Endothelial nitric oxide synthase gene sequence variations and vascular disease

Genes contribute significantly to interpopulation differences in vascular disease. Endothelial nitric oxide synthase (eNOS)-a key regulator of vascular nitric oxide production-has been investigated extensively to determine the relevance of DNA variants in the eNOS gene and vascular diseases. Variants in the promoter region, introns, and exons have been explored in a large number of populations but findings have been inconsistent. This paper reviews the current status of functional significance for reported sequence variations in the eNOS gene and the relevance of these variants to different forms of vascular diseases.     

Haplotype analysis of the endothelial nitric oxide synthase gene in asthma

Nitric oxide (NO) is an important mediator of physiologic processes in the airways. Evidence exists that genetic factors affect NO formation and contribute to the pathophysiology of asthma. The aims of this study were to determine the endothelial NO synthase (eNOS) haplotypes in Czech asthmatics and control subjects and examine their relation to asthma. We analyzed a total of six polymorphisms. Two SNPs in the promoter (C-786T and C-691T), two variants in the introns (27-bp repeat in intron 4 and G11T in intron 23), and two others in the exons (C774T in exon 6 and G894T in exon 7) were genotyped in 610 subjects (asthma, n = 294; healthy controls, n = 316), and a case-control association study was conducted. No significant differences in allele or genotype frequencies for individual polymorphisms were observed between patients with asthma and controls after correction for multiple comparisons. Nevertheless, a G to T exchange in intron 23 was related with specific sensitization for feather (p = 0.008, p(corr) < 0.05). However, the common haplotype -786T/-691C/27-bp 5 repeat variant/774C/894G/11T was associated with lower risk of asthma (p = 0.001, p(corr) < 0.05, odds ratio = 0.58, 95% confidence interval = 0.46-0.73). These findings suggest that endothelial NOS variants may be one of the factors participating in protection or susceptibility to asthma in our population.     

Nitric oxide synthase expression and nitric oxide toxicity in oligodendrocytes

Oligodendrocytes (OLG) have more complex interactions with nitric oxide (NO) than initially suspected. Historically, OLG were seen only as targets of high NO levels released from other cells. Expression of nitric oxide synthase type II (NOS-2) in primary cultures of OLGs stimulated by cytokines led to controversy due to the presence of small numbers of microglia, cells also inducible for NOS-2 expression. The present review summarizes the findings that immature OLG express NOS-2, but that they do not in their most mature stage in culture as membrane sheet-bearing cells. This raises questions about the regulation of NOS-2 expression in OLG. Additionally, novel data are presented on NOS-3 expression in cultured OLG. If confirmed in vivo, this finding suggests that constitutive NOS-3 expression may play a key role in OLG injury due to its activation by calcium, in interaction with pathways mediating glutamate toxicity. The authors discuss in vivo NO levels to place in vitro findings in context, and compare OLG sensitivity to NO with that of other brain cells. Lastly, the multiple interactions of NO are considered with regard to glutamate cytotoxicity, the antioxidant glutathione, mitochondrial function, and myelin architecture.     

Corticosterone and phenytoin reduce neuronal nitric oxide synthase messenger RNA expression in rat hippocampus.

The production and release of the corticosteroids, namely the glucocorticoids and the mineralocorticoids, are regulated by various stimuli, including stress. Previous studies from our laboratory have shown that chronic exposure to stress or to stress levels of glucocorticoids produces atrophy of the apical dendrites of CA3 pyramidal neurons in the hippocampus. This stress-induced dendritic remodeling is blocked by the anti-epileptic drug phenytoin, which suppresses glutamate release, and also by N-methyl-D-aspartate receptor antagonists. These results suggest an interaction between glucocorticoids and excitatory amino acids in the development of stress-induced atrophy of CA3 pyramidal neurons. Since nitric oxide is proposed to play an important role in mediating both the physiological and pathophysiological actions of excitatory amino acids, we examined the regulation of neuronal nitric oxide synthase messenger RNA expression by corticosterone and phenytoin in the rat hippocampus. The expression of neuronal nitric oxide synthase messenger RNA in hippocampal pyramidal neurons and granule neurons of the dentate gyrus was unaffected by 21-day administration of corticosterone (40 mg/kg), phenytoin (40 mg/kg) or the combination of corticosterone and phenytoin. However, in hippocampal interneurons, corticosterone/ phenytoin co-administration led to a significant reduction in neuronal nitric oxide synthase messenger RNA levels when compared with vehicle controls. These results suggest that, during exposure to stress levels of corticosterone, phenytoin inhibits glucocorticoid-induced atrophy of CA3 pyramidal neurons by reducing neuronal nitric oxide synthase expression in hippocampal interneurons. Moreover, these results may provide another example of synaptic plasticity in the hippocampus mediated by nitric oxide synthase.     

