The influence of NO synthase inhibitor and free oxygen radicals scavenger--methylene blue--on streptozotocin-induced diabetes in rats

The excessive production of nitric oxide (NO) and the subsequent increase of local oxidative stress is suggested as one of the pathophysiological mechanisms of streptozotocin-induced diabetes. It was reported that the administration of NO synthase inhibitors partially attenuated the development of streptozotocin-induced diabetes and reduced hyperglycaemia. Here we have studied the influence of methylene blue, which combines the properties of NO synthase inhibitor with antioxidant effects. The experiments were performed on male rats divided into four groups: control, diabetic (single dose of 70 mg of streptozotocin/kg i.p.), methylene blue (50 mg/kg in the food) and diabetic simultaneously fed with methylene blue. After 45 days the experiments were discontinued by decapitation. Serum glycaemia, glycated haemoglobin and oxidative stress parameters (plasma malondialdehyde concentration and erythrocyte superoxide dismutase activity) were significantly higher in the diabetic group. Simultaneous methylene blue administration partially reduced glycaemia and glycated haemoglobin, but did not decrease oxidative stress. We conclude that NO synthase inhibitor methylene blue partially attenuates the development of streptozotocin-induced diabetes in male rats, but does not reduce the development of oxidative stress in the diabetic group.     

Modulation of nitric oxide (NO) biosynthesis in lactobacilli

We characterized effects of nitric oxide synthase (NOS) substrate L-arginine and classical inhibitors of mammalian NOS on nitric oxide (NO) biosynthesis in probiotic bacteria Lactobacillus plantarum 8P-A3. NO-synthase origin of nitric oxide detected by fluorescent NO indicator 1,2-diaminoanthraquinone (DAA) was confirmed by induction of NO production by exogenous L-arginine. None of the used inhibitors of three isoforms of mammalian NOSs (L-NAME, L-NIL, nNOS inhibitor I) showed significant inhibitory effect of lactobacillar NO-synthase activity.     

Selective inhibition of inducible NO-synthase by nonselective inhibitor

The study has shown that Nw-nitro-L-arginine, a nonselective nitric oxide (NO) inhibitor, in low non-vasoactive doses (10 mg/kg) exerted a protective effect in heat shock as demonstrated by a decrease in the mortality rate and prevention of acute hypotension in rats. The L-NNA in the same dose inhibited the basal NO production but left unaffected a carbachol-activated NO production. The findings suggest a possibility in principle of preferential inhibition of inducible NO-synthase in pathological conditions related to the NO overproduction using non-vasoactive doses of L-NNA the nonselective NO-synthase inhibitor.     

Role of endogenous nitric oxide in endotoxin-induced alteration of hypoxic pulmonary vasoconstriction in mice

Pulmonary vasoconstriction in response to alveolar hypoxia (HPV) is frequently impaired in patients with sepsis or acute respiratory distress syndrome or in animal models of endotoxemia. Pulmonary vasodilation due to overproduction of nitric oxide (NO) by NO synthase 2 (NOS2) may be responsible for this impaired HPV after administration of endotoxin (LPS). We investigated the effects of acute nonspecific (N(G)-nitro-L-arginine methyl ester, L-NAME) and NOS2-specific [L-N6-(1-iminoethyl)lysine, L-NIL] NOS inhibition and congenital deficiency of NOS2 on impaired HPV during endotoxemia. The pulmonary vasoconstrictor response and pulmonary vascular pressure-flow (P-Q) relationship during normoxia and hypoxia were studied in isolated, perfused, and ventilated lungs from LPS-pretreated and untreated wild-type and NOS2-deficient mice with and without L-NAME or L-NIL added to the perfusate. Compared with lungs from untreated mice, lungs from LPS-challenged wild-type mice constricted less in response to hypoxia (69 +/- 17 vs. 3 +/- 7%, respectively, P < 0.001). Perfusion with L-NAME or L-NIL restored this blunted HPV response only in part. In contrast, LPS administration did not impair the vasoconstrictor response to hypoxia in NOS2-deficient mice. Analysis of the pulmonary vascular P-Q relationship suggested that the HPV response may consist of different components that are specifically NOS isoform modulated in untreated and LPS-treated mice. These results demonstrate in a murine model of endotoxemia that NOS2-derived NO production is critical for LPS-mediated development of impaired HPV. Furthermore, impaired HPV during endotoxemia may be at least in part mediated by mechanisms other than simply pulmonary vasodilation by NOS2-derived NO overproduction.     

