Inhibition Of Nitric Oxide Synthase Activity Research Articles

Increased sensitivity of Fancc-deficient hematopoietic cells to nitric oxide and evidence that this species mediates growth inhibition by cytokines

AbstractFanconi anemia complementation group C (Fancc)–deficient murine bone marrow progenitors demonstrate increased sensitivity to growth inhibition by interferon γ (IFNγ), tumor necrosis factor α (TNFα), and macrophage inflammatory protein 1α (MIP-1α). This property has been proposed as a possible pathogenic factor in the marrow failure seen in Fanconi anemia. Supporting our hypothesis that nitric oxide (NO) production might be a common effector in this sensitivity, we found that cytokine-mediated growth inhibition ofFancc−/− bone marrow cells was prevented by inhibiting NO synthase activity. Interestingly,Fancc−/− hematopoietic cells also exhibited increased growth inhibition on exposure to 2 distinct NO-generating agents, S-nitroso-N-acetyl-D, L-penicillamine (SNAP) and diethylenetriamine nitric oxide adduct (DETA/NO). In keeping with the sensitivity of Fancc−/− cells to IFNγ, inducible nitric oxide synthase (iNOS) levels and nitrite release were both increased following stimulation ofFancc−/− macrophages with this cytokine, either alone or in combination with bacterial lipopolysaccharide. Suggesting a plausible mechanism for the increased expression of iNOS, IFNγ-stimulated Fancc−/− macrophages generated higher levels of phospho-Stat1, a positive regulator ofinos (nos2) gene expression. These observations, while confined to C57BL/6 Fancc−/−hematopoietic cells, raise the possibility that nitric oxide has a role in the pathogenesis of Fanconi anemia.

Neuronal nitric oxide synthase modulates basal catecholamine secretion in bovine chromaffin cells.

The role of endogenously produced nitric oxide (NO) in the regulation of basal catecholamine (CA) secretion was studied in chromaffin cells. Treatment of chromaffin cells with nitric oxide synthase (NOS) inhibitors produced a dose-dependent increase in basal catecholamine secretion, which paralleled their ability to inhibit NOS activity. This inhibitory profile was similar to that found in neurons, suggesting the constitutive expression of neuronal NOS (nNOS) in these cells, which was confirmed by Western blot analysis. A study of the kinetics and pharmacology of nNOS activity expressed in chromaffin cells in culture indicated that NOS activity is calcium-dependent, increases with time, and is highly dependent on both intracellular concentrations of L-arginine (K(m) approximately 4 microM, V(max) = 908 +/- 60 pmol/hr x 10(6) cells) and transport of L-arginine into the cells (exhibiting two affinity constants of k(1) = 3.2 +/- 0.3 microM and k(2) = 126 +/- 5.5 microM). The effects of NOS inhibitors on CA secretion were mediated by the L-arginine-NO-cGMP pathway, insofar as exogenous L-arginine was able to partially block the increase in CA secretion evoked by them, and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ), a specific inhibitor of guanylate cyclase, and zaprinast, an inhibitor of the cGMP phosphodiesterase, were able to increase and inhibit, respectively, basal CA secretion in a dose-dependent manner. These results suggest that chromaffin cells exhibit a tonic production of NO by nNOS that keeps the basal CA secretion at low levels, and this could be necessary for maintaining a normotensive state.

Activation of Constitutive Nitric-oxide Synthase Activity Is an Early Signaling Event Induced by Ionizing Radiation

Ionizing radiation at clinical dose levels activates both pro- and anti-proliferative signal transduction pathways, the balance of which determines cell fate. The initiating and amplifying mechanisms involved in the activation are poorly understood. We demonstrate that one mechanism involves stimulation of constitutive nitric-oxide synthase (NOS) activity. NOS activity of Chinese hamster ovary cells was measured by the arginine --> citrulline conversion assay. Irradiation stimulated a transient activation of NOS with maximal activity at 5 min of post-irradiation. Western blot analysis and genetic manipulation by overexpression of wild type or dominant negative NOS mutant identify the radiation-induced isoform as NOS-1. Further evidence that NOS-1 is activated by radiation was the demonstration of radiation-induced cGMP formation in cells transiently transfected with the NO-dependent soluble guanylate cyclase. Protein Tyr nitration, a footprint of peroxynitrite formation, followed radiation exposure and was inhibited by expression of a dominant negative NOS-1 mutant. Radiation-induced ERK1/2 kinase activity, a cytoprotective response to radiation, was also blocked by inhibiting NOS activity. These experiments establish NO-dependent signal transduction pathways as being radio-responsive. Given the lipophilic and relatively stable properties of NO, these results also suggest a possible mechanism by which ionization events in one cell may activate signaling processes in adjacent cells.

