Role of nitric oxide and protein kinase C in ACh-induced cardioprotection

We examined the roles of nitric oxide and protein kinase C (PKC) in ACh-pro duced protection of cultured cardiomyocytes during simulated ischemia and r eoxygenation. Cell viability was quantified using propidium iodide in chick embryonic ventricular myocytes. O-2 radicals were quantified using 2', 7'- dichlorofluorescin diacetate. After a 10-min infusion of ACh (0.5 or 1 mM) and a 10-min drug-free period, we simulated ischemia for 1 h and reoxygenat ion for 3 h. ACh reduced cardiocyte death [32 +/- 4%; n = 6 and 23 +/- 4%; n = 7 (P< 0.05)] and attenuated oxidant stress during ischemia and reoxygen ation in a concentration-dependent manner compared with controls (47 +/- 4% ; n = 8; P< 0.05). The increase in O-2 radicals before simulated ischemia [ 357 +/- 49; n = 4 and 528 +/- 52; n = 8 vs. 211 +/- 34; n = 8; P< 0.05 (arb itrary units)] was abolished by the specific nitric oxide synthase inhibito r N-G-nitro-L-arginine methyl ester (L-NAME) and was markedly attenuated by N-G-monomethyl-L-arginine (L-NMMA). L-NAME or L-NMMA blocked the protectiv e effects of ACh, which selectively increased PKC-<epsilon> isoform activit y in the particulate fraction. The PKC inhibitor Go-6976 had no effect on O -2 radical production before simulated ischemia but it abolished the protec tion; therefore nitric oxide is a large component of ACh-generated O-2 radi cals. Nitric oxide and O-2 radicals activate the PKC-epsilon isoform by whi ch ACh protects against injury.