The 1991 Ulf von Euler Lecture. The L-arginine: nitric oxide pathway.
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Amino Acid Oxidoreductases↗
Biomedical subjects
S Moncada
Publications and source records attributed to S Moncada.
Biosynthesis and role of the endothelium-derived vasodilator, nitric oxide, in the gastric mucosa.
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Amino Acid Oxidoreductases↗
Role of basal release of nitric oxide on coronary flow and mechanical performance of the isolated rat heart.
1. The role of nitric oxide (NO) in the regulation of coronary flow and mechanical function was studied in isolated working rat hearts. 2. The inhibition of basal release of NO by L-Ng-monomethylarginine (L-NMMA; 500 microM), a specific inhibitor of its synthesis, induced a reduction of coronary flow to 39.1% (+/- 3.4) of its basal level. 3. Associated with this was a drop of cardiac output to 58.10% (+/- 5.42) of control values. 4. This mechanical dysfunction, which was more pronounced in hypertrophied hearts, appears to be related to ischaemia, as it was prevented by simultaneously administered glyceryl trinitrate. Furthermore, L-NMMA did not alter the contractile activity of isolated cardiac myocytes, thereby excluding a direct toxic effect. 5. These findings provide evidence that NO plays a crucial role in the maintenance of basal coronary flow and appears to be essential for sustaining mechanical activity.
Animals↗
Increased concentrations of nitrite in synovial fluid and serum samples suggest increased nitric oxide synthesis in rheumatic diseases.
Cytokines induce nitric oxide synthesis by endothelial cells, macrophages and polymorphonuclear leucocytes, indicating a role for nitric oxide in inflammatory processes. Nitric oxide production was therefore measured indirectly as nitrite in serum and synovial fluid samples from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) together with serum samples from healthy volunteers matched for age and sex. Serum nitrite concentrations in patients with RA and OA were significantly higher than in controls. In both disease groups synovial fluid nitrite was significantly higher than serum nitrite, implying nitric oxide synthesis by the synovium. Serum and synovial fluid nitrite concentrations in RA were also significantly higher than those in OA. These data show increased nitric oxide production in RA and OA and suggest a role for nitric oxide as an inflammatory mediator in rheumatic diseases.
Arthritis, Rheumatoid↗
Role of endothelium-derived relaxing factor in parasympathetic coronary vasodilation.
Vasodilation following the infusion of acetylcholine is due to the release of endothelium-derived relaxing factor (EDRF). However, the role of EDRF in neurogenic coronary vasodilation, when acetylcholine is released outside the vessel at the adventitial-medial junction, has not been established. The action of EDRF in parasympathetic coronary vasodilation was tested in the present study using a specific inhibitor of EDRF synthesis, nitro-L-arginine methyl ester (L-NAME). Experiments were conducted on closed-chest, alpha-chloralose-anesthetized dogs with the heart paced at a constant rate. Phentolamine and propranolol were administered to block alpha- and beta-adrenergic receptors, and ibuprofen was given to inhibit prostaglandin synthesis. Intracoronary infusion of L-NAME decreased the coronary vasodilation in response to intracoronary acetylcholine or vagal stimulation. The coronary response to the endothelium-independent vasodilator nitroglycerin was unaffected by L-NAME. These data demonstrate that L-NAME specifically inhibits coronary vasodilation caused by acetylcholine and vagal stimulation, indicating that parasympathetic coronary vasodilation is dependent on EDRF.
Animals↗
Nitric oxide functions as an inhibitor of platelet adhesion under flow conditions.
