Non-selective Nitric Oxide Synthase Inhibitor L-NAME Research Articles

Ethnomedicinal Study and Evaluation of the Anxiolytic-like and Diuretic Effects of the Orchid Stanhopea tigrina Bateman ex Lindl-(Orchidaceae).

Stanhopea tigrina Bateman ex Lindl. (Orchidaceae) is an orchid endemic to Mexico, known as "Calavera" or "calaverita", in the Huasteca Potosina (central region of Mexico). This plant species is used for the folk treatment of mental disorders and urological kidney disorders, according to the ethnomedicinal information obtained in this study. Ethanolic extracts of leaves (HE) and pseudobulb (PE) were obtained by microwave-assisted extraction (MAE). Gas Chromatography coupled with Mass Spectrometry (GC-MS) was used to carry out the chemical characterization of HE and PE. The pharmacological effects (antioxidant, diuretic, anxiolytic, locomotor, hypnotic, and sedative) of HE and PE were evaluated. The possible mechanism of action of the anxiolytic-like activity induced by HE was assessed using inhibitors of the GABAergic, adrenergic, and serotonergic systems. The possible mechanism of the diuretic action of HE was assessed using prostaglandin inhibitory antagonists and nitric oxide synthase (NOS) blockers. HE at 50 and 100 mg/kg exerted anxiolytic-like activity without inducing hypnosis or sedation. Flumazenil, prazosin, and ketanserin inhibited the anxiolytic-like activity shown by HE, which suggests the participation of GABA, α1-adrenergic receptors, and 5-HT2 receptors, respectively. The diuretic effect was reversed by the non-selective NOS inhibitor L-NAME, which caused the reduction in nitric oxide (NO). These results demonstrate that the ethanolic extract of S. tigrina leaves exhibited anxiolytic-like activity and diuretic effects without inducing hypnosis or sedation. This work validates the medicinal uses of this orchid species.

Stenosis coexists with compromised \u03b11-adrenergic contractions in the ascending aorta of a mouse model of Williams-Beuren syndrome

Williams-Beuren syndrome (WBS) is a rare disorder caused by a heterozygous deletion of 26–28 contiguous genes that affects the brain and cardiovascular system. Here, we investigated whether WBS affects aortic structure and function in the complete deletion (CD) mouse model harbouring the most common deletion found in WBS patients. Thoracic aortas from 3–4 months-old male CD mice and wild-type littermates were mounted in wire myographs or were processed for histomorphometrical analysis. Nitric oxide synthase (NOS) isoforms and oxidative stress levels were assessed. Ascending aortas from young adult CD mice showed moderate (50%) luminal stenosis, whereas endothelial function and oxidative stress were comparable to wild-type. CD mice showed greater contractions to KCl. However, α1-adrenergic contractions to phenylephrine, but not with a thromboxane analogue, were compromised. Decreased phenylephrine responses were not affected by selective inducible NOS blockade with 1400 W, but were prevented by the non-selective NOS inhibitor L-NAME and the selective neuronal NOS inhibitor SMTC. Consistently, CD mice showed increased neuronal NOS expression in aortas. Overall, aortic stenosis in CD mice coexists with excessive nNOS-derived NO signaling that compromises ascending aorta α1-adrenergic contractions. We suggest that increased neuronal NOS signaling may act as a physiological ‘brake’ against the detrimental effects of stenosis.

Anticonvulsant effect of dextrometrophan on pentylenetetrazole-induced seizures in mice: Involvement of nitric oxide and N-methyl-d-aspartate receptors.

Dextrometrophan (DM), widely used as an antitussive, has recently generated interest as an anticonvulsant drug. Some effects of dextrometrophan are associated with alterations in several pathways, such as inhibition of nitric oxide synthase (NOS) enzyme and N-methyl d-aspartate (NMDA) receptors. In this study, we aimed to investigate the anticonvulsant effect of acute administration of dextrometrophan on pentylenetetrazole (PTZ)-induced seizures and the probable involvement of the nitric oxide (NO) pathway and NMDA receptors in this effect. For this purpose, seizures were induced by intravenous PTZ infusion. All drugs were administrated by intraperitoneal (i.p.) route before PTZ injection. Our results demonstrate that acute DM treatment (10-100mg/kg) increased the seizure threshold. In addition, the nonselective NOS inhibitor L-NAME (10mg/kg) and the neural NOS inhibitor, 7-nitroindazole (40mg/kg), at doses that had no effect on seizure threshold, augmented the anticonvulsant effect of DM (3mg/kg), while the inducible NOS inhibitor, aminoguanidine (100mg/kg), did not affect the anticonvulsant effect of DM. Moreover, the NOS substrate l-arginine (60mg/kg) blunted the anticonvulsant effect of DM (100mg/kg). Also, NMDA antagonists, ketamine (0.5mg/kg) and MK-801 (0.05mg/kg), augmented the anticonvulsant effect of DM (3mg/kg). In conclusion, we demonstrated that the anticonvulsant effect of DM is mediated by a decline in neural nitric oxide activity and inhibition of NMDA receptors.

