Inhibition of nitric oxide synthase reduces ultrasonic vocalizations of rat pups.

The present study investigated the effects of drugs acting on the brain nitric oxide pathway on ultrasonic vocalizations, body temperature and locomotion in 7-8-day-old rat pups. Both a selective neuronal nitric oxide synthase (NOS) inhibitor (7-nitroindazole) and a non-selective NOS inhibitor (nitro-L-arginine-methyl ester, L-NAME) decreased the number of ultrasonic vocalizations in a dose-dependent manner. The non-selective NOS inhibitor, L-NAME, suppressed not only ultrasonic vocalizations but also locomotion. The inactive isomer of the NOS inhibitor, nitro-D-arginine-methyl ester (D-NAME), and the biological precursor of nitric oxide, L-arginine, had no effect on ultrasonic vocalizations or locomotion. These data indicate that drugs suppressing nitric oxide synthesis produced an anxiolytic effect in rat pups. However, only the selective NOS inhibitor, 7-nitroindazole, was 'anxioselective', i.e., reduced ultrasonic vocalizations without causing sedation. Increased synthesis of nitric oxide in the brain had no apparent behavioral effect in this model.     

Tinospora tuberculata suppresses nitric oxide synthesis in mouse macrophages

We have obtained evidence that co-incubation of thioglycollate-elicited peritoneal macrophages with an aqueous extract of Tinospora tuberculata inhibits lipopolysaccharide-stimulated excessive production of nitric oxide (NO) in vitro. This effect is concentration-dependent and appears to involve suppression of both inducible nitric oxide synthase (iNOS) activity and NADPH-diaphorase activity, thus altering NO production. As NO is one of the critical mediators in various disorders and iNOS inhibitors may have therapeutic potential, these results may explain some aspects of the multifunctional properties of Tinospora tuberculata, which has been used in various folk remedies in southeast Asia and China.     

The effect of the nitric oxide synthesis inhibitor L-NAME on amitriptyline-induced hypotension in rats

OBJECTIVE: Hypotension induced by tricyclic antidepressants is multifactorial. Previous animal experiments suggest a contribution from nitric oxide production. Our study aimed to evaluate the role of nitric oxide in amitriptyline-induced hypotension using N-nitro-L-arginine methyl ester, a nitric oxide synthesis inhibitor, and 3-morpholino sydnonimine, a nitric oxide donor, in anesthetized rats. METHODS: Amitriptyline intoxication was induced by the continuous infusion of amitriptyline 0.625 mg/kg/min throughout the experiment in anesthetized rats. Fifteen and 25 minutes after amitriptyline infusion began, two bolus doses of 10 mg/kg of N-nitro-L-arginine methyl ester (n = 8) or an equivalent volume of 5% dextrose solution (n = 8) was administered to each rat (Protocol 1). To investigate whether the effect of N-nitro-L-arginine methyl ester on blood pressure is counteracted by 3-morpholino sydnonimine, after the same protocol of amitriptyline infusion and 5 minutes after an N-nitro-L-arginine methyl ester bolus, a bolus of 3000 nmol/kg of 3-morpholino sydnonimine was administered (n = 8) to each rat (Protocol 2). To investigate the effect of N-nitro-L-arginine methyl ester on 3-morpholino sydnonimine induced hypotension, a group of rats received a continuous infusion of 0.54 mg/kg/h of 3-morpholino sydnonimine until 50% reduction was observed in mean arterial blood pressure followed by a bolus dose of 10 mg/kg of N-nitro-L-arginine methyl ester (n = 6) or 5% dextrose solution (n = 6) (Protocol 3). Outcome measures included mean arterial blood pressure, heart rate, and QRS duration in electrocardiogram. Student's t test and survival analysis were used for selected comparisons. RESULTS: For all parameters, the treatment groups were similar at baseline and at postamitriptyline periods before therapy was rendered. Amitriptyline infusion significantly reduced mean arterial blood pressure by 50.8 +/- 2.2% and prolonged QRS by 23.9 +/- 7.2% after 15 minutes. In Protocol 1, N-nitro-L-arginine methyl ester significantly increased mean arterial blood pressure compared to dextrose-treated control animals within 30 minutes (77.9 +/- 8.5% vs. 49.7 +/- 5.0% mmHg, p < 0.01, 95% CI 57.1-98.7%). QRS duration progressively increased during the amitriptyline infusion; however, there was no significant difference in QRS width between N-nitro-L-arginine methyl ester and control groups at any time point. N-nitro-L-arginine methyl ester increased survival time compared to controls (33.4 +/- 4.1 vs. 19.9 +/- 2.7 minutes, p < 0.01, 95% CI 25.4-41.3) but did not affect mortality. In Protocol 2 of continuous infusion of amitriptyline, 3-morpholino sydnonimine counteracted the N-nitro-L-arginine methyl ester-induced increase in mean arterial blood pressure. In both protocols, heart rate decreased significantly during amitriptyline infusion but there was no difference between treatment and control groups. In Protocol 3, N-nitro-L-arginine methyl ester bolus reversed 3-morpholino sydnonimine-induced hypotension compared to dextrose bolus. (83.8 +/- 5.7% vs. 54.6 +/- 4.8%, p < 0.01, 95% CI 69.2-98.4). CONCLUSION: N-nitro-L-arginine methyl ester is found to be effective in temporarily improving hypotension and prolonging survival time but does not affect overall mortality. Because this effect was antagonized by 3-morpholino sydnonimine, nitric oxide production appears to contribute to the pathophysiology of amitriptyline-induced hypotension.     

