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S Moncada

Publications and source records attributed to S Moncada.

At least 91 records · Page 5Linked to original sources
PubMed1995

Molecular genetic analysis of the duplication of human inducible nitric oxide synthase (NOS2) sequences.

In previous studies, we found multiple copies of inducible nitric oxide synthase gene (NOS2)-like sequences in the human genome and mapped them to the pericentric region of chromosome 17. Here, we describe the cloning and sequencing of exon22 regions from three of these NOS2-like sequences. We have also mapped another NOS2-like sequence to human chromosome 14. Since there are multiple NOS2-like sequences present in the human genome, we have also carried out Zoo Blot hybridisation analysis using a NOS2 cDNA probe. Our data suggest that duplication of NOS2-like sequences occurred very recently in primate evolution.

Amino Acid Oxidoreductases↗
PubMed1995

Direct measurement of nitric oxide in human beings.

Nitric oxide is a short-lived radical involved in various biological processes. We have used an electrochemical microsensor to detect nitric oxide signals in blood vessels of healthy volunteers. The sensor was inserted into a hand vein, and the vessel was stimulated with acetylcholine or bradykinin. Dose-dependent signals were detected and were attenuated by an inhibitor of nitric oxide synthase. The results provide further evidence that endothelium-derived relaxing factor is nitric oxide and demonstrate a method for monitoring the L-arginine/nitric-oxide pathway in human beings.

Acetylcholine↗
PubMed1995

Altered immune responses in mice lacking inducible nitric oxide synthase.

Nitric oxide (NO) is important in many biological functions. It is generated from L-arginine by the enzyme NO synthase (NOS). The cytokine-inducible NOS (iNOS) is activated by several immunological stimuli, leading to the production of large quantities of NO which can be cytotoxic. To define the biological role of iNOS further, we generated iNOS mutant mice. These are viable, fertile and without evident histopathological abnormalities. However, in contrast to wild-type and heterozygous mice, which are highly resistant to the protozoa parasite Leishmania major infection, mutant mice are uniformly susceptible. The infected mutant mice developed a significantly stronger Th1 type of immune response than the wild-type or heterozygous mice. The mutant mice showed reduced nonspecific inflammatory response to carrageenin, and were resistant to lipopolysaccharide-induced mortality.

Amino Acid Oxidoreductases↗
PubMed1995

Purification to homogeneity and characterisation of rat brain recombinant nitric oxide synthase.

We have previously demonstrated high expression of rat neuronal nitric oxide synthase (NO synthase) in a baculovirus system [Charles, I. G., Chubb, A., Gill, R., Clare, J., Lowe, P. N., Holmes, L. S., Page, M., Keeling, J. G., Moncada, S. & Riveros-Moreno, V. (1993) Biochem. Biophys. Res. Commun. 196, 1481-1489], where a small proportion of the expressed enzyme was soluble and active, but the majority was insoluble (approximately 15% of the total insoluble proteins). NO synthase is a complex enzyme, requiring several cofactors for full activity. These include tightly bound FAD, FMN, heme and tetrahydrobiopterin, in addition to calmodulin and NADPH. Here, we report that a substantial proportion of the total NO synthase produced becomes soluble following addition of hemin (2.5 micrograms/ml) to the culture medium. However, the enzyme purified under these conditions had very low specific activity, 50 nmol.min-1.mg-1, after ADP-Sepharose affinity purification. Full activity (approximately 800 nmol.min-1.mg-1) could, however, be obtained by including precursors for the cofactors, nicotinic acid, riboflavin, and sepiapterin in the culture medium. We demonstrate that the enzyme activity is exclusively associated with the dimeric form of the enzyme, which had the following molar ratios for the cofactors: heme, 0.92; FAD, 0.57; FMN, 0.34; H4biopterin, 0.32, with a specific activity of 1500 nmol.min-1.mg-1. The provision of substantial quantities of good quality enzyme, as described here, will facilitate the studies on the relationship between enzyme structure and its mechanism of catalysis.

Amino Acid Oxidoreductases↗
PubMed1995

Roles of nitric oxide in tumor growth.

A subclone of the human colon adenocarcinoma cell line DLD-1, which grew reproducibly as subcutaneous tumors in nude mice, was isolated. Such cells, when engineered to generate nitric oxide (NO) continuously, grew more slowly in vitro than the wild-type parental cells. This growth retardation was reversed by the addition of N-iminoethyl-L-ornithine. In nude mice, however, the tumors from these cells grew faster than those derived from wild-type cells and were markedly more vascularized, suggesting that NO may act as part of a signaling cascade for neovascularization. Recent observations that the generation of NO in human breast and gynecological cancers correlates positively with tumor grade are consistent with this hypothesis. We suggest that NO may have a dual pro- and antitumor action, depending on the local concentration of the molecule.