Enhanced XOR activity in eNOS-deficient mice: Effects on the nitrate-nitrite-NO pathway and ROS homeostasis

Xanthine oxidoreductase (XOR) is generally known as the final enzyme in purine metabolism and as a source of reactive oxygen species (ROS). In addition, this enzyme has been suggested to mediate nitric oxide (NO) formation via reduction of inorganic nitrate and nitrite. This NO synthase (NOS)-independent pathway for NO generation is of particular importance during certain conditions when NO bioavailability is diminished due to reduced activity of endothelial NOS (eNOS) or increased oxidative stress, including aging and cardiovascular disease. The exact interplay between NOS- and XOR-derived NO generation is not fully elucidated yet. The aim of the present study was to investigate if eNOS deficiency is associated with changes in XOR expression and activity and the possible impact on nitrite, NO and ROS homeostasis.Plasma levels of nitrate and nitrite were similar between eNOS deficient (eNOS−/−) and wildtype (wt) mice. XOR activity was upregulated in eNOS−/− compared with wt, but not in nNOS−/−, iNOS−/− or wt mice treated with the non-selective NOS inhibitor L-NAME. Following an acute dose of nitrate, plasma nitrite increased more in eNOS−/− compared with wt, and this augmented response was abolished by the selective XOR inhibitor febuxostat. Livers from eNOS−/− displayed higher nitrite reducing capacity compared with wt, and this effect was attenuated by febuxostat. Dietary supplementation with nitrate increased XOR expression and activity, but concomitantly reduced superoxide generation. The latter effect was also seen in vitro after nitrite administration. Treatment with febuxostat elevated blood pressure in eNOS−/−, but not in wt mice. A high dose of dietary nitrate reduced blood pressure in naïve eNOS−/− mice, and again this effect was abolished by febuxostat. In conclusion, eNOS deficiency is associated with an upregulation of XOR facilitating the nitrate-nitrite-NO pathway and decreasing the generation of ROS. This interplay between XOR and eNOS is proposed to play a significant role in NO homeostasis and blood pressure regulation.

Protective Effect of Estradiol on Acute Lung Inflammation Induced by an Intestinal Ischemic Insult is Dependent on Nitric Oxide

It has been shown that the innate immune system mediates acute lung inflammation triggered by intestinal trauma. Sexual dimorphism modulates the profile of TH1 and TH2 lymphocytes, and accordingly sex hormones may modulate acute lung inflammation by intestinal ischemia/reperfusion (I/R). Studies indicate that female rats are relatively resistant to organ injury caused by hemorrhagic shock and that the gut of female is more resistant than that of the male to deleterious effects of ischemic injury. At the present study, we investigated the effect of estradiol (E(2)) on the lung inflammation after intestinal I/R and its interaction with the nitric oxide (NO) pathway. Anesthetized female rats submitted or not to 7 days ovariectomy (OVx) were subjected to occlusion of the superior mesenteric artery during 45 min, followed by 2 h of reperfusion. Groups of rats were treated with E(2) (17β-estradiol, 280 μg/kg, s.c.) 24 h before ischemia and/or with the nonselective NO synthase inhibitor L-NAME (Nω-nitro-L-arginine methyl ester hydrochloride) (5 mg/kg, i.v.). In a parallel set of experiments, the selective NO synthase inhibitor, aminoguanidine (50 mg/kg i.v.), was given 1 h before ischemia. In all groups, lung vascular permeability (LVP) was assessed using the Evans blue dye extravasation method, neutrophil recruitment to the tissues by the standard myeloperoxidase (MPO) method, and endothelial NO synthase (eNOS) protein expression by Western blot. In OVx rats, LVP and MPO were increased after intestinal I/R as compared with intact controls. Estradiol reverted the LVP, but not MPO. Aminoguanidine reduced LVP in OVx rats. The E(2) protective effect on LVP was abolished by L-NAME; moreover, an increase in LVP even when compared with OVx rats treated only with L-NAME was observed. In addition, lung eNOS protein expression was reduced in OVx-I/R rats in comparison to intact controls and the E(2) inhibited this effect. Estradiol treatment is able to reduce lung inflammation due to intestinal I/R, but with the concomitant blockade of NOS activity, this effect is abolished. Nitric oxide probably reduces the vascular deleterious effects of intestinal I/R, and E(2) pretreatment reduces lung inflammation after intestinal I/R and exerts these effects by modulating eNOS protein expression in the lungs.

