S pe c i a l R e p o r tT h e ne w e ngl a nd jou r na l o f m e dicine n engl j med nejm.org T h e ne w e ngl a nd jou r na l o f m e dicine n engl j med nejm.org

Endothelium-derived relaing factor (EDRF) activity has been attributed to the highly labile nitric oxide radical (NO). In view of the fact that the plasma and cellular mifleux contain reactive species that can rapidly inactivate NO, it has been postulated that NO is stabilized by a carrier molecule that preserves its biological activity. Reduced thiol species are candidates for this role, reacting readily in the presence of NO to yield biologically active S-nitrosothiols that are more stable than NO itself. Because sulfhydryl groups in proteins represent an abundant source of reduced thiol in biologic systems, we examined the reaction of several sulfhy--I dryl-containing proteins of diverse nature and function upon exposure to authentic NO and EDRF. We demonstrate that S-nitroso proteins form readily under physiologic conditions and possess EDRF-like effects of vasodilation and platelet inhibition. These observations suggest that S-nitrosothiol groups in proteins may serve as intermediates in the cellular metabolism ofNO and raise the possibility ofan additional type of cellular regulatory mechanism.The richest source of reduced thiol in plasma (and a particularly prevalent source in cellular cytosol) is protein sulflydryl groups (19). The reaction ofNO with protein thiols has not been previously studied, and the potential biological significance ofthis reaction has been neglected because ofthe exclusion of proteins from (bio)assays of the functional activity and half-life of EDRF and from analyses of its chemical characteristics. We therefore investigated the reaction of protein thiols exposed to NO, and we present data showing that a variety of proteins of biological significance and relative abundance can be S-nitrosylated. S-Nitrosylation of proteins endows these molecules with potent and long-lasting EDRF-like effects of vasodilation and platelet inhibition that are mediated by guanylate cyclase activation. These observations raise the possibility that S-nitrosothiol groups in proteins may serve as intermediates in the cellular metabolism or bioactivity ofNO and that their formation may represent an important cellular regulatory mechanism.The endothelium-dependent relaxation of vascular smooth muscle first observed by Furchgott and Zawadski (1) has been largely attributed to nitric oxide (NO) derived from L-arginine through the action of NO synthase (2)(3)(4). This free radical ultimately stimulates guanylate cyclase by the formation of a nitrosyl-heme complex at the activator site of the enzyme (5, 6); however, the molecular mechanism(s) by which NO is transferred from synthase to cyclase remains poorly understood. The rapidity of the reaction of NO with molecular oxygen (7), superoxide anion (8), and heme (2) as well as nonheme iron (9) and the ready availability of these inactivating reactants in the plasma and cellular milieux militate against simple diffusion-limited transport of free NO in this medium. That endothelium-derived relaxing factor (EDRE) has the relatively long half-life of the ord...

The vascular endothelium is a critical regulator of vascular function. Diverse stimuli such as proinflammatory cytokines and hemodynamic forces modulate endothelial phenotype and thereby impact on the development of vascular disease states. Therefore, identification of the regulatory factors that mediate the effects of these stimuli on endothelial function is of considerable interest. Transcriptional profiling studies identified the Kruppel-like factor (KLF)2 as being inhibited by the inflammatory cytokine interleukin-1 ␤ and induced by laminar shear stress in cultured human umbilical vein endothelial cells. Overexpression of KLF2 in umbilical vein endothelial cells robustly induced endothelial nitric oxide synthase expression and total enzymatic activity. In addition, KLF2 overexpression potently inhibited the induction of vascular cell adhesion molecule-1 and endothelial adhesion molecule E-selectin in response to various proinflammatory cytokines. Consistent with these observations, in vitro flow assays demonstrate that T cell attachment and rolling are markedly attenuated in endothelial monolayers transduced with KLF2. Finally, our studies implicate recruitment by KLF2 of the transcriptional coactivator cyclic AMP response element-binding protein (CBP/p300) as a unifying mechanism for these various effects. These data implicate KLF2 as a novel regulator of endothelial activation in response to proinflammatory stimuli.

