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The mammalian myocardium expresses four adenosine receptor (AR) subtypes: [A.sub.1]AR, [A.sub.2a]AR, [A.sub.2b]AR, and [A.sub.3]AR. The [A.sub.1]AR is well known for its profound antiadrenergic effects, but the roles of other AR subtypes in modulating contractility remain inconclusive. Thus, the objective of this study was to determine the direct and indirect effects of [A.sub.2a]AR and [A.sub.2b]AR on cardiac contractility. Experiments were conducted in paced, constant pressure-perfused isolated hearts from wild-type (WT), [A.sub.2a]AR knockout (KO), and [A.sub.2b]AR KO mice. The [A.sub.2a]AR agonist CGS-21680 did not alter basal contractility or [beta]-adrenergic receptor agonist isoproterenol (Iso)-mediated positive inotropic responses, and Iso-induced effects were unaltered in [A.sub.2a]AR KO hearts. However, [A.sub.2a]AR gene ablation resulted in a potentiation of the antiadrenergic effects mediated by the [A.sub.1]AR agonist 2-chloro-N-cyclopentyladenosine. The nonselective AR agonist 5'-N-ethylcarboxamido adenosine and the selective [A.sub.2b]AR agonist BAY 60-6583 induced coronary flow-independent increases in contractility, but BAY 60-6583 did not alter Iso-induced contractile responses. The [A.sub.1]AR antiadrenergic effect was not potentiated in [A.sub.2b]AR KO hearts. The expression of all four AR subtypes in the heart and ventricular myocytes was confirmed using real-time quantitative PCR. Taken together, these results indicate that [A.sub.2a]AR does not increase cardiac contractility directly but indirectly alters contractility by modulating the [A.sub.1]AR antiadrenergic effect, whereas [A.sub.2b]AR exerts direct contractile effects but does not alter [beta]-adrenergic or [A.sub.1]AR antiadrenergic effects. These results indicate that multiple ARs differentially modulate cardiac function. [A.sub.2a] adenosine receptor; [A.sub.2b] adenosine receptor; knockout mice; cardiac contractility doi: 10.1152/ajpheart.00511.2010.

The mechanism by which distinct stimuli activate the same mitogen-activated protein kinases (MAPKs) is unclear. We examined compartmentalized MAPK signaling and altered redox state as possible mechanisms. Adult rat cardiomyocytes were exposed to the adenosine [A.sub.1] receptor agonist 2-chloro-[N.sup.6]-cyclopentyladenosine (CCPA; 500 nM) or [H.sub.2][O.sub.2] (100 [micro]M) for 15 min. Nuclear/myofilament, cytosolic, Triton-soluble membrane, and Triton-insoluble membrane fractions were generated. CCPA and [H.sub.2][O.sub.2] activated p38 MAPK and p44/p42 ERKs in cytosolic fractions. In Triton-soluble membrane fractions, [H.sub.2][O.sub.2] activated p38 MAPK and p42 ERK, whereas CCPA had no effect on MAPK activation in this fraction. The greatest difference between [H.sub.2][O.sub.2] and CCPA was in the Triton-insoluble membrane fraction, where [H.sub.2][O.sub.2] increased p38 and p42 activation and CCPA reduced MAPK activation. CCPA also increased protein phosphatase 2A activity in the Triton-insoluble membrane fraction, suggesting that the activation of this phosphatase may mediate CCPA effects in this fraction. The Triton-insoluble membrane fraction was enriched in the caveolae marker caveolin-3, and >85% of p38 MAPK and p42 ERK was bound to this scaffolding protein in these membranes, suggesting that caveolae may play a role in the divergence of MAPK signals from different stimuli. The antioxidant N-2-mercaptopropionyl glycine (300 [micro]M) reduced [H.sub.2][O.sub.2]-mediated MAPK activation but failed to attenuate CCPA-induced MAPK activation. [H.sub.2][O.sub.2] but not CCPA increased reactive oxygen species (ROS). Thus the adenosine [A.sub.1] receptor and oxidative stress differentially modulate subcellular MAPKs, with the main site of divergence being the Triton-insoluble membrane fraction. However, the adenosine A1 receptor-mediated MAPK activation does not involve ROS formation. signal transduction; compartmentation

