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Nitric Oxide Synthase Activity (nitric + oxide_synthase_activity)
Selected AbstractsSplice-isoform specific immunolocalization of neuronal nitric oxide synthase in mouse and rat brain reveals that the PDZ-complex-building nNOS, ,-finger is largely exposed to antibodiesDEVELOPMENTAL NEUROBIOLOGY, Issue 4 2007Kristina Langnaese Abstract Knock out mice deficient for the splice-isoform ,, of neuronal nitric oxide synthase (nNOS,,) display residual nitric oxide synthase activity and immunosignal. To attribute this signal to the two minor neuronal nitric oxide synthase splice variants, ,, and ,,, we generated isoform-specific anti-peptide antibodies against the nNOS,, specific ,,-finger motif involved in PDZ domain scaffolding and the nNOS,, specific N-terminus. The nNOS,, ,,-finger-specific antibody clearly recognized the 160-kDa band of recombinant nNOS,, on Western blots. Using immunocytochemistry, this antibody displayed, in rats and wild-type mice, a labeling pattern similar to but not identical with that obtained using a commercial pan-nNOS antibody. This similarity indicates that the majority of immunocytochemically detectable nNOS is not likely to be complexed with PDZ-domain proteins via the ,,-finger motif. This conclusion was confirmed by the inhibition of PSD-95/nNOS interaction by the nNOS,, ,,-finger antibody in pull-down assays. By contrast, nNOS,, ,,-finger labeling was clearly reduced in hippocampal and cortical neuropil areas enriched in NMDA receptor complex containing spine synapses. In nNOS,, knock out mice, nNOS,, was not detectable, whereas the pan-nNOS antibody showed a distinct labeling of cell bodies throughout the brain, most likely reflecting ,,/,,-isoforms in these cells. The nNOS,, antibody clearly detected bacterial expressed nNOS,, fusion protein and nNOS,, in overexpressing HEK cells by Western blotting. Immunocytochemically, individual cell bodies in striatum, cerebral cortex, and in some brain stem nuclei were labeled in knock out but not in wild-type mice, indicating an upregulation of nNOS,, in nNOS,, deficient animals. © 2007 Wiley Periodicals, Inc. Develop Neurobiol, 2007 [source] The effect of modulation of , -glutamyl transpeptidase and nitric oxide synthase activity on GSH homeostasis in HepG2 cellsFUNDAMENTAL & CLINICAL PHARMACOLOGY, Issue 1 2007Inga Kwiecie Abstract High glutathione (GSH) level and elevated , -glutamyl transpeptidase (,GT) activity are hallmarks of tumor cells. Toxicity of drugs and radiation to the cells is largely dependent on the level of thiols. In the present studies, we attempted to inhibit ,GT activity in human hepatoblastoma (HepG2) cells to examine whether the administration of ,GT inhibitors, acivicin (AC) and 1,2,3,4-tetrahydroisoquinoline (TIQ) influences cell proliferation and enhances cytostatic action of doxorubicin (DOX) and cisplatin (CP) on HepG2 cells. The effects of these inhibitors were determined by 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT), BrdU and lactate dehydrogenase (LDH) tests and by estimation of GSH level. Additionally, we investigated the changes in caspase-3 activity, which is a marker of apoptosis. The obtained results showed that the ,GT inhibitors introduced to the medium alone elicited cytotoxic effect, which was accompanied by an increase in GSH level in the cells. TIQ concomitantly increased caspase-3 activity. Doxorubicin and CP proved to be cytotoxic, and both inhibitors augmented this effect. As well DOX as CP radically decreased GSH levels, whereas ,GT inhibitors had diverse effects. Therefore, the obtained results confirm that ,GT inhibitors can enhance pharmacological action of DOX and CP, which may permit clinicians to decrease their doses thereby alleviating side effects. Aminoguanidine (nitric oxide synthase inhibitor) given alone was little cytotoxic to HepG2 cells, while its introduction to the medium together with DOX and CP significantly increased their cytotoxicity. Aminoguanidine on its own did not show any effect on GSH level in HepG2 cells, but markedly and significantly elevated its concentration when added in combination with CP but not with DOX. This indicates that when CP was used as a cytostatic, GSH level rose after treatment with its combination with both AC and aminoguanidine. [source] Increasing dimethylarginine levels are associated with adverse clinical outcome in severe alcoholic hepatitis,HEPATOLOGY, Issue 1 2007Rajeshwar P. Mookerjee Previous studies suggest reduced hepatic endothelial nitric oxide synthase activity contributes to increased intrahepatic resistance. Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor, undergoes hepatic metabolism via dimethylarginine-dimethylamino-hydrolase, and is derived by the action of protein-arginine-methyltransferases. Our study assessed whether ADMA, and its stereo-isomer symmetric dimethylarginine (SDMA), are increased in alcoholic hepatitis patients, and determined any relationship with severity of portal hypertension (hepatic venous pressure gradient measurement) and outcome. Fifty-two patients with decompensated alcoholic cirrhosis were studied, 27 with acute alcoholic hepatitis and cirrhosis, in whom hepatic venous pressure gradient was higher (P = 0.001) than cirrhosis alone, and correlated with ADMA measurement. Plasma ADMA and SDMA were significantly higher in alcoholic hepatitis patients and in nonsurvivors. Dimethylarginine-dimethylamino-hydrolase protein expression was reduced and protein-arginine-methyltransferase-1 increased in alcoholic hepatitis livers. ADMA, SDMA and their combined sum, which we termed a dimethylarginine score, were better predictors of outcome compared with Pugh score, MELD and Maddrey's discriminant-function. Conclusion: Alcoholic hepatitis patients have higher portal pressures associated with increased ADMA, which may result from both decreased breakdown (decreased hepatic dimethylarginine-dimethylamino-hydrolase) and/or increased production. Elevated dimethylarginines may serve as important biological markers of deleterious outcome in alcoholic hepatitis. (HEPATOLOGY 2007;45:62,71.) [source] Effects of some synthetic kynurenines on brain amino acids and nitric oxide after pentylenetetrazole administration to ratsJOURNAL OF PINEAL RESEARCH, Issue 4 2004Leila Bikjdaouene Abstract:, We have previously proven that some synthetic kynurenines behave as antagonists of the N-methyl- d -aspartate receptor inhibiting neuronal subtype of nitric oxide synthase activity. We now investigate the anticonvulsant activity of four of these kynurenines in pentylenetetrazole (PTZ)-treated rats. The rats were treated with each kynurenine (10,160 mg/kg, s.c.) 30 min before PTZ administration (100 mg/kg, s.c.). Then, latency, duration and intensity of the first seizure and the percent animal survival were noted. PTZ-induced death was counteracted by high doses of kynurenines. Latency of the first seizure was significantly increased and its intensity reduced at the same doses, whereas the duration of the first seizure significantly decreased with doses of 20 mg/kg in most of the kynurenines tested. Three hours after PTZ administration, the surviving animals were sacrificed and the levels of brain amino acids and nitrite were measured. PTZ administration increased glutamate, glutamine, serine and taurine levels in different brain areas. High doses of kynurenines generally counteracted the effects of PTZ on excitatory amino acids, but they also reduced inhibitory aminoacids. However, the most consistent effect of kynurenines was the dose-dependent reduction of brain nitrite levels induced by PTZ. These results reveal a new family of anticonvulsant drugs that affect mainly to nitric oxide production in the brain. [source] Ethanol Consumption Increases Nitric Oxide Production in Rats, and Its Peroxynitrite-Mediated Toxicity Is Attenuated by PolyenylphosphatidylcholineALCOHOLISM, Issue 6 2002Enrique Baraona Background: Nitric oxide generally mediates beneficial responses but becomes deleterious when coexistence with enhanced superoxide formation leads to the synthesis of peroxynitrite, a potent oxidant and nitrating agent. Methods: To study the effects of ethanol and polyenylphosphatidylcholine on nitric oxide metabolism and toxicity, 36 rats were pair-fed liquid diets with 36% of energy either as ethanol or as additional carbohydrate for 24 days and were killed 90 min after intragastric feeding. Half received polyenylphosphatidylcholine in the diet (3 g/liter), and the other half equivalent amounts of essential fatty acids and choline. Nitric oxide was measured by chemiluminescence in arterial blood and liver cytosol and as a product of the inducible nitric oxide synthase activity. Peroxynitrite formation was assessed by the increase in nitrotyrosine protein residues, measured immunochemically. Results: In blood, administration of ethanol with or without polyenylphosphatidylcholine doubled nitric oxide levels. In the liver, ethanol increased nitric oxide by 52% (p < 0.01), and polyenylphosphatidylcholine attenuated this effect. Ethanol consumption increased the cytosolic activity of the inducible nitric oxide synthase and induced microsomal cytochromes P-450 capable of producing both nitric oxide and superoxide. This was associated with an 18% (p < 0.01) increase in