Increased iNOS Expression Research Articles

Exhaled NO and iNOS expression in sputum cells of healthy, obese and OSA subjects

Obstructive sleep apnoea (OSA) is associated with airways inflammation; a key role in this regard seems to be played by nitric oxide (NO). The aim of this study was to measure exhaled NO and expression of its enzyme, the inducible nitric oxide synthase (iNOS) in cells of induced sputum in OSA patients and in obese subjects without sleep apnoea and to correlate these inflammatory markers with severity of OSA. We enrolled 18 obese patients with OSA (10 men, age 48.2 +/- 8.4 years), 15 obese patients without OSA (eight men, age 52.8 +/- 11 years) and 10 healthy subjects (five men, age 42 +/- 4 years). Exhaled NO was measured using a chemiluminescence analyser; iNOS expression was measured in the sputum cells by immunocytochemistry. Exhaled NO resulted significantly increased in OSA and in obese patients (23.1 +/- 2.1 and 17.9 +/- 2.1 p.p.b.) than in healthy subjects (7.2 +/- 0.6 p.p.b.; P < 0.001). OSA and obese patients showed a higher percentage of neutrophils and a lower percentage of macrophages in the induced sputum compared to healthy subjects. In addition, OSA and obese patients showed higher iNOS expression in neutrophils and in macrophages with respect to healthy subjects. A positive correlation between exhaled NO, iNOS expression and AHI was observed. These data confirm the presence of airway inflammation in OSA and in obese patients, and suggest the possible role for NO and iNOS expression in neutrophils of the induced sputum as noninvasive markers to identify and monitor the airway inflammation in these subjects.

Differential expression of neuronal and inducible nitric oxide synthase in rat brain after subchronic administration of 3-monochloro-1,2-propanediol

The compound 3-monochloro-1,2-propanediol (3-MCPD) is a contaminant of acid-hydrolyzed vegetable protein foodstuffs. Several reports have suggested that chronic exposure to 3-MCPD can produce neurotoxicity or neurobehavioral effects in experimental animals. We sought to further explore the neurotoxic effects of 3-MCPD (10 or 30 mg/kg) administered for 13 weeks on the expression of two forms of nitric oxide synthase (NOS), neuronal NOS (nNOS), and inducible NOS (iNOS), in rat cerebral cortex and striatum. Using immunocytochemistry, the number of nNOS-expressing neurons or the optical density of iNOS staining in sections from three coronal levels (bregma 1.0, −0.4, and −2.3 mm) were compared between 3-MCPD-treated and control rats. At bregma level 1.0 mm, the number of nNOS-expressing neurons was significantly decreased in the 10 and 30 mg/kg groups. At bregma level −0.4 mm, nNOS expression was significantly decreased only in the 30 mg/kg group, in the cortex and striatum. However, at bregma level −2.3 mm, 3-MCPD administration produced no significant difference in the number of nNOS-expressing neurons in the cortex or striatum. In contrast, iNOS expression was significantly increased in the neocortex and striatum at all three rostrocaudal levels following subchronic 3-MCPD administration. These data suggest that subchronic 3-MCPD exposure may involve compensatory mechanisms acting on nNOS and iNOS expression to maintain nitric oxide homeostasis in the rostral part of the neocortex and striatum. However, in the caudal brain, increased iNOS expression did not profoundly suppress nNOS expression. Thus, the present study suggests that 3-MCPD-induced neurotoxicity is mediated, at least in part, through disturbances in the nitric oxide signaling pathway and exhibits a rostrocaudal difference, through differential expression of nNOS and iNOS in the neocortex and striatum.

