Oxide Synthase Inhibitor L-nitroarginine Research Articles

Experimental Evidence that 3-Methylglutaric Acid Disturbs Mitochondrial Function and Induced Oxidative Stress in Rat Brain Synaptosomes: New Converging Mechanisms.

3-Methylglutaric acid (3MGA) is an organic acid that accumulates in various organic acidemias whose patients present neurodegeneration events in children coursing with metabolic acidurias. Limited evidence describes the toxic mechanisms elicited by 3MGA in the brain. Herein, we explored the effects of 3MGA on different toxic endpoints in synaptosomal and mitochondrial-enriched fractions of adult rat brains to provide novel information on early mechanisms evoked by this metabolite. At 1 and 5mM concentration, 3MGA increased lipid peroxidation, but decreased mitochondrial function only at 5mM concentration. Despite less intense effects were obtained at 1mM concentration, its co-administration with the kynurenine pathway (KP) metabolite and N-methyl-D-aspartate receptor (NMDAr) agonist, quinolinic acid (QUIN, 50 and 100µM), produced toxic synergism on markers of oxidative stress and mitochondrial function. The toxicity of 3MGA per se (5mM) was prevented by the cannabinoid receptor agonist WIN55,212-2 and the NMDAr antagonist kynurenic acid (KYNA), suggesting cannabinoid and glutamatergic components in the 3MGA pattern of toxicity. The synergic model (3MGA + QUIN) was also sensitive to KYNA and the antioxidant S-allylcysteine, but not to the nitric oxide synthase inhibitor L-nitroarginine methyl ester. These findings suggest various underlying mechanisms involved in the neurotoxicity of 3MGA that may possibly contribute to the neurodegeneration observed in acidemias.

Evaluation of low-dose endotoxin administration during pregnancy as a model of preeclampsia.

Recent evidence implicates nitric oxide (*NO) in the pathogenesis of preeclampsia. The authors tested the hypothesis that administration of low-dose endotoxin to pregnant rats mimics the signs of preeclampsia in humans and that *NO and *NO-derived species play a role in that animal model. Endotoxin was infused at doses of 1, 2 and 10 microg/kg over 1 h to rats on day 14 of pregnancy. Mean arterial pressure, urinary protein, urinary and plasma nitrite plus nitrate (NO2- + NO3-) concentrations, and platelet count were measured before and after the endotoxin infusion. In another group of pregnant rats, the nitric oxide synthase inhibitor L-nitroarginine methyl ester (L-NAME) was administered in drinking water at a dose of 3 mg x kg(-1) x d(-1) starting on day 7 of pregnancy. Endotoxin was then infused at 10 microg/kg on day 14 of pregnancy. Kidneys and uteroplacental units were examined histologically and analyzed immunohistochemically for 3-nitrotyrosine. Endotoxin administration at doses of 2 and 10 microg/kg caused proteinuria and thrombocytopenia in pregnant rats, but did not result in hypertension. Urinary NO2- + NO3- concentration, reflective of tissue *NO production rates, was significantly elevated in pregnant rats that received endotoxin at 10 microg/kg. Ingestion of L-NAME caused hypertension. Tissues from pregnant rats treated with L-NAME, endotoxin at 10 microg/kg, and a combination of L-NAME and endotoxin had increased 3-nitrotyrosine immunoreactivity. Nitric oxide either directly or through secondary species plays a significant role in the biochemical and physiologic changes that occur in a rodent model of endotoxin-induced injury.

Lamotrigine and carbamazepine affect differently the release of D-[3H]aspartate from mouse cerebral cortex slices: involvement of NO.

The effects of lamotrigine and carbamazepine on the release of preloaded D-[3H]aspartate and the involvement of nitric oxide were studied with mouse cerebral cortical slices in a superfusion system. Lamotrigine inhibited the veratridine-evoked release, whereas the K+-stimulated release was attenuated more strongly by carbamazepine than by lamotrigine. These effects were accentuated by the N-methyl-D-aspartate receptor antagonist L-2-amino-5-phosphonovalerate and the nitric oxide synthase inhibitor L-nitroarginine, but diminished by the nitric oxide donor sodium nitroprusside. The results show that in addition to the blockade of voltage-sensitive Na+ (and Ca2+) channels, NO-mediated mechanisms are probably involved in the anticonvulsant actions of carbamazepine and, in particular, those of lamotrigine.

