Nitric Oxide Synthase Type Research Articles

The Development of Classically and Alternatively Activated Macrophages Has Different Effects on The Varied Stages of Radiation-induced Pulmonary Injury in Mice

The classical and alternative activation of macrophages has been proposed to play a role in radiation-induced pneumonitis and fibrosis, respectively. To test this hypothesis, the thoraces of C57BL/6 mice were irradiated with 12 Gy X-rays, and irradiated and control mice were euthanized at 1, 8, 12, 24 and 72 hours, and 2, 4, 8, 16 and 24 weeks after irradiation. The expression of inducible nitric oxide synthase (iNOS) and arginase type 1 (Arg-1) was evaluated at the mRNA and protein levels at different stages post-irradiation. We demonstrated that the enhanced mRNA and protein expression of iNOS occurred within the pneumonic stage, whereas the high levels of Arg-1 expression occurred within the fibrotic phase. Immunohistochemistry revealed that iNOS and Arg-1 were mainly expressed in macrophages. The expression of iNOS and Arg-1 may be associated with acute radiation pneumonitis and the development of radiation fibrosis, respectively. Although the function of macrophages cannot explain the whole process of radiation-induced pulmonary injury development, it may play an important regulatory role during this process.

Biomechanical signals and the C-type natriuretic peptide counteract catabolic activities induced by IL-1β in chondrocyte/agarose constructs

IntroductionThe present study examined the effect of C-type natriuretic peptide (CNP) on the anabolic and catabolic activities in chondrocyte/agarose constructs subjected to dynamic compression.MethodsConstructs were cultured under free-swelling conditions or subjected to dynamic compression with low (0.1 to 100 pM) or high concentrations (1 to 1,000 nM) of CNP, interleukin-1β (IL-1β), and/or KT-5823 (inhibits cyclic GMP-dependent protein kinase II (PKGII)). Anabolic and catabolic activities were assessed as follows: nitric oxide (NO) and prostaglandin E2 (PGE2) release, and [3H]-thymidine and 35SO4 incorporation were quantified by using biochemical assays. Gene expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), aggrecan, and collagen type II were assessed with real-time quantitative PCR (qPCR). Two-way ANOVA and the post hoc Bonferroni-corrected t tests were used to examine data.ResultsCNP reduced NO and PGE2 release and partially restored [3H]-thymidine and 35SO4 incorporation in constructs cultured with IL-1β. The response was dependent on the concentration of CNP, such that 100 pM increased [3H]-thymidine incorporation (P < 0.001). This is in contrast to 35SO4 incorporation, which was enhanced with 100 or 1000 nM CNP in the presence and absence of IL-1β (P < 0.001). Stimulation by both dynamic compression and CNP and/or the PKGII inhibitor further reduced NO and PGE2 release and restored [3H]-thymidine and 35SO4 incorporation. In the presence and absence of IL-1β, the magnitude of stimulation for [3H]-thymidine and 35SO4 incorporation by dynamic compression was dependent on the concentration of CNP and the response was inhibited with the PKGII inhibitor. In addition, stimulation by CNP and/or dynamic compression reduced IL-1β-induced iNOS and COX-2 expression and restored aggrecan and collagen type II expression. The catabolic response was not further influenced with the PKGII inhibitor in IL-1β-treated constructs.ConclusionsTreatment with CNP and dynamic compression increased anabolic activities and blocked catabolic effects induced by IL-1β. The anabolic response was PKGII mediated and raises important questions about the molecular mechanisms of CNP with mechanical signals in cartilage. Therapeutic agents like CNP could be administered in conjunction with controlled exercise therapy to slow the OA disease progression and to repair damaged cartilage. The findings from this research provide the potential for developing novel agents to slow the pathophysiologic mechanisms and to treat OA in the young and old.

