of change from immediate pretreatment values. For the isolated heart studies differences within groups were determined by repeated-measures ANOVA followed by Dunnett's t-test for comparison to baseline values or Newman-Keuls multiple- comparison test. IB-MECA results in the pig were analyzed by repeated-measures ANOVA and Dunnett's t-test. A P value < 0.05 was considered statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Isolated heart experiments. The results of the initial studies with IB-MECA (50 nM) in isolated rat and rabbit hearts are shown in Fig. 1. In isolated rabbit hearts, IB-MECA exerted no effect on coronary flow, but in rat hearts IB-MECA increased coronary flow from 13.3 ± 0.9 to 16.5 ± 0.8 ml/min, a 25% increase, which rapidly returned to control values at the end of the infusion. In neither rabbit nor rat myocardium did IB-MECA have any effect on heart rate or LVDP, and LVDP values remained stable throughout all of the experimental protocols (data not shown). The next series of experiments was performed to determine whether the IB-MECA-induced increase in coronary flow exhibited tachyphylaxis, an effect known to occur with repeated adenosine A₃-receptor activation (17, 29). Figure 2A illustrates that the effects of multiple IB-MECA infusions in the same heart are reversible and reproducible. The three 2-min IB-MECA infusions increased coronary flow 25 ± 5, 24 ± 3, and 20 ± 3%, respectively. These results are qualitatively similar to those produced by the adenosine A_2a-receptor agonist CGS-21680 in another group of hearts (Fig. 2B). Although the magnitude of the CGS-21680-induced coronary flow increase was much greater (73 ± 12, 83 ± 14, and 70 ± 16% increases in the 3 exposures), similar reversible and reproducible effects were observed.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Effects of adenosine A3-receptor agonist IB-MECA (IBM) on coronary flow (CF) in isolated rat and rabbit hearts. IBM (50 nM) was infused for 2 min in hearts perfused at 70 mmHg. * P < 0.05 vs. baseline (base) CF values.

View larger version (38K): [in this window] [in a new window]   Fig. 2.   Reproducibility and reversibility of adenosine A3-receptor agonist IBM (A) and adenosine A2a-receptor agonist CGS-21680 (B) effects on CF in isolated rat hearts. IBM (50 nM) and CGS-21680 (5 nM) were infused for 2 min followed by a 12- to 15-min wash before the next exposure. * P < 0.05 vs. base and wash CF values.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Effects of the adenosine A3-receptor antagonist MRS-1191 (MRS) on IBM-induced increase in CF in isolated rat hearts. Hearts were treated with IBM (50 nM) as described for Figs. 1 and 2. Subsequent to the first IBM and wash period, hearts were pretreated for 5 min with MRS (50 nM) before second IB-MECA exposure in the continued presence of MRS-1191. * P< 0.05 vs. base, # P < 0.05 vs. MRS CF value.

View larger version (32K): [in this window] [in a new window]   Fig. 4.   Effects of adenosine A2a-receptor antagonist Sch-58261 on IB-MECA (A) and CGS-21680 (B) CF increases. Hearts were first treated with IBM or CGS-21680 as previously described. After a 12- to 15-min washout, hearts were pretreated with Sch-58261 (SCH, 50 nM) for 5 min before second exposure to IBM or CGS-21680. * P < 0.05 vs. base, # P < 0.05 vs. immediate wash CF value.

View larger version (27K): [in this window] [in a new window]   Fig. 5.   Representative experiments illustrating the antagonism of IB-MECA (IBM, 50 nM)-induced CF increases by MRS-1191 (A) and Sch-58261 (B). In contrast to the protocols used in Figs. 3 and 4, rat hearts in this protocol were first exposed to IB-MECA for 2 min then treated with a combination of IB-MECA + MRS-1191 (MRS, 50 nM) or SCH (50 nM).