Adenocarcinoma↗
PubMed1995

The L-arginine:nitric oxide pathway is the major source of plasma nitrite in fasted humans.

15N guanidino-labelled L-arginine was infused into fasted human volunteers giving, at equilibrium, a stable 1:10 ratio of 15N to 14N arginine in the plasma. Separate GC-MS assays were used to compare the degree of enrichment of plasma arginine, nitrite and nitrate and thus define the quantitative relationship between the L-arginine:nitric oxide (NO) pathway and the formation of these oxides of nitrogen. 15N nitrite enrichments rose to 8.3% (SD 0.5), five hours after the start of the infusion. In contrast, 15N nitrate enrichments apparently rose to only 1.6% (SD 0.4) at this time. This discrepancy could be explained by our finding that the commonly used Tesch GC-MS nitrate assay is subject to considerable interference from non-nitrate sources in plasma. Taking this into account, nitrate enrichments were similar to those observed for plasma nitrite. These results therefore indicate that the measurement of these compounds in plasma is a valid indicator of NO generation in fasted humans.

Adult↗
PubMed1995

Enzymatic characterisation of recombinant murine inducible nitric oxide synthase.

A complementary DNA (cDNA) encoding murine inducible nitric oxide synthase was cloned from activated J774 macrophages. Expression of this cDNA in a baculovirus-insect cell system allowed comparison of the recombinant enzyme with the native homologue. Western blot analysis of activated J774 and baculovirus-infected insect cell cytosols demonstrated reactivity against a protein of 135 kDa. Kinetic studies on the recombinant and native enzymes revealed an absolute requirement for L-arginine and NADPH in order to achieve full activity. In addition, both enzymes were found to have similar maximum velocities and Km values for these two substrates. The nitric oxide synthase antagonists N-guanidino monomethyl L-arginine and N-iminoethyl L-ornithine inhibited both enzymes at a similar rate. Furthermore, comparable concentrations of inhibitor were required to achieve half maximal enzyme inhibition. These results indicate that recombinant inducible NO synthase appears to be pharmacologically indistinguishable from the native enzyme.

Amino Acid Oxidoreductases↗
PubMed1995

The modulation of IL-6 and TNF-alpha release by nitric oxide following stimulation of J774 cells with LPS and IFN-gamma.

Lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) induced nitric oxide synthase (NOS) activity, tumour necrosis factor-alpha (TNF-alpha), interleukin (IL)-6 and prostaglandin (PG)E2 release in J774 cells, a murine macrophage cell line. The role of endogenous NO in modulating TNF-alpha and IL-6 release was investigated using N-iminoethyl-L-ornithine (L-NIO), a specific inhibitor of NOS. L-NIO (10-1000 microM) produced a concentration-dependent potentiation of LPS and IFN-gamma induced IL-6 release. Time-course studies demonstrated a significant potentiation of IL-6 release at 12 h with a maximum effect at 48 h. By contrast to its effects on IL-6, L-NIO significantly attenuated TNF-alpha release, and at 48 h reduced PGE2 release. The NO-donor S-nitroso-N-acetyl-penicillamine (SNAP,300 microM), significantly inhibited LPS and IFN-gamma induced IL-6 release, but potentiated TNF-alpha release. In addition, SNAP prevented the potentiation of IL-6 and the inhibition of TNF-alpha release by L-NIO. Stimulation of J774 cells with a combination of LPS and IFN-gamma for 24 h or 48 h reduced cell viability which was prevented by L-NIO. Furthermore, SNAP also reduced cell viability determined after 24 h incubation. These results indicate that NO can differentially modulate LPS and IFN-gamma-induced cytokine release from J774 cells, up-regulating TNF-alpha but down-regulating IL-6, and that NO is cytotoxic to these cells.

Analysis of Variance↗
PubMed1995

Induction of nitric oxide synthase in colonic smooth muscle from patients with toxic megacolon.

BACKGROUND & AIMS: Colonic inflammation may lead to motility disturbances, including severe atony. Nitric oxide is released by inflamed tissue and induces smooth muscle relaxation. The aim of this study was to analyze NO generation pathways in colonic tissue from patients who had ulcerative colitis with or without toxic megacolon and in tumor-free samples from patients with colonic neoplasm. METHODS: Enzymatic activity was determined by transformation of [14C]arginine to [14C]citrulline in mucosa and muscular layer samples. Immunostaining of tissue sections with antibody against inducible NO synthase was investigated. The effects of endotoxin on NO synthase activity was tested in muscle strips from human colon. RESULTS: Ca(2+)-independent NO synthase was undetectable or very low in muscularis propria from tumor and colitis controls. In contrast, specimens from patients with toxic megacolon had high activity (P < 0.05). Positive immunostaining for inducible NO synthase was found in muscular layers from patients with megacolon but not in tumor and colitis controls. Finally, endotoxin induced Ca(2+)-independent NO synthase activity in colonic muscle. CONCLUSIONS: Toxic megacolon is associated with the appearance of inducible NO synthase in the colonic muscularis propria. Local generation of excessive amounts of NO may be responsible for the colonic dilatation that is the hallmark of this syndrome.

