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Biomedical subjects

S Moncada

Publications and source records attributed to S Moncada.

At least 73 records · Page 4Linked to original sources
PubMed1997

Induction and role of NO synthase in hypotensive hepatic failure.

Nitric oxide (NO) plays an important role in the physiological and pathophysiological control of the vascular system. NO is synthesized by isoforms of the enzyme NO synthase (NOS). Hepatic failure is complicated by hypotension, low systemic vascular resistance, and resistance to vasoconstrictor drugs. The potential role of NO in these abnormalities was investigated by using in vitro pharmacological interventions on hepatic arteries obtained from both donor and recipient patients at the time of liver transplantation. The presence of NOS mRNA was investigated by reverse transcription polymerase chain reaction (RT-PCR) with primers designed from human endothelial NOS (eNOS) and inducible NOS (iNOS) cDNA sequences. Arteries from patients with hepatic failure had an impaired constrictor response to phenylephrine compared with those of donor arteries. The constrictor effect of phenylephrine was potentiated by NG-monomethyl-L-arginine, an inhibitor of NOS, which had no effect in donor control arteries. RT-PCR identified human eNOS mRNA in donor and recipient arteries and human iNOS mRNA in recipient arteries only. Induction of NOS in the vasculature with subsequent NO-induced vasodilatation may therefore contribute to the hemodynamic abnormalities observed in hepatic failure and potentially in other pathologies associated with endotoxemia. Whether selective inhibitors of iNOS will improve hemodynamic control or clinical outcome in these conditions requires further study.

Acetylcholine↗
PubMed1997

Nitric oxide synthase is expressed in human macrophages during foreign body inflammation.

Although nitric oxide (NO) is a well documented effector molecule in rodent macrophages, its significance in human mononuclear phagocytic cells has been controversial. The foreign body inflammatory reaction around loosened joint replacement implants leads to formation of an osteolytic granulomatous pseudo-synovial membrane rich in activated macrophages. We studied 13 specimens of interface membrane tissue collected from revision surgery of aseptically loosened hip and knee prostheses for the presence of inducible NO synthase (iNOS). The presence of iNOS was demonstrated immunohistochemically in 10 of these specimens. Within the tissue this enzyme was confined to macrophages and vascular endothelial cells. iNOS activity was demonstrated biochemically by measuring the calcium-independent generation of citrulline from L-arginine, and the presence of iNOS mRNA was demonstrated using reverse transcriptase polymerase chain reaction. NO synthesis in the interface tissue may be an important factor in the maintenance of the inflammatory and osteolytic processes.

Adult↗
PubMed1996

Inhibition of gastric acid secretion by stress: a protective reflex mediated by cerebral nitric oxide.

Moderate somatic stress inhibits gastric acid secretion. We have investigated the role of endogenously released NO in this phenomenon. Elevation of body temperature by 3 degrees C or a reduction of 35 mmHg (1 mmHg = 133 Pa) in blood pressure for 10 min produced a rapid and long-lasting reduction of distension-stimulated acid secretion in the rat perfused stomach in vivo. A similar inhibitory effect on acid secretion was produced by the intracisternal (i.c.) administration of oxytocin, a peptide known to be released during stress. Intracisternal administration of the NO-synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) reversed the antisecretory effect induced by all these stimuli, an action prevented by intracisternal coadministration of the NO precursor, L-arginine. Furthermore, microinjection of L-NAME into the dorsal motor nucleus of the vagus nerve reversed the acid inhibitory effects of mild hyperthermia, i.v. endotoxin, or i.c. oxytocin, an action prevented by prior microinjection of L-arginine. By contrast, microinjection of L-NAME into the nucleus tractus solitarius failed to affect the inhibitory effects of hyperthermia, i.v. endotoxin, or i.c. oxytocin. Immunohistochemical techniques demonstrated that following hyperthermia there was a significant increase in immunoreactivity to neuronal NO synthase in different areas of the brain, including the dorsal motor nucleus of the vagus. Thus, our results suggest that the inhibition of gastric acid secretion, a defense mechanism during stress, is mediated by a nervous reflex involving a neuronal pathway that includes NO synthesis in the brain, specifically in the dorsal motor nucleus of the vagus.

Animals↗
PubMed1996

Visualisation of nitric oxide generated by activated murine macrophages.

We have visualised the release and approximate diffusion profile of nitric oxide (NO) from activated murine macrophages using a high transmission microscope coupled to a high sensitivity photon counting camera. The images generated by NO were cell-associated and spread over an area of approximately 175 micrometers from the activated macrophage. The signals obtained were dependent on the presence of exogenous L-arginine in the medium and followed a time course similar to that previously described for the generation of NO by the inducible form of NO synthase. The light signal was attenuated by the inhibitor of NO synthase, N omega-nitro-L-arginine methyl ester. Studies using superoxide-deficient macrophages further confirmed that the signals detected were generated by NO rather than reactive oxygen intermediates.