Nitric oxide generation by hemocytes of the mussel Mytilus galloprovincialis.

The phagocytic activity of Mytilus galloprovincialis hemocytes is thought to be associated with NADPH-oxidase activity of the plasma membrane, thus producing superoxide anions. Few studies, however, have been devoted to nitric oxide release by these haemocytes. We investigated NO generation in M. galloprovincialis in order to understand its role in the defensive mechanisms of these organisms. The presence of NO-synthase-like enzymatic activity in protein homogenates from M. galloprovincialis hemocytes was revealed by the conversion of radiolabelled L-arginine to L-citrulline. We observed partial inhibition of the luminol-dependent chemiluminescence of stimulated M. galloprovincialis hemocytes by both NO-synthase inhibitors and superoxide dismutase, indicating that peroxynitrite (which results from the reaction between nitric oxide and superoxide anions) partially mediated this chemiluminescence. Furthermore, we confirmed the production of nitric oxide by M. galloprovincialis by highlighting the nitric oxide-synthase-dependence of the nitrate and nitrite production of stimulated hemocytes.     

The antimicrobial activity of nitric oxide and its role in the infectious process

In this article information on an important role of nitric oxide (NO) in inhibiting the growth of a number of pathogenic microorganisms, including intracellular parasites, and their elimination from the host body is presented. Differences between the mechanisms of the production of NO and free-radical compounds having antimicrobial action are given. The regulation of the activity of constitutive NO-synthase and inducible NO-synthase and the relationship between the latter and the phagocytic activity and production of anti-inflammatory cytokines are described. An important role of NO in the development of the nonspecific resistance of the body is mentioned.     

Mechanisms underlying enhanced contractile response to endothelin-1 in diabetic rat basilar artery

We investigated the influence of streptozotocin-induced diabetes on the responsiveness of the rat basilar artery to endothelin-1 (ET-1) and nitric oxide (NO), which is known to counteract ET-1. In basilar arteries isolated from diabetic rats: (a) the ET-1-induced contraction was enhanced, (b) the contraction induced by N(G)-nitro-l-arginine [a nitric oxide synthase (NOS) inhibitor] was weaker, and (c) the levels of the mRNAs for ET(A)/ET(B) receptors and prepro-ET-1, but not for NOS, were significantly elevated (all versus age-matched controls). These data indicate that ET-1-induced vasoconstriction may be increased in the diabetic rat basilar artery, and that this hyper-reactivity to ET-1 may be due to an overproduction of ET-1, an up-regulation of ET(A)/ET(B) receptors, and a defect in the bioavailability of NO.     

Role of Nitric Oxide in the Progression of Pneumoconiosis

Conflicting evidence has been reported as to whether nitric oxide (NO) possesses anti-inflammatory or inflammatory properties. Data are presented indicating that in vitro or in vivo exposure to selected occupational dusts, i.e., crystalline silica, organic dust contaminated with endotoxin, or asbestos, results in upregulation of inducible nitric oxide synthase (iNOS) and the production of NO by alveolar macrophages and pulmonary epithelial cells. Nitric oxide production is associated temporally and anatomically with pulmonary damage, inflammation, and disease progression in response to occupational dusts. Blockage of inducible nitric oxide synthase by administration of NOS inhibitors or in iNOS knockout mice decreases the magnitude of injury and inflammation following in vivo exposure to silica, endotoxin, or asbestos. Therefore, NO may play an important role in the initiation and progression of pneumoconiosis.     

Nitric oxide is involved in the protective effects of short-term adaptaion to hypoxia in the course of stress-induced disorders in Krushinsky-Molodkina rats

A possible involvement of nitric oxide in the protective effect of short-term adaptation of Krushinsky-Molodkina rats to mild hypoxia simulating 5000 m above sea level was studied. Nitric oxide proved to have a considerable protective effect on stress-induced disorders in Krushinsky-Molodkina rats as demonstrated using NO-synthase inhibitors and NO monitoring by electron spin resonance under different experimental conditions.     