The complex role of nitric oxide in the regulation of glomerular ultrafiltration

The complex role of nitric oxide in the regulation of glomerular ultrafiltration. Nitric oxide is an important neurohumoral modulator of glomerular ultrafiltration and renal hemodynamics. Multiple nitric oxide synthase (NOS) isoforms are present within the kidney. However, it is difficult to discern which NOS is most active from prior studies using non-selective NOS blockers. It is recently apparent that NOS activity is important to the activity of tubuloglomerular feedback (TGF) systems, systems that relate tubular reabsorption to the regulation of glomerular ultrafiltration. Neuronal (nNOS) or brain NOS (bNOS; NOS I) is present within the macula densa, the sensing element of TGF systems. Inhibition of NOS activity and specifically bNOS enhances TGF activity. The TGF system also adapts temporally and these events appear to be dependent upon up-regulation of activity of bNOS within the kidney. Temporal adaptation occurs within one to three hours whereby activation of TGF is followed by a gradual return of the glomerular filtration rate (GFR) and renal blood flow toward normal levels. After 24 hours of benzolamide treatment and withdrawal of this agent, glomerular filtration actually increases to supranormal levels and this is prevented by inhibition of bNOS activity. Factors regulating bNOS activity have not been fully clarified. We have recently observed N-methyl-d-aspartate (NMDA) receptors within the kidney that, when inhibited, result in major reductions in renal blood flow and GFR, suggesting an important role for the NMDA receptor in regulation of renal hemodynamics. Future studies will determine whether NMDA receptor is also an important regulator of bNOS activity and the TGF system.

Protective effects of noradrenaline against tumor necrosis factor-α-induced apoptosis in cultured rat brown adipocytes: role of nitric oxide-induced heat shock protein 70 expression

To elucidate the effects and molecular mechanism(s) by means of which noradrenaline (NA) protects against the tumor necrosis factor (TNF)-alpha-induced apoptosis of brown adipocytes. Brown fat precursor cells were isolated from young rats; 2.5 million cells were added to each 24-well culture plate and cultured in a defined culture medium. On day 8, the cultured cells were exposed to murine recombinant TNF-alpha and/or cycloheximide (CHX; 10 microg/ml) and/or NA and/or nitric oxide (NO) donors and/or the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) and/or 10 microM heat shock protein 70 (HSP70) antisense or sense oligomers. Analysis of DNA fragmentation on agarose gel as a marker of apoptosis; reverse transcriptase-polymerase chain reaction analysis of mRNA levels; Western blotting analysis of protein levels. Pretreatment of primary cultures of rat brown fat cells with micromolar concentrations of NA or the NO-donor S-nitroso-N-acetylpenicillamine (SNAP) induced the expression of HSP70 mRNA and protein, which was associated with cytoprotection against TNF-alpha plus CHX-induced apoptosis. The L-NAME inhibitor of NO synthase activity inhibited both NA-stimulated HSP70 expression and cytoprotection. Furthermore, pretreatment of brown adipocytes with an antisense oligonucleotide to HSP70 antagonized both SNAP- and NA-induced cytoprotection. These findings demonstrate that the NO produced by NA stimulation can induce resistance to the TNF-alpha-induced apoptosis of brown adipocytes, possibly by means of the expression of HSP70.

Regulation of inhibitor of apoptosis expression by nitric oxide and cytokines: relation to apoptosis induction in rat mesangial cells and raw 264.7 macrophages.

