Abstract

Opioids generate free radicals that mediate protection in isolated cultured cardiomyocytes. We hypothesize that the nature of these radicals is nitric oxide, and that nitric oxide activates the protein kinase C (PKC) δ isoform. Through this signal transduction pathway, opiates protect cardiomyocytes during hypoxia and reoxygenation. Cell viability was quantified in chick embryonic ventricular myocytes with propidium iodide. Oxygen radicals were quantified using a molecular probe, 2′,7′-dichlorofluorescin diacetate (DCFH-DA). After a 10-min infusion of the opioid δ receptor agonist BW373U86 (BW; 2 or 20 pM) and a 10-min drug-free period, cells were subjected to hypoxia for 1 h followed by reoxygenation for 3 h. BW produced a concentration-dependent reduction in cardiomyocyte death (2 pM, 35.3 ± 3.9%, n = 5; 20 pM, 21.5 ± 4.0%, n = 8, p < 0.05 versus controls) and attenuated oxidant stress compared with controls (43.3 ± 4.2%, n = 8). The increase in DCFH-DA oxidation with BW before hypoxia was abolished by the specific nitric-oxide synthase inhibitors nitro-l-arginine methyl ester (l-NAME) orN^G-monomethyl-l-arginine (l-NMMA) (100 μM each). l-NAME orl-NMMA blocked the protective effects of BW. BW selectively increased the activity of PKC δ isoform in the particulate fraction, and its protection was abolished by the selective PKC δ inhibitor rottlerin (1 μM). Similar to BW, infusion with 5 μM of the nitric oxide donorS-nitroso-N-acetylpenicillamine (SNAP) reduced cardiomyocyte death (24.6 ± 3.7, n = 8), and this protection was blocked by chelerythrine or rottlerin. Chelerythrine and rottlerin had no effect on BW-generated oxygen radicals before hypoxia, but they abolished the protection of SNAP. The nature of DCFH oxidation produced by opioid δ receptor stimulation is nitric oxide. Nitric oxide mediates cardioprotection via activating PKC δ in isolated myocytes.