Animals↗
PubMed1996

Nitric oxide and peroxynitrite exert distinct effects on mitochondrial respiration which are differentially blocked by glutathione or glucose.

Nitric oxide (NO) and peroxynitrite both inhibit respiration by brain submitochondrial particles, the former reversibly at cytochrome c oxidase, the latter irreversibly at complexes I-III. Both GSH (IC50 =10 microM) and glucose (IC50 = 8 mM) prevented inhibition of respiration by peroxynitrite (ONOO-), but neither glucose (100 mM) nor GSH (100 microM) affected that by NO. Thus, unless ONOO- is formed within mitochondria it is unlikely to inhibit respiration in cells directly, because of reactions with cellular thiols and carbohydrates. However, the reversible inhibition of respiration cytochrome c oxidase by NO is likely to occur (e.g. in the brain during ischaemia) and could be responsible for cytotoxicity.

Animals↗
PubMed1996

Molecular cloning and structural organization of the human inducible nitric oxide synthase gene (NOS2).

Previously, we reported the isolation and molecular cloning of human inducible nitric oxide synthase gene (NOS2) sequences from human chromosome 17 cosmid libraries. Here we describe the further characterization and sequencing of the NOS2 gene. The genomic structure of the NOS2 gene was determined from two overlapping cosmid clones, namely, pcos4A and pcos20. the NOS2 open reading frame is encoded by 27 exons, with translation initiation and termination in exon 2 and exon 27, respectively. These results differ from the previously reported organization of the iNOS gene, where 26 exons were reported for the genomic structure of NOS2.

Alternative Splicing↗
PubMed1996

cGMP mediates the vascular and platelet actions of nitric oxide: confirmation using an inhibitor of the soluble guanylyl cyclase.

The L-arginine:nitric oxide (NO) pathway is believed to exert many of its physiological effects via stimulation of the soluble guanylyl cyclase (SGC); however, the lack of a selective inhibitor of this enzyme has prevented conclusive demonstration of this mechanism of action. We have found that the compound 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ) inhibits the elevation of cGMP induced by the NO donor S-nitroso-DL-penicillamine in human platelets and rat vascular smooth muscle (IC50 = 10-60 nM and <10 nM, respectively) and that this is accompanied by prevention of the platelet inhibitory and vasodilator actions of NO donors. ODQ also inhibited the antiaggregatory action of NO generated by the platelets but did not affect the action of prostacyclin or that of a cGMP mimetic. In addition, ODQ inhibited the vasodilator actions of endogenously released NO and of NO generated after induction of NO synthase in vascular preparations. It did not, however, affect the increase in vascular smooth muscle cGMP or the dilatation induced by atrial natriuretic factor. ODQ had no effect on NO synthase activity, nor did it react with NO. It did, however, potently (IC50 approximately 10 nM) inhibit the activity of the SGC in cytosol obtained from crude extract of rat aortic smooth muscle. Thus ODQ prevents the actions of NO on platelets and vascular smooth muscle through its potent inhibitory effect on the SGC.

Animals↗
PubMed1996

Identification of the domains of neuronal nitric oxide synthase by limited proteolysis.

Nitric oxide synthase (EC 1.14.13.39) binds arginine and NADPH as substrates, and FAD, FMN, tetrahydrobiopterin, haem and calmodulin as cofactors. The protein consists of a central calmodulin-binding sequence flanked on the N-terminal side by a haem-binding region, analogous to cytochrome P-450, and on the C-terminal side by a region homologous with NADPH:cytochrome P-450 reductase. The structure of recombinant rat brain nitric oxide synthase was analysed by limited proteolyis. The products were identified by using antibodies to defined sequences, and by N-terminal sequencing. Low concentrations of trypsin produced three fragments, similar to those in a previous report [Sheta, McMillan and Masters (1994) J. Biol. Chem. 269, 15147-15153]: that of Mr approx. 135000 (N-terminus Gly-221) resulted from loss of the N-terminal extension (residues 1-220) unique to neuronal nitric oxide synthase. The fragments of Mr 90000 (haem region) and 80000 (reductase region, N-terminus Ala-728) were produced by cleavage within the calmodulin-binding region. With more extensive trypsin treatment, these species were shown to be transient, and three smaller, highly stable fragments of Mr 14000 (N-terminus Leu-744 within the calmodulin region), 60000 (N-terminus Gly-221) and 63000 (N-terminus Lys-856 within the FMN domain) were formed. The species of Mr approx. 60000 represents a domain retaining haem and nitroarginine binding. The two species of Mr 63000 and 14000 remain associated as a complex. This complex retains cytochrome c reductase activity, and thus is the complete reductase region, yet cleaved at Lys-856. This cleavage occurs within a sequence insertion relative to the FMN domain present in inducible nitric oxide synthase. Prolonged proteolysis treatment led to the production of a protein of Mr approx. 53000 (N-terminus Ala-953), corresponding to a cleavage between the FMN and FAD domains. The major products after chymotryptic digestion were similar to those with trypsin, although the pathway of intermediates differed. The haem domain was smaller, starting at residue 275, yet still retained the arginine binding site. These data have allowed us to identify stable domains representing both the arginine/haem-binding and the reductase regions.