Endothelium-Derived Nitric Oxide Supports Renin Cell Recruitment Through the Nitric Oxide–Sensitive Guanylate Cyclase Pathway

Chronic challenge of renin-angiotensin causes recruitment of renin-producing cells in the kidney along the media layer of afferent arterioles and hypertrophy of cells in the juxtaglomerular apparatus. This study aimed to define the role of nitric oxide (NO) with regard to the recruitment pattern of renin-producing cells and to the possible pathways along which NO could act. We considered the hypothesis that endothelium-derived NO acts via NO-sensitive guanylate cyclase. Mice were treated with low-salt diet in combination with the angiotensin I-converting enzyme inhibitor enalapril for 3 weeks, which led to a 13-fold increase in renin expression associated with marked recruitment of renin cells in afferent arterioles and hypertrophy of the juxtaglomerular apparatus in wild-type mice. In wild-type mice additionally treated with the nonselective NO synthase inhibitor L-NAME, the recruitment of renin-expressing cells along the afferent arterioles was absent and juxtaglomerular hypertrophy was diminished. An almost identical attenuation of renin cell recruitment as with L-NAME treatment in wild-type mice was found in mice lacking the endothelial isoform of NO synthase. Treatment of mice lacking NO-sensitive guanylate cyclase in renin-expressing cells and preglomerular smooth muscle cells with low-salt diet in combination with the angiotensin I-converting enzyme inhibitor enalapril for 3 weeks produced juxtaglomerular hypertrophy like in wild-type mice, but no recruitment in afferent arterioles. These findings suggest that endothelium-derived NO and concomitant formation of cGMP in preglomerular renin cell precursors supports recruitment of renin-expressing cells along preglomerular vessels, but not in the juxtaglomerular apparatus.

Endogenous Nitric Oxide Can Act as Beneficial or Deleterious in the Hypoxic Lung Depending on the Reoxygenation Time

Nitric oxide (NO) has been implicated in many pathophysiological situations in the lung, including hypoxia/reoxygenation. This work seeks to clarify the current controversy concerning the double protective/toxic role of endogenous NO under hypoxia/reoxygenation situations in the lung by using a nitric oxide synthase (NOS) inhibitor, in a novel approach to address the problems raised from assaults under such circumstances. A follow-up study was conducted in Wistar rats submitted to hypoxia/reoxygenation (hypoxia for 30 min; reoxygenation of 0 h, 48 h, and 5 days), with or without prior treatment using the nonselective NOS inhibitor L-NAME (1.5 mM, in drinking water). Lipid peroxidation, apoptosis level, protein nitration, in situ NOS activity and NO production (NOx) were analyzed. This is the first work to focus on the time-course effects of L-NAME in the adult rat lung submitted to hypoxia/reoxygenation. The results showed that after L-NAME administration, in situ NOS activity was almost completely eliminated and consequently, NOx levels fell. Lipid peroxidation and the percentage of apoptotic cells rose at the earliest reoxygenation time (0 h), but decreased in the later period (48 h and 5 days). Also nitrated protein expression decreased at 48 h and 5 days posthypoxia. These results suggest that NOS-derived NO exerts two different effects on lung hypoxia/reoxygenation injury depending on the reoxygenation time: NO has a beneficial role just after the hypoxic stimulus and a deleterious effect in the later reoxygenation times. Moreover, we propose that this dual role of NO depends directly on the producer NOS isoform.

