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 (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 mu M W-7 and partially inhibited by the phosphatidylinositol 3'-kinase (PI-(3)K) inhibitor 10 mu M 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 mu M) decreased cell shortening and NOi production without concomitant changes in L-type Ca2+ current (I-Ca,I-L) or [Ca2+](i) transients. The positive inotropic agents EMD 57033 or CGP 48506 (1 mu M) increased cell shortening and NOi production without concomitant changes in I-Ca,I-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 beta-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 mu M), decreased by the eNOS inhibitor L-NIO (1 mu M) 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.