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.

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1 The role of L-arginine in the basal and stimulated generation of nitric oxide (NO) for endothelium-dependent relaxation was studied by use of NG-monomethyl L-arginine (L-NMMA), a specific inhibitor of this pathway. 2 L-Arginine (10-100MM), but not D-arginine (100 MM), induced small but significant endotheliumdependent relaxations of rings of rabbit aorta. In contrast, L-NMMA (1-300pM) produced small, endothelium-dependent contractions, while its enantiomer N -monomethyl-D-arginine (D-NMMA; 100M) had no effect.3 L-NMMA (1-300pM) inhibited endothelium-dependent relaxations induced by acetylcholine (ACh), the calcium ionophore A23187, substance P or L-arginine without affecting the endotheliumindependent relaxations induced by glyceryl trinitrate or sodium nitroprusside. 4 The inhibition of endothelium-dependent relaxation by L-NMMA (30Mm) was reversed by Larginine (3-300 yM) but not by D-arginine (300 gM) or a number of close analogues (100 pM).5 The release of NO induced by ACh from perfused segments of rabbit aorta was also inhibited by L-NMMA (3-300pM), but not by D-NMMA (1001Mm) and this effect of L-NMMA was reversed by L-arginine (3-300 Mm). 6 These results support the proposal that L-arginine is the physiological precursor for the basal and stimulated generation of NO for endothelium-dependent relaxation.

The mechanism of the increased sensitivity to nitrovasodilators after removal of endothelial nitric oxide (NO) was investigated in vitro and in vivo. The vasoconstrictor potency of phenylephrine and the force of contraction of rat isolated aortic rings were significantly enhanced after endothelium removal or treatment with inhibitors ofendothelial NO synthase. Furthermore, these procedures led to a significant decrease in the basal levels of cGMP in the vascular rings. Moreover, the potency of glyceryl trinitrate (n3Gro) and sodium nitroprusside (SNP) as relaxing agents and the ability of SNP to induce increases in cGMP in aortic rings were significantly enhanced in those rings denuded of endothelium or treated with the inhibitors. These procedures did not affect the vasodilator actions of isoprenaline or 8-bromo-cGMP. In the anesthetized rat, treatment with the inhibitors enhanced significantly the hypotensive responses to n3Gro without affecting those to isoprenaline. These data indicate that the removal of the basal NO-mediated vasodilator tone in the cardiovascular system leads, at the level of the soluble guanylate cyclase, to a specific supersensitivity to nitrovasodilators in vivo. The existence of such a phenomenon has iimportant implications for understanding the local physiological control of blood flow, its pathological disturbances, and the mechanism of action of nitrovasodilators.The vascular endothelium synthesizes nitric oxide (NO) from L-arginine (1,2). This NO accounts for the actions of endothelium-derived relaxing factor (3) and acts via the stimulation of the soluble guanylate cyclase in the vascular smooth muscle (4). The soluble guanylate cyclase may be considered the intracellular receptor of NO (5). Inhibition of the synthesis of NO by NG-monomethyl-L-arginine refs. 6 and 7) and other arginine analogues (8, 9) induces endothelium-dependent contraction of vascular tissue in vitro and a dose-related hypertensive response (for review, see ref. 10). Furthermore, L-NMMA decreases conductance in several vascular beds in animals (11) and reduces blood flow in the arterial circulation of humans (12). These observations show that the basal release of endothelium-derived relaxing factor/NO (7, 13) is responsible for maintaining a vasodilator tone in the cardiovascular system (14). The removal ofa basal mediator tone in other tissues, most notably innervated structures, frequently leads to a specific supersensitivity to the exogenous application of that mediator (15, 16), a phenomenon recognized over 100 years ago (17). Based on this, it is possible to hypothesize that the removal of NO in the vasculature and the consequent up-regulation of its receptor might lead to an increase in the sensitivity to those vasodilators that act by stimulating the soluble guanylate cyclase.Increased sensitivity to sodium nitroprusside (SNP), glyceryl trinitrate (n3Gro), or 3-morpholinosydnonimine (SIN-1) has been described in vitro in deendothelialized vascular tissue (18) or after treatment with nonspeci...

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1 The effect of S-nitroso-glutathione (GSNO), a stable nitrosothiol, on platelet activation was examined in vitro and in vivo. 2 The adhesion of human platelets to fibrillar collagen and human endothelial cell monolayers was inhibited by GSNO. 3 GSNO caused a concentration-dependent inhibition of collagen-induced platelet aggregation in vitro and decreased ADP-induced aggregation in the conscious rat. 4 Inhibition of platelet aggregation in vitro correlated with the increase in intraplatelet cyclic GMP levels. 5 The release of NO from GSNO was enhanced by platelet lysate, native glutathione and ascorbate. 6 The results show that GSNO is a carrier of NO and therefore has pharmacological activity as an inhibitor of platelet activation.