There is increasing evidence for interactions among adenosine receptor subtypes in the brain and heart. The purpose of this study was to determine whether the adenosine [A.sub.2a] receptor modulates the infarct size-reducing effect of preischemic administration of adenosine receptor agonists in intact rat myocardium. Adult male rats were submitted to in vivo regional myocardial ischemia (25 min) and 2 h reperfusion. Vehicle-treated rats were compared with rats pretreated with the [A.sub.1] agonist 2-chloro-[N.sup.6]-cyclopentyladenosine (CCPA, 10 [micro]g/kg), the nonselective agonist 5'-N-ethylcarboxamidoadenosine (NECA, 10 [micro]g/kg), or the [A.sub.2a] agonist 2-[4-(2-carboxyethyl)phenethylamino]-5' -N-methylcarboxamidoadenosine (CGS-21680, 20 [micro]g/kg). Additional CCPA- and NECA-treated rats were pretreated with the [A.sub.1] antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 100 [micro]g/kg), the [A.sub.2a]/[A.sub.2b] antagonist 4-(-2-[7-amino-2-{2-furyl}{1,2,4} triazolo{2,3-a}{1,3,5}triazin-5-y1-amino/ethyl)phenol (ZM-241385, 1.5 mg/kg) or the [A.sub.3] antagonist 3-propyl-6-ethyl-5[(ethylthio) carbonyl]-2-phenyl-4-propyl-3-pyridine carboxylate (MRS-1523, 2 mg/kg). CCPA and NECA reduced myocardial infarct size by 50% and 35%, respectively, versus vehicle, but CGS-21680 had no effect. DPCPX blunted the bradycardia associated with CCPA and NECA, whereas ZM-241385 attenuated their hypotensive effects. Both DPCPX and ZM-241385 blocked the protective effects of CCPA and NECA. The [A.sub.3] antagonist did not alter the hemodynamic effects of CCPA or NECA, nor did it alter adenosine agonist cardioprotection. None of the antagonists alone altered myocardial infarct size. These findings suggest that although preischemic administration of an [A.sub.2a] receptor agonist does not induce cardioprotection, antagonism of the [A.sub.2a] and/or the [A.sub.2b] receptor blocks the cardioprotection associated with adenosine agonist pretreatment. adenosine receptor; ischemia-reperfusion; infarct reduction

Ischemia-reperfusion activates ERK and p38 MAPK in cardiac membranes, but the role of caveolae in MAPK signaling during this stress has not been studied. The purpose of this study was to determine the effect of in vivo myocardial ischemia-reperfusion on the level and distribution of caveolin-1 and -3 and cholesterol as well as MAPK activation in caveolin-enriched fractions. Adult male rats were subjected to in vivo regional myocardial ischemia induced by 25 min of coronary artery occlusion and 10 min (n = 5) or 2 h (n = 4) of reperfusion. Another group of rats served as appropriate nonischemic time controls (n = 4). A discontinuous sucrose density gradient was used to isolate caveolae/lipid rafts from ischemic and nonischemic heart tissue. Caveolin-1 and -3, as well as cholesterol, were enriched in the light fractions. A redistribution of caveolin-3 and a reduction in caveolin-1 and cholesterol levels in the light fractions occurred after 10 min of reperfusion. The ERKs were activated in ischemic zone light and heavy fractions by 10 min of reperfusion, p44 ERK was activated alter 2 h of reperfusion only in the light fractions, whereas p42 ERK phosphorylation was increased in the light and heavy fractions. Although no p38 MAPK activation occurred after 10 min of reperfusion, 2 h of reperfusion caused significant activation of p38 MAPK in nonischemic zone light and heavy fractions. These results show the importance of caveolar membrane/lipid rafts in MAPK signaling and suggest that subcellular compartmentation of p44/p42 ERKs and p38 MAPK may play distinct roles in the response to myocardial ischemia-reperfusion. caveolae; caveolin-3; caveolin-1 doi:10.1152/ajpheart.01354.2005