Blockade of Nitric Oxide Overproduction and Oxidative Stress by <Emphasis Type="Italic">Nigella sativa</Emphasis> Oil Attenuates Morphine-Induced Tolerance and Dependence in Mice

The effects of Nigella sativa oil on morphine-induced tolerance and dependence in mice and possible mechanism(s) of these effects were investigated, for the first time, in this study. Repeated administration of Nigella sativa oil (4 ml/kg, p.o.) along with morphine (5 mg/kg, s.c.) attenuated the development of tolerance, as measured by the hot plate test, and dependence, as assessed by naloxone (5 mg/kg, i.p.)-precipitated withdrawal manifestations. Concomitantly, nitric oxide overproduction and increase in brain malondialdehyde level induced by repeated administration of morphine to mice or by administration of naloxone to morphine-dependent mice were inhibited by co-administration of the oil. Also, the decrease in brain intracellular reduced glutathione level and glutathione peroxidase activity induced by both treatments were inhibited by co-administration of the oil. The increase in brain glutamate level induced by both treatments was not inhibited by concurrent administration of the oil. The inhibitory effect of the oil on morphine-induced tolerance and dependence and on naloxone-induced biochemical alterations in morphine-dependent mice was enhanced by concurrent i.p. administration of the NMDA receptor antagonist, dizocilpine (0.25 mg/kg). Similarly, concurrent i.p. administration of the NO synthase inhibitors; L-N (G)-nitroarginine methyl ester (10 mg/kg), aminoguanidine (20 mg/kg) and 7-nitroindazole (25 mg/kg) or the antioxidant, N-acetylcysteine (50 mg/kg) enhanced this inhibitory effect of the oil. On the other hand, this effect was antagonized by concurrent i.p. administration of the nitric oxide precursor, L-arginine (300 mg/kg). These results provide evidence that Nigella sativa oil, through inhibition of morphine-induced NO overproduction and oxidative stress, appears to have a therapeutic potential in opioid tolerance and dependence.     

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.     

Statins restore ischemic limb blood flow in diabetic microangiopathy via eNOS/NO upregulation but not via PDGF-BB expression

3-Hydroxy-3-methyl-glutaryl CoA reductase inhibitors, or statins, have pleiotropic effects and can protect the vasculature in a manner independent of their lipid-lowering effect. The effectiveness of statins in reducing the risk of coronary events has been shown even in patients with diabetes, and their effects on diabetic complications have been reported. Using a model of severe hindlimb ischemia in streptozotocin-induced diabetic mice (STZ-DM), we investigated the effects and mechanisms of statin therapy in diabetic angiopathy in ischemic hindlimbs. As a result, STZ-DM mice frequently lost their hindlimbs after induced ischemia, whereas non-DM mice did not. Supplementation with statins significantly prevented autoamputation. We previously showed that diabetic vascular complications are caused by impaired expression of PDGF-BB, but statin therapy did not enhance PDGF-BB expression. Statins helped enhance endogenous endothelial nitric oxide (NO) synthase (eNOS) expression. Furthermore, the inhibition of NO synthesis by the administration of N(omega)-nitro-l-arginine methyl ester impaired the ability of statins to prevent STZ-DM mouse limb autoamputation, indicating that the therapeutic effect of statins in hindlimb ischemia in STZ-DM mice occurs via the eNOS/NO pathway. A combination therapy of statins and PDGF-BB gene supplementation was more effective for diabetic angiopathy than either therapy alone. In conclusion, these findings indicate that statin therapy might be useful for preventing intractable diabetic foot disease in patients with diabetic angiopathy.     

Nitric oxide synthase: models and mechanisms

The overproduction or underproduction of nitric oxide has been implicated in pathological symptoms such as endotoxic shock, diabetes, allograft rejection, and myocardial ischimia/reperfusion injury. A thorough understanding of the biosynthesis of nitric oxide is necessary to probe and manipulate these signaling events. There is also considerable pharmacological interest in developing selective inhibitors of the several isoforms of nitric oxide synthase. The recently determined crystal structures of complexes between nitric oxide synthase and substrate, the mechanisms of the enzymatic reaction that generate nitric oxide and chemical precedents and models for these reactions are now coming into focus, but there are still numerous fascinating and unanswered questions regarding nitric oxide biosynthesis.     