Mesangial cells and RAW 264.7 macrophages respond to different nitric oxide (NO) donors within 16 to 24 h or 6 to 8 h, respectively, with apoptotic cell death. RAW 264.7 macrophages also die in response to endogenous NO production. In contrast, endogenous NO production fails to significantly induce cell death in mesangial cells. It was hypothesized that differences in the expression of antiapoptotic proteins, in particular the inhibitor of apoptosis (IAP) protein family, might be responsible for this cell type-specific behavior. Therefore, IAP expression was investigated in relation to apoptosis induction in response to NO and cytokines in both cell types. In mesangial cells, interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha induced cellular inhibitor of apoptosis 1 (cIAP1) mRNA expression within 3 h. In contrast, X chromosome-linked inhibitor of apoptosis (XIAP) mRNA levels remained unaffected by cytokines. Although coincubation of cells with IL-1beta and tumor necrosis factor-alpha or IL-1beta and basic fibroblast growth factor resulted in synergistic induction of inducible NO synthase, comparable potentiating effects on cIAP1 induction were absent. Exogenously released NO from NO donors promoted cIAP1 mRNA upregulation in mesangial cells, whereas XIAP mRNA was downregulated. However, the changes observed on the mRNA level were not adequately translated to the protein level, and corresponding values for cIAP1 and XIAP were only slightly affected. In contrast, in lipopolysaccharide/interferon-gamma-stimulated RAW 264.7 macrophages, massive NO-dependent downregulation of cIAP1 and XIAP protein levels, which correlated temporally with the induction of apoptosis, was observed. This effect was at least partially reversed by N(G)-monomethyl-L-arginine, an inhibitor of NO synthase activity. In summary, a direct correlation between the downregulation of IAP protein levels and the induction of apoptosis by endogenous NO was observed in macrophages. In contrast, a stable level of IAP protein in mesangial cells might represent a mechanism for the resistance of the cells to endogenously produced NO.

Failure of L-Nitroarginine to Inhibit the Activity of Aortic Inducible Nitric Oxide Synthase

Nitric oxide (NO) is produced by a family of three isoenzymes: the endothelial, inducible and neuronal NO synthases. L-Nitroarginine methyl ester (L-NAME) is the most commonly used inhibitor of NO synthase activity. The goal of the present study was to evaluate to what extent L-nitroarginine (L-NA), the in vivo circulating metabolite of L-NAME, blocks NO production in the rat aorta depending on the NO synthase isoform expressed (and evidenced by Western blotting) and on the presence or absence of the extracellular NO synthase substrate L-arginine (100 µM, i.e. the plasma concentration). Intact [endothelium present (E+)] control aortic rings express mainly endothelial NO synthase. L-NA (30–100 µM) induced a dose-dependent contraction (due to blockade of the relaxant properties of NO) irrespective of the presence or absence of L-arginine. In deendothelialized (E–) control aortic rings, the three isoforms of NO synthase are virtually absent (as demonstrated by Western blotting) and L-NA does not elicit any contractile effect. E– aortic rings from lipopolysaccharide (LPS)-treated rats express mainly inducible NO synthase. In these rings, L-NA induced a dose-dependent (0–100 µM) contraction in the absence of extracellular L-arginine, whereas L-arginine (100 µM) completely abrogated the contractile effect of the NO synthase inhibitor. Chronic L-NAME administration (50 mg/kg/day for 4 weeks) elicited the aortic expression of inducible NO synthase, but to a lesser extent (about 5-fold) than in LPS-treated rat aorta. The average plasma concentration of L-NA was 50 ± 10 µM in these rats. In E– rings from these L-NAME-treated rats, L-NA induced a similar contractile response (but smaller in magnitude) to that observed in LPS-treated rat aorta. Altogether, these data suggest that (1) in the presence of a physiological concentration of extracellular L-arginine, L-NA fails to inhibit inducible NO synthase, and (2) chronic L-NAME administration, at a dose commonly given to block NO production in vivo, leaves the activity of inducible NO synthase unaffected.

Regulation of metamorphosis in ascidians involves NO/cGMP signaling and HSP90.

