Role of nitric oxide on diaphragmatic contractile failure in Escherichia coli endotoxemic rats

Contractile dysfunction of the respiratory muscles plays an important role in the genesis of respiratory failure during sepsis. Nitric oxide (NO), a free radical that is cytotoxic and negatively inotropic in the heart and skeletal muscle, is produced in large amounts during sepsis by a NO synthase inducible (iNOS) by LPS and/or cytokines. The aim of this study was to investigate whether iNOS was induced in the diaphragm of Escherichia coli endotoxemic rats and whether inhibition of iNOS induction or of NOS synthesis attenuated diaphragmatic contractile dysfunction. Rats were inoculated intravenously (IV) with 10 mg/kg of E. coli endotoxin (LPS animals) or saline (C animals). Six hours after LPS inoculation animals showed a significant increase in diaphragmatic NOS activity (L-citrulline production, P < 0.005). Inducible NOS protein was detected by Western-Blot in the diaphragms of LPS animals, while it was absent in C animals. LPS animals had a significant decrease in diaphragmatic force (P < 0.0001) measured in vitro. In LPS animals, inhibition of iNOS induction with dexamethasone (4 mg/kg IV 45 min before LPS) or inhibition of NOS activity with N(G)-methyl-L-arginine (8 mg/kg IV 90 min after LPS) prevented LPS-induced diaphragmatic contractile dysfunction. We conclude that increased NOS activity due to iNOS was involved in the genesis of diaphragmatic dysfunction observed in E. coli endotoxemic rats.     

The effects of n-stearoylethanolamine on the NO-synthase pathway of NO generation in the aorta and heart of streptozotocin-induced diabetic rats

The aim of the presented experiments was to study the influence of suturated NAE--N-stearoylethanolamine (NSE) on the NO synthesis by NO-synthases in aorta and heart tissues of rats with developmental (12-week) streptozotocin-induced (50 mg/kg of body weight) diabetes. Also we evaluated the state of endothelium-dependent relax reactions of aorta smooth muscles. It was shown that the development of diabetes is accompanied with disbalance of NO-synthesis wich consist in inducible NOS (iNOS) activation and inhibition of constitutive NOS (cNOS) and arginase activities. The aorta smooth muscle endothelium-dependent relax reactions were decreased in diabetic rats. The NSE administration to rats with development streptozotocin-induced diabetes resulted in inhibition of iNOS activity and elevation of cNOS and arginase activities in these tissues. Normalization of NO-synthesis under NSE action was accompanied with restoration of aorta smooth muscle endothelium-dependent relax reactions in diabetic rats.     

iNOS activity in the aged rat liver tissue

Free radical damage to many cellular components has been proposed as the main mechanism underlying the aging process. In the liver, NO can be generated by iNOS, but also by the constitutively expressed endothelial NOS (eNOS). iNOS enzyme appears to be expressed in liver disease such as cirrhosis and fulminant hepatitis, while the eNOS is expressed in physiological conditions. Ten young and ten old Wistar rats were sacrificed and their livers were excised. Liver sections were incubated with an anti-iNOS antibody of rabbit origin. RT-PCR and Western blot analysis were performed and nitric oxide activity was calculated. A significant increase of iNOS immunoreactivity was seen in the aged liver sections versus young liver sections. iNOS protein is expressed in greater quantities in the aged group, compared to the young group. In this study we show, for the first time, that aging in the rat liver is accompanied by a spontaneous induction of iNOS mRNA, high levels of iNOS protein and immunohistochemistry/image analysis.     