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Endothelial nitric-oxide synthase (eNOS) generates the key signaling molecule nitric oxide in response to intralumenal hormonal and mechanical stimuli. We designed studies to determine whether eNOS is localized to plasmalemmal microdomains implicated in signal transduction called caveolae. Using immunoblot analysis, eNOS protein was detected in caveolar membrane fractions isolated from endothelial cell plasma membranes by a newly developed detergent-free method; eNOS protein was not found in noncaveolar plasma membrane. Similarly, NOS enzymatic activity was 9.4-fold enriched in caveolar membrane versus whole plasma membrane, whereas it was undetectable in noncaveolar plasma membrane. 51-86% of total NOS activity in postnuclear supernatant was recovered in plasma membrane, and 57-100% of activity in plasma membrane was recovered in caveolae. Immunoelectron microscopy showed that eNOS heavily decorated endothelial caveolae, whereas coated pits and smooth plasma membrane were devoid of gold particles. Furthermore, eNOS was targeted to caveolae in COS-7 cells transfected with wild-type eNOS cDNA. Studies with eNOS mutants revealed that both myristoylation and palmitoylation are required to target the enzyme to caveolae and that each acylation process enhances targeting by 10-fold. Thus, acylation targets eNOS to plasmalemmal caveolae. Localization to this microdomain is likely to optimize eNOS activation and the extracellular release of nitric oxide.The endothelial isoform of nitric-oxide synthase (eNOS) 1 is one of three isoenzymes that converts L-arginine to L-citrulline plus the key signaling molecule nitric oxide (NO). eNOS is acutely activated by increases in endothelial intracellular calcium induced by the stimulation of diverse G-protein-coupled cell surface receptors and by physical stimuli such as hemodynamic shear stress and varying oxygenation. Endotheliumderived NO regulates blood pressure, platelet aggregation, and vascular smooth muscle mitogenesis. Diminished NO production has been implicated in the pathogenesis of a variety of vascular disorders including atherosclerosis and pulmonary hypertension (1-4).eNOS is a unique isoform of the enzyme in that it is primarily localized to the particulate subcellular fraction (1-4). Studies evaluating NOS enzymatic activity have demonstrated that functional eNOS is primarily associated with the plasma membrane (5). Since eNOS activity is acutely regulated by intralumenal factors and the NO produced is a labile, cytotoxic messenger molecule (1-4), the intracellular site of NO synthesis is likely to have a major influence on its biological activity in the vascular wall. We therefore sought to determine the specific subcellular location to which eNOS is targeted and designed studies to delineate whether the enzyme is localized to plasmalemmal caveolae. Caveolae are plasmalemmal microdomains that have been implicated in the transcytosis of macromolecules, the uptake of small molecules by potocytosis, and the compartmentalization of signaling molecules (6 -9). The ...

