Richard M.J. Palmer

Characterization of three inhibitors of endothelial nitric oxide synthase in vitro and in vivo

1 Three analogues of l‐arginine were characterized as inhibitors of endothelial nitric oxide (NO) synthase by measuring their effect on the endothelial NO synthase from porcine aortae, on the vascular tone of rings of rat aorta and on the blood pressure of the anaesthetized rat. 2 NG‐monomethyl‐l‐arginine (l‐NMMA), N‐iminoethyl‐l‐ornithine (l‐NIO) and NG‐nitro‐l‐arginine methyl ester (l‐NAME; all at 0.1–100 μm) caused concentration‐dependent inhibition of the Ca2+‐dependent endothelial NO synthase from porcine aortae. 3 l‐NMMA, l‐NIO and l‐NAME caused an endothelium‐dependent contraction and an inhibition of the endothelium‐dependent relaxation induced by acetylcholine (ACh) in aortic rings. 4 l‐NMMA, l‐NIO and l‐NAME (0.03–300 mg kg−1, i.v.) induced a dose‐dependent increase in mean systemic arterial blood pressure accompanied by bradycardia. 5 l‐NMMA, l‐NIO and l‐NAME (100 mg kg−1, 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.

L-arginine is the physiological precursor for the formation of nitric oxide in endothelium-dependent relaxation

The formation of nitric oxide (NO) from L-arginine by vascular endothelial cells and its relationship to endothelium-dependent relaxation of vascular rings was studied. The release of NO, measured by bioassay or chemiluminescence, from porcine aortic endothelial cells stimulated with bradykinin was enhanced by infusions of L-, but not D-arginine. The release of 15NO, determined by high resolution mass spectrometry, from L-guanidino 15N (99%) arginine was also observed, indicating that NO is formed from the terminal guanidino nitrogen atom(s) of L-arginine. L-NG-monomethyl arginine (L-NMMA), but not D-NMMA, inhibited both the generation of NO by endothelial cells in culture and the endothelium-dependent relaxation of rabbit aortic rings. Both these effects were reversed by L-arginine. These data indicate that L-arginine is the physiological precursor for the formation of NO which mediates endothelium-dependent relaxation.

The anti‐aggregating properties of vascular endothelium: interactions between prostacyclin and nitric oxide

1 The interactions between endothelium‐derived nitric oxide (NO) and prostacyclin as inhibitors of platelet aggregation were examined. 2 Porcine aortic endothelial cells treated with indomethacin and stimulated with bradykinin (10–100 nm) released NO in quantities sufficient to account for the inhibition of platelet aggregation attributed to endothelium‐derived relaxing factor (EDRF). 3 In the absence of indomethacin, stimulation of the cells with bradykinin (1–3 nm) released small amounts of prostacyclin and EDRF which synergistically inhibited platelet aggregation. 4 EDRF and authentic NO also caused disaggregation of platelets aggregated either with collagen or with U46619. 5 A reciprocal potentiation of both the anti‐and the dis‐aggregating activity was also observed between low concentrations of prostacyclin and authentic NO or EDRF released from endothelial cells. 6 It is likely that interactions between prostacyclin and NO released by the endothelium play a role in the homeostatic regulation of platelet‐vessel wall interactions.

NMDA RECEPTOR ACTIVATION INDUCES NITRIC-OXIDE SYNTHESIS FROM ARGININE IN RAT-BRAIN SLICES

Activation of N-methyl-D-aspartate (NMDA) receptors in rat cerebellum leads to the release of endothelium-derived relaxing factor, now identified as nitric oxide (NO), a stimulator of soluble guanylate cyclase. L-NG-monomethylarginine (L-NMMA), which blocks NO synthesis from L-arginine in several tissues, including a crude synaptosomal preparation from brain, inhibited the elevation of cyclic GMP induced by NMDA in rat cerebellar slices. D-NMMA was ineffective. L-Arginine, but not its D enantiomer, augmented the response to NMDA and reversed the inhibition by L-NMMA. The results indicate that stimulation of NMDA receptors results in the activation of the enzyme which catalyzes the formation of NO from L-arginine.