Adult↗
PubMed1995

Cardiomyopathy: a role for nitric oxide?

The negative inotropism, myocardial dilatation and cytotoxicity in inflammatory heart disorders may be due to increased generation of nitric oxide (NO) by immunological induction of a high output NO pathway. This short review discusses the initial experiments which lead to this hypothesis, and evaluates data that this pathway exists in animal and human cardiomyopathic disorders. It is proposed that manipulation of this pathway may prove to be beneficial in patients with these disorders.

Acute Disease↗
PubMed1995

Calcium supplementation prevents pregnancy-induced hypertension by increasing the production of vascular nitric oxide.

Pregnancy-induced hypertension (PIH) remains a common cause of maternal and fetal morbidity and mortality. During the past 7 years, some progress has been made in the prevention of PIH. Specifically, clinical studies have shown that supplementation with calcium can significantly reduce the frequency of PIH, specially in populations with a low calcium intake. We have suggested that, in such a population, calcium supplementation is a safe and effective measure for reducing the frequency of PIH. Thus, the purpose of this article is to advance a hypothesis about the mechanism by which calcium supplementation reduces the risk of PIH. We propose that dietary calcium supplementation reduces the frequency of PIH by maintaining the serum ionized calcium level which is crucial for the production of endothelial nitric oxide, the increased generation of which maintains the vasodilatation that is characteristic of normal pregnancy.

Calcium↗
PubMed1995

Nitric oxide synthase activity in human breast cancer.

Nitric oxide (NO) is generated by a family of isoenzymes (NO synthases) expressed in a wide range of mammalian cells. We have recently reported NO synthase expression in human gynaecological cancers. In this study we have assessed the activity and distribution of NO synthase in a series of human breast tumours and in normal breast tissue. Calcium-dependent (constitutive) and -independent (inducible) NO synthase activity, as well as NO biosynthesis, was high in invasive tumours compared with benign or normal tissue. Furthermore, for invasive ductal carcinomas, NO biosynthesis was significantly greater for grade III compared with grade II tumours. Immunohistochemical investigations revealed immunolabelling with a monoclonal antibody to murine inducible NO synthase predominantly within tumour-associated macrophages. Immunolabelling with a polyclonal antiserum raised against rat brain NO synthase was also observed in vascular endothelial and myoepithelial cells. Thus NO synthase is expressed in human breast tumours, where its presence correlates with tumour grade.

Amino Acid Oxidoreductases↗
PubMed1995

Inhibition by lamotrigine of the generation of nitric oxide in rat forebrain slices.

Depolarization of adult rat forebrain slices with veratrine induced the release of excitatory amino acids (glutamate and aspartate), the synthesis of nitric oxide (NO), and increases in cyclic GMP (cGMP). The NO synthase inhibitors N omega-monomethyl-L-arginine and N omega-nitro-L-arginine methyl ester decreased the release of NO and the levels of cGMP without affecting the release of excitatory amino acids. In contrast, the antiepileptic drug lamotrigine inhibited the release of excitatory amino acids and of NO, and decreased the levels of cGMP without causing a significant direct inhibition of the NO synthase. Furthermore, the synthesis of NO and the increases in cGMP induced by veratrine were partially blocked by the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 but not by 6-nitro-7-sulphamobenzo (f) quinoxaline-2,3-dione, a non-NMDA receptor antagonist. Neither of these compounds inhibited directly the NO synthase or the release of excitatory amino acids. Thus, these three types of compound act as an inhibitor of voltage-sensitive sodium channels (lamotrigine), as a receptor antagonist (MK-801), or as direct inhibitors of the NO synthase, to block the pathway leading to increased cGMP after veratrine depolarization. It is likely that some of the pharmacological and therapeutic actions shared by these three types of compound are, at least in part, a consequence of inhibition of the synthesis of NO.

Amino Acids↗
PubMed1995

L-arginine: nitric oxide pathway in endotoxemia and human septic shock.