Diacerein suppresses the increase in plasma nitric oxide in rat adjuvant-induced arthritis.

We investigated the effects of rhein, an active metabolite of diacerein, on the interleukin-1alpha-stimulated production of nitric oxide (NO) in rabbit articular chondrocytes, and the effects of diacerein on NO production in rat adjuvant-induced arthritis. At doses of 10 and 30 microM, rhein significantly inhibited the interleukin-1alpha-stimulated NO production in chondrocytes. In the rat adjuvant-induced arthritis model, diacerein was administered for 21 days, starting at the time of adjuvant injection. Paw swelling and plasma NO level were measured in order to assess the effect of diacerein on arthritis and NO biosynthesis in the whole body. At doses of 30 and 100 mg/kg/day, diacerein significantly suppressed the development of adjuvant-induced arthritis and the increase in plasma NO. These results suggest that the inhibitory effect of diacerein on rat adjuvant-induced arthritis is partly related to its reduction of the NO production induced by adjuvant-induced arthritis.     

Inhibition of nitric oxide synthesis in vascular smooth muscle by retinoids.

1. These studies examine the effect of retinoids on interleukin 1 beta (IL-1 beta)-induced nitric oxide synthase (NOS) activity in cultured rat aortic vascular smooth muscle (VSM) cells and isolated rat aortic rings. 2. All-trans-retinoic acid (all-trans-RA, 0.1-10 microM) and its active analogues produced concentration-dependent inhibition of IL-1 beta (0.1-10 ng ml-1)-induced nitrite production in cultured VSM cells. In contrast, the inactive retinoid, Ro 14-6113 (0.1-10 microM), had no effect on IL-1 beta-induced nitrite production. 3. Since some of the actions of retinoids are mediated by induction of transforming growth factor beta (TGF-beta), its effect on inducible NOS activity in VSM cells was examined. TGF-beta produced concentration-dependent (0.1-10 ng ml-1) inhibition of IL-1 beta-induced nitrite production and the maximum effect (approximately 90% inhibition) was significantly greater than that seen with all-trans-RA (approximately 70% with 10 microM). However, an anti-TGF-beta antibody (50 micrograms ml-1) which blocked the effect of exogenous TGF-beta (5 ng ml-1) did not significantly reverse the inhibitory action of all-trans-RA (10 microM). 4. In addition to inhibiting IL-1 beta-induced nitrite production, all-trans-RA (10 microM) reduced substantially inducible NOS mRNA and protein levels in IL-1 beta-induced VSM cells (P < 0.01). 5. Incubation of isolated rat aortic rings with IL-1 beta (10 ng ml-1) caused a progressive resistance of the rings to the vasoconstrictor action of phenylephrine (10 nM to 10 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of nitric oxide synthesis by NG-nitro-L-arginine methyl ester (L-NAME): requirement for bioactivation to the free acid, NG-nitro-L-arginine.