Modulation of the Nitric Oxide Metabolism Overcomes the Unresponsiveness of the Diabetic Human Myocardium to Protection Against Ischemic Injury

We have demonstrated that diabetic human myocardium cannot be protected by ischemic preconditioning (IP) and identified a dysfunction of the mitochondria as the cause of the defect. Here we have investigated whether modulation of the nitric oxide (NO) metabolism can overcome the unresponsiveness of the diabetic myocardium to cardioprotection. Myocardial slices (30-40 mg) obtained from the right atrial appendage of patients with diabetes undergoing elective cardiac surgery were randomized to the following protocol (n=6/group): NO donor SNAP (100 μM), nonselective nitric oxide synthase (NOS) inhibitor L-NAME (100 μM), and selective neuronal NOS (nNOS) inhibitor TRIM (100 μM) for 20 min prior to 90 min ischemia followed by 120 min reoxygenation (37°C). Some preparations were subjected to ischemic/reoxygenation alone or to IP (5 min ischemia/5 min reoxygenation) to act as control. Tissue injury was assessed by creatine kinase (CK) released (IU/mg wet wt), and cell necrosis and apoptosis by propidium iodide and TUNEL (% of aerobic control). IP did not decrease CK release, cell necrosis or apoptosis in diabetic myocardium. However, NO donor SNAP, the nonspecific NOS inhibitor L-NAME, and the specific nNOS inhibitor TRIM significantly reduced CK leakage, cell necrosis, and apoptosis in diabetic myocardium. These results demonstrate that both the provision of exogenous NO and the suppression of endogenous NO production result in potent protection of diabetic human myocardium overcoming the unresponsiveness of these tissues to cardioprotective therapies.

Neutrophil elastase contributes to the development of ischemia/reperfusion-induced liver injury by decreasing the production of insulin-like growth factor-I in rats

Neutrophil elastase (NE) decreases the endothelial production of prostacyclin (PGI(2)) through the inhibition of endothelial nitric oxide synthase (NOS) activation and thereby contributes to the development of ischemia/reperfusion (I/R)-induced liver injury. We previously demonstrated that calcitonin gene-related peptide (CGRP) released from sensory neurons increases the insulin-like growth factor- I (IGF-I) production and thereby reduces I/R-induced liver injury. Because PGI(2) is capable of stimulating sensory neurons, we hypothesized that NE contributes to the development of I/R-induced liver injury by decreasing IGF-I production. In the present study, we examined this hypothesis in rats subjected to hepatic I/R. Ischemia/reperfusion-induced decreases of hepatic tissue levels of CGRP and IGF-I were prevented significantly by NE inhibitors, sivelestat, and L-658, 758, and these effects of NE inhibitors were reversed completely by the nonselective cyclooxygenase inhibitor indomethacin (IM) and the nonselective NOS inhibitor L-NAME but not by the selective inducible NOS inhibitor 1400W. I/R-induced increases of hepatic tissue levels of caspase-3, myeloperoxidase and the number of apoptotic cells were inhibited by NE inhibitors, and these effects of NE inhibitors were reversed by IM and L-NAME but not by 1400W. Administration of iloprost, a stable PGI(2) analog, produced effects similar to those induced by NE inhibitors. Taken together, these observations strongly suggest that NE may play a critical role in the development of I/R-induced liver injury by decreasing the IGF-I production through the inhibition of sensory neuron stimulation, which may lead to an increase of neutrophil accumulation and hepatic apoptosis through activation of caspase-3 in rats.

Different mechanisms underlie the effects of acute and long-term inhibition of nitric oxide synthases in antigen-induced pulmonary eosinophil recruitment in BALB/C mice

Nitric oxide synthase (NOS) inhibitors are largely used to evaluate the NO contribution to pulmonary allergy, but contrasting data have been reported. In this study, pharmacological, biochemical and pharmacokinetic assays were performed to compare the effects of acute and long-term treatment of BALB/C mice with the non-selective NOS inhibitor l-NAME in ovalbumin (OVA)-challenged mice. Acute l-NAME treatment (50 mg/kg, gavage) significantly reduced the eosinophil number in bronchoalveolar lavage fluid (BALF). The inducible NOS (iNOS) inhibitor aminoguanidine (20 mg/kg/day in the drinking water) also significantly reduced the eosinophil number in BALF. In contrast, 3-week l-NAME treatment (50 and 150 mg/kg/day in the drinking water) significantly increased the pulmonary eosinophil influx. The constitutive NOS (cNOS) activity in brain and lungs was reduced by both acute and 3-week l-NAME treatments. The pulmonary iNOS activity was reduced by acute l-NAME (or aminoguanidine), but unaffected by 3-week l-NAME treatment. Acute l-NAME (or aminoguanidine) treatment was more efficient to reduce the NO x − levels compared with 3-week l-NAME treatment. The pharmacokinetic study revealed that l-NAME is not bioavailable when given orally. After acute l-NAME intake, serum concentrations of the metabolite N ω-nitro- l-arginine decreased from 30 min to 24 h. In the 3-week l-NAME treatment, the N ω-nitro- l-arginine concentration was close to the detection limit. In conclusion, 3-week treatment with l-NAME yields low serum N ω-nitro- l-arginine concentrations, causing preferential inhibition of cNOS activity. Therefore, eosinophil influx potentiation by 3-week l-NAME treatment may reflect removal of protective cNOS-derived NO, with no interference on the ongoing inflammation due to iNOS-derived NO.

