Increased iNOS activity is essential for pulmonary epithelial tight junction dysfunction in endotoxemic mice

A murine endotoxemia model and cultured Calu-3 monolayers were used to test the hypothesis that excessive nitric oxide (NO) production secondary to induction of inducible NO synthase (iNOS) is a key factor leading to altered tight junction (TJ) protein expression and function in the pulmonary epithelium. C57Bl/6J mice were injected with either Escherichia coli 0111:B4 lipopolysaccharide (LPS; 2 mg/kg) or vehicle. Twelve hours later, leakage of FITC-dextran (M(r) 4 kDa; FD4) from blood into bronchoalveolar lavage fluid was significantly increased in endotoxemic but not control mice. This decrease in bronchoalveolar barrier function was associated with upregulation of iNOS protein expression and NF-kappaB activation in lung tissue. Expression of the TJ proteins, zonula occludens (ZO)-1, ZO-2, ZO-3, and occludin, as assessed by immunoblotting and/or immunofluorescence, decreased in lung after the injection of mice with LPS. Treatment of endotoxemic mice with an isoform-selective iNOS inhibitor [l-N(6)-(1-iminoethyl)lysine; l-NIL] ameliorated LPS-induced changes in TJ protein expression and preserved bronchoalveolar epithelial barrier function. Incubating Calu-3 bronchiolar epithelial monolayers with cytomix (a mixture of 1,000 U/ml IFN-gamma, 10 ng/ml TNF-alpha, and 1 ng/ml IL-1beta) increased permeability to FD4, but adding l-NIL prevented this effect. These results suggest that decreased expression and mistargeting of TJ proteins in lung after systemic inflammation may be NO dependent.     

Involvement of Syk kinase in TNF-induced nitric oxide production by airway epithelial cells

We have recently found that Syk is widely expressed in lung epithelial cells (EC) and participates in beta1 integrin signaling. In this study, we assessed the role of Syk in regulation of NO production. Stimulation of human bronchial EC line HS-24 by TNF caused an increased expression of inducible nitric oxide synthase (iNOS). Inhibition of Syk using siRNA or piceatannol down-regulated the iNOS expression and reduced NO production. This effect occurred in EC simultaneously stimulated via beta1 integrins, suggesting that TNF and beta1 integrins provide co-stimulatory signals. Inhibition of Syk down-regulated TNF-induced p38 and p44/42 MAPK phosphorylation and nuclear translocation of p65 NF-kappaB. Thus, TNF-induced activation of pro-inflammatory signaling in EC leading to enhanced expression of iNOS and NO production was dependent on Syk. Syk-mediated signaling regulates NO production at least partly via activating the MAPK cascade. Understanding the role of Syk in airway EC may help in developing new therapeutic tools for inflammatory lung disorders.     

Cytokine-induced arginase activity in pulmonary endothelial cells is dependent on Src family tyrosine kinase activity

We hypothesized that the Src family tyrosine kinases (STKs) are involved in the upregulation of arginase and inducible nitric oxide synthase (iNOS) expression in response to inflammatory stimuli in pulmonary endothelial cells. Treatment of bovine pulmonary arterial endothelial cells (bPAEC) with lipopolysaccharide and tumor necrosis factor-alpha (L/T) resulted in increased urea and nitric oxide (NO) production, and this increase in urea and NO production was inhibited by the STK inhibitor PP1 (10 microM). The STK inhibitors PP2 (10 microM) and herbimycin A (10 microM) also prevented the L/T-induced expression of both arginase II and iNOS mRNA in bPAEC. Together, the data demonstrate a central role of STK in the upregulation of both arginase II and iNOS in bPAEC in response to L/T treatment. To identify the specific kinase(s) required for the induction of urea and NO production, we studied human pulmonary microvascular endothelial cells (hPMVEC) so that short interfering RNA (siRNA) techniques could be employed. We found that hPMVEC express Fyn, Yes, c-Src, Lyn, and Blk and that the protein expression of Fyn, Yes, c-Src, and Lyn could be inhibited with specific siRNA. The siRNA targeting Fyn prevented the cytokine-induced increase in urea and NO production, whereas siRNAs specifically targeting Yes, c-Src, and Lyn had no appreciable effect on cytokine-induced urea and NO production. These findings support our hypothesis that inflammatory stimuli lead to increased urea and NO production through a STK-mediated pathway. Furthermore, these results indicate that the STK Fyn plays a critical role in this process.     

