Investigation of a neuronal nitric oxide synthase gene (NOS1) polymorphism in a multiple sclerosis population

Multiple Sclerosis (MS) is a chronic neurological disease characterized by demyelination associated with infiltrating white blood cells in the central nervous system (CNS). Nitric oxide synthases (NOS) are a family of enzymes that control the production of nitric oxide. It is possible that neuronal NOS could be involved in MS pathophysiology and hence the nNOS gene is a potential candidate for involvement in disease susceptibility. The aim of this study was to determine whether allelic variation at the nNOS gene locus is associated with MS in an Australian cohort. DNA samples obtained from a Caucasian Australian population affected with MS and an unaffected control population, matched for gender, age and ethnicity, were genotyped for a microsatellite polymorphism in the promoter region of the nNOS gene. Allele frequencies were compared using chi-squared based statistical analyses with significance tested by Monte Carlo simulation. Allelic analysis of MS cases and controls produced a chi-squared value of 5.63 with simulated P = 0.96 (OR(max) = 1.41, 95% CI: 0.926-2.15). Similarly, a Mann-Whitney U analysis gave a non-significant P-value of 0.377 for allele distribution. No differences in allele frequencies were observed for gender or clinical course subtype (P > 0.05). Statistical analysis indicated that there is no association of this nNOS variant and MS and hence the gene does not appear to play a genetically significant role in disease susceptibility.

Chromosome 17 and the inducible nitric oxide synthase gene in human essential hypertension

Essential hypertension is a common multifactorial trait that results in a significantly increased risk for heart attack and stroke. The condition has a genetic basis, although at present the number of genes is unknown. In order to identify such genes, we are utilising a linkage scanning approach using microsatellite markers and affected sibships. Here we provide evidence for the location of at least one hypertension susceptibility locus on chromosome 17. Analysis of 177 affected sibpairs gave evidence for significant excess allele sharing to D17S949 (SPLINK: P=0.0029; MAPMAKER SIBS: P=0.0033; ASPEX: P=0.0061; GENEHUNTER: P=0.0096; ANALYZE (SIBPAIR): P=0.0025) on 17q22–24, with significant allele sharing also indicated for an additional marker, D17S799 (SPLINK: P=0.025; MAPMAKER SIBS: P= 0.025) located close to the centromere. Since these two genomic regions are well separated, our results indicate that there may be more than one chromosome 17 locus affecting human blood pressure. Moreover, further investigation of this chromosome, utilizing a polymorphism within the promoter of the iNOS candidate gene, NOS2A, revealed both increased allele sharing among sibpairs (SPLINK: P=0.02; ASPEX: P=0.00004) and positive association (P= 0.034) of NOS2A to essential hypertension. Hence these results indicate that chromosome 17 and, more specifically, the NOS2A gene may play a role in human essential hypertension.

Migraine association and linkage studies of an endothelial nitric oxide synthase (NOS3) gene polymorphism

Migraine shows strong familial aggregation. However, the number of genes involved in the disorder is unknown and not identified. Nitric oxide is involved in the central processing of pain stimuli and plays an important role in the regulation of basal or stimulated vasodilation. Nitric oxide synthase, which controls the synthesis of nitric oxide, could possibly be a cause, or candidate gene, in migraine etiology. In this study, we detected a polymorphism for endothelial nitric oxide synthase by polymerase chain reaction and tested this for association and linkage to migraine. Results from the study did not show an association of the nitric oxide synthase microsatellite when tested in 91 affected and 85 unaffected individuals. Using the FASTLINK program for parametric linkage analysis, the polymorphism did not show significant linkage to migraine when tested in four migraine pedigrees composed of 116 individuals, 52 affected. Total LOD scores excluded linkage up to 8.5 cM between the nitric oxide synthase polymorphism and migraine. Results using the nonparametric affected pedigree member form of analysis also did not support a role for this gene in migraine etiology.

Effects of hyperbaric oxygen and inducible nitric oxide synthase inhibitor treatment on femoral head osteonecrosis in a rat model