NMDA and non-NMDA ionotropic glutamate receptors modulate striatal acetylcholine release via pre- and postsynaptic mechanisms.

The effects of NMDA and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) on endogenous acetylcholine release from rat striatal slices and synaptosomes were investigated. Both agonists (1-300 microM) facilitated acetylcholine release from slices in a dose-dependent manner. NMDA (100-300 microM) and AMPA (30-300 microM), however, subsequently inhibited acetylcholine release. NMDA (100 microM)-induced facilitation was antagonized by 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) and dizocilpine (both 1-10 microM), whereas the 10 microM AMPA effect was antagonized by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 1-30 microM). NMDA (100 microM)-induced inhibition was counteracted by CPP, but not dizocilpine, and by the nitric oxide synthase inhibitor L-nitroarginine (1-100 microM). Tetrodotoxin (0.5 microM) prevented the facilitatory effect of 3 microM NMDA and AMPA, but left unchanged that of 30 microM NMDA and 100 microM AMPA. Acetylcholine release from synaptosomes was stimulated by KCl (7.5-100 mM) in a dose-dependent manner. NMDA and AMPA maximally potentiated the 20 mM KCl effect at 1 microM and 0.01 microM, but were ineffective at 100 microM and 10 microM, respectively. Inhibition of acetylcholine release was never found in synaptosomes. The effects of 1 microM NMDA and 0.01 microM AMPA were antagonized by CPP (0.0001-1 microM) or dizocilpine (0.0001-10 microM) and by CNQX (0.001-1 microM), respectively. These data suggest that glutamatergic control of striatal acetylcholine release is mediated via both pre- and postsynaptic NMDA and non-NMDA ionotropic receptors.

Involvement of nitric oxide and serotonin in modulation of antinociception and pressor responses evoked by stimulation in the dorsolateral region of the periaqueductal gray matter in the rat

In rats anaesthetized with alphaxalone/alphadolone, electrical stimulation in the periaqueductal gray matter in the region lying lateral and dorsolateral to the aqueduct produced a pressor response and an increase in the latency of the tail flick response to noxious heat applied to the tail. The antinociception and the pressor response were significantly attenuated following microinjection of 15 nmol 5-hydroxytryptamine at the site of stimulation in the periaqueductal gray matter. Microinjection of an equal volume of 165 mM saline had no effect. The inhibitory effects of 5-hydroxytryptamine were blocked by prior intracerebroventricular administration of 100 μg of the nitric oxide synthase inhibitor l-nitroarginine methyl ester. Neither 5-hydroxytryptamine or l-nitroarginine methyl ester had any effect on resting arterial pressure or on the baseline latency of the tail flick reflex. It is suggested that the inhibitory effects of 5-hydroxytryptamine in the dorsolateral periaqueductal gray matter are normally dependent on the functional integrity of local nitric oxide synthase-containing interneurons. Nitric oxide may act in association with 5-hydroxytryptamine to control the excitability of the aversive system in the midbrain.

Actions of diaspirin cross-linked hemoglobin on isolated rat and dog vessels

The objectives of these studies were to investigate the responses of isolated blood vessels from rats and dogs to the administration of diaspirin cross-linked hemoglobin (DCLHb) and to determine the mechanisms of these responses. Isolated vascular rings (3 to 5 mm) were suspended at optimal passive tension in Krebs-filled (37° C) tissue baths and bubbled with 95% O 2-5% CO 2, and isometric tension was recorded. With the vessels under basal conditions increasing concentrations of DCLHb (10 -8-3 × 10 -6 mol/L) were added. DCLHb addition was repeated during a submaximal contraction with norepinephrine and again during acetylcholine relaxation. The effects of the nitric oxide synthase inhibitor L-nitro arginine (10 -5 mol/L) on the responses to DCLHb were also determined. Dog vessels developed very little tension (1% to 5% of norepinephrine maximum), whereas rat arteries contracted between 9% and 15% when exposed to DCLHb under basal conditions. However, both the dog and rat vessels developed significant tension to DCLHb when they were precontracted (5% to 54%) and also when they were relaxed with acetylcholine (21% to 93%). L-nitro arginine eliminated the contractile responses to DCLHb but did not cause contraction of any of the vessels under basal conditions. We conclude that in this model the mechanism of DCLHb-induced contractions of in vitro dog and rat vessels is dependent on interference with nitric oxide. This is similar to the mechanism of DCLHb action in isolated pig vessels reported previously. Differences in responses of dog, rat, and pig vessels under basal conditions in vitro are the result of active generation of nitric oxide by pig but not by dog or rat vessels.