Inducible nitric oxide synthase inhibitors suppress airway inflammation in mice through down-regulation of chemokine expression

Growing evidence demonstrates that inducible NO synthase (iNOS) is induced in the airways of asthmatic patients. However, the precise role of NO in the lung inflammation is unknown. This study investigated the effect of both selective and nonselective iNOS inhibitors in an allergen-driven murine lung inflammation model. OVA challenge resulted in an accumulation of eosinophils and neutrophils in the airways. Expression of iNOS immunostaining in lung sections together with an increase in calcium-independent NOS activity in lung homogenates was also observed after OVA challenge. Treatment with iNOS inhibitors from the day of challenge to the day of sacrifice resulted in an inhibition of the inflammatory cell influx together with a down-regulation of macrophage inflammatory protein-2 and monocyte chemoattractant protein-1 production. In contrast, eosinophilic and neutrophilic inhibition was not observed with treatment during the sensitization. Both treatments induced an increased production of Th2-type cytokines (IL-4 and IL-5) with a concomitant decrease in production of Th1-type cytokine (IFN-gamma). In vitro exposure of primary cultures of murine lung fibroblasts to a NO donor, hydroxylamine, induced a dose-dependent release of macrophage inflammatory protein-2 and monocyte chemoattractant protein-1. Our results suggest that lung inflammation after allergen challenge in mice is partially dependent on NO produced mainly by iNOS. NO appears to increase lung chemokine expression and, thereby, to facilitate influx of inflammatory cells into the airways.     

Phosphatidylinositol 3-kinase inhibitor suppresses inducible nitric oxide synthase expression in bronchiole epithelial cells in asthmatic rats

Inducible nitric oxide synthase (iNOS) is known to produce nitric oxide (NO), which is a main contributor to asthmatic airway inflammation. Recent studies have shown that phosphatidylinositol 3-kinase (PI3K) is ubiquitously expressed in airway epithelial cells and its inhibition could relieve airway inflammation and hyperresponsiveness. This study aimed to explore the interaction of PI3K and NO signaling in allergic asthma. We investigated the effects of PI3K inhibitor wortmannin on iNOS expression in bronchiole epithelial cells and NO, IL-4 and IFN-γ levels in lung tissues of asthmatic rat model, which was prepared by 10% OVA solution sensitization and 1% OVA aerosol challenge. Our results showed that the ratio of eosinophils to total cells in BALF, PI3K activity, NO and IL-4 levels in lung tissues was increased after OVA sensitization and challenge, but then was attenuated by the administration of wortmannin. In contrast, IFN-γ level in lung tissues was decreased after OVA sensitization and challenge and increased after the administration of wortmannin. The expression of iNOS protein in bronchiole epithelial cells, iNOS mRNA level and iNOS activity in lung tissues was markedly upregulated after OVA sensitization and challenge, but the upregulation was significantly antagonized by wortmannin. Taken together, these data provide evidence that PI3K functions upstream to modulate iNOS/NO signaling, which then promotes the development of airway inflammation in asthmatic animal model. PI3K inhibitor wortmannin could lead to reduced iNOS expression and NO production, therefore inhibiting airway inflammatory responses.     

Dual role of inducible nitric oxide synthase in acute asbestos-induced lung injury

Reactive oxygen and nitrogen species have been implicated in the pathogenesis of asbestos fibers-associated pulmonary diseases. By comparing the responses of inducible nitric oxide synthase (iNOS) knockout and wild-type mice we investigated the consequences of iNOS expression for the development of the inflammatory response and tissue injury upon intratracheal instillation of asbestos fibers. Exposure to asbestos fibers resulted in an increased iNOS mRNA and protein expression in the lungs from wild-type mice. Moreover, iNOS knockout mice exhibited an exceeded pulmonary expression and production of TNF-alpha as well as a higher influx of neutrophils into the alveolar space than wild-type mice. In contrast, iNOS knockout animals displayed an attenuated oxidant-related tissue injury reflected in a decrease in protein leakage and LDH release into the alveolar space as well as weaker nitrotyrosine staining of lung tissue compared to wild-type mice. Data presented here indicate that iNOS-derived NO exerts a dichotomous role in acute asbestos-induced lung injury in that iNOS deficiency resulted in an exacerbated inflammatory response but improved oxidant-promoted lung tissue damage.     

