Acetylcholine leads to free radical production dependent on K(ATP) channels, G(i) 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.


CONCLUSIONS
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).
Oldenburg
Olaf
O
Department of Physiology, MSB 3024, College of Medicine, University of South Alabama, Mobile 36688, USA.
Qin
Qining
Q
Sharma
Ana R
AR
Cohen
Michael V
MV
Downey
James M
JM
Benoit
Joseph N
JN
D000109:Acetylcholine / Q000494:pharmacology*; D000704:Analysis of Variance; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D001011:Aorta; D001285:Atropine / Q000494:pharmacology; D002460:Cell Line; D003652:Decanoic Acids / Q000494:pharmacology; D003981:Diazoxide / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D016166:Free Radical Scavengers / Q000494:pharmacology; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019833:Genistein / Q000494:pharmacology; D006880:Hydroxy Acids / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D019194:Ischemic Preconditioning / Q000379:methods; D008297:Male; D016210:Methacholine Chloride / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D018721:Muscarinic Agonists / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D010477:Perfusion; D037342:Pertussis Toxin / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010880:Piperidines / Q000494:pharmacology; D026902:Potassium Channel Blockers / Q000494:pharmacology; D015221:Potassium Channels / Q000187:drug effects / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism*; D011950:Receptors, Cholinergic / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D008625:Tiopronin / Q000494:pharmacology; D014634:Valinomycin / Q000494:pharmacology; D000077191:Wortmannin
D000109:Acetylcholine; D000730:Androstadienes; D001285:Atropine; D003652:Decanoic Acids; D003981:Diazoxide; D004791:Enzyme Inhibitors; D016166:Free Radical Scavengers; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D019833:Genistein; D006880:Hydroxy Acids; D007476:Ionophores; D016210:Methacholine Chloride; D018721:Muscarinic Agonists; D037342:Pertussis Toxin; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010880:Piperidines; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D011505:Protein-Tyrosine Kinases; D017382:Reactive Oxygen Species; D011950:Receptors, Cholinergic; D008625:Tiopronin; D014634:Valinomycin; D000077191:Wortmannin; C052853:5-hydroxydecanoic acid; C042375:4-diphenylacetoxy-1,1-dimethylpiperidinium