Role Of Nitric Oxide Synthase Research Articles

Differential roles of nitric oxide synthases in regulation of ultraviolet B light-induced apoptosis

Ultraviolet B light (UVB) activates nitric oxide synthase(s) (NOSs) and nitric oxide (NO ) production, which plays a role in regulation of apoptosis. However, the role of NO in UVB-induced apoptosis remains controversial. In this study, we analyzed expression and activation of constitutive NOSs (cNOSs) and their roles in UV-induced apoptosis of HaCaT keratinocytes. Our data showed that the expression of neuronal NOS (nNOS) was increased while endothelial NOS (eNOS) was uncoupled in the early phase (0–6 h) post-UVB. The expression of both cNOSs peaked at 12 h post-UVB and NO was transiently elevated with 30 min and then steadily rose from 6 to 18 h post-UVB. The expression of iNOS was detected at 6 h post-UVB and then sturdily increased. Inhibition of cNOSs with l-NAME reduced the inducibility of NO in the early and late phases of irradiation. Along with the eNOS uncoupling, an increased level of peroxynitrite (ONOO −) was detected in the early phase, but not in the late phase post-UVB. Inhibition of cNOSs reduced the production of ONOO − in the early time, but led to an increase of ONOO − in the late time after UVB-irradiation. The results indicate that cNOSs regulate NO /ONOO − imbalance after UVB-irradiation. Our data suggested that the activation of cNOSs in the early phase post-UVB leads to NO /ONOO − imbalance and promotes apoptosis via a caspase 3-independent pathway. The elevation of NO in the late phase of UVB-irradiation is mainly produced by inducible NOS (iNOS). However, cNOSs also contribute to the NO production and to maintain a higher NO /ONOO − ratio, which reduces caspase 3 activity and protects cells from UVB-induced apoptosis.

Nitric oxide synthase 3 contributes to ventilator-induced lung injury.

Nitric oxide synthase (NOS) depletion or inhibition reduces ventilator-induced lung injury (VILI), but the responsible mechanisms remain incompletely defined. The aim of this study was to elucidate the role of endothelial NOS, NOS3, in the pathogenesis of VILI in an in vivo mouse model. Wild-type and NOS3-deficient mice were ventilated with high-tidal volume (HV(T); 40 ml/kg) for 4 h, with and without adding NO to the inhaled gas. Additional wild-type mice were pretreated with tetrahydrobiopterin and ascorbic acid, agents that can prevent NOS-generated superoxide production. Arterial blood gas tensions, histology, and lung mechanics were evaluated after 4 h of HV(T) ventilation. The concentration of protein, IgM, cytokines, malondialdehyde, and 8-isoprostane were measured in bronchoalveolar lavage fluid (BALF). Myeloperoxidase activity, total and oxidized glutathione levels, and NOS-derived superoxide production were measured in lung tissue homogenates. HV(T) ventilation induced VILI in wild-type mice, as reflected by decreased lung compliance, increased concentrations of protein and cytokines in BALF, and oxidative stress. All indices of VILI were ameliorated in NOS3-deficient mice. Augmenting pulmonary NO levels by breathing NO during mechanical ventilation did not increase lung injury in NOS3-deficient mice. HV(T) ventilation increased NOS-inhibitable superoxide production in lung extracts from wild-type mice but not in those from NOS3-deficient mice. Administration of tetrahydrobiopterin and ascorbic acid ameliorated VILI in wild-type mice. Our results indicate that NOS3 contributes to ventilator-induced lung injury via increased production of superoxide.

Abstract 1400: Mitigation of radiation-induced injury with the nitric oxide synthase co-factor, tetrahydrobiopterin

Abstract The purpose of this study was to determine the effect of modulation of the nitric oxide (NO) and the cGMP/PKG signaling pathway on radiation-induced myofibroblast differentiation and radiation-induced injury in mice. Nitric oxide (NO) is a diverse second messenger critical for a variety of cell functions produced by nitric oxide synthases (NOS). NOS activity is regulated in part by the availability of co-factors including tetrahydrobiopterin (BH4). In the absence of BH4, NOS can become ‘uncoupled’ to produce superoxide anion which upon reaction with NO can form peroxynitrite, a highly reactive molecule known to contribute to protein tyrosine nitration. BH4 is approved for human consumption and therefore may represent a safe, viable strategy to reduce tissue injury from radiation. We show here that BH4 administration to mice prior to whole body irradiation with 9.5 Gy significantly increases mouse survival. In contrast, inhibition of NOS activity either before or after irradiation significantly reduced survival. We also show that the phosphodiesterase 5 inhibitor, sildenafil, inhibits the radiation-induced differentiation of human myofibroblasts. The mechanisms underlying the protective effects of BH4 and the role of NOS in radiation-induced tissue injury will be discussed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1400.