The purpose of this study was to determine whether aged myocardium exhibits decreased responsiveness to adenosine [A.sub.1] and [A.sub.2a] receptor activation. Studies were conducted in adult (4-6 months) and aged (24-26 months) Fischer 344 x Brown Norway hybrid (F344 x BN) rats. Effects of the adenosine [A.sub.1]/[A.sub.2], agonist AMP579 were measured in isolated hearts and in rats submitted to in vivo regional myocardial ischemia. Aged isolated hearts exhibited lower spontaneous heart rates and higher coronary resistance, as well as normal [A.sub.1]- and [A.sub.2a]-mediated responses. There was no difference in control infarct size between adult and aged rats; however, AMP579 treatment resulted in a 50% greater infarct size reduction in aged rats (18 [+ or -] 4% of risk area) compared to adult rats (37 [+ or -] 3%). These findings suggest that adenosine [A.sub.1] and [A.sub.2a] receptor-mediated effects are not diminished in normal aged myocardium, and that aged hearts exhibit increased adenosine agonist-induced infarct reduction.

Adenosine [A.sub.1] receptor delayed preconditioning (PC) against myocardial infarction has been well described; however, there have been limited investigations of the signaling mechanisms that mediate this phenomenon. In addition, there are multiple conflicting reports on the role of inducible nitric oxide synthase (iNOS) in mediating [A.sub.1] late-phase PC. The purpose of this study was to determine the roles of the p38 and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases (MAPKs) in in vivo delayed [A.sub.1] receptor PC and whether this protection at the myocyte level is due to upregulation of iNOS. Myocardial infarct size was measured in open-chest anesthetized rats 24 h after treatment with vehicle or the adenosine [A.sub.1] agonist 2-chloro[N.sup.6]-cyclopentyladenosine (CCPA; 100 [micro]g/kg ip). Additional rats receiving CCPA were pretreated with the p38 inhibitor SB-203580 (1 mg/kg ip) or the MAPK/ERK kinase (MEK) inhibitor PD-098059 (0.5 mg/kg ip). At 24 h after CCPA administration, a group of animals was given the iNOS inhibitor 1400W 10 min before ischemia. Treatment with CCPA reduced infarct size from 48 [+ or -] 2 to 28 [+ or -] 2% of the area at risk, an effect that was blocked by both SB-203580 and PD-098059 but not 1400W. Ventricular myocytes isolated 24 h after CCPA injection exhibited significantly reduced oxidative stress during [H.sub.2][O.sub.2] exposure compared with myocytes from vehicle-injected animals, and this effect was not blocked by the iNOS inhibitor 1400W. Western blot analysis of whole heart and cardiac myocyte protein samples revealed no expression of iNOS 6 or 24 h after CCPA treatment. These results indicate that adenosine [A.sub.1] receptor delayed PC in rats is mediated by MAPK-dependent mechanisms, but this phenomenon is not associated with the early or late expression of iNOS. agonist; reperfusion; infarct size; p38; extracellular signal-related kinase; cardiac myocytes; mitogen-activated protein kinase; inducible nitric oxide synthase

