Role Of Nitric Oxide Synthase Research Articles

The Inducible Nitric-Oxide Synthase Modulates Endothelin-1-Dependent Release of Prostacyclin and Inhibition of Platelet Aggregation ex Vivo in the Mouse

Nitric oxide and other reactive oxygen species generated by nitric-oxide synthases (NOS) modulate, among several other cellular responses, the production of eicosanoids and platelet aggregation. The roles of specific NOS in these two phenomena remain to be determined. Thus, the present study assessed whether inducible NOS (iNOS) and endothelial NOS (eNOS) modulate in a similar manner the production of eicosanoids and platelet aggregation. Mice knocked out for eNOS (eNOS-/-) or iNOS (iNOS-/-) and their wild-type (WT) congeners were used to analyze agonist-induced increases in plasma levels of eicosanoids as well as inhibition of platelet aggregation ex vivo. Systemically administered endothelin-1 (ET-1) triggered an increase in plasma levels of 6-keto prostaglandin F(1alpha) (6-keto PGF(1alpha)) in WT and eNOS-/- but not in iNOS-/- mice. ET-1 (0.01-1 nmol/kg) also induced a dose-dependent inhibition of platelet aggregation in WT and eNOS-/- but not in iNOS-/- mice. Another agonist, bradykinin (10 nmol/kg), triggered the release of 6-keto PGF(1alpha) and inhibited platelet aggregation in all strains of mice studied. In addition, ADP-induced platelet aggregation in vitro was similarly reduced by iloprost (100 nM) in iNOS-/- mice and WT congeners. In another series of experiments, ET-1 (0.1 nmol/kg) significantly increased 8-isoprostane plasma levels in WT but not in iNOS-/- mice. Finally, a 3-week treatment with anti-oxidants inhibited the capacity of ET-1 to significantly increase plasma 6-keto PGF(1alpha) in WT mice. We show for the first time that iNOS is involved in the control of ET-1-induced prostacyclin release and related inhibition of platelet aggregation in the murine model.

Time-related changes in constitutive and inducible nitric oxide synthases in the rat striatum in a model of Huntington's disease

Excitotoxicity and oxidative stress are mechanisms involved in the neuronal cell death induced by the intrastriatal injection of quinolinic acid (QUIN) as a model of Huntington's disease. Production of nitric oxide by nitric oxide synthase (NOS) has been proposed to participate in QUIN-induced neurotoxicity; however, the precise role of NOS in QUIN-induced toxicity still remains controversial. In order to provide further information on the role of NOS isoforms in QUIN toxicity, we performed real time RT-PCR and immunohistochemistry of inducible NOS (iNOS), endothelial NOS (eNOS) and neuronal NOS (nNOS) and determined Ca 2+-dependent and Ca 2+-independent NOS activity in a temporal course (3–48 h), after an intrastriatal injection of QUIN to rats. NOS isoforms exhibited a transitory expression of mRNA and protein after QUIN infusion: eNOS increased between 3 and 24 h, iNOS between 12 and 24 h, while nNOS at 35 and 48 h. Ca 2+-independent activity (iNOS) did not show any change, while Ca 2+-dependent activity (constitutive NOS: eNOS/nNOS) exhibited increased levels at 3 h. Our results support the participation of Ca 2+-dependent NOS isoforms during the toxic events produced at early times after QUIN injection.

Aminoguanidine produces beneficial haemodynamic effects in a canine model of acute pulmonary thromboembolism