Protective role of 17 -estradiol against the development of Helicobacter pylori-induced gastric cancer in INS-GAS mice

The incidence of gastric cancer is higher in men than women. Epidemiological studies suggest that female hormones reduce gastric cancer risk. We examined the effect of ovarian-dependent female hormones on Helicobacter pylori-induced gastric cancer in hypergastrinemic INS-GAS mice. Male and female sexually intact or ovariectomized (OVX) mice were inoculated with H.pylori SS1 or vehicle-only at 10 weeks of age, and tissues were evaluated at 16 or 28 weeks post-infection (WPI). A subset of OVX females were supplemented with 17beta-estradiol (E2), beginning at 16 WPI. Stomachs were evaluated by histopathology, Ki-67 proliferation index, H.pylori quantitative culture and quantitative polymerase chain reaction for messenger RNA expression of inducible nitric oxide synthase (iNOS) and inflammatory cytokines. Infected OVX females developed significantly more severe gastritis (P < 0.05) than infected intact females at both time points. E2 treatment in infected OVX females attenuated the severity of gastritis. Gastrointestinal intraepithelial neoplasia (GIN) developed in 42% of infected males and 10% of infected OVX females by 28 WPI, whereas infected intact females and E2-treated OVX females did not develop GIN. Infected OVX females showed significantly increased iNOS expression and epithelial cell proliferation when compared with intact, infected females. Likewise, interferon-gamma, tumor necrosis factor-alpha and interleukin-1beta (IL-1beta) expression in infected OVX females were significantly increased at 28 WPI when compared with intact counterparts. E2 treatment in infected OVX females significantly decreased IL-1beta expression, increased IL-10 expression and reduced epithelial cell proliferation. These results demonstrate a protective effect of E2 in H.pylori-induced gastric cancer in a mouse model.

Inducible NO synthase expression in endomyocardial biopsies after heart transplantation in relation to the postoperative course

Ischemia and reperfusion during heart transplantation cause damage to cardiomyocytes and endothelial cells and may initiate later acute rejection. Free oxygen radicals generated by iNOS are widely accepted to be responsible for ischemic injury. Increased iNOS expression on cardiac tissue may represent a more intensive tissue injury during ischemia and reperfusion in heart transplantation. The aim of this study was, therefore, to test the hypothesis that increased iNOS expression in early postoperative endomyocardial biopsies correlates with rejection or infection episodes in the later postoperative course. Right ventricular endomyocardial biopsies were obtained from heart transplantation recipients at transplantation and during the first 2 weeks postoperatively. The recipients were divided into three groups depending on the postoperative course during the first year after transplantation: patients in group 1 had an uncomplicated postoperative course, patients in group 2 developed significant signs of postoperative infection, while patients in group 3 presented with acute rejection (< or =grade 2R ISHLT). The expression was analyzed in a semi-quantitative score. iNOS expression was found in cardiomyocytes, endothelial cells, infiltrating cells, and vascular smooth muscle cells. At the time of heart transplantation, the expression was significantly increased in the rejection group compared to the other groups. This increase was even more pronounced in week 2. The present study shows that an increased iNOS expression at the time of heart transplantation could precede an acute rejection in the later postoperative course. Thus, measurements of iNOS expression may be of predictive value for an increased rejection risk and therefore offer the possibility of earlier therapeutic intervention.

Ultrasound Induces Hypoxia-inducible Factor-1 Activation and Inducible Nitric-oxide Synthase Expression through the Integrin/Integrin-linked Kinase/Akt/Mammalian Target of Rapamycin Pathway in Osteoblasts

It has been shown that ultrasound (US) stimulation accelerates fracture healing in the animal models and clinical studies. Nitric oxide (NO) is a crucial early mediator in mechanically induced bone formation. Here we found that US stimulation increased NO formation and the protein level of inducible nitric-oxide synthase (iNOS). US-mediated iNOS expression was attenuated by anti-integrin alpha5beta1 or beta1 antibodies but not anti-integrin alphavbeta3 or beta3 antibodies or focal adhesion kinase mutant. Integrin-linked kinase (ILK) inhibitor (KP-392), Akt inhibitor (1L-6-hydroxymethyl-chiro-inositol-2-[(R)-2-O-methyl-3-O-octadecylcarbonate]) or mammalian target of rapamycin (mTOR) inhibitor (rapamycin) also inhibited the potentiating action of US. US stimulation increased the kinase activity of ILK and phosphorylation of Akt and mTOR. Furthermore, US stimulation also increased the stability and activity of HIF-1 protein. The binding of HIF-1alpha to the HRE elements on the iNOS promoter was enhanced by US stimulation. Moreover, the use of pharmacological inhibitors or genetic inhibition revealed that both ILK/Akt and mTOR signaling pathway were potentially required for US-induced HIF-1alpha activation and subsequent iNOS up-regulation. Taken together, our results provide evidence that US stimulation up-regulates iNOS expression in osteoblasts by an HIF-1alpha-dependent mechanism involving the activation of ILK/Akt and mTOR pathways via integrin receptor.