Borna disease virus P protein inhibits nitric oxide synthase gene expression in astrocytes

Borna disease virus (BDV) is one of the potential infectious agents involved in the development of central nervous system (CNS) diseases. Neurons and astrocytes are the main targets of BDV infection, but little is known about the roles of BDV infection in the biological effects of astrocytes. Here we reported that BDV inhibits the activation of inducible nitric oxide synthase (iNOS) in murine astrocytes induced by bacterial LPS and PMA. To determine which protein of BDV is responsible for the regulation of iNOS expression, we co-transfected murine astrocytes with reporter plasmid iNOS-luciferase and plasmid expressing individual BDV proteins. Results from analyses of reporter activities revealed that only the phosphoprotein (P) of BDV had an inhibitory effect on the activation of iNOS. In addition, P protein inhibits nitric oxide production through regulating iNOS expression. We also reported that the nuclear factor kappa B (NF-kappaB) binding element, AP-1 recognition site, and interferon-stimulated response element (ISRE) on the iNOS promoter were involved in the repression of iNOS gene expression regulated by the P protein. Functional analysis indicated that sequences from amino acids 134 to 174 of the P protein are necessary for the regulation of iNOS. These data suggested that BDV may suppress signal transduction pathways, which resulted in the inhibition of iNOS activation in astrocytes.     

Effect of cAMP on inducible nitric oxide synthase gene expression: its dual and cell-specific functions

The effects of some cAMP-elevating agents on the induction of nitric oxide synthase II (NOS II) were investigated for a macrophage-derived cell line, RAW264.7, stimulated with lipopolysaccharide (LPS) or interferon-gamma (IFN-gamma) and the results were compared for the case of vascular smooth muscle cells (VSMC) stimulated with interleukin-1 beta (IL-1 beta). Forskolin, dibutyryl cAMP, and a phosphodiesterase inhibitor, 3-isobutyl-1-methyl xanthine, resulted in an elevated production of nitrite and nitrate, NOS II activities, NOS II mRNA accumulation, and the protein level in RAW264.7 cells stimulated with LPS or IFN-gamma. However, the addition of combinations of these reagents decreased these levels in RAW264.7 cells, but enhanced them in VSMC that had been stimulated with IL-1 beta. When intracellular cAMP levels in VSMC were measured, they were elevated by about 100 times more in the forskolin-treated cells, compared to the untreated cells. Stimulated RAW264.7 cells, on the other hand, produced much lower levels of cAMP than VSMC. It is likely that cAMP functions in two opposing directions in terms of NOS II gene induction in RAW264.7 cells in a dose-dependent manner. The effects of cAMP-elevating agents on promoter activities of the 5'-flanking region of the mouse NOS II gene were then examined. The promoter activities were enhanced in RAW264.7 cells, even in the presence of all three cAMP-elevating agents. Although the binding of NF-kappa B to responsive elements is essential for the induction of the NOS II gene, cAMP-elevating agents had no effect on NF-kappa B binding to the element, thus eliminating the involvement of NF-kappa B in the suppression of the NOS II gene by high concentrations of cAMP. These data suggest that a putative responsive element to high levels of cAMP is present outside of the region examined in this study. The inhibitory effects of cAMP in RAW264.7 cells would be due to the presence of a negative regulatory factor that is absent in VSMC.     