OBJECTIVE: To investigate the relationship between nitric oxide production, endotoxemia, and hemodynamic alterations in human septic shock. DESIGN: Prospective study. SETTING: A 32-bed intensive care unit in a university referral hospital. PATIENTS: Two groups of septic patients with shock (n = 13) or without shock (n = 16) and an additional group of nonseptic patients as control group (n = 25). MEASUREMENTS: Plasma nitrite and nitrate concentrations were measured as an index of nitric oxide generation. Nitrite and nitrate concentrations were correlated with plasma endotoxin and hemodynamic variables. MAIN RESULTS: Increased plasma nitrite and nitrate concentrations were found in patients with septic shock (p < .01). Nitrite and nitrate correlated directly with endotoxin concentration (r2 = .21, p < .05) and cardiac output (r2 = .49, p < .05), and inversely with systolic blood pressure (r2 = .24, p < .01). CONCLUSIONS: This study demonstrated the activation of the L-arginine:nitric oxide pathway in human endotoxemic septic shock, suggesting that nitric oxide may be an important mediator of the hemodynamic disturbances in this pathophysiologic situation.

Arginine↗
PubMed1995

Impairment of nitrergic-mediated relaxation of rat isolated duodenum by experimental diabetes.

1. Diabetes mellitus is associated with changes in gastrointestinal motility. The effects of experimental diabetes, induced by streptozotocin administration to rats 3-4 weeks previously, on the nitric oxide (NO)-mediated (nitrergic) relaxation of the duodenum have now been investigated. 2. The non-adrenergic, non-cholinergic (NANC) relaxation of the isolated duodenum induced by nicotine (0.3-10 microM) or the nicotinic agonist, 1,1-dimethyl-4-phenylpiperazinium (DMPP; 10 microM) was inhibited by the NO synthase inhibitor, NG-nitro-L-arginine (3-100 microM). 3. This nitrergic relaxation induced by nicotine or DMPP of the duodenum from diabetic rats was substantially smaller than that of the tissue from control rats. 4. By contrast, the relaxation of the duodenum from diabetic rats to the NO donor, nitroprusside (0.3-10 microM) was similar to that of control tissue, whereas the relaxation to ATP (0.1-3 microM) was enhanced to a small but significant degree. 5. Incubation of duodenal tissue from control rats at 4 degrees C for 72 h, which leads to neuronal disruption, significantly attenuated the relaxation to nicotine or DMPP whereas the relaxation induced by nitroprusside or ATP was not affected. Comparable cold-storage did not affect the endothelium-dependent relaxation of rat aortic rings induced by acetylcholine (0.01-2 microM). 6. The calcium-dependent NO synthase activity in duodenal tissue, determined by the conversion of radiolabelled L-arginine to citrulline, was significantly reduced in cold-stored tissue and in tissue obtained from diabetic rats. 7. These findings in the rat duodenum indicate that a reduction in intestinal NO synthase activity is associated with an impairment of the NANC relaxation. A defect in the intestinal nitrergic innervation could thus contribute to the motility dysfunction observed in diabetes.

Adenosine Triphosphate↗
PubMed1995

Endotoxin inhibition of distension-stimulated gastric acid secretion in rat: mediation by NO in the central nervous system.

1. The involvement of nitric oxide in the acute inhibitory effects of low doses of endotoxin, following intracerebroventricular (i.c.v.) or intravenous (i.v.) administration, on gastric acid secretion stimulated by distension or i.v. infusion of pentagastrin has been investigated in the continuously perfused stomach of the anaesthetized rat. 2. The i.c.v. administration of E. coli endotoxin (800 ng kg-1) abolished the acid secretory response induced by gastric distension (20 cm water intragastric pressure) within 30 min of administration. 3. By contrast, submaximal rates of acid secretion induced by i.v. infusion of pentagastrin (8 micrograms kg-1 h-1) were not inhibited by i.c.v. administration of endotoxin (800 ng kg-1). 4. Prior i.c.v. administration of the NO synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME, 800 micrograms kg-1) restored the acid secretory responses to distension in rats treated with endotoxin (i.c.v.). 5. Likewise, i.v. administration of endotoxin (5 micrograms kg-1) abolished the acid secretory response induced by gastric distension within 30 min of administration. Prior i.c.v. injection of L-NAME (800 micrograms kg-1) or its i.v. administration (10 mg kg-1) restored acid secretory responses in rats receiving i.v. endotoxin. 6. The reversal by L-NAME (i.v.) of the acid inhibitory effects of endotoxin (i.v.) was prevented by L-arginine (12 mg kg-1, i.c.v. or 100 mg kg-1, i.v.), but not by its enantiomer D-arginine. 7. The present results imply the existence of an acute response to endotoxin involving NO synthesis in the brain. NO may act as a neuromodulator or neurotransmitter in a nervous reflex leading to the inhibition of acid secretion stimulated by gastric distension.

Animals↗