Signalling mechanisms in contraction-mediated stimulation of intracellular NO production in cat ventricular myocytes.
In this study we sought to determine whether contractile activity has a role as a signalling mechanism in the activation of intracellular nitric oxide (NO(i)) production induced by electrical stimulation of cat ventricular myocytes. Field stimulation (FS) of single ventricular myocytes elicited frequency-dependent increases in NO(i) that were blocked by the calmodulin (CaM) inhibitor 10 microM W-7 and partially inhibited by the phosphatidylinositol 3'-kinase (PI-(3)K) inhibitor 10 microMm LY294002. Increasing extracellular [Ca(2+)] caused a concentration-dependent increase in FS-induced NO(i) that was partially inhibited by LY294002. The negative inotropic agents BDM (5 mm) or blebbistatin (10 microM) decreased cell shortening and NO(i) production without concomitant changes in L-type Ca(2+) current (I(Ca,L)) or [Ca(2+)](i) transients. The positive inotropic agents EMD 57033 or CGP 48506 (1 microM) increased cell shortening and NO(i) production without concomitant changes in I(Ca,L) or [Ca(2+)](i) transients. FS-induced NO(i) production was decreased in myocytes infected (100 multiplicity of viral infection (MOI); 24 h) with a replication-deficient adenovirus expressing a dominant-negative mutant of protein kinase B (Akt) compared with cells infected with a control adenovirus expressing beta-galactosidase. FS-induced NO(i) was partially inhibited by either endothelial (eNOS) or neuronal nitric oxide synthase (nNOS) inhibitors and completely blocked by simultaneous exposure to both. FS-induced [Ca(2+)](i) transients were increased by the nNOS inhibitor nNOS-I (0.24 microM), decreased by the eNOS inhibitor L-NIO (1 microM) and unchanged by exposure to both inhibitors. We conclude that in cat ventricular myocytes, FS-induced NO(i) production requires both Ca(2+)-dependent CaM signalling and Ca(2+)-independent PI-(3)K-Akt signalling activated by contractile activity. FS activates NO(i) production from both eNOS and nNOS, and each source of NO(i) exerts opposing effects on [Ca(2+)](i) transient amplitude. These findings are important for understanding the regulation of NO(i) signalling in the normal and mechanically failing heart.
Dedkova
E N
EN
Department of Physiology, Loyola University Medical Center, 2160 S. First Avenue, Maywood, IL 60153, USA.
Wang
Y G
YG
Ji
X
X
Blatter
L A
LA
Samarel
A M
AM
Lipsius
S L
SL
D000818:Animals; D000889:Anti-Arrhythmia Agents / Q000494:pharmacology; D002118:Calcium / Q000502:physiology; D020013:Calcium Signaling / Q000502:physiology; D002316:Cardiotonic Agents / Q000494:pharmacology; D002415:Cats; D002867:Chromones / Q000494:pharmacology; D004558:Electric Stimulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006352:Heart Ventricles / Q000166:cytology; D007150:Immunohistochemistry; D007546:Isoquinolines / Q000494:pharmacology; D008297:Male; D009025:Morpholines / Q000494:pharmacology; D009200:Myocardial Contraction / Q000502:physiology*; D032383:Myocytes, Cardiac / Q000378:metabolism*; D009569:Nitric Oxide / Q000096:biosynthesis*; D052248:Nitric Oxide Synthase Type I / Q000037:antagonists & inhibitors; D052250:Nitric Oxide Synthase Type III / Q000037:antagonists & inhibitors; D051058:Oncogene Protein v-akt / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D015398:Signal Transduction / Q000502:physiology*; D013449:Sulfonamides / Q000494:pharmacology; D016276:Ventricular Function
D000889:Anti-Arrhythmia Agents; D002118:Calcium; D002316:Cardiotonic Agents; D002867:Chromones; D004791:Enzyme Inhibitors; D007546:Isoquinolines; D009025:Morpholines; D009569:Nitric Oxide; D052248:Nitric Oxide Synthase Type I; D052250:Nitric Oxide Synthase Type III; D051058:Oncogene Protein v-akt; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D013449:Sulfonamides; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide