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Biomedical subjects

S Moncada

Publications and source records attributed to S Moncada.

At least 127 records · Page 7Linked to original sources
PubMed1994

Paradoxical fate and biological action of peroxynitrite on human platelets.

Peroxynitrite (ONOO-), which is formed from the reaction of nitric oxide (NO) and superoxide (O2-), has been suggested to be responsible for some of the cytotoxic effects of these molecules. When protonated, ONOO- gives rise to hydroxyl (OH.) and nitrogen dioxide (NO2) radicals, which are capable of inducing tissue damage. We have investigated the effects of ONOO- on human platelets in vitro in order to explore the potential of this oxidant to contribute to tissue damage. ONOO- caused aggregation of washed platelets and reversed the inhibition of aggregation induced by S-nitroso-N-acetyl-DL-penicillamine (SNAP), prostacyclin, and indomethacin. However, in platelet-rich plasma, ONOO- not only did not possess proaggregatory properties but acted as an inhibitor of platelet aggregation. This reversal of the aggregatory effect of ONOO- could also be achieved in washed platelets by adding low concentrations of plasma, human serum albumin, or glutathione and was inhibited by hemoglobin. An analysis of the reaction products of ONOO- and glutathione revealed the presence of both NO and S-nitrosoglutathione in quantities sufficient to account for the antiaggregatory effects observed. Thus the fate and therefore the actions of ONOO- in biological systems are critically dependent on the biological environment in which this oxidant is present.

Antigens, CD↗
PubMed1994

Nitric oxide is present in exhaled breath in humans: direct GC-MS confirmation.

The physiological role of nitric oxide (NO) is being investigated in many experimental and clinical settings. There is considerable evidence that NO is involved in the regulation of lung vascular function. In addition there are many studies reporting the beneficial effect of NO inhalation. NO formed from L-Arginine has been detected in exhaled breath using indirect mass spectrometry and chemiluminescence. Both methods provided good evidence for the presence of NO in breath samples but were not unequivocal. We therefore developed a method using gas-chromatography-mass spectrometry which allowed us to measure trace levels of NO in air and breath. Eight healthy volunteers supplied numerous breath samples for analysis. A clear peak for nitric oxide was observed in seven volunteers. The mean level was 13ppb (n = 7, range < 2 to 19ppb). This data is in good agreement with our previous data and unequivocally confirms the presence of nitric oxide in human breath.

Female↗
PubMed1994

Arrest of preterm labour and prolongation of gestation with glyceryl trinitrate, a nitric oxide donor.

13 women admitted consecutively in preterm labour were recruited to an observational study of the effect of glyceryl trinitrate (GTN) patches on uterine contractions and prolongation of pregnancy. All twenty episodes of preterm labour (at 23-33 weeks' gestation) responded. 1 patient delivered because of cervical incompetence despite cessation of uterine contractions. The mean prolongation of pregnancy was 34 days. 8 babies have been born so far, and are progressing well. GTN patches appear to be a safe, well-tolerated, and non-invasive method of suppressing uterine contractions in preterm labour.

Administration, Cutaneous↗
PubMed1994

Induction of calcium-dependent nitric oxide synthases by sex hormones.

We have examined the effects of pregnancy and sex hormones on calcium-dependent and calcium-independent nitric oxide synthases (NOSs) in the guinea pig. Pregnancy (near term) caused a > 4-fold increase in the activity of calcium-dependent NOS in the uterine artery and at least a doubling in the heart, kidney, skeletal muscle, esophagus, and cerebellum. The increase in NOS activity in the cerebellum during pregnancy was inhibited by the estrogen-receptor antagonist tamoxifen. Treatment with estradiol (but not progesterone) also increased calcium-dependent NOS activity in the tissues examined from both females and males. Testosterone increased calcium-dependent NOS only in the cerebellum. No significant change in calcium-independent NOS activity was observed either during pregnancy or after the administration of any sex hormone. Both pregnancy and estradiol treatment increased the amount of mRNAs for NOS isozymes eNOS and nNOS in skeletal muscle, suggesting that the increases in NOS activity result from enzyme induction. Thus both eNOS and nNOS are subject to regulation by estrogen, an action that could explain some of the changes that occur during pregnancy and some gender differences in physiology and pathophysiology.