1. The L-arginine derivatives NG-nitro-L-arginine (L-NOARG) and NG-nitro-L-arginine methyl ester (L-NAME) have been widely used to inhibit constitutive NO synthase (NOS) in different biological systems. This work was carried out to investigate whether L-NAME is a direct inhibitor of NOS or requires preceding hydrolytic bioactivation to L-NOARG for inhibition of the enzyme. 2. A bolus of L-NAME and L-NOARG (0.25 micromol) increased coronary perfusion pressure of rat isolated hearts to the same extent (21 +/- 0.8 mmHg; n = 5), but the effect developed more rapidly following addition of L-NOARG than L-NAME (mean half-time: 0.7 vs 4.2 min). The time-dependent onset of the inhibitory effect of L-NAME was paralleled by the appearance of L-NOARG in the coronary effluent. 3. Freshly dissolved L-NAME was a 50 fold less potent inhibitor of purified brain NOS (mean IC50 = 70 microM) than L-NOARG (IC50 = 1.4 microM), but the apparent inhibitory potency of L-NAME approached that of L-NOARG upon prolonged incubation at neutral or alkaline pH. H.p.l.c. analyses revealed that NOS inhibition by L-NAME closely correlated with hydrolysis of the drug to L-NOARG. 4. Freshly dissolved L-NAME contained 2% of L-NOARG and was hydrolyzed with a half-life of 365 +/- 11.2 min in buffer (pH 7.4), 207 +/- 1.7 min in human plasma, and 29 +/- 2.2 min in whole blood (n = 3 in each case). When L-NAME was preincubated in plasma or buffer, inhibition of NOS was proportional to formation of L-NOARG, but in blood the inhibition was much less than expected from the rates of L-NAME hydrolysis. This was explained by accumulation of L-NOARG in blood cells. 5. These results suggest that L-NAME represents a prodrug lacking NOS inhibitory activity unless it is hydrolyzed to L-NOARG. Bioactivation of L-NAME proceeds at moderate rates in physiological buffers, but is markedly accelerated in tissues such as blood or vascular endothelium.     

Tetrahydrobiopterin synthesis inhibitors induce nitric oxide synthesis in rat aorta

I. Incubation of rato aortic rings with tetrahydrobiopterin synthesis inhibitors (NAS or DAHP) significantly decreased contractions to phenylephrine. These two compounds significantly potentiated the vascular hyporeactivity induced by endotoxin. Inhibitors of nitric oxide synthesis (NLA or MLA) restored the contractile responses to this α₁-agonist in NAS- or DAHP-treated control rings and abolished the NAS- or DAHP-induced increased hyporeactivity to PE in endotoxin-treated aortic rings. These observations suggest that treatment of isolated blood vessels with BH₄ synthesis inhibitors induces the production of NO' synthesis, resulting in turn in a vascular hyporeactivity to PE potentiated in endotoxin-treated preparations.     

Inhibition of nitric oxide synthesis reduces the hypotension induced by bacterial lipopolysaccharides in the rat in vivo

E. coli lipopolysaccharide (LPS; 15 mg kg-1 i.v.) produced a long-lasting reduction in mean arterial blood pressure (MAP) in the anaesthetized rat. Inhibition of nitric oxide endothelium-derived relaxing factor (EDRF) synthesis with NG-monomethyl-L-arginine (MeArg, 1 mg kg-1 min-1 i.v. for 30 min) produced an increase in MAP and largely attenuated the LPS-induced hypotension; both effects were significantly reversed with L-arginine (6 mg kg-1 min-1 i.v.). When compared to MeArg, phenylephrine (300 mg kg-1 h-1 i.v.) produced a similar pressor response, but much less attenuation of the hypotensive response to LPS. Thus, a stimulation of EDRF release contributes to the LPS-induced hypotension in the anaesthetized rat.     

L-NAME, a nitric oxide synthase inhibitor, modulates cholinergic antinociception.