Activating the nitric oxide (NO)-cyclic guanosine 3',5'-monophosphate (cGMP) pathway improves haemodynamics following acute pulmonary thromboembolism (APT). However, the role of NO synthase (NOS) isoforms in the responses to APT has not been determined. We examined the effects of selective and non-selective inducible NOS (iNOS) inhibition. Haemodynamic evaluations were performed in non-embolized dogs treated with saline (control group; n = 4), L-NAME (NAME group; n = 3), or aminoguanidine (AG group; n = 3), and in dogs that received the same drugs and were embolized with 5 mL kg(-1) of clots made with autologous blood (Emb group, n = 9; NAME + Emb group, n = 4 and AG + Emb group, n = 7). The lung concentrations of nitrite/nitrate (NOx) and cGMP were determined by chemiluminescence and ELISA respectively. Acute pulmonary thromboembolism increased mean pulmonary arterial pressure (MPAP) and pulmonary vascular resistance index (PVRI) by 21.4 +/- 1.7 mmHg and by 843 +/- 34 dyn s cm(-5) m(-2), respectively, in Emb group. MPAP and PVRI increased to higher levels in the NAME + Emb group 15 min after APT and all dogs in this group died 15-30 min after APT. Conversely, lower MPAP and PVRI levels were found in the AG + Emb group 2 h after APT compared with the Emb group (both P < 0.05). Higher NOx concentrations were found in the Emb group compared with the other groups (all P < 0.05). Higher cGMP concentrations were found in the Emb and AG + Emb groups compared with the other groups (all P < 0.05). These results indicate that endogenous NO protects against APT-induced cardiovascular responses. Moreover, iNOS-derived NO possibly produces unfavourable effects, which are counteracted by aminoguanidine. However, non-NO-related mechanisms may also be involved.

Hemodynamic Responses Elicited by Systemic Injections of Flavin Adenine Dinucleotide in Anesthetized Rats

Flavin adenine dinucleotide (FAD) elicits an endothelium-dependent vasodilation in isolated rat mesenteric beds via activation of P2Y-purinoceptors. The aims of this study were to characterize the hemodynamic responses elicited by systemic injections of FAD and flavin mononucleotide (FMN) in anesthetized rats and to determine the role of nitric oxide synthase (NOS), cyclooxygenase, P2Y/P2X-purinoceptors, and muscarinic receptor in these responses. FAD (0.05-1.0 micromol/kg, iv) elicited dose-dependent decreases in heart rate (HR), mean arterial blood pressure (MAP), and hindquarter vascular resistance (HQR), whereas it elicited an initial increase and then a decrease in mesenteric (MR) vascular resistance. The FAD-induced responses were not affected by the P2Y/P2X-purinoceptor antagonist suramin, the muscarinic receptor antagonist methyl-atropine, or the cyclooxygenase inhibitor indomethacin. The vasodilator actions of FAD were unaffected by the NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME), whereas the bradycardia elicited by higher doses of FAD were diminished by L-NAME. FMN did not elicit hemodynamic responses in the absence or presence of L-NAME. In summary, FAD-induced bradycardia depends, in part, on the activation of NOS, whereas the vasodilator actions of FAD are not obviously due to newly synthesized nitrosyl factors. These findings and those in our companion manuscript support the concepts that the adenine moiety confers biological activity to FAD, which releases preformed pools of nitrosyl factors.

Reduced Photoreceptor Damage after Photodynamic Therapy through Blockade of Nitric Oxide Synthase in a Model of Choroidal Neovascularization

To investigate the role of nitric oxide synthase (NOS) in photoreceptor degeneration associated with photodynamic therapy (PDT) in a laser-induced model of choroidal neovascularization (CNV). PDT was performed in monkey and Brown Norway rats with laser-induced CNV. L-NAME, a NOS inhibitor, or saline was injected intraperitoneally in rats with CNV. An NO donor, or saline, was injected intravitreously into normal rats. Photoreceptor apoptosis was evaluated by TUNEL and electron microscopy. NOS, ED-1, and cleaved-caspase-3 (c-casp-3) expression were determined by immunohistochemistry. CNV lesions were examined by fluorescence angiography and choroidal flat mount. TUNEL and electron microscopy showed photoreceptor apoptosis after PDT. In rats, there were significantly more TUNEL-positive cells in the photoreceptors 24 hours after PDT, whereas in the CNV lesions there were more TUNEL-positive cells 6 hours after PDT. C-casp-3 was detected in the CNV lesions but not in the photoreceptors after PDT. There was no difference in the numbers of ED-1-positive macrophages before and after PDT. However, inducible NOS (iNOS) was increased after PDT in macrophages. Intravitreous injection of the NO donor without PDT also induced substantial photoreceptor apoptosis. L-NAME-treated animals had significantly fewer TUNEL-positive cells in the photoreceptors than saline-treated animals after PDT (P < 0.05). There were no differences in CNV size and leakage between L-NAME- and saline-treated groups. iNOS expression in macrophages contributes to PDT-induced photoreceptor degeneration. NOS inhibition reduces PDT-induced photoreceptor degeneration without compromising the treatment effect of PDT in an experimental model of CNV.

