The endothelial isoform of nitric-oxide synthase (eNOS) is regulated by a complex pattern of post-translational modifications. In these studies, we show that eNOS is dynamically regulated by S-nitrosylation, the covalent adduction of nitric oxide (NO)-derived nitrosyl groups to the cysteine thiols of proteins. We report that eNOS is tonically S-nitrosylated in resting bovine aortic endothelial cells and that the enzyme undergoes rapid transient denitrosylation after addition of the eNOS agonist, vascular endothelial growth factor. eNOS is thereafter progressively renitrosylated to basal levels. The receptor-mediated decrease in eNOS S-nitrosylation is inversely related to enzyme phosphorylation at Ser 1179 , a site associated with eNOS activation. We also document that targeting of eNOS to the cell membrane is required for eNOS S-nitrosylation. Acylation-deficient mutant eNOS, which is targeted to the cytosol, does not undergo S-nitrosylation. Using purified eNOS, we show that eNOS S-nitrosylation by exogenous NO donors inhibits enzyme activity and that eNOS inhibition is reversed by denitrosylation. We determine that the cysteines of the zinc-tetrathiolate that comprise the eNOS dimer interface are the targets of S-nitrosylation. Mutation of the zinc-tetrathiolate cysteines eliminates eNOS S-nitrosylation but does not eliminate NO synthase activity, arguing strongly that disruption of the zinc-tetrathiolate does not necessarily lead to eNOS monomerization in vivo. Taken together, these studies suggest that eNOS S-nitrosylation may represent an important mechanism for regulation of NO signaling pathways in the vascular wall.

Sphingosine 1-phosphate (S1P) is a platelet-derived sphingolipid that binds to S1P 1 (EDG-1) receptors and activates the endothelial isoform of NO synthase (eNOS). S1P and the polypeptide growth factor vascular endothelial growth factor (VEGF) act independently to modulate angiogenesis and activate eNOS. In these studies, we explored the cross-talk between S1P and VEGF signaling pathways. When cultured bovine aortic endothelial cells were treated with VEGF (10 ng͞ml), the expression of S1P 1 protein and mRNA increased by Ϸ4-fold. S1P1 up-regulation by VEGF was seen within 30 min of VEGF addition and reached a maximum after 1.5 h. By contrast, expression of neither bradykinin B2 receptors nor the scaffolding protein caveolin-1 was altered by VEGF treatment. The EC 50 for VEGF-promoted induction of S1P 1 expression was Ϸ2 ng͞ml, within its physiological concentration range. S1P1 induction by VEGF was attenuated by the tyrosine kinase inhibitor genistein and by the PKC inhibitor calphostin C. Preincubation of bovine aortic endothelial cells with VEGF (10 ng͞ml for 90 min) markedly enhanced subsequent S1P-dependent eNOS activation. VEGF pretreatment of cultured endothelial cells also markedly potentiated S1P-promoted eNOS phosphorylation at Ser-1179, as well as S1P-mediated activation of kinase Akt. In isolated rat arteries, VEGF pretreatment markedly potentiated S1P-mediated vasorelaxation and eNOS Ser-1179 phosphorylation. Taken together, these data indicate that VEGF specifically induces expression of S1P 1 receptors, associated with enhanced intracellular signaling responses to S1P and the potentiation of S1P-mediated vasorelaxation. We suggest that VEGF acts to sensitize the vascular endothelium to the effects of lipid mediators by promoting the induction of S1P 1 receptors, representing a potentially important point of cross-talk between receptor-regulated eNOS signaling pathways in the vasculature.

Endothelial nitric-oxide synthase (eNOS) undergoes a complex pattern of post-translational modifications that regulate its activity. We have recently reported that eNOS is constitutively S-nitrosylated in endothelial cells and that agonists promote eNOS denitrosylation concomitant with enzyme activation (Erwin, P. A., Lin, A. J., Golan, D. E., and Michel, T. (2005) , modifications that are dependent on the prior co-translational and irreversible N-myristoylation of eNOS Gly 2 (4). Upon agonist stimulation, eNOS is rapidly depalmitoylated and translocates from peripheral membrane caveolae to internal membrane structures; over time, the enzyme is retargeted to caveolae and repalmitoylated as eNOS returns to basal activity levels (2, 5, 6). We have previously reported that S-nitrosylation reversibly inhibits eNOS activity, that eNOS is tonically S-nitrosylated in vascular endothelial cells, and that the enzyme undergoes rapid, transient denitrosylation upon agonist stimulation, followed by progressive renitrosylation as the enzyme returns to resting activity levels (7). We also showed that endogenous eNOS S-nitrosylation in transfected cells is abolished when the zinc-tetrathiolate cysteines (residues 96 and 101 in the bovine eNOS sequence) of eNOS are changed to serine by PCR-directed mutagenesis (7). We used mass spectrometry to analyze the S-nitrosylation pattern of purified recombinant eNOS and showed directly that the zinc-tetrathiolate of eNOS is preferentially S-nitrosylated in the intact enzyme. We also report that subcellular targeting is a determinant of eNOS S-nitrosylation and that translocation between cellular compartments is necessary for agonist-modulated eNOS denitrosylation. Finally, we extended our work from cultured bovine aortic endothelial cells (BAEC) to show that dynamic S-nitrosylation occurs in intact mouse blood vessels. Taken together, these data suggest that S-nitrosylation is a dynamic and physiologically relevant regulator of NO signaling pathways in the vascular endothelium.