Treatment of larvae of the ascidians Boltenia villosa (Family: Pyuridae) and Cnemidocarpa finmarkiensis (Family: Styelidae) with drugs that inhibit the function of the molecular chaperone HSP90 increased the frequency of tail resorption, the primary morphogenetic event of metamorphosis. If treatment was initiated at hatching, metamorphic events subsequent to tail resorption failed to occur, indicating an ongoing role for HSP90 during morphogenesis. Removal of tails from heads of mature, but not newly hatched larvae, induced metamorphosis of the head. Decapitation experiments indicate that the capacity of tails to shorten in response to inhibition of HSP90 function requires communication with heads. To identify candidate proteins with which HSP90 may interact to regulate metamorphosis, we noted that in mammalian cells, nitric oxide synthase (NOS) interacts with HSP90 and its activity is sensitive to drugs that inhibit HSP90 function. In addition, nitric oxide (NO) signaling in the marine snail Ilyanassa obsoleta is an important regulator of metamorphosis. Inhibition of NOS activity in these ascidian larvae with L-NAME increased the frequency of metamorphosis, consistent with a putative interaction of NOS and HSP90. NOS is present in tail muscle cells, implicating them as targets for the drug treatments, consistent with the decapitation experiments. Inhibition of soluble guanylyl cyclase, the most common effector of NO signaling, also increased the frequency of metamorphosis. In contrast to treatment with anti-HSP90 drugs, metamorphosis induced with L-NAME or ODQ was complete. The results presented suggest that an HSP90-dependent, NO-based regulatory mechanism localized in tails represses ascidian metamorphosis. We discuss these results in relation to the induction of ascidian metamorphosis by several unrelated agents.

Ceramide reduces endothelium-dependent vasodilation by increasing superoxide production in small bovine coronary arteries.

Ceramide serves as a second messenger in a variety of mammalian cells. Little is known regarding the role of ceramide in the regulation of vascular endothelial function. The present study was designed to determine whether ceramide affects endothelium-dependent vasodilation in coronary arteries and to explore the mechanism of action of ceramide. In isolated and pressurized small bovine coronary arteries, cell-permeable C(2)-ceramide (10(-)(5) mol/L) markedly attenuated vasodilator responses to bradykinin and A23187 (by 40% and 60%, respectively). In the presence of K(G)-nitro-L-arginine methyl ester, ceramide produced no further inhibition on the vasodilation induced by these vasodilators. Ceramide had no effect on DETA NONOate-induced vasodilation. By use of a fluorescence NO indicator (4,5-diaminofluorescein diacetate), intracellular NO was measured in the endothelium of freshly isolated small coronary arteries. It was found that ceramide significantly inhibited bradykinin-induced NO increase within endothelial cells. However, it had no effect on the activity of arterial or endothelial NO synthase. Pretreatment of the arteries with sodium dihydroxybenzene disulfonate (Tiron, 10(-)(3) mol/L), a cell-permeable superoxide scavenger, or polyethylene glycol superoxide dismutase (100 U/mL) largely restored the inhibitory effects of ceramide on the vasodilation and NO increase induced by bradykinin or A23187. Moreover, ceramide time-dependently increased intracellular superoxide (O(2)(-. )) in the endothelium, as measured by a fluorescent O(2)(-. )indicator, dihydroethidium. These results demonstrate that ceramide inhibits endothelium-dependent vasodilation in small coronary arteries by decreasing NO in vascular endothelial cells and that this decrease in NO is associated with increased O(2)(-. ) but not with the inhibition of NO synthase activity within these cells.

The Contribution of Nitric Oxide to Vestibular Compensation: Are There Species Differences?

Nitric oxide (NO) has been implicated in the processes by which animals recover from peripheral vestibular damage (''vestibular compensation''). However, there is little systematic data available on the effects of NO inhibition on the vestibular compensation process. In the present study we administered the nitric oxide synthase (NOS) inhibitor N G -nitro-L-arginine methyl ester (L-NAME) using a subcutaneous osmotic minipump and examined its effects on the compensation of spontaneous nystagmus (SN), yaw head tilt (YHT) and roll head tilt (RHT) in guinea pigs. Following unilateral labyrinthectomy (UL), treatment with 5, 10, 50 or 100 mM L-NAME had no effect on the expression of any of these symptoms or their rate of compensation. By contrast, pre-UL treatment with 100 mM L-NAME resulted in a decrease in SN frequency at 10 h post-UL and an increase in its rate of compensation. Lower concentrations had no effect on SN. Pre-UL treatment with L-NAME had no significant effect on YHT or RHT at any particular time point. Analysis of NOS activity demonstrated that the highest concentration of L-NAME inhibited NOS activity in the contralateral vestibular nucleus complex, bilateral cerebellum and bilateral cortices. These results suggest that L-NAME may have different effects on vestibular compensation in guinea pigs compared to other species, such as the rat and frog.

Retrograde signalling with nitric oxide at neocortical synapses.