Amino Acid Oxidoreductases↗
PubMed1994

Reversible inhibition of cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, by nitric oxide. Implications for neurodegenerative diseases.

Incubation of rat skeletal muscle mitochondria with the nitric oxide generator, S-nitrosoglutathione (GSNO) reversibly inhibited oxygen utilisation with all substrates tested. The visible absorption spectra of the inhibited mitochondria showed that cytochromes c+c1, b and a+a3 were reduced, indicating a block at the distal end of the respiratory chain. Analysis of the respiratory chain enzyme activities in the presence of GSNO localised the site of inhibition of cytochrome c oxidase alone. These results indicate that nitric oxide is capable of rapidly and reversibly inhibiting the mitochondrial respiratory chain and may be implicated in the cytotoxic effects of nitric oxide in the CNS and other tissues.

Animals↗
PubMed1994

Mapping of the genes encoding human inducible and endothelial nitric oxide synthase (NOS2 and NOS3) to the pericentric region of chromosome 17 and to chromosome 7, respectively.

Nitric oxide (NO) is an important molecular messenger regulating the functions of a wide variety of cells and tissues. NO is synthesized from L-arginine by a variety of isoforms of the enzyme nitric oxide synthase (NOS). We have used Southern blotting analysis on DNAs obtained from a panel of human-rodent hybrid cell lines to map the gene encoding the inducible NOS (NOS2) to chromosome 17cen-17q11 and the gene encoding the endothelial form of NOS (NOS3) to chromosome 7. Fluorescence in situ hybridization using a NOS2 probe gave several signals in the 17p11-q11 pericentromeric region.

Amino Acid Oxidoreductases↗
PubMed1994

A rapid and simple method for the measurement of nitrite and nitrate in plasma by high performance capillary electrophoresis.

Quantification of nitric oxide in vivo is difficult because of its short half-life. Instead the oxidised products, nitrite and nitrate, are commonly used as indices of nitric oxide generation. We describe here a simple method for the measurement of nitrite and nitrate in plasma using high performance capillary electrophoresis. Plasma standard curves gave regression coefficients of 0.98 for nitrite and 0.99 for nitrate with an intra-assay coefficient of variance of 4.6% for 50 microM nitrite (n = 10) and 1.2% for 50 microM nitrate (n = 10). The assay allows measurement of nitrite and nitrate in a single analysis requiring minimal sample preparation, and with the sensitivity to detect both basal and physiological changes in plasma nitrite and nitrate.

Biomarkers↗
PubMed1994

Understanding the controversy over the identity of EDRF.

Thirteen years after its discovery, there is still controversy over the chemical identity of endothelium-derived relaxing factor (EDRF). Although pharmacological and chemical evidence indicates that EDRF is nitric oxide, other candidates, including S-nitrosocysteine, dinitrosyl-iron-cysteine complex, nitroxyl and hydroxylamine, have been proposed to account for the vasorelaxant properties of EDRF. Such diverse compounds should differ in their stability and in reactivity with oxyhaemoglobin and with redox-active nucleophiles such as thiols. Here we use a bioassay to compare the pharmacodynamic profiles of these and other compounds with those of nitric oxide and EDRF. We find that some S-nitrosothiols, dinitrosyl-iron-cysteine complex, sodium nitroxyl and hydroxylamine can be eliminated as candidates as they are more stable than EDRF and less susceptible to inhibition by oxyhaemoglobin. Co-infusion of cysteine revealed major differences between the remaining candidates because it reduced the effect of authentic nitric oxide and EDRF on the bioassay tissues but enhanced the survival of S-nitrosocysteine and S-nitrosocysteamine. Our results further support the evidence that EDRF, the pharmacological entity described by Furchgott and Zawadzki, is nitric oxide.

