Endothelium-derived relaxing factor (EDRF) is a labile humoral agent which mediates the action of some vasodilators. Nitrovasodilators, which may act by releasing nitric oxide (NO), mimic the effect of EDRF and it has recently been suggested by Furchgott that EDRF may be NO. We have examined this suggestion by studying the release of EDRF and NO from endothelial cells in culture. No was determined as the chemiluminescent product of its reaction with ozone. The biological activity of EDRF and of NO was measured by bioassay. The relaxation of the bioassay tissues induced by EDRF was indistinguishable from that induced by NO. Both substances were equally unstable. Bradykinin caused concentration-dependent release of NO from the cells in amounts sufficient to account for the biological activity of EDRF. The relaxations induced by EDRF and NO were inhibited by haemoglobin and enhanced by superoxide dismutase to a similar degree. Thus NO released from endothelial cells is indistinguishable from EDRF in terms of biological activity, stability, and susceptibility to an inhibitor and to a potentiator. We suggest that EDRF and NO are identical.

Nitric oxide (NO) released by vascular endothelial cells accounts for the relaxation of strips of vascular tissue and for the inhibition of platelet aggregation and platelet adhesion attributed to endothelium-derived relaxing factor. We now demonstrate that NO can be synthesized from L-arginine by porcine aortic endothelial cells in culture. Nitric oxide was detected by bioassay, chemiluminescence or by mass spectrometry. Release of NO from the endothelial cells induced by bradykinin and the calcium ionophore A23187 was reversibly enhanced by infusions of L-arginine and L-citrulline, but not D-arginine or other close structural analogues. Mass spectrometry studies using 15N-labelled L-arginine indicated that this enhancement was due to the formation of NO from the terminal guanidino nitrogen atom(s) of L-arginine. The strict substrate specificity of this reaction suggests that L-arginine is the precursor for NO synthesis in vascular endothelial cells.

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Endothelium-derived vascular relaxing factor (EDRF) is a humoral agent that is released by vascular endothelium and mediates vasodilator responses induced by various substances including acetylcholine and bradykinin. EDRF is very unstable, with a half-life of between 6 and 50 s, and is clearly distinguishable from prostacyclin. The chemical structure of EDRF is unknown but it has been suggested that it is either a hydroperoxy- or free radical-derivative of arachidonic acid or an unstable aldehyde, ketone or lactone. We have examined the role of superoxide anion (O-2) in the inactivation of EDRF released from vascular endothelial cells cultured on microcarrier beads and bioassayed using a cascade of superfused aortic smooth muscle strips. With this system, we have now demonstrated that EDRF is protected from breakdown by superoxide dismutase (SOD) and Cu2+, but not by catalase, and is inactivated by Fe2+. These findings indicate that O-2 contributes significantly to the instability of EDRF.

0.03-300mgkg-1, i.v.) induced a dose-dependent increase in mean systemic arterial blood pressure accompanied by bradycardia. 5L-NMMA, L-NIO and L-NAME (100 mgkg-',i.v.) inhibited significantly the hypotensive responses to ACh and bradykinin. 6 The increase in blood pressure and bradycardia produced by these compounds were reversed by L-arginine (30-100 mg kg-1, i.v.) in a dose-dependent manner. 7 All of these effects were enantiomer specific. 8 These results indicate that L-NMMA, L-NIO and L-NAME are inhibitors of NO synthase in the vascular endothelium and confirm the important role of NO synthesis in the maintenance of vascular tone and blood pressure.

