Endothelial nitric-oxide synthase (eNOS) and caveolin-1 are associated within endothelial plasmalemmal caveolae. It is not known, however, whether eNOS and caveolin-1 interact directly or indirectly or whether the interaction affects eNOS activity. To answer these questions, we have cloned the bovine caveolin-1 cDNA and have investigated the eNOS-caveolin-1 interaction in an in vitro binding assay system using glutathione S-transferase (GST)-caveolin-1 fusion proteins and baculovirusexpressed bovine eNOS. We have also mapped the domains involved in the interaction using an in vivo yeast two-hybrid system. Results obtained using both in vitro and in vivo protein interaction assays show that both Nand C-terminal cytosolic domains of caveolin-1 interact directly with the eNOS oxygenase domain. Interaction of eNOS with GST-caveolin-1 fusion proteins significantly inhibits enzyme catalytic activity. A synthetic peptide corresponding to caveolin-1 residues 82-101 also potently and reversibly inhibits eNOS activity by interfering with the interaction of the enzyme with Ca 2؉

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Ascorbic acid enhances NO bioactivity in patients with vascular disease through unclear mechanism(s).We investigated the role of intracellular ascorbic acid in endothelium-derived NO bioactivity. Incubation of porcine aortic endothelial cells (PAECs) with ascorbic acid produced time-and dose-dependent intracellular ascorbic acid accumulation that enhanced NO bioactivity by 70% measured as A23187-induced cGMP accumulation. This effect was due to enhanced NO production because ascorbate stimulated both PAEC nitrogen oxide (NO 2 ؊ ؉ NO 3 Ϫ ) production and L-arginine to L-citrulline conversion by 59 and 72%, respectively, without altering the cGMP response to authentic NO. Ascorbic acid also stimulated the catalytic activity of eNOS derived from either PAEC membrane fractions or baculovirus-infected Sf9 cells. Ascorbic acid enhanced bovine eNOS V max by ϳ50% without altering the K m for L-arginine. The effect of ascorbate was tetrahydrobiopterin (BH 4 )-dependent, because ascorbate was ineffective with BH 4 concentrations >10 M or in PAECs treated with sepiapterin to increase intracellular BH 4 . The effect of ascorbic acid was also specific because A23187-stimulated cGMP accumulation in PAECs was insensitive to intracellular glutathione manipulation and only ascorbic acid, not glutathione, increased the intracellular concentration of BH 4 . These data suggest that ascorbic acid enhances NO bioactivity in a BH 4 -dependent manner by increasing intracellular BH 4 content.Nitric oxide is produced from L-arginine in the vascular endothelium by the endothelial isoform of nitric-oxide synthase (NOS).1 Endothelial production of NO is crucial in the control of vascular tone (1), arterial pressure (2-4), smooth muscle cell proliferation (5, 6), and platelet adhesion to the endothelial surface (7). Impaired endothelium-derived NO bioactivity is a common feature of many vascular diseases (8 -10) that is thought to contribute to their clinical manifestations (11,12).The action of NO is particularly sensitive to the local availability of superoxide. Both endothelial elaboration of NO and arterial relaxation in response to nitrovasodilators are dependent upon intact copper-zinc superoxide dismutase (SOD) activity (13,14). Animal models of hypercholesterolemia (15, 16) and hypertension (17) demonstrate an excess vascular superoxide flux that is linked to reduced NO bioactivity. Conversely, increasing vascular SOD activity enhances NO-mediated arterial relaxation in experimental models of atherosclerosis (18,19) and hypertension (17). Thus, scavenging superoxide has important implications for NO bioactivity under both normal and pathologic conditions. Ascorbic acid also efficiently scavenges superoxide (20) and numerous studies in a host of pathologic conditions such as diabetes (21), hypercholesterolemia (22), smoking (23), and hypertension (24) indicate that NO bioactivity is improved by parenteral ascorbic acid at supraphysiologic concentrations (ϳ10 mM). We have observed enhanced NO bioactivity in atherosclerotic patients aft...

and Ser 635 regulates eNOS activity and contributes to the agonist-stimulated eNOS activation process. Endothelial nitric-oxide synthase (eNOS)1 is an important enzyme in the cardiovascular system producing nitric oxide (NO), a key regulator of blood pressure, platelet function, and vessel remodeling. Endothelial NOS is regulated by multiple mechanisms involving both protein-protein interactions with several different proteins, including caveolin-1 and Hsp90 (1), and post-translational modifications that include Nmyristoylation, cysteine palmitoylation, and multisite phosphorylation. The two most thoroughly studied phosphorylation sites have been the activation site, human Ser 1177

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Neuronal nitric-oxide synthase (nNOS) has been shown previously to interact with ␣ 1 -syntrophin in the dystrophin complex of skeletal muscle. In the present study, we have examined whether nNOS also interacts with caveolin-3 in skeletal muscle. nNOS and caveolin-3 are coimmunoprecipitated from rat skeletal muscle homogenates by antibodies directed against either of the two proteins. Synthetic peptides corresponding to the membrane-proximal caveolin-3 residues 65-84 and 109 -130 and homologous caveolin-1 residues 82-101 and 135-156 potently inhibit the catalytic activity of purified, recombinant nNOS. Purified nNOS also binds to a glutathione S-transferase-caveolin-1 fusion protein in in vitro binding assays. In vitro binding is completely abolished by preincubation of nNOS with either of the two caveolin-3 inhibitory peptides. Interactions between nNOS and caveolin-3, therefore, appear to be direct and to involve two distinct caveolin scaffolding/inhibitory domains. Other caveolin-interacting enzymes, including endothelial nitric-oxide synthase and the c-Src tyrosine kinase, are also potently inhibited by each of the four caveolin peptides. Inhibitory interactions mediated by two different caveolin domains may thus be a general feature of enzyme docking to caveolin proteins in plasmalemmal caveolae.