The role of nitric oxide in the development of streptozotocin-induced diabetes mellitus: experimental and clinical implications

The overproduction of the free radical nitric oxide (NO) by activated immunocompetent cells with subsequent development of local oxidative stress is supposed to be one of the possible pathophysiological mechanisms of beta-cell damage during streptozotocin-induced diabetes. The blockade of increased NO production by simultaneous administration of NO-synthase inhibitors partially suppresses the hyperglycemia and the increase of glycated hemoglobin concentration. Here we summarize the current state of knowledge concerning the modulation of streptozotocin-induced diabetes development by treatment with NO-synthase inhibitors including the partial inhibition of the changes in serum leptin levels. The differences in the reaction to streptozotocin administration between wild type mice and inducible NO-synthase knockout mice are also discussed. The overproduction of NO during the development of streptozotocin-induced diabetes is probably an important part of the complex autoimmune reaction which leads to the destruction of pancreatic beta-cells. Further clarification of the role of nitric oxide in streptozotocin-induced diabetes development could have important clinical implications.     

Nitrogen oxide storage as a factor of adaptive defence

Adaptation to environmental factors possesses multiple NO-dependent protective effects and stimulates the NO storage. An adaptation to a mild stress was shown to reduce the death rate in rats from 57% to 8% and to prevent a heat shock-induced hypotension and endothelial overactivation. Treatment of the rats with the NO-synthase inhibitor L-NNA interfered with the NO storage and formation of protective effects, while the NO donor dinitrosyl iron complex facilitated the NO storage and simulated the adaptive defence. The data obtained suggest an important role of the NO storage in adaptive defence of the organism.     

Antioxidative protection in the central nervous ganglia of a mollusc Lymnaea stagnalis at modulation of activity of the NO-ergic system

In experiments on molluscs Lymnnaea stagnalis the state of antioxidative protection is studied in central nervous ganglia during a long-term activation (inhibition) of synthesis of nitric monoxide (NO) in the body. Effect of the blocker of NO-synthase N(G)-nitro-L-arginine (L-NNA) at the background of enhancement of pulmonary respiration has been established to be associated with a rise of levels of reduced glutathione and TBK-active products in the nervous tissue at preservation of a relatively high superoxide dismutase activity and a low glutathione peroxidase activity as compared with control group and the animals treated with the metabolic precursor of NO synthesis L-arginine. In spite of the revealed disturbances of balance of the body pro- and antioxidative system, DNA electrophoresis detected no products of its degradation, which can indicate the absence of massive programmed death of the nervous tissue cells in Lymnaea stagnalis during modulation of activity of the NO-ergic system.     

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.     

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.     

The N-stearoylethanolamine effect on the NO-synthase way of nitrogen oxide formation and phospholipid composition of erythrocyte membranes in rats with streptozotocine diabetes

The influence of N-stearoylethanolamine (NSE) on the NO-synthase way of NO generation and phospholipids composition of erythrocyte membranes of rats with streptozotocine-induced diabetes has been studied. It has been shown that the activation of iNOS activity, cNOS activity inhibition and increase of the stable NO metabolites content takes place in the red blood cells (RBC) of diabetic rats. The alterations were also found in the RBC membrane phospholipid content: a decrease of phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, sphingomieline content and increase of phosphatidylethanolamine, phosphatidylcholine lysoforms level. The NSE suspension administration (50 mg/kg of body weight) to diabetic rats (3 months after the diabetes induction) resulted in iNOS activity inhibition, recovering of cNOS activity and normalization of NO stable metabolites level in RBC. The decrease of phospholipids lysoform levels, normalization of phosphatidylethanolamine, phosphatidylcholine content and increase of phosphatidylinositol level were found after NSE action.     

Nitric oxide effect on the hemoglobin-oxygen affinity.

The biological roles of nitric oxide (NO)-hemoglobin (Hb) derivatives are obscure. It is proposed that NO can function as an allosteric regulator of hemoglobin oxygen-binding properties. We aimed to estimate the effects of NO donors and NO-synthase substrate (L-arginine) on hemoglobin-oxygen affinity (HOA) in experiments in vitro with the various ratios between NO formed and Hb and various oxygen pressures. HOA index (p50), blood pH, plasma and red blood cell (RBC) concentrations of nitrite/nitrate and methemoglobin amounts were measured after the experiments. In our experiments, blood incubation with NO donors (glyceryltrinitrate, molsidomine, sodium nitroprusside, S-nitrosocysteine) or NO-synthase substrate (L-arginine) did not change HOA even at NO:Hb ratio of 1:1. At the same time our results showed that oxygenated blood incubation with S-nitrosocysteine induced an oxyhemoglobin dissociation curve shift leftwards. This indicates a leading role of met-Hb in a modification of Hb oxygen-binding properties. However other NO-modified forms of hemoglobin (S-nitroso- and nitrosylhemoglobin) also may be involved in the regulation of HOA. The results obtained indicate that nitric oxide can be the allosteric effector of hemoglobin, increasing or decreasing its oxygen affinity - possibly, through the generation of different NO-Hb derivatives.     