Animals↗
PubMed1994

Nitric oxide synthase activity in human gynecological cancer.

Nitric oxide is generated by the NO synthases, a family of isoenzymes expressed in a wide range of mammalian cells. In the vascular and nervous systems distinct isoforms generate NO to act as a signal transduction mechanism. The isoform induced by cytokines, on the other hand, provides a sustained release of NO which mediates some cytotoxic and cytostatic effects of the immune system. Solid tumors are a heterogeneous population of cell types, including tumor, vascular, and infiltrating immune cells. Studies in vitro show that NO synthase can be present in many of these cells. However, its presence in situ in solid human tumors has not been reported. In this study, we have investigated NO synthase activity and its cellular localization in malignant and nonmalignant human gynecological tissue. Nitric oxide synthase activity was observed in malignant tissue, was highest (> or = 250 pmol/min/g tissue) in poorly differentiated tumors, and was below detectable levels in normal gynecological tissue. Furthermore, investigations with a polyclonal NO synthase antibody revealed immunoreactivity only in malignant tissue. This was associated with NO synthase activity and localized to tumor cells. Thus NO synthase is present in human gynecological tumors, and its presence seems to correlate inversely with the differentiation of the tumor.

Amino Acid Oxidoreductases↗
PubMed1994

Release of nitric oxide by nerve stimulation in the human urogenital tract.

Nerve-induced release of the nitric oxide (NO) breakdown products nitrite (NO2-) and nitrate (NO3-) and the histochemical localization of NO synthase in the human penile corpus cavernosum and urethra were studied. Relaxations induced by nerve stimulation were inhibited by N omega-nitro-L-arginine methyl ester (L-NAME), an effect which was reversed by L-arginine. Relaxations elicited either by nerve stimulation or exogenous NO were accompanied by the appearance of equivalent amounts of NO2- and NO3- over a very similar time course. Nerve-induced release of NO2- and NO3- was inhibited by L-NAME. Histochemical studies showed NADPH diaphorase and NO-positive nerve fibres surrounding the arteries and smooth muscle bundles in the corpus cavernosum and the urethra. The results suggest that NO is a mediator for non-adrenergic non-cholinergic relaxation in the human urogenital tract.

Amino Acid Oxidoreductases↗
PubMed1994

Inducible nitric oxide synthase is increased in murine lung epithelial cells by cytokine stimulation.

Nitric oxide (NO) is detectable in exhaled air. To elucidate whether airway epithelial cells could be a source of NO, we investigated the expression of inducible nitric oxide synthase (iNOS) by the murine lung epithelial cell line, LA-4, in response to cytokine stimulation and the ability of corticosteroids to modulate this effect. Stimulation with cytomix, a combination of interleukin-1 beta, tumor necrosis factor-alpha, and interferon-gamma, elevated nitrite levels by 873% in the culture supernatants and enhanced the conversion of arginine to citrulline by 273% at 24 h. An increased number of cells stained for iNOS and an increase in iNOS mRNA was also observed. Dexamethasone decreased the cytokine-induced increase in nitrite levels, NOS activity, iNOS immunoreactivity, and mRNA but did not change the half life of iNOS mRNA. These results show that lung epithelial cells can release NO, a process which can be inhibited by dexamethasone.

Amino Acid Oxidoreductases↗
PubMed1994

Production of nitric oxide and superoxide by activated macrophages and killing of Leishmania major.