内皮型、诱导型一氧化氮合酶在乳腺癌中的表达

目的 :研究内皮型一氧化氮合酶 (eNOS)、诱导型一氧化氮合酶 (iNOS)在乳癌中表达及与淋巴结转移的关系。方法 :采用免疫组化S P法检测 60例乳癌中eNOS和iNOS的表达。结果 :eNOS和iNOS阳性在乳癌中表达率分别为 75 0 %和71 7%。在淋巴结转移组和无淋巴结转移组中eNOS阳性表达率分别为 66 7%和 83 3 % ,两组间差异无统计学意义 (χ2 =2 2 2 ,P >0 0 5) ,而iNOS在淋巴结转移和无转移组中阳性表达率分别为 53 3 %和 90 0 % ,两组间差异有统计学意义 (χ2 =9 93 ,P <0 0 1 )。结论 :内皮型、诱导型一氧化氮合酶在乳腺癌中高表达 ;iNOS的表达与乳腺癌的淋巴转移相关     

Neuronal nitric oxide synthase (bNOS) messenger RNA and protein expression in developing rat brainstem nuclei

Neuronal nitric oxide synthase (bNOS) messenger RNA expression and immunoreactivity were mapped in series of cryosections through the developing rat brainstem nuclei. Between embryonic day E16 and postnatal day P16, brainstem nuclei expressed both bNOS messenger RNA (mRNA) in situ hybridization signals and protein immunoreactivity. However, NOS mRNA signals were absent from the Edinger Westphal, facial or motory trigeminal nucleus. Strong patterns of mRNA signals and immunoreactivity occurred in neurons located in the substantia nigra pars compacta and the laterodorsal tegmental nuclei. Between E24 and P16, altered patterns of bNOS mRNA positive and immunoreactive neurons, e.g. superior and inferior colliculi, raphe nuclei, solitary tract or pontine nucleus were documented. Altered NOS expression patterns thus may reflect developmental processes within distinct neuronal populations such as cell phenotype discrimination or synaptogenesis within efferent or afferent brainstem pathways. The NOS/NO system therefore appears to be a modulator for intra-/intercellular adjustment processes in normal development.     

Inducible and endothelial nitric oxide synthase genes polymorphism in inflammatory bowel disease

To assess the influence of inducible and endothelial nitric oxide synthase gene (NOS2A and NOS3) polymorphisms in susceptibility to Crohn's disease (CD) and ulcerative colitis (UC). A total of 505 inflammatory bowel disease (IBD) patients (221 with UC and 284 with CD) and 332 ethnically matched controls were studied. Patients and controls were genotyped by polymerase chain reaction -based techniques for a multiallelic (CCTTT)(n) repeat and biallelic marker (TAAA)(n) in the promoter region of the NOS2A gene and for a T/C polymorphism at position -786 in the promoter region and a polymorphism in exon 7(298Glu/Asp) of the NOS3 gene. There was not association between NOS2A and NOS3 genotypes, alleles or haplotypes frequencies with UC, CD and controls. Our data suggest that NOS2A and NOS3 polymorphisms do not play a major role in IBD predisposition.     

Cell-specific effects of pentoxifylline on nitric oxide production and inducible nitric oxide synthase mRNA expression.

Cytokine-stimulated astrocytes and macrophages are potent producers of nitric oxide (NO), a free radical proposed to play an important role in organ-specific autoimmunity, including demyelinating diseases of the central nervous system. The aim of this study was to investigate effects of pentoxifylline (PTX), a phosphodiesterase inhibitor with immunomodulatory properties, on NO production and inducible NO synthase (iNOS) mRNA expression in rat astrocytes and macrophages. We have shown that PTX affects cytokine (interferon-gamma, IFN-gamma; interleukin-1, IL-1; tumour-necrosis factor-alpha, TNF-alpha)-induced NO production in both cell types, but in the opposite manner--enhancing in astrocytes and suppressive in macrophages. While PTX did not have any effect on enzymatic activity of iNOS in activated cells, expression of iNOS mRNA was elevated in astrocytes and decreased in macrophages treated with cytokines and PTX. Treatment with PTX alone affected neither NO production nor iNOS mRNA levels in astrocytes or macrophages. This study indicates involvement of different signalling pathways associated with iNOS induction in astrocytes and macrophages, thus emphasizing complexity of regulation of NO synthesis in different cell types.     