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Effects of adenosine A3-receptor agonist Cl-IB-MECA on CF in isolated perfused rat hearts. Left, effect of 50 nM Cl-IB-MECA; right, effect of 100 nM Cl-IB-MECA. * P < 0.05 vs. base.

In situ pig experiments. Two of the four pigs treated with IB-MECA died within the first 1-3 min of the intravenous bolus. In the remaining four pigs IB-MECA decreased mean arterial pressure (MAP) from 90 ± 8 to 60 ± 6 mmHg (P < 0.05). Approximately 10 min later MAP recovered to 81 ± 6 mmHg. A subsequent treatment with the same dose of IB-MECA only marginally decreased MAP (73 ± 5 mmHg). Exposure to IB-MECA was associated with parallel decreases in LVDP, LV +dP/dt, and segment shortening. Baseline heart rates were 110 ± 11 beats/min and only exhibited a nonsignificant increase in the first 10 min after IB-MECA (134 ± 5 beats/min). Coronary blood flow decreased from 24.8 ± 1.4 to 5.8 ± 1.4 ml/min in the initial minute after IB-MECA infusion, and recovered completely within 10 min to 26.2 ± 4.9 ml/min. The second IB-MECA treatment had no significant effect on LAD blood flow (19.8 ± 1.6 to 14.3 ± 2.9 ml/min). The first IB-MECA infusion was accompanied by observable increases in right ventricular volume, and in the final two pigs pulmonary artery pressure was monitored. In these two animals mean pulmonary pressure increased from 14 and 10 mmHg to 43 and 33 mmHg, respectively, within 2-3 min after exposure to IB-MECA.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The results of the present study indicate that the adenosine A₃-receptor agonist IB-MECA, at a concentration as low as 50 nM, produces a 30% increase in coronary flow in the isolated rat heart. This effect did not exhibit tachyphylaxis and was only partially blunted by the adenosine A₃-receptor antagonist MRS-1191 (50 nM). In fact the IB-MECA-induced coronary vasodilatation was completely blocked by the adenosine A_2a-receptor blocker Sch-58261 (50 nM). The same concentration of Cl-IB-MECA did not alter coronary flow in the rat heart, but doubling the dose to 100 nM produced ~15% increase in coronary flow, a flow increase that was blocked by Sch-58261. Neither IB-MECA nor Cl-IB-MECA altered flow in the rabbit heart, and in neither species did either agonist alter ventricular function or heart rate. In the pig, both IB-MECA (5 µg/kg) and Cl-IB-MECA (25 µg/kg) transiently decreased systemic blood pressure and increased pulmonary artery pressure. These results indicate that the adenosine A₃-receptor agonists IB-MECA and Cl-IB-MECA can exert species-dependent effects in the cardiovascular system. In the normal rat heart these effects appear to be caused primarily by A_2a-receptor activation.

On the basis of both pharmacological and molecular biology studies, there is significant evidence of the existence of the adenosine A₃ receptor. Cloning studies indicate that whereas the A₃-receptor message is present in the heart, it is expressed at levels generally much lower than in tissues such as the lung, testes, and brain (14, 22, 31). However, neither cardiac adenosine A₃-receptor density nor the physiological effects of A₃-receptor activation have been determined. Studies of A₃-receptor agonist effects in the rat have been limited to the systemic effects of APNEA, IB-MECA, and Cl-IB-MECA, all of which produce histamine release and profound hypotension, presumably caused by activation of mast cell adenosine A₃ receptors (6, 8, 20, 29). In contrast to the results observed in rats, in the only study in the intact rabbit, IB-MECA did not exert any systemic effect and did not increase histamine levels (3). These differences in A₃-receptor agonist effects in the rat and rabbit could be the result of species differences in the adenosine-receptor subtype, which promotes mast cell degranulation. This hypothesis is supported by work of Auchampach et al. (2), which reports that canine mast cell degranulation is induced not by the adenosine A₃ receptor but by the A_2b receptor. The lack of studies in the rat heart, in intact animals larger than rats and rabbits, and in the above-noted species differences provided the basis for this study.