Murine macrophages can be activated to produce nitric oxide (NO) and superoxide and these two radicals can react to form peroxynitrite, a powerful oxidant which may be involved in parasite killing. We now show that murine macrophages activated with zymosan and interferon-gamma (ZYM/IFN-gamma) produced both superoxide (peaking 1-2 h after stimulation, then rapidly declining) and NO (barely detectable at 6 h, peaking by 24 h). Macrophages activated with ZYM alone produced only superoxide, while stimulation with lipopolysaccharide (LPS) and IFN-gamma induced NO but not superoxide. Cells stimulated with ZYM/IFN-gamma or LPS/IFN-gamma killed Leishmania major to a similar degree, an effect that was completely blocked by the addition of N-iminoethyl-L-ornithine. However, macrophages stimulated with ZYM alone were unable to kill L. major. S-nitroso-acetyl-penicillamine, which releases NO, was highly leishmanicidal when added directly to the parasites. 3-morpholino-sydnonimine hydrochloride which releases both NO and superoxide simultaneously, was also efficient at killing L. major and this cytotoxicity was greatly enhanced by the addition of superoxide dismutase. Finally, authentic peroxynitrite failed to induce any cytotoxic effect, even at a high concentration. Thus macrophages can produce either NO, superoxide or both, depending on the stimulus. However, the killing of L. major is dependent only on the production of NO.

Animals↗
PubMed1994

Induction of nitric oxide synthases early in pregnancy.

OBJECTIVE: We have previously demonstrated that calcium-dependent nitric oxide synthase is induced by estrogen and that by the end of pregnancy nitric oxide synthase of both endothelial and neuronal origin is increased in various maternal tissues. This rise in activity may be crucial for the alterations in muscle activity necessary for a successful pregnancy. If so, the increase in nitric oxide synthase activity must occur early in gestation. STUDY DESIGN: We tested the hypothesis that pregnancy increases nitric oxide synthase activity early in gestation by measuring in heart, kidney, skeletal muscle, and esophagus of time-mated guinea pigs the conversion by nitric oxide synthase of carbon 14-labeled L-arginine to carbon 14-labeled citrulline and the concentration of cyclic guanosine monophosphate, the second messenger of nitric oxide. RESULTS: Calcium-dependent nitric oxide synthase activity was increased twofold to fourfold by pregnancy in each tissue examined. The rise began by 0.14 gestation (9 of 63 +/- 2 days) and reached a plateau by 0.30 gestation (19 days), which was then maintained until term. The treatment of pregnant animals with tamoxifen decreased nitric oxide synthase activity to nonpregnant values in the heart, where tamoxifen is an estrogen receptor antagonist, but not in kidney, skeletal muscle, and esophagus. Cyclic guanosine monophosphate also rose progressively in each tissue studied until about 0.70 gestation before declining in skeletal muscle, kidney, and heart. It remained elevated in the esophagus. CONCLUSION: These studies demonstrate that nitric oxide synthase activity in maternal tissues rises early in pregnancy and is associated with an increase in cyclic guanosine monophosphate, the second messenger of nitric oxide. These findings are consistent with the hypothesis that an increase in nitric oxide synthase plays a role in smooth muscle adaptations of pregnancy.

Amino Acid Oxidoreductases↗
PubMed1994

Human colon cancer cell lines show a diverse pattern of nitric oxide synthase gene expression and nitric oxide generation.

A panel of human colonic adenocarcinoma cell lines was examined both for expression of mRNAs of the nitric oxide synthase (NOS) gene family and for evidence of enzymic activity based on citrulline and nitrite (NO2-) formation. Reverse transcription-polymerase chain reaction (RT-PCR), revealed that all lines (SW480, SW620, DLD-1 and WiDr) expressed mRNA for the Ca(2+)-dependent endothelial (e)NOS, while SW480 cells also expressed the Ca(2+)-dependent neuronal (n)NOS. The mRNA for the Ca(2+)-independent inducible (i)NOS was expressed both by cytokine-stimulated and by unstimulated SW480, SW620 and DLD-1 cells, but none was seen at any time in the WiDr cells. There was, however, little correlation between mRNA expression and enzymic activity based on citrulline and NO2- formation. Thus none of the cell lines exhibited measurable Ca(2+)-dependent NOS activity, while Ca(2+)-independent NOS activity was seen in all but the WiDr cells. Furthermore, DLD-1 cells generated citrulline with resultant NO2- formation only after stimulation with lipopolysaccharide (LPS) and/or cytokines, while SW480 and SW620 did so constitutively. Thus RT-PCR studies indicate that tumour cells of similar epithelial origin display a diverse pattern of NOS gene family expression, and parallel biochemical studies clearly indicate that such expression does not always result in measurable enzymic activity leading to the generation of NO.