Red wine polyphenolic compounds inhibit tracheal smooth muscle contraction during allergen-induced hyperreactivity of the airways

The aims of the study were to investigate the short and long-term effects of Provinol (red wine polyphenolic compounds) on tracheal smooth muscle reactivity using an in-vitro model of ovalbumin-induced airway inflammation in guinea-pig trachea, and to evaluate the role of nitric oxide (NO) in the bronchodilatory effect of Provinol. The amplitude of tracheal smooth muscle contraction in response to mediators of bronchoconstriction - histamine (10 nM-1 mM), acetylcholine (10 nM-1 mM) and to allergen (ovalbumin 10(-5)-10(-3) g mL(-1)) was used as a parameter of tracheal smooth muscle reactivity. To test the short-term effects of Provinol, isolated tracheal strips were pre-treated for 30 min with Provinol (10(-4) mg mL(-1)) alone or in combination with Nomega-nitro-L-arginine methyl ester (L-NAME; 10(-6) mol L(-1)). To test the long-term effects of Provinol, isolated tracheal strips were prepared from guinea pigs that had been treated for 14 days with Provinol (20 mg kg(-1) per day) alone or in combination with L-NAME (40 mg kg(-1) per day). Incubation of tracheal smooth muscle with Provinol decreased the amplitude of contraction in response to ovalbumin, histamine and acetylcholine. The non-selective NO synthase inhibitor L-NAME partially abolished the effect of Provinol on acetylcholine and ovalbumin-induced but not histamine-induced bronchoconstriction. A similar profile was observed after 14 days' oral administration of Provinol. In conclusion, Provinol inhibited the allergen- and spasmogen-induced contraction of tracheal smooth muscle in ovalbumin-sensitized guinea pigs via a mechanism that was mediated at least partially through the metabolism of NO.

Pentagastrin‐induced nitric oxide‐dependent protein secretion from the parotid gland of the anaesthetized rat

Infusion of pentagastrin (20 microg kg(-1) h(-1), i.v.) for 10 min evokes protein output but no overt fluid secretion from the parotid gland of the rat, as revealed by increased protein concentration in a subsequent wash-out flow of saliva in response to a bolus injection of methacholine (5 microg kg(-1), i.v.) 10 min later. Using this experimental set-up, the contribution of nitric oxide (NO) generation to the protein and amylase response evoked by pentagastrin was investigated. Neither the neuronal type NO synthase inhibitor N(omega)-propyl-L-arginine (N-PLA; 30 mg kg(-1), i.v.) nor the non-selective NO synthase inhibitor L-NAME (30 mg kg(-1), i.v.) as such affected the methacholine-evoked volume response or the outputs of protein and amylase. However, when preceeded by the pentagastrin infusion, the expected increases in concentrations of protein (145%) and amylase activity (127%) of the methacholine-evoked response (compared to a pre-infusion methacholine response) were reduced to 68 and 74%, respectively, in the presence of N-PLA, and to 70 and 63%, respectively, in the presence of L-NAME. Thus, NO generation resulting from the activity of the neuronal type NO synthase, most probably of parenchymal origin, plays an important role in the pentagastrin-induced protein and amylase secretion of the rat parotid gland.