Systemic administration of sumatriptan and buspirone (20 mg/kg: 5-HT1A agonists) produced antinociception against acetic acid-induced writhing. The antinociceptive effect was potentiated by cholinomimetic physostigmine (0.05 mg/kg i.p.) and blocked by the muscarinic antagonist atropine (5 mg/kg i.p.). Naloxone, an opiate antagonist, failed to reverse the sumatriptan- or buspirone-induced antinociception, but pindolol (10 mg/kg), a nonselective 5-HT1A antagonist, blocked this response. Sumatriptan- or buspirone-induced antinociception was significantly potentiated by L-NAME (a nitric oxide [NO] synthase inhibitor) although L-NAME (20 mg/kg) given alone had no effect on the nociceptive threshold. Recent studies have suggested that the L-arginine/NO/cGMP pathway is involved in the modulation of pain perception. The present results suggest that NO may play a role in cholinergic antinociception-mediated 5-HT1A receptor stimulation and that NO exerts an inhibitory action on cholinergic analgesia.     

The nitric oxide synthesis inhibitor L-NAME produces anxiogenic-like effects in the rat elevated plus-maze test, but not in the social interaction test

The effects of the nitric oxide synthase inhibitor, Nw-nitro-L-arginine methyl ester (L-NAME) were investigated in two animal models of anxiety: the elevated plus-maze and the social interaction test. In the elevated plus-maze, L-NAME (12.5-50 mg/kg) had an anxiogenic-like profile as indicated by dose-dependent reductions in the time spent on the open arms, open arm entries, the percentage of open arm entries and head dips, but there was no significant effect on the number of stretch attend postures. In contrast, L-NAME (12.5-50 mg/kg) failed to modify time spent in social interaction but did reduce a measure of vertical activity. The differential effects of L-NAME in the two anxiety paradigms are discussed.     

Inhibition of nitric oxide synthesis reveals non-cholinergic excitatory neurotransmission in the canine proximal colon.

1. Neuromuscular transmission in the circular muscle of the canine proximal colon was examined, in the presence and absence of nitric oxide synthase inhibitors, by use of mechanical and intracellular microelectrode recording techniques. 2. Electrical field stimulation (EFS; 0.1-20 HZ) produced frequency-dependent contractions of circular muscle strips which reached a maximum at 15 Hz. These responses were enhanced by NG-monomethyl-L-arginine (L-NMMA; 300 microM) and reduced by atropine (1 microM). The effects of L-NMMA were reversed by L-arginine (3 mM). All responses to EFS were abolished by tetrodotoxin (1 microM). 3. In the presence of atropine, phentolamine and propranolol (all at 1 microM; 'non-adrenergic, non-cholingergic (NANC) conditions'), EFS evoked frequency-dependent inhibition of phasic contractions which reached a maximum at 5 Hz. At higher frequencies of EFS, inhibition diminished, and these responses were followed by post-stimulus excitation. 4. Under NANC conditions and in the presence of L-NG-nitroarginine methyl ester (L-NAME; 200 microM), EFS evoked contractions at frequencies of 5 Hz or greater. These contractions were reduced by co-incubation with L-arginine (2 mM) and abolished by tetrodotoxin (1 microM). 5. In the presence of atropine (1 microM), EFS (5-20 Hz) caused frequency-dependent inhibition of electrical slow waves. In the presence of L-NAME (100 microM) and atropine, the inhibitory response to EFS was abolished and an increase in slow wave duration was seen at stimulation frequencies greater than 5 Hz. The effects of EFS on slow wave duration were abolished by tetrodotoxin (1 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of inducible nitric oxide synthesis by catalposide from Catalpa ovata

Catalposide (1) and two related iridoids were isolated from the stem of Catalpa ovata (Bignoniaceae) by bioassay guided fractionation. Catalposide (1) significantly inhibited the production of nitric oxide (NO) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages in a dose-dependent manner. RT-PCR and Western blot analyses demonstrated that catalposide (1) suppressed the expression of inducible nitric oxide synthase (iNOS) gene and iNOS protein. Catalposide (1) also inhibited the activation of LPS-induced NF-kappaB as analyzed by electrophoretic mobility shift assay (EMSA). In addition to the inhibitory effect on NO production in LPS-stimulated RAW 264.7 cells, catalposide (1) significantly inhibited the NO production in cytokine-stimulated human DLD-1 and rat vascular smooth muscle (VSM) cells in a dose-dependent manner.     

