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 Red, which is oxidized to a fluorescent form after exposure to ROS. The mitochondrial
ATP-sensitive
potassium (mK(
ATP)) channel opener
diazoxide (50 microM) 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 microM 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 microM
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 microM) (-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 microM 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.