Long-term changes of synaptic transmission in slices of rat visual cortex were induced by intracellular tetanization: bursts of short depolarizing pulses applied through the intracellular electrode without concomitant presynaptic stimulation. Long-term synaptic changes after this purely postsynaptic induction were associated with alterations of release indices, thus providing a case for retrograde signalling at neocortical synapses. Both long-term potentiation and long-term depression were accompanied by presynaptic changes, indicating that retrograde signalling can achieve both up- and down-regulation of transmitter release. The direction and the magnitude of the amplitude changes induced by a prolonged intracellular tetanization depended on the initial properties of the input. The inputs with initially high paired-pulse facilitation (PPF) ratio, indicative of low release probability, were most often potentiated. The inputs with initially low PPF ratio, indicative of high release probability, were usually depressed or did not change. Thus, prolonged postsynaptic activity can lead to normalization of the weights of nonactivated synapses. The dependence of polarity of synaptic modifications on initial PPF disappeared when plastic changes were induced with a shorter intracellular tetanization, or when the NO signalling pathway was interrupted by inhibition of NO synthase activity or by application of NO scavengers. This indicates that the NO-dependent retrograde signalling system has a relatively high activation threshold. Long-term synaptic modifications, induced by a weak postsynaptic challenge or under blockade of NO signalling, were nevertheless associated with presynaptic changes. This suggests the existence of another retrograde signalling system, additional to the high threshold, NO-dependent system. Therefore, our data provide a clear case for retrograde signalling at neocortical synapses and indicate that multiple retrograde signalling systems, part of which are NO-dependent, are involved.

L-arginine prevents bone loss and bone collagen breakdown in cyclosporin A-treated rats.

Cyclosporin A is implicated in the pathogenesis of post-transplantation bone disease. Because of recent evidence that cyclosporin A may cause renal and cardiovascular toxicity by inhibiting nitric oxide (NO) activity, and that NO slows bone remodeling and bone loss in animal and human studies, we investigated a possible link between NO production and beneficial effects on bone health in cyclosporin A-treated rats. Thirty-six 10-week-old male rats were assigned to six groups of six animals each, and treated for 4 weeks with: vehicle; cyclosporin A; l-arginine; N G-nitro- l-arginine methylester ( l-NAME, a general inhibitor of NO synthase activity); a combination of cyclosporin A+ l-arginine; and a combination of cyclosporin A+ l-NAME. Whole body and regional (spine and pelvis) bone mineral content of rats were measured under basal conditions and at the end of the treatment period by dual-energy X-ray absorptiometry (DXA) scanning. Femur weights and serum concentrations of pyridinoline, a reliable marker of bone resorption, were measured at the end of the study period. Cyclosporin A-, l-NAME-, and cyclosporin A+ l-NAME-treated rats had significantly lower bone mineral content and femur weights, and significantly higher pyridinoline levels than did control animals. The administration of l-arginine appeared to prevent bone loss caused by cyclosporin A, suggesting that this amino acid, which can be converted to produce NO, might prove useful in preventing disturbed bone modeling and inhibition of bone growth associated with cyclosporin A therapy.

The effects of l-NAME on vestibular compensation and NOS activity in the vestibular nucleus, cerebellum and cortex of the guinea pig

Nitric oxide (NO) has been implicated in the processes by which animals recover from peripheral vestibular damage (‘vestibular compensation’). However, few data exist on the dose–response effects of systemic administration of the nitric oxide synthase (NOS) inhibitor, N G-nitro- l-arginine methyl ester ( l-NAME), on the vestibular compensation process. The aim of this study was to investigate the effects on compensation of 5, 10, 50 or 100 mM l-NAME administered by s.c osmotic minipump for 50 h following unilateral vestibular deafferentation (UVD) in guinea pig, either commencing the drug treatment at 4 h pre-UVD or at the time of the UVD (i.e., post-UVD). Post-UVD treatment with l-NAME, at any of the four concentrations used, had no effect on the compensation of spontaneous nystagmus (SN), yaw head tilt (YHT) or roll head tilt (RHT). By contrast, pre-UVD treatment with 100 mM l-NAME resulted in a significant decrease in SN frequency ( P<0.05) and a change in the rate of its compensation ( P<0.0005). Pre-UVD l-NAME resulted in a significant increase in the overall magnitude of YHT ( P<0.005); however, post-hoc comparisons revealed no significant differences between any specific l-NAME and vehicle groups. Pre-UVD l-NAME had no effect on RHT at any concentration. Analysis of NOS activity in the pre-UVD l-NAME treatment groups at 50 h post-UVD showed that only 100 mM l-NAME resulted in a significant decrease in NOS activity in the contralateral medial vestibular nucleus (MVN)/prepositus hypoglossi (PH) ( P<0.05) and that NOS activity in the ipsilateral MVN/PH was not significantly affected. However, NOS activity was significantly inhibited in the bilateral cerebellum and cortices for several concentrations of l-NAME. These results suggest that pre-UVD systemic administration of l-NAME can significantly increase the rate of SN compensation in guinea pig and that this effect is correlated with inhibition of NOS activity in several regions of the CNS.