nitrotyrosine protein residues, products of peroxynitrite toxicity, which occurred predominantly in steatotic hepatocytes. Polyenylphosphatidylcholine attenuated these changes by decreasing the ethanol effect on both the cytosolic and the microsomal activities, in addition to acting as a powerful antioxidant. Acute administration of the same ethanol dose increased nitric oxide levels, but did not affect nitrotyrosine protein residues. Conclusions: Chronic, but not acute, ethanol administration increases peroxynitrite hepatotoxicity by enhancing concomitant production of nitric oxide and superoxide, both of which are prevented by polyenylphosphatidylcholine. [source] Naphthoquinones and bioactive compounds from tobacco as modulators of neuronal nitric oxide synthase activityPHYTOTHERAPY RESEARCH, Issue 12 2009Priya Venkatakrishnan Abstract Studies were conducted with extracts of several varieties of tobacco in search of neuronal nitric oxide synthase (nNOS) inhibitors which may be of value in the treatment of stroke. Current therapies do not directly exploit modulation of nNOS activity due to poor selectivity of the currently available nNOS inhibitors. The properties of a potentially novel nNOS inhibitor(s) derived from tobacco extracts, and the concentration-dependent, modulatory effects of the tobacco-derived naphthoquinone compound, 2,3,6-trimethyl-1,4-naphthoquinone (TMN), on nNOS activity were investigated, using 2-methyl-1,4-naphthoquinone (menadione) as a control. Up to 31 µM, both TMN and menadione stimulated nNOS-catalysed l -citrulline production. However, at higher concentrations of TMN (62.5,500 µM), the stimulation was lost in a concentration-dependent manner. With TMN, the loss of stimulation did not decrease beyond the control activity. With menadione (62.5,500 µM), the loss of stimulation surpassed that of the control (78 ± 0.01% of control activity), indicating a true inhibition of nNOS activity. This study suggests that potential nNOS inhibitors are present in tobacco, most of which remain to be identified. Copyright © 2009 John Wiley & Sons, Ltd. [source] Panax notoginseng saponins attenuate acute lung injury induced by intestinal ischaemia/reperfusion in ratsRESPIROLOGY, Issue 6 2009Ling RONG ABSTRACT Background and objective: Acute lung injury remains a challenge for both clinicians and scientists. The effects of Panax notoginseng saponins (PNS) on acute lung injury induced by intestinal ischaemia/reperfusion (II/R) were studied in rats. Methods: Forty-eight Wistar rats were randomly assigned to four groups: (1) a sham-operated group that received laparotomy without II/R (n= 12); (2) a sham + PNS group, which was identical to group 1 except for PNS treatment (n= 12); (3) an II/R group that had 1 h of intestinal ischaemia followed by 3 h of reperfusion (n= 12); and (4) an II/R + PNS group that received 100 mg/kg of PNS, i.v., 15 min before reperfusion (n= 12). The effects of PNS administration on lung tissue histology, activities of oxidant and antioxidant enzymes, levels of malondialdehyde, nitric oxide and inducible nitric oxide synthase activity were examined. Levels of surfactant protein B, cell numbers in BAL fluid and plasma levels of pro-inflammatory cytokines were also examined. Results: Compared with the II/R group, pulmonary parenchymal damage, activities of oxidant enzymes, levels of malondialdehyde and nitric oxide, inducible nitric oxide synthase activity in lung tissue, and plasma levels of pro-inflammatory cytokines were significantly reduced by PNS treatment. In addition, the decreases in antioxidant enzyme activities were prevented in the II/R + PNS group. Total leukocyte and neutrophil counts were significantly decreased by PNS treatment. The decline in surfactant protein B levels in BAL fluid was reduced in the II/R + PNS group compared with the II/R group. Conclusions: Administration of PNS before reperfusion injury alleviates acute lung injury induced by II/R, and this is attributable to the antioxidant and anti-inflammatory effects of PNS. [source] Neuronal nitric oxide synthase activity in rat urinary bladder detrusor: participation in M3 and M4 muscarinic receptor functionAUTONOMIC & AUTACOID PHARMACOLOGY, Issue 3 2005B. Orman Summary 1,The aim of this paper was to determine the different signalling cascades involved in contraction of the rat urinary bladder detrusor muscle mediated via muscarinic acetylcholine receptors (muscarinic AChR). Contractile responses, phosphoinositides (IPs) accumulation, nitric oxide synthase (NOS) activity and cyclic GMP (cGMP) production were measured to determine the reactions associated with the effect of cholinergic agonist carbachol. The specific muscarinic AChR subtype antagonists and different inhibitors of the enzymatic pathways involved in muscarinic receptor-dependent activation of NOS and cGMP were tested. 