The results of the present study confirm the apparent species difference between rat and rabbit myocardium in terms of their responses to IB-MECA. In the isolated rat heart, IB-MECA (50 nM) produced a ~30% increase in coronary flow, whereas the same dose had no effect in the isolated rabbit heart. Preliminary studies indicated that 10 nM IB-MECA increased coronary flow in the rat by 10% (data not shown). The A₃-receptor agonist Cl-IB-MECA (50 nM) had no effect on coronary flow in the rat, but doubling the concentration to 100 nM was also associated with coronary vasodilation. Because adenosine A₃-receptor activation in the intact rat is associated with mast cell-induced histamine release and peripheral vascular dilatation (6, 8, 20, 29), and because histamine is a coronary dilator in the rat (27), the simplest explanation for the present findings in the rat heart is that IB-MECA is activating the adenosine A₃ receptor on resident cardiac mast cells promoting degranulation and the release of histamine. The lack of effect of IB-MECA on coronary flow in the rabbit could be caused by the aforementioned differences in adenosine-receptor subtype involvement in mast cells. Alternatively, histamine can produce coronary constriction in the rabbit (21), and the net effect of histamine is determined by the contributions of H₁ and H₂ histamine receptors, which may have opposing effects (5, 21, 30).

Three observations in our data, however, argue against a role for adenosine A₃-receptor activation in the IB-MECA/Cl-IB-MECA-induced increases in coronary flow in the rat. First, the coronary flow increase with IB-MECA showed no evidence of tachyphylaxis or desensitization, a characteristic of adenosine A₃-receptor activation (17, 29). In the multiple-exposure group, IB-MECA produced flow increases of 25 ± 5, 24 ± 3, and 20 ± 3%, respectively. It is possible that the 2-min exposure was too short to promote tachyphylaxis, but Palmer et al. (17) reported that the cloned rat adenosine A₃ receptor exhibited a rapid phosphorylation and functional desensitization with a half-time of <1 min. Second, Cl-IB-MECA, reported to be more selective for the A₃ receptor than IB-MECA (13), did not produce coronary dilation in the rat heart at a concentration of 50 nM but did increase coronary flow (approximately one-half that of flow produced by 50 nM IB-MECA) at 100 nM. Because both of these agents have been reported to bind to cloned adenosine A₃ receptors at doses <10 nM (7, 9, 13), doses of 50-100 nM may not be selective for rat cardiac adenosine A₃ receptors.

The most compelling evidence that the IB-MECA-induced coronary dilation was not the result of A₃-receptor activation is based on the results obtained with the adenosine-receptor antagonists MRS-1191 and Sch-58261. In the pretreatment protocol, the adenosine A₃ receptor antagonist MRS-1191 reduced the IB-MECA coronary flow increases by only 30%. When IB-MECA was infused first, the subsequent infusion of MRS-1191 did not antagonize coronary vasodilation (Fig. 5). MRS-1191, a relatively new A₃-receptor antagonist, is a 1,4-dihydropyridine derivative with a inhibitor constant (K_i) value of 31 nM for human adenosine A₃ receptors, and it has been reported to be 1,000-fold selective for cloned human A₃ versus rat A₁ and A_2a receptors (11). The K_i value of this antagonist for the cloned rat A₃ receptor has been reported to be 1.42 µM (12), but even at this dose it would be expected to have little effect on rat A_2a receptors (K_i > 100 µM) (11). Although there have been few studies with this agent, MRS-1191 at the dose used in the present study blocked Cl-IB-MECA-induced cardioprotection in cultured chick ventricular myocytes without significantly altering the protective effects of the adenosine A₁-receptor agonist 2-chloro-N⁶-cyclopentyladenosine (24).