Endothelial stunning and myocyte recovery after reperfusion of jeopardized muscle: A role of l-arginine blood cardioplegia

Ischemia and reperfusion may damage myocytes and endothelium in jeopardized hearts. This study tested whether (1) endothelial dysfunction (reduced nitric oxide release) exists despite good contractile performance and (2) supplementation of blood cardioplegic solution with nitric oxide precursor l-arginine augments nitric oxide and restores endothelial function. Among 30 Yorkshire-Duroc pigs, 6 received standard glutamate/aspartate blood cardioplegic solution without global ischemia. Twenty-four underwent 20 minutes of 37° C global ischemia. Six received normal blood reperfusion. In 18, the aortic clamp remained in place 30 more minutes and all received 3 infusions of blood cardioplegic solution. In 6, the blood cardioplegic solution was unaltered; in 6, the blood cardioplegic solution contained l-arginine (a nitric oxide precursor) at 2 mmol/L; in 6, the blood cardioplegic solution contained the nitric oxide synthase inhibitor l-nitro arginine methyl ester ( l-NAME) at 1 mmol/L. Complete contractile and endothelial recovery occurred without ischemia. In jeopardized hearts, complete systolic recovery followed infusion of blood cardioplegic solution and of blood cardioplegic solution plus l-arginine. Conversely, contractility recovered approximately 40% after infusion of normal blood and blood cardioplegic solution plus l-NAME. Postischemic nitric oxide production fell 50% in the groups that received blood cardioplegic solution and blood cardioplegic solution plus l-NAME but was increased in the group that received blood cardioplegic solution l-arginine. In vivo endothelium-dependent vasodilator responses to acetylcholine recovered 75% ± 5% of baseline in the blood cardioplegic solution plus l-arginine group, but less than 20% of baseline in other jeopardized hearts. Endothelium-independent smooth muscle responses to sodium nitroprusside were relatively unaltered. Myeloperoxidase activity (neutrophil accumulation) was similar in the blood cardioplegic solution (without ischemia) and blood cardioplegic solution plus l-arginine groups (0.01 ± 0.002 vs 0.013 ± 0.003 μg/gm tissue). Myeloperoxidase activity was raised substantially to 0.033 ± 0.002 μg/gm after exposure to normal blood and to 0.025 ± 0.003 μg/gm after infusion of blood cardioplegic solution and was highest at 0.053 ± 0.01 μg/gm with exposure to blood cardioplegic solution plus l-NAME in jeopardized hearts. The discrepancy between contractile recovery and endothelial dysfunction in jeopardized muscle can be reversed by adding l-arginine to blood cardioplegic solution. (J Thorac Cardiovasc Surg 1997;113:379-89)

Actions of S-nitrosocysteine in the nucleus tractus solitarii are unrelated to release of nitric oxide

Cardiovascular effects elicited by microinjection of l- S-nitrosocysteine in the nucleus tractus solitarii (NTS) were compared and contrasted with those produced by the dextroisomer, other nitric oxide donors and nitric oxide itself. l- S-nitrosocysteine produced dose-related decreases of arterial pressure and heart rate. In contrast, d- S-nitrosocysteine, S-nitrosoglutathione, glyceryl trinitrate, and sodium nitroprusside produced minimal responses that were not dose-related. Likewise, injection of cystine and nitric oxide, two products of S-nitrosocysteine breakdown, produced no significant response. Headspace analysis using chemiluminescence revealed that l- and d- S-nitrosocysteine released identical amounts of nitric oxide when exposed to homogenates of whole rat brain. Responses to l- S-nitrosocysteine were not affected by local injection of oxyhemoglobin or the nitric oxide synthase inhibitor l-nitroarginine methylester. Although injection of l-cysteine into the NTS produced responses similar to those seen with injection of l- S-nitrosocysteine, blockade of excitatory amino acid receptors with kynurenic acid inhibited responses to cysteine but not those to the nitrosothiol. The study demonstrates that S-nitrosocysteine is biologically active in the NTS. Its action is independent of release of nitric oxide from the nitrosothiol but may be mediated through stereoselective sites on target neurons.