Histamine Upregulates the Expression of Inducible Nitric Oxide Synthase in Human Intimal Smooth Muscle Cells via Histamine H1 Receptor and NF-κB Signaling Pathway

Histamine increases endothelial nitric oxide (NO) production as an endothelium-dependent vasodilator, which acts as a vasoconstrictor in atherosclerotic coronary arteries. To investigate the relation between histamine and NO production in intimal smooth muscle cells (SMCs), we studied the effect of histamine on inducible NO synthase (iNOS) expression in the SMCs. In cultured human intimal SMCs, histamine increased NO production, iNOS expression, and NF-kappaB nuclear translocation, which were inhibited by histamine H1 blocker and NF-kappaB inhibitor. Luciferase assay using -8.3 kb upstream of human iNOS promoter region and electrophoretic mobility shift assay suggested that a NF-kappaB motif located at -3922 to -3914 would be necessary for histamine-inducible promoter activity. In addition, H1 blocker, NF-kappaB inhibitor, and dominant negative IkappaB alpha or IkappaB kinase beta downregulated the histamine-induced iNOS promoter activity. In the human aorta, histamine content was estimated to be 310+/-66 pmol/mg protein in the atherosclerotic intima, while that was to be 43+/-22 pmol/mg protein in the media (P<0.001). Histamine stimulates intimal SMCs to increase iNOS expression via H1 receptors and NF-kappaB signaling pathway. Histamine could be one of NO-regulating factors, by inducing iNOS expression in intimal SMCs, and may be related to atherogenesis.

Airway nitric oxide output is reduced in bronchiectasis

Increased concentrations of exhaled nitric oxide (NO) have been detected in inflammatory lung diseases including asthma and have been attributed to increased expression and activity of inducible nitric oxide synthase (iNOS) within the airways. However, previous studies of exhaled NO in patients with bronchiectasis have yielded conflicting results, with reports of both increased and normal NO values. Recent evidence from animal models suggests that chronic airway infection reduces NO production within the lung, despite causing increased iNOS expression. We tested the hypothesis that, in human subjects with bronchiectasis, chronic airway infection reduces NO output from the conducting airways. Using a recently described two-compartment model, we measured separately the contributions of the conducting airways and the alveoli to exhaled NO in nine patients with stable bronchiectasis and eight control subjects before and after inhaled glucocorticoid therapy. We found that airway NO output was significantly lower in bronchiectasis than in normal airways whereas NO output from the alveoli was similar to that of control subjects. High-dose inhaled glucocorticoid therapy did not alter airway or alveolar NO production. These findings demonstrate that, in patients with bronchiectasis, airway NO output is reduced and that iNOS does not contribute significantly to airway NO production.

INOS expression in rat aorta is increased after spinal cord transection: A possible cause of orthostatic hypotension in man

Orthostatic hypotension commonly occurs in persons with spinal cord injury (SCI), limiting rehabilitation and independence. Findings of increased production of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) after exposure to simulated microgravity suggest that increased iNOS expression contributes to OH in persons with SCI. To test this possibility, male Wistar rats underwent surgical transection of the spinal cord (T10) or sham-SCI surgery followed by euthanasia 3, 7 or 14 days later. Expression in thoracic aortic of inducible (iNOS), endothelial (eNOS) and neuronal (nNOS) NOS was then determined. In SCI rats, expression of iNOS mRNA was decreased at 3 days, had returned to normal levels of expression at 7 days and was increased at 14 days post-SCI (1.8-fold). In contrast, levels of eNOS mRNA were increased at 3 days (1.4-fold), then declined over time reaching levels by day 14 that were reduced compared to sham-SCI (0.23-fold). There were no significant effects of SCI on nNOS expression. These findings suggest a possible role for increased iNOS expression in the pathogenesis of OH in persons with SCI.