Nitric oxide formation in the oropharyngeal tract: possible influence of cigarette smoking.

Cigarette smoking reduces the level of nitric oxide (NO) in exhaled air by an unknown mechanism. The view that part of the effect of cigarette smoking on NO production should occur in the oropharyngeal tract is supported by several studies. We have therefore compared smokers and non-smokers regarding non-enzymatic formation of NO from nitrite in the oral cavity since this is a primary candidate target for cigarette smoke. We have also looked at NO synthase-dependent NO formation in the mucosa of the oropharyngeal tract as an alternative target for the inhibitory effect induced by cigarette smoke. Smokers exhaled 67% lower levels of NO than controls (p<0.01, n=15 each group). We could not detect any significant difference in salivary nitrite, nitrate or ascorbate between smokers and non-smokers. Mouthwash with the antibacterial agent chlorhexidine reduced salivary nitrite (-65%) and exhaled NO levels (-10%) similarly in the two groups. Immunohistochemical techniques revealed dense expression of inducible (but not endothelial or neuronal) NO synthase in the squamous epithelium of non-inflamed tonsillar and gingival tissue biopsies. In the same biopsies, significant Ca2+ -independent citrulline-forming activity was detected. We found no difference between smoking and non-smoking subjects regarding NO-synthase expression and in vitro activity. In another group of non-smoking subjects (n=10), spraying the oropharyngeal tract with the NO-synthase inhibitor NG-monomethyl-L-arginine (250 mg) significantly reduced exhaled NO levels for at least 30 min (-18%, p<0.01). Our data suggest that cigarette smoking does not affect non-enzymatic NO formation from nitrite in saliva. However, NO is also formed by inducible NO synthase in the squamous epithelium of the normal oropharyngeal tract. We suggest that cigarette smoking may down-regulate enzymatic NO formation in the oropharyngeal compartment as well as in the bronchial compartment.     

Arginase activity in the frog urinary bladder epithelial cells and its involvement in regulation of nitric oxide production

The activity of arginase converting arginine into ornithine and urea is of particular interest among many factors regulating NO production in the cells. It is known that by competing with NO-synthase for common substrate, arginase can affect the NO synthesis. In the present work, the properties of arginase from the frog Rana temporaria L. urinary bladder epithelial cells possessing the NO-synthase activity were characterized, and possible contribution of arginase to regulation of NO production by epithelial cells was studied. It has been shown that the enzyme had the temperature optimum in the range of 55-60 degrees C, K(m) for arginine 23 mM, and V(max) about 10 nmol urea/mg protein/min, and its activity was effictively inhibited by (S)-(2-boronoethyl)-L-cysteine (BEC), an inhibitor of arginase, at concentrations from 10(-6) to 10(-4) M. The comparison of arginase activity in various frog tissues revealed the following pattern: liver > kidney > brain > urinary bladder (epithelium) > heart > testis. The arginase activity in the isolated urinary bladder epithelial cells was 3 times higher than that in the intact urinary bladder. To evaluate the role of arginase in the regulation of NO production, epithelial cells were cultivated in the media L-15 or 199 containing different amounts of arginine; the concentration of NO2-, the stable NO metabolite, was determined in the culture fluid after 18-20 h of cells incubation. The vast majority of the produced nitrites are associated with the NOS activity, as L-NAME, the NOS-inhibitor, decreased their accumulation by 77.1% in the L-15 medium and by 80% in 199 medium. BEC (10(-4) M) increased the nitrite production by 18.0 % +/- 2.7 in the L-15 medium and by 24.2 +/- 3.5 in the 199 medium (p < 0.05). The obtained data indicate a relatively high arginase activity in the frog urinary bladder epithelium and its involvement in regulation of NO production by epithelial cells.     

Oxidative stress in pathogenesis of arterial hypertension in ISIAH rats]

The NO-synthase activity and the rate of NO production in the rat aortic wall and cerebellum were 2-1.5-fold higher in the ISIAH rats than in normotensive WAG rat strain. In contrast, the superoxide dismutase (SOD) activity was significantly reduced in the ISIAH rats. The blood level of reduced thiols was also much lower in the ISIAH rats. The findings suggest that oxidative stress may play a significant role in pathogenesis of stress-sensitive hypertension in the ISIAH rat strain.     