Ibuprofen attenuates cardiopulmonary dysfunction by modifying vascular tone in endotoxemia.

Ibuprofen, a cyclooxygenase inhibitor, improves pulmonary and cardiovascular injury in endotoxemia. We studied the mechanism of the beneficial effects of ibuprofen in relation to production of inflammatory mediators which influence vascular tone in endotoxemia. Rats were randomly assigned to one of three groups: (1) control, (2) endotoxemia alone; and (3) ibuprofen pretreatment and endotoxemia. Plasma and lung lavage concentrations of tumor necrosis factor, thromboxane B2 (TXB2), leukotriene (LT) C4,D4,E4 and nitric oxide (NO) were determined over a 2 h period. Pretreatment with ibuprofen resulted in increased survival, and attenuation of pulmonary and cardiovascular dysfunction when compared to the rats receiving endotoxin alone. The marked elevation in plasma TXB2 concentration in endotoxemic rats was prevented by pretreatment with ibuprofen. Similarly, pretreatment with ibuprofen prevented the decrease in lung lavage NO levels in endotoxemic rats. The improved survival and cardiopulmonary protection in endotoxemic rats pretreated with ibuprofen appears to be related to decreased thromboxane production and preservation of endothelial production of nitric oxide.     

Roles of iNOS and nNOS in sepsis-induced pulmonary apoptosis

Apoptosis(programmed cell death) is induced in pulmonary cells and contributes to the pathogenesis of acute lung injury in septic humans. Previous studies have shown that nitric oxide (NO) is an important modulator of apoptosis; however, the functional role of NO derived from inducible NO synthase (iNOS) in sepsis-induced pulmonary apoptosis remains unknown. We measured pulmonary apoptosis in a rat model of Escherichia coli lipopolysaccharide (LPS)-induced sepsis in the absence and presence of the selective iNOS inhibitor 1400W. Four groups were studied 24 h after saline (control) or LPS injection in the absence and presence of 1400W pretreatment. Apoptosis was evaluated using DNA fragmentation, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining, and caspase activation. LPS administration significantly augmented pulmonary cell apoptosis and caspase-3 activity in airway and alveolar epithelial cells. Pretreatment with 1400W significantly enhanced LPS-induced pulmonary apoptosis and increased caspase-3 and -7 activation. The antiapoptotic effect of iNOS was confirmed in iNOS-/- mice, which developed a greater degree of pulmonary apoptosis both under control conditions and in response to LPS compared with wild-type mice. By comparison, genetic deletion of the neuronal NOS had no effect on LPS-induced pulmonary apoptosis. We conclude that NO derived from iNOS plays an important protective role against sepsis-induced pulmonary apoptosis.     

Increased nitric oxide production accompanies blunted hypoxic pulmonary vasoconstriction in hyperoxic rat lung

Hyperoxia may affect lung physiology in different ways. We investigated the effect of hyperoxia on the protein expression of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS), nitric oxide (NO) production, and hypoxic pulmonary vasoconstriction (HPV) in rat lung. Twenty-four male rats were divided into hyperoxic and normoxic groups. Hyperoxic rats were placed in > 90% F1O2 for 60 h prior to experiments. After baseline in vitro analysis, the rats underwent isolated, perfused lung experiments. Two consecutive hypoxic challenges (10 min each) were administered with the administration of a non-specific NOS inhibitor, N-nitro-L-arginine methyl ester (L-NAME), in between. We measured intravascular NO production, pulmonary arterial pressure, and protein expression of eNOS and iNOS by immunohistochemistry. We found that hyperoxia rats exhibited increased baseline NO production (P < 0.001) and blunted HPV response (P < 0.001) during hypoxic challenges compared to normoxia rats. We also detected a temporal association between the attenuation in HPV and increased NO production level with a negative pre-L-NAME correlation between HPV and NO (R = 0.52, P < 0.05). After L-NAME administration, a second hypoxic challenge restored the HPV response in the hyperoxic group. There were increased protein expression of eNOS (12.6 +/- 3.1-fold, n = 3) (X200) and iNOS (8.1 +/- 2.6-fold, n = 3) (X200) in the hyperoxia group. We conclude that hyperoxia increases the protein expression of eNOS and iNOS with a subsequent increased release of endogenous NO, which attenuates the HPV response.     

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.     