Decreased Transplant Arteriosclerosis in Endothelial Nitric Oxide Synthase-Deficient Mice

Occlusive vascular changes, characterized by the formation of a neointima with lumen obstruction, are key histologic findings of allograft arteriosclerosis. Vascular integrity of the graft is critically dependent on nitric oxide (NO), synthesized by NO synthases (NOS), of which three isoforms have been located in the arterial wall: endothelial NOS (eNOS), inducible NOS, and neuronal NOS (nNOS). We have studied the role of NOS in a murine model of aortic allograft rejection. The descending thoracic aorta of donor mice (BALB/c mice) was transplanted into two groups of recipients: (a) C57BL/6J and (b) C57BL/6J mice homozygous (-/-) for a knockout of the eNOS gene (eNOS(-/-)). After 4 weeks, pronounced neointima formation, upregulated expression of adhesion molecules, and increased infiltration by inflammatory cells were demonstrated in wild-type recipient mice, whereas eNOS(-/-) recipient mice were protected from neointima development by a significantly increased synthesis of NO, as shown by increased formation of cGMP; this was mainly explained by upregulation of inducible NOS and nNOS. Upregulation of inducible NOS and nNOS isoforms may be beneficial in preventing allograft arteriosclerosis in the early posttransplant period.

KMUP‐1 attenuates isoprenaline‐induced cardiac hypertrophy in rats through NO/cGMP/PKG and ERK1/2/calcineurin A pathways

To determine whether KMUP-1, a novel xanthine-based derivative, attenuates isoprenaline (ISO)-induced cardiac hypertrophy in rats, and if so, whether the anti-hypertrophic effect is mediated by the nitric oxide (NO) pathway. In vivo, cardiac hypertrophy was induced by injection of ISO (5 mg.kg(-1).day(-1), s.c.) for 10 days in Wistar rats. In the treatment group, KMUP-1 was administered 1 h before ISO. After 10 days, effects of KMUP-1 on survival, cardiac hypertrophy and fibrosis, the NO/guanosine 3'5'-cyclic monophosphate (cGMP)/protein kinase G (PKG) and hypertrophy signalling pathways [calcineurin A and extracellular signal-regulated kinase (ERK)1/2] were examined. To investigate the role of nitric oxide synthase (NOS) in the effects of KMUP-1, a NOS inhibitor, N(omega)-nitro-L-arginine (L-NNA) was co-administered with KMUP-1. In vitro, anti-hypertrophic effects of KMUP-1 were studied in ISO-induced hypertrophic neonatal rat cardiomyocytes. In vivo, KMUP-1 pretreatment attenuated the cardiac hypertrophy and fibrosis and improved the survival of ISO-treated rats. Plasma NOx (nitrite and nitrate) and cardiac endothelial NOS, cGMP and PKG were all increased by KMUP-1. The activation of hypertrophic signalling by calcineurin A and ERK1/2 in ISO-treated rats was also attenuated by KMUP-1. All these effects of KMUP-1 were inhibited by simultaneous administration of L-NNA. Similarly, in vitro, KMUP-1 attenuated hypertrophic responses and signalling induced by ISO in neonatal rat cardiomyocytes. KMUP-1 attenuates the cardiac hypertrophy in rats induced by administration of ISO. These effects are mediated, at least in part, by NOS activation. This novel agent, which targets the NO/cGMP pathway, has a potential role in the prevention of cardiac hypertrophy.