2,Carbachol stimulation of M3 and M4 muscarinic AChR increased contractility, IPs accumulation, NOS activity and cGMP production. All of these effects were selectively blunted by 4-DAMP and tropicamide, M3 and M4 antagonists respectively. 3,The inhibitors of phospholipase C (PLC), calcium/calmodulin (CaM), neuronal NOS (nNOS) and soluble guanylate cyclase, but not of protein kinase C and endothelial NOS (eNOS), inhibited the carbachol action on detrusor contractility. These inhibitors also attenuated the muscarinic receptor-dependent increase in cGMP and activation of NOS. 4,In addition, sodium nitroprusside and 8-bromo-cGMP, induced negative relaxant effect. 5,The results obtained suggest that carbachol activation of M3 and M4 muscarinic AChRs, exerts a contractile effect on rat detrusor that is accompanied by an increased production of cGMP and nNOS activity. The mechanism appears to occur secondarily to stimulation of IPs turnover via PLC activation. This in turn, triggers cascade reactions involving CaM, leading to activation of nNOS and soluble guanylate cyclase. They, in turn, exert a modulator inhibitory cGMP-mediated mechanism limiting the effect of muscarinic AChR stimulation of the bladder. [source] Neuroprotective effect of asymmetric dimethylarginine against 1-methyl-4-phenylpyridinium ion-induced damage in PC12 cellsCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 5-6 2010Xiao-Qing Tang Summary 1. Asymmetric dimethylarginine (ADMA) is a well-known endogenous nitric oxide synthase (NOS) inhibitor. Although it has been shown to be a novel risk marker in cardiovascular medicine and chronic kidney disease, we speculated that in some states associated with excess of nitric oxide (NO), such as 1-methyl-4-phenylpyridinium ion (MPP+)-induced neuronal injury, ADMA might be protective by limiting the toxic effect of high concentrations of NO. 2. The aim of the present study is to explore the protection of ADMA against MPP+ -induced apoptosis and the molecular mechanisms underlying in PC12 cells. 3. We found that exogenous application of ADMA obviously protected PC12 cells against MPP+ -induced cytotoxicity and apoptosis not only by reducing the loss of mitochondrial membrane potential, but also by attenuating an increase in intracellular reactive oxygen species. Moreover, ADMA attenuated MPP+ -induced excessive activation of nitric oxide synthase and overproduction of NO. 4. The results of the present study suggest that the protection caused by ADMA is related to preserving mitochondrial membrane potential and attenuating the MPP+ -induced intracellular reactive oxygen species generation through inhibiting nitric oxide synthase activity and limiting NO generation. [source] pH and nitric oxide synthase activity and expression in bovine aortic endothelial cellsACTA PAEDIATRICA, Issue 7 2006Sandor Nagy Abstract Aim: Nitric oxide (NO) plays an important role in the transition from intrauterine to extrauterine life. If this transition fails, a condition called persistent pulmonary hypertension of the neonate (PPHN) may develop. The current treatment modalities for this disease include induction of alkalosis by hyperventilation or alkali infusion, inhaled nitric oxide (iNO) and extracorporeal membrane oxygenation. There is evidence from animal studies that the elevated pH, not the low pCO2 is responsible for the resultant pulmonary vasodilatation. In this study, we examined the effect of pH on the activity and expression of endothelial nitric oxide synthase (eNOS) in cultured bovine aortic endothelial cells (BAEC) as a possible explanation for the pH dependent drop in pulmonary vascular resistance. Methods: BAEC were exposed to a pH gradient of 7.1,7.6 for 4 h (short-term) and 16 h (long-term). Standard Western blotting technique was used to detect expression of eNOS. Activity was measured by an indirect assay using bovine aortic smooth muscle cells (BASM) as reporter cells and measuring cGMP levels as a marker of NO production. The cells were exposed to the pH gradient for a total of 4 h and measurement were made at 30, 60 and 90 min, and 2, 3 and 4 hours. Results: eNOS activity and expression remained unchanged during the four and sixteen hours of exposure. Conclusion: In this in vitro experiment, we could not demonstrate an alkalosis-induced increase in eNOS activity and expression. The clinically observed pH dependent vasodilatation does not appear to be directly mediated through the induction of eNOS. [source] | |||||||||||||