These results suggest that direct stimulation of cardiocyte opioid delta(1) receptors leads to activation of mitochondrial K(ATP) channels. The resultant increase of intracellular free radical signals may be an important component of the signaling pathways by which morphine mimics preconditioning in cardiomyocytes.

Oxygen radicals and protein kinase C (PKC) mediate ischemic preconditioning. Using a cultured chick embryonic cardiomyocyte model of hypoxia and reoxygenation, we found that the oxygen radicals generated by ischemic preconditioning were H 2O2. Like preconditioning, H 2O2 selectively activated the ⑀-isoform of PKC in the particulate compartment and increased cell viability after 1 h of hypoxia and 3 h of reoxygenation. The glutathione peroxidase ebselen (converting H 2O2 to H2O) and the superoxide dismutase inhibitor diethyldithiocarbamic acid abolished the increased H 2O2 and the protection of preconditioning. PKC activation with phorbol 12-myristate 13-acetate increased cell survival; the protection of preconditioning was blocked by ⑀V 1-2, a selective PKC-⑀ antagonist. Similar to preconditioning, the protection of PKC activation was abolished by mitochondrial K ATP channel blockade with 5-hydroxydecanoate or by GABA receptor stimulation with midazolam or diazepam. In addition, PKC, mitochondrial ATP-sensitive K ϩ (KATP) channels, and GABA receptors had no effects on H 2O2 generated by ischemic preconditioning before prolonged hypoxia and reoxygenation. We conclude that H 2O2 opens mitochondrial KATP channels and inhibits GABA receptors via activating PKC-⑀. Through this signal transduction, preconditioning protects ischemic cardiomyocytes.␥-aminobutyric acid receptors; preconditioning OXYGEN RADICALS are important intracellular second messengers that mediate cardioprotection (7, 9). Preconditioning and flumazenil generate oxygen radicals and protect cardiomyocytes during ischemia-reperfusion (42). Flumazenil inhibits the GABA receptor complex (20). The GABA receptor antagonist is used clinically for reversal of apnea and loss of consciousness associated with oversedation (2) and might be beneficial in protecting against cerebral ischemia (20). Rapid administration of a GABA complex antagonist produces minimal coronary and left ventricular hemodynamic responses (24). GABA receptor antagonists might have a role in the management of patients with ischemic heart disease if they can mimic preconditioning to protect the heart and thus have cardiac protective properties. The first objective of our study was to examine the role of GABA receptors in the development of preconditioning protection.GABA receptors have been identified in mitochondria (2,19,27,28). Reactive oxygen species originating from mitochondria, ATP-sensitive K ϩ (K ATP ) channels, and protein kinase C (PKC) are second messengers in cardioprotection produced by hypoxic preconditioning, acetylcholine, opioids, and flumazenil (25,31,34,37,38). A second objective of this study was to examine the role of GABA receptors on preconditioning-induced oxygen radicals before the start of ischemia and to determine the sequence of GABA receptors, PKC, and oxygen radicals in the signal transduction pathway of ischemic preconditioning.Cardiocyte injury in ischemia-reperfusion models correlates with the amount of free radicals produced (33), especially during the fir...

Preconditioning reduces cardiomyocyte necrosis in vivo and in vitro, but it is unknown whether preconditioning blocks apoptosis. We wanted to compare the effects of preconditioning on necrosis and apoptosis in cardiomyocytes. Necrosis was detected with propidium iodide, and apoptosis was quantified by three complementary techniques: flow cytometry, TdT-mediated dUTP nick-end labeling assay, and DNA-laddering electrophoresis. Apoptosis increased with simulated ischemia time (6 h, 19 +/- 1%; 12 h, 27 +/- 2%; 18 h, 40 +/- 4%; 24 h, 54 +/- 4%; and 36 h, 83 +/- 4%; n = 6 for each group). Simulated ischemia and reoxygenation contributed equally to apoptosis (12-h ischemia, 27 +/- 2%, n = 6; 12-h ischemia and 12-h reoxygenation, 51 +/- 4%, n = 6; and 24-h ischemia, 54 +/- 5%, n = 8). Necrosis occurred primarily during reoxygenation; none was detected during simulated ischemia. Preconditioning with 10 min of simulated ischemia reduced necrosis (18 +/- 6%, n = 8) but had no effect on apoptosis. However, three 1-min cycles of simulated ischemia separated by 5 min of reoxygenation reduced necrosis and apoptosis similarly. The protein kinase C (PKC) inhibitors Go6976 (0.1 microM) or chelerythrene (4 microM) abolished the effect of preconditioning. Preconditioning selectively activated PKC epsilon but had no effect on PKC delta and on total PKC enzyme activity. Preconditioning protected against necrosis and apoptosis, but the preconditioning ischemia required for blocking apoptosis was less than that for reducing necrosis. Activation of PKC epsilon isoform is important in mediating the protection.

We examined the roles of nitric oxide and protein kinase C (PKC) in ACh-produced protection of cultured cardiomyocytes during simulated ischemia and reoxygenation. 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 reoxygenation 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 reoxygenation 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 (arbitrary units)] was abolished by the specific nitric oxide synthase inhibitor 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 protective effects of ACh, which selectively increased PKC-epsilon isoform activity in the particulate fraction. The PKC inhibitor Gö-6976 had no effect on O(2) radical production before simulated ischemia but it abolished the protection; therefore nitric oxide is a large component of ACh-generated O(2) radicals. Nitric oxide and O(2) radicals activate the PKC-epsilon isoform by which ACh protects against injury.

Stimulation of delta1-opioid receptors generates oxygen radicals via mitochondrial KATP channels. This signaling pathway attenuates oxidant stress and cell death in cardiomyocytes.