Nitric oxide synthase activation and oxidative stress, but not intracellular zinc dyshomeostasis, regulate ultraviolet B light-induced apoptosis

Aims To investigate the role of nitric oxide synthase (NOS) and intracellular free zinc ion (Zn 2+) in regulation of ultraviolet B light (UVB)-induced cell damage and apoptosis. Main methods Real-time confocal microscopy measurement was used to determine the changes of intracellular free zinc concentration under different conditions. Cell apoptotic death was determined using fluorescein isothiocyanate (FITC) conjugated-annexin V (ANX5)/PI labeling followed by flow cytometry. Western analysis was used to determine cell apoptosis and eNOS uncoupling. Key findings UVB induced an elevation of Zn 2+ within 2 min of exposure. The UVB-induced intracellular Zn 2+ elevation was dependent on the increase of constitutive nitric oxide synthase (cNOS) activity and production of superoxide. Removal of Zn 2+ with a lower concentration (< 25 μM) of N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), a Zn 2+-specific chelator, did not induce cell death or prevent cells from UVB-induced apoptosis. However, a higher [TPEN] (> 50 μM) was cytotoxic to cells, but prevented cells from further UVB-induced apoptosis. The higher [TPEN] also induced cNOS uncoupling. Furthermore, treating the cells with a membrane permeable superoxide dismutase (PEG-SOD) inhibited Zn 2+ release and reduced apoptotic cell death after UVB treatment. The results demonstrated a complex and dynamic regulation of UVB-induced cell damage. Significance Our findings not only advance our understanding of the correlations between cNOS activation and Zn elevation, but also elucidated the role of cNOS in regulation of oxidative stress and apoptosis upon UVB-irradiation.

Role of inducible nitric oxide synthase on the development of virus-associated asthma exacerbation which is dependent on Th1 and Th17 cell responses

Asthma is characterized by airway inflammation induced by immune dysfunction to inhaled antigens. Although respiratory viral infections are the most common cause of asthma exacerbation, immunologic mechanisms underlying virus-associated asthma exacerbation are controversial. Clinical evidence indicates that nitric oxide (NO) levels in exhaled air are increased in exacerbated asthma patients compared to stable patients. Here, we evaluated the immunologic mechanisms and the role of NO synthases (NOSs) in the development of virus-associated asthma exacerbation. A murine model of virus-associated asthma exacerbation was established using intranasal challenge with ovalbumin (OVA) plus dsRNA for 4 weeks in mice sensitized with OVA plus dsRNA. Lung infiltration of inflammatory cells, especially neutrophils, was increased by repeated challenge with OVA plus dsRNA, as compared to OVA alone. The neutrophilic inflammation enhanced by dsRNA was partly abolished in the absence of IFN-gamma or IL-17 gene expression, whereas unaffected in the absence of IL-13. In terms of the roles of NOSs, dsRNA-enhanced neutrophilic inflammation was significantly decreased in inducible NOS (iNOS)-deficient mice compared to wild type controls; in addition, this phenotype was inhibited by treatment with a non-specific NOS inhibitor (L-NAME) or an specific inhibitor (1400 W), but not with a specific endothelial NOS inhibitor (AP-CAV peptide). Taken together, these findings suggest that iNOS pathway is important in the development of virus-associated exacerbation of neutrophilic inflammation, which is dependent on both Th1 and Th17 cell responses.

Central role of nitric oxide synthase in AICAR and caffeine-induced mitochondrial biogenesis in L6 myocytes