Investigation of Helicobacter pylori in tonsillary tissue with Pronto Dry ® test and pathologic examination

Objective The objectives of this clinical study were to identify, by means of the Pronto Dry ® test and pathologic examination, Helicobacter pylori (HP) in tonsillary tissue and to establish the role of HP in tonsillary microbiology by identifying that bacterium in the tonsillary mucosa or within the tonsil core. Methods The subjects consisted of 52 patients (25 men and 27 women; age range, 3–65 years; mean age, 15.1 ± 14.5 years) who were scheduled to undergo tonsillectomy for the treatment of chronic tonsillitis and who had not been treated with an antibiotic or a bismuth-containing compound for 6 months before the initiation of the study. In each patient, two specimens (one 4 mm × 4 mm tissue sample from the nonmucosal tonsil core and one 4 mm × 4 mm sample of mucosal tissue) were excised from both tonsils immediately after tonsillectomy. The specimens were placed in the Pronto Dry ® test kit, and the test results were obtained 1 h later. The remaining tonsillary tissues were submitted for pathologic analysis via hematoxylin–eosin stain, Giemsa stain, Warthin-Starry silver stain, and staining for inducible nitric oxide synthase (iNOS). Results The results of the Pronto Dry ® test were positive for HP in 42% ( n = 22) of the excised mucosal tissue and in 47% ( n = 24) of the excised core tissue. In 27% ( n = 14) of the patients, both the core and the mucosal tissues tested positive for HP. There was no significant difference between the positive Pronto Dry ® test ratios of the biopsies obtained from the mucosa and those obtained from the core ( P = 0.693). iNOS staining showed that macrophage iNOS activity was significantly higher ( P = 0.025) in biopsied mucosal tissues with a positive Pronto Dry ® test result than in those with a negative result. Light microscopy revealed no HP in samples stained with hematoxylin–eosin stain, Giemsa stain, or Warthin-Starry silver stain. Conclusion Positive Pronto Dry ® test results and the results of iNOS staining showed that HP contributes to chronic tonsillitis, especially at the mucosal layer. Although HP does not colonize, it contributes to the chronic tonsillary inflammatory process as a triggering agent by affecting macrophages in the tonsil and thus increasing iNOS expression.

Nitrosative Stress and Pathogenesis of Insulin Resistance

Insulin resistance is a major causative factor for type 2 diabetes and is associated with increased risk of cardiovascular disease. Despite intense investigation for a number of years, molecular mechanisms underlying insulin resistance remain to be determined. Recently, chronic inflammation has been highlighted as a culprit for obesity-induced insulin resistance. Nonetheless, upstream regulators and downstream effectors of chronic inflammation in insulin resistance remain unclarified. Inducible nitric oxide synthase (iNOS), a mediator of inflammation, has emerged as an important player in insulin resistance. Obesity is associated with increased iNOS expression in insulin-sensitive tissues in rodents and humans. Inhibition of iNOS ameliorates obesity-induced insulin resistance. However, molecular mechanisms by which iNOS mediates insulin resistance remain largely unknown. Protein S-nitrosylation, a covalent attachment of NO moiety to thiol sulfhydryls, has emerged as a major mediator of a broad array of NO actions. S-nitrosylation is elevated in patients with type 2 diabetes, and increased S-nitrosylation of insulin signaling molecules, including insulin receptor, insulin receptor substrate-1, and Akt/PKB, has been shown in skeletal muscle of obese, diabetic mice. Akt/PKB is reversibly inactivated by S-nitrosylation. Based on these findings, S-nitrosylation has recently been proposed to play an important role in the pathogenesis of insulin resistance.