Introduction: Apoptosis is the final destiny of many cells in the body, though this process has been observed in some pathological processes. One of these pathological processes is femoral head non-traumatic osteonecrosis. Among many pro/anti-apoptotic factors, nitric oxide has recently been an area of further interest. Osteocyte apoptosis and its relation to pro-apoptotic action invite further research, and the inducible form of nitric oxide synthase (iNOS)—which produces a high concentration of nitric oxide—has been flagged. The aim of this study was to investigate the effect of hyperbaric oxygen (HBO) and inducible NOS suppressor (Aminoguanidine) in prevention of femoral head osteonecrosis in an experimental model of osteonecrosis in spontaneous hypertensive rats (SHRs). Methods: After animal ethic approval 34 SHR rats were divided into four groups. Ten rats were allocated to the control group without any treatment, and eight rats were allocated to three treatment groups namely: HBO, Aminoguanidine (AMG), and the combination of HBO and AMG treatments (HBO+AMG). The HBO group received 250 kPa of oxygen via hyperbaric chamber for 30 days started at their 5th week of life; the AMG group received 1mg/ml of AMG in drinking water from the fifth week till the 17th week of life; and the last group received a combination of these treatments. Rats were sacrificed at the end of the 17th week of life and both femurs were analysed for evidence of osteonecrosis using Micro CT scan and H&E staining. Also, osteocyte apoptosis and the presence of two different forms of NOS (inducible (iNOS) and endothelial (eNOS)) were analysed by immunostaining and apoptosis staining (Hoechst and TUNEL). Results: Bone morphology of metaphyseal and epiphyseal area of all rats were investigated and analysed. Micro CT findings revealed significantly higher mean fractional trabecular bone volume (FBV) of metaphyseal area in untreated SHRs compared with all other treatments (HBO, P<0.05, HBO+AMG, P<0.005, and AMG P<0.001). Bone surface to volume ratio also significantly increased with HBO+AMG and AMG treatments when compared with the control group (18.7 Vs 20.8, P<0.05, and 18.7 Vs 21.1, P<0.05). Epiphyseal mean FBV did not change significantly among groups. In the metaphyseal area, trabecular thickness and numbers significantly decreased with AMG treatment, while trabecular separation significantly increased with both AMG and HBO+AMG treatment. Histological ratio of no ossification and osteonecrosis was 37.5%, 43.7%, 18.7% and 6.2% of control, HBO, HBO+AMG and AMG groups respectively with only significant difference observed between HBO and AMG treatment (P<0.01). High concentration of iNOS was observed in the region of osteonecrosis while there was no evidence of eNOS activity around that region. In comparison with the control group, the ratio of osteocyte apoptosis significantly reduced in AMG treatment (P<0.005). We also observed significantly fewer apoptotic osteocytes in AMG group comparing with HBO treatment (P<0.05). Conclusion: None of our treatments prevents osteonecrosis at the histological or micro CT scan level. High concentration of iNOS in the region of osteonecrosis and significant reduction of osteocyte apoptosis with AMG treatment were supportive of iNOS modulating osteocyte apoptosis in SHRs.

Novel role of the nitrite transporter NirC in Salmonella pathogenesis: SPI2-dependent suppression of inducible nitric oxide synthase in activated macrophages

Activation of macrophages by interferon gamma (IFN- ) and the subsequent production of nitric oxide (NO) are critical for the host defence against Salmonella enterica serovar Typhimurium infection. We report here the inhibition of IFN- -induced NO production in RAW264.7 macrophages infected with wild-type Salmonella. This phenomenon was shown to be dependent on the nirC gene, which encodes a potential nitrite transporter. We observed a higher NO output from IFN- -treated macrophages infected with a nirC mutant of Salmonella. The nirC mutant also showed significantly decreased intracellular proliferation in a NO-dependent manner in activated RAW264.7 macrophages and in liver, spleen and secondary lymph nodes of mice, which was restored by complementing the gene in trans. Under acidified nitrite stress, a twofold more pronounced NO-mediated repression of SPI2 was observed in the nirC knockout strain compared to the wild-type. This enhanced SPI2 repression in the nirC knockout led to a higher level of STAT-1 phosphorylation and inducible nitric oxide synthase (iNOS) expression than seen with the wild-type strain. In iNOS knockout mice, the organ load of the nirC knockout strain was similar to that of the wild-type strain, indicating that the mutant is exclusively sensitive to the host nitrosative stress. Taken together, these results reveal that intracellular Salmonella evade killing in activated macrophages by downregulating IFN- -induced NO production, and they highlight the critical role of nirC as a virulence gene.

Inducible nitric oxide synthase and control of intracellular bacterial pathogens

Inducible nitric oxide synthase (iNOS) has important functions in innate immunity and regulation of immune functions. Here, the role of iNOS in the pathogenesis of various intracellular bacterial infections is discussed. These pathogens have also evolved a broad array of strategies to repair damage by reactive nitrogen intermediates, and to suppress or inhibit functions of iNOS.