Adenocarcinoma↗
PubMed1994

Induction of nitric oxide synthase in rat immune complex glomerulonephritis.

Nitric oxide (NO) is a biological mediator which is synthesized from L-arginine by a family of nitric oxide synthases (NOS). Previously we have shown that NO is synthesized ex vivo by glomeruli obtained from animals with acute immune complex glomerulonephritis. We have now sought evidence for the in vivo induction of NOS in glomeruli by immunohistochemistry using specific antisera raised against a peptide sequence of inducible mouse macrophage NOS and by in situ hybridization. The expression of the enzyme was studied in kidneys of rats with acute unilateral immune complex glomerulonephritis, induced by cationized IgG, by immunohistochemistry. Inducible NOS (iNOS) was present in glomeruli in nephritic (left) kidneys at the time of maximum macrophage infiltration, both within intraglomerular mononuclear cells and cells emigrating into Bowman's space. iNOS expressing cells were also present in interstitial infiltrates. There was no expression in normal rat kidneys or in glomeruli in the non-nephritic (right) kidneys of experimental rats. In situ hybridization confirmed the immunohistochemical localization. These results provide the first direct evidence for the presence and localization of inducible NOS in glomeruli and support a significant role for NO in the pathogenesis of immune complex glomerulonephritis.

Amino Acid Oxidoreductases↗
PubMed1994

Effects of a nitric oxide synthase inhibitor in humans with septic shock.

OBJECTIVE: Studies in animals have indicated that increased production of nitric oxide from an inducible isoform of nitric oxide synthase contributes to the pathophysiology of endotoxic and cytokine induced shock. The aim of this study was to determine the role of nitric oxide in septic shock in humans. METHODS: The study was a randomised, double blind, placebo controlled investigation of the effects of the nitric oxide synthase inhibitor NG monomethyl-L-arginine (L-NMMA) in 12 patients with severe sepsis associated with hypotension. Measurements of haemodynamic, haematological, and biochemical variables were made. RESULTS: L-NMMA (0.3 and 1 mg.kg-1) produced a dose dependent increase in mean arterial pressure, systemic vascular resistance, pulmonary vascular resistance, central venous pressure, and pulmonary artery occlusion pressure, and a decrease in cardiac output and heart rate. In response to the highest dose of L-NMMA systemic vascular resistance increased from 547(SEM 92) to 889(143) dyne.s.cm-5, mean arterial blood pressure increased from 80.9(2.9) to 100.5(6.1) mm Hg, and cardiac output decreased from 11.2(2.1) to 8.9(1.9) litres.min-1. Continuous infusion of L-NMMA (1 mg.kg-1 x h-1) produced sustained haemodynamic changes. Platelet numbers decreased during the course of the study in both the L-NMMA and the placebo group and did not differ significantly between the groups. CONCLUSIONS: Low doses of L-NMMA cause a widespread increase in vascular tone and raise blood pressure in patients with septic shock. Overproduction of nitric oxide appears to be an important mechanism in septic shock in patients, and inhibition of nitric oxide synthesis might provide a novel therapeutic approach to this condition. However, L-NMMA produced a fall in cardiac output and it is possible that this would worsen tissue perfusion. Larger studies examining the effects of L-NMMA on mortality and morbidity are now required.

Adult↗