Muscle-derived nitric oxide synthase expression, differences associated with muscle fiber-type, and disease susceptibility in a rat model of myasthenia gravis

Reports from this laboratory suggested that expression of skeletal muscle-derived, inducible nitric oxide synthase (iNOS), is associated with resistance of a particular rat strain to the autoimmune model of myasthenia gravis (MG). The study reported below demonstrates a similar association between iNOS induction in skeletal muscle and disease-resistance when comparing different skeletal muscles originating from the same rat strain. Thus, soleus muscles, shown previously to be relatively resistant to disease even when obtained from disease-susceptible Lewis rats, were observed to express high levels of iNOS following exposure to antibody reactive with the nicotinic acetylcholine receptor (AChR). Increased iNOS expression appears to be associated with slow-twitch, type 1 fibers and would explain the relatively high iNOS expression in soleus muscles since they are dominated by this fiber type, compared to disease-susceptible EDL muscles which are dominated by fast-twitch, type 2 fibers.

Hypothermic preconditioning reduces Purkinje cell death possibly by preventing the over-expression of inducible nitric oxide synthase in rat cerebellar slices after an in vitro simulated ischemia

We showed that hypothermic preconditioning (HPC) increased survival of Purkinje neurons in rat cerebellar slices after oxygen–glucose deprivation (OGD). HPC also reduced the OGD-increased expression of high mobility group I (Y) proteins, a transcription factor that can enhance inducible nitric oxide synthase (iNOS) expression. iNOS is a putatively damaging protein that contributes to ischemic brain injury. Heat shock proteins (HSPs) can be induced by various stimuli to protect cells. We hypothesize that HPC induces neuroprotection by reducing the expression of putatively damaging proteins such as iNOS and/or by increasing the expression of putatively protective proteins such as HSPs. Cerebellar slices were prepared from adult male Sprague–Dawley rats and incubated in circulating artificial cerebrospinal fluid. OGD was for 20 min at 37 °C and was followed by a 5-h recovery at 37 °C before slices were used for morphological, immunohistochemical and Western analyses. HPC was performed by incubating slices at 33 °C for 20 min at 1 h before the OGD. HPC and aminoguanidine, an iNOS inhibitor, prevented OGD-induced Purkinje cell death/injury. OGD increased the expression of iNOS and nitrosylated proteins. These increases were abolished by aminoguanidine and HPC. Interestingly, the expression of HSP70 was increased by OGD but not by HPC. Our results suggest that an increased iNOS expression contributes to the pathophysiology of OGD-induced Purkinje neuronal death in our model. Our results also suggest the involvement of inhibiting the expression of the putatively damaging iNOS proteins in the HPC-induced neuroprotection. HSP70 may not contribute to the HPC-induced neuroprotection.

Inducible nitric oxide synthase up‐regulation and mitochondrial glutathione depletion mediate cyanide‐induced necrosis in mesencephalic cells

We have previously shown in rat primary cultured mesencephalic cells that cyanide induces a high level of oxidative stress and necrotic death. To evaluate the mechanism of the cytotoxicity, the effects of cyanide on intracellular glutathione (GSH) pools and inducible nitric oxide synthase (iNOS)-mediated reactive nitrogen species (RNS) generation were studied. Cyanide rapidly depleted intracellular GSH. Restoration of GSH blocked cell death, whereas depletion of GSH by synthesis inhibition increased the necrosis. Selective depletion of mitochondrial GSH (mtGSH) increased oxidative stress and enhanced cell death, whereas the cytoplasmic pool was not critical to cell survival. These actions were accompanied by increased iNOS expression as determined by Western blot analysis, RT-PCR and immunohistochemistry. Up-regulation of iNOS led to increased generation of NO as reflected by elevated nitrite levels (an end product of NO metabolism). It was determined by use of a selective inhibitor that up-regulation of iNOS expression was transcriptionally regulated by activation of nuclear factor-kappaB, a redox-sensitive transcription factor. It was concluded that, in cyanide-mediated neurotoxicity, mtGSH is a vital component of the cellular antioxidant defense, and its depletion can lead to oxidative stress-mediated iNOS up-regulation, thus enhancing RNS generation and necrosis.