大鼠肢体缺血/再灌注后肺损伤及一氧化氮合酶的变化与意义

目的：研究大鼠肢体缺血／再灌注后急性肺损伤时,内皮型一氧化氮合酶（eNOS）和诱导型一氧化氮合酶（i-NOS）的表达及其在急性肺损伤发生中的作用。方法：雄性Wistar大鼠于后肢根部阻断血流后松解（4h／4h）,分别给予L-Arg和氨基胍（AG）预先干预,分为control、IR、L-Arg和AG组,免疫组织化学方法检测肺组织中iNOS和eNOS的表达,同时检测肺组织中MDA、MPO、W／D和NO2^-／NO3^-值,肺组织形态学观察以评价肺损伤的程度。结果：与control组比较,I／R组eNOS表达降低,iNOS表达增强,MDA、MPO、W／D和NO2^-／NO3^-值增加。肺组织充血、炎细胞浸润,肺泡腔渗液;与I／R组比较,L-Arg组eNOS、iNOS表达无明显变化,NO2^-／NO3^-增加。MDA、MPO、W／D降低,肺组织损伤有减轻趋势,AG组eNOS表达无明显变化,iNOS活性降低,NO2^-／NO3^-减少,MDA、MPO、W／D增加,肺组织损伤有加重趋势。结论：肢体缺血／再灌注急性肺损伤过程中,iNOS表达增加,NO生成增多,在肺损伤发生中有一定的保护作用。     

In situ assessment of oxidant and nitrogenic stress in bleomycin pulmonary fibrosis

Reactive oxygen species (ROS) and nitric oxide (NO) have a role in the development of pulmonary fibrosis after bleomycin administration. The ROS production induces an antioxidant response, involving superoxide dismutases (SODs), catalase, and glutathione peroxidases. We compared in situ oxidative burden and antioxidant enzyme activity in bleomycin-injured rat lungs and normal controls. ROS expression and catalase, glucose-6-phosphate-dehydrogenase (G6PHD), and NOS/NADPH-diaphorase activity were investigated by using histochemical reactions. Nitric oxide synthase (e-NOS and i-NOS) and SOD (MnSOD, Cu/ZnSOD, ECSOD) expression was investigated immunohistochemically. After treatment ROS production was enhanced in both phagocytes and in type II alveolar epithelial cells. Mn, Cu/Zn, and ECSOD were overexpressed in parenchymal cells, whereas interstitium expressed ECSOD. Catalase and G6PHD activity was moderately increased in parenchymal and inflammatory cells. NOS/NADPH-d activity and i-NOS expression increased in alveolar and bronchiolar epithelia and in inflammatory cells. It can be suggested that the concomitant activation of antioxidant enzymes is not adequate to scavenge the oxidant burden induced by bleomycin lung damage. Inflammatory cells and also epithelial cells are responsible of ROS and NO production. This oxidative and nitrosative stress may be a substantial trigger in TGF-β1 overexpression by activated type II pneumocytes, leading to fibrotic lesions.     

Induction of inducible nitric oxide (NO) synthase mRNA and NO production in macrophages infected with influenza A/PR/8 virus and stimulated with its ether-split product

We investigated the inductive activity of infective influenza A/PR/8/34 (PR8) virus and its ether-split product (ESP) on the expression of inducible nitric oxide (NO) synthase (iNOS) and NO production in RAW264.7 (RAW) cells, a murine macrophage (M psi) cell line, and thioglycolate-elicited peritoneal M psi (TPM). In both cells, PR8 virus infection induced iNOS mRNA between 4 hr and 24 hr, attaining a peak value at 12 hr. In correlation with induction of iNOS mRNA, NO amounts increased significantly from 12 to 24 hr. Moreover, this study demonstrated that ESP with the same hemagglutination titer as PR8 virus could induce iNOS mRNA and NO production, although the inductive activity of ESP was weaker than that of PR8 virus. Considering the dual role (beneficial and detrimental roles) of NO on certain inflammatory disorders and virus infections, the inductive activity of influenza virus on the iNOS-mediated NO production independent of its infectivity might contribute to a modification of influenza virus infection.     