Biological significance of expression of nitric oxide synthases in neurogenic bladder

Objective To explore the expression of nitric oxide synthases including neuronal nitric oxide synthases(nNOS), inducible nitric oxide synthases(iNOS), endothelial nitric oxide synthases (eNOS) in neurogenic bladder tissues, and analyze it's producing feature and significance. Methods There were 30 cases with neurogenic bladder(18 males, 12 females). The average age was 6.3±3.1 years. All patients appeared with myelodysplasia, urinary and fecal incontinence in different degree. Twenty-six cases were manifested with hyperreflexia bladders, and all patients were treated with surgical procedures. During operation, collected bladder tissue samples including tissues of apex vesicae and tissues of bladder neck, and all tissues were enveloped with mineral wax. All tissues were detected for nNOS, iNOS, and eNOS respectively in tissues of apex vesicae and tissues of bladder neck,and with normal bladder tissues as control group (bladder tissues of hypospadia, 10 cases), and according to clinical features, to explore the expression of NOS, and to analyze the relationship among them. Results In normal apex vesicae tissues, all cases stained with nNOS, and distributed among bundles of smooth muscles, and surface of smooth muscles and interstitial tissue, histochemica;score (HS) 2.8-4.0 and 1.2-2.7. There were no stained cells in bladder tissues of iNOS, and HS was very low, HS:0-0. 4 and 0-0.1 ;eNOS mainly distributed in interstitial tissues in rarefaction manners, and mainly in vascular endothelial cell (VEC), and smooth muscles had no stainings the most expression among them was nNOS, and mainly distributed in bladder neck tissues. In neurogenic bladder tissues, the main expression of NOS type was iNOS, and nNOS decreased significantly. eNOS mainly expressed in VEC among interstitial tissues, and had no staining in smooth muscle cells and collagenoblast and rarefaction of microvessel in bladder tissues, and microvessel density decreased significantly than normal bladder tissues. Microvessal density(MVD) in bladder tisssus (6. 8± 3.2/100per square) was less than that in normal tissues (16.7±6.3/100 per square). Conclusions In normal bladder tissues, nNOS mainly distributes in bladder neck and urethra, and nitric oxide mainly derives from nNOS. Much more matrix fibers, fewer nitrogenergic nerves, and less nNOS expression are seen in neurogenic bladder interstitial tissue. There are more iNOS expressions in bladder tissues,and NO is mainly derived from iNOS, and it may play an important role in pathological bladder tissues, especially in fibrosis of bladder wall. eNOS may be considered as angiopoietic labeling, and may evaluate the blood supply of bladder. Key words: Urinary bladder, neurogenic; Nitric oxide synthase type Ⅰ ; Nitric oxide synthase typeⅡ; Nitric oxide synthase type Ⅲ

Association of the Pentanucleotide Repeat Polymorphism in NOS2 Promoter Region with Susceptibility to Migraine in a Chinese Population

Genes involved in the production of nitric oxide (NO) have been suggested as genetic factors for migraine. It has been studied whether polymorphisms in the genes encoding for different types of NO synthase (NOS) could be involved in the liability to migraine; however, most studies yield negative results. The pentanucleotide repeat microsatellite in the promoter region of inducible NOS (NOS2) shows highly significant differences in allelic frequencies among ethnically diverse populations. Thus, variation in the number of pentanucleotide repeats may have some significance in the predisposition to migraine among different human populations. The aim of this study was to investigate the possible association between pentanucleotide repeat polymorphism and the risk for migraine in Chinese population. We studied the genotypic and allelic frequencies of the pentanucleotide repeat polymorphism in the promoter region of NOS2 in 504 patients with migraine and 512 healthy controls, using polymerase chain reaction amplification and polyacrylamide gel electrophoresis analyses. Comparison of global allele counts between patients and controls showed that the difference was significant (p = 0.0014). The carriage of 9-repeat and 10-repeat alleles was significantly more common in controls, whereas 11-repeat allele was more common in patients after Bonferroni correction for multiple comparisons. A specific analysis of the different cutoffs for number of repeats showed that allelic and genotypic frequencies for the 9-repeat and 10-repeat cutoff were significantly different between cases and controls (p = 0.007 and p = 0.005 for allelic frequencies, respectively; p = 0.0086 and p = 0.0033 for genotypic frequencies, respectively). Our results implied an association between NOS2 pentanucleotide repeat polymorphism and migraine susceptibility in a Chinese population. Considering the significant allelic frequency differences in ethnically diverse populations, further replication studies, especially in ethnically different groups, were necessary to fully establish the role of NOS2 polymorphism in migraine susceptibility.