A specific inhibitor of nitric oxide formation from l-arginine attenuates endothelium-dependent relaxation

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 (10–100 μm), but not d‐arginine (100 μm), induced small but significant endothelium‐dependent relaxations of rings of rabbit aorta. In contrast, l‐NMMA (1–300 μm) produced small, endothelium‐dependent contractions, while its enantiomer NG‐monomethyl‐d‐arginine (d‐NMMA; 100 μ) had no effect. 3 l‐NMMA (1–300 μm) inhibited endothelium‐dependent relaxations induced by acetylcholine (ACh), the calcium ionophore A23187, substance P or l‐arginine without affecting the endothelium‐independent relaxations induced by glyceryl trinitrate or sodium nitroprusside. 4 The inhibition of endothelium‐dependent relaxation by l‐NMMA (30 μm) was reversed by l‐arginine (3–300 μm) but not by d‐arginine (300 μm) or a number of close analogues (100 μm). 5 The release of NO induced by ACh from perfused segments of rabbit aorta was also inhibited by l‐NMMA (3–300 μm), but not by d‐NMMA (100 μm) and this effect of l‐NMMA was reversed by l‐arginine (3–300 μm). 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.

Comparative pharmacology of endothelium-derived relaxing factor, nitric oxide and prostacyclin in platelets

1 The pharmacological effects of endothelium‐derived relaxing factor (EDRF), nitric oxide (NO) and prostacyclin on human and rabbit platelets were examined. 2 EDRF is released from porcine aortic endothelial cells, cultured on microcarriers and treated with indomethacin, in sufficient quantities to inhibit platelet aggregation induced by 9,11‐dideoxy‐9α,11α‐methano epoxy‐prostaglandin F2α (U46619) and collagen. 3 The anti‐aggregating activity of EDRF was potentiated by M&B 22948, a selective inhibitor of cyclic GMP phosphodiesterase, and by superoxide dismutase (SOD) and was inhibited by haemoglobin and Fe2+. 4 Both NO and prostacyclin inhibited platelet aggregation. 5 The anti‐aggregatory activity of NO, but not that of prostacyclin, was potentiated by M&B 22948 and by SOD and was inhibited by haemoglobin and Fe2+. Thus NO is a potent inhibitor of platelet aggregation whose activity on platelets mimics that of EDRF. 6 It is likely that the inhibitory effect of NO on platelets represents the action of endogenous EDRF and therefore this substance, together with prostacyclin, is a regulator of platelet‐vessel wall interactions.

The role of nitric oxide and cGMP in platelet adhesion to vascular endothelium.

The inhibition of platelet adhesion by nitric oxide (NO) and prostacyclin and their mechanism of action was studied. Platelet adhesion to collagen fibrils and endothelial cell matrix was inhibited completely by NO but only partially by prostacyclin. Adhesion of platelets to endothelial cell monolayers was inhibited by bradykinin. This effect of bradykinin was unaffected by aspirin, and was accounted for by the amounts of NO released by the endothelial cells. Inhibition of platelet adhesion by NO and prostacyclin was potentiated by selective inhibitors of cGMP phosphodiesterase, but not of cAMP phosphodiesterase, indicating that elevation of cGMP regulates platelet adhesion.

A novel citrulline-forming enzyme implicated in the formation of nitric oxide by vascular endothelial cells

An enzyme in homogenates of porcine vascular endothelial cells forms L-citrulline from L-arginine. This enzyme is soluble and NADPH-dependent. In addition, the enzyme is inhibited by NG-monomethyl-L-arginine, suggesting that it is involved in the formation of nitric oxide by vascular endothelial cells.

Characterization of the L-arginine:nitric oxide pathway in human platelets.