Cyclophosphamide Induced Cystitis: Role of Nitric Oxide Synthase, Cyclooxygenase-1 and 2, and NK 1 Receptors

The role of substance P, inducible nitric oxide synthase, and cyclooxygenase-1 and 2 on the pathogenesis of cyclophosphamide induced cystitis was investigated in rats. Sprague-Dawley male rats received 1 of certain treatments, including 1) 0.9 weight per volume saline (0.10 ml/100 gm intraperitoneally), 2) cyclophosphamide (75 mg/kg intraperitoneally), 3) cyclophosphamide plus the NK(1) receptor antagonist Win-51.708 (20 mg/kg intraperitoneally), 4) cyclophosphamide plus the inducible nitric oxide synthase inhibitor S-methylthiourea (20 mg/kg intraperitoneally), 5) cyclophosphamide plus the highly selective cyclooxygenase-2 inhibitor rofecoxib (15 mg/kg intraperitoneally), 6) cyclophosphamide plus the selective cyclooxygenase-2 inhibitor meloxicam (15 mg/kg intraperitoneally), 7) cyclophosphamide plus the nonselective cyclooxygenase inhibitor ketoprofen (20 mg/kg intraperitoneally) or 8) cyclophosphamide plus methylthiourea plus meloxicam. Parameters were evaluated 6 hours after cyclophosphamide administration, including plasma protein extravasation, histological changes, myeloperoxidase and inducible nitric oxide synthase activities in the bladder, plasmatic nitric oxide metabolites and urinary nitric oxide metabolites, and prostaglandin E(2) levels. Cyclophosphamide produced inflammatory and cytotoxic changes in the bladder, accompanied by increased nitric oxide metabolites, urinary prostaglandins, myeloperoxidase and inducible nitric oxide synthase activity. Pretreatment with Win-51.708 and with methylthiourea prevented all of these effects except myeloperoxidase activity, which was only prevented by Win-51.708. All inducible cyclooxygenases were able to prevent prostaglandin synthesis and increases in myeloperoxidase activity. Combined inhibition of inducible nitric oxide synthase and cyclooxygenase-2/cyclooxygenase-1 (methylthiourea plus meloxicam) did not provide any additional protection against bladder damage, increased inducible nitric oxide synthase activity or prostaglandin E(2) synthesis. Additionally, this combination was unable to prevent increased myeloperoxidase activity. The results of this study suggest that there is crosstalk between nitric oxide and the cyclooxygenase enzyme with cyclooxygenase-1/cyclooxygenase-2 isoforms having an important role in this relationship. Augmented myeloperoxidase activity seems to be associated with NK(1) receptor activation and low levels of nitric oxide with cyclooxygenase-1 having an important role.

Rolle des L-Arginin/NO-Stoffwechselweges im akuten Nierenversagen

Role of the L-arginine/NO-pathway in acute renal failure ■ Summary Regulation of renal hemodynamics – in particular intraglomerluar hemodynamics -is closely related to the L-arginine/ NO-pathway. Almost all forms of acute renal failure (ARF) are characterized by a reduction in renal plasma flow (RPF) and increased renal vascular resistance. NO, metabolized from L-arginine, is capable of improving renal function due to its vasorelaxing properties in almost all models of ARF, whereas unselective inhibition of nitric oxide synthase (NOS) generally exerts contrary effects. Increased tubular generation of oxygen radicals in the early course of ischemic ARF indicates increased oxidative stress. Simultaneously regulatory changes in expression of iNOS (inducible NOS) and eNOS (endothelial NOS) are observed. Apart from the positive functional effects with L-arginine supplementation a remarkable reduction in the formation of oxygen radicals and reduced overexpression of iNOS are observed. This is important, because in ischemic ARF increased expression of iNOS is accompanied by increased formation of oxygen radicals and peroxynitrite. Both substances are highly reactive and exert many cytotoxic effects. eNOS-derived NO probably plays such an important role in maintaining and improving renal and intraglomerular hemodynamics that negative effects from iNOS-derived NO are overcome. The roles of NOS are different for different types of NOS and also depend on the time course of ARF. Future management strategies should focus on the interplay between antioxidants and NO regulators and the correlation of reactive oxygen species and NO.

