Glu298Asp polymorphism of the endothelial nitric oxide synthase gene in Turkish patients with ischemic stroke

The low plasma nitric oxide concentrations and reduced vascular reactivity are considered major proatherogenic mechanisms in cardiovascular diseases. The present study aimed to assess the allelic frequency and the genotypic distribution of the Glu298Asp gene polymorphism at exon 7 of endothelial nitric oxide synthase (eNOS) gene in Turkish ischemic stroke patients compared to appropriate healthy controls, and to correlate the genetic findings with stroke subtypes. The study population included 146 (75 males, 71 females) patients with ischemic stroke which were categorized according to the Trial of ORG 10172 in Acute Stroke Treatment (TOAST) and 133 (34 males, 99 females) healthy subjects. The eNOS polymorphism was identified with a PCR followed by RFLP with the restriction enzyme BanII. Genotypes were defined as GG, GT, and TT according to the presence of the G and T alleles. In this case-control study, we did not find any significant difference in either the genotypic distribution or allelic frequency of Glu298Asp gene polymorphism between the patients and the controls. In addition, there was also no significant difference for the genotype distribution and the allelic frequency among the stroke subtypes. The results suggested the lack of the association between the Glu298Asp gene polymorphism and ischemic stroke or subtypes of ischemic stroke in the Turkish population.     

The missense Glu298Asp variant of the endothelial nitric oxide synthase gene is strongly associated with placental abruption

We recently identified a missense variant (Glu298Asp) that lies within exon 7 of the endothelial nitric oxide synthase (eNOS) gene, and that is associated with severe preeclampsia (proteinuric hypertension that develops as a consequence of pregnancy). Maternal hypertension is the most consistently identified factor predisposing to placental abruption. Our objective, therefore, was to analyze the association between the Glu298Asp eNOS gene variant and placental abruption. The study participants included 35 patients with histories of placental abruption and 170 control subjects. Screening for the Glu298Asp eNOS gene variant was carried out by analysis of polymerase chain reaction/restriction fragment length polymorphism. The analyses revealed that the frequency of the Glu298Asp variant (Glu298Asp homozygotes and heterozygotes) was significantly (P<0.001) higher in the placental abruption group (n=14; 40%) than in the control group (n=24; 14%). We conclude that the presence of the Glu298Asp eNOS gene variant could be a marker of increased risk of developing placental abruption.     

Association of the Glu298Asp polymorphism in the endothelial nitric oxide synthase gene with essential hypertension resistant to conventional therapy

Endothelial nitric oxide synthase (eNOS) produces nitric oxide (NO) which, after diffusing into vascular smooth muscle cells, activates guanylate cyclase leading to vasodilatation. A polymorphism (894G to T) in exon 7 of the eNOS gene causes the conversion of Glu to Asp in position 298. The recently described crystal structure of the heme domain of eNOS protein shows that Glu298 is fully solvent accessible and distant from regions integral to enzyme function. Searching for phenotypic expression of eNOS gene variants, we genotyped a group of patients with essential hypertension (H, n = 119) for the Glu298Asp polymorphism and compared them with age- and sex-matched healthy normals (N, n = 85). To specify phenotypic expression further, the hypertensive patients were subdivided into one group that responded well to regular antihypertensive therapy (CH, n = 45) and one group that was resistant to the therapy (RH, n = 74). Patients with BP higher than 140/90 mmHg when on adequate lifestyle modification and triple-combination therapy (including diuretics) were considered resistant. In RH and H groups, a significantly higher frequency of T alleles (P = 0.022 and P = 0.046, respectively) was found compared to normotonics (N). In well-controlled hypertonics, the same tendency was found, but did not reach statistical significance. The Glu298Asp polymorphism may contribute to the complex pathogenesis of essential hypertension and may be a factor in the resistance of these patients to conventional antihypertensive therapy. The presence of this allele may thus be predictive of the patients' therapeutic response.     

A missense Glu298Asp variant in the endothelial nitric oxide synthase gene is associated with coronary spasm in the Japanese

Coronary spasm plays an important role in the pathogenesis of not only variant angina but also ischemic heart disease in general. However, the precise mechanism(s) by which coronary spasm occurs remains to be elucidated. Coronary spasm may arise from interactions between environmental and genetic factors. Endothelial derived nitric oxide (NO) has been implicated in the control of vascular tone. We have recently shown that both basal and acetylcholine (ACh)-induced NO activities are impaired in the coronary arteries of patients with coronary spasm. The purpose of this study has been to elucidate the possible variants that occur in the coding region of the endothelial nitric oxide synthase (eNOS) gene and that may be associated with coronary spasm. After initial screening in the entire 26 coding regions of the eNOS gene, we found a missense Glu298Asp variant in exon 7 in patients with coronary spasm. We subsequently performed a larger scale study involving 113 patients with coronary spasm and 100 control subjects, who were all diagnosed by intracoronary injection of ACh. The analysis revealed a significant difference in the distribution of the variant between the coronary spasm group (21.2%) and control group (9.0%; P=0.014 for dominant effect). Thus, we have found the missense Glu298Asp variant in the eNOS gene by the analysis of its entire 26 coding regions. The variant is significantly associated with coronary spasm. Received: 2 February 1998 / Accepted: 9 April 1998     