The expression and activity of renal nitric oxide synthase and circulating nitric oxide in polycystic kidney disease rats

Nitric oxide (NO) influences tubular fluid and electrolyte transport, and hence possibly also fluid accumulation in renal cysts. The expression and activity of intrarenal constitutive NO synthase (cNOS) [neuronal NOS, nNOS and endothelial NOS, eNOS] and inducible NOS (iNOS) and plasma nitrite/nitrate (PNOx) concentration were assessed in homozygous Han:SPRD polycystic kidney disease (PKD) rats (cy/cy), heterozygous Han:SPRD PKD rats (cy/+), homozygous normal Han:SPRD littermates (+/+) and Sprague Dawley rats (sd). The results showed: 1) nNOS expression was decreased in proximal tubules and thick ascending limbs of the loop of Henle in cy/cy and cy/+ rats compared to +/+ and sd rats (p<0.05). nNOS was weakly expressed in the epithelium of small cysts and unexpressed in epithelium of large cysts. 2) iNOS expression was increased in proximal tubular epithelial cells in cy/+ rats compared to +/+ rats and sd rats (p<0.01). iNOS expression in cyst epithelium was decreased in cy/+ rats (p<0.05) and absent in cy/cy rats. 3) eNOS expression was similar in the endothelium of intrarenal arteries in all groups. 4) The activity of renal cNOS was decreased in cy/cy and cy/+ rats; the activity of iNOS was decreased only in cy/cy rats, with no significant difference among the other three groups. 5) PNOx concentration was higher in cy/cy rats than in the other three groups, and correlated positively with plasma creatinine and urea. In conclusion, NOS expression and activity decreased as cysts developed, suggesting that NO downregulation is involved in the pathogenesis of PKD.     

Sex difference in the expression and regulation of nitric oxide synthase gene in the rat preoptic area

Neuronal nitric oxide synthase (nNOS) mRNA-positive cells were visualized by non-isotopic in situ hybridization histochemistry in the organum vasculosum of the lamina terminalis (OVLT) and the preoptic area (POA) in gonadectomized juvenile female and male rats. In the rostral POA (rPOA) at the level of the anteroventral periventricular nucleus, nNOS mRNA-positive cells were distributed in an inverted V-shaped area over the third ventricle and were in close proximity to cell bodies of gonadotropin-releasing hormone (GnRH)-immunoreactive neurons. In the caudal POA (cPOA) at the level of the medial preoptic nucleus, no topological association existed between GnRH and nNOS. Throughout the rPOA, both the number and the area of nNOS mRNA positive cells were significantly larger in the gonadectomized females than in the gonadectomized males. Treatment with estradiol for 2 days, followed by progesterone in the next morning, which caused an increase in serum luteinizing hormone 6 h later, induced a significant reduction of the nNOS mRNA expression in the rPOA in the female but not in the male rat at the time of sacrifice. In the OVLT and the cPOA, ovarian steroids had no effect on nNOS mRNA expression of both sexes. The results indicate that nNOS mRNA expression in the rPOA is sexually dimorphic and regulated by ovarian steroids in a sex specific manner.     

Microenvironmental regulation of inducible nitric oxide synthase expression and nitric oxide production in mouse bone marrow-derived mast cells