Expression of nitric oxide synthase isoforms in the dorsal horn of monoarthritic rats: effects of competitive and uncompetitive N-methyl-D-aspartate antagonists

Chronic pain is associated with N-methyl-D-aspartate (NMDA) receptor activation and downstream production of nitric oxide, which has a pivotal role in multisynaptic local circuit nociceptive processing in the spinal cord. The formation of nitric oxide is catalyzed by three major nitric oxide synthase (NOS) isoforms (neuronal, nNOS; inducible, iNOS; endothelial, eNOS), which are increased in the spinal cord of rodents subjected to some tonic and chronic forms of experimental pain. Despite the important role of NOS in spinal cord nociceptive transmission, there have been no studies exploring the effect of NMDA receptor blockade on NOS expression in the dorsal horn during chronic pain. Furthermore, NOS isoforms have not been fully characterized in the dorsal horn of animals subjected to arthritic pain. The aim of this work was therefore to study the expression of nNOS, iNOS and eNOS in the dorsal horns of monoarthritic rats, and the modifications in NOS expression induced by pharmacological blockade of spinal cord NMDA receptors. Monoarthritis was produced by intra-articular injection of complete Freund's adjuvant into the right tibio-tarsal joint. At week 4, monoarthritic rats were given either the competitive NMDA antagonist (±)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) or the uncompetitive NMDA antagonist ketamine. After 6 and 24 hours, animals were killed and posterior quadrants of the lumbar spinal cord were dissected. Sample tissues were homogenized and subjected to immunoblotting with anti-nNOS, anti-iNOS or anti-eNOS monoclonal antibodies. The nNOS isoform, but not the iNOS and eNOS isoforms, were detected in the dorsal horns of control rats. Monoarthritis increased the expression of nNOS, iNOS and eNOS in the dorsal horns ipsilateral and contralateral to the inflamed hindpaw. Intrathecal administration of CPP and ketamine reduced nNOS expression in monoarthritic rats but increased the expression of iNOS and eNOS. Results suggest that blockade of spinal cord NMDA receptors produces complex regulatory changes in the expression of NOS isoforms in monoarthritic rats that may be relevant for nitridergic neuronal/glial mechanisms involved in the pathophysiology of monoarthritis and in the pharmacological response to drugs interacting with NMDA receptors.

Regional age-related changes in neuronal nitric oxide synthase (nNOS), messenger RNA levels and activity in SAMP8 brain

BackgroundNitric oxide (NO) is a multifunctional molecule synthesized by three isozymes of the NO synthase (NOSs) acting as a messenger/modulator and/or a potential neurotoxin. In rodents, the role of NOSs in sleep processes and throughout aging is now well established. For example, sleep parameters are highly deteriorated in senescence accelerated-prone 8 (SAMP8) mice, a useful animal model to study aging or age-associated disorders, while the inducible form of NOS (iNOS) is down-regulated within the cortex and the sleep-structures of the brainstem. Evidence is now increasing for a role of iNOS and resulting oxidative stress but not for the constitutive expressed isozyme (nNOS). To better understand the role of nNOS in the behavioural impairments observed in SAMP8 versus SAMR1 (control) animals, we evaluated age-related variations occurring in the nNOS expression and activity and nitrites/nitrates (NOx-) levels, in three brain areas (n = 7 animals in each group). Calibrated reverse transcriptase (RT) and real-time polymerase chain reaction (PCR) and biochemical procedures were used.ResultsWe found that the levels of nNOS mRNA decreased in the cortex and the hippocampus of 8- vs 2-month-old animals followed by an increase in 12-vs 8-month-old animals in both strains. In the brainstem, levels of nNOS mRNA decreased in an age-dependent manner in SAMP8, but not in SAMR1. Regional age-related changes were also observed in nNOS activity. Moreover, nNOS activity in hippocampus was found lower in 8-month-old SAMP8 than in SAMR1, while in the cortex and the brainstem, nNOS activities increased at 8 months and afterward decreased with age in SAMP8 and SAMR1. NOx- levels showed profiles similar to nNOS activities in the cortex and the brainstem but were undetectable in the hippocampus of SAMP8 and SAMR1. Finally, NOx- levels were higher in the cortex of 8 month-old SAMP8 than in age-matched SAMR1.ConclusionConcomitant variations occurring in NO levels derived from nNOS and iNOS at an early age constitute a major factor of risk for sleep and/or memory impairments in SAMP8.

