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J Physiol. 2007 Apr 1;580(Pt 1):327-45. Epub 2007 Jan 18.

Signalling mechanisms in contraction-mediated stimulation of intracellular NO production in cat ventricular myocytes.

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Department of Physiology, Loyola University Medical Center, 2160 S. First Avenue, Maywood, IL 60153, USA.


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.

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