The objective of this study was to determine whether nitric oxide (NO) is responsible for the vascular smooth muscle relaxation elicited by endothelium-derived relaxing factor (EDRF). EDRF is an unstable humoral substance released from artery and vein that mediates the action of endothelium-dependent vasodilators. NO is an unstable endothelium-independent vasodilator that is released from vasodilator drugs such as nitroprusside and glyceryl trinitrate. We have repeatedly observed that the actions of NO on vascular smooth muscle closely resemble those of EDRF. In the present study the vascular effects of EDRF released from perfused bovine intrapulmonary artery and vein were compared with the effects of NO delivered by superfusion over endotheliumdenuded arterial and venous strips arranged in a cascade. EDRF was indistinguishable from NO in that both were labile (1i/2 = 3-5 sec), inactivated by pyrogallol or superoxide anion, stabilized by superoxide dismutase, and inhibited by oxyhemoglobin or potassium. Both EDRF and NO produced comparable increases in cyclic GMP accumulation in artery and vein, and this cyclic GMP accumulation was inhibited by pyrogallol, oxyhemoglobin, potassium, and methylene blue. EDRF was identified chemically as NO, or a labile nitroso species, by two procedures. First, like NO, EDRF released from freshly isolated aortic endothelial cells reacted with hemoglobin to yield nitrosylhemoglobin. Second, EDRF and NO each similarly promoted the diazotization of sulfanilic acid and yielded the same reaction product after coupling with N-(1-naphthyl)-ethylenediamine. Thus, EDRF released from artery and vein possesses identical biological and chemical properties as NO.Both artery and vein are capable of releasing endotheliumderived relaxing factor (EDRF) in response to chemically diverse vasodilators (1-9). Endothelium-dependent relaxation of artery and vein appears to be mediated by increases in tissue cyclic GMP levels (10-13), and such effects are inhibited by methylene blue, hemoglobin, and myoglobin (13-15). Nitroso compounds, organic nitrate and nitrite esters, and inorganic nitrite cause vascular smooth muscle relaxation and cyclic GMP accumulation by endotheliumindependent mechanisms, and these actions are attributed to the release of nitric oxide (NO) (16)(17)(18)(19). NO itself is a labile substance that causes transient relaxation and cyclic GMP accumulation in both artery and vein (16)(17)(18)(19)(20) MATERIALS AND METHODSReagents. Acetylcholine chloride, phenylephrine hydrochloride, A23187, pyrogallol, hemoglobin (human), and superoxide dismutase (bovine liver) were obtained from Sigma. Glyceryl trinitrate (10% wt/wt triturate in lactose) was a gift from Imperial Chemical Industries (Macclesfield, England), and propylbenzylylcholine mustard was provided by the National Institute for Medical Research (Mill Hill, London). NO (99o pure) was obtained from Matheson. A saturated solution of NO (1-2 mM) in oxygen-free water (prepared by vacuum evacuation and nitrogen flushing) was pre...

In retrospect, basic research in the fields of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) during the past two decades appears to have followed a logical course, beginning with the findings that NO and cGMP are vascular smooth muscle relaxants, that nitroglycerin relaxes smooth muscle by metabolism to NO, progressing to the discovery that mammalian cells synthesize NO, and finally the revelation that NO is a neurotransmitter mediating vasodilation in specialized vascular beds. A great deal of basic and clinical research on the physiologic and pathophysiologic roles of NO in cardiovascular function has been conducted since the discovery that endothelium-derived relaxing factor (EDRF) is NO. The new knowledge on NO should enable investigators in this field to develop novel and more effective therapeutic strategies for the prevention, diagnosis, and treatment of numerous cardiovascular disorders. The goal of this review was to highlight the early research that led to our current understanding of the pathophysiologic role of NO in cardiovascular medicine. Furthermore, we discussed the possible mechanism of some drugs interfering with NO signaling cascade.