State of antioxidative protection in central nervous ganglia of the mollusc <Emphasis Type="Italic">Lymnaea stagnalis</Emphasis> at modulation of activity of the NO-ergic system

In experiments on mollusc Lymnaea stagnalis, the state of antioxidative protection is studied in central nervous ganglia during a long-term activation (inhibition) of synthesis of nitrogen monoxide (NO) in the body. The effect of the blocker of NO-synthase N^G-nitro-L-arginine (L-NNA) at the background of enhancement of pulmonary respiration has been found to be associated with a rise of levels of reduced glutathione and TBK-active products in the nervous tissue at preservation of a relatively high superoxide dismutase activity and a low glutathione peroxidase activity compared with the control group and the animals treated with the metabolic precursor of NO synthesis L-arginine. In spite of the revealed disturbances of balance of the body proand antioxidative system, DNA electrophoresis detected no products of its degradation, which can indicate the absence of massive programmed death of the nervous tissue cells in Lymnaea stagnalis during modulation of activity of the NO-ergic system.     

Effect of N-stearoylethanolamine on the level of stable NO metabolites in different pathological conditions which are accompanied by oxidative stress

The effects of N-stearoylethanolamine (NSE) at the level of stable NO metabolites--NO2- NO3- under different pathological conditions which were accompanied by oxidative stress and NO disbalance were studied. It was found that NSE promoted the increasing of NO metabolites level on the animal models with deficit of NO compared to control and evoked decreasing content of NO2- and NO3- under pathological conditions with NO overproduction. It is suppoused, that NSE effects could be determined, by its ability to modulate the the activity of NO-synthase izoformes.     

The role of polymorphic variants of gene of inducible NO-synthase NOS2 in brain infarction in patients with acute ischemic stroke

Cerebrovascular diseases including stroke are an important problem of public health. Stroke development depends on external factors and individual genetic specificity of patient. Excessive NO production by inducible NO-synthase (iNOS) damages brain tissue at various stages of the disease. The goal of this work was to study the role of 4 polymorphic variants of gene of inducible NO-synthase iNOS (-2447C/G, -1659C/T, -0,7(OTTA)n I/D, S608L (150C/T)) in brain infarction in patients with acute ischemic stroke. A statistically significant correlation between S608L (150C/T) polymorphism and infarction dynamics was observed during days 1-3 and 7-21 after infarction. These parameters correlated with neurological status estimated using the Orgogozo scale during days 1-7 of the disease development. It was demonstrated that genotype N150N was associated with ischemic focus propagation regardless of its volume and neurological status by Orgogozo scale in patients with acute stroke. It was also observed that genotype N150N had effect on ischemic damage during days 1-3 in case of low initial volume.     

Nitroxidergic neurons in nuclei of rat and human medulla oblongata

Four types of neurons distinguished by NADPH-diaphorase reaction have been identified in seven nuclei of human and rat medulla oblongata. It was found that nitrogen oxide (NO)-positive neurons had similar distributions; neurons with high NO-synthase activity predominated in vasomotor nuclei and, in sensitive nuclei, most neurons had low NO-syntase activity.     

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.     

5,6-Dihydroxyindole-2-carboxylic acid,a diffusible melanin precursor,is a potent stimulator of lipopolysaccharide-induced production of nitric oxide by J774 macrophages

Pre-incubation of J774 murine macrophages with 5,6-dihydroxyindole-2-carboxylic acid (DHICA), a diffusible intermediate in the biosynthesis of eumelanins, leads to a marked increase in the levels of nitric oxide (NO) produced by lipopolysaccharide (LPS)-induced NO-synthase (iNOS). The effect varies with DHICA concentratior being maximum at a concentration of 1 × 10⁻⁶M, and is suppressed by the NOS inhibitor N^G-monomethyl-L-arginine (L-NMMA). No stimulation is observed when macrophages are exposed to DHICA after activation with LPS, indicating that the indole does not affect the catalytic activity of iNOS. These results point to a hitherto unrecognized role of DHICA as a chemical messenger mediating interaction between active melanocytes and macrophages in epidermal inflammatory and immune responses.     

Hypothalamic monoamines in the cold stress during changes in activity of nitric oxide system

Effects of NO-synthase inhibitor N(omega)-nitro-L-arginine (LNA) and donor sodium nitroprusside (SNP) on alteration in body temperature, plasma corticosterone level and hypothalamic monoamines in response to cold exposure, were studied. Drop of the body temperature in cold exposure in rats treated with LNA or SNP was the same as in the control group. Administration of SNP (2 mg/kg i.p.) significantly increased the basal level of corticosterone (CS). Cold exposure elevated CS in all groups of rats. LNA did not markedly alter the hypothalamic noradrenaline (NA) while SNP significantly decreased the NA. Cold exposure resulted in additional decrease of the NA in SNP-treated rats. NA was found to significantly increase within 48 hrs following the cold exposure in the LNA as well as in the SNP groups. SNP significantly increased basal dopamine and DOPAC levels. Cold exposure did not affect hypothalamic dopamine. In the experiments, NO changes of serotonin and 5-hydroxyindoleacetic acid were observed. The findings suggest that antagonistic effects of the NO-synthase inhibitor and NO donor postulated in literature for various kinds of stress do not occur in experiments with cold stress.     

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.