In contrast to these results with MRS-1191, two adenosine A₂- receptor antagonists, CSC and Sch-58261, completely blocked the increase in coronary flow associated with IB-MECA. Additionally, Sch-58261 blocked the coronary flow increase associated with 100 nM Cl-IB-MECA. Sch-58261 is a relatively new and selective adenosine A_2a-receptor antagonist (4, 15, 32). The ability of Sch-58261 to block adenosine A_2a receptor-mediated coronary dilation in the present study was verified by showing that Sch-58261 completely blocked the CGS-21680-induced increase in coronary flow. In contrast to the very limited studies with MRS-1191, there have been several studies that demonstrate the ability of Sch-58261 to block adenosine- and adenosine A_2a agonist-induced vasodilatation in vivo (15) and in vitro (4, 32). Belardinelli et al. (4) reported that Sch-58261 blocked adenosine-induced decreases in coronary perfusion pressure in the constant flow-perfused isolated guinea pig heart with an IC₅₀ of 6.8 ± 0.6 nM. The same authors reported that this antagonist also potently blocked the coronary effects of the adenosine A_2a agonist CGS-21680 without altering adenosine A₁ agonist prolongation of the S-H interval. Zocchi et al. (32) reported that Sch-58261 did not bind to adenosine A₃ receptors at concentrations up to 1 µM. These results strongly suggest that IB-MECA-induced coronary dilation in the rat is mediated via adenosine A_2a-receptor activation.

Although both IB-MECA and Cl-IB-MECA produced coronary dilation in the rat heart, neither agent at the doses used in the present study altered coronary flow in the rabbit heart. There are two possible explanations for this difference. First, as mentioned previously there appear to be species differences in adenosine-receptor subtype mediation of mast cell degranulation (2, 3, 6, 8, 20, 29) and possible species differences in histamine effects on the coronary vasculature (5, 21, 28, 30). Alternatively, because the IB-MECA flow increase was completely blocked by the adenosine A_2a-receptor antagonist Sch-58261, it is possible that the rabbit heart has a higher threshold for adenosine A_2a-receptor activation. This could be caused by differences in rat and rabbit adenosine A_2a receptors, receptor density, or efficiency of receptor coupling to intracellular signal transduction pathways. Shryock et al. (24) recently reported that the guinea pig heart had a high adenosine A_2a-receptor reserve based in part on the observation that CGS-21680 produced a half-maximal increase in coronary conductance with only 1.3% occupation of adenosine A_2a receptors. Although we did not perform complete dose-response curves with CGS-21680 in either species, the rat heart did show much greater dilation with 5 nM CGS-21680 (~50% increase in coronary flow) than did the rabbit heart (8 ± 4% in 3 hearts). Even at a 10-fold higher dose (50 nM), CGS-21680-induced dilation in the rabbit (30 ± 5%, n = 3) was less than that observed with 5 nM CG-21680 in the rat heart. It is thus possible that the rabbit heart has a lower adenosine A_2a-receptor reserve for coronary dilation than rat and guinea pig myocardium.

Although our observation that IB-MECA produced coronary dilation in the rat is the first such report in cardiac tissue, there are other reports of IB-MECA effects on the vasculature. Prentice et al. (18, 19) reported that IB-MECA relaxed isolated rat aortas and mesenteric arteries, respectively. In their latter study (19) they concluded that this effect of IB-MECA was not caused by A₃-receptor activation based on pharmacological criteria. They also could not block this relaxation with the adenosine A_2a-receptor antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4]-triazolo[2,3-a][1,3,5] triazin-5-ylamino]ethyl)phenol (ZM-241385). These findings contrast with our present results in which IB-MECA coronary dilation in the rat was blocked completely by the A_2a-receptor antagonist Sch-58261. Although our findings were unexpected, they are not completely without precedent. Shearman and Weaver (23) reported that the A₃-receptor radioligand ¹²⁵I-labeled AB-MECA primarily labeled A₁ and A_2a adenosine receptors in the rat brain striatum, an observation they attributed to the low expression of adenosine A₃ receptors combined with the fact that ¹²⁵I-labeled AB-MECA has been reported to have an affinity as low as 25 nM for recombinant canine A_2a adenosine receptors (16). Results similar to our present findings were reported by Sullivan and Linden (26), who observed that IB-MECA inhibition of tumor necrosis factor release in human monocytes was mediated by the adenosine A_2a, not the A₃, receptor.