Endotoxin-induced vasodilatation in anaesthetized rat skin involves nitric oxide and prostaglandin synthesis.

1. The effect of intradermally injected endotoxin on skin blood flow was investigated in anaesthetized male Wistar rats in vivo. 2. Local skin blood flow changes were measured hourly for 6 h in the shaved dorsal skin with a laser-Doppler flow probe and compared to changes in control sites which had been injected with 100 microliters of phosphate-buffered saline. By 3 h, skin blood flow increased above basal by 129 +/- 27% and 186 +/- 29% with 1 and 10 micrograms of endotoxin respectively. Blood flow remained significantly elevated at 6 h, the corresponding figures being 129 +/- 24% and 154 +/- 31% (P less than 0.05, n = 6 rats, mean +/- s.e.mean). 3. In further experiments, the response to 3 micrograms of endotoxin was measured at 4 h and treatment with a cyclo-oxygenase inhibitor, nitric oxide synthase inhibitors or a topical steroid all significantly inhibited this response (P less than 0.05 in each case, n = 6 rats in each group with duplicate sites in each animal). 4. Indomethacin 3 x 10(-9) mol per site injected 3.5 h after injection of endotoxin suppressed the mean 4 h response to endotoxin by 78%; NG-nitro-L-arginine methyl ester (L-NAME) 10(-7) mol per site suppressed the response by 95%; NG-monomethyl-L-arginine (L-NMMA) 10(-7) mol per site suppressed the response by 50%; whereas the D-isomer of NG-monomethyl-arginine 10(-7) mol per site had no significant effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition by nitric oxide and nitric oxide-producing vasodilators of DNA synthesis in vascular smooth muscle cells

Effects of nitric oxide (NO) and NO-producing vasodilators such as glyceryl trinitrate and sodium nitroprusside were tested on DNA synthesis in the clonal rat aortic smooth muscle cells, RACS-1. DNA synthesis was estimated by [3H]thymidine incorporation to DNA. NO and NO-producing vasodilators inhibited the DNA synthesis that was induced by 10% fetal calf serum. NO and NO-producing vasodilators also inhibited the basal level of DNA synthesis that occurred possibly as a result of autocrine mechanisms. NO-producing vasodilators also inhibited the fetal calf serum-induced proliferation of cells. Sodium nitroprusside inhibited the endothelin-mediated DNA synthesis. In another mesenchymal cell line, Chinese hamster fibroblast V79 cells, NO and NO-producing vasodilators failed to inhibit DNA synthesis, excluding the possibility of general cell toxicity. An exposure to NO and NO-producing vasodilators resulted in an increase of cyclic GMP (cGMP) content in the RACS-1 cells. A cGMP analog, 8-bromo-cGMP, inhibited DNA synthesis in the RACS-1 cells. These results suggest that EDRF/nitric oxide released from endothelium possibly contributes to inhibition of the DNA synthesis in vascular smooth muscle cells.     

Inhibition of nitric oxide formation reduces voluntary ethanol consumption in the rat

Brain nitric oxide is involved in the mechanisms that regulate ingestive behaviour. To test whether this compound plays a role in alcohol preference, we studied the effects of different doses ofN ^G-nitro-L-arginine (L-NO arg), an inhibitor of nitric oxide synthase (NOS), on voluntary consumption of ethanol and on blood alcohol levels produced by a single intraperitoneal dose of alcohol in the rat. L-NO arg produced a significant and dose-dependent reduction of ethanol intake (P<0.001) without influencing total fluid consumption or feeding behaviour. L-NO arg did not influence the kinetics of alcohol. Our data show that inhibition of nitric oxide formation accompanies reduction of ethanol intake and suggest a possible role for nitric oxide in ethanol self-administration.     

Inhibition of inducible nitric oxide synthase gene expression and nitric oxide synthesis in vascular smooth muscle cells by granulocyte-colony stimulating factor in vitro.