Co-induction of nitric oxide and tetrahydrobiopterin synthesis in the myocardium in vivo

Induction of the inducible isoform of nitric oxide (NO) synthase (iNOS) in the myocardium is implicated as a mechanism in the development of cardiac depression in immune activated states associated with an enhanced release of cytokines, such as septic shock. We evaluated the in vivo synthesis of NO and tetrahydrobiopterin (BH4), a cofactor of NOS, in the heart tissue using a model of LPS injection in rats (LPS: 10 mg/kg, i.v.). In control rats, iNOS activity or iNOS mRNA in the heart was negligible. Three hours after LPS administration, a marked induction of iNOS mRNA and activity was observed in the heart. A significant increase in BH4 content and GTP cyclohydrolase mRNA abundance was also observed in the heart from LPS-treated rats. Our results demonstrate induction of NO synthesis and parallel increase in BH4 concentration in the heart of rats after LPS treatment in vivo and may provide molecular evidence responsible for the increased production of BH4 which may up-regulate iNOS activity in the heart in vivo. (Mol Cell Biochem 166: 177-181, 1997)     

Age-related decline of inducible nitric oxide synthase gene expression in primary cultured rat hepatocytes

Hepatocytes exhibit a non-specific immune response by expressing the enzyme inducible nitric oxide (NO) synthase (iNOS, NOS2) through the stimulation of a mixture of cytokines, or a single cytokine such as interleukin-1beta. We examined the age-dependent inducibility of the iNOS gene expression and the capacity of NO production in response to lipopolysaccharide (LPS) or interleukin-1beta (IL-1beta) in primary cultured rat hepatocytes that were isolated from the livers of rats, 3 (young) and 24 (aging) months of age. NO production (NO2-), indicating iNOS activity, was much higher in the young rat hepatocytes following stimulation with LPS or IL-1beta. Likewise, in the young hepatocytes, Western blot analyses showed much higher protein levels in the iNOS expression; it was also a little higher in mRNA levels that were analyzed by RT-PCR. Furthermore, after stimulation with IL-1beta, the levels of transactivation of nuclear factor-KB (NF-kappaB) that were involved in the induction of the iNOS gene were reduced without a significant difference in the aged cells. Therefore, the decrease of NO formation in the aged hepatocytes was due to the belated and incomplete inducibility of the iNOS protein expression, together with a minor contribution of the reduced-transactivation of NF-kappaB. These results suggest that the age-related decline of the iNOS gene expression in primary rat hepatocytes may be associated with the increased incidence of many infective diseases with aging.     

Paradoxical effects of two oximes on nitric oxide production by purified NO synthases, in cell culture and in animals.

We have studied the impact of two novel compounds TO-85 (2,6-di-(alpha-aziridino-alpha-hydroxyiminomethyl)pyridine and TO-133 (bis-(diaziridinoglyoximato)copper), designed as NO donors, on nitrite production by cell cultures, NO production in rat tissues and their ability to inhibit purified NO synthases (NOS). Both substances induced considerable increase of nitrite production in cell cultures. When NO production was assayed in rat organs by means of ESR using Fe(DETC) as a spin trap the anticipated NO-increasing activity of TO-85 was observed only in kidneys; the NO level increasing almost 10-fold. Treatment of rats with TO-133, decreased the NO concentration in brain cortex, cerebellum and liver. When the drugs were administered to animals with high level of iNOS expression induced by LPS, TO-85 did not significantly modify the LPS-induced NO production; administration of TO-133 caused a significant decrease of NO production in blood, brain cortex and cerebellum. Only high concentrations of TO-85 were capable of inhibiting iNOS (IC50=7 mM), the substance inhibited eNOS at lower concentrations (IC50=250 microM). Inhibitory activities of TO-85 on nNOS were dependent on BH4 concentrations, suggesting eventual competition of TO-85 with BH4 when the substance interacts with nNOS. TO-133 reduced eNOS activity with IC50=200 microM, nNOS activity with IC50=200 microM, iNOS activity was not much affected by this substance. Thus, the two tested compounds manifest opposite effects on NO production by purified enzymes and in cell culture. The pattern of the NO synthesis modification in a living animal appears to be even more complex. Our results stress the importance of direct measurements of NO in the tissues using the ESR method.     

In vivo and in vitro effects of lysine clonixinate on nitric oxide synthase in LPS-treated and untreated rat lung preparations.