Amino Acid Sequence↗
PubMed1996

Myometrial arginase activity increases with advancing pregnancy in the guinea pig.

OBJECTIVES: Arginase has been suggested to play an important role in cellular growth and development, particularly important to the fetus, by supplying L-ornithine for the synthesis of polyamines. The purpose of this investigation was to determine whether pregnancy alters myometrial arginase activity and whether estradiol was responsible for the change. STUDY DESIGN: Myometrium and kidney were obtained from nonpregnant and pregnant guinea pigs of known gestational age. Arginase activity was measured under physiologic conditions by the conversion carbon 14-labeled guanidino-L-arginine to carbon 14-labeled urea. The concentrations of the enzyme's substrate, L-arginine, and its principal metabolite, L-ornithine, were measured in myometrium from near-term pregnant animals by use of an amino acid analyzer. Finally, a group of random cycle guinea pigs received 500 microgram/kg estradiol for 5 days before the myometrium was removed. RESULTS: Myometrial arginase activity in pregnant animals was more than double that of myometrium from nonpregnant animals by the time the first measurement was made at 0.14 gestation. It continued to rise, peaking at values >25-fold higher than the nonpregnant activity by 0.90 gestation. Arginase activity in the myometrium underlying the placental implantation site was >25 fold higher (p<0.05) than myometrium from nonpregnant animals when first studied at 0.63 gestation and 10-fold higher than the contralateral fundal myometrium at the same time of gestation. Myometrial arginase activity in the sterile horn of six pregnant animals was half that of the horn containing one or more pups, but still five times higher than that of nonpregnant animals. Renal arginase activity also rose with advancing pregnancy, but the magnitude of the increase (up to 2-fold) was much smaller than that observed in either the fundal or placental implantation site myometrium. Estradiol had no significant effect on myometrial arginase activity. CONCLUSIONS: These studies demonstrate that pregnancy increases myometrial arginase activity and that the presence of placenta or fetus is necessary for the maximal effect.

Animals↗
PubMed1996

Inhibition of nitrergic relaxations by a selective inhibitor of the soluble guanylate cyclase.

1. The actions of 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ), a specific inhibitor of the soluble guanylate cyclase (SGC), were investigated in the rabbit anococcygeus muscle. 2. ODQ (1 nM-1 microM) inhibited in a concentration-dependent manner the relaxations induced by electrical field stimulation (EFS; 50 V, 0.3 ms duration, 1 Hz, for 5 s, every 120 s). 3. ODQ (1 microM) also inhibited the relaxations elicited by EFS (50 V, 0.3 ms duration, 1, 2.5, 5, 10 Hz, for 5 s) and sodium nitroprusside (SNP; 1 microM) without affecting those induced by isoprenaline (1 microM), atrial natriuretic peptide (ANP; 100 nM) or an analogue of cyclic GMP (8-pCPT-cyclic GMP; 500 microM). 4. ODQ (1 microM) inhibited the elevations in the concentration of cyclic GMP induced by SNP or EFS, but not by ANP. ODQ did not affect the concentrations of cyclic AMP. 5. Nitrergic relaxation in this tissue appears, therefore, to be mediated via activation of SGC.

Animals↗
PubMed1996

Nitric oxide and the regulation of blood pressure in the hypertension-prone and hypertension-resistant Sabra rat.

We examined the role of nitric oxide (NO) in the inherited resistance or susceptibility to hypertension in the Sabra hypertension-prone (SBH) and hypertension-resistant (SBN) rat. Basal mean arterial blood pressure was significantly greater in SBH than in SBN rats. Phenylephrine elevated blood pressure to a similar extent in both substrains, whereas the NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA) had a greater pressor effect in SBN rats. The vasoconstrictor potency of phenylephrine was significantly higher in endothelium-intact aortic rings from the SBH rat, whereas the vasoconstrictor potency of L-NMMA was higher in those from the SBN substrain. Acetylcholine-induced endothelium-dependent relaxation was greater in aortic rings from SBN rats. The vasodilator potency of glyceryl trinitrate was significantly higher in aortic rings from SBH rats and was enhanced after endothelium removal. Both the activity of calcium-dependent NO synthase from aortic endothelial cells and the basal concentration of nitrite/nitrate in plasma were significantly greater in SBN than in SBH rats. In normotensive Wistar rats, basal mean arterial blood pressure, the pressor effect of L-NMMA, endothelial NO synthase activity, and plasma nitrite/ nitrate concentrations were all between the values in SBH and SBN rats. These results indicate that a decrease in NO generation plays a role in the susceptibility of SBH rats to hypertension. Furthermore, the resistance to hypertension in the SBN strain may be related to increased NO generation.