Metformin Attenuates Production of Nitric Oxide in Response to Lipopolysaccharide by Inhibiting MyD88-Independent Pathway

Metformin is reported to ameliorate inflammation in diabetic patients. The effect of metformin on lipopolysaccharide-induced nitric oxide production was studied by using RAW 264.7 macrophage-like cells. The action of metformin was analyzed by dividing lipopolysaccharide signaling into the MyD88-dependent and -independent pathways. Metformin significantly reduced the expression of an inducible type of nitric oxide synthase and inhibited lipopolysaccharide-induced nitric oxide production. On the other hand, metformin did not inhibit lipopolysaccharide-induced tumor necrosis factor-alpha production. The expression levels of interferon-beta protein and mRNA, which is a key molecule in MyD88-independent pathway, were significantly inhibited by metformin. Compound C, a specific AMP-activated protein kinase inhibitor, did not affect the inhibitory action of metformin. Metformin was suggested to inhibit lipopolysaccharide-induced nitric oxide production via inhibition of interferon-beta production in MyD88-independent pathway. Metformin might exhibit an anti- inflammatory action on diabetic complications as well as the antidiabetic action.

Role of endothelial nitric oxide synthase in the pathogenesis and development of diabetic cystopathy in STZ-induced diabetic rat model

Objective To investigate the role of endothelial nitric oxide synthase(eNOS)in the pathogenesis and development of diabetic cystopathy.Methods Diabetic model was established by a single intra-peritoneal injection of 60 mg/kg STZ in male Sprague-Dawley rats.Thirty-six rats were randomly divided into 3 groups:normal control,diuresis control and diabetes groups each(n=12).Examinations were performed at 6W and 12W after injection of STZ.Cystometry was taken to evaluate the bladder function.Expression levels of eNOS mRNA and protein were assessed with RT-PCR and Western blot assays respectively and their correlations with bladder function index were analyzed.Results The bladder capacity,single voiding volume and post-voiding volume at 6W were(0.75±0.04),(0.70±0.02)and(0.06±0.00)ml in normal group,(1.62±0.15),(1.52±0.30)and (0.11±0.01)ml in diuresis group,and(2.29±0.16),(2.06±0.25)and(0.23±0.01)ml in diabetic group.Compared with normal and diuresis groups,they were significantly increased in diabetic group(P＜0.01).At 12W,they were(0.86±0.06),(0.80±0.04)and(0.07±0.00)ml in normal group,(1.93±0.10),(1.77±0.11)and(0.13±0.02)ml in diuresis group,and(2.65±0.26),(2.09±0.21)and(0.56±0.06)ml in diabetic group.Post-voiding volume increased significantly compared with that at 6W in diabetic group(P＜0.01).At 6W,voiding efficiencies of each group were(93.3±2.1)%,(93.2±2.7)%and(90.0±2.5)0A,with no significant difference.While at 12W they were(93.1±2.2),(93.8±3.2)and(78.1±2.6)%.Voiding efficiency was significantly reduced in diabetic group compared with other 2 groups.No significant difference was observed among bladder pressures of each group.Expression levels of eNOS mRNA from each group at 6W were 0.81±0.12,0.90±0.12,and 1.98±0.16.Compared with normal and diuresis groups,eNOS mRNA expression level significantly increased in diabetic group(P＜0.01).while at 12W they were 0.87±0.17,1.05±0.11,and 2.89±0.23 respectively.Compared with that at 6W,expression level of eNOS mRNA significantly increased at 12W in diabetic group(P＜0.01).Expression levels of eNOS protein were 0.98±0.12,0.93±0.14,and 2.28±0.16.eNOS protein expression level significantly increased in diabetic group,compared with normal and diuresis groups(P＜0.01).At 12 W they were 1.03±0.13,1.14±0.11,and 3.12±0.20,respectively.Compared with that at 6W,expression level of eNOS protein significantly increased at 12W in diabetic group(P＜0.01).The expression level of eNOS mRNA was negatively correlated with voiding efficiency in diabetic group(r=-0.669,P＜0.01).Conclusion The up-regulated expression levels of both eNOS mRNA and protein may participate in the pathogenesis and development of diabetic cystopathy. Key words: Diabetes mellitus; Urinary bladder diseases; Nitric oxide synthase type Ⅲ; Rats

Increased iNOS, MMP-2, and HSP-72 in Skeletal Muscle Following High-Intensity Exercise Training

