K S Wood

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 endothelium-denuded arterial and venous strips arranged in a cascade. EDRF was indistinguishable from NO in that both were labile (t1/2 = 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.

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.

The mechanism of activation of soluble guanylate cyclase purified from bovine lung by high molecular weight, nitrosyl-hemoprotein complexes is reported. Heme-containing, heme-deficient, and heme-reconstituted forms of guanylate cyclase were studied. Nitric oxide (NO) and nitroso compounds activated heme-containing and heme-reconstituted enzymes (over 50-fold), with an accompanying shift in the Soret absorption peak from 431 to 398 nm, but failed to activate or alter the spectral characteristics of heme-deficient enzyme. In contrast, preformed NO-hemoprotein complexes as well as low molecular weight NO-heme activated all forms of guanylate cyclase. Heme-deficient guanylate cyclase was first reacted with excess amounts of NO-hemoglobin, NO-myoglobin, or NO-catalase and then rapidly separated from the NO-hemoprotein by column chromatography. Spectrophotometric analysis indicated that the NO-heme moiety was transferred from each of the NO-hemoproteins to heme-deficient guanylate cyclase. Approximately 1 mol of NO-heme was bound per mol of holoenzyme and the specific activity of this enzyme form was over 50-fold greater than that of unreacted, heme-deficient enzyme. NO-heme was tightly bound to guanylate cyclase as no transfer of enzyme-bound NO-heme to apohemoglobin was evident. Enzyme activated by NO-hemoproteins closely resembled, kinetically, that activated by NO or NO-heme. In contrast, reactions between heme-deficient guanylate cyclase and hemoproteins did not result in heme transfer, whereas heme alone rapidly reconstituted the enzyme. These observations indicate that soluble guanylate cyclase can be readily reconstituted with, and thereby activated by, NO-heme through an exchange reaction with NO-hemoproteins.