Acetylcholine-induced production of reactive oxygen species in adult rabbit ventricular myocytes is dependent on phosphatidylinositol 3-and Src-kinase activation and mitochondrial KATP channel opening

Acetylcholine (ACh), like ischemic preconditioning (PC), protects against infarction and is dependent on generation of reactive oxygen species (ROS). To investigate the mechanism by which ACh causes ROS production, isolated adult rabbit cardiomyocytes underwent a timed incubation in reduced MitoTracker(TM) Red, which is oxidized to a fluorescent form after exposure to ROS. The mitochondrial ATP-sensitive potassium (mK(ATP)) channel opener diazoxide (50 muM) increased fluorescence by 47 +/- 9% (P = 0.007), indicating that opening of mK(ATP) leads to ROS generation. and that increase was blocked by the mK(ATP) blocker 5-hydroxydecanoate (5HD, 1 mM); 250 muM ACh caused a similar increase in ROS generation (+45 +/- 6% for all experiments, P < 0.001). ACh-induced ROS production was prevented by (1) blockade of muscarinic surface receptors with 100 mu M atropine (-6 +/- 2%, P = n.s.) or 250 nM 4-DAMP (+5 +/- 13%, P = n.s.), indicating that ACh's effect was receptor mediated; (2) closing K-ATP channels with either the non-selective channel closer glibenclamide (50 mu M) (-1.2 +/- 17%, P = n.s.) or the selective mK(ATP) closer 5HD (-1.8 +/- 9%, P = n.s.), indicating that increased ROS production involved opening of mK(ATP); (3) blockade of mitochondrial electron transport chain with 200 nM myxothiazol (-4 +/- 9%, P = n.s.), indicating ROS came from the mitochondria; (4) addition of 100 nM wortmannin (-13 +/- 12%, P = n.s.), indicating that phosphatidylinositol 3-(PI3)-kinase was involved; and (5) blockade of Src-kinase with 1 mu M PP2 (-2 +/- 5%, P = n.s.), indicating the involvement of an Src-kinase. These results support the hypothesis that occupation of muscarinic surface receptors by ACh causes activation of PI3- and Src-kinases that then open mK(ATP) resulting in mitochondrial ROS generation and triggering of the preconditioned state. (C) 2003 Elsevier Science Ltd. All rights reserved.