The role of endothelium-derived nitric oxide in the regulation of blood pressure in the anesthetized rabbit was studied with Na-monomethyl-L-arginine (L-NMMA), a specific inhibitor of its formation from L-arginine. L-NMMA (3-100 mg-kg-'), but not its D-enantiomer, induced a dosedependent long-lasting (15-90 min) increase in mean systemic arterial blood pressure. L-NMMA (100 mg kg-') also inhibited significantly the hypotensive action of acetylcholine, without affecting that of glyceryl trinitrate. Both these actions of L-NMMA were reversed by L-arginine (300 mg-kg-'), but not by D-arginine (300 mg-kg-'), indomethacin (1 mg-kg-'), prazosin (0.3 mg-kg-'), or by vagotomy. The effects of L-NMMA in vivo were associated with a significant inhibition of the release of nitric oxide from perfused aortic segments ex vivo. This inhibition was reversed by infusing L-arginine through the aortic segments. These results indicate that nitric oxide formation from L-arginine by the vascular endothelium plays a role in the regulation of blood pressure and in the hypotensive actions of acetylcholine.Although endothelium-dependent vascular relaxation and the release of endothelium-derived relaxing factor in vitro has been clearly established (1-4), there is only circumstantial evidence to indicate their occurrence in vivo. Alterations in vessel diameter that follow changes in blood flow are endothelium dependent (5). Furthermore, damage to the endothelium (6, 7) or treatment with methylene blue (8) or gossypol (9), two nonspecific inhibitors of endothelium-dependent relaxation, all abolish the response to endothelium-dependent vasodilators in vivo without affecting the response to the endothelium-independent vasodilators, sodium nitroprusside or glyceryl trinitrate (n3Gro).Nitric oxide (NO) accounts for the biological actions of endothelium-derived relaxing factor (10-15) and is formed by vascular endothelial cells from the terminal guanido nitrogen atom(s) of the amino acid L-arginine (16,17). This biosynthetic process, the endothelium-dependent relaxation of vascular rings, and the vasodilatation induced by acetylcholine (ACh) in the coronary circulation of the rabbit heart are inhibited by the L-arginine analogue, Na-monomethyl-L-arginine . These results indicate that L-arginine is the physiological precursor for NO synthesis by the vascular endothelium.We have now used L-NMMA to investigate the role of NO in the regulation of blood pressure in the anesthetized rabbit.MATERIALS AND METHODS Methods. Male New Zealand White rabbits (2.0-2.2 kg) were anesthetized with sodium pentobarbitone (40-50 mg-kg-1). Anesthesia was then maintained by a continuous infusion of sodium pentobarbitone (15 mg kg-' hr-1) via the left marginal ear vein and the rabbits were ventilated with room air via a tracheotomy tube.

A soluble enzyme obtained from rat forebrain catalyzes the NADPH-dependent formation of nitric oxide (NO) and citrulline from L-arginine. The NO formed stimulates the soluble guanylate cyclase and this stimulation is abolished by low concentrations of hemoglobin. The synthesis of NO and citrulline is dependent on the presence of physiological concentrations of free Ca2+ and is inhibited by NG-monomethyl-L-arginine, but not by its enantiomer NGmonomethylDarginin e or by Lcanavanine. L-Homoarginine, L-argyl-L-apartate, or L-arginine methyl ester can replace L-ginine as substrates for the enzyme. These results indicate that NO is formed from Larginine in the brain through an enzymic reaction similar to that in vascular endothelial cells, neutrophils, and macrophages, adding support to our hypothesis that the formation of NO from L-arginine is a widespread transduction mechanism for the stimulation of the soluble guanylate cyclase. Activated macrophages also synthesize NOj and NO3 from the terminal guanido nitrogen atom(s) of L-arginine (5). This reaction, which occurs via the formation of NO (6), is involved in the cytotoxic activities of these cells (7). We have demonstrated recently that rat peritoneal neutrophils form NO from L-arginine (8).The endothelial cell and macrophage enzyme that forms NO from L-arginine is soluble, is NADPH-dependent, forms citrulline as a coproduct, and is inhibited by NI-monomethyl-L-arginine refs. 6 and 9). Furthermore, in both cells the enzyme requires a divalent cation, which in the case of the macrophage has been suggested to be Mg2+ (6).Some years ago, L-arginine was identified as an endogenous activator of the soluble guanylate cyclase in brain tissue (10). Since this activation resembled that of the nitrovasodilators (11) and NO is known to stimulate soluble guanylate cyclase in the brain (12), we have investigated the existence in the central nervous system of an enzymic system capable of converting L-arginine into NO and citrulline.While this work was in progress Garthwaite et al. (13) Preparation of Crude Synaptosomal Cytosol. Male rats (200-300 g, four for each preparation) were killed by cervical dislocation and the forebrains were rapidly removed and cooled in ice-cold washing buffer (0.32 M sucrose/10 mM Hepes/0.1 mM EDTA, pH 7.4); subsequent procedures were carried out at 0-40C. The tissue was placed in fresh washing buffer, finely minced, washed once with 50 ml of washing buffer and twice with homogenization buffer (0.32 M sucrose/10 mM Hepes, 1 mM DL-dithiothreitol, pH 7.4) to remove contaminating erythrocytes, and homogenized with 20 strokes of a Dounce homogenizer. The homogenate was diluted to 50 ml with homogenization buffer and centrifuged (1400 x g, 10 min); the supernatant obtained was centrifuged (18,000 x g, 10 min) to obtain a crude synaptosomal pellet. After aspiration of the supernatant, 8 ml of 1 mM DLdithiothreitol (dissolved in distilled water) was added to the pellet to cause hypotonic swelling of the synaptosomes, which were then lysed by ho...

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