Aryl hydrocarbon receptor activation during influenza virus infection unveils a novel pathway of IFN-gamma production by phagocytic cells

The contribution of environmental factors is important as we consider reasons that underlie differential susceptibility to influenza virus. Aryl hydrocarbon receptor (AhR) activation by the pollutant dioxin during influenza virus infection decreases survival, which correlates with a 4-fold increase in pulmonary IFN-gamma levels. We report here that the majority of IFN-gamma-producing cells in the lung are neutrophils and macrophages not lymphocytes, and elevated IFN-gamma is associated with increased pulmonary inducible NO synthase (iNOS) levels. Moreover, we show that even in the absence of dioxin, infection with influenza virus elicits IFN-gamma production by B cells, gammadelta T cells, CD11c(+) cells, macrophages and neutrophils, as well as CD3(+) and NK1.1(+) cells in the lung. Bone marrow chimeric mice reveal that AhR-mediated events external to hemopoietic cells direct dioxin-enhanced IFN-gamma production. We also show that AhR-mediated increases in IFN-gamma are dependent upon iNOS, but elevated iNOS in lung epithelial cells is not driven by AhR-dependent signals from bone marrow-derived cells. Thus, the lung contains important targets of AhR regulation, which likely influence a novel iNOS-mediated mechanism that controls IFN-gamma production by phagocytic cells. This suggests that AhR activation changes the response of lung parenchymal cells, such that regulatory pathways in the lung are cued to respond inappropriately during infection. These findings also imply that environmental factors may contribute to differential susceptibility to influenza virus and other respiratory pathogens.     

Selective in vivo inhibition of inducible nitric oxide synthase in a rat model of sepsis.

Elevated production of nitric oxide (NO) by the inducible NO synthase (type II, iNOS) may contribute to the vascular hyporesponsiveness and hemodynamic alterations associated with sepsis. Selective inhibition of this isoenzyme is a possible therapeutic intervention to correct these pathophysiological alterations. Aminoguanidine has been shown to be a selective iNOS inhibitor and to correct the endotoxin-mediated vascular hypocontractility in vitro. However, to date aminoguanidine has not been shown to selectively block iNOS activity in vivo. The in vivo effects of aminoguanidine were assessed in the cecal ligation and perforation model of sepsis in rats. Aminoguanidine (1.75-175 mg/kg) was administered to septic and sham-operated rats for 3 h before euthanasia and harvest of tissues. NOS activities were determined in the thoracic aorta and lung from these animals. Aminoguanidine (17.5 mg/kg) did not alter the mean arterial pressure; however, it did inhibit induced iNOS (but not constitutive NOS) activity in the lung and thoracic aorta from septic animals. Only the higher dose of aminoguanidine (175 mg/kg) was able to increase the mean arterial pressure in septic and sham-operated animals. Thus selective inhibition of iNOS in vivo with aminoguanidine is possible, but our data suggest that other mechanisms, in addition to iNOS induction, are responsible for the loss of vascular tone characteristic of sepsis.     

Nitric oxide suppression of cellular proliferation depends on cationic amino acid transporter activity in cytokine-stimulated pulmonary endothelial cells

Inducible nitric oxide (NO) synthase (iNOS) is a stress response protein upregulated in inflammatory conditions, and NO may suppress cellular proliferation. We hypothesized that preventing L-arginine (L-arg) uptake in endothelial cells would prevent lipopolysaccharide/tumor necrosis factor-α (LPS/TNF)-induced, NO-mediated suppression of cellular proliferation. Bovine pulmonary arterial endothelial cells (bPAEC) were treated with LPS/TNF or vehicle (control), and either 10 mM L-leucine [L-leu; a competitive inhibitor of L-arg uptake by the cationic amino acid transporter (CAT)] or its vehicle. In parallel experiments, iNOS or arginase II were overexpressed in bPAEC using an adenoviral vector (AdiNOS or AdArgII, respectively). LPS/TNF treatment increased the expression of iNOS, arginase II, CAT-1, and CAT-2 mRNA in bPAEC, resulting in greater NO and urea production than in control bPAEC, which was prevented by L-leu. LPS/TNF treatment resulted in fewer viable cells than in controls, and LPS/TNF-stimulated bPAEC treated with L-leu had more viable cells than LPS/TNF treatment alone. LPS/TNF treatment resulted in cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase expression, which was attenuated by L-leu. AdiNOS reduced viable cell number, and treatment of AdiNOS transfected bPAEC with L-leu preserved cell number. AdArgII increased viable cell number, and treatment of AdArgII transfected bPAEC with L-leu prevented the increase in cell number. These data demonstrate that iNOS expression in pulmonary endothelial cells leads to decreased cellular proliferation, which can be attenuated by preventing cellular L-arg uptake. We speculate that CAT activity may represent a novel therapeutic target in inflammatory lung diseases characterized by NO overproduction.     