Nitric oxide protects nitric oxide synthase function from hydroxyl radical-induced inhibition

The interdependent relationships among nitric oxide synthase (NOS), its coenzyme, cofactors and nitric oxide (NO ) were studied using electron paramagnetic resonance spectroscopy. It was found that superoxide-dependent hydroxyl free radical (OH ), derived from NOS coenzyme and cofactors, inhibits NOS activity, and that endogenous NO generated by NOS scavenges OH and protects NOS function. These results reveal a new role for NO that may be important in NOS function and cellular free radical homeostasis.

Alterations of nitric oxide synthase expression and activity during rat lung transplantation.

Decreased nitric oxide (NO) production has been reported during lung transplantation in patients. To study the effects of ischemia and reperfusion on endogenous NO synthase (NOS) expression, both an ex vivo and an in vivo lung injury model for transplantation were used. Donor rat lungs were flushed with cold low-potassium dextran solution and subjected to either cold (4 degrees C for 12 h) or warm (21 degrees C for 4 h) ischemic preservation followed by reperfusion with an ex vivo model. A significant increase in inducible NOS and a decrease in endothelial NOS mRNA was found after reperfusion. These results were confirmed in a rat single-lung transplant model after warm preservation. Interestingly, protein contents of both inducible NOS and endothelial NOS increased in the transplanted lung after 2 h of reperfusion. However, the total activity of NOS in the transplanted lungs remained at very low levels. We conclude that ischemic lung preservation and reperfusion result in altered NOS gene and protein expression with inhibited NOS activity, which may contribute to the injury of lung transplants.

Transient changes in the synthesis of nitric oxide result in long-term as well as short-term changes in acetic acid-induced writhing in mice

A single injection of nitric oxide (NO) synthase (NOS) inhibitors prevents the development of persistent hyperalgesia induced by various manipulations, suggesting that NO precipitates long-term changes in nociception. We examined the possibility that inhibition of NOS may also be sufficient to produce long-term decreases in nociceptive assays, such as writhing, that are known to be sensitive to the short-term effects of NOS inhibitors. We characterized short- and long-term effects of NOS inhibitors, N ω-nitro- l-arginine ( l-NAME) or 7-nitro indazole (7-NI) injected intrathecally (i.t.) in mice on acetic acid-induced writhing. Doses of l-NAME that had no effect on hot plate or tail flick latencies inhibited writhing (0.01–30 nmol) as well as spinal nNOS activity (5 and 100 nmol) when injected i.t. 60–90 min before testing. Anti-nociception was not mimicked by d-NAME but was prevented by co-administration with the NO precursor, l-arginine. Injection i.t. of 7-NI (30 min), a selective inhibitor of neuronal NOS (nNOS), inhibited NOS activity in the spinal cord and produced anti-nociception, confirming that writhing is sensitive to inhibition of nNOS. Although the acute action of both NOS inhibitors dissipated completely by 3–6 h, a delayed and prolonged inhibition of writhing was again observed 24 h after l-NAME (5–100 nmol), a time when spinal NOS activity was no longer inhibited by l-NAME (5 and 100 nmol) or 7-NI (25 nmol). This novel effect appears to be initiated by the transient inhibition of nNOS as delayed anti-nociception was mimicked by 7-NI at doses (10–100 nmol) that no longer inhibited spinal nNOS (25 nmol) at 24 h. Co-administration with l-arginine prevented the delayed (24 h) anti-nociceptive effects of l-NAME (30 nmol). l-Arginine (30 and 100 nmol) was without effect on nociception when administered alone 60 min or 24 h prior to testing. Together these data indicate that brief changes in the activity of nNOS induce both long- as well as short-term changes in nociception.

