u-0126 and 1-3-dipropyl-8-cyclopentylxanthine

Ischemic preconditioning (IPC) is thought to protect by activating survival kinases during reperfusion. We tested whether binding of adenosine receptors is also required during reperfusion and, if so, how long these receptors must be populated. Isolated rabbit hearts were subjected to 30 min of regional ischemia and 2 h of reperfusion. IPC reduced infarct size from 32.1 +/- 4.6% of the risk zone in control hearts to 7.3 +/- 3.6%. IPC protection was blocked by a 20-min pulse of the nonselective adenosine receptor blocker 8-(p-sulfophenyl)-theophylline when started either 5 min before or 10 min after the onset of reperfusion but not when started after 30 min of reperfusion. Protection was also blocked by either 8-cyclopentyl-1,3-dipropylxanthine, an adenosine A1-selective receptor antagonist, or MRS1754, an A2B-selective antagonist, but not by 8-(3-chlorostyryl)caffeine, an A2A-selective antagonist. Blockade of phosphatidylinositol 3-OH kinase (PI3K) with a 20-min pulse of wortmannin also aborted protection when started either 5 min before or 10 or 30 min after the onset of reperfusion but failed when started after 60 min of reflow. U-0126, an antagonist of MEK1/2 and therefore of ERK1/2, blocked protection when started 5 min before reperfusion but not when started after only 10 min of reperfusion. These studies reveal that A1 and/or A2B receptors initiate the protective signal transduction cascade during reperfusion. Although PI3K activity must continue long into the reperfusion phase, adenosine receptor occupancy is no longer needed by 30 min of reperfusion, and ERK activity is only required in the first few minutes of reperfusion.

Topics: Acetamides; Adenosine A1 Receptor Antagonists; Adenosine A2 Receptor Antagonists; Androstadienes; Animals; Butadienes; Caffeine; Female; Hemodynamics; Ischemic Preconditioning, Myocardial; Male; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Myocardial Infarction; Nitriles; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Purinergic P1 Receptor Antagonists; Purines; Rabbits; Receptors, Purinergic P1; Theophylline; Wortmannin; Xanthines

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,4a(S*)]-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 +/- 3% to 29 +/- 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 A1 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.

Topics: Animals; Butadienes; Enzyme Inhibitors; Imidazoles; Ischemic Preconditioning, Myocardial; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Myocardial Infarction; Nitriles; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Pyridines; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P1; Reperfusion Injury; Subcellular Fractions; Triazines; Triazoles; Xanthines