Skeletal muscle adapts to exercise by an upregulation of cellular defenses, such as inducible nitric oxide synthase (iNOS) and matrix metalloproteinase type 2 (MMP-2) and heat shock protein type-72 (HSP-72). The aims of the study were to examine iNOS, MMP-2, and HSP-72 mRNA and protein expression after high-intensity exercise training and to examine whether the expression levels are fiber type dependent. Young Wistar rats were assigned to either 2 or 4 weeks of a high-intensity (32 m/min) running exercise for 40 minutes 5 day per week. A non-running group served as a control. Western blotting and reverse transcriptase-polymerase chain reaction of muscle mRNA and protein levels were assessed in the medial gastrocnemius, quadriceps, soleus, crural, and sternal head of diaphragm muscles. High-intensity exercise training for 4 weeks but not for 2 weeks resulted in a significant increase in both RNA and protein levels of iNOS, MMP-2, and HSP-72 in all muscles examined except the sternal head of diaphragm. High-intensity exercise training is required to promote the expression of iNOS, MMP-2, and HSP-72 in hind limb muscles regardless their muscle fiber type, whereas in the diaphragm the changes are fiber-type dependent.

Sustained Gonadotropin-Releasing Hormone Stimulation Mobilizes the cAMP/PKA Pathway to Induce Nitric Oxide Synthase Type 1 Expression in Rat Pituitary Cells In Vitro and In Vivo at Proestrus1

Previous in vivo studies have established that pituitary nitric oxide synthase type 1 (NOS1) is regulated by gonadotropin-releasing hormone (GnRH). The aim of our study was to elucidate the mechanisms of NOS1 regulation by GnRH in rat pituitary cells. Using a perifused cell system, we demonstrated that NOS1 induction was sensitive to GnRH pulse frequency and was maximally induced under continuous GnRH stimulation. In primary cultures of rat pituitary cells, sustained stimulation with the GnRH agonist triptorelin (GnRHa) increased NOS1 protein levels, whereas NOS2 and NOS3 levels were unaffected. NOS1 up-regulation occurred in gonadotroph cells only, in a time-dependent and concentration-dependent manner (maximum increase, 2.5-fold; half-maximal concentration, 0.17 nM). GnRHa effect was mimicked by cAMP pathway activators and, most importantly, was blocked by disruption of the protein kinase A (PKA) pathway using pharmacological inhibitors such as Rp-cAMP or drug phosphatase technology-protein kinase inhibitor (DPT-PKI), a cell-permeant PKI peptide. In contrast, modulation of the PKC pathway and inhibition of the MAPK cascade were ineffective. Overall, these experiments demonstrated that GnRH-induced up-regulation of pituitary NOS1 is mediated notably by the cAMP/PKA pathway. Last, in vivo administration of a GnRH antagonist markedly inhibited the pituitary cAMP rise at proestrus in addition to suppressing NOS1 increase. Altogether, our data suggest that the cAMP/PKA signaling pathway is preferentially recruited under sustained GnRH stimulation in vivo during proestrus, allowing the expression of a specific set of PKA-regulated proteins, including NOS1, in gonadotroph cells.

In-vitro anti-inflammatory activity of Pinus sylvestris and Plantago lanceolata extracts: effect on inducible NOS, COX-1, COX-2 and their products in J774A.1 murine macrophages

Extracts of the plant species Pinus sylvestris L. and Plantago lanceolata L. have been used in traditional medicine for the treatment of certain respiratory diseases, but little is known about their precise effects and mechanisms of action. In this study, we investigated the effect of these plant extracts on the production of nitric oxide (NO) and prostaglandin E(2), NO synthase (NOS) type II, cyclooxygenase-1 (COX-1) and COX-2 mRNA expression in the murine macrophage cell line J774A.1. We found that Pinus sylvestris and Plantago lanceolata extracts inhibited NO production in a concentration-dependent manner in this cell line, without obvious cytotoxic effects as tested by MTT assay. The Plantago lanceolata extract at all doses used, and the Pinus sylvestris extract at high doses, showed significant scavenging of NO radicals released by the NO donor PAPA-NONOate. Our data also show that pre-treatment with these extracts significantly inhibits inducible NOS (iNOS) mRNA production in this cell line, without affecting COX-1 mRNA expression. COX-2 mRNA levels and PGE(2) levels induced by lipopolysaccharide/interferon-gamma were not modified upon pre-treatment with the extracts. Thus, our results suggest that the anti-inflammatory properties of Pinus sylvestris and Plantago lanceolata extracts may reflect decreased NO production, possibly due to inhibitory effects on iNOS gene expression or to NO-scavenging activity.