Recent studies have shown that some nonsteroidal antiinflammatory drugs (NSAIDS) inhibited the inducible NO synthase (iNOS) without direct effect on the catalytic activity of this enzyme. This study was conducted to investigate the in vitro and in vivo effects of lysine clonixinate (LC) and indomethacin (INDO) on NOS activity in rat lung preparation. LC is a drug with antiinflammatory, antipyretic, and analgesic action. In the in vitro experiments, rats were injected with saline or lipopolysaccharide (LPS) and killed 6 h after treatment. Lung preparations were incubated with LC at 2.3 x 10(-5) M or 3.8 x 10(-5) M. The minimum concentration did not modify NOS activity in control or LPS-treated rats but the maximum dose inhibited increased NO production induced by LPS. Furthermore, INDO at 10(-6) M had no effect on enzymatic activity in control or LPS-treated rats. In the in vivo experiments, 40 mg/kg of LC were injected ip. Such a dose did not affect basal production of NO. When LC and LPS were injected simultaneously 6 h before sacrifice, a significant decrease in LPS-induced NOS activity was observed. INDO 10 mg/kg injected in control animals had no effect on NOS activity and did not block LPS induced stimulation of NO production when injected simultaneously. Finally, when LC (40 mg/kg) was injected 3 h after LPS, the enzymatic activity remained unchanged. Expression of iNOS was detected by Western blotting in rats treated with LPS plus 4, 10, 20, and 40 mg/kg of LC. The lowest dose was the only one showing no effect on LPS-induced increase of iNOS. In short, LC is a NSAID with inhibitory action on the expression of LPS-induced NOS, effect that was not seen with INDO in our experimental conditions.     

Up-regulation of rat adrenomedullin gene expression by endotoxin: Relation to nitric oxide synthesis

The effect of bacterial endotoxin (LPS) on adrenomedullin (AM) gene expression was investigated in cultured rat aortic vascular smooth muscle (VSM) cells and in tissues from anesthetized rats. The addition of LPS together with interferon-y to VSM cells resulted in a marked increase in the abundance of AM mRNA as well as the appearance of mRNA for the inducible isoform of nitric oxide (NO) synthase (iNOS). Intravenous injection of LPS into rats also increased AM mRNA abundance and induced iNOS mRNA in lung, heart, liver, and kidney. AM significantly enhanced NO synthesis evoked by LPS and interferon-v in cultured VSM cells. These data suggest that AM may contribute to circulatory failure during endotoxin shock, in part, by modulating NO synthesis.     

Induction of endothelial nitric-oxide synthase in rat brain astrocytes by systemic lipopolysaccharide treatment

In the brain, three isoforms of nitric oxide (NO) synthase (NOS), namely neuronal NOS (nNOS, NOS1), inducible NOS (iNOS, NOS2), and endothelial NOS (eNOS, NOS3), have been implicated in biological roles such as neurotransmission, neurotoxicity, immune function, and blood vessel regulation, each isoform exhibiting in part overlapping roles. Previous studies showed that iNOS is induced in the brain by systemic treatment with lipopolysaccharide (LPS), a Gram-negative bacteria-derived stimulant of the innate immune system. Here we found that eNOS mRNA is induced in the rat brain by intraperitoneal injection of LPS of a smaller amount than that required for induction of iNOS mRNA. The induction of eNOS mRNA was followed by an increase in eNOS protein. Immunohistochemical analysis revealed that eNOS is located in astrocytes of both gray and white matters as well as in blood vessels. Induction of eNOS in response to a low dose of LPS, together with its localization in major components of the blood-brain barrier, suggests that brain eNOS is involved in early pathophysiologic response against systemic infection before iNOS is induced with progression of the infection.     

Endotoxaemia in rats: role of leukocyte sequestration in rapid pulmonary nitric oxide synthase-2 expression.

