. In vivo adenosine receptor preconditioning reduces myocardial infarct size via subcellular ERK signaling. Am J Physiol Heart Circ Physiol 288: H2253-H2259, 2005. First published January 14, 2005 doi:10.1152/ajpheart.01009.2004.-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, cytosolic, and membrane fractions. Adenosine receptor PC with the A1/A2a agonist 1S-[1a,2b,3b,4acyclopentane carboxamide (AMP-579) reduced infarct size from 49 Ϯ 3% to 29 Ϯ 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 fraction. 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 1 antagonist 8-cyclopentyl-1,3-dipropylxanthine and, surprisingly, the A2a antagonist ZM-241385. These results indicate that the unique adenosine receptor agonist AMP-579 exerts its beneficial effects in vivo via both A1 and A2a receptor modulation of subcellular ERK isoform signaling.

Although acute adenosine preconditioning (PC) is well established, the signaling pathways mediating this cardioprotection remain unclear. Because adenosine receptor agonists activate p38 MAPK and this kinase has been implicated in ischemic and pharmacological PC, the purpose of this study was to determine the role of p38 MAPK in acute adenosine receptor PC. The role of p38 MAPK activation in discrete subcellular compartments during ischemia-reperfusion was also determined. The following groups were used in an in vivo rat ischemia-reperfusion model: 1) control (10% DMSO i.v.), 2) the A(1)/A(2a) adenosine receptor AMP-579 (50 microg/kg i.v.), 3) AMP-579 + the A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 100 microg/kg i.v.), 4) AMP-579 + the p38 MAPK inhibitor SB-203580 (1 mg/kg i.v.), and 5) SB-203580 alone. p38 MAPK activation was measured by Western blot analysis in cytosolic, mitochondrial, membrane, and nuclear/myofilament fractions obtained from hearts at preischemic, ischemic, and reperfusion time points. A significant reduction in infarct size was observed with AMP-579 PC, an effect blocked by DPCPX or SB-203580 pretreatment. AMP-579 treatment was associated with a significant increase in p38 MAPK activation in the nuclear/myofilament fraction before ischemia, whereas no activation of this kinase occurred during ischemia or reperfusion. In contrast, p38 MAPK was activated in the mitochondrial fraction by ischemia and in the cytosolic, mitochondrial, and membrane fractions by reperfusion in the control group. SB-203580 blocked the AMP-579-induced increase in phosphorylation of the downstream p38 substrate activating transcription factor-2. These results suggest a role for p38 MAPK activation in discrete subcellular compartments in acute adenosine A(1) receptor PC.

Lasley RD, Kristo G, Keith BJ, Mentzer RM, Jr. The A2a/A2b receptor antagonist ZM-241385 blocks the cardioprotective effect of adenosine agonist pretreatment in in vivo rat myocardium. Am J Physiol Heart Circ Physiol 292: H426 -H431, 2007. First published September 15, 2006; doi:10.1152/ajpheart.00675.2006.-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 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 1 agonist 2-chloro-N 6 -cyclopentyladenosine (CCPA, 10 g/kg), the nonselective agonist 5Ј-N-ethylcarboxamidoadenosine (NECA, 10 g/kg), or the A 2a agonist 2-[4-(2-carboxyethyl)phenethylamino]-5Ј-N-methylcarboxamidoadenosine (CGS-21680, 20 g/kg). Additional CCPA-and NECA-treated rats were pretreated with the A 1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 100 g/kg), the A 2a/ A 2b antagonist 4-(-2-[7-amino-2-{2-furyl}{1,2,4}triazolo{2,3-a} {1,3,5}triazin-5-yl-amino]ethyl)phenol (ZM-241385, 1.5 mg/kg) or the A 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 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 A2a receptor agonist does not induce cardioprotection, antagonism of the A2a and/or the A2b receptor blocks the cardioprotection associated with adenosine agonist pretreatment. adenosine receptor; ischemia-reperfusion; infarct reduction IT IS NOW WELL-RECOGNIZED that the administration of adenosine before myocardial ischemia (i.e., pretreatment) protects against both reversible and irreversible injury, an effect that appears to be mediated via the activation of cardiomyocyte A 1 and/or A 3 adenosine receptor subtypes (14, 21). The results of a very limited number of reports indicate that the administration of adenosine A 2a agonists before ischemia is not cardioprotective (1,13,32,33). However, there is now significant evidence that A 2a receptor activation during reperfusion (i.e., postconditioning) reduces myocardial infarct size (7,11,12,(35)(36)(37). The results of in vivo studies suggest that A 2a receptor postconditioning is mediated primarily via inhibition of inflammatory cell adhesion to vascular endothelial cells (7, 37). Additional reports...

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 after 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.

Adenosine A1 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 A1 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 A1 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 A1 agonist 2-chloro-N6-cyclopentyladenosine (CCPA; 100 microg/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 1400 W 10 min before ischemia. Treatment with CCPA reduced infarct size from 48 +/- 2 to 28 +/- 2% of the area at risk, an effect that was blocked by both SB-203580 and PD-098059 but not 1400 W. Ventricular myocytes isolated 24 h after CCPA injection exhibited significantly reduced oxidative stress during H2O2 exposure compared with myocytes from vehicle-injected animals, and this effect was not blocked by the iNOS inhibitor 1400 W. 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 A1 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.