Nitric oxide produced by endothelial nitric oxide synthase promotes diuresis

Extracellular fluid volume is highly regulated, at least in part, by peripheral resistance and renal function. Nitric oxide (NO) produced by NO synthase type 3 (NOS 3) in the nonrenal vasculature may promote fluid retention by reducing systemic vascular resistance and arterial pressure. In contrast, NO produced by renal NOS 3 promotes water excretion by reducing renal vascular resistance, increasing glomerular filtration, and inhibiting reabsorption along the nephron. Thus, the net effect of NO from NOS 3 on urinary volume (UV) is unclear. We hypothesized that NO produced by NOS 3 promotes water excretion primarily due to renal tubular effects. We gave conscious wild-type and NOS 3 -/- mice an acute volume load and measured UV, blood pressure, plasma renin concentration (PRC), Na(+), vasopressin, and urinary Na(+) and creatinine concentrations. To give the acute volume load, we trained mice to drink a large volume of water while in metabolic cages. On the day of the experiment, water was replaced with 1% sucrose, and mice had access to it for 1 h. Volume intake was similar in both groups. Over 3 h, wild-type mice excreted 62 +/- 10% of the volume load, but NOS 3 -/- excreted only 42 +/- 5% (P < 0.05). Blood pressure in NOS 3 -/- was 118 +/- 3 compared with 110 +/- 2 mmHg in wild-type mice (P < 0.05), but it did not change following volume load in either strain. PRC, vasopressin, and glomerular filtration rate were similar between groups. Urinary Na(+) excretion was 49.3 +/- 7.0 in wild-type vs. 37.8 +/- 6.4 mumol/3 h in NOS 3 -/- mice (P < 0.05). Bumetanide administration eliminated the difference in volume excretion between wild-type and NOS 3 -/- mice. We conclude that 1) NO produced by NOS 3 promotes water and Na(+) excretion and 2) the renal epithelial actions of NO produced by NOS 3 supersede the systemic and renal vascular actions.

The time course of vasoconstriction and endothelin receptor A expression in pulmonary arterioles of mice continuously exposed to ambient urban levels of air pollution

The present study aimed to verify the time course of the effects of environmental levels of urban air pollution toxicity on lung arterioles. BALB/c mice (n=56) were continuously exposed to selective chambers equipped with (filtered, F) or without (non-filtered, NF) filter devices for particles and toxic gases for 24h/day, over 14, 21, 30 or 45 days. After exposure, we evaluated the lumen-wall relationship (an estimator of arteriolar narrowing), endothelial nitric oxide synthase (eNOS) and endothelin type A receptor (ETAr) expression in the vascular wall and inflammatory influx of the peribronchiolar area. Concentrations of fine particulate matter (PM<or=2.5 microg/m(3)), nitrogen dioxide (NO(2)), black smoke (BS), humidity and temperature in both the environment and inside the chambers were measured daily. Filters cleared 100% of BS and 97% of PM inside the F chamber. The arteriole wall of the lungs of mice from NF chamber had an increased ETAr expression (p<or=0.042) concomitant to a decrease in the lumen/wall ratio (p=0.02) on the early days of exposure, compared to controls. They also presented a progressive increment of inflammatory influx in the peribronchiolar area during the study (p=0.04) and decrement of the eNOS expression on the 45th day of exposure in both vascular layers (p<or=0.03). We found that after 14 days of exposure, the ambient levels of air pollutants in Sao Paulo induced vasoconstriction that was associated with an increase in ETAr expression. These vascular results do not appear to be coupled to the progressive inflammatory influx in lung tissue, suggesting a down-regulation of vasoconstrictive mechanisms through an imbalance in the cytokines network. It is likely that these responses are protective measures that decrease tissue damage brought about by continuous exposure to air pollutants.

B1 cells produce nitric oxide in response to a series of toll-like receptor ligands

The effect of a series of toll-like receptor (TLR) ligands on the production of nitric oxide (NO) in mouse B1 cells was examined by using CD5 + IgM + WEHI 231 cells. The stimulation with a series of TLR ligands, which were Pam3Csk4 for TLR1/2, poly I:C for TLR3, lipopolysaccharide (LPS) for TLR4, imiquimod for TLR7 and CpG DNA for TLR9, resulted in enhanced NO production via augmented expression of an inducible type of NO synthase (iNOS). LPS was most potent for the enhancement of NO production, followed by poly I:C and Pam3Csk4. Imiquimod and CpG DNA led to slight NO production. The LPS-induced NO production was dependent on MyD88-dependent pathway consisting of nuclear factor (NF)-κB and a series of mitogen-activated protein kinases (MAPKs). Further, it was also dependent on the MyD88-independent pathway consisting of toll-IL-1R domain-containing adaptor-inducing IFN-β (TRIF) and interferon regulatory factor (IRF)-3. Physiologic peritoneal B1 cells also produced NO via the iNOS expression in response to LPS. The immunological significance of TLR ligands-induced NO production in B1 cells is discussed.

