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...

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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Soluble guanylate cyclase [GTP pyrophosphatelyase (cyclizing), EC 4.6.1.2] purified from bovine lung is markedly activated (30-to 40-fold) byprotoporphyrin IX (Ka,(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25) and is inhibited by hematin (Ki, 3.7 MATERIALS AND METHODS Purification ofGuanylate Cyclase. Details ofthe purification procedures will be described elsewhere. Briefly, fresh bovine lung (0.5 kg) was homogenized in 3 vol of 25 mM triethanolamine HCl (Et3N.HCl), pH 7.8/5 mM dithiothreitol. Homogenates were centrifuged at 105,000 X g for 60 min, and the supernatant was stirred with 400 g of DEAE-Sepharose CL-6B (Pharmacia), preequilibrated with Et3N-HCVdithiothreitol. The resin was extensively washed with Et3N-HCVdithiothreitol and packed into a column (2.5 x 80 cm). Guanylate cyclase was eluted with 1.3 liters ofa linear NaCl gradient (0-0.4 M) in Et3N-HCVdithiothreitol. Fractions with peak activity (0.20-0.23 M NaCl) were pooled, concentrated, and applied to a column (2.5 x 80 cm) ofUltrogel (LKB) equilibrated with Et3N.HCVdithiothreitol. Guanylate cyclase was eluted with 300 ml of Et3N-HCVdithiothreitol and active fractions were pooled, concentrated, and applied to a column (1.5 X 20 cm) of Matrex gel blue A (Amicon). After incorporating the sample into the resin, flow was stopped for 45 min, and the column was then washed extensively with Et3N-HCVdithiothreitol followed with 0.5 M NaCl in Et3N-HCVdithiothreitol. Guanylate cyclase was eluted with 400 ml of a linear NaCl gradient (0.5-2 M) in Et3N-HCVdithiothreitol. Fractions with peak enzymatic activity (1.2-1.3 M NaCI) were pooled and concentrated. Glycerol was added to 30% (vol/vol), and 0.2-to 0.5-ml aliquots were equilibrated with oxygen-free N2 and stored at -60'C. Basal specific activities (tkmol of cGMP per min/mg of protein) were 0.1-0.2 in the presence of 1 mM GTP and 3 mM Mg2+, and 0.3-0.5 with 1 mM GTP and 3 mM Mn2+.Guanylate cyclase was purified 6,000-to 10,000-fold from the starting crude supernatant (0. Thepublication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

The role of the endothelium in modulating cyclic nucleotide levels and intrinsic smooth muscle tone was studied in isolated rings of bovine intrapulmonary artery and vein. Cyclic 3',5'-guanosine monophosphate (cGMP) levels were threefold to fourfold higher in unrubbed artery and vein than in vessels that had been denuded of endothelium. Cyclic 3',5'-adenosine monophosphate (cAMP) levels were twofold higher in unrubbed than in endothelium-denuded artery, but no differences were observed in veins. Methylene blue, an inhibitor of guanylate cyclase, decreased cGMP but not cAMP levels, and this was accompanied by increases in smooth muscle tone. M&B 22,948, an inhibitor of cGMP-phosphodiesterase, increased cGMP but not cAMP levels, and this was accompanied by decreases in smooth muscle tone. Unrubbed vessels were more sensitive than endothelium-denuded vessels to the actions of both methylene blue and M&B 22,948, and this may be attributed to endothelium-dependent increases in cGMP turnover. Moreover, unrubbed vessels were more sensitive than endothelium-denuded vessels to contractile responses to phenylephrine and potassium, and these responses were potentiated by methylene blue and attenuated by M&B 22,948. Although indomethacin lowered cAMP levels in unrubbed artery, no changes in tone or contractile responsiveness were observed. A consistent observation was that the smaller branches of unrubbed but not endothelium-denuded intrapulmonary artery and vein had higher levels of cGMP but not cAMP, were sensitive to endothelium-dependent vasodilators, were more sensitive to methylene blue, and would not maintain a steady level of submaximal tone to phenylephrine when compared with larger branches from a common vascular bed.(ABSTRACT TRUNCATED AT 250 WORDS)