The protective effects of adenosine receptor acute preconditioning (PC) are well known; however, the signaling mechanism mediating this effect has not been determined in in vivo models. The purpose of this study was to determine the role of the extracellular signal-regulated kinase (ERK) pathway in mediating adenosine PC in in vivo rat myocardium. Open-chest rats were submitted to 25 min of coronary artery occlusion and 2 h of reperfusion. ERK activation was assessed by measuring total and dually phosphorylated p44/42 ERK isoforms in nuclear and/or myofilament, mitochondrial, cytosotic, and membrane fractions. Adenosine receptor PC with the [A.sub.1]/[A.sub.2a], agonist 1S-[1a,2b,3b,4a([S.sup.*])]-4- [7-[[2-(3-chloro-2-thienyl)-1-methylpropyl]amino] -3H-imidazo[4,5-b]pyridyl-3-yl]cyclopentane carboxamide (AMP-579) reduced infarct size from 49 [+ or -] 3% to 29 [+ or -] 3%, an effect that was blocked by the mitogen-activated protein kinase-ERK inhibitor U-0126. ERK isoforms were present in all fractions, with the greatest expression in the cytosolic fraction and the least in the mitochondrial traction. AMP-579 treatment increased preischemic p44/42 ERK phosphorylation in all fractions 2.7- to 6.9-fold. Reperfusion increased ERK isoform activation in all fractions, but there were no differences between control and AMP-579 hearts. Preischemic increases in phospo-p44/p42 ERK with AMP-579 were blunted by U-0126, although only in mitochondrial and membrane compartments. The PC effects of AMP-579 on infarct size and ERK were blunted by both the [A.sub.1] antagonist 8-cyclopentyl-1,3-dipropylxanthine and, surprisingly, the [A.sub.2a] antagonist ZM-241385. These results indicate that the unique adenosine receptor agonist AMP-579 exerts its beneficial effects in vivo via both [A.sub.1] and [A.sub.2a] receptor modulation of subcellular ERK isoform signaling. myocardium; ischemia-reperfusion; compartmentation; adenosine

The purpose of this study was to determine whether the adenosine [A.sub.1]/[A.sub.2a] receptor agonist AMP-579 induces acute and delayed preconditioning against in vivo myocardial stunning. Regional stunning was produced by 15 min of coronary artery occlusion and 3 h of reperfusion (RP) in anesthetized open-chest pigs. In acute protection studies, animals were pretreated with saline, low-dose AMP-579 (15 [micro]g/kg iv bolus 10 min before ischemia), or high-dose AMP-579 (50 [micro]g/kg iv at 14 [micro]g/kg bolus + 1.2 [micro]g x [kg.sup.-1] x [min.sup.-1] for 30 min before coronary occlusion). The delayed preconditioning effects of AMP-579 were evaluated 24 h after administration of saline vehicle or high-dose AMP-579 (50 [micro]g/kg iv). Load-insensitive contractility was assessed by measuring regional preload recruitable stroke work (PRSW) and PRSW area. Acute preconditioning with AMP-579 dose dependently improved regional PRSW: 129 [+ or -] 5 and 100 [+ or -] 2% in high- and low-dose AMP-579 groups, respectively, and 78 [+ or -] 5% in the control group at 3 h of RP. Administration of the adenosine Aa receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (0.7 mg/kg) blocked the acute protective effect of high-dose AMP-579, indicating that these effects are mediated through Aa receptor activation. Delayed preconditioning with AMP-579 significantly increased recovery of PRSW area: 64 [+ or -] 5 vs. 33 [+ or -] 5% in control at 3 h of RP. In isolated perfused rat heart studies, kinetics of the onset and washout of AMP-579 [A.sub.1] and [A.sub.2a] receptor-mediated effects were distinct compared with those of other adenosine receptor agonists. The unique nature of the adenosine agonist AMP-579 may play a role in its ability to induce delayed preconditioning against in vivo myocardial stunning. adenosine receptors; ischemia-reperfusion

