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...

We have recently shown that nitric oxide or authentic endothelium-derived relaxing factor generated in a biologic system reacts in the presence of specific protein thiols to form S-nitrosoprotein derivatives that have endotheliumderived relaxing factor-like properties. The single free cysteine of serum albumin, Cys-34, is particularly reactive toward nitrogen oxides (most likely nitrosonium ion) under physiologic conditions, primarily because of its anomalously low pK; given its abundance in plasma, where it accounts for -0.5 mM thiol, we hypothesized that this plasma protein serves as a reservoir for nitric oxide produced by the endothelial cell. To test this hypothesis, we developed a methodology, which involves UV photolytic cleavage of the S-NO bond before reaction with ozone for chemiluminescence detection, with which to measure free nitric oxide, S-nitrosodiols, and S-nitrosoproteins in biologic systems. We found that human plasma contains =7 pIM S-nitrosothiols, of which 96% are S-nitrosoproteins, 82% of which is accounted for by S-nitroso-erum albumin. By contrast, plasma levels of free nitric oxide are only in the 3-nM range. In rabbits, plasma S-nitrosothiols are present at -1 #M; 60 min after administration of NG-monomethyl-Larginine at 50 mg/ml, a selective and potent inhibitor of nitric oxide synthetases, S-nitrosothiols decreased by "40% (>95% of which were accounted for by S-nitrosoproteins, and ""80% of which was S-nitroso-serum albumin); this decrease was accompanied by a concomitant increase in mean arterial blood pressure of 22%. These data suggest that naturally produced nitric oxide circulates in plasma primarily complexed in S-nitrosothiol species, principal among which is S-nitroso-serum albumin. This abundant, relatively long-lived adduct likely serves as a reservoir with which plasma levels of highly reactive, short-lived free nitric oxide can be regulated for the maintenance of vascular tone.Endothelium-derived relaxing factor (EDRF), first described by Furchgott and Zawadzki (1), is a product of the normal endothelial cell having both vasodilatory (2) and antiplatelet (3,4) properties. Pharmacologic studies suggest that disease states as varied as septic shock (5), atherosclerosis (6), and hypoxia-induced pulmonary hypertension (7) may be associated with abnormal concentrations of EDRF in the vascular milieu. As a result of the seminal work of two groups (8, 9), this bioactive substance is believed to be equivalent to nitric oxide or a chemical congener or adduct thereof. Among the species thought of potential importance as adducts of nitric oxide are S-nitrosothiols-adducts with the sulfhydryl groups of amino acids, peptides, and proteins. We have recently shown that nitric oxide and authentic EDRF react with free thiol groups of proteins under physiologic conditions in vitro to form S-nitrosoproteins (10)-nitric oxide adducts with bioactivities comparable to EDRF but with half-lives of the order of hours. Although the facile formation of such species is intrinsically interest...

Elevated levels of homocysteine are associated with an increased risk of atherosclerosis and thrombosis. The reactivity of the sulfhydryl group of homocysteine has been implicated in molecular mechanisms underlying this increased risk. There is also increasingly compelling evidence that thiols react in the presence of nitric oxide (NO) and endothelium-derived relaxing factor (EDRF) to form S-nitrosothiols, compounds with potent vasodilatory and antiplatelet effects. We, therefore, hypothesized that S-nitrosation of homocysteine would confer these beneficial bioactivities to the thiol, and at the same time attenuate its pathogenicity. We found that prolonged (> 3 h) exposure of endothelial cells to homocysteine results in impaired EDRF responses. By contrast, brief (15 min) exposure of endothelial cells, stimulated to secrete EDRF, to homocysteine results in the formation of S-NO-homocysteine, a potent antiplatelet agent and vasodilator. In contrast to homocysteine, S-NO-homocysteine does not support H202 generation and does not undergo conversion to homocysteine thiolactone, reaction products believed to contribute to endothelial toxicity. These results suggest that the normal endothelium modulates the potential, adverse effects of homocysteine by releasing EDRF and forming the adduct S-NO-homocysteine. The adverse vascular properties of homocysteine may result from an inability to sustain S-NO formation owing to a progressive imbalance between the production of NO by progressively dysfunctional endothelial cells and the levels of homocysteine. (J.

IntroductionRecent evidence suggests that sulfhydryl species can react with oxides of nitrogen under physiologic conditions and thereby stabilize endothelium-derived relaxing factor (EDRF) activity, but the presence of a specific in vivo thiol carrier for nitric oxide (NO) remains controversial.

Tissue-type plasminogen activator (t-PA) reacts upon exposure to endothelium-derived relaxing factor (EDRF) by way of the enzyme's single free sulfhydryl to form a stable S-nitrosothiol protein adduct. S-nitrosylation endows t-PA with potent vasodilatory and antiplatelet properties that are accompanied by elevations in intracellular cyclic GMP analogous to those induced by low molecular weight (e.g., S-nitroso amino acid) S-nitrosothiols. Moreover, this chemical modification does not adversely affect the catalytic efficiency of t-PA, the fibrin stimulation of this activity, the binding of t-PA to fibrinogen, or the interaction of the enzyme with its physiologic serine protease inhibitor, plasminogen-activator inhibitor type I. The coupling of vasodilatory, antiplatelet, and fibrinolytic properties in one molecule makes the S-nitrosylated t-PA a unique molecular species and may provide insight into the mechanisms by which the endothelium maintains vessel patency. These data also suggest a pharmacologic approach to treatment of thromboocclusive disorders.