Protective Role of Cys-178 against the Inactivation and Oligomerization of Human Insulin-degrading Enzyme by Oxidation and Nitrosylation

Insulin-degrading enzyme (IDE), a 110-kDa metalloendopeptidase, hydrolyzes several physiologically relevant peptides, including insulin and amyloid-beta (Abeta). Human IDE has 13 cysteines and is inhibited by hydrogen peroxide and S-nitrosoglutathione (GSNO), donors of reactive oxygen and nitrogen species, respectively. Here, we report that the oxidative burst of BV-2 microglial cells leads to oxidation or nitrosylation of secreted IDE, leading to the reduced activity. Hydrogen peroxide and GSNO treatment of IDE reduces the V(max) for Abeta degradation, increases IDE oligomerization, and decreases IDE thermostability. Additionally, this inhibitory response of IDE is substrate-dependent, biphasic for Abeta degradation but monophasic for a shorter bradykinin-mimetic substrate. Our mutational analysis of IDE and peptide mass fingerprinting of GSNO-treated IDE using Fourier transform-ion cyclotron resonance mass spectrometer reveal a surprising interplay of Cys-178 with Cys-110 and Cys-819 for catalytic activity and with Cys-789 and Cys-966 for oligomerization. Cys-110 is near the zinc-binding catalytic center and is normally buried. The oxidation and nitrosylation of Cys-819 allow Cys-110 to be oxidized or nitrosylated, leading to complete inactivation of IDE. Cys-789 is spatially adjacent to Cys-966, and their nitrosylation and oxidation together trigger the oligomerization and inhibition of IDE. Interestingly, the Cys-178 modification buffers the inhibition caused by Cys-819 modification and prevents the oxidation or nitrosylation of Cys-110. The Cys-178 modification can also prevent the oligomerization-mediated inhibition. Thus, IDE can be intricately regulated by reactive oxygen or nitrogen species. The structure of IDE reveals the molecular basis for the long distance interactions of these cysteines and how they regulate IDE function.

Nitric oxide dysfunction in vascular endothelium and platelets: role in essential hypertension

Both endothelial and platelet-derived nitric oxide have important vasculoprotective properties. Increasing evidence suggests a dysfunctional platelet nitric oxide synthase type 3 (NOS3) pathway in essential hypertension, whereas for endothelial-derived nitric oxide the picture is more complicated, with many studies suggesting an impairment of endothelial nitric oxide generation, whilst others have suggested that the endothelial nitric oxide pathway is preserved. Controversy also exists as to whether any observed reduction in endothelial or platelet-derived nitric oxide exerts a pathogenetic role or is simply the result of the raised blood pressure. In this review, we examine the evidence that endothelial and/or platelet-derived nitric oxide are disturbed in essential hypertension, and whether such disturbances are cause or effect.

Malaria severity and human nitric oxide synthase type 2 (NOS2) promoter haplotypes

Nitric oxide (NO) mediates host resistance to severe malaria and other infectious diseases. NO production and mononuclear cell expression of the NO producing enzyme-inducible nitric oxide synthase (NOS2) have been associated with protection from severe falciparum malaria. The purpose of this study was to identify single nucleotide polymorphisms (SNPs) and haplotypes in the NOS2 promoter, to identify associations of these haplotypes with malaria severity and to test the effects of these polymorphisms on promoter activity. We identified 34 SNPs in the proximal 7.3 kb region of the NOS2 promoter and inferred NOS2 promoter haplotypes based on genotyping 24 of these SNPs in a population of Tanzanian children with and without cerebral malaria. We identified 71 haplotypes; 24 of these haplotypes comprised 82% of the alleles. We determined whether NOS2 promoter haplotypes were associated with malaria severity in two groups of subjects from Dar es Salaam (N = 185 and N = 250) and in an inception cohort of children from Muheza-Tanga, Tanzania (N = 883). We did not find consistent associations of NOS2 promoter haplotypes with malaria severity or malarial anemia, although interpretation of these results was potentially limited by the sample size of each group. Furthermore, cytokine-induced NOS2 promoter activity determined using luciferase reporter constructs containing the proximal 7.3 kb region of the NOS2 promoter and the G-954C or C-1173T SNPs did not differ from NOS2 promoter constructs that lacked these polymorphisms. Taken together, these studies suggest that the relationship between NOS2 promoter polymorphisms and malaria severity is more complex than previously described.

