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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.
Nitric oxide (NO) was produced from sodium nitroprusside in the presence of vascular tissue but was not released spontaneously from the nitroprusside anion. In the absence of tissue in the dark nitroprusside did not release NO. When solutions of nitroprusside alone were irradiated with visible light, nitric oxide was released at rates linearly proportional to nitroprusside concentration and light intensity. Nitric oxide was produced from solutions of nitroprusside in the dark after the addition of vascular tissue, including lengths of rabbit aorta, subcellular fractions of aorta, and human plasma. NO was also released from nitroprusside after reaction with various reducing agents including cysteine and other thiols, ascorbic acid, sodium dithionite, ferrous chloride, hemoglobin, myoglobin, and partially purified cytochrome P450 with an NADPH-regenerating system. HCN was simultaneously produced in these solutions, and addition of KCN blocked NO release. Iodine oxidized intermediate cyanoferrates and blocked nitric oxide release. KCN or iodine also blocked NO production by tissue, but had no effect upon photochemical NO release. These results show that, apart from photolysis which makes no physiological contribution, release of nitric oxide from nitroprusside, in simple solutions and in biological tissue, occurs after nitroprusside has undergone reduction and lost cyanide.
BACKGROUND - The endothelium-derived relaxing factor has been shown to be nitric oxide or a related nitroso compound, synthesized by the enzyme nitric oxide synthetase, which oxidizes the guanidono nitrogens of arginine. This enzyme is activated by increases in cytosolic calcium. The effect of the clinically used calcium channel antagonists on this process is controversial. The present study was performed to determine whether calcium channel blockade with these pharmacologic agents would alter the activity of nitric acid synthetase in intact endothelial cells.
METHODS AND RESULTS - A specific and sensitive chemiluminescence assay was used to measure the release of nitrogen oxides (nitric oxide and one-electron oxidation products of nitric oxide) from bovine aortic endothelial cells grown in culture. Under basal conditions, the release of nitrogen oxides was about 0.2 nmol/100 micrograms protein/hr. Bradykinin doubled this response. Removal of extracellular calcium abolished basal and bradykinin-stimulated release of nitrogen oxides. Neither diltiazem, verapamil, nor nifedipine in concentrations that are encountered clinically altered the release of nitrogen oxides.
CONCLUSIONS - These experiments show that although the production of nitrogen oxides is dependent on extracellular calcium, the clinically used calcium channel antagonists do not inhibit the release of the endothelium-derived relaxing factor.