Melatonin Decreases Nitric oxide Production, Inducible Nitric oxide Synthase Expression and Lipid Peroxidation Induced by Venezuelan Encephalitis Equine Virus in Neuroblastoma Cell Cultures

Increased expression of inducible nitric oxide synthase has been shown in murine Venezuelan equine encephalitis (VEE) virus infection. In this experimental model, melatonin (MTL) treatment has shown to be beneficial. The aim of this study was to determine the effect of VEE virus on the nitric oxide (NO) production and lipid peroxidation in neuroblastoma cell cultures, and to investigate the role of MTL during cell-virus interaction. Neuroblastoma cells were co-cultured with VEE virus and treated with MTL at doses ranging from 0 to 1.8 mM, for 6, 12, 24 and 48 h. NO and lipid peroxidation were measured in culture supernatants and in the cellular content by nitrite concentration and thiobarbituric acid assay, respectively. Expression of inducible nitric oxide synthase (iNOS) was determined by indirect immunofluorescence. Increased production of NO and lipid peroxidation products were found in supernatants and cellular contents of VEE virus treated cultures. Both NO and lipid peroxidation were decreased by MTL treatment in a time dependent manner. Increased iNOS expression was observed in VEE virus infected cultures that was reduced by MTL treatment. These results could be related to the beneficial role of MTL in the VEE experimental disease and address the possible therapeutic potential of the hormone in human VEE virus infection.

The effect of an adenosine A1 receptor agonist in the treatment of experimental subarachnoid hemorrhage-induced cerebrovasospasm

Adenosine is a potent vasodilator and an important modulator of cardiovascular function. It has been postulated that nitric oxide (NO) is involved in adenosine-induced vasodilation. This study was designed to examine the effect of an adenosine A1 agonist, N6-cyclopentyladenosine (CPA), in the prevention of subarachnoid haemorrhage (SAH)-induced vasospasm. Method. Experimental SAH was induced in Sprague-Dawley rats by injecting 0.3 mL autogenous blood into the cisterna magna. Intraperitoneal injections of CPA (0.003 mg/kg), or vehicle were administered 5 min and 24 hours after induction of SAH. The degree of vasospasm was determined by averaging the cross sectional areas of the basilar artery 2 days after SAH. Expressions of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) in basilar artery were evaluated. Findings. There were no significant differences among the control and treated groups in physiological parameters recorded before sacrifice. When compared with animals in the control group, cross-sectional area of basilar arteries areas in the SAH only, SAH plus vehicle and SAH plus CPA groups were reduced by 19% (p < 0.01), 22% (p < 0.01), and 9% (p = 0.133), respectively. The cross-sectional areas of the CPA-treated group differed significantly from those of the SAH only and SAH plus vehicle group (p < 0.05). Induction of iNOS-mRNA and protein in basilar artery by SAH was not significantly diminished by CPA. The SAH-induced suppression of eNOS-mRNA and protein were relieved by CPA treatment. Conclusions. This is the first evidence to show an adenosine A1 receptor agonist is effective in partially preventing SAH-induced vasospasm without significant cardiovascular complications. The mechanisms of adenosine A1 receptor agonists in attenuating SAH-induced vasospasm may be, in part, related to preserve the normal eNOS expression after SAH. Inability in reversing the increased iNOS expression after SAH may lead to the incomplete anti-spastic effect of CPA.

