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Departments of 1 Anesthesia and Critical Care and 2 Medicine, University of Chicago, Chicago, Illinois 60637; and 3 Department of Anesthesia and Critical Care, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114
We examined the ability of ACh to mimic ischemic preconditioning in cardiomyocytes and the role of ATP-sensitive potassium (KATP) channels and mitochondrial reactive oxygen species (ROS) in mediating this effect. Chick embryonic ventricular myocytes were studied in a flow-through chamber while flow rate, pH, PO2, and PCO2 were controlled. Cell viability was quantified with propidium iodide (5 µM), and production of ROS was measured using 2',7'-dichlorofluorescin diacetate. Data were expressed as means ± SE. Preconditioning with 10 min of ischemia followed by 10 min of reoxygenation or 10 min of ACh (1 mM) followed by a drug-free period before 1 h of ischemia and 3 h of reoxygenation reduced cell death to the same extent [preconditioning 19 ± 2% (n = 6, P < 0.05) ACh 21 ± 5% (n = 6, P < 0.05) vs controls 42 ± 5% (n = 9)]. Like preconditioning, ACh increased ROS production threefold before ischemia [0.60 ± 0.16 (n = 7, P < 0.05) vs. controls, 0.16 ± 0.03 (n = 6); arbitrary units]. Protection and increased ROS production during ACh preconditioning were abolished with 5-hydroxydecanoate (5-HD, 100 µM), a selective mitochondrial KATP channel antagonist, and the thiol reductant 2-mercaptopropionyl glycine (2-MPG, 1 mM), an antioxidant [cell death: 5-HD+ACh 37 ± 7% (n = 5), 2-MPG+ACh 47 ± 6% (n = 6); ROS signals: 5-HD+ACh 0.09 ± 0.03 (n = 5), 2-MPG+ACh 0.01 ± 0.04 (n = 4)]. In addition, ACh-induced ROS signaling was blocked by the mitochondrial site III electron transport inhibitor myxothiazol (0.02 ± 0.07, n = 5). These results demonstrate that activation of mitochondrial KATP channels and increased ROS production from mitochondria are important intracellular signals that participate in ACh-induced preconditioning in cardiomyocytes.
signal transduction; adenosine 5'-triphosphate-dependent potassium channels; reactive oxygen species; mitochondria
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