Nitric oxide (NO), depending on the amount, time and source of generation may exert both, protective and deleterious actions during endotoxic acute lung injury (ALI). Evaluation of the expression and localization of NOS isoforms in the lung of lipopolysaccharide (LPS)-treated rats may contribute to understanding the role of NO in pathogenesis of ALI. Tissue samples (lung, heart, liver, kidney and spleen) as well as peripheral blood polymorphonuclear cells (PMNs) were collected from control male Wistar rats and LPS - treated animals, 15, 30, 60, 120 and 180 min after LPS injection (2 mg kg(-1) min(-1) for 10 minutes, i.v.). Levels of NOS-2 and NOS-3 mRNA and protein in tissues and PMNs were estimated by RT-PCR, Northern blotting and Western blotting. Additionally, myeloperoxidase (MPO) activity in tissue samples was assayed. NOS-3 mRNA as well as protein were detected in lungs of control animals; pulmonary NOS-3 expression was not influenced by LPS. The induction of NOS-2 mRNA in rat lungs and in PMNs isolated from peripheral blood was observed 15 minutes after LPS challenge. In contrast, increase of NOS-2 mRNA in the heart, kidneys, liver and spleen was observed 2-3 hours after LPS injection. In all tissues rise in NOS-2 mRNA was followed after 1-2 hours by increase of NOS-2 protein. Importantly, progressive leukocyte sequestration in the lung parenchyma that started as early as 15 min after LPS injection was revealed only in the lungs; in other organs no significant changes in MPO activity were detected up to 180 min after LPS injection. In conclusion, infusion of LPS caused much more rapid expression of NOS-2 in lungs as compared to the heart, kidneys, liver and spleen. Early induction of NOS-2 may depend on the LPS-stimulated rapid neutrophil sequestration within lung vasculature and fast induction of NOS-2 in sequestrated neutrophils.     

Differential expression and localization of nitric oxide synthases in cirrhotic livers of bile duct-ligated rats.

Increased vascular nitric oxide (NO) production has been implicated in the pathogenesis of the hyperdynamic circulation in liver cirrhosis. This study investigated the expression of three isoforms of NO synthase (NOS) in rat cirrhotic livers. Cirrhosis was induced by chronic bile duct ligation (BDL). NOS enzyme activity was assessed by L-citrulline generation. Competitive RT-PCR was performed to detect the mRNA levels of NOS. In situ hybridization was done to localize NOS mRNA. Protein expression of NOS was evaluated by Western blotting and immunohistochemistry. The L-citrulline assay showed that constitutive NOS (cNOS) enzymatic activity was decreased, while inducible NOS (iNOS) activity was increased in BDL livers. Both endothelial NOS (eNOS) and neuronal NOS (nNOS) mRNA were detected in BDL and sham rats, but with enhanced expression in BDL rats. eNOS protein was redistributed with less expression in sinusoidal endothelial cells, but the total levels in liver were not changed. nNOS was induced in hepatocytes of BDL rats, in contrast to only a weak signal observed around some blood vessels in sham livers. Intense mRNA and protein expression of iNOS was induced in livers of BDL rats and was localized in hepatocytes, with no or a negligible amount in control livers. In conclusion, iNOS was induced in cirrhotic liver with its activity increased. In contrast, cNOS activity was impaired, regardless of unchanged eNOS protein levels and enhanced nNOS expression. These results suggest that all three types of NOS have a role in cirrhosis, but their expression and regulation are different.     

Effects of a selective endothelin a receptor antagonist on the expressions of iNOS and eNOS in the heart of early streptozotocin-induced diabetic rats

