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S-nitrosylation, the selective modification of cysteine residues in proteins to form S-nitrosocysteine, is a major emerging mechanism by which nitric oxide acts as a signaling molecule. Even though nitric oxide is intimately involved in the regulation of vascular smooth muscle cell functions, the potential protein targets for nitric oxide modification as well as structural features that underlie the specificity of protein S-nitrosocysteine formation in these cells remain unknown. Therefore, we used a proteomic approach using selective peptide capturing and site-specific adduct mapping to identify the targets of S-nitrosylation in human aortic smooth muscle cells upon exposure to S-nitrosocysteine and propylamine propylamine NONOate. This strategy identified 20 unique S-nitrosocysteine-containing peptides belonging to 18 proteins including cytoskeletal proteins, chaperones, proteins of the translational machinery, vesicular transport, and signaling. Sequence analysis of the S-nitrosocysteine-containing peptides revealed the presence of acid/base motifs, as well as hydrophobic motifs surrounding the identified cysteine residues. High-resolution immunogold electron microscopy supported the cellular localization of several of these proteins. Interestingly, seven of the 18 proteins identified are localized within the ER/Golgi complex, suggesting a role for S-nitrosylation in membrane trafficking and ER stress response in vascular smooth muscle.
We report on developmental changes of pulmonary and systemic nitric oxide (NO) metabolites in a baboon model of chronic lung disease with or without exposure to inhaled NO. The plasma levels of nitrite and nitrate, staining for S-nitrosothiols and 3-nitrotyrosine in the large airways, increased between 125 d and 140 d of gestation (term 185 d) in animals developing in utero. The developmental increase in NO-mediated protein modifications was not interrupted by delivery at 125 d of gestation and mechanical ventilation for 14 d, whereas plasma nitrite and nitrate levels increased in this model. Exposure to inhaled NO resulted in a further increase in plasma nitrite and nitrate and an increase in plasma S-nitrosothiol without altering lung NO synthase expression. These data demonstrate a developmental progression in levels of pulmonary NO metabolites that parallel known maturational increases in total NO synthase activity in the lung. Despite known suppression of total pulmonary NO synthase activity in the chronic lung disease model, pulmonary and systemic NO metabolite levels are higher than in the developmental control animals. Thus, a deficiency in NO production and biological function in the premature baboon was not apparent by the detection and quantification of these surrogate markers of NO production.
Human coronary and peripheral arteries show endothelial dysfunction in a variety of conditions, including atherosclerosis, hypercholesterolemia, smoking, and hypertension. This dysfunction manifests as a loss of endothelium-dependent vasodilation to acetylcholine infusion or sheer stress, and is typically associated with decreased generation of nitric oxide (NO) by the endothelium. Vitamin C, or ascorbic acid, when acutely infused or chronically ingested, improves the defective endothelium-dependent vasodilation present in these clinical conditions. The mechanism of the ascorbic acid effect is unknown, although it has been attributed to an antioxidant function of the vitamin to enhance the synthesis or prevent the breakdown of NO. In this review, multiple mechanisms are considered that might account for the ability of ascorbate to preserve NO. These include ascorbate-induced decreases in low-density lipoprotein (LDL) oxidation, scavenging of intracellular superoxide, release of NO from circulating or tissue S-nitrosothiols, direct reduction of nitrite to NO, and activation of either endothelial NO synthase or smooth muscle guanylate cyclase. The ability of ascorbic acid supplements to enhance defective endothelial function in human diseases provides a rationale for use of such supplements in these conditions. However, it is first necessary to determine which of the many plausible mechanisms account for the effect, and to ensure that undesirable toxic effects are not present.
Studies of cultured bovine aortic endothelial cells using quantitative chemiluminescence techniques have shown that the amount of nitric oxide released under basal conditions, or in response to either bradykinin or the calcium ionophore A23187 is insufficient to account for the vasorelaxant activities of the endothelium-derived relaxing factor (EDRF) derived from the same source. This observation contradicts previous suggestions that nitric oxide and EDRF are the same compound, but may be explained if EDRF is a compound that contains nitric oxide within its structure but is a much more potent vasodilator than nitric oxide. Such a molecule could be one of several nitrosothiols which may yield nitric oxide after a one-electron reduction. The present experiments were carried out to test the possibility that the biological activities of the endothelium-derived relaxing factor might more closely resemble those of one of these compounds, S-nitrosocysteine, than nitric oxide. Nitric oxide release from cultured bovine aortic endothelial cells was detected by chemiluminescence and bioassay experiments compared the vasodilator potencies of nitric oxide, S-nitrosocysteine, and EDRF. The results suggest that EDRF is much more likely to be a nitrosylated compound such as a nitrosothiol than authentic nitric oxide.
