Nitric oxide circulates in mammalian plasma primarily as an S-nitroso adduct of serum albumin.

Abstract

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...

NO Inhibits Cytokine-Induced iNOS Expression and NF-κB Activation by Interfering With Phosphorylation and Degradation of IκB-α

“…[31][32][33] NO can also modify proteins by nitrosylation of Cys residue to form S-nitrosothiols. 34 Therefore, it is possible to speculate that the inhibitory effect of NO on cytokine-induced NF-B activation may be due to its direct nitrosylation of Cys residue(s) of I B-␣ kinase to decrease its enzymatic activity. It is also possible that NO may inhibit NF-B by dephosphorylation of I B-␣, because NO has been shown to activate protein phosphatases in monocytes.…”

Nitric oxide metabolites induced in Anopheles stephensi control malaria parasite infection

“…Free and photolyzable · NO were measured by photolysis-chemiluminescence detection [11]. Midguts (10 per sample) were dissected into 0.1M sodium phosphate buffer (0.09M Na 2 HPO 4 , 0.006M NaH 2 PO 4 ; pH 8) containing 100μM DTPA or into 0.15M NaCl (pH 7.4) containing 100μM DTPA and injected into the manual injection valve of an Isco 2350 HPLC pump connected to a photolysis chamber (Nitrolite, Thermedics, Inc., Woburn, MA).…”

“…Based on the detection of significant levels of SNOs within the blood-filled A. stephensi midgut we chose to examine the formation of SNO-proteins as a function of time post-feeding because the nature of SNO-modified peptides and proteins could influence the toxicity of · NO in the midgut [6,11]. To assess the profile of S-nitrosylated midgut proteins in A. stephensi, lysates were assayed using the "SNO-biotin switch" method [15].…”

Nitric oxide and arthritis

“…The short half-life of NO limits its immediate sphere of influence to autocrine and local paracrine effects; the high affinity of hemoglobin for NO imposes further limits. Nevertheless, the longevity and range of its physiologic effects may be extended by the formation of stable, biologically active adducts with proteins and other molecules (40). As a small lipophilic molecule, NO is well suited to its role in local, intercellular communication.…”