Inhibitors of inducible nitric oxide (NO) synthase are more effective than an NO donor in reducing carbon-tetrachloride induced acute liver injury.

The exact functional role of nitric oxide (NO) in liver injury is currently a source of controversy. NO is enzymatically synthesized by nitric oxide synthase (NOS). In this study, we assessed the role of inducible NOS (iNOS) in carbon tetrachloride (CCl4)-induced acute liver injury using inhibitors of iNOS, and an NO donor. Adult ICR mice were injected with CCl4 with or without the iNOS inhibitors (5-methylisothiourea hemisulfate [SMT] and l-N6-(1-iminoethyl)-lysine [L-NIL]) and an NO donor (Sodium Nitroprusside [SNP]). Blood and liver tissues were collected for analysis. Immunohistochemistry (IHC), serum alanine aminotransferase (ALT), serum total 8-isoprostane analysis, RT-PCR, Western Blotting (WB) and EMSA were done. Our results showed increased levels of ALT, necrosis, total 8-isoprostane and nitrotyrosine after CCl4 administration. iNOS inhibitors and SNP abrogated these effects but the effect was more pronounced with SMT and L-NIL. RT-PCR, WB and IHC in CCl4-treated mice demonstrated upregulation of TNF-alpha, iNOS, and COX-2. The administration of iNOS inhibitors with CCl4 diminished the expression of these proinflammatory mediators. NF-kappaB was also upregulated in CCl4-treated mice and was reversed in mice pretreated with iNOS inhibitors. SNP pretreated mice also showed a lower expression of COX-2 when compared with CCl4 treated mice but TNF-alpha, iNOS and NF-kappaB activity were unaffected. We propose that a high level of nitric oxide is associated with CCl4-induced acute liver injury and the liver injury can be ameliorated by decreasing the NO level with iNOS inhibitors and an NO donor with the former more effective in reducing CCl4-induced liver injury.

Brain natriuretic peptide limits myocardial infarct size dependent of nitric oxide synthase in rats

BackgroundBrain natriuretic peptide (BNP) has recently been shown to have a cardioprotective effect in animal models of myocardial ischemia-reperfusion (I-R) injury. We hypothesized that exogenous BNP limits myocardial infarction on nitric oxide synthase pathway. MethodsA rat model of myocardial I-R injury was established by ligating the left descending coronary artery for 30 min and then reperfusing for 2 h. BNP was injected with different dose 5 min after the ligation and lasting for 145 min. The myocardial infarct size and the area at risk of ischemia were measured by staining with triphenyltetrazolium chloride (TTC) and Evans blue dye. To examine the role of nitric oxide synthase (NOS), expression of eNOS in the left ventricle was analyzed by western blotting. Nω-nitro-l-arginine methyl ester (l-NAME; 30 ug/kg), or S-methylisothiourea (SMT; 3 ug/kg) was administrated before I-R with or without BNP. ResultsThe control infarct-to-risk ratio was 45.1±1.72% (means±SE). BNP infused 5 min after ischemia limited infarct size in a dosage-dependent manner, with maximal protection observed at 0.01 ug/(kg min) (infarct-to-risk: 24.7±1.69%, P<0.01 vs. control), associated with a 10-fold increase of myocardial endothelial nitric oxide synthase above the control value. Protection afforded by BNP was abolished by l-NAME but not by SMT, suggesting the involvement of putative endothelial but not inducible nitric oxide synthase activation. ConclusionsWe conclude that natriuretic peptide/NOS/NO signaling may constitute an important injury-limiting mechanism in myocardium.