Animals↗
PubMed1996

The ontogeny of cerebral and cerebellar nitric oxide synthase in the guinea pig and rat.

The appearance of nitric oxide synthase (NOS, EC 1.14.13.39) activity in the brain of fetal and neonatal guinea pigs and rats was studied. In the guinea pig, NOS increased from an almost undetectable level at 0.49 of gestation (31 d), reaching adult levels before birth and peaking at 140% of the adult activity (forebrain) or 250% of the adult activity (cerebellum) in the week after birth. The rise in fetal NOS activity followed the reported rise in the estrogen receptor concentration in the brain and could be reduced by treatment of the guinea pig at full term with tamoxifen, implicating estrogens in the expression of fetal NOS activity. In the rat, brain NOS activity did not rise significantly until after birth, reaching adult levels approximately 2 wk after birth, and rising to 150 or 130% of the adult activity in the forebrain and cerebellum, respectively, at 4 wk after birth. The appearance of NOS activity in the rat also followed the reported appearance of estrogen receptors in the brain. In both species the appearance of high NOS activity in the brain immediately precedes the period in which maximal synaptogenesis occurs: immediately before birth in the guinea pig and 2-3 wk after birth in the rat. Thus the appearance of a functional estrogen-estrogen receptor system in the brain may be responsible, at least in part, for the expression of a high activity of NOS, which in turn may play important roles in promoting cerebral blood flow and synaptogenesis in the developing brain.

Age Factors↗
PubMed1995

Association of microvascular leakage with induction of nitric oxide synthase: effects of nitric oxide synthase inhibitors in various organs.

Endotoxin (Escherichia coli lipopolysaccharide 0111:B4, 3 mg/kg i.v.) induced the expression of a calcium-independent nitric oxide (NO) synthase, determined after 5 h in cardiac, hepatic, pulmonary and renal tissues, as assessed by the conversion of radiolabelled L-arginine to L-citrulline. This widespread induction of NO synthase in these conscious rats was associated with microvascular injury, as assessed by the vascular leakage of radiolabelled human serum albumin. Concurrent administration of the NO synthase inhibitor. NG-nitro-L-arginine methyl ester (L-NAME, 1-5 mg/kg s.c.) with endotoxin, provoked acute vascular leakage within 2 h in the various organs. By contrast, the delayed injection of L-NAME (1-5 mg/kg s.c.) or NG-monomethyl-L-arginine (12.5-50 mg/kg s.c.) until 3 h after endotoxin challenge inhibited the subsequent microvascular leakage in these organs. These effects of NO synthase inhibitors were reversed by L-arginine (300 mg/kg s.c.) pretreatment. These results support a protective role of constitutive NO synthase in the early phase of endotoxin shock. Such actions contrast with the aggressive actions of the products of inducible NO synthase in the development of widespread microvascular injury in endotoxemic states.

Animals↗
PubMed1995

The killing of Leishmania major by human macrophages is mediated by nitric oxide induced after ligation of the Fc epsilon RII/CD23 surface antigen.

Serum IgE concentrations and the expression of the low-affinity receptor for IgE (Fc epsilon RII/CD23) are increased in cutaneous leishmaniasis or after immune challenge with Leishmania antigens. In vitro, the ligation of CD23 by IgE-anti-IgE immune complexes (IgE-IC) or by anti-CD23 monoclonal antibody (mAb) induces nitric oxide (NO) synthase and the generation of various cytokines by human monocytes/macrophages. The present study shows that IgE-IC, via CD23 binding, induce intracellular killing of Leishmania major in human monocyte-derived macrophages through the induction of the L-arginine:NO pathway. This was demonstrated by increased generation of nitrite (NO2-), the stable oxidation product of NO, and by the ability of NG-monomethyl-L-arginine to block both NO generation and parasite killing. A similar NO-dependent effect was observed with interferon gamma-treated cells. Tumor necrosis factor alpha is involved in this process, since both the induction of NO synthase and the killing of parasites caused by anti-CD23 mAb were inhibited by an anti-tumor necrosis factor alpha mAb. Treatment of noninfected CD23+ macrophages with IgE-IC provided protection against subsequent in vitro infection of these cells by Leishmania major promastigotes. Thus, IgE-IC promote killing of L. major by inducing NO synthase in human macrophages.

Amino Acid Oxidoreductases↗