Vascular tone is regulated through the actions of locally produced agents. Among the vasoconstrictors, the most potent agent is endothelin (ET), which exerts its vasoconstrictor actions principally through ET type A (ET(A)) receptors. Of the vasodilators, nitric oxide (NO) seems to be the most important contributor to the acute regulation of vascular tone. Vasculopathy is an important feature of diabetes mellitus (DM). Endogenous ET-mediated vasoconstrictor tone is augmented in diabetic states, and conflicting results persist concerning the NO system in diabetes. The present study investigated the expressions of inducible NO synthases (iNOS) and endothelial NOS (eNOS) in the heart of diabetic animals and the effects of a selective ET(A) receptor antagonist on these alterations. Type I diabetes was induced by intraperitoneal injection of streptozotocin (65 mg/kg) in Sprague-Dawley rats, while control (Con) rats received only citrate buffer. After 1 week, the streptozotocin-administered rats were randomly divided into two groups: the selective ET(A) receptor antagonist-administered group (DM+TA-0201, 1 mg/kg/day, by osmotic minipump for 2 weeks) and the DM+vehicle group (comprising the diabetic rats that received saline). The random blood glucose level was 405 +/- 103 mg/dl in DM animals, and this level was unchanged by ET antagonism. Body weight was more greatly decreased in DM rats than in Con rats, but the left ventricle to body weight ratio was increased in the DM group and was unaffected by ET antagonism. Protein expressions of eNOS and iNOS were assessed in the left ventricular tissues. eNOS expression was significantly increased in DM heart and was greatly inhibited by the treatment with ET antagonist. The expression of iNOS was also increased in early DM heart but was reversed by the ET antagonist. Thus, endothelin antagonism might be beneficial for DM heart by reversing the upregulated eNOS and iNOS expressions.     

Therapeutic effect of green tea extract on oxidative stress in aorta and heart of streptozotocin diabetic rats

Hyperglycemia induced oxidative stress has been proposed as a cause of many complications of diabetes including cardiac dysfunction. The present study depicts the therapeutic effect of green tea extract on oxidative stress in aorta as well as heart of streptozotocin diabetic rats. Six weeks after diabetes induction, green tea was administered orally for 4 weeks [300 mg (kg body weight)(-1) day (-1)]. In aorta and heart of diabetic rats there was a significant increase in the activity of superoxide dismutase, catalase and glutathione peroxidase with an increase in lipid peroxides. Diabetic rats showed a significant decrease in the levels of serum and cardiac glutathione. Green tea administration to diabetic rats reduced lipid peroxides and activity of antioxidant enzymes whereas increased glutathione content. The results demonstrate that the induction of antioxidant enzymes in diabetic rats is not efficient and sufficient to reduce the oxidative stress. But green tea by providing a competent antioxidative mechanism ameliorates the oxidative stress in the aorta and heart of diabetic rats. The study suggests that green tea may provide a useful therapeutic option in the reversal of oxidative stress induced cardiac dysfunction in diabetes mellitus.     

Comparison of effects of nitric oxide synthase (NOS) inhibitors on plasma nitrite/nitrate levels and tissue NOS activity in septic organs

An excessive production of nitric oxide (NO) by NO synthase (NOS) is considered to contribute to circulatory disturbance, tissue damage, and refractory hypotention, which are often observed in septic disorders. It is anticipated that a selective inducible NOS (iNOS) inhibitor with excellent pharmacokinetics may be potentially effective as a novel and potent therapeutic intervention in sepsis. We examined whether or not a selective iNOS inhibitor shows iNOS selectivity at the tissue level, when administered systemically. The effects of four NOS inhibitors on plasma nitrite/nitrate (NOx) and tissue NOS levels were compared in major organs (lungs, liver, heart, kidneys, and brain) 6 hr after the injection of E. coli lipopolysaccharide (LPS) into male Wistar-King rats. The rats treated with the three iNOS inhibitors (N-(3-(aminomethyl)benzyl)acetamidine (1400W), (1 S, 5 S, 6 R, 7 R )-2-aza-7-chloro-3-imino-5-methylbicyclo [4.1.0] heptane hydrochloride (ONO-1714), and aminoguanidine) administered 1 hr after LPS injection, showed dose-dependent decreases in plasma NOx levels and NOS activity in the lungs. The non-selective NOS inhibitor (N(G)-methyl-L-arginine (L-NMMA)) had an effect only at the maximum dose. The differences in in vitro iNOS selectivity among these drugs did not correlate with iNOS selectivity at the tissue level. The relationship between plasma NOx levels and NOS activity in the lungs showed a linear relationship with or without the NOS inhibitors. In conclusion, the iNOS selectivity of these drugs does not seem to differ at the tissue level. Plasma NOx levels may be a useful indicator of lung NOS activity.     