Nitric Oxide Signaling via Nuclearized Endothelial Nitric-oxide Synthase Modulates Expression of the Immediate Early Genes iNOS and mPGES-1

Stimulation of freshly isolated rat hepatocytes with lysophosphatidic acid (LPA) resulted in LPA1 receptor-mediated and nitricoxide-dependent up-regulation of the immediate early genes iNOS (inducible nitric-oxide synthase (NOS)) and mPGES-1 (microsomal prostaglandin E synthase-1). Because LPA is a ligand for both cell surface and intracellular receptor sites and a potent endothelial NOS (eNOS) activator, we hypothesized that NO derived from activated nuclearized eNOS might participate in gene regulation. Herein we show, by confocal microscopy performed on porcine cerebral endothelial cells expressing native LPA1-receptor and eNOS and on HTC4 rat hepatoma cells co-transfected with recombinant human LPA1-receptor and fused eNOS-GFP cDNA, a dynamic eNOS translocation from peripheral to nuclear regions upon stimulation with LPA. Nuclear localization of eNOS and its downstream effector, soluble guanylate cyclase, were demonstrated in situ in rat liver specimens by immunogold labeling using specific antibodies. Stimulation of this nuclear fraction with LPA and the NO donor sodium nitroprusside resulted, respectively, in increased production of nitrite (and eNOS phosphorylation) and cGMP; these separate responses were also correspondingly blocked by NOS inhibitor L-NAME and soluble guanylate cyclase inhibitor ODQ. In addition, sodium nitroprusside evoked a sequential increase in nuclear Ca2+ transients, activation of p42 MAPK, NF-kappaB binding to DNA consensus sequence, and dependent iNOS RNA. This study describes a hitherto unrecognized molecular mechanism by which nuclear eNOS through ensuing NO modulates nuclear calcium homeostasis involved in gene transcription-associated events. Moreover, our findings strongly support the concept of the nucleus as an autonomous signaling compartment.

Cross talk between nitric oxide and ERK1/2 signaling pathway in the spinal cord mediates naloxone-precipitated withdrawal in morphine-dependent rats

Our recent study has shown activation of spinal extracellular signal-regulated kinase-1 and -2 (ERK1/2), a member of the mitogen-activated protein kinase (MAPK) family, contributes to naloxone-precipitated withdrawal and withdrawal-induced spinal neuronal sensitization in morphine-dependent rats. However, the mechanism and significance of the spinal ERK1/2 activation during morphine dependence and withdrawal remain unknown. In this study, we reported that intrathecal (i.t.) pretreatment with either the non-selective nitric oxide synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME), neuronal NOS (nNOS) inhibitor 7-nitro indazole (7-NI), or the inducible NOS (iNOS) inhibitor aminoguanidine (AG), could reduce morphine withdrawal-induced increase of phospho-ERK1/2 (pERK1/2) expression in the rat spinal cord. On the other hand, attenuation of the spinal ERK phosphorylation by the MAPK kinase (MEK) inhibitor U0126 also could inhibit the increase of nNOS and iNOS expression in the spinal cord of morphine withdrawal rats. Inhibitory expression of pERK1/2 by i.t. NOS inhibitor L-NAME, 7-NI or AG and of nNOS and iNOS by i.t. U0126 in the spinal cord were accompanied by decreased scores of morphine withdrawal and the inhibited spinal Fos protein (a maker for neuronal excitation or activation) expression induced by morphine withdrawal. These findings suggest cross talk between nitric oxide (NO) and the ERK1/2 signaling pathway mediates morphine withdrawal and withdrawal-induced spinal neuronal sensitization in morphine-dependent rats.

P.2.026 Change in brain-derived neurotrophic factor immunoreactivity in nucleus accumbens following repetitive electroconvulsive shocks

Our recent study has shown activation of spinal extracellular signal-regulated kinase-1 and -2 (ERK1/2), a member of the mitogen-activated protein kinase (MAPK) family, contributes to naloxone-precipitated withdrawal and withdrawal-induced spinal neuronal sensitization in morphine-dependent rats. However, the mechanism and significance of the spinal ERK1/2 activation during morphine dependence and withdrawal remain unknown. In this study, we reported that intrathecal (i.t.) pretreatment with either the non-selective nitric oxide synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME), neuronal NOS (nNOS) inhibitor 7-nitro indazole (7-NI), or the inducible NOS (iNOS) inhibitor aminoguanidine (AG), could reduce morphine withdrawal-induced increase of phospho-ERK1/2 (pERK1/2) expression in the rat spinal cord. On the other hand, attenuation of the spinal ERK phosphorylation by the MAPK kinase (MEK) inhibitor U0126 also could inhibit the increase of nNOS and iNOS expression in the spinal cord of morphine withdrawal rats. Inhibitory expression of pERK1/2 by i.t. NOS inhibitor L-NAME, 7-NI or AG and of nNOS and iNOS by i.t. U0126 in the spinal cord were accompanied by decreased scores of morphine withdrawal and the inhibited spinal Fos protein (a maker for neuronal excitation or activation) expression induced by morphine withdrawal. These findings suggest cross talk between nitric oxide (NO) and the ERK1/2 signaling pathway mediates morphine withdrawal and withdrawal-induced spinal neuronal sensitization in morphine-dependent rats.