1 The activation of the l‐arginine:nitric oxide (NO) pathway during aggregation of human platelets by adenosine 5′‐diphosphate (ADP), arachidonic acid, thrombin and the calcium ionophore A23187 and its inhibition by NG‐monomethyl‐l‐arginine (l‐NMMA), NG‐nitro‐l‐arginine methyl ester (l‐NAME) and N‐iminoethyl‐l‐ornithine (l‐NIO) were studied. The inhibition of the cytosolic platelet NO synthase by these compounds was also examined. 2 Platelet aggregation induced by ADP (1–10 μm) and arachidonic acid (0.1–10 μm), but not that induced by thrombin (1–30 mu ml−1) or A23187 (1–10 nm), was inhibited by l‐, but not d‐arginine (1–30 μm). However, in the presence of a subthreshold concentration of prostacyclin (0.1 nm) or of M & B 22948 (1 μm), a selective inhibitor of guanosine 3′:5′‐cyclic monophosphate (cyclic GMP) phosphodiesterase, l‐arginine caused concentration‐dependent inhibition of aggregation induced by all of these aggregating agents. 3 l‐NMMA, l‐NAME and l‐NIO (all at 1–30 μm), but not their d‐enantiomers, enhanced to the same extent platelet aggregation induced by ADP, arachidonic acid and thrombin without affecting that induced by A23187. 4 In the presence of 300 μm l‐arginine, the NO synthase in platelet cytosol was inhibited by l‐NMMA, l‐NAME and l‐NIO with IC50s of 74 ± 9, 79 ± 8 and 8.5 ± 1.5 μm (n = 3), respectively. 5 These results indicate that the l‐arginine: NO pathway in human platelets plays a role in the modulation of platelet aggregation.

Identification of N-iminoethyl-L-ornithine as an irreversible inhibitor of nitric oxide synthase in phagocytic cells.

1 The synthesis of nitric oxide (NO) from l‐arginine by rat peritoneal neutrophils (PMN) and the murine macrophage cell‐line J774 and the inhibition of this synthesis by N‐iminoethyl‐l‐ornithine (l‐NIO), NG‐monomethyl‐l‐arginine (l‐NMMA), NG‐nitro‐l‐arginine (l‐NNA) and its methyl ester (l‐NAME) were investigated. 2 l‐NIO was the most potent inhibitor in both types of cells while l‐NMMA was less active. l‐NNA and l‐NAME had no significant effect in PMN and l‐NNA produced only approximately 40% inhibition of the generation of NO in the J774 cells at the highest concentration tested (300 μm). 3 The inhibitory effect of l‐NIO was rapid in onset, requiring 10 min pre‐incubation to achieve its full inhibitory activity, while the other compounds required 20–60 min pre‐incubation to achieve their full effect. 4 The inhibitory effect of l‐NIO (10 μm) on intact cells could not be reversed by l‐arginine (300 μm) but could be prevented by concomitant incubation with this compound (300 μm), while the effect of the other inhibitors could be reversed by a 3–5 fold molar excess of l‐arginine. 5 The NO synthase from both PMN and J774 cells was cytosolic and NADPH‐ but not Ca2+‐dependent, with Km values for l‐arginine of 3.3 ± 0.8 and 4.2 ± 1.1 μm respectively. 6 l‐NIO was the most potent inhibitor of the neutrophil and J774 enzymes with IC50 values of 0.8 ± 0.1 and 3 ± 0.5 μm respectively. Furthermore, the effect of l‐NIO was irreversible. The other three compounds were less potent, reversible inhibitors. 7 The inhibitory effects of all these compounds were enantiomerically specific. 8 These data indicate that l‐NIO is a novel, potent, rapid in onset and irreversible inhibitor of NO synthase in phagocytic cells. The rapid uptake of l‐NIO compared with the other compounds indicates that phagocytic cells have different uptake mechanisms for l‐arginine analogues.