title: Regulation of endothelial nitric-oxide synthase (NOS) S-glutathionylation by neuronal NOS: evidence of a functional interaction between myocardial constitutive NOS isoforms.
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.
authors.affiliation: Department of Cardiovascular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom.
authors.forename: Winifred O
authors.initials: WO
authors.lastname: Idigo
authors.forename: Svetlana
authors.initials: S
authors.lastname: Reilly
authors.forename: Mei Hua
authors.initials: MH
authors.lastname: Zhang
authors.forename: Yin Hua
authors.initials: YH
authors.lastname: Zhang
authors.forename: Raja
authors.initials: R
authors.lastname: Jayaram
authors.forename: Ricardo
authors.initials: R
authors.lastname: Carnicer
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Crabtree
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Balligand
authors.forename: Barbara
authors.initials: B
authors.lastname: Casadei
mesh_terms: D000818:Animals; D001119:Arginase / Q000235:genetics / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002956:Citrulline / Q000031:analogs & derivatives / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006352:Heart Ventricles / Q000166:cytology / Q000201:enzymology; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D009124:Muscle Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009206:Myocardium / Q000166:cytology / Q000201:enzymology*; D032383:Myocytes, Cardiac / Q000166:cytology / Q000201:enzymology*; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D052248:Nitric Oxide Synthase Type I / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D022702:Receptors, Adrenergic, beta-3 / Q000235:genetics / Q000276:immunology; D013481:Superoxides / Q000378:metabolism; D013890:Thiourea / Q000031:analogs & derivatives / Q000494:pharmacology; D014968:Xanthine Dehydrogenase / Q000235:genetics / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D007527:Isoenzymes; D008562:Membrane Glycoproteins; D009124:Muscle Proteins; D022702:Receptors, Adrenergic, beta-3; D013481:Superoxides; D002956:Citrulline; D052248:Nitric Oxide Synthase Type I; D052250:Nitric Oxide Synthase Type III; C496321:Nos1 protein, mouse; C496315:Nos3 protein, mouse; D014968:Xanthine Dehydrogenase; C488842:Cybb protein, mouse; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D001119:Arginase; D013890:Thiourea; C086717:S-methylthiocitrulline; D019331:NG-Nitroarginine Methyl Ester