Interferon-Gamma and Nitric Oxide Synthase 2 Mediate the Aggregation of Resident Adherent Peritoneal Exudate Cells: Implications for the Host Response to Pathogens

Interferon-gamma (Ifn gamma), a key macrophage activating cytokine, plays pleiotropic roles in host immunity. In this study, the ability of Ifn gamma to induce the aggregation of resident mouse adherent peritoneal exudate cells (APECs), consisting primarily of macrophages, was investigated. Cell-cell interactions involve adhesion molecules and, upon addition of Ifn gamma, CD11b re-localizes preferentially to the sites of interaction on APECs. A functional role of CD11b in enhancing aggregation is demonstrated using Reopro, a blocking reagent, and siRNA to Cd11b. Studies with NG-methyl-L-arginine (LNMA), an inhibitor of Nitric oxide synthase (Nos), NO donors, e.g., S-nitroso-N-acetyl-DL-penicillamine (SNAP) or Diethylenetriamine/ nitric oxide adduct (DETA/NO), and Nos2(-/-) mice identified Nitric oxide (NO) induced by Ifn gamma as a key regulator of aggregation of APECs. Further studies with Nos2(-/-) APECs revealed that some Ifn. responses are independent of NO: induction of MHC class II and CD80. On the other hand, Nos2 derived NO is important for other functions: motility, phagocytosis, morphology and aggregation. Studies with cytoskeleton depolymerizing agents revealed that Ifn gamma and NO mediate the cortical stabilization of Actin and Tubulin which contribute to aggregation of APECs. The biological relevance of aggregation of APECs was delineated using infection experiments with Salmonella Typhimurium (S. Typhimurium). APECs from orally infected, but not uninfected, mice produce high amounts of NO and aggregate upon ex vivo culture in a Nos2-dependent manner. Importantly, aggregated APECs induced by Ifn gamma contain fewer intracellular S. Typhimurium compared to their single counterparts post infection. Further experiments with LNMA or Reopro revealed that both NO and CD11b are important for aggregation; in addition, NO is bactericidal. Overall, this study elucidates novel roles for Ifn gamma and Nos2 in regulating Actin, Tubulin, CD11b, motility and morphology during the aggregation response of APECs. The implications of aggregation or ``group behavior'' of APECs are discussed in the context of host resistance to infectious organisms.

Changes of tissue endothelin-1 and nitric oxide synthase in a sheep model of large intestinal obstruction

Large intestinal obstruction (LIO) in farm animals can cause a ischaemic necrosis of intestinal tissue, eventually leading to death. The roles of endothelin-1 (ET-1) and nitric oxide (NO) are not well understood in the process of LIO, but evidence suggests that endothelial-derived mediators may participate. In the present study, ET-1 concentration and total nitric oxide synthase (NOS) activity were measured in heart, liver, pancreas, lung and kidney in a model of LIO in sheep. Our data demonstrated that ET-1 concentration and NOS activity were altered, with significant increases of ET-1 in heart, lung and kidney and of NOS activity in pancreas and kidney, but a marked decline of NOS activity in liver (p<0.05). It is postulated that these alterations in NOS activity and ET-1 concentration may contribute to the progressive loss of organ function, and finally lead to death in LIO in sheep.

Functional comparison of the endothelial nitric oxide synthase Glu298Asp polymorphic variants in human endothelial cells

The G894T endothelial nitric oxide synthase (eNOS) polymorphism results in a Glu to Asp substitution at position 298. This position is located externally on the protein and as the regulation of eNOS is dependent on its subcellular localization and interaction with modulatory proteins, we aimed to address whether the substitution of Asp at 298 had any effect on these mechanisms. Initially, we developed a novel method to accurately determine molar quantities of each variant by expressing them as green fluorescent protein (GFP) fusion proteins and using recombinant adenoviruses to facilitate transient infection of human microvascular endothelial cells. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting of eNOS298Asp revealed a 135-kDa proteolytic fragment which was not present with eNOS298Glu. This proteolysis was prevented by using LDS buffer confirming that this differential cleavage is an artefact of sample preparation and unlikely to occur intracellularly. Nitric oxide was measured following stimulation with calcium ionophore or oestrogen in the presence of varying sepiapterin concentrations. GFP fluorescence was used to quantify the amount of fusion protein and calculate intracellular specific activity. There was no significant difference in intracellular specific activity between Glu298 and Asp298 eNOS in response to calcium ionophore or oestrogen. Tetrahydrobiopterin supplementation increased eNOS activity of both variants in an identical manner. The presence of the GFP also facilitated the visualization of the variants by confocal microscopy and demonstrated that both localized to the plasma membrane and the Golgi. These findings demonstrate that the Asp substitution at 298 does not have a major effect in modulating eNOS activity in vivo.