Erythropoietin and hypoxia increase erythropoietin receptor and nitric oxide levels in lung microvascular endothelial cells

Acute lung exposure to low oxygen results in pulmonary vasoconstriction and redistribution of blood flow. We used human microvascular endothelial cells from lung (HMVEC-L) to study the acute response to oxygen stress. We observed that hypoxia and erythropoietin (EPO) increased erythropoietin receptor (EPOR) gene expression and protein level in HMVEC-L. In addition, EPO dose- and time-dependently stimulated nitric oxide (NO) production. This NO stimulation was evident despite hypoxia induced reduction of endothelial NO synthase (eNOS) gene expression. Western blot of phospho-eNOS (serine1177) and eNOS and was significantly induced by hypoxia but not after EPO treatment. However, iNOS increased at hypoxia and with EPO stimulation compared to normal oxygen tension. In accordance with our previous results of NO induction by EPO at low oxygen tension in human umbilical vein endothelial cells and bone marrow endothelial cells, these results provide further evidence in HMVEC-L for EPO regulation of NO production to modify the effects of hypoxia and cause compensatory vasoconstriction.     

Cytokines and lipopolysaccharides induce inducible nitric oxide synthase but not enzyme activity in adult rat cardiomyocytes

There is evidence that nitric oxide (NO) formation in adult cardiomyocytes stimulated with lipopolysaccharide (LPS) is not commensurate with iNOS levels. Tetrahydrobiopterin (BH(4)) is a key factor in the stabilization and NO production by iNOS homodimer. Thus we hypothesized that BH(4) is a limiting factor for NO production in adult cardiomyocytes in response to LPS and cytokines (TNF-alpha, IL-1, IFN-gamma alone, or mixed). It was verified that LPS and cytokines induced iNOS expression which did not translate into increased nitrite or [(14)C]citrulline production. This response coincided with defective BH(4) synthesis and low GTP cyclohydrolase activity. Furthermore, supplementation with BH(4) and ascorbate failed to increase iNOS activity. This effect was related to preferential accumulation of BH(2) rather than BH(4) in these cells. Uncoupled iNOS activity in stimulated cells was examined using mitochondrial aconitase activity as an endogenous marker of superoxide anion radical (O(2)(-)) formation, and found not to be significantly inhibited. 2-Hydroxyethidium also was not significantly increased. We conclude that adult cardiomyocytes are an unlikely source of NO and O(2)(-) in inflammatory conditions. This finding adds a new and unexpected layer of complexity to our understanding of the responses of the adult heart to inflammation.     

Nitric oxide (NO) pretreatment increases cytokine-induced NO production in cultured rat hepatocytes by suppressing GTP cyclohydrolase I feedback inhibitory protein level and promoting inducible NO synthase dimerization

Nitric oxide (NO) regulates the biological activity of many enzymes and other functional proteins as well as gene expression. In this study, we tested whether pretreatment with NO regulates NO production in response to cytokines in cultured rat hepatocytes. Hepatocytes were recovered in fresh medium for 24 h following pretreatment with the NO donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP) and stimulated to express the inducible NO synthase (iNOS) with interleukin-1beta and interferon-gamma or transfected with the human iNOS gene. NO pretreatment resulted in a significant increase in NO production without changing iNOS expression for both conditions. This effect, which did not occur in macrophages and smooth muscle cells, was inhibited when NO was scavenged using red blood cells. Pretreatment with oxidized SNAP, 8-Br-cGMP, NO(2)(-), or NO(3)(-) did not increase the cytokine-induced NO production. SNAP pretreatment increased cytosolic iNOS activity measured only in the absence of exogenous tetrahydrobiopterin (BH(4)). SNAP pretreatment suppressed the level of GTP cyclohydrolase I (GTPCHI) feedback regulatory protein (GFRP) and increased GTPCHI activity without changing GTPCHI protein level. SNAP pretreatment also increased total cellular levels of biopterin and active iNOS dimer. These results suggest that SNAP pretreatment increased NO production from iNOS by elevating cellular BH(4) levels and promoting iNOS subunit dimerization through the suppression of GFRP levels and subsequent activation of GTPCHI.