The intermediary metabolite pyruvate has been shown to exert significant beneficial effects in in vitro models of myocardial oxidative stress and ischemia-reperfusion injury. However, there have been few reports of the ability of pyruvate to attenuate myocardial stunning or reduce infarct size in vivo. This study tested whether supraphysiological levels of pyruvate protect against reversible and irreversible in vivo myocardial ischemia-reperfusion injury. Anesthetized, open-chest pigs (n = 7/group) underwent 15 min of left anterior descending coronary artery (LAD) occlusion and 3 h of reperfusion to induce stunning. Load-insensitive contractility measurements of regional preload recruitable stroke work (PRSW) and PRSW area (PRSWA) were generated. Vehicle or pyruvate (100 mg/kg iv bolus + 10 mg * [kg.sub.-1] * [min.sub.-1] intra-atrial infusion) was administered during ischemia and for the first hour of reperfusion. In infarct studies, pigs (n = 6/group) underwent 1 h of LAD ischemia and 3 h of reperfusion. Group I pigs received vehicle or pyruvate for 30 min before and throughout ischemia. In group II, the infusion was extended through 1 h of reperfusion. In the stunning protocol, pyruvate significantly improved the recovery of PRSWA at 1 h (50 [+ or -] 4% vs. 23 [+ or -] 3% in controls) and 3 h (69 [+ or -] 5% vs. 39 [+ or -] 3% in controls) reperfusion. Control pigs exhibited infarct sizes of 66 [+ or -] 1% of the area at risk. The pyruvate I protocol was associated with an infarct size of 49 [+ or -] 3% (P < 0.05), whereas the pyruvate II protocol was associated with an infarct size of 30 [+ or -] 2% (P < 0.05 vs. control and pyruvate I). These findings suggest that pyruvate attenuates stunning and decreases myocardial infarction in vivo in part by reduction of reperfusion injury. Metabolic interventions such as pyruvate should he considered when designing the optimal therapeutic strategies for limiting myocardial ischemia-reperfusion injury. energetics; myocardial ischemia; reperfusion

Adenosine [A.sub.2a] receptors are found in coronary vascular tissue although, their presence in myocardium is subject to investigation. Although there have been numerous studies on adenosine [A.sub.2a] receptor agonist effects on contractility and cAMP levels in ventricular myocytes, these have yielded conflicting results. Negative pharmacological studies have even led to the conclusion that [A.sub.2a] receptors are not present in cardiac myocytes. The purpose of this study was to determine whether [A.sub.2a] receptors are expressed in rat ventricular myocytes and what physiological effects are mediated via activation of these receptors. Western blot analysis with a polyclonal antibody raised against a peptide sequence specific to the carboxy terminus of the [A.sub.2a] receptor revealed the presence of a band at ~45 kDa. However, the immunoreactivity was located in the nonmembrane fraction of the cell lysate. The membrane fraction only exhibited an immunoreactive band [greater than or equal to] 50 kDa. Treatment of isolated myocytes with the adenosine [A.sub.2a] agonist 2-[4-[(2-carboxyethyl)-phenyl]ethylamino]-5'-N-ethylcarboxamidoadeno-sine (CGS-21680) exerted no effects on cAMP levels or myocyte twitch amplitude. These results indicate that although rat ventricular myocytes appear to express adenosine [A.sub.2a] receptors, stimulation with an [A.sub.2a] agonist exerts no functional effects, possibly because of the subcellular localization of the [A.sub.2a] receptor. phosphodiesterase inhibitor; Western blot

Research was conducted to examine the species-dependent hemodynamic effects of adenosine A3-receptor agonists N6-(3-iodobenzyl)-9-(5-(methylcarbamoyl)-beta-D-ribofuranosyl)adenine (IB-MECA) and 2-chloro-N6-(3-iodobenzyl)-9(5-(methylcarbamoly)-beta-D-ribofuranosyl)adenin e (CI-IB-MECA). The objective was to compare the hemodynamic effects of IB-MECA and Cl-IB-MECA in isolated rat and rabbit hearts and in the intact, open-chest pig. Results demonstrate that adenosine A3-receptor agonists generate species-dependent effects, which in the rat heart may be caused by adenosine A(sub 2a)-receptor activation.

