Abstract-Nitrates such as nitroglycerin (GTN) and nitric oxide donors such as S-nitrosothiols are clinically vasoactive through stimulation of soluble guanylyl cyclase (sGC), which produces the second messenger cGMP. Development of nitrate tolerance, after exposure to GTN for several hours, is a major drawback to a widely used cardiovascular therapy.We recently showed that exposure to nitric oxide and to S-nitrosothiols causes S-nitrosylation of sGC, which directly desensitizes sGC to stimulation by nitric oxide. We tested the hypothesis that desensitization of sGC by S-nitrosylation is a mechanism of nitrate tolerance. Our results established that vascular tolerance to nitrates can be recapitulated in vivo by S-nitrosylation through exposure to cell membrane-permeable S-nitrosothiols and that sGC is S-nitrosylated and desensitized in the tolerant, treated tissues. We next determined that (1) GTN treatment of primary aortic smooth muscle cells induces S-nitrosylation of sGC and its desensitization as a function of GTN concentration; (2) S-nitrosylation and desensitization are prevented by treatment with N-acetyl-cysteine, a precursor of glutathione, used clinically to prevent development of nitrate tolerance; and (3) S-nitrosylation and desensitization are reversed by cessation of GTN treatment. Finally, we demonstrated that in vivo development of nitrate tolerance and crosstolerance by 3-day chronic GTN treatment correlates with S-nitrosylation and desensitization of sGC in tolerant tissues. These results suggest that in vivo nitrate tolerance is mediated, in part, by desensitization of sGC through GTN-dependent S-nitrosylation. (Circ Res. 2008;103:606-614.)Key Words: cGMP Ⅲ nitric oxide Ⅲ nitrosation Ⅲ S-nitrosothiols Ⅲ vascular tolerance S ince the 19th century, nitroglycerin (glyceryl trinitrate [GTN]) has been used in clinical medicine to treat angina pectoris and, more recently, congestive heart failure, acute myocardial infarction, and other cardiovascular diseases. 1 It is used because of its excellent therapeutic profile, although it induces nitrate tolerance. Nitrate tolerance is defined as the attenuation or loss of vascular responsiveness to nitrate (eg, GTN) after continuous exposure to GTN or other organic nitrates. 2 Crosstolerance corresponds to the lack of vascular responsiveness to nitric oxide (NO) and NO donors (eg, nitrosothiols) and is characterized by an increase in reactive oxygen species. 3 NO and nitrates exert their vasorelaxing effects through stimulation of soluble guanylyl cyclase (sGC), which produces cGMP. 4 At the molecular level, tolerance corresponds to an absence of increased vascular cGMP production in response to NO or nitrate, highlighting the central role of sGC activity in vascular tolerance. The mechanism of development of nitrate tolerance remains a mystery; the prevalent actual model is impairment of GTN bioconversion through inhibition of mitochondrial aldehyde dehydrogenase, thereby reducing the release of NO or its derivatives. 5,6 Other frequently cited mechani...

The molecular mechanisms of endothelial nitric oxide synthase (eNOS) regulation of microvascular permeability remain unresolved. Agonist-induced internalization may have a role in this process. We demonstrate here that internalization of eNOS is required to deliver NO to subcellular locations to increase endothelial monolayer permeability to macromolecules. Using dominant-negative mutants of dynamin-2 (dyn2K44A) and caveolin-1 (cav1Y14F), we show that anchoring eNOS-containing caveolae to plasma membrane inhibits hyperpermeability induced by plateletactivating factor (PAF), VEGF in ECV-CD8eNOSGFP (ECV-304 transfected cells) and postcapillary venular endothelial cells (CVEC). We also observed that anchoring caveolar eNOS to the plasma membrane uncouples eNOS phosphorylation at Ser-1177 from NO production. This dissociation occurred in a mutant-and cell-dependent way. PAF induced Ser-1177-eNOS phosphorylation in ECVCD8eNOSGFP and CVEC transfected with dyn2K44A, but it dephosphorylated eNOS at Ser-1177 in CVEC transfected with cav1Y14F. Interestingly, dyn2K44A eliminated NO production, whereas cav1Y14F caused reduction in NO production in CVEC. NO production by cav1Y14F-transfected CVEC occurred in caveolae bound to the plasma membrane, and was ineffective in causing an increase in permeability. Our study demonstrates that eNOS internalization is required for agonist-induced hyperpermeability, and suggests that a mechanism by which eNOS is activated by phosphorylation at the plasma membrane and its endocytosis is required to deliver NO to subcellular targets to cause hyperpermeability.caveolae ͉ endothelial nitric oxide synthase ͉ endothelial permeability ͉ inflammation ͉ protein traffic N itric oxide (NO) is the signal for several outcomes in the control of circulation. Its main source in the cardiovascular system is endothelial (e)NOS. The enzyme is modified by N-myristoylation and palmitoylation, which targets it to the caveolae in the plasma membrane where it is kept in a basal state by binding to caveolin-1 through a consensus site (1, 2). Agonists activate eNOS through multiple mechanisms: phosphorylation/ dephosphorylation of specific residues, interaction with different proteins, S-nitrosylation, and specific subcellular localization (1, 3-7). Agonists such as platelet-activating factor (PAF) and VEGF phosphorylate eNOS via Akt (8, 9). Despite advances in our understanding of the biochemistry of eNOS, the mechanisms by which these molecular modifications determine the functional outcome of eNOS activation remain unexplored.NO derived from eNOS is a key mediator in the hyperpermeability response to PAF and VEGF (7,10,11). We previously showed that eNOS internalization (endocytosis) is a required step in the signaling cascade leading to PAF-induced hyperpermeability (7). In this study, we tested the hypothesis that caveolar internalization of eNOS is a requirement for localized NO production and NO delivery to a subcellular target to cause hyperpermeability. To address this hypothesis, we used ECV-304 cells (...

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