Signaling mechanisms that mediate nitric oxide production induced by acetylcholine exposure and withdrawal in cat atrial myocytes.
Fluorescence microscopy and the NO-sensitive indicator 4,5-diaminofluorescein were used to determine the effects of acetylcholine (ACh) on intracellular NO (NOi) in cat atrial myocytes. Field stimulation (1 Hz) of cells or exposure of quiescent cells to ACh (1 to 10 micromol/L) had no effect on NOi. However, in field-stimulated cells, ACh exposure increased NOi, and ACh withdrawal elicited an additional, prominent increase in NOi production. During ACh exposure, addition of 1 micromol/L atropine increased NOi production similar to ACh withdrawal. ACh-induced increases in NOi were reduced by prior exposure to 1 mmol/L extracellular Ca2+ ([Ca2+]o) and prevented by 0.5 mmol/L [Ca2+]o, 1 micromol/L verapamil, 1 micromol/L atropine, 10 micromol/L L-N5-(1-iminoethyl)ornithine, 10 micromol/L W-7, or incubating cells in pertussis toxin or 10 micromol/L LY294002 (inhibits phosphatidylinositol 3-kinase). Switching to 0.5 mmol/L [Ca2+]o during ACh withdrawal prevented the additional increase in NOi. ACh exposure increased phosphorylation (Ser473) of protein kinase B (Akt), and this effect was blocked by LY294002 and unaffected in low (0.5 mmol/L) [Ca2+]o. Confocal microscopy revealed that ACh exposure increased NOi at local subsarcolemmal sites, and ACh withdrawal additionally increased NOi by recruiting additional subsarcolemmal release sites. Disruption of caveolae by 2 mmol/L methyl-beta-cyclodextrin abolished ACh-induced NOi production. We conclude that in cat atrial myocytes, ACh stimulates NOi release from local subsarcolemmal sites. ACh-induced increases in NOi requires both muscarinic receptor-mediated Gi protein/phosphatidylinositol 3-kinase/Akt signaling and voltage-activated Ca2+ influx for stimulation of calmodulin-dependent endothelial NO synthase activity. Increases in NOi elicited by ACh withdrawal result from the recovery of Ca2+ influx after ACh inhibition. NO signaling elicited by ACh withdrawal stimulates rapid recovery from cholinergic atrial inhibition.
Dedkova
Elena N
EN
Department of Physiology, Loyola University Medical Center, 2160 S First Ave, Maywood, Ill 60153, USA.
Ji
Xiang
X
Wang
Yong Gao
YG
Blatter
Lothar A
LA
Lipsius
Stephen L
SL
D000109:Acetylcholine / Q000494:pharmacology*; D000818:Animals; D001285:Atropine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D002415:Cats; D002867:Chromones / Q000494:pharmacology; D003505:Cyclodextrins / Q000494:pharmacology; D004558:Electric Stimulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D006325:Heart Atria / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008297:Male; D009025:Morpholines / Q000494:pharmacology; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis*; D037342:Pertussis Toxin / Q000494:pharmacology; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D011976:Receptors, Muscarinic / Q000378:metabolism; D015398:Signal Transduction; D013449:Sulfonamides / Q000494:pharmacology; D014665:Vasodilator Agents / Q000494:pharmacology*; D047392:beta-Cyclodextrins
D002867:Chromones; D003505:Cyclodextrins; D004791:Enzyme Inhibitors; D009025:Morpholines; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011518:Proto-Oncogene Proteins; D011976:Receptors, Muscarinic; D013449:Sulfonamides; D014665:Vasodilator Agents; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D009569:Nitric Oxide; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C017967:W 7; D001285:Atropine; D037342:Pertussis Toxin; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D000109:Acetylcholine; D002118:Calcium