In addition to its well-known role in relaxation of vascular smooth muscle, NO modulates immune responses in a concentration- and location-specific manner. For MC, it is well accepted that exogenous NO regulates their function. However, there are inconsistencies in the literature of whether MC express NOS and make NO. MC progenitors mature in peripheral tissues, but the factors that influence MC maturation and their specific phenotype, such as whether they express NOS, are not well understood. To study microenvironmental conditions that could be "permissive" for NOS expression, we cultured BMMC in various conditions--BMMC(IL-3), BMMC(SCF/IL-3), or BMMC(SCF/IL-4)-for >3 weeks and examined NOS expression. We detected Nos2 mRNA in BMMC(SCF/IL-4) but not BMMC(IL-3) or BMMC(SCF/IL-3). After stimulation with IFN-γ and/or LPS, NOS2 expression and NO production were detected in BMMC(SCF/IL-4) but rarely detected in BMMC cultured with other conditions. Confocal microscopic analysis showed that NOS2 expression induced by IFN-γ colocalized in CD117(+) BMMC. NO production, after activation with IFN-γ and LPS in BMMC(SCF/IL-4), was abrogated by pretreatment with the NOS2-specific inhibitor. In addition to NOS2 expression, BMMC(SCF/IL-4) were distinguished from BMMC(IL-3) in heparin and MMCP expression. Thus, MC progenitors that develop in SCF + IL-4 can be induced to express NOS2 after receiving appropriate signals, such as IFN-γ, and subsequently produce NO. Microenvironmental conditions during their development can influence whether MC are capable of NOS expression and of NO production.     

A new role for monoamine oxidases in the modulation of macrophage-inducible nitric oxide synthase gene expression

This report focuses on the modulatory role of endogenous H(2)O(2) on lipopolysaccharide (LPS)/interferon-gamma (IFN-gamma)-induced inducible nitric oxide synthase (NOS2) gene expression in rat peritoneal macrophages. Exogenously added H(2)O(2) was initially found to inhibit the synthesis of NOS2, which prompted us to assess the effect of the activity of monoamine oxidase (MAO) and semicarbazide-sensitive amine oxidase (SSAO) as H(2)O(2)-forming enzymes on NOS2 gene expression. In the presence of their substrates, tyramine for MAO and benzylamine for SSAO, intracellular synthesis of H(2)O(2) took place with concomitant inhibition of LPS/IFN-gamma-induced NOS2 protein synthesis, as detected by Western blotting, flow cytometry, and immunofluorescence microscopy analyses. Pargyline and semicarbazide, specific inhibitors of MAO and SSAO, respectively, canceled this negative effect of MAO substrates on NOS2 expression. In the presence of Fe(2+) and Cu(2+) ions, inhibition of NOS2 expression was enhanced, suggesting the participation in this regulation of species derived from Fenton chemistry. In addition, the negative effect of H(2)O(2), generated by MAOs, was found to be exerted on NOS2 mRNA levels. These data offer a new insight in the control of NOS2 expression through the intracellular levels of H(2)O(2) and other reactive oxygen species (ROS). The hypothesis can be raised that the inhibition of NOS by H(2)O(2) could constitute a protective mechanism against the cytotoxic consequences of the activation of ROS-generating enzymes, thus providing a new, singular role for the MAO family of proteins.     

Nitric oxide-mediated mechanism of neuronal nitric oxide synthase and inducible nitric oxide synthase expression during hypoxia in the cerebral cortex of newborn piglets