Antioxidant levels in the rat brain after nitric oxide synthase inhibition: a preliminary report

Protective effects of NOS inhibitors and free radical scavengers in cerebral ischemia are well documented. The present study was undertaken to determine the possible effects of NOS inhibition on brain antioxidants. Levels of both enzymatic [glutathione peroxidase (GPx), catalase and superoxide dismutase (SOD)] and non-enzymatic [reduced glutathione (GSH)] antioxidants following nitric oxide synthase (NOS) inhibition by NG-nitro-L-arginine methyl ester (L-NAME), D-NAME or 7-nitro-indazole (7-NI) have been investigated. NOS activity and antioxidant levels in the rat cerebellum and medulla were estimated 1 h after treatment with L-NAME (10, 30 and 100 mg/kg, i.p.), D-NAME (100 mg/kg, i.p.) or 7-NI (25 mg/kg, i.p.). L-NAME and 7-NI inhibited NOS activity in a dose-dependent manner. D-NAME also exhibited significant NOS inhibition. The activity of SOD and the GSH level remained unaltered following NOS inhibition. However, L-NAME and D-NAME at 100 mg/kg attenuated GPx activity in the cerebellum, though 7-NI had no effect. L-NAME inhibited catalase activity in medulla only at 30 mg/kg, but had no effect in cerebellum. However, 7-NI (25 mg/kg), D-NAME and L-NAME at 100 mg/kg did not affect catalase activity in the rat brain. Thus, NOS inhibition by the three agents did not have major effects on brain antioxidant levels.

Differential activation of nitric oxide synthase through muscarinic acetylcholine receptors in rat salivary glands

Muscarinic receptors play an important role in secretory and vasodilator responses in rat salivary glands. Nitric oxide synthase (NOS) appears to be one of the multiple effectors coupled to muscarinic receptors in both submandibular and sublingual glands although some differences have been found depending on the gland studied. First, submandibular glands had a lower basal activity of nitric oxide synthase than sublingual glands and the concentration–response curve for carbachol was bell-shaped in the former but not in sublingual glands. Second, cGMP levels displayed a similar profile to that observed for NOS activity in both glands. Third, protein kinase C also coupled to muscarinic receptor activation in the glands might have a regulatory effect on nitric oxide production since its activity was higher in basal conditions in submandibular than sublingual glands and it also increased in the presence of the agonist at a concentration that inhibited NOS activity in submandibular glands. The effects appear to be partly related to the expression of a minor population of M 1 receptors in submandibular glands absent in sublingual as determined in binding and signaling experiments with the muscarinic receptor antagonist pirenzepine.

Inhibition of NO Synthase Activity in Nervous Tissue Leads to Decreased Motor Activity in the Rat

The nitric oxide/cGMP system has been shown to play a crucial role in the mechanism of learning and memory. The aim of the present study was to investigate whether the inhibition of NO synthase in brain regions leads to alterations of spontaneous behavior in rats. Male Wistar rats were treated with NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) at the dose of 40 mg/kg/day. After 4 weeks of L-NAME treatment, NO synthase activity was significantly decreased by 75 % in the cerebellum, by 71 % in the cerebral cortex and by 72 % in the thoracic spinal cord. Decreased NO synthase activity in the nervous tissue was associated with decreased motor horizontal and vertical activities as well as by lowered frequency of sniffing, cleaning and defecation. It is concluded that the inhibition of NO synthase activity has a suppressive effect on spontaneous behavior of rats.

Differential expression of nitric oxide synthase in human stomach cancer

The level of expression and cellular localization of isoenzymes of nitric oxide synthase (NOS) was detected in human stomach tumor tissues. Tumor tissues showed 70% higher activity of NOS than that of normal tissues (P<0.01). Poorly differentiated adenocarcinoma tend to have higher activity (P<0.05) than well differentiated and moderately differentiated tumor tissues. Aminoguanidine (AG), 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT), NG-monomethyl-l-arginine (l-NMMA), and Nω-nitro-l-arginine (l-NNA) inhibited NOS activity in tumor tissues by 18, 14, 11 and 13%, respectively. The TNF-α mRNA expression was correlated with the inducible NOS (iNOS) level, which was high in adenocarcinomas and low in normal tissues. Tumor tissues showed higher expression of iNOS in gland epithelial cells but the level of eNOS was significantly decreased with an exception of concentrated localization in the proliferating capillary endothelium. These results revealed that isoforms of NOS might contribute differentially to growth and progression of human stomach tumor.