Cardioprotective PKG-independent NO signaling at reperfusion

Abstract

Cell models of ischemic preconditioning (IPC) indicate nitric oxide (NO) is involved in protection accruing during reoxygenation but disagree whether it acts through PKG. Using a more relevant intact heart model, we studied isolated rabbit hearts subjected to 30-min coronary artery occlusion/120-min reperfusion. We previously found protection from PKG activator 8-(4-chlorophenylthio)-guanosine 3′,5′-cyclic monophosphate (CPT-cGMP) at reperfusion was blocked by A2b adenosine receptor (A2bAR), ERK, or phosphatidylinositol 3-kinase (PI3-kinase) blockers. In this investigation A2bAR agonist BAY 60-6583 or CPT-cGMP at reperfusion reduced infarction comparably to IPC. Their protection was abrogated by Nω-nitro-l-arginine methyl ester (l-NAME), suggesting a PKG-independent NO synthase in IPC's mediator pathway downstream of PKG and A2bAR. NO donor S-nitroso- N-acetyl-d,l-penicillamine (SNAP) at reperfusion also protected. This protection was not blocked by PI3-kinase inhibitor wortmannin or ERK antagonist PD-98059, suggesting NO acted downstream of these kinases. Protection from SNAP was not affected by mitochondrial ATP-sensitive K+ channel closer 5-hydroxydecanoate, PKC antagonist chelerythrine, reactive oxygen species scavenger N-2-mercaptopropionylglycine, or soluble guanylyl cyclase antagonist 1 H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). Absence of ODQ effect indicated NO was acting independently of PKG. BAY 58-2667, a soluble guanylyl cyclase activator, was protective, and l-NAME blocked its infarct-sparing effect, indicating a second signaling event dependent on NO generation but independent of PKG. SB216763, a blocker of glycogen synthase kinase-3β (GSK-3β), decreased infarct size, and its infarct-sparing effect was not affected by l-NAME, suggesting GSK-3β acted downstream or independently of NO. Hence, NO signaling occurs in IPC's mediator pathway downstream of Akt and ERK, and its protection is independent of PKG.