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 A(2b) adenosine receptor (A(2b)AR), ERK, or phosphatidylinositol 3-kinase (PI3-kinase) blockers. In this investigation A(2b)AR agonist BAY 60-6583 or CPT-cGMP at reperfusion reduced infarction comparably to IPC. Their protection was abrogated by N(ω)-nitro-l-arginine methyl ester (l-NAME), suggesting a PKG-independent NO synthase in IPC's mediator pathway downstream of PKG and A(2b)AR. 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 1H-[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.

Fig. 1.

Proposed signaling scheme for ischemic preconditioning. Note that in the schematic, nitric oxide synthase (NOS) signals through protein kinase G (PKG) upstream in the trigger pathway but acts in a PKG-independent manner downstream in the mediator pathway at reperfusion. The role of glycogen synthase kinase-3β (GSK-3β) is controversial, but if it is in the pathway, then it must be downstream of the newly described NOS site. AR, adenosine receptor; PI3-K, phosphatidylinositol 3-kinase; PLC, phospholipase C; PLD, phospholipase D; ERK, extracellular signal-regulated kinase; GC, guanylyl cyclase; PKC, protein kinase C; mK_ATP, mitochondrial ATP-dependent K⁺ channel; mPTP, mitochondrial permeability transition pore; RISK, reperfusion injury survival kinases.

Fig. 2.

Experimental protocols. BAY 60, BAY 60-6583; Chel, chelerythrine; 5-HD, 5-hydroxydecanoic acid; IPC, ischemic preconditioning; l-NAME, N^ω-nitro-l-arginine methyl ester; MPG, N-2-mercaptopropionylglycine; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; PD, PD-98059; SB, SB216763; Wort, wortmannin. S-nitroso-N-acetyl-d,l-penicillamine (SNAP) was present alone at 2 μM for either 15 or 35 min, and doses of 4 and 6 μM were infused for 35 min.

Fig. 3.

Infarct size as a percentage of risk zone for control group 1 rabbits and those treated with either 1 or 3 cycles of IPC (IPC × 1 or 3), BAY 60, or 8-(4-chlorophenylthio)-cGMP (CPT) alone or with coadministered l-NAME, Wort, or PD. Open circles represent individual hearts, and solid circles indicate group means ± SE; shaded circles for IPC × 1 represent historical data (43). Whereas IPC, BAY 60, and CPT were individually very protective, l-NAME aborted that protection. Wort and PD each also blocked BAY 60's protective effect. *P < 0.001.

Fig. 4.

Infarct size as a percentage of risk zone for control group 1 rabbits and those treated with 2 μM SNAP for either 15 (SNAP 15) or 35 min (SNAP 35) and rabbits treated simultaneously with the 35-min SNAP infusion and Wort, PD, Chel, 5-HD, MPG, or ODQ. Open circles represent individual hearts, and solid circles indicate group means ± SE. The longer infusion of SNAP was protective, and this protection could not be abrogated by any of the antagonists (Wort, PD, Chel, 5-HD, ODQ), the reactive oxygen species scavenger (MPG), or the K_ATP channel closer (5-HD). *P < 0.001.

Fig. 5.

SNAP was administered at 3 different doses in the presence and absence of l-NAME. l-NAME blocked the protection from low-dose but not high-dose SNAP. Loss of protection at the low dose of SNAP was presumably due to the loss of endogenous NO, which would have been additive with that from SNAP. Open circles represent individual hearts, and solid circles indicate group means ± SE. *P < 0.001.

Fig. 6.

Infarct size as a percentage of risk zone for control group 3 rabbits and those treated with BAY 58-2667 (BAY 58) alone or simultaneously with l-NAME. Open circles represent individual hearts, and solid circles indicate group means ± SE. BAY 58 was protective, and this protection was completely blocked by l-NAME. *P < 0.001.

Fig. 7.

Infarct size as a percentage of risk zone for control group 2 rabbits and those treated with SB alone or simultaneously with l-NAME. Open circles represent individual hearts, and solid circles indicate group means ± SE. SB was protective, and this protection was not affected by l-NAME. *P < 0.001.