Blockade of neuronal nitric oxide synthase abolishes the toxic effects of Tx2-5, a lethal Phoneutria nigriventer spider toxin

The primary goal of this study was to determine whether Tx2-5, a sodium channel selective toxin obtained from the venom of the spider Phoneutria nigriventer, produced penile erection by means of nitric oxide mechanism. Toxin identity was analyzed by MALDI-TOF, ES-MS and N-terminal amino acid sequencing. Pretreating mice with the non-selective nitric oxide synthase (NOS) inhibitor N ω-Nitro- l-arginine methyl ester hydrochloride ( l-NAME) and the selective neuronal-NOS inhibitor 7-Nitroindazole (7-NI) prior to Tx2-5 i.p. (10 μg/25 g mouse) injection challenged the hypothesis above. Controls were injected with the d-isomer or DMSO or saline. Results demonstrated that l-NAME inhibited penile erections in about half the animals treated, while 7-NI completely abolished this effect. Interestingly 7-NI also abolished all the other symptoms of intoxication induced by Tx2-5, including salivation, respiratory distress and death. Tx2-5 killed all the animals of the control group and no one in the 7-NI-treated group. We conclude that (1) intraperitoneal injections of Tx2-5 induce a toxic syndrome that include penile erection, hypersalivation and death by respiratory distress or pulmonary edema; (2) pretreatment with the non-selective NOS inhibitor l-NAME reduces the penile erection and partially protects from the lethal effects of Tx2-5; (3) pretreatment with the nNOS-selective inhibitor 7-NI completely abolishes all the toxic effects of Tx2-5, including penile erection and death suggesting that nNOS is the major player in this intoxication; (4) toxins from other animals that affect sodium channels in the same way as Tx2-5 and induce similar toxic syndromes may have as a major common target, the activation of nitric oxide synthases.

Pharmacological assessment of the nitric-oxide synthase isoform involved in eosinophilic inflammation in a rat model of sephadex-induced airway inflammation.

Excessive local production of nitric oxide (NO) has been suggested to play a role in rodent models of airway inflammation and in pulmonary diseases such as asthma. However, even given the plethora of data available including gene expression data, pharmacological data, and gene deletion studies in animal models, it is still not clear which nitric-oxide synthase (NOS) isoform is involved in eosinophilic airway inflammation. In this rat study, the nonselective NOS inhibitor L-NAME (N(G)-nitro-L-arginine methyl ester), but not a selective inducible NOS (iNOS) inhibitor 1400W (N-3-(aminomethyl)benzyl)acetamidine), impacted on Sephadex-induced inflammation by significantly inhibiting lung edema, eosinophil infiltration, tumor necrosis factor alpha, interleukin-13, and eotaxin levels in the lung tissue. Furthermore, iNOS gene expression was not induced following Sephadex administration, which confirms that iNOS does not play a role in this model. To demonstrate that this phenomenon was not restricted to this model of asthma, L-NAME, but not 1400W, was shown to reduce eosinophilia in an antigen-induced model. However, in contrast to the Sephadex model, there was an induction of iNOS gene expression after antigen challenge. In a model of aerosolized lipopolysaccharide-induced inflammation, where iNOS gene expression is increased, 1400W inhibited the increased neutrophilia. These data suggest that the compound has been administered using an appropriate dosing regimen for iNOS inhibition in the rat lung. In conclusion, it appears that constitutive, not inducible, NOS isoforms are important in NO production in models of allergic inflammation, which questions whether there is a role for iNOS inhibitors as therapy for the treatment of asthma.