Neuroprotective effect of s-methylisothiourea in transient focal cerebral ischemia in rat

Over production of NO by nitric oxide synthase (NOS) in the brain parenchyma has been demonstrated to contribute to tissue damage. NO may be toxic by formation of peroxinitrite after a reaction between NO and superoxide appears to be one of the major pathways leading to cell death. Of three types of NOS, nNOS is neurotoxic in early and iNOS in late stage of transient cerebral ischemia (TFCI), while eNOS is neuroprotective in all stages. We examined the neuroprotective effect of a preferential iNOS inhibitor s-methylisothiourea (SMT) at 0, 8, 24 and 48 h as multiple injections (30 and 100 mg/kg, i.p.) in ischemia and reperfusion injury in a rat model of middle cerebral artery occlusion (2 h) and reperfusion (72 h). After 2 h of ischemia and 72 h of reperfusion, animals were sacrificed for studying the infarct volume, brain edema and apoptosis and neuro-behavioral abnormality was assessed at 24, 48 and 72 h of reperfusion. SMT reduced significantly the infarct volume, neuro-behavioral abnormality, brain edema, number of apoptotic cells in penumbra and NOx levels in plasma and brain both at 30 and 100 mg/kg in dose-dependent manner. The amount of peroxynitrite measured by rhodamine assay was significantly reduced by SMT, as compared to control group. SMT protected Neuro 2a cells against sodium azide-induced damage. It is concluded that, SMT may possibly targeting both constitutive as well as inducible NOS at varying time interval to elicit neuroprotection in TFCI rats.

Nitric Oxide Synthase Isoenzyme Expression and Activity in Peripheral Lung Tissue of Patients with Chronic Obstructive Pulmonary Disease

Nitric oxide (NO) is increased in the lung periphery of patients with chronic obstructive pulmonary disease (COPD). However, expression of the NO synthase(s) responsible for elevated NO has not been identified in the peripheral lung tissue of patients with COPD of varying severity. Protein and mRNA expression of nitric oxide synthase type I (neuronal NOS [nNOS]), type II (inducible NOS [iNOS]), and type III (endothelial NOS [eNOS]) were quantified by Western blotting and reverse transcription-polymerase chain reaction, respectively, in specimens of surgically resected lung tissue from nonsmoker control subjects, patients with COPD of varying severity, and smokers without COPD, and in a lung epithelial cell line (A549). The effects of nitrative/oxidative stress on NOS expression and activity were also evaluated in vitro in A549 cells. nNOS nitration was quantified by immunoprecipitation and dimerization of nNOS was detected by low-temperature SDS-PAGE/Western blot in the presence of the peroxynitrite generator, 3-morpholinosydnonimine-N-ethylcarbamide (SIN1), in vitro and in vivo. Lung tissue from patients with severe and very severe COPD had graded increases in nNOS (mRNA and protein) compared with nonsmokers and normal smokers. Hydrogen peroxide (H(2)O(2)) and SIN1 as well as the cytokine mixture (IFN-gamma, IL-1beta, and tumor necrosis factor-alpha) increased mRNA expression and activity of nNOS in A549 cells in a concentration-dependent manner compared with nontreated cells. Tyrosine nitration resulted in an increase in nNOS activity in vitro, but did not affect its dimerization. Patients with COPD have a significant increase in nNOS expression and activity that reflects the severity of the disease and may be secondary to oxidative stress.