Improved postischemic function following acute exercise is not mediated by nitric oxide synthase in the rat heart

The mediators of acute exercise-induced preconditioning against ischemia-reperfusion injury are not understood. This study assesses the role of nitric oxide synthase (NOS), a reported mediator of other forms of preconditioning. Male Fischer 344 rats were divided into five groups (n = 6-7): sedentary (Sed); exercised 2 days on a treadmill at 20 m/min, 6 degrees grade, for 60 min (Run); sedentary, perfused with 100 microM N(omega)-nitro-l-arginine methyl ester hydrochloride (l-NAME) to inhibit NOS (Sed/L-N); exercised, perfused with l-NAME (Run/L-N); and exercised in a 4 degrees C environment, perfused with l-NAME (CRun/L-N). Twenty-four hours following exercise, isolated, perfused working hearts were subjected to 22.5 min of global ischemia plus 30 min of normoxic reperfusion. Left ventricle contents of several putative preconditioning mediators were determined. Postischemic recovery of cardiac output times systolic pressure was better in Run than Sed (78.4 vs. 50.2% of preischemia, P < 0.05). Inhibition of NOS did not abrogate the improved recovery in the exercise groups or alter recovery in Sed. All exercise groups also displayed improved myocardial efficiency (cardiac output times systolic pressure/oxygen consumption) postischemia and less lactate dehydrogenase release (P < 0.05). l-NAME appeared to lower lactate dehydrogenase release independent of exercise. The only change in antioxidant enzyme activity was a decrease in manganese superoxide dismutase in CRun/L-N (P < 0.05). Heat shock protein 72 expression increased only in Run and Run/L-N and endothelial NOS only in CRun/L-N (P < 0.05). Acute exercise-induced preconditioning of the Fischer 344 rat heart is not mediated by NOS and does not require increases in heat shock protein 72 or antioxidant enzymes.

Effects of hydroxyurea and L-arginine on the production of nitric oxide metabolites in cultures of normal and sickle erythrocytes

Previous in vitro studies suggest that erythrocytes may be a source of nitric oxide (NO) produced by nitric oxide synthase (NOS) or by oxyhemoglobin-mediated oxidation of hydroxyurea (HU). This study was performed to determine the roles of HU and NOS in the production of NO by normal and sickle erythrocytes. Red blood cells (RBCs) from normal adult hemoglobin (HbAA) and homozygous sickle cell subjects (HbSS) were incubated with PBS containing 0.2 mM hydrogen peroxide (control) for 2 h at 37°C in the presence and absence of L-arginine, the substrate for NOS, and with L-arginine plus HU in the presence and absence of L-NMMA, a specific inhibitor of NOS. The nitrate and nitrite metabolites of NO, expressed as [NOx], were measured. [NOx] in the HbAA and HbSS RBC cultures was not significantly different in the presence and absence of 1.0 mM L-arginine (p > 0.1). [NOx] in the HbAA and HbSS cultures treated with a clinically relevant dose of HU (1.0 mM) plus 1.0 mM L-arginine was significantly greater than that in controls incubated with PBS and with L-arginine p < 0.01. However, [NOx] in the HbAA and HbSS cultures treated with 50 μg/ml L-NMMA was not significantly different than that in the cultures treated with HU plus L-arginine in the absence of L-NMMA. These findings suggest that NOx production by erythrocytes may be increased by treatment with HU and may not be decreased by inhibiting NOS. Therefore, we conclude that a therapeutic dose of HU may increase the plasma concentration of NO by a mechanism that does not require erythrocytes NOS activity

Hyperoxic and hyperbaric-induced cardioprotection: Role of nitric oxide synthase 3