Regulation Of B16F1 melanoma cell metastasis by inducible functions of the hepatic microvasculature

We have previously shown that circulating intravascular cells generally arrest by mechanical restriction in the hepatic sinusoids, causing rapid release of nitric oxide (NO) which is cytotoxic to these cells and inhibits their growth into metastatic tumours. Here, we present evidence that these NO-dependent cytotoxic mechanisms are susceptible to upregulation by lipopolysaccharide (LPS). Five×105 fluorescently labelled melanoma cells were injected into the mesenteric vein of C57BL/6 mice to effect their localisation in the hepatic microvasculature. Test mice were then given 1 mg/kg LPS intraperitoneally (i.p.) to activate the microvascular cells. By electron paramagnetic resonance (EPR) spectroscopy, the expression of NO in the liver was significantly increased by 8 h in the LPS-treated mice. The non-selective NO synthase inhibitor L-NAME inhibited the induction of NO by LPS, while its inactive enantiomer D-NAME had no significant effect. Using immunohistochemistry (IHC), iNOS-positive microvascular cells were detected in the terminal portal venule (TPV) region of the liver 8 h after LPS stimulation. LPS treatment also increased the retention of melanoma cells in the liver between 8 and 24 h, especially in the TPV region. Eight hours after cell injection, local expression of VCAM-1 and ICAM-1 was detected by double-label immunohistochemistry at the sites of tumour cell arrest. Expression of these adhesion molecules was enhanced in mice treated with LPS. Using flow cytometry, 98% of the B16F1 melanoma cells expressed VLA-4, the counter receptor of VCAM-1, and approximately 1.5% expressed LFA-1, the counter receptor of ICAM-1. LPS did not significantly alter the expression of either counter receptor on melanoma cells in vitro or in vivo. By DNA end-labelling, the rates of melanoma cell apoptosis were significantly increased from 8 to 24 h in the TPV region (but not in the sinusoids) of LPS-treated mice. Fourteen days after tumour cell injection, the LPS-treated mice had a significantly smaller hepatic metastatic tumour burden than the control mice. These data suggest that LPS can inhibit the metastasis of melanoma cells in the liver by inducing the expression of NO and adhesion molecules by the hepatic endothelium. The induction of iNOS and the inducible cytotoxic effect of LPS appear to be primarily located within the TPV region of the liver acinus.

Inhibitors of nitric oxide synthase attenuate nerve growth factor-mediated increases in choline acetyltransferase expression in PC12 cells.

NGF can regulate nitric oxide synthase (NOS) expression and nitric oxide (NO) can modulate NGF-mediated neurotrophic responses. To investigate the role of NO in NGF-activated expression of cholinergic phenotype, PC12 cells were treated with either the nonselective NOS inhibitor L-NAME (N (omega)-nitro-L-arginine methylester) or the inducible NOS selective inhibitor MIU (s-methylisothiourea), and the effect on NGF-stimulated ChAT mRNA levels and ChAT specific activity was determined. NGF increased steady-state levels of mRNA and protein for both inducible and constitutive isozymes of NOS in PC12 cells, and led to enhanced NOS activity and NO production. MIU and, to a lesser extent, L-NAME blocked neurite outgrowth in nerve growth factor (NGF)-treated PC12 cells. Both L-NAME and MIU attenuated NGF-mediated increases in choline transferase (ChAT)-specific activity and prevented the increase in expression of ChAT mRNA normally produced by NGF treatment of PC12 cells. The present study indicates that NO may be involved in the modulation of signal transduction pathways by which NGF leads to increased ChAT gene expression in PC12 cells.

Nitric oxide synthase inhibitors impair reference memory formation in a radial arm maze task in rats

We investigated the role of nitric oxide (NO) in learning and memory formation in a radial maze test, by using the NO synthase (NOS) inhibitors, N G-nitro- l-arginine methyl ester ( l-NAME) and 7-nitroindazole (7-NI), and an NO precursor, l-arginine. Rats were trained on a working and reference memory task in an eight arm radial maze for 30 days, wherein the same four arms were baited for each daily training trial. Daily administration of the specific neuronal NOS inhibitor 7-NI, as well as the non-selective NOS inhibitor l-NAME, impaired the acquisition of the task. Analysis of memory categories affected by NOS inhibitors revealed that reference memory formation was impaired by the treatment with l-NAME and 7-NI. l-NAME also impaired working memory although 7-NI had no effect. l-Arginine significantly increased the choice accuracy, by reducing the reference memory errors, of the radial arm maze task during the last ten days although it had no effect during the first 20 trials. These results suggest that NO plays an important role in spatial reference memory formation.