5-Aminoimidazole-4-carboxamide-ribonucleoside (AICAR) and caffeine, which activate AMP-activated protein kinase (AMPK) and cause sarcoplasmic reticulum calcium release, respectively, have been shown to increase mitochondrial biogenesis in L6 myotubes. Nitric oxide (NO) donors also increase mitochondrial biogenesis. Since neuronal and endothelial NO synthase (NOS) are calcium dependent and are also phosphorylated by AMPK, we hypothesized that NOS inhibition would attenuate the activation of mitochondrial biogenesis in response to AICAR and caffeine. L6 myotubes either were not treated (control) or were exposed acutely or for 5 h/day over 5 days to 100 microM of N(G)-nitro-L-arginine methyl ester (L-NAME, NOS inhibitor), 100 microM S-nitroso-N-acetyl-penicillamine (SNAP) (NO donor) +/- 100 microM L-NAME, 2 mM AICAR +/- 100 microM L-NAME, or 5 mM caffeine +/- 100 microM L-NAME (n = 12/treatment). Acute AICAR administration increased (P < 0.05) phospho- (P-)AMPK, but also increased P-CaMK, with resultant chronic increases in peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1 alpha), cytochrome-c oxidase (COX)-1, and COX-4 protein expression compared with control cells. NOS inhibition, which had no effect on AICAR-stimulated P-AMPK, surprisingly increased P-CaMK and attenuated the AICAR-induced increases in COX-1 and COX-4 protein. Caffeine administration, which increased P-CaMK without affecting P-AMPK, increased COX-1, COX-4, PGC-1 alpha, and citrate synthase activity. NOS inhibition, surprisingly, greatly attenuated the effect of caffeine on P-CaMK and attenuated the increases in COX-1 and COX-4 protein. SNAP increased all markers of mitochondrial biogenesis, and it also increased P-AMPK and P-CaMK. In conclusion, AICAR and caffeine increase mitochondrial biogenesis in L6 myotubes, at least in part, via interactions with NOS.

The Role of Nitric Oxide Synthase and Heme Oxygenase in the Protective Effect of Hypothermia in Ischemia-Reperfusion Injury

Ischemia-reperfusion injury plays an important role in limb salvage following limb ischemia. The purpose of the present study was to evaluate the effect of local hypothermia and chemical modulators on microvascular permeability following ischemia-reperfusion injury in skeletal muscle. Sprague-Dawley rats were randomized into nine groups. Postcapillary venules of the extensor digitorum longus muscle were visualized with use of intravital microscopy. Following an intravenous bolus of fluorescein isothiocyanate-labeled albumin, the intravascular and extravascular space was examined for leak. Rats in the sham group underwent a one-hour mock ischemic period without the application of a femoral artery tourniquet, followed by one hour of mock reperfusion. The treatment groups (n = 5 in each group) had the tourniquet applied for one hour, followed by one hour of reperfusion at 10 degrees C (cold) alone, at 10 degrees C with nitric oxide synthase inhibitor, at 10 degrees C with heme oxygenase inhibitor, at 10 degrees C with a combination of inhibitors, at 34 degrees C (warm) alone, at 34 degrees C with a heme oxygenase inducer, at 34 degrees C with a nitric oxide synthase inducer, or at 34 degrees C with a combination of inducers. Rats in the sham group did not show a significant increase in microvascular permeability. Rats in the warm ischemia/reperfusion group displayed significant increases in microvascular permeability, as did the rats that received inhibitors of heme oxygenase and nitric oxide synthase at 10 degrees C. No significant increase in microvascular permeability was observed in the animals in the cold ischemia/reperfusion group or in animals that received inducers of heme oxygenase and nitric oxide synthase at 34 degrees C. Local hypothermia protects skeletal muscle from increased microvascular permeability following ischemia-reperfusion injury. This protective effect is also seen with the induction of the nitric oxide synthase and heme oxygenase systems at physiologic temperature. We also have shown that the protective effects of hypothermia are blocked by giving heme oxygenase and nitric oxide synthase inhibitors while keeping the muscle hypothermic. These findings demonstrate that heme oxygenase and nitric oxide synthase play a combined role in ischemia-reperfusion injury, suggesting possible pathways for clinical intervention to modulate injury seen following trauma, tourniquet use, vascular surgery, and microvascular surgery.

The Role of Nitric Oxide Synthase and Heme Oxygenase I in Radiation-induced Lung Injury

The Role of Nitric Oxide Synthase and Heme Oxygenase I in Radiation-induced Lung Injury

Aminoguanidine administered during the induction of oral tolerance alters the systemic response of the tolerised rats