Nitric oxide (NO) in oxygen-containing aqueous solution has a short half-life that is often attributed to a rapid oxidation to both NOj-and NOT. The chemical fate ofNO in aqueous solution is often assumed to be the same as that in air, where NO is oxidized to NO2 followed by dimerization to N204. Water then reacts with N204 to form both NO-and NO-. We report here that NO in aqueous solution containing oxygen is oxidized primarily to NO -with little or no formation of NO3. In the presence of oxyhemoglobin or oxymyoglobin, however, NO and NO-were oxidized completely to N03. Methemoglobin was inactive in this regard. The unpurified cytosoLic fraction from rat cerebellum, which contains constitutive NO synthase activity, catalyzed the conversion of L-arginine primarily to NO3-(NOiT/NOiT ratio = 0.25). After chromatography on DEAE-Sephacel or affinity chromatography using 2',5'-ADP-Sepharose 4B, active fractions containing NO synthase activity catalyzed the conversion of L-arginine primarily to NO-(NOj-/NOj ratio = 5.6) or only to NOT, Sufficient evidence has been amassed to indicate a wide biological role for endogenous nitric oxide (NO) in modulating physiological and pathophysiological processes (1). NO is synthesized in various cell types by a family ofisoforms ofNO synthase (2). Some isoforms are constitutive and activated by calcium, whereas other isoforms are inducible and regulated by transcriptional mechanisms. Both isoforms catalyze the same complex oxidation of L-arginine to NO plus L-citrulhne (3-6). The mechanism of catalysis of NO synthase is similar to that for the cytochrome P450 monooxygenases in that molecular oxygen is incorporated into the substrate by reactions involving NADPH, flavins, and heme (7).To appreciate the diverse biological actions of NO, it is essential to understand not only the biosynthesis but also the metabolism of NO and the chemistry of NO in aqueous solution. NO pure aqueous solution, however, display half-lives of 500 sec or longer (10). This means that, in the presence of biological tissues, NO is rapidly converted to a less-active or inactive product. The chemical lability of NO in cells and tissues has been attributed to a rapid oxidation to both NOj and NO3 (11)(12)(13)(14). The common belief that NO is oxidatively metabolized to both NOj and NO3 derives largely from experiments with intact cells, tissues, and whole animals rather than pure aqueous systems. For example, macrophages that have been activated in culture to induce NO synthase activity generate both . Moreover, endogenous NO-production in whole animals cannot be observed by assaying plasma or urine because of the nearly complete oxidation of NO or NO -to NO-(12). NO gas reacts with oxygen to form NO2 gas, which dimerizes to N204. N204 dismutates spontaneously in water to form NO-(as HNO2) and NO-(as HNO3) (16). The assumption is commonly made that NO in an aqueous solution containing oxygen generates NO-and NO-. This assumption is inconsistent with chemical studies showing that pure aqueous solutions...

The objective of this study was to elucidate the close similarity in properties between endothelium-derived relaxing factor (EDRF) and nitric oxide radical (NO). Whenever possible, a comparison was also made between arterial and venous EDRF. In vascular relaxation experiments, acetylcholine and bradykinin were used as endothelium-dependent relaxants of isolated rings of bovine intrapulmonary artery and vein, respectively, and NO was used to relax endothelium-denuded rings. Oxyhemoglobin produced virtually identical concentration-dependent inhibitory effects on both endothelium-dependent and NO-elicited relaxation. Oxyhemoglobin and oxymyoglobin lowered cyclic guanosine monophosphate (cGMP) levels, increased tone in unrubbed artery and vein, and abolished the marked accumulation of vascular cGMP caused both by endothelium-dependent relaxants and by NO. The marked inhibitory effects of oxyhemoglobin on arterial and venous relaxant responses and cGMP accumulation as well as its contractile effects were abolished or reversed by carbon monoxide. These observations indicate that EDRF and NO possess identical properties in their interactions with oxyhemoproteins. Both EDRF from artery and vein and NO activated purified soluble guanylate cyclase by heme-dependent mechanisms, thereby revealing an additional similarity in heme interactions. Spectrophotometric analysis disclosed that the characteristic shift in the Soret peak for hemoglobin produced by NO was also produced by an endothelium-derived factor released from washed aortic endothelial cells by acetylcholine or A23187. Pyrogallol, via the action of superoxide anion, markedly inhibited the spectral shifts, relaxant effects, and cGMP accumulating actions produced by both EDRF and NO. Superoxide dismutase enhanced the relaxant and cGMP accumulating effects of both EDRF and NO. Thus, EDRF and NO are inactivated by superoxide in a closely similar manner. We conclude, therefore, that EDRF from artery and vein is either NO or a chemically related radical species.

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