Previously, we have shown that hypoxia results in increased generation of nitric oxide free radicals in the cerebral cortex of newborn piglets that may be due to up-regulation of nitric oxide synthases, neuronal nitric oxide synthase and inducible nitric oxide synthase. The present study tests the hypothesis that hypoxia results in increased expression of neuronal nitric oxide synthase and inducible nitric oxide synthase in the cerebral cortex of newborn piglets and that the increased expression is nitric oxide-mediated. Newborn piglets, 2-4 days old, were divided to normoxic (n=4), hypoxic (n=4) and hypoxic-treated with 7-nitro-indazole-sodium salt, a selective neuronal nitric oxide synthase inhibitor (hypoxic-7-nitro-indazole-sodium salt, n=6, 1 mg/kg, 60 min prior to hypoxia). Piglets were anesthetized, ventilated and exposed to an FiO2 of 0.21 or 0.07 for 60 min. Cerebral tissue hypoxia was documented biochemically by determining ATP and phosphocreatine. The expression of neuronal nitric oxide synthase and inducible nitric oxide synthase was determined by Western blot using specific antibodies for neuronal nitric oxide synthase and inducible nitric oxide synthase. Protein bands were detected by enhanced chemiluminescence, analyzed by imaging densitometry and the protein band density expressed as absorbance (OD x mm(2)). The density of neuronal nitric oxide synthase in the normoxic, hypoxic and hypoxic-7-nitro-indazole-sodium salt groups was: 41.56+/-4.27 in normoxic, 61.82+/-3.57 in hypoxic (P<0.05) and 47.80+/-1.56 in hypoxic-7-nitro-indazole-sodium salt groups (P=NS vs normoxic), respectively. Similarly, the density of inducible nitric oxide synthase in the normoxic, hypoxic and hypoxic-7-nitro-indazole-sodium salt groups was: 105.21+/-9.09, 157.71+/-13.33 (P<0.05 vx normoxic), 117.84+/-10.32 (p=NS vx normoxic), respectively. The data show that hypoxia results in increased expression of neuronal nitric oxide synthase and inducible nitric oxide synthase proteins in the cerebral cortex of newborn piglets and that the hypoxia-induced increased expression is prevented by the administration of 7-nitro-indazole-sodium salt. Furthermore, the neuronal nitric oxide synthase inhibition prevented the inducible nitric oxide synthase expression for a period of 7 days after hypoxia. Since administration of 7-nitro-indazole-sodium salt prevents nitric oxide generation by inhibiting neuronal nitric oxide synthase, we conclude that the hypoxia-induced increased expression of neuronal nitric oxide synthase and inducible nitric oxide synthase is mediated by neuronal nitric oxide synthase derived nitric oxide. We speculate that during hypoxia nitric oxide-mediated up-regulation of nitric oxide synthases will continue the perpetual cycle of nitric oxide generation-->NOS up-regulation-->nitric oxide generation resulting in hypoxic neuronal death.     

Genetic analysis of nitric oxide synthase isoforms: targeted mutation in mice

Sine the discovery that nitric oxide is an endogenous vasodilator responsible for endothelium-derived relaxing factor activity, nitric oxide has been found in many different cell types and implicated in many diverse biological processes. Because pharmacological blockade does not distinguish between the three major isoforms of nitric oxide synthase, the tissue and enzyme source of nitric oxide is unclear in many situations. Targeted disruption of the genes for the various isoforms of nitric oxide synthase offers a useful genetic approach to study the roles of each isoform and to examine the effects of their deletion on physiological processes in intact animals. Here we review the phenotypes of the various nitric oxide synthase mutant mice and examine what they reveal about the complexities of the nitric oxide signaling system and about molecular and physiological compensations brought into play in the absence of individual isoforms.Abbreviations rCBF Relative cerebral blood flow - EDRF Endothelium-dependent relaxing factor - IJP Inhibitory junction potentials - LTP Long-term potentiation - L-NAME l-N-Arginine-methyl ester - L-NMMA l N-Monomethyl arginine - L-NA lNitro arginine - LPS Lipopolysaccharide - NOS Nitric oxide synthase - nNOS Neuronal NOS - iNOS Inducible NOS - eNOS Endothelial NOS - VIP Vasoactive intestinal peptide     

巨噬细胞诱导型一氧化氮合酶的表达调节机制

一氧化氮是一种重要的巨噬细胞免疫效应分子,它参与免疫调节和宿主防御反应.一氧化氮的生成主要由诱导型一氧化氮合酶调节,然而诱导型一氧化氮合酶表达的调节机制及信号通路尚不完全清楚.     

Polymorphism of the inducible nitric oxide synthase gene in celiac disease

The aim of this study was to investigate the possible association between the inducible nitric oxide synthase (NOS2) gene promoter polymorphism, CCTTTn microsatellite, with celiac disease susceptibility. We carried out a familial study in which 53 Spanish families were genotyped by a polymerase chain reaction (PCR)-based method combined with fluorescent technology. A transmission disequilibrium test was performed to investigate the transmission pattern of the different CCTTTn alleles from parents to affected offspring. The test did not reach any statistically significant difference because none of the CCTTTn repeats was shown to be significantly transmitted to the affected siblings. Our data suggest that the CCTTTn pentanucleotide microsatellite in the NOS2 gene promoter does not play a major role in celiac disease development.