The responses of small (60-100 microns), medium (101-190 microns), and large (191-300 microns) porcine coronary microvessels to nitroglycerin were examined in vitro using a video-imaging apparatus. Large coronary microvessels, preconstricted with acetylcholine, relaxed by 90% in response to nitroglycerin, whereas small microvessels relaxed only 20% to nitroglycerin. Responses to putative metabolites of nitroglycerin, S-nitrosocysteine, and nitric oxide, were also examined. S-Nitrosocysteine produced equal relaxations in all sizes of coronary microvessels. Nitric oxide was 10 times more potent in large coronary arteries than in small but produced greater than 90% relaxation of all sizes of coronary microvessels at the highest concentrations. Bradykinin and the calcium ionophore A23187, which release endothelium-derived relaxing factor (EDRF), produced similar relaxation in small, medium, and large microvessels. The compound LY 83583 (which depletes vascular guanylate cyclase) reduced responses to nitroglycerin, nitric oxide, S-nitrosocysteine, bradykinin, and the calcium ionophore A23187 in microvessels of all sizes. Our data are compatible with the concept that nitroglycerin must undergo reductive processing to exert its vasodilator effect, likely through the formation of nitrosothiols. In small coronary microvessels, this biotransformation of nitroglycerin is diminished compared with larger coronary arteries. This may be caused by a relative deficiency of available sulfhydryl groups or a lack of enzymes necessary for conversion of nitroglycerin to its active metabolites in small coronary resistance vessels.
Endothelium-derived factor (EDRF) from bovine aortic endothelial cells was compared to solutions of authentic nitric oxide (NO) and to solutions of the nitrosothiol S-nitroso-L-cysteine. EDRF was produced from endothelial cells by basal release or by stimulation with the calcium ionophore A23187. Biological activity was measured as relaxation of porcine coronary arteries preconstricted with prostaglandin F2 alpha, and chemical analysis was made of the nitrosyl content by measurement of NO released after chemical reduction with 1% sodium iodide in glacial acetic acid. EDRF, NO, and nitrosocysteine had identical half-lives, were all inactivated by hemoglobin and methylene blue, and were all augmented in their biological activity by superoxide dismutase. When solutions were analyzed for their biological activity as a function of the NO content (after NaI/acetic acid reduction), nitrosocysteine showed more vasodilation per amount of contained NO than did authentic NO. Solutions containing EDRF (basal release or by stimulation with A23187) subjected to the same analysis appeared similar to nitrosocysteine, and were distinct from solutions of NO. These experiments show that nitrosyl compounds other than NO can have properties very similar or identical to EDRF, and that in this system EDRF appears more similar to nitrosocysteine than to NO.
Nitroglycerin dilates large (greater than or equal to 100 microns) but not small coronary arterial microvessels, and a putative metabolite of nitroglycerin, S-nitroso-L-cysteine, has been shown in vitro to dilate both large and small coronary microvessels. Based on this evidence, we tested the hypothesis that the lack of response of small coronary microvessels was due to an inability of small coronary microvessels to convert nitroglycerin into its vasoactive metabolite and examined possible explanations for this phenomenon. We studied left ventricular epicardial microvessels in vivo using video microscopy and stroboscopic epi-illumination in anesthetized, open-chest dogs. Diameters were determined while the epicardium was suffused with nitroglycerin, S-nitroso-L-cysteine, or S-nitroso-D-cysteine (all 10 microM) and nitroglycerin in the presence of L- or D-cysteine (100 microM). None of the agents affected systemic hemodynamics. Nitroglycerin dilated large arterioles (20 +/- 2%) but not small arterioles (1 +/- 1%). Both S-nitroso-L-cysteine and S-nitroso-D-cysteine were potent dilators of all size classes of microvessels. Concomitant application of L-cysteine and nitroglycerin evoked dilation in small microvessels (22 +/- 4%, p less than 0.5 versus nitroglycerin alone) and larger microvessels (27 +/- 6%, p = NS versus nitroglycerin alone). D-Cysteine did not alter the microvascular response to nitroglycerin in either small (7 +/- 4%, p = NS versus nitroglycerin alone) or large (18 +/- 3%, p = NS versus nitroglycerin alone) microvessels. Neither L-cysteine nor D-cysteine had a direct effect on microvascular diameter. These findings suggest that 1) sulfhydryl groups are required for the conversion of nitroglycerin to its vasoactive metabolite; 2) the interaction between nitroglycerin and sulfhydryl residues is a stereospecific process, indicating either an intracellular mechanism or a membrane-associated enzymatic reaction; and 3) a lack of available sulfhydryl groups may be responsible for the lack of response of small coronary arterioles to nitroglycerin.