BACKGROUND: Nitric oxide (NO) has been identified as endothelium-derived relaxing factor (EDRF), which, in addition to its relaxant effects on vascular smooth muscle cells, is also a potent inhibitor of platelet aggregation. An inhibitory role on platelet adhesion has been suggested from experiments with washed platelets under static conditions. We have determined whether endothelium-derived and exogenous NO also regulates platelet adhesion in whole blood under flow conditions. METHODS AND RESULTS: The effect of endothelium-derived NO was studied by the addition of specific inhibitors of NO production, L-N-monomethyl arginine (L-NMMA) and N-iminoethyl-L-ornithine (L-NIO), to a perfusion system in which both endothelial cells and their matrices were present. A concentration-dependent increase in platelet adhesion to the matrix was found with a maximum inhibition at a concentration of 2 mM L-NMMA and 0.1 mM L-NIO. The effect was dependent on the presence of endothelial cells, because no increase in platelet adhesion was observed in their absence. The effect of exogenous NO was tested in a specially devised perfusion system in which the NO was introduced at the site of adhesion by means of a porous membrane on which an extracellular matrix of endothelial cells was present. Inhibition of platelet adhesion by NO was found at all shear rates tested and after all perfusion periods. CONCLUSIONS: These results demonstrate that NO is a potent inhibitor of platelet adhesion under flow conditions and thereby contributes to the regulatory role of vascular endothelial cells on platelet-vessel wall interaction.
Animals↗
The simultaneous generation of superoxide and nitric oxide can initiate lipid peroxidation in human low density lipoprotein.
Oxidation of low density lipoprotein (LDL) has been shown to occur in the artery wall of atherosclerotic lesions in both animal models and human arteries. The oxidant(s) responsible for initiating this process are under intensive investigation and 15-lipoxygenase has been suggested in this context. Another possibility is that nitric oxide and superoxide, generated by cells present in the artery wall, react together to form peroxynitrite which decomposes to form the highly reactive hydroxyl radical. In the present study we have modelled the simultaneous generation of superoxide and nitric oxide by using the sydnonimine, SIN-1 and have investigated its effects on LDL. SIN-1 liberates both superoxide and nitric oxide during autooxidation resulting in the formation of hydroxyl radicals. We have demonstrated that superoxide generated by SIN-1 is not available to take part in a dismutation reaction since it reacts preferentially with nitric oxide. It follows, therefore, that during the autooxidation of SIN-1 little or no superoxide, or perhydroxyl radical will be available to initiate lipid peroxidation. We have shown that SIN-1 is capable of initiating the peroxidation of LDL and also converts the lipoprotein to a more negatively charged form. The SIN-1-dependent peroxidation of LDL is completely inhibited by superoxide dismutase which scavenges superoxide. Neither sodium nitroprusside or S-nitroso-N-acetyl penicillamine, which only produce nitric oxide, are able to modify LDL. These results are consistent with the hypothesis that a product of superoxide and nitric oxide could oxidize lipoproteins in the artery wall and so contribute to the pathogenesis of atherosclerosis in vivo.
Antioxidants↗
Effect of local intra-arterial NG-monomethyl-L-arginine in patients with hypertension: the nitric oxide dilator mechanism appears abnormal.
OBJECTIVE: There is indirect evidence that the nitric oxide system may be impaired in hypertensive patients. The objective of this study was to examine basal nitric oxide-mediated dilation in hypertensive patients. DESIGN: The forearm blood flow (FBF) response to noradrenaline and NG-monomethyl-L-arginine (L-NMMA), a stereospecific inhibitor of nitric oxide synthesis, was compared in seven untreated hypertensive patients and 17 normotensive controls. METHODS: Drugs were infused locally into the brachial artery and FBF measured using venous occlusion plethysmography. RESULTS: In normotensives noradrenaline (60, 120 and 240 pmol/min) and L-NMMA (1,2 and 4 mumol/min) produced similar reductions in resting FBF. In the hypertensives L-NMMA was significantly less effective than noradrenaline, such that the threshold dose for L-NMMA vasoconstriction was increased and the overall response to L-NMMA reduced. Furthermore, when noradrenaline was used as an internal control there was a significant negative relationship between the response to L-NMMA and blood pressure. When the responses to L-NMMA and noradrenaline were compared between groups, the response to L-NMMA was significantly less in hypertensives compared with normotensives, whereas there was no statistical difference in the response to noradrenaline between the two groups. CONCLUSIONS: The results suggest an abnormality of basal nitric oxide-mediated dilation in the forearm arteriolar bed of patients with untreated essential hypertension.
Adult↗
Constitutive and inducible nitric oxide synthases in human megakaryoblastic cells.
