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

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Abstract
In this study we sought to determine whether contractile activity has a role as a signalling mechanism in the activation of intracellular nitric oxide (NOi) production induced by electrical stimulation of cat ventricular myocytes. Field stimulation (FS) of single ventricular myocytes elicited frequency‐dependent increases in NOi that were blocked by the calmodulin (CaM) inhibitor 10 μm W‐7 and partially inhibited by the phosphatidylinositol 3′‐kinase (PI‐(3)K) inhibitor 10 μm LY294002. Increasing extracellular [Ca2+] caused a concentration‐dependent increase in FS‐induced NOi that was partially inhibited by LY294002. The negative inotropic agents BDM (5 mm) or blebbistatin (10 μm) decreased cell shortening and NOi production without concomitant changes in L‐type Ca2+ current (ICa,L) or [Ca2+]i transients. The positive inotropic agents EMD 57033 or CGP 48506 (1 μm) increased cell shortening and NOi production without concomitant changes in ICa,L or [Ca2+]i transients. FS‐induced NOi 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 β‐galactosidase. FS‐induced NOi was partially inhibited by either endothelial (eNOS) or neuronal nitric oxide synthase (nNOS) inhibitors and completely blocked by simultaneous exposure to both. FS‐induced [Ca2+]i transients were increased by the nNOS inhibitor nNOS‐I (0.24 μm), decreased by the eNOS inhibitor L‐NIO (1 μm) and unchanged by exposure to both inhibitors. We conclude that in cat ventricular myocytes, FS‐induced NOi production requires both Ca2+‐dependent CaM signalling and Ca2+‐independent PI‐(3)K–Akt signalling activated by contractile activity. FS activates NOi production from both eNOS and nNOS, and each source of NOi exerts opposing effects on [Ca2+]i transient amplitude. These findings are important for understanding the regulation of NOi signalling in the normal and mechanically failing heart.