Differences in the Ability to Generate Type 1 T Helper Cells Need Not Determine Differences in the Ability to ResistMycobacterium tuberculosisInfection among Mouse Strains

C57BL/6 (B6) and BALB/c mice are considerably more resistant to infection with Mycobacterium tuberculosis than DBA/2 mice. To determine whether the difference in resistance is because DBA/2 mice generate a type 1 T helper (Th1) immune response of lower magnitude, the Th1 response to airborne infection of mice of all 3 strains was measured in terms of the number of interferon (IFN)-gamma-producing CD4 and CD8 T cells generated. Despite the superior resistance of BALB/c mice compared with DBA/2 mice, both strains generated a similarly low number of Th1 cells. On the other hand, B6 mice, despite being approximately equal in resistance compared with BALB/c mice, generated a much larger number of Th1 cells. In DBA/2 mice, a higher level of lung infection was associated with larger numbers of M. tuberculosis bacilli in individual macrophages at sites of infection, indicating lower levels of macrophage mycobacteriostatic function. Despite this, infected macrophages from DBA/2 mice stained positive for nitric oxide synthase type 2 (NOS2) by immunocytochemistry as intensely as did infected macrophages from B6 and BALB/c mice, indicating the acquisition of NOS2-dependent mycobacteriostatic function in all cases. The ability to generate a large number of Th1 cells need not determine the ability to resist M. tuberculosis infection.

Erythropoietin activates nitric oxide synthase in murine erythrocytes

Erythropoietin (Epo) is the main regulator of erythrocyte production and a potent cytoprotective factor. It was suggested that some of Epo cytoprotective properties are due to its regulation of nitric oxide (NO) production. Recently, functionally active endothelial type NO synthase (eNOS) was discovered in mature murine and human red blood cells (RBC-eNOS). The goal of the present study was to characterize the effect of physiological and therapeutic doses of Epo on RBC-eNOS function. We found that recombinant human Epo (rHuEpo) binds specifically to mouse erythrocytes. Epo binding sites are not equally distributed through the RBC population but prevail in reticulocytes and young erythrocytes with about 105 receptors/cell, compared with adult and old erythrocytes containing 1-4 receptors/cell. The treatment of mouse erythrocytes with rHuEpo resulted in a time- and dose-dependent upregulation of NO production mediated via activation of the phosphatidylinositol-3-kinase /Akt pathway and RBC-eNOS phosphorylation at Ser-1177. Finally, when erythrocytes were incubated in L-arginine-free medium, rHuEpo treatment resulted in upregulation of superoxide radical production with concomitant shifting of the cellular redox state toward more oxidized state. Epo-induced changes in erythrocyte redox potential were absent in erythrocytes from eNOS-deficient mice.

Aspirin acetylates nitric oxide synthase type 3 in platelets thereby increasing its activity

Acute administration of aspirin increases nitric oxide (NO) synthesis by platelets, an effect not shared by other non-steroidal anti-inflammatory drugs. The aim of the present study was to determine the mechanism by which aspirin acutely increases the activity of NO synthase type 3 (NOS-3), the predominant NOS isoform expressed by platelets, and specifically whether this occurs through an increase in its acetylation. Platelets isolated from the blood of healthy human subjects were exposed in vitro to vehicle or aspirin at different concentrations (5 micromol/L-4 mmol/L). Changes in intraplatelet Ca(2+) concentration were determined from fura-2 fluorescence. Following immunoprecipitation of NOS-3 from platelet lysates, its activity was determined from l-[(3)H]arginine to l-[(3)H]citrulline conversion, and its serine phosphorylation quantified by western blotting. Acetylation of NOS-3 in platelets was assessed by the incorporation of radioactivity into the immunoprecipitated enzyme from [acetyl-(14)C]aspirin. Following transfection of HeLa cells with NOS-3, NO biosynthesis in response to aspirin was determined from cyclic GMP measurement, and sites of NOS-3 acetylation were ascertained by liquid chromatography-tandem mass spectrometry. At all concentrations tested, aspirin increased the activity of NOS-3 from platelets. This was not associated with any measurable change in intraplatelet Ca(2+) concentration. Serine phosphorylation of NOS-3 in platelets was decreased, and this was especially marked for serine-1177 phosphorylation, whereas acetylation of NOS-3 was increased, by aspirin incubation. HeLa cells transfected with NOS-3 exhibited an increase in NO biosynthesis following aspirin exposure, and this was associated with acetylation of the enzyme on both serine-765 and serine-771. Aspirin acetylates NOS-3 acutely in platelets, and this causes an increase in its activity as well as a decrease in its phosphorylation. It is also possible that aspirin indirectly affects NOS-3 activity by acetylating other substrates within the platelet, but this remains to be determined.