Induction of stress proteins by tobacco smoke in human monocytes: modulation by antioxidants.

Tobacco smoke (TS) induced in human monocytes the synthesis of both the classical heat shock proteins (HSP) (Hsp70, Hsp90, Hsp110) and the oxidation-specific stress protein (SP) heme oxygenase (HO). To determine the role of reactive oxygen species in SP induction by TS, we incubated the monocytes with various antioxidants before exposure to TS. Quercetin and N-acetylcysteine (NAC) both prevented the induction of HO by TS but not, or less so, than that of the classical HSP, while the nitric oxide synthase inhibitor L-nitroarginine had no effect. Thus, at least two mechanisms appear involved in SP induction by TS; (i) the induction of HO (oxidation-dependent), which was prevented by quercetin and NAC; and (ii) the induction of Hsp70, which was, at least in part, oxidation-independent. SP induction might represent an adequate biosensor for TS and other radical-mediated environmental exposures.

The influence of nitric oxide on perigeniculate GABAergic cell activity in the anaesthetized cat.

We have tested the effect of iontophoretic application of the nitric oxide synthase inhibitor L-nitroarginine on the activity of a population of 53 perigeniculate (PGN) cells, recorded extracellularly in the anaesthetized paralysed cat. In all cells tested with visual stimulation during L-nitroarginine application (n = 15), the visually elicited responses were markedly reduced, on average by 63 +/- 15%, and there was a reduction in spontaneous activity too. This effect was blocked by co-application of the substrate for nitric oxide synthase, L-arginine, but not by the inactive D-isoform, although application of L-arginine alone was without effect. Pressure application of the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP) elevated both visual responses and spontaneous discharge, an effect also seen with a second nitric oxide donor, sodium nitroprusside (n = 12). The nitric oxide synthase inhibitor L-nitroarginine was applied to a sub-population of seven cells and it selectively decreased NMDA mediated excitation (reduction 80 +/- 14%) with little or no effect on the excitation mediated by alpha-amino-3-hydroxy-5-5-methyl-4-isoxazole-propionic acid (AMPA) or quisqualate (effects not statistically significant), and it had no effect (n = 7) on excitation mediated by the metabotropic agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD). Furthermore, application of SNAP also increased the magnitude of excitatory responses mediated by NMDA receptors. On a different population of seven cells, application of the new NO donor diethylamine-nitric oxide (DEA-NO) enhanced the actions of NMDA without an effect on responses to AMPA. These effects are qualitatively and quantitatively similar to those we have previously described for X and Y type cells in the dorsal lateral geniculate nucleus (dLGN), despite the known opposite effects of acetylcholine (ACh) application in the dLGN and PGN (ACh is co-localized with nitric oxide synthase at both sites). We propose that within the PGN nitric oxide acts to enhance transmission utilizing NMDA receptors selectively (thereby interacting with the globally inhibiting effect of ACh at this site) to enhance visual responses, reducing or removing the non-specific inhibitory drive from PGN to dLGN seen in the spindling activity of slow-wave sleep. These effects will act in concert with the facilitatory actions of both ACh and nitric oxide within the dLGN proper, and will thereby enhance the faithful transmission of visual information from retina to cortex.

The role of nitric oxide generation in interferon-evoked cytochrome P450 down-regulation

The down-regulation of cytochrome P450 during host defence involves one or more mediators of unknown source and identity. Owing to striking similarities in the features that are involved in the production of NO and the features involved in the down-regulation of cytochrome P450 we hypothesized that NO could be a mediator in the interferon-evoked loss of cytochrome P450. In these experiments the nitric oxide synthase inhibitor l-nitroarginine (5 mg/kg) failed to protect mice from the down-regulation of cytochrome P450, ethoxyresorufin dealkylase and para-nitrophenol hydroxylase in the liver following administration of the interferon inducer polyinosinic.polycytidylic acid. A higher dose of L-nitroarginine (30 mg/kg) given every 12 h for 3 days also did not protect against cytochrome P450 losses. In addition, no down-regulation of the enzyme was observed in animals treated with multiple doses of the NO-generating drugs glyceryl trinitrate (16 and 80 mg/kg given every 4 h for 16 h) and nitroprusside (20 and 100 mg/kg given every 4 h for 16 h). These results indicate that the down-regulation of cytochrome P450 by interferon or its inducers probably does not involve NO as a required intermediary.