A Study of the Pathogenesis and Prevention of Central Pontine Myelinolysis in a Rat Model

The development of central pontine myelinolysis was studied in rats. Severe hyponatraemia was induced using vasopressin tannate and 2.5% dextrose in water and then rapidly corrected with hypertonic saline alone, hypertonic saline and dexamethasone simultaneously, or hypertonic saline plus dexamethasone 24 h later. The permeability of the blood-brain barrier was evaluated using the extravasation of Evans blue dye and the expression of inducible nitric oxide synthase (iNOS) in the brain was examined using Western blot analysis. Histological sections were examined for demyelinating lesions. In rats receiving hypertonic saline alone, Evans blue dye content and expression of iNOS began to increase 6 and 3 h, respectively, after rapid correction of hyponatraemia and demyelinating lesions were seen. When dexamethasone was given simultaneously with hypertonic saline, these increases were inhibited and demyelinating lesions were absent. These effects were lost if dexamethasone injection was delayed. Disruption of the blood-brain barrier and increased iNOS expression may be involved in the pathogenesis of central pontine myelinolysis, and early treatment with dexamethasone may help prevent the development of central pontine myelinolysis.

Effects of Select PM-Associated Metals on Alveolar Macrophage Phosphorylated ERK1 and -2 and iNOS Expression During Ongoing Alteration in Iron Homeostasis

It was hypothesized that relative mass relationships among select constituent metals and iron (Fe3+) govern the pulmonary immunotoxic potential of any PM2.5 sample, as these determine the extent to which Fe3+ binding by transferrin is affected (resulting in altered alveolar macrophage [AM] Fe status and subsequent antibacterial function). Iron response protein (IRP) binding activity is a useful indirect measurement of changes in Fe status, as reductions in cell Fe levels lead to increases in IRP binding. However, AM IRP activity can be affected by an increased presence of nitric oxide generated by inducible nitric oxide synthase (iNOS). This study sought to determine if any changes in AM IRP activity induced by PM2.5 constituents V, Mn, or Al were independent from effects of the metals on cell NO formation. NR8383 rat AM were exposed to Fe3+ alone or combined with V, Mn, or Al at metal:Fe ratios representative of those in PM2.5 collected in New York City, Los Angeles, and Seattle during fall 2001. Cells were then assessed for changes in IRP activity and iNOS expression. Phosphorylated extracellular signal-regulated kinase (ERK) 1 and 2 levels were also measured since activated ERKs are involved in signaling pathways that lead to increased iNOS expression. The results indicate that V and Al, and to a lesser extent Mn, altered IRP activity, though the effects were not consistently concentration dependent. Furthermore, while V and Mn treatments did not induce iNOS expression, Al did. These results confirmed our hypothesis that certain metals associated with PM2.5 might alter the pulmonary immunocompetence of exposed hosts by affecting the Fe status of AM, a major class of deep lung defense cells.This study was supported by funds from the USEPA/PM Center Grant R82735101. The authors are also grateful to services/assistance provided, in part, by the Center Program in the NYU Department of Environmental Medicine that is supported by NIEHS (grant ES00260). The authors also acknowledge the support provided from the U.S. EPA/PM Center Grant R82735501 at the Northwest Center for Particulate Matter and Health in Seattle, WA, and by U.S. EPA grants R82735201 and CR8280260-01-0 at the Southern California Particle Center and Supersite in Los Angeles.

Evidence for the participation of nitric oxide in pemphigus

Pemphigus is an inflammatory autoimmune disorder of the skin. Nitric oxide (NO) is an inflammatory mediator linked to a variety of physiological and pathophysiological phenomena that include skin tumors, psoriasis, urticaria, and atopic dermatitis. Inflammatory cells present in pemphigus lesions are important sources of NO production. We investigated whether NO is involved in pemphigus. A prospective cohort study was conducted at the Dermatology Service of the Hospital Universitário Walter Cantídio of the Federal University of Ceará. All patients seen at the outpatient clinic between August 2000 and July 2001, with a clinically and histologically confirmed diagnosis of pemphigus were included. The median age was 42.5 years (range: 12-69 years) with a male to female ratio of 3:2. Total serum nitrite levels, used as a marker for NO production, were determined by the Griess reaction. Skin biopsies from pemphigus and breast surgery (control) patients were used for the detection of the inducible NO synthase (iNOS) by immunohistochemistry. Twenty-two (22) patients with pemphigus and eight (8) controls who did not differ in demographic characteristics were included. Total serum nitrite levels were significantly higher (>7 micromol/L) in pemphigus patients compared to controls (<6 micromol/L), regardless of the severity of the clinical activity of pemphigus (P < 0.0001). All pemphigus biopsies presented increased immunostaining for iNOS that was not detected in normal skin samples. These data are the first to demonstrate that pemphigus patients display increased serum NO levels that are associated with increased iNOS expression in the affected skin.