Research was conducted to show that the positive inotropic effect of pyruvate can be characterized in single isolated rat ventricular myocytes. Ca2+ tolerant myocytes were prepared from rat hearts by enzymatic dispersion while myocytes were treated with the fluorescent Ca2+ indicator fura 2-acetoxymethyl ester. Results suggested that positive effect is the direct result of the augmentation of systolic intracellular Ca2+ concentrations.

The effects of in vivo myocardial stunning on sarcoplasmic reticulum (SR) function was investigated. Regional myocardial reticulum stunning was induced in open chest pigs. Results of the study revealed that a depressed activity of the SR function in postischemic myocardium is a mechanism underlying myocardial stunning. Furthermore, such interventions that prevent stunning-induced decreases in SR function can effectively preserve postischemic myocardial function.

The effects of R-75231-induced elevations on interstitial fluids (ISF) and plasma adenosine on myocardial infarct size were analyzed in pentobarbital-anesthetized swine with coronary artery occlusions (CAO). Pretreatment of swine with R-75231 before CAO increased the levels of ISF adenosine during ischemia. R75231 also reduced the levels of ISF inosine, hypoxanthine and infarct size in swine with CAO. Furthermore, R-75231-induced elevations on adenosine were correlated with infarct size reductions.

Transient infusion of adenosine intravenously increases interstitial fluid (ISF) adenosine levels and decreases myocardial infarct size, but they attain basal levels after the culmination of infusion. After washouts, adenosine preconditioning has negligible influence on prolonged ischemia indicating that other mediators are necessary for cardioprotective processes. The increase in ISF adenosine during short infusion may be mediated by adenosine A1 receptors.

Adenosine regulates myocardical protein kinase C (PKC) activity via negative inotropic and vasoconstrictive effects of phorbol, PMA in isolated rat hearts by potentiating the actions of adenosine A1 and A2 receptor-agonists. The attenuation of negative inotropic effect of PMA occurs due to inhibition of PKC activity or attenuation of PKC-mediated alteration of Ca2+. Adenosine prevents ischemia by inhibiting ischemia-induced activation of PKC by attenuating the effects of increased PKC activity on intracellular calcium.

Adenosine [A.sub.3] agonists have been shown to protect ischemic rat and rabbit myocardium. However, these agonists have been reported to exert [A.sub.3] independent effects, and no cardiac [A.sub.3] receptor has yet been identified. We thus tested whether [A.sub.3] agonist protection is due to [A.sub.1] receptor activation. Isolated rat and rabbit hearts were subjected to 25 and 45 min of global ischemia, respectively. Rat hearts pretreated with adenosine (100 [micro]M), the [A.sub.3] agonist 2-chloro-[N.sup.6]-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IB-MECA, 50 nM), and vehicle recovered 73 [+ or -] 2%, 75 [+ or -] 4%, and 46 [+ or -] 4%, respectively, of preischemic left ventricular developed pressure (LVDP) after 30 min of reperfusion. The [A.sub.1] antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 100 nM) blocked the beneficial effects of Cl-IB-MECA (51 [+ or -] 5%) and adenosine (47 [+ or -] 6%). In rabbit hearts, the beneficial effects of the [A.sub.3] agonist [N.sup.6]-(3-iodobenzyl)adenosine-5'-N-methyluronamide (50 nM) and the [A.sub.1] agonist 2-chloro-[N.sup.6]-cyclopentyladenosine (100 nM) on postischemic LVDP (75 [+ or -] 4 and 74 [+ or -] 5%, respectively) were blocked by DPCPX (34 [+ or -] 4 and 36 [+ or -] 3%, respectively). The reduction in infarct size with both agonists was also completely blocked by DPCPX. These results suggest that these [A.sub.3] agonists protect ischemic myocardium via [A.sub.1] receptor activation. ischemia; reperfusion; cardiac receptor

