Abstract
Myocardial constitutive No production depends on the activity of both endothelial and neuronal NOS (eNOS and nNOS, respectively). Stimulation of myocardial β(3)-adrenergic receptor (β(3)-AR) produces a negative inotropic effect that is dependent on eNOS. We evaluated whether nNOS also plays a role in β(3)-AR signaling and found that the β(3)-AR-mediated reduction in cell shortening and [Ca(2+)](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)(·-)) production was increased in nNOS(-/-) mice and reduced by L-N(ω)-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)(·-), only XOR inhibition reduced eNOS S-glutathionylation and Ser-1177 phosphorylation and restored both eNOS coupled activity and the negative inotropic and [Ca(2+)](i) transient response to β(3)-AR stimulation in nNOS(-/-) mice. In summary, our data show that increased O(2)(·-) production by XOR selectively uncouples eNOS activity and abolishes the negative inotropic effect of β(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.
Highlights
Whether neuronal nitric-oxide synthase plays a role in the endothelial NOS-dependent negative inotropic effect of 3-adrenergic stimulation remains to be established
The Effect of 3-AR Stimulation Is Abolished in the Presence of neuronal nitric-oxide synthase (nNOS) Inhibition or Gene Deletion—3-AR stimulation with BRLϩNAD resulted in a small but significant reduction in cell shortening in left ventricular (LV) myocytes from both eNOSϩ/ϩ and nNOSϩ/ϩ mice (Fig. 1)
BRLϩNAD had no effect on contraction in myocytes from eNOSϪ/Ϫ mice (Fig. 1A); the negative inotropic effect of 3-AR stimulation was abolished in nNOSϪ/Ϫ myocytes and in nNOSϩ/ϩ myocytes pretreated with the nNOS-specific inhibitor, SMTC (100 M; Fig. 1B)
Summary
Whether neuronal nitric-oxide synthase (nNOS) plays a role in the endothelial NOS (eNOS)-dependent negative inotropic effect of 3-adrenergic stimulation remains to be established. Results: nNOS knock-out or inhibition leads to increased superoxide production, eNOS uncoupling, and abrogation of 3-adrenergic responses. Conclusion: Disabling nNOS disrupts eNOS function and downstream signaling. Significance: nNOS plays a crucial role in preserving myocardial nitroso-redox balance and coupled eNOS activity. Myocardial constitutive No production depends on the activity of both endothelial and neuronal NOS (eNOS and nNOS, respectively). Stimulation of myocardial 3-adrenergic receptor (3-AR) produces a negative inotropic effect that is dependent on eNOS. We evaluated whether nNOS plays a role in
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