Human megakaryoblastic cells (Meg-01) were found to possess constitutive and express inducible nitric oxide (NO) synthase activities. The constitutive NO synthase was Ca+(+)- and NADPH-dependent, as is the NO synthase found previously in human platelets. Stimulation of Meg-01 cells by the cytokines interleukin-1 beta (0.15-32.5 ng/ml) and tumor necrosis factor-alpha (0.15-10 ng/ml) resulted in expression of the inducible, Ca+(+)-independent, NO synthase. This activity was increased by the addition of NADPH, tetrahydrobiopterin and sepiapterin as cofactors. Induction of this enzyme was accompanied by a decrease in the constitutive NO synthase activity, a phenomenon which was prevented or reversed by dexamethasone (1 microM). Thus, human early differentiated megakaryocytic cells can synthesize NO from L-arginine by both the constitutive and the inducible NO synthases. These findings indicate that these enzymes may play an important biological role in megakaryocyte and platelet functions.
Amino Acid Oxidoreductases↗
Nitric oxide gas: mediator, modulator, and pathophysiologic entity.
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Animals↗
Endogenous nitric oxide is present in the exhaled air of rabbits, guinea pigs and humans.
The presence of nitric oxide (NO) in the exhaled air of humans and of anaesthetized rabbits and guinea pigs was demonstrated by chemiluminescence, diazotization and mass spectrometry. This NO is endogenously produced in the lung by an NO synthase, since its generation in guinea pigs and rabbits was inhibited by N omega-nitro-L-arginine methyl ester and NG-monomethyl-L-arginine, inhibitors of this enzyme. The effect of the inhibitors was reversed by the precursor of NO synthesis, L-arginine. Since NO is produced by normal vascular endothelium for the physiological regulation of blood flow and pressure and also by activated macrophages to contribute to non-specific immunity, our experiments suggest that NO may play both vascular regulatory and host defence roles in pulmonary physiology and pathophysiology.
Amino Acid Oxidoreductases↗
Endogenous nitric oxide modulates morphine-induced constipation.
Administration of morphine in mice causes inhibition of the gastrointestinal transit of a charcoal meal. Morphine-induced constipation in mice seems to depend predominantly on action(s) on the central nervous system since N-methyl morphine, a quaternary derivative, inhibits intestinal transit only when administered intracerebroventricularly (i.c.v.). L- but not D-arginine, given intraperitoneally, reversed the constipation induced by both morphine and its quaternary analogue. L-arginine was ineffective when given i.c.v. and did not reverse atropine-induced constipation. These results suggest that L-arginine preferentially modulates opioid-induced constipation through a stereospecific and peripheral action(s). It is possible that the effect of L-arginine is achieved by increasing the amount of nitric oxide released by non-adrenergic, non-cholinergic nerves in the gut. Thus, L-arginine may represent a useful agent for the treatment of undesirable constipation associated with the use of narcotic analgesics.
Animals↗
Constitutive and inducible nitric oxide synthases incorporate molecular oxygen into both nitric oxide and citrulline.
Nitric oxide (NO) is synthesized by a number of cells from a guanidino nitrogen atom of L-arginine by the action of either constitutive or inducible NO synthases, both of which form citrulline as a co-product. We have determined the source of the oxygen in both NO and in citrulline formed by the constitutive NO synthase from the vascular endothelium and brain and by the inducible NO synthase from the murine macrophage cell line J774. All these enzymes incorporate molecular oxygen both into NO and into citrulline. Furthermore, activated J774 cells form NO from omega-hydroxyl-L-arginine, confirming the proposal that this compound is an intermediate in the biosynthesis of NO.
Amino Acid Oxidoreductases↗
Human colorectal adenocarcinoma cells: differential nitric oxide synthesis determines their ability to aggregate platelets.