Herewith we investigated the role of nitric oxide synthase (NOS)-II in the establishment of oral tolerance induced by low antigen dose. To accomplish this, we used a rat model of oral tolerance induced by intragastric administration of low doses of ovalbumin (OVA). NOS-II was inhibited in vivo during the onset of tolerance by intraperitoneal (i.p.) treatment with aminoguanidine (AMG), a selective NOS-II inhibitor. Four experimental groups were generated: (TOL), tolerised rats, receiving OVA but no AMG; (TAG), rats tolerised with OVA and simultaneously receiving AMG i.p.; (CAG), controls treated with AMG but no oral antigen; and (CONT), controls receiving neither OVA nor AMG treatment. The state of oral tolerance was evaluated in all groups by analysing several immune parameters upon subcutaneous administration of OVA in Freund’s complete adjuvant. First, we were able to determine that NOS-II inhibition altered the TH1/TH2 balance in tolerised rats, driving the TH2 anti-OVA response in TOL rats towards TH1 in TAG animals, which showed enhanced delayed hypersensitivity responses. Second, splenocyte cultures from TAG rats showed lower levels of IL-10 production compared to TOL samples as determined by ELISA analysis. Last, we detected the presence of a functional distinct Tr1 regulatory T cell population in spleen samples recovered from TAG animals. Contrary to what happened with TOL Tr1 cells, the levels of Tr1 cells in TAG samples were modified by in vitro stimulation with OVA. All together, these data indicate a preponderant role for NOS-II in the process of oral tolerance induced by low antigen dose.

Resveratrol attenuates lipopolysaccharide-induced hepatitis in d-galactosamine sensitized rats: Role of nitric oxide synthase 2 and heme oxygenase-1

The goal of study was directed to investigate the effects of resveratrol (RES) pretreatment on the enhancing action of d-galactosamine ( d-GalN; 800 mg/kg) on lipopolysaccharide (LPS; 0.5 μg/kg) inducing liver failure in rats. Liver function was assessed by determination of plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), α-glutathione S-transferase (alpha GST) and bilirubin (BILI). Plasma NO 2 − was assessed by NO 2 −/NO 3 − colorimetric kit. The estimation of nonenzymatic and enzymatic antioxidants (glutathione and catalase) was performed in plasma and liver homogenate. Lipid peroxidation was evaluated by the thiobarbituric acid reacting substances (TBARS) and the conjugated dienes (CD). Morphological examinations using light and electron microscopy were performed. Observations related to pharmacological increases of inducible nitric oxide synthase (NOS-2)/nitric oxide (NO) and inducible heme oxygenase (HO-1) in fulminant hepatic failure and modulation by resveratrol were followed up by real-time reverse transcription PCR (RT-PCR) in liver tissue. In the present study we found that among the mechanisms responsible for the hepatoprotective effect of resveratrol in the LPS/ d-GalN liver toxicity model are reduction in NO, downregulation of NOS-2, modification of oxidative stress parameters and modulation of HO-1 which led to overall improvement in hepatotoxic markers and morphology after the hepatic insult.

The Role of Nitric-oxide Synthase in the Regulation of UVB Light-induced Phosphorylation of the α Subunit of Eukaryotic Initiation Factor 2

UV light induces phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIF2alpha) and inhibits global protein synthesis. Both eIF2 kinases, protein kinase-like endoplasmic reticulum kinase (PERK) and general control of nonderepressible protein kinase 2 (GCN2), have been shown to phosphorylate eIF2alpha in response to UV irradiation. However, the roles of PERK and GCN2 in UV-induced eIF2alpha phosphorylation are controversial. The one or more upstream signaling pathways that lead to the activation of PERK or GCN2 remain unknown. In this report we provide data showing that both PERK and GCN2 contribute to UV-induced eIF2alpha phosphorylation in human keratinocyte (HaCaT) and mouse embryonic fibroblast cells. Reduction of expression of PERK or GCN2 by small interfering RNA decreases phosphorylation of eIF2alpha after UV irradiation. These data also show that nitric-oxide synthase (NOS)-mediated oxidative stress plays a role in regulation of eIF2alpha phosphorylation upon UV irradiation. Treating the cells with the broad NOS inhibitor N(G)-methyl-l-arginine, the free radical scavenger N-acetyl-l-cysteine, or the NOS substrate l-arginine partially inhibits UV-induced eIF2alpha phosphorylation. The results presented above led us to propose that NOS mediates UV-induced eIF2alpha phosphorylation by activation of both PERK and GCN2 via oxidative stress and l-arginine starvation signaling pathways.