Clinical and experimental evidence suggests that granulocyte-colony stimulating factor (G-CSF) acts as an anti-inflammatory modulator with beneficial effects in severe inflammatory diseases, e.g., sepsis and septic shock. Excessive production of nitric oxide (NO) is regarded as a potent mediator of the vascular changes leading to systemic hypotension that occurs during sepsis. Therefore, the aim of the present study was to investigate the influence of G-CSF on inducible nitric oxide synthase (iNOS) gene expression and NO synthesis in vascular smooth muscle cells (VSMC). Qualitative and quantitative analyses of iNOS cDNA revealed that G-CSF significantly reduced interferon-gamma/lipopolysaccharide (IFN-gamma/LPS) dependent iNOS gene expression (P < 0.05) following 6, 18, 24, and 48 h incubation periods. In addition, the co-application of G-CSF resulted in a decreased IFN-gamma/LPS mediated iNOS protein generation as detected by immunoblotting methods after 24 and 48 h. Measurement of the stable NO metabolites showed a significant reduction of nitrite/nitrate concentrations following co-incubation of VSMC with G-CSF + IFN-gamma/LPS (242.57 +/- 10.73 nmol NO2-/NO3-/mg cell protein, n = 8) as compared to IFN-gamma/LPS treatment (306.20 +/- 19.26 nmol NO2-/NO3-/mg cell protein, n = 8, P < 0.05) following a 24-h incubation protocol. This inhibitory effect of G-CSF was still present after a 48 h incubation period (G-CSF + IFN-gamma/LPS: 319.56 +/- 6.26 nmol NO2-/NO3-/mg cell protein; IFN-gamma/LPS: 489.20 +/- 27.15 nmol NO2-/NO3-/mg cell protein (P < 0.05), n = 8, respectively). The present findings suggest that inhibition of iNOS gene expression and NO generation in VSMC might be one of the protective anti-inflammatory effects of G-CSF during sepsis.     

Suppression of nitric oxide synthesis by L-NAME reverses the beneficial effects of pioglitazone on scopolamine-induced memory impairment in mice

Pioglitazone, an agonist of peroxisome proliferator-activated receptor gamma (PPARγ), which is widely used in treatment of type 2 diabetes, has shown some therapeutic effect in Alzheimer's disease. In this study, effects of acute pioglitazone on acquisition, consolidation and retrieval of memory, and also the involvement of nitric oxide (NO) in the effects of pioglitazone on spatial recognition memory has been investigated in a two-trial recognition Y-maze test and passive avoidance in mice. Memory impairment was induced by scopolamine (1 mg/kg, i.p.). Pioglitazone (10 and 20 mg/kg, p.o.) was administrated prior to either acquisition, consolidation or retention trials, while L-NAME (N-nitro-l-arginine methyl ester), a non-specific NO synthase inhibitor, was administered (10 mg/kg, i.p.) 30 min before each trial. Results: 1) pioglitazone improved the acquisition of recognition spatial memory-impaired by scopolamine; L-NAME dramatically reversed improving effects of pioglitazone on memory acquisition; 2) pioglitazone did not change the consolidation of spatial memory, impaired by scopolamine; 3) pioglitazone improved the retrieval of spatial memory and L-NAME did not alter the beneficial effect of pioglitazone; 4) pioglitazone did not affect scopolamine-induced cognitive impairments in the passive avoidance test.The present study demonstrates the beneficial effect of acute pioglitazone administration on acquisition and retrieval of scopolamine-induced cognitive deficits. This effect was reversed only in acquisition phase by nitric oxide synthase inhibitor, L-NAME, therefore, it could be concluded that NO might be involved in the pioglitazone beneficial effect of spatial memory acquisition.     

Inhibition by sodium nitroprusside of the expression of inducible nitric oxide synthase in rat neutrophils.

A well-known nitric oxide (NO)-releasing compound, sodium nitroprusside (SNP), decreases in a dose-dependent manner NO synthase (NOS) activity induced in rat neutrophils by treatment with lipopolysaccharide (LPS). This inhibitory action of SNP seems not to be due to its direct effect on the enzyme activity. The strong nitrosonium ion (NO+) character of SNP could be responsible for its inhibition of NOS induction in neutrophils.