The influence of nitric oxide synthase inhibitor L-NAME on bones of male rats with streptozotocin-induced diabetes

The pathophysiological processes underlying the development of diabetic osteopenia has not hitherto been elucidated. Induction of streptozotocin diabetes leads in our experiments to decrease of bone density, ash, mineral content and to thinner cortical width compared to control male rats. In order to investigate the pathogenetic role of bone resorption by osteoclasts in streptozotocin-induced diabetes, we determined the circulating levels of tartrate-resistant acid phosphatase (TRAP), a biochemical marker for bone resorption. Plasma TRAP values in diabetic rats did not differ from their corresponding controls. Streptozotocin diabetes by itself did not have any effect on the weight of seminal vesicles which are highly testosterone-dependent. Low doses of nitric oxide cause bone resorption, but higher doses of NO inhibit bone resorbing activity. We examined the effect of L-NAME (inhibitor of nitric oxide production) after six weeks of administration to diabetic rats. There was no further significant loss of bone mineral density, ash and mineral content or tibia weight in diabetic rats treated with L-NAME. L-NAME itself did not decrease bone metabolism. In our study no evidence of an increased bone resorption was found. Our results have indicated that a predominance of bone resorption over bone formation is not involved in the pathogenesis of diabetes-associated osteopenia. Inhibition of NO neither increased osteoclastic activity (TRAP) nor induced osteopenia in L-NAME-treated rats. This suggests a possibility that NO is not involved in the pathogenesis of diabetic osteopenia.     

Variation of nitric oxide content regulates the development of apoptosis in the retina

It was shown that retinal ischemia entailed apoptosis in the inner layers of the retina. Administration of an NO-synthase inhibitor suppressed the development of ischemic apoptosis. To ascertain whether nitric oxide could induce the retinal apoptosis by itself, a nontoxic NO donor—dinitrosyl iron complex (DNIC) with glutathione—was injected into the vitreous body. DNIC at low concentrations induced apoptosis in the same retinal layers as in ischemia. However, with increasing DNIC doses, the number of apoptotic nuclei decreased markedly. Simultaneous administration of excess glutathione prevented apoptosis at any DNIC dose. The obtained data demonstrate the neurotoxic properties of the excess of nitric oxide in the retina.     

Influence of Nitric Oxide Synthase Inhibitors on Embryo Destruction and Morphological Features in Pre- and Postimplantation Mouse Embryos

Nitric oxide is an important intraovarian regulatory factor. The periimplantation period is a critical phase in mouse development. Although it was shown that nitric oxide plays an essential role during gestation, its role in the preimplantation period is not yet fully clear. We studied the involvement of nitric oxide in developmental competence (embryonic defects and morphology of pre- and postimplantation embryos) using nitric oxide synthase inhibitors, which suppress all forms of nitric oxide synthase, and female mice, to which the inhibitors had been administered before their mating with intact males. The level of mortality of pre- and postimplantation embryos in females mated to intact males increased soon after the administration of inhibitors. Studies of the morphology of embryos have shown that there was a delay in embryogenesis at the stages of cleavage and gastrulation. The results obtained suggest that nitric oxide is a potent regulator of embryonic differentiation, specifically in pre- and postimplantation mouse embryos.     

Influence of nitric oxide synthase inhibitors on embryonic destructions and morphological features in pre- and postimplantation mouse embryos

Nitric oxide is an important intraovarian regulatory factor. The periimplantation period is a critical phase in mouse development. Although it was shown that nitric oxide plays an essential role during gestation, its role in the preimplantation period is not yet fully clear. We studied the involvement of nitric oxide in developmental competence (embryonic defects and morphology of pre- and postimplantation embryos) using nitric oxide synthase inhibitors, which suppress all forms of nitric oxide synthase, and female mice, to which the inhibitors had been administered before their mating with intact males. The level of mortality of pre- and postimplantation embryos in females mated to intact males increased soon after the administration of inhibitors. Studies of the morphology of embryos have shown that there was a delay in embryogenesis at the stages of cleavage and gastrulation. The results obtained suggest that nitric oxide is a potent regulator of embryonic differentiation, specifically in pre- and postimplantation mouse embryos.     