Antifibrotic role of inducible nitric oxide synthase.

Long-term treatment in rats with l-NAME, an isoform-non-specific inhibitor of nitric oxide synthase (NOS), leads to fibrosis of the heart and kidney, suggesting that nitric oxide (NO) may play a role in preventing tissue fibrosis. In this process, a likely target of NO is the quenching of reactive oxygen species (ROS) through peroxynitrite formation, and one possible source for this NO is inducible NOS (iNOS). Using Peyronie's disease (PD) tissue from both human specimens and from a rat model of PD as the source of fibrotic tissue, we investigated if NO derived from iNOS could act as such an antifibrogenic defense mechanism by determining whether: (a) tunical ROS and iNOS are increased in PD; and (b) the long-term inhibition of iNOS activity decreases the NO/ROS balance in the tunica albuginea thereby promoting collagen deposition. It was determined that in the human PD plaque, iNOS mRNA and protein, ROS, collagen, and the peroxynitrite marker, nitrotyrosine, were all increased in comparison to the normal tunica. In the rat model of PD, the fibrotic plaque also showed significant increases in iNOS mRNA and protein, nitrotyrosine, ROS as measured by heme oxygenase-1, and collagen when compared with the normal control tunica. When a selective inhibitor of iNOS, L-NIL, was given to rats with the PD-like plaque, this resulted in a decrease in nitrotyrosine levels but intensified ROS levels and collagen deposition. These data demonstrate that: (a) iNOS induction occurs in both the human and rat PD fibrotic plaque; and (b) that the NO derived from iNOS appears to counteract ROS formation and collagen deposition. Because the inhibition of iNOS activity leads to a decrease in the NO/ROS ratio, thereby favoring the development of fibrosis, it is proposed that iNOS induction in this tissue may be a protective mechanism against fibrosis and abnormal wound healing.     

Characterization of Oxidative Stress in Various Tissues of Diabetic and Galactose-fed Rats

Rats fed a galactose-rich diet have been used for several years as a model for diabetes to study, particularly in the eye, the effects of excess blood hexoses. This study sought to determine the utility of galactosemia as a model for oxidative stress in extraocular tissues by examining biomarkers of oxidative stress in galactose-fed rats and experimentally-induced diabetic rats. Sprague-Dawley rats were divided into four groups: experimental control; streptozotocin-induced diabetic; insulin-treated diabetic; and galactose-fed. The rats were maintained on these regimens for 30 days, at which point the activities of catalase, glutathione peroxidase, glutathione reductase, and superoxide dismutase, as well as levels of lipid peroxidation and reduced and oxidized glutathione were determined in heart, liver, and kidney. This study indicates that while there are some similarities between galactosemic and diabetic rats in these measured indices of oxidative stress (hepatic catalase activity levels and hepatic and renal levels of oxidized glutathione in both diabetic and galactosemic rats were significantly decreased when compared to normal), overall the galactosemic rat model is not closely parallel to the diabetic rat model in extra-ocular tissues. In addition, several effects of diabetes (increased hepatic glutathione peroxidase activity, increased superoxide dismutase activity in kidney and heart, decreased renal and increased cardiac catalase activity) were not mimicked in galactosemic rats, and glutathione concentration in both liver and heart was affected in opposite ways in diabetic rats and galactose- fed rats. Insulin treatment reversed/prevented the activity changes in renal and cardiac superoxide dismutase, renal and cardiac catalase, and hepatic glutathione peroxidase as well as the hepatic changes in lipid peroxidation and reduced and oxidized glutathione, and the increase in cardiac glutathione. Thus, prudence should be exercised in the use of experimentally galactosemic rats as a model for diabetes until the correspondence of the models has been more fully characterized.     