Nitric oxide (NO) is a ubiquitous intercellular messenger molecule synthesized from the amino acid L-arginine by NO synthases in diverse cells and tissues. NO is synthesized in vascular endothelial cells and appears to play an important role in the control of blood pressure and platelet aggregation. A detailed understanding of the regulation of NO synthesis by endothelial cells has been hampered by the lack of molecular clones for endothelial NO synthase; the isolation and characterization ofsuch clones is reported herein. The constitutive NO synthases present in endothelial cells and in brain share common biochemical and pharmacologic features. We purified NO synthase from bovine brain and determined the amino acid sequence of several tryptic peptides. The sequence ofthe bovine brain peptides is nearly identical to the deduced amino acid sequence previously determined for the rat brain NO synthase.These sequence data were utilized to design PCR-generated NO synthase cDNA probes, which were used to isolate clones encoding NO synthase from a bovine aortic endothelial cell (BAEC) cDNA library. A full-length NO synthase cDNA clone was isolated, representing a protein of 1205 amino acids with a molecular mass of 133 kDa; transfection of this clone in a heterologous expression system demonstrated the expected enzymatic activity. The deduced amino acid sequence of the BAEC NO synthase cDNA differs at numerous residues from the sequence determined for the purified bovine brain protein and shows 50-60% sequence identity with recently isolated molecular clones for murine macrophage and rat brain NO synthase isoforms. Bovine genomic Southern blots probed with bovine brain and BAEC NO synthase cDNA probes identify distinct bands, indicating that these cDNAs are the products of different genes. Prolonged treatment of BAECs with the cytokine tumor necrosis factor a, which we have previously shown to result in a marked increase in NO synthase activity, is associated with a decrease in the abundance of the 4.8-kilobase BAEC NO synthase transcript. The increase in BAEC NO synthase activity induced by tumor necrosis factor a is thus likely to involve posttranscriptional mechanisms or the induction of a distinct endothelial NO synthase isoform.The vascular endothelium forms the lining of the circulatory system in all vertebrates and comprises a dynamic monocellular interface between the surrounding vascular cells and the soluble and cellular components of the blood. In response to diverse stimuli, the vascular endothelium synthesizes and secretes vasoactive agents that control vascular tone (1, 2). In 1980, a labile endothelium-derived compound was discovered to play an important role in relaxing vascular smooth muscle and was termed endothelium-derived relaxing factor (EDRF) (3). EDRF was subsequently shown to be equivalent to nitric oxide (NO) or a NO-containing compound (for review, see ref. 4). NO synthesis is being discovered in a striking diversity of tissues and cultured cells (5).Catalysis by NO synthase, in all e...

The endothelial nitric-oxide synthase (eNOS) is a key determinant of vascular homeostasis. Like all known nitric-oxide synthases, eNOS enzyme activity is dependent on Ca 2؉ -calmodulin. eNOS is dynamically targeted to specialized cell surface signal-transducing domains termed plasmalemmal caveolae and interacts with caveolin, an integral membrane protein that comprises a key structural component of caveolae. We have previously reported that the association between eNOS and caveolin is quantitative and tissue-specific (Feron, O., Belhassen, L., Kobzick, L., Smith, T. W., Kelly, R. A., and Michel, T. (1996) J. Biol. Chem. 271, 22810 -22814). We now report that in endothelial cells the interaction between eNOS and caveolin is importantly regulated by Ca 2؉ -calmodulin. Addition of calmodulin disrupts the heteromeric complex formed between eNOS and caveolin in a Ca 2؉ -dependent fashion. In addition, overexpression of caveolin markedly attenuates eNOS enzyme activity, but this inhibition is reversed by purified calmodulin. Caveolin overexpression does not affect the activity of the other NOS isoforms, suggesting eNOSspecific inhibition of NO synthase by caveolin. We propose a model of reciprocal regulation of eNOS in endothelial cells wherein the inhibitory eNOS-caveolin complex is disrupted by binding of Ca 2؉

The complex regulation of eNOS (endothelial nitric oxide synthase) in cardiovascular physiology occurs at multiple stages. eNOS mRNA levels are controlled both at the transcriptional and post-transcriptional phases, and epigenetic mechanisms appear to modulate tissue-specific eNOS expression. The eNOS enzyme reversibly associates with a diverse family of protein partners that regulate eNOS sub-cellular localization, catalytic function, and biological activity. eNOS enzyme activity and sub-cellular localization are intimately controlled by post-translational modifications including phosphorylation, nitrosylation, and acylation. The multiple extra-cellular stimuli affecting eNOS function coordinate their efforts through these key modifications to dynamically control eNOS and NO bioactivity in the vessel wall. This review will focus on the biochemical partners and perturbations of the eNOS protein as this vital enzyme undergoes modulation by diverse signal transduction pathways in the vascular endothelium.