© 2002 by European Society of Cardiology
Copyright © 2002, European Society of Cardiology
Acetylcholine leads to free radical production dependent on KATP channels, Gi proteins, phosphatidylinositol 3-kinase and tyrosine kinase
aDepartment of Physiology, MSB 3024, College of Medicine, University of South Alabama, Mobile, AL 36688, USA
bDepartment of Medicine, College of Medicine, University of South Alabama, Mobile, AL, USA
* Corresponding author. Tel.: +1-251-460-6818; fax: +1-251-460-6464 jdowney{at}usouthal.edu
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 KATP channels (mito KATP) 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 Gi 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 KATP channel opener, and valinomycin, a potassium ionophore, also significantly increased ROS production, and these effects were not blocked by PTX, while the KATP channel closer 5-hydroxydecanoate blocked ACh-induced ROS production (92.3±3.8%). These results suggest ROS production is directly influenced by KATP 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 KATP (presumably that in mitochondria).
KEYWORDS Acetylcholine; Free radicals; K-ATP channel; Mitochondria; Preconditioning; Protein kinases; Signal transduction
1 Present address: Department of Pharmacology, Physiology and Therapeutics, University of North Dakota, Grand Forks, ND, USA.
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