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Acetylcholine leads to free radical production dependent on KATP channels, Gi proteins, phosphatidylinositol 3-kinase and tyrosine kinase

OBJECTIVE Acetylcholine (ACh) mimics ischemic preconditioning (PC) and therefore protects the heart against lethal ischemia. Steps common to both ischemic and drug-induced PC are opening of mitochondrial K(ATP) channels (mito K(ATP)) and generation of reactive oxygen species (ROS). The aim of this study was to test whether ACh-induced ROS production could be seen in a vascular smooth muscle cell line, and, if so, to investigate the underlying signaling pathway. METHODS Mitochondrial ROS generation was quantified by measuring changes in fluorescence of ROS-sensitive intracellular markers in vascular smooth muscle cells (A7r5). RESULTS Fluorescence, and, therefore, ROS production, was increased to 197.5+/-8.5% of baseline after 45 min of exposure of cells to 2 mM ACh (P<0.001 vs. untreated controls). This effect was blocked by co-treatment with a muscarinic receptor antagonist (atropine 102.8+/-2.9%, 4-DAMP 92.6+/-7.4%) or by inhibition of G(i) with pertussis toxin (PTX) (90.5+/-4.4%), implicating a receptor-mediated rather than non-specific effect of ACh. The increased fluorescence induced by ACh was also abrogated by the free radical scavenger N-(2-mercaptopropionyl) glycine (104.2+/-10.1%), documenting that ROS were indeed the cause of the enhanced fluorescence. Both diazoxide, a K(ATP) channel opener, and valinomycin, a potassium ionophore, also significantly increased ROS production, and these effects were not blocked by PTX, while the K(ATP) channel closer 5-hydroxydecanoate blocked ACh-induced ROS production (92.3+/-3.8%). These results suggest ROS production is directly influenced by K(ATP) activity and K(+) movements in the cell. The tyrosine kinase inhibitor genistein (102.8+/-6.6%) and the phosphatidylinositol 3 (PI3)-kinase inhibitor wortmannin (90.7+/-4.1%) also inhibited the ability of ACh to increase ROS production. CONCLUSION The signaling pathway by which ACh leads to ROS generation in A7r5 cells involves a muscarinic surface receptor, a pertussis toxin-sensitive G protein, PI3-kinase, at least one tyrosine kinase, and a 5-hydroxydecanoate (5-HD)-dependent K(ATP) (presumably that in mitochondria).

The relative order of mKATP channels, free radicals and p38 MAPK in preconditioning's protective pathway in rat heart

OBJECTIVES Ischemic preconditioning (PC) reduces myocardial infarction by a mechanism that involves opening of mitochondrial ATP-dependent potassium channels (mK(ATP)), reactive oxygen species (ROS), and possibly activation of p38 mitogen-activated protein kinase (p38 MAPK). The actual order of these steps, however, is a matter of current debate. This study examined whether protection afforded by menadione, which protects by causing mitochondria to produce ROS, requires mK(ATP) opening. In addition, we tested whether protection from anisomycin, a p38 MAPK activator, is dependent on ROS production. METHODS AND RESULTS Isolated, buffer-perfused rat hearts were pretreated with menadione, and infarction was assessed after 30 min of regional ischemia and 120 min of reperfusion. Menadione reduced infarction in a dose-dependent manner with an EC(50) of 270 nM. Menadiones infarct-limiting effect was insensitive to 200 microM 5-hydroxydecanoate (5HD), an mK(ATP) channel blocker, whereas protection by diazoxide and PC were blocked by 5HD. Anisomycin caused hearts to resist infarction and this protective effect was abrogated by SB203580, a p38 MAPK inhibitor, and 2-mercaptopropionylglycine (MPG), a free radical scavenger. CONCLUSIONS These results indicate that mK(ATP) opening occurs upstream of mitochondrial ROS generation in the protective pathway. Furthermore, protection afforded by anisomycin was p38 MAPK- and ROS-dependent.