<Emphasis Type="SmallCaps">l</Emphasis>-Arginine Uptake in Normal and Diabetic Rat Retina and Retinal Pigment Epithelium

Diabetes-induced increase in oxidative stress is postulated as playing a significant role in the development of retinopathy. The retinal pigment epithelium (RPE) which forms part of the retinal blood barrier has been reported to be affected in diabetes. Besides functioning as a neurotransmitter, the radical nitric oxide (NO) can act as a cytotoxic agent. NO is synthesized by nitric oxide synthase (NOS) that oxidizes arginine to citrulline producing NO. Given that intracellular concentration of arginine depends mainly on its transport, we studied arginine transport in RPE and retina from normal and streptozotocin-induced diabetic rats. Retina and RPE take up arginine by a saturable system with an apparent K_M of 70–80 μM. Tissue incubation in the presence of insulin or high glucose concentrations significantly increased arginine transport in RPE but not in retina from control rats. Similarly, arginine uptake was enhanced in RPE, but not in the retina from streptozotocin-induced diabetic rats. However, NO content was two-fold higher in diabetic retina and RPE compared to that in the control rats. Such findings may suggest that diabetes induced an increase in NO levels in RPE, which may have brought about alterations in its functioning and in turn manifestations of diabetic retinopathy. Special issue article in honor of Dr. Ricardo Tapia.     

The influence of N-stearoylethanolamine on the activity of antioxidant enzymes and on the level of stable NO metabolites in the rat testes and blood plasma at the early stages of streptozotocine-induced diabetes

The influence of N-stearoylethanolamine was investigated on the activity of enzymes of antioxidant protection and content of stable metabolites of nitric oxide (NO) in the testes and plasma of rats at the early stages of development of streptozotocine-induced diabetes mellitus. It was shown that the activity of superoxide dismutase, catalase is reduced in the plasma and testes of animals with streptozotocin-induced (50 mg/kg) diabetes (blood glucose 8-10 mmol/L). A significant increase in the amount of nitrite and nitrate anions was revealed in the plasma of rats, while only the level of nitrite was significantly changed in the testes of animals. The per os administration of the NSE aqueous suspension in a dose of 50 mg/kg during 10 days to the rats with induced diabetes contributed to the normalization of catalase activity in the testis, which correlated with a decrease in the amount of TBA-reacting products and activity of superoxide dismutase and catalase in the blood plasma of animals; the use of NSE also contributed to the reduction of nitrite content in the gonads and to normalization of both nitrite and nitrate in the blood plasma of rats. The NSE administration to intact animals caused an increase in superoxide dismutase activity and significantly reduced the content of stable NO metabolites in the blood plasma of animals.     

Influence of nitric oxide donor,NO-synthase substrate,and inhibitor on leucocyte ROS-generating activity during ascites tumor growth

The influence of nitric oxide (NO) donor, NO-synthase substrate (L-arginine), and inhibitor (nitroarginine) on the reactive oxygen species (ROS)-generating activity of blood plasma polymorphonuclear leucocytes and ascitic fluid macrophages was studied during tumor growth in animal organisms. It was found that, in the initial period of tumor growth, 8 × 10⁻⁵ M sodium nitroprusside (SNP), which is an NO donor, reduced the potential ROS-generating activity of macrophages by 38.5 ± 9% and plasma polymorphicnuclear leucocytes by 27.6 ± 7%. However, the dynamics of this process during the tumor growth was conservative and variations in ROS production by phagocytes were 10 ± 3%. L-arginine induced a decrease in the ROS-generating activity of granulocytes and mononucleares by 25–30%. The results point to inducible inhibition effect of NO-synthase on the ROS-generating activity of NADPH-oxidase in the course of tumor growth. Nitroarginine, an inhibitor of NO-synthase, produced stable increase in the ROS-generating activity of phagocytes isolated from the tumor at different periods of its growth. The use NO-synthase inhibitors to increase the ROS level in the area of tumor growth may favor the suppression of tumor-cell growth in vivo.