Regulation of Endothelial Nitric-oxide Synthase (NOS) S-Glutathionylation by Neuronal NOS EVIDENCE OF A FUNCTIONAL INTERACTION BETWEEN MYOCARDIAL CONSTITUTIVE NOS ISOFORMS

Myocardial constitutive No production depends on the activity of both endothelial and neuronal NOS (eNOS and nNOS, respectively). Stimulation of myocardial beta(3)-adrenergic receptor (beta(3)-AR) produces a negative inotropic effect that is dependent on eNOS. We evaluated whether nNOS also plays a role in beta(3)-AR signaling and found that the beta(3)-AR-mediated reduction in cell shortening and [Ca2+](i) transient amplitude was abolished both in eNOS(-/-) and nNOS(-/-) left ventricular (LV) myocytes and in wild type LV myocytes after nNOS inhibition with S-methyl-L-thiocitrulline. LV superoxide (O-2(radical anion)) production was increased in nNOS(-/-) mice and reduced by L-N-omega-nitroarginine methyl ester (L-NAME), indicating uncoupling of eNOS activity. eNOS S-glutathionylation and Ser-1177 phosphorylation were significantly increased in nNOS(-/-) myocytes, whereas myocardial tetrahydrobiopterin, eNOS Thr-495 phosphorylation, and arginase activity did not differ between genotypes. Although inhibitors of xanthine oxidoreductase (XOR) or NOX2 NADPH oxidase caused a similar reduction in myocardial O-2(radical anion), only XOR inhibition reduced eNOS S-glutathionylation and Ser-1177 phosphorylation and restored both eNOS coupled activity and the negative inotropic and [Ca2+] i transient response to beta(3)-AR stimulation in nNOS(-/-) mice. In summary, our data show that increased O-2(radical anion) production by XOR selectively uncouples eNOS activity and abolishes the negative inotropic effect of beta(3)-AR stimulation in nNOS(-/-) myocytes. These findings provide unequivocal evidence of a functional interaction between the myocardial constitutive NOS isoforms and indicate that aspects of the myocardial phenotype of nNOS(-/-) mice result from disruption of eNOS signaling.