Identification of a regulatory cis-element within the 3′-untranslated region of the murine inducible nitric oxide synthase (iNOS) mRNA; interaction with heterogeneous nuclear ribonucleoproteins I and L and role in the iNOS gene expression

The aim of this study was to investigate the role of heterogeneous nuclear ribonucleoprotein I (hnRNPI) and hnRNPL in the regulation of the murine inducible nitric oxide synthase (iNOS) gene during inflammation. Treatment of mice with lipopolysaccharide (LPS)/ d-galactosamine, or of RAW 264.7 cells with LPS/interferon-γ (IFN-γ), strongly increased iNOS expression while reducing hnRNPI levels and complex formation between hnRNPI/hnRNPL and the 3′-untranslated region (3′-UTR) of iNOS mRNA. Introduction of the iNOS 3′-UTR to a luciferase reporter gene reduced its expression in RAW 264.7 cells. However, when hnRNPI and hnRNPL binding sites were deleted, luciferase expression was recovered. LPS/IFN-γ increased the luciferase activity of the full-length 3′-UTR construct compared to control, while its effects on the deletion constructs were modest. The results indicate that LPS/IFN-γ induce iNOS through a mechanism involving hnRNPI and hnRNPL binding to iNOS 3′-UTR. Our data suggest that iNOS mRNA degradation is promoted upon binding of hnRNPI and hnRNPL to a destabilizing region within its 3′-UTR, while inflammatory stimuli causing dissociation of the mRNA–protein complex, yield a more stable transcript. This appears to be particularly significant during extended inflammatory stimuli, resulting in sustained nitric oxide production. The critical event launching this process appears to be the degradation of hnRNPI.

N-acetylcysteine Prevents Nitrosative Stress-Associated Depression of Blood Pressure and Heart Rate in Streptozotocin Diabetic Rats

Previous studies have indicated that cardiovascular abnormalities such as depressed blood pressure and heart rate occur in streptozotocin (STZ) diabetic rats. Chronic diabetes, which is associated with increased expression of inducible nitric oxide synthase (iNOS) and oxidative stress, may produce peroxynitrite/nitrotyrosine and cause nitrosative stress. We hypothesized that nitrosative stress causes cardiovascular depression in STZ diabetic rats and therefore can be corrected by reducing its formation. Control and STZ diabetic rats were treated orally for 9 weeks with N-acetylcysteine (NAC), an antioxidant and inhibitor of iNOS. At termination, the mean arterial blood pressure (MABP) and heart rate (HR) were measured in conscious rats. Nitrotyrosine and endothelial nitric oxide synthase (eNOS) and iNOS expression were assessed in the heart and mesenteric arteries by immunohistochemistry and Western blot experiments. Untreated diabetic rats showed depressed MABP and HR that was prevented by treatment with NAC. In untreated diabetic rats, levels of 15-F(2t)-isoprostane, an indicator of lipid peroxidation increased, whereas plasma nitric oxide and antioxidant concentrations decreased. Furthermore, decreased eNOS and increased iNOS expression were associated with elevated nitrosative stress in blood vessel and heart tissue of untreated diabetic rats. N-acetylcysteine treatment of diabetic rats not only restored the antioxidant capacity but also reduced the expression of iNOS and nitrotyrosine and normalized the expression of eNOS to that of control rats in heart and superior mesenteric arteries. The results suggest that nitrosative stress depress MABP and HR following diabetes. Further studies are required to elucidate the mechanisms involved in nitrosative stress mediated depression of blood pressure and heart rate.