Lasley, Robert D., M. Salik Jahania, and Robert M. Mentzer, Jr. Beneficial effects of adenosine [A.sub.2a] agonist CGS-21680 in infarcted and stunned porcine myocardium. Am J Physiol Heart Circ Physiol 280: H1660-H1666, 2001.--Although there are conflicting results on whether adenosine infusion during reperfusion alters infarct size, there are several reports that indicate adenosine [A.sub.2a] agonists reduce infarct size. There are also reports that the [A.sub.2a] agonist CGS-21680 increases cAMP and contractility in ventricular myocytes. The purpose of this study was to determine whether low-dose intracoronary infusions of CGS-21680 during reperfusion exert any beneficial effects in irreversibly and reversibly injured myocardium. Open-chest pigs were submitted to 60 min of coronary artery occlusion and 3 h of reperfusion. Treated pigs were administered intracoronary CGS-21680 (0.2 [micro]g [multiplied by] [kg.sup.-1] [multiplied by] [min.sup.-1]) for the first 60 min of reperfusion. Pigs submitted to regional stunning (15 min ischemia) were treated with intracoronary CGS-21680 (0.15 [micro]g [multiplied by] [kg.sup.-1] [multiplied by] [min.sup.-1]) after 2 h of reperfusion. In the infarct protocol, CGS-21680 reduced infarct size from 62 [+ or -] 2% of the region at risk to 36 [+ or -] 2%. In stunned myocardium, CGS increased load-independent regional preload recruitable stroke work and area by [is greater than or equal to] 70%, but the same infusion in normal myocardium was associated with no inotropic effect. Both beneficial effects were associated with little systemic hemodynamic effects. These findings suggest that reperfusion infusions of low doses of the [A.sub.2a] agonist CGS-21680 exert beneficial effects in reversibly and irreversibly injured myocardium. adenosine [A.sub.2a] receptor; myocardial stunning; preload recruitable stroke work; myocardial infarction

Narayan, Prakash, Robert M. Mentzer, Jr., and Robert D. Lasley. Phosphatase inhibitor cantharidin blocks adenosine [A.sub.1] receptor anti-adrenergic effect in rat cardiac myocytes. Am. J. Physiol. Heart Circ. Physiol. 278: H1-H7, 2000.--Experiments were performed to examine whether the protein phosphatase inhibitor cantharidin blocks the anti-adrenergic effect of adenosine [A.sub.1] receptor stimulation. In electrically stimulated adult rat ventricular myocytes loaded with the intracellular calcium concentration ([[[Ca.sup.2+].sub.i]) indicator fluo-3, isoproterenol (10 nM) increased systolic [[[Ca.sup.2+].sub.1] by 46%, increased twitch amplitude by 56%, and increased total cellular cAMP content by 140%. The adenosine [A.sub.1] receptor agonist 2-chloro-[N.sup.6]-cyclopentlyadenosine (CCPA) reduced isoproterenol-stimulated [[[Ca.sup.2+].sub.i] and contractility by 87 and 80%, respectively, but reduced cAMP content by only 18%. Cantharidin had no effects on myocyte [[[Ca.sup.2+].sub.i], contractility, or cAMP in the absence or presence of isoproterenol but blocked the effects of CCPA on [[[Ca.sup.2+].sub.i] and contractility by ~44%. Cantharidin had no effect on CCPA attenuation of isoproterenol-induced increases in cAMP. Pretreatment with CCPA also reduced the increase in contractile parameters produced by the direct cAMP-dependent protein kinase A (PKA) activator 8-bromocAMP. These results suggest that activation of protein phosphatases mediate, in part, the anti-adrenergic effect of adenosine [A.sub.1] receptor activation in ventricular myocardium. calcium; contractility; adenosine 3',5'-cyclic monophosphate