Mechanisms for preserved cerebrovascular autoregulation during hypertension in rats after sinoaortic denervation.

1. Cerebral blood flow (CBF) and cerebrovascular resistance (CVR) autoregulate to higher levels of arterial pressure (AP) in rats after sinoaortic denervation (SAD) than in intact rats. 2. Potential mechanisms for this phenomenon were studied by monitoring CBF by laser flowmetry while increasing AP in rats after: (i) SAD; (ii) SAD plus bilateral removal of the superior cervical ganglia; (iii) SAD plus interruption of all renal vessels and nerves (renal isolation); (iv) SAD plus sympathectomy and renal isolation; and (v) intravenous treatment with the nitric oxide synthase inhibitor L-nitroarginine (LNA). 3. Compared to intact control rats autoregulation persisted to higher absolute levels of AP in SAD rats with isolation of the kidneys and resistance did not fall significantly below that in rats with SAD alone. 4. Effects of SAD on autoregulation were not altered by combining renal isolation with an interruption of sympathetics. 5. LNA did not affect the baroreflex but blocked a breakthrough of autoregulation even at a maximal mean AP (MAP) of 189 +/- 2 mmHg. 6. Breakthrough occurred in animals pretreated with LNA plus L-arginine but not in animals given LNA plus D-arginine. 7. These data are consistent with a role of the baroreflex in the expression of breakthrough and suggest that breakthrough may result from release of nitric oxide or a nitric oxide donor.

Inhibition of nitric oxide synthesis extends cerebrovascular autoregulation during hypertension

In anesthetized intact rats, cerebral blood flow is autoregulated until mean arterial blood pressure (MAP) exceeds 150 mmHg. At higher pressures cerebral blood flow breaks through autoregulation and rapidly increases. However, interruption of the arterial baroreceptor reflex eliminates breakthrough of autoregulation. Thus, breakthrough may reflect active rather than passive vasodilatation. We, therefore, sought to determine if breakthrough depends upon synthesis of the vasodilator nitric oxide. Thirty-eight anesthetized adult male Sprague-Dawley rats were studied. In all, MAP was raised by slow i.v. infusion of phenylephrine. In rats pretreated with the nitric oxide synthase inhibitor L-nitroarginine (LNA; 22 mg/kg i.v.) or with a combination of LNA plus D-arginine (DArg; 240 mg/kg i.v.), breakthrough did not occur even when MAP exceeded 185 mmHg (LNA) and 165 mmHg (DArg). In contrast, breakthrough occurred in rats treated with LNA plus L-arginine (LArg; 240 mg/kg i.v.) and in rats whose basal vascular tone had been increased by pretreatment with arginine vasopressin prior to infusion of phenylephrine. Removal of sympathetic innervation to cerebral vessls attenuated, but did not eliminate, effects of LNA on breakthrough. Thus, vasodilatation seen with breakthrough of autoregulation depends upon release of nitric oxide or a nitric oxide donor.

The contribution of calcitonin gene-related peptide (CGRP) to neurogenic vasodilator responses.

The neuropeptide calcitonin gene-related peptide (CGRP) is a potent vasodilator in the microcirculation of many tissues including the skin and joint. In order to elucidate the mechanism of endogenous CGRP release, we have used a multiple site 133Xe clearance technique to measure local blood flow changes in response to agents injected intradermally in the rabbit. Capsaicin (100 nmol/site) and human alpha CGRP (3 pmol/site) stimulated similar increases in blood flow and, in both cases, the effect was totally abolished by the CGRP antagonist, CGRP8-37 (1 nmol/site). By contrast, the nitric oxide synthase inhibitor L-nitro arginine methyl ester (L-NAME, 30 nmol/site) had little effect on human alpha CGRP-induced vasodilation, but caused significant inhibition of the response to capsaicin (p < 0.05). These results show that increased blood flow in rabbit skin caused by exogenous CGRP is independent of nitric oxide. In addition, however, they suggest that nitric oxide is required for either the release of endogenous CGRP from capsaicin-sensitive nerves or its subsequent activity.