Changes In Erythrocyte Deformability In Alzheimer's Disease: A Role For Nitric Oxide Synthase

Changes In Erythrocyte Deformability In Alzheimer's Disease: A Role For Nitric Oxide Synthase

Role of NO Synthase in the Development of Trypanosoma cruzi–Induced Cardiomyopathy in Mice

Trypanosoma cruzi infection results in an increase in myocardial NO and intense inflammation. NO modulates the T. cruzi-induced myocardial inflammatory reaction. NO synthase (NOS)1-, NOS2-, and NOS3-null mice were infected with T. cruzi (Brazil strain). Infected NOS1-null mice had increased parasitemia, mortality, and left ventricular inner diameter (LVID). Chronically infected NOS1- and NOS2-null and wild-type mice (WT) exhibited increased right ventricular internal diameter (RVID), although the fold increase in the NOS2-null mice was smaller. Infected NOS3-null mice exhibited a significant reduction both in LVID and RVID. Reverse transcriptase-polymerase chain reaction showed expression of NOS2 and NOS3 in hearts of infected NOS1-null and WT mice, whereas infected NOS2-null hearts showed little change in expression of other NOS isoforms. Infected NOS3-null hearts showed an increase only in NOS1 expression. These results may indicate different roles for NOS isoforms in T. cruzi-induced cardiomyopathy.

The Potential Role of Nitric Oxide Synthase in Survival and Regeneration of Magnocellular Neurons of Hypothalamo-Neurohypophyseal System

Previous investigations from this laboratory have demonstrated that hypophysectomy induces up-regulation of neuronal nitric oxide synthase (nNOS) in magnocellular neurons of the mammalian hypothalamo-neurohypophyseal system (HNS). Accompanied by this upregulation of nNOS, both neuronal regeneration and degeneration are also observed in this system following hypophysectomy. The specific aim of this study was to determine the potential role of nNOS upregulation in neuronal survival and regeneration after hypophysectomy in the adult Sprague-Dawley (SD) rat by using a competitive nitric oxide synthase blocker, N(G)-nitrol-L: -arginine methyl ester (L: -NAME). We found that L: -NAME treatment effectively blocked the regeneration of magnocellular neurons of the rodent hypothalamus as observed in the lumen of the third cerebral ventricle following hypophysectomy. However, L: -NAME had no effect on the survival of magnocellular neurons in the supraoptic (SON) and paraventricular (PVN) nuclei after hypophysectomy. These results suggest that the induced increase of nNOS expression enhance the regenerative ability of magnocellular neurons of the HNS following hypophysectomy.

The role of nitric oxide synthase in regulation of ultraviolet light‐induced phosphorylation of the alpha‐subunit of eukaryotic initiation factor 2

Ultraviolet light (UV) induces phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIF2α) and inhibits global protein synthesis. Both eIF2 kinases PKR (RNA‐dependent protein kinase)‐like ER (endoplasmic reticulum) kinase (PERK) and general control non‐derepressible‐2 (GCN2) have been shown to phosphorylate eIF2α in response to UV‐irradiation. However, the roles of PERK and GCN2 in UV‐induced translation inhibition are controversial. The upstream signaling pathway(s) that leads to the activation of PERK or GCN2 remains unknown. This report contains data that show PERK and GCN2 contribute equally for UV‐induced eIF2α phosphorylation in human keratinocyte (HaCat) cells. Reducing expression of PERK or GCN2 by small interfering RNA (siRNA) decreases phosphorylation of eIF2α after UV‐irradiation. The data also show that nitric oxide synthase (NOS) mediated oxidative stress plays a role in regulation of eIF2α phosphorylation in HaCat cells upon UV‐irradiation. Treating the cells with a broad NOS inhibitor NG‐methyl‐L‐arginine, a free radical scavenger N‐acetyl‐L‐cysteine or an NOS substrate L‐arginine partially inhibits the UV‐induced eIF2α phosphorylation. The same treatments also partially rescued the translation inhibition induced by UV‐irradiation. The results presented above led to a hypothesis that NOS mediates UV‐induced eIF2α phosphorylation by activation of both PERK and GCN2 via oxidative‐stress and L‐arginine starvation signaling pathways.