The relative contributions of the fraction of inspired oxygen (FIO2) and atmospheric pressure (ATM) to cardioprotection are unknown. We determined whether the product of FIO2 x ATM (oxygen partial pressure) controls the extent of hyperoxic+hyperbaric-induced cardioprotection and involves activation of nitric oxide synthase (NOS). Adult Sprague Dawley rats (n = 10/gp) were treated for 1 h with (1) normoxia+normobaria (21% O2 at 1 ATM), (2) hyperoxia+normobaria (100% O2 at 1 ATM), (3) normoxia+hyperbaria (21% O2 at 2 ATM) and (4) hyperoxia+hyperbaria (100% O2 at 2 ATM). Infarct size following 25 min ischemia and 180 min reperfusion was decreased following hyperoxia+normobaria and normoxia+hyperbaria compared with normoxia+normobaria and further decreased following hyperoxia+hyperbaria treatment. l-NAME (200 microM) reversed the cardioprotective effects of hyperoxia+hyperbaria. Nitrite plus nitrate content was increased 2.2-fold in rats treated with normoxia+hyperbaria and hyperoxia+hyperbaria. NOS3 protein increased 1.2-fold and association of hsp90 with NOS3 four-fold in hyperoxic+hyperbaric rats. Cardioprotection conferred by hyperoxia+hyperbaria is directly dependent on oxygen availability and mediated by NOS.

Nitric oxide synthase inhibitor attenuates number of regenerating spinal motoneurons in adult rats

Neuronal survival and death-related effects of nitric oxide synthase are widely studied, yet its potential involvement in regeneration remains largely unexplored. In the present study, the regenerative role of nitric oxide synthase in injured motoneurons was investigated. A ventral root was avulsed and a piece of peripheral nerve was implanted into the spinal cord. Results showed that nitric oxide synthase inhibitor reduced the number of regenerating motoneurons to half compared with sham-operated control at 2 weeks and 4 weeks after injury, but the rate of axonal regeneration was not affected. Our study adds a new line of evidence that expression of nitric oxide synthase is beneficial to the axonal regeneration of the injured spinal motoneurons.

Nitric oxide synthases are involved in the modulation of cardiovascular adaptation in hemorrhaged rats

Aim Nitric oxide has been implicated in the cardiovascular adaptation to hemorrhagic shock. Our aim was to study the potential role of nitric oxide synthases (NOS) in the cardiovascular response in hemorrhagic hypotension produced experimentally in anesthetized rats. Methods Groups of animals ( n = 14, per group): (a) normotensive; (b) hypovolemic (20% blood loss); (c) normotensive and pretreatment with N G-nitro- l-arginine methyl ester ( l-NAME); (d) hypovolemic and pretreatment with l-NAME. Results l-NAME restored the hypotension induced by hemorrhage. Blood loss decreased heart rate in the first stage increasing at 60 and 120 min. l-NAME blunted this effect. Right atria and left ventricle histochemical NOS activities increased at 60 and 120 min (atria 8% and 24%, respectively; ventricle 21% and 45%, respectively). This activity increased 17% in smooth muscle at 120 min. Heart endothelial NOS protein levels increased in heart at 60 min being attenuated at 120 min. Inducible NOS protein levels raised significantly in right atria, left ventricle and aorta at 120 min. Conclusion Hemorrhagic shock induced a time-dependent and specific NOS activation modulating cardiovascular function. The inhibition of nitric oxide system appears to prevent the acceleration of heart rate during late phases after acute hypovolemic state induced by blood loss.

A Comparative Study of Inducible and Neuronal Nitric Oxide Synthases

Nitric oxide synthases (NOS) independent of the isozyme, produce nitric oxide (·NO), superoxide (O2·−), and hydrogen peroxide (H2O2). Since ·NO has been implicated in many physiological processes, the importance of O2·− and H2O2 in regulating cell signaling by ·NO cannot be overlooked. Before addressing these questions, we investigated the production of ·NO, O2·−, and H2O2 by purified NOS. NOS 1 and NOS 2 were chosen, as the flux of ·NO from each isozyme supports differential biological activity. We found that the initial rate and sustained production of ·NO was considerably greater for NOS 2 as compared to NOS 1. In the absence of L-arginine, however, NOS 1 generation of O2·− and H2O2 was found to be substantially greater than that measured for NOS 2. In addition, one of the potential outcomes of NOS 1 and NOS 2 production of ·NO and O2·− is the formation of peroxynitrite (ONOO−). For NOS 2, the nearly simultaneous generation of O2·− and ·NO at the reductase and heme domains, respectively, increases the likelihood of ONOO− formation while NOS 1 produces ·NO and O2·− at the same domain sequentially– the heme, minimizing ONOO− formation. Differences between NOS 1 and NOS 2 production of ·NO, O2·−, H2O2, and ONOO− may define the specific physiologic function of each isozyme. To this end, we are continuing to examine NOS-derived products and their possible role in physiological processes with emphasis on the role of NOS 2 in the control of infection of the spore forming Gram-positive bacterium Bacillus anthracis.