The existence and role of an L-arginine:nitric oxide (NO) pathway in two human colorectal adenocarcinoma cell lines, SW-480 and SW-620, were investigated. Both cell lines, which derive from the same patient, SW-480 from the primary tumor and SW-620 from its metastatic lesion, were shown to have a cytosolic, Ca(2+)-independent, NADPH-dependent NO synthase, the activity of which was lower in the cytosol of SW-620. These cells were more potent inducers of platelet aggregation. In contrast, SW-480, which had more NO synthase activity, were less potent inducers of platelet aggregation. Pretreatment of both cell lines with NG-monomethyl-L-arginine, an inhibitor of NO synthase, potentiated their proaggregating effect and made them equally active. Exogenous L-arginine, NO, and related nitrovasodilators all inhibited platelet aggregation induced by SW-620. The antiaggregating activity of NO was further potentiated by prostacyclin and by M&B22948, a selective inhibitor of cyclic GMP phosphodiesterase. We propose that the generation of NO by tumor cells inversely correlates with their metastatic potential. Furthermore, we show that the lower activity of NO synthase in metastatic cells is due to the presence in these cells of a low molecular weight inhibitor of the NO synthase. In addition, agents which modulate platelet function by a cyclic GMP-dependent mechanism may be useful in the prevention of tumor metastasis.
Adenocarcinoma↗
Synthesis of nitric oxide in the bovine retina.
In the absence of light, high concentrations of cGMP open ion channels in the plasma membranes of rod outer segments. The source of stimulation of retinal guanylate cyclase is not known. Nitric oxide is a potent stimulator of guanylate cyclase in other cell systems. The present data demonstrate that nitric oxide synthase, an enzyme responsible for the production of nitric oxide, is present in retina, and specifically in the rod outer segments. This enzyme uses L-arginine as a substrate and is NADPH- and calcium- dependent. L-arginine-derived nitric oxide may be a source of activation of guanylate cyclase in the retina.
Amino Acid Oxidoreductases↗
Bradykinin and ATP stimulate L-arginine uptake and nitric oxide release in vascular endothelial cells.
The effects of bradykinin and ATP on L-arginine transport and nitric oxide (NO) production were studied in porcine aortic endothelial cells cultured and perfused on microcarriers and deprived of L-arginine for 24 h. Stimulation of cells with bradykinin (100 nM) or ATP (100 microM) resulted in a rapid increase in L-arginine uptake and NO release. In the presence of nitro-L-arginine (100 microM), an inhibitor of NO synthase, the stimulatory effect of bradykinin on L-arginine uptake was partially inhibited while NO release was completely abolished. Nitro-L-arginine alone was not an inhibitor of basal L-arginine transport, suggesting that its inhibitory action was not directly on the L-arginine transporter but a result of the inhibition of NO generation. These data indicate that during agonist-stimulated NO production there is a concomitant increase in the transport of L-arginine into endothelial cells providing a mechanism for the continual generation of NO.
Adenosine Triphosphate↗
Glucocorticoids inhibit the induction of nitric oxide synthase and the related cell damage in adenocarcinoma cells.
Lipopolysaccharide (LPS) induced a time-dependent synthesis of nitric oxide (NO) in EMT6 adenocarcinoma cells, assayed by accumulation of NO-derived nitrite in the medium. The induction of NO synthesis was inhibited in a concentration-dependent manner by the glucocorticoids dexamethasone (IC50 = 5 nM) and hydrocortisone (IC50 = 20 nM) and this effect was partially antagonized by progesterone and cortexolone. If addition of dexamethasone was delayed 6 h or more, inhibition of nitrite accumulation over 24 h was substantially reduced, indicating a lack of direct effect of glucocorticoids on the NO synthase. Nitrite accumulation was accompanied by cell damage, which was increased by L-arginine and inhibited by NG-monomethyl-L-arginine (L-NMMA) and dexamethasone. These data show that NO is a primary cytotoxic mediator and that suppression of its formation by glucocorticoids explains some of their anti-inflammatory and cytoprotective effects.
Amino Acid Oxidoreductases↗
Widespread tissue distribution, species distribution and changes in activity of Ca(2+)-dependent and Ca(2+)-independent nitric oxide synthases.
The distribution of Ca(2+)-dependent and Ca(2+)-independent nitric oxide synthase (NOS) was studied in rabbits and in control and endotoxin-treated rats and guinea-pigs. There was a widespread localization of NOS which differed for the two forms of the enzyme and which showed marked differences between species. Endotoxin induced the activity of the Ca(2+)-independent NOS in many tissues and also increased the activity of Ca(2+)-dependent NOS in the rat ileum and caecum. These results demonstrate the differential distribution of NOSs in control and endotoxin-treated animals and emphasize the widespread biological role of nitric oxide (NO).
Amino Acid Oxidoreductases↗