The Differential Role of Nitric Oxide Synthases in Ultraviolet Light B‐induced Apoptosis

Ultraviolet light (UV) activates nitric oxide synthase(s) (NOSs), which produces nitric oxide (NO). However the involvement of each NOS in UV‐induced signaling is not clear. The role of NO in UV‐induced apoptosis remains controversial. In this study, we analyzed expression and activation of NOSs in UVB‐irradiated keratinocytes HaCaT. We also determined the roles of NOSs in UV‐induced apoptosis. Our data showed that the expression of nNOS was increased and then decreased at 12 h and 24 h post‐UV respectively. While eNOS expression was not changed by UV, a decreased amount of coupled eNOS with an increased amount of uncoupled eNOS was observed within half‐hour of UV‐treatment. There was no increased level of iNOS was detected after UV‐irradiation. These results suggested that UV‐induced NO elevation is mainly regulated by cNOS.To determine the role of each cNOS and NO in UV‐induced apoptosis, we analyzed the extent of effect of a broad NOS inhibitor N‐nitro‐L‐arginine methyl ester (L‐NAME) on apoptotic death of HaCaT upon UV‐irradiation. Our data showed that NO was increased almost immediately and peaked at 18 h after UV‐irradiation. A pulse inhibition of NOS in the early phase (0‐0.5 h) partially inhibits UV‐induced apoptosis. However, a prolonged inhibition of NOS (0‐18 h) promotes apoptosis after UV‐treatment. In addition, the caspase 3 activity, but not amount, was changed in the treated cells and was correlated with the PARP cleavage and apoptosis of the HaCaT cells. Base on the results, we propose that in the early stage of UV‐irradiation, eNOS is uncoupled and produces O2‐ and promotes apoptosis. In the late stage of UV‐irradiation, the expression of nNOS is increased and the elevated [NO] produced by nNOS inhibits caspase‐3 activity and apoptosis of the irradiated cells.

Structure, reactivity and function of bacterial nitric oxide synthases

Nitric oxide synthases (NOSs) from higher organisms are heme‐based mono‐oxygenases that catalyze the conversion of L‐arginine (L‐arg) to nitric oxide (NO), a potent signaling molecule and cytotoxic agent. Bacterial homologs of mammalian NOSs (mNOSs) have recently been characterized in a subset of mostly gram‐positive bacteria. Crystallographic and spectroscopic analyses of bacterial NOSs bound to substrates, intermediates and inhibitors provide insights into structural and chemical properties important for this enzyme family to produce NO. In particular, NOSs isolated from thermophilic bacteria have slower reaction profiles than their mesophilic counterparts. This property has proved useful for the characterization of oxy‐heme species. The biological roles of bacterial NOS appear to differ from those of mNOS, but overall, they are not well understood. Nevertheless, studies of bacterial NOSs show great promise for unraveling new functions for NO in prokaryotes.

Differential roles of nitric oxide synthase isozymes in cardiotoxicity and mortality following chronic doxorubicin treatment in mice

The roles of individual nitric oxide synthases (NOS) in anthracycline-related cardiotoxicity are not completely understood. We investigated the effects of a chronic treatment with doxorubicin (DOX) on knockouts of the individual NOS isozymes and on transgenic mice with myocardial overexpression of eNOS. Fractional shortening (FS) was reduced in untreated homozygous nNOS and iNOS knockouts as well as in eNOS transgenics. DOX-induced FS decrease in wild-type mice was attenuated only in eNOS knockouts, which were found to overexpress nNOS. No worsening of contractility was observed in DOX-treated eNOS transgenics and iNOS knockouts. Although the surviving DOX-treated nNOS knockouts exhibited no further impairment in contractility, most (70%) animals died within 7 weeks after treatment onset. In comparison to untreated wild-type hearts, the nitric oxide (NO) level was lower in hearts from DOX-treated wild-type mice and in all three untreated knockouts. DOX treatment had no effect on NO in the knockouts. These data indicate differential roles of the individual NOS in DOX-induced cardiotoxicity. Protection against DOX effects conferred by eNOS deletion may be mediated by a compensatory overexpression of nNOS. NOS inhibition-based prevention of anthracycline-induced cardiotoxicity should be eNOS-selective, simultaneously avoiding inhibiting nNOS.