TNF-α、IL-1β及LPS对血管平滑肌细胞一氧化氮合酶基因表达的影响及作用机制研究

通过ＲＮＡ印迹分析和亚硝酸盐含量测定检查ＴＮＦ－α、ＩＬ－１β和ＬＰＳ对大鼠血管平滑肌细胞（ＶＳＭＣ）诱导型一氧化氮合酶（ｉＮＯＳ）基因表达及ＮＯ生成的影响．结果表明,ＴＮＦ－α、ＩＬ－１β和ＬＰＳ均能显著诱导ＶＳＭＣｉＮＯＳ基因表达和促进ＮＯ生成,其作用强度与浓度和作用时间有关;双因素（ＴＮＦ－α＋ＬＰＳ,ＬＰＳ＋ＩＬ－１β）对诱导ｉＮＯＳ基因表达及ＮＯ生成产生协同作用．ＰｏｌｙｍｙｘｉｎＢ和地塞米松可部分抑制ＴＮＦ－α对ｉＮＯＳ基因表达的诱导作用及ＮＯ生成     

Impaired glucose-stimulated insulin secretion in the GK rat is associated with abnormalities in islet nitric oxide production

We investigated implications of nitric oxide (NO) derived from islet neuronal constitutive NO synthase (ncNOS) and inducible NOS (iNOS) on insulin secretory mechanisms in the mildly diabetic GK rat. Islets from GK rats and Wistar controls were analysed for ncNOS and iNOS by HPLC, immunoblotting and immunocytochemistry in relation to insulin secretion stimulated by glucose or l-arginine in vitro and in vivo. No obvious difference in ncNOS fluorescence in GK vs control islets was seen but freshly isolated GK islets displayed a marked iNOS expression and activity. After incubation at low glucose GK islets showed an abnormal increase in both iNOS and ncNOS activities. At high glucose the impaired glucose-stimulated insulin release was associated with an increased iNOS expression and activity and NOS inhibition dose-dependently amplified insulin secretion in both GK and control islets. This effect by NOS inhibition was also evident in depolarized islets at low glucose, where forskolin had a further amplifying effect in GK but not in control islets. NOS inhibition increased basal insulin release in perfused GK pancreata and amplified insulin release after glucose stimulation in both GK and control pancreata, almost abrogating the nadir separating first and second phase in controls. A defective insulin response to l-arginine was seen in GK rats in vitro and in vivo, being partially restored by NOS inhibition. The results suggest that increased islet NOS activities might contribute to the defective insulin response to glucose and l-arginine in the GK rat. Excessive iNOS expression and activity might be deleterious for the beta-cells over time.     

Identification of copper/zinc superoxide dismutase as a novel nitric oxide-regulated gene in rat glomerular mesangial cells and kidneys of endotoxemic rats.

To define the mechanism of nitric oxide (NO) action in the glomerulus, we attempted to identify genes that are regulated by NO in rat glomerular mesangial cells. We identified a Cu/Zn superoxide dismutase (SOD) that was strongly induced in these cells by treatment with S-nitroso-glutathione as a NO-donating agent. Bacterial lipopolysaccharide (LPS) acutely decreased Cu/Zn SOD mRNA levels. The LPS-mediated decrease in Cu/Zn SOD is reversed by endogenously produced NO, as LPS also induced a delayed strong iNOS expression in these cells in vitro, which is accompanied by increased Cu/Zn SOD expression. NO dependency of Cu/Zn SOD mRNA recovery could be demonstrated by inhibition of this process by L-NG-monomethylarginine, an inhibitor of NOS enzymatic activity. To demonstrate the in vivo relevance of our observations, we have chosen LPS-treated rats as a model for induction of a systemic inflammatory response. In these animals, we demonstrate a direct coupling of Cu/Zn SOD expression levels to the presence of NO, as Cu/Zn SOD mRNA levels declined during acute inflammation in the presence of a selective inhibitor of iNOS. We propose that the up-regulation of Cu/Zn SOD by endogenous NO may serve as an adaptive, protective mechanism to prevent the formation of toxic quantities of peroxynitrite in conditions associated with iNOS induction during endotoxic shock.