The present intact open-chest pig studies are the first reports, to our knowledge, on IB-MECA and Cl-IB-MECA effects in an intact animal preparation other than the rat or rabbit. Our results are essentially identical to those observed in the rat administered with APNEA, IB-MECA, and Cl-IB-MECA (6, 8, 20, 29). We observed a rapid decrease in arterial blood pressure that normalized within 10 min and only a slight nonsignificant increase in heart rate. Two pigs died within the first minute of the IB-MECA bolus. Consistent with reports of rapid desensitization of the adenosine A₃ receptor (17), we observed a much smaller hemodynamic effect in the pig with a second IB-MECA infusion. An observation that was visible in all animals receiving IB-MECA and Cl-IB-MECA was a large increase in right ventricular volume concurrent with the decrease in systemic blood pressure. In two pigs treated with IB-MECA and one pig with Cl-IB-MECA, we measured an increase in pulmonary artery pressure. Because histamine has been reported to increase pulmonary pressure and decrease systemic pressure (5, 30), these observations are consistent with reports of adenosine A₃ receptor-mediated activation of mast cells resulting in degranulation and histamine release.

Although the effects of both agonists in the pig were consistent with adenosine A₃-receptor activation, there are two points that must be mentioned. First, the IB-MECA hemodynamic effects were produced with a 5 µg/kg dose, but a 10 µg/kg Cl-IB-MECA dose exerted no effect, and the hemodynamic effects that were elicited by Cl-IB-MECA were associated with a 25 µg/kg dose. If this effect were mediated by A₃-receptor activation, one would expect that the more selective Cl-IB-MECA would also be more, not less, potent in evoking these responses. Thus the dose-dependent effects of these agents in the intact pig were similar to those in the isolated rat heart, where coronary dilation was only observed with a higher concentration of Cl-IB-MECA. A second important point is that in two separate pigs neither the mast cell inhibitor sodium cromolyn nor the histamine H₁-receptor blocker diphenhydramine blocked the effects of IB-MECA (data not shown). We used the same dose of cromolyn (20 mg/kg) that Hannon et al. (8) used to block APNEA-induced hypotension in the rat. The effect of the histamine blocker diphenhydramine (30 mg/kg) was difficult to assess because administration of this agent alone produced profound hemodynamic effects, similar to those produced by IB-MECA, which were not completely reversible. Histamine exerts its effects via two distinct-receptor subtypes (H₁ and H₂) the expressions may be of which may be species and tissue specific. Cooper et al. (5) reported that in the pig histamine produced dose-dependent effects with lower doses eliciting systemic hypotension and pulmonary hypertension, most likely via histamine H₁-receptor activation. It must also be pointed out that mast cell degranulation likely results in the release of other vasoactive agents, such as thromboxanes and ATP.

In summary, the results of this study indicate that the adenosine A₃-receptor agonists IB-MECA and Cl-IB-MECA exert species-dependent effects. Although neither agent exerted effects in the rabbit heart, both agonists produced coronary vasodilation in the rat heart. In addition, this dilatory effect was minimally altered by the A₃-receptor antagonist MRS-1191 but was completely blocked by the adenosine A_2a-receptor antagonist Sch-58261. In the intact pig IB-MECA and Cl-IB-MECA both produced profound, but reversible, systemic hypotension and pulmonary hypertension. The results of this study indicate that although these agents have been reported to be protective in the ischemic-reperfused rabbit heart (10, 27), further studies on the exact role of cardiac adenosine A₃ receptors need to be performed in additional species and intact animal preparations.