The role of nitric oxide synthase in exercise induced changes in endothelial progenitor cell number

The role of nitric oxide synthase in exercise induced changes in endothelial progenitor cell number

Methylmercury intoxication activates nitric oxide synthase in chick retinal cell culture

The visual system is a potential target for methylmercury (MeHg) intoxication. Nevertheless, there are few studies about the cellular mechanisms of toxicity induced by MeHg in retinal cells. Various reports have indicated a critical role for nitric oxide synthase (NOS) activation in modulating MeHg neurotoxicity in cerebellar and cortical regions. The aim of the present study is to describe the effects of MeHg on cell viability and NOS activation in chick retinal cell cultures. For this purpose, primary cultures were prepared from 7-day-old chick embryos: retinas were aseptically dissected and dissociated and cells were grown at 37 degrees C for 7-8 days. Cultures were exposed to MeHg (10 microM, 100 microM, and 1 mM) for 2, 4, and 6 h. Cell viability was measured by MTT method and NOS activity by monitoring the conversion of L-[H3]-arginine to L-[H3]-citrulline. The incubation of cultured retina cells with 10 and 100 microM MeHg promoted an increase of NOS activity compared to control (P < 0.05). Maximum values (P < 0.05) were reached after 4 h of MeHg incubation: increases of 81.6 +/- 5.3 and 91.3 +/- 3.7%, respectively (data are reported as mean +/- SEM for 4 replicates). MeHg also promoted a concentration- and time-dependent decrease in cell viability, with the highest toxicity (a reduction of about 80% in cell viability) being observed at the concentration of 1 mM and after 4-6 h of incubation. The present study demonstrates for the first time the modulation of MeHg neurotoxicity in retinal cells by the nitrergic system.

Early pharmacological preconditioning by erythropoietin mediated by inducible NOS and mitochondrial ATP‐dependent potassium channels in the rat heart

Administration of recombinant human erythropoietin (rhEPO) is known to induce protection against cardiac ischaemia injury improving functional recovery and reducing apoptosis. But the underlying mechanisms are not elucidated. We determined the role of nitric oxide synthases (NOS) as well as ATP-dependent (K(ATP)) and calcium-activated (K(Ca)) potassium channels in the early cardioprotection induced by rhEPO. Wistar male rats were divided into two experimental groups treated by rhEPO (5,000 IU/kg, i.p.) or saline (control group). One hour later, rats were anaesthetized, hearts isolated, retrogradely perfused and submitted to a 30-min no-flow global ischaemia followed by 120 min of reperfusion sequence. Cardiac functional recovery (left ventricular developed pressure, LVDP) was significantly higher in the group treated by rhEPO (LVDP at 30 min reperfusion: 71.7 +/- 2.3 mmHg) compared with the control group (57.4 +/- 5.8 mmHg). We observed the same significant effect on its derivative (dP/dt). The rhEPO-induced improvement in ventricular function was abolished by perfusion prior to ischaemia with either N-nitro-l-arginine methyl ester (l-NAME, a nonspecific NOS inhibitor) or N-(3-(aminomethyl)benzyl)acetamidine (1,400W, a specific inducible NOS inhibitor) or 5-hydroxydecanoic acid (5HD, a mitochondrial K(ATP) channel blocker) but not with paxilline (a K(Ca) channel inhibitor). Thus, in vivo rhEPO administration provides early preconditioning against ischaemic injury in the isolated perfused rat heart that is dependent on iNOS and mitochondrial K(ATP) channels.