Relationship between hemorheology and Glu(298)Asp polymorphism of endothelial nitric oxide synthase gene in patients with coronary artery disease

This study aimed to investigate the relationship between endothelial nitric oxide synthase Glu(298)Asp gene polymorphism and hemorheological parameters. Red blood cell (RBC) deformability, aggregation were measured using an ectacytometry, whole blood, plasma viscosities were determined by a viscometer. Restriction fragment length polymorphism was used to detect polymorphism. Plasma nitrite, nitrate concentrations were determined by Griess method. The genotype distribution of the control group was as follows: 50 (67.5%) GG, 21 (28.4%) GT, 3 (4.1%) TT. A 48 (57.8%) of the patients with CAD had GG, 28 (33.7%) GT, 7 (8.5%) of them TT genotype. RBC aggregation index of CAD patients with G allele was higher and t½ lower compared to controls carrying the same allele. The amplitude of RBC aggregation of healthy subjects with T allele, who are under increased cardiovascular risk was lower compared to control subjects with G allele. The results of this study indicate that, alterations in RBC aggregation seem to be a consequence of CAD, more than being a preexisting cause. Additionally, some compensatory mechanisms by causing decrements in RBC aggregation, may help regulation of circulation in healthy individuals with high cardiovascular risk.     

Redox properties of human endothelial nitric-oxide synthase oxygenase and reductase domains purified from yeast expression system

Characterization of the redox properties of endothelial nitric-oxide synthase (eNOS) is fundamental to understanding the complicated reaction mechanism of this important enzyme participating in cardiovascular function. Yeast overexpression of both the oxygenase and reductase domains of human eNOS, i.e. eNOS(ox) and eNOS(red), has been established to accomplish this goal. UV-visible and electron paramagnetic resonance (EPR) spectral characterization for the resting eNOS(ox) and its complexes with various ligands indicated a standard NOS heme structure as a thiolate hemeprotein. Two low spin imidazole heme complexes but not the isolated eNOS(ox) were resolved by EPR indicating slight difference in heme geometry of the dimeric eNOS(ox) domain. Stoichiometric titration of eNOS(ox) demonstrated that the heme has a capacity for a reducing equivalent of 1-1.5. Additional 1.5-2.5 reducing equivalents were consumed before heme reduction occurred indicating the presence of other unknown high potential redox centers. There is no indication for additional metal centers that could explain this extra electron capacity of eNOS(ox). Ferrous eNOS(ox), in the presence of l-arginine, is fully functional in forming the tetrahydrobiopterin radical upon mixing with oxygen as demonstrated by rapid-freeze EPR measurements. Calmodulin binds eNOS(red) at 1:1 stoichiometry and high affinity. Stoichiometric titration and computer simulation enabled the determination for three redox potential separations between the four half-reactions of FMN and FAD. The extinction coefficient could also be resolved for each flavin for its semiquinone, oxidized, and reduced forms at multiple wavelengths. This first redox characterization on both eNOS domains by stoichiometric titration and the generation of a high quality EPR spectrum for the BH(4) radical intermediate illustrated the usefulness of these tools in future detailed investigations into the reaction mechanism of eNOS.     

Preliminary computational modeling of nitric oxide synthase 3 interactions with caveolin-1： influence of exon 7 Glu298Asp polymorphism

The significance of endothelial nitric oxide synthase 3 （NOS3） activity has been recognized for many years, however it was only recently that the complicated regulation of this constitutively expressed enzyme in endothelial cells was identified. A critical component of the NOS3 regulatory cyde in endothelial cells is its intracellnlar localization to caveolae. The caveolar coordination of NOS3, more specifically its interaction with caveolin-1 （Cav-1）, plays a major role in normal endothelial NOS3 activity and vascular bioavailability of nitric oxide. We have recently shown that the presence of NOS3 exon 7 Glu298Asp polymorphism caused diminished shear-dependent NOS activation, was less extensively associated with caveolae, and had a decreased degree of interaction with Cav-1. Here, we carried out preliminary investigations to identify possible mechanisms of the genotype-dependent endothelial cell responses we observed in our previous investigations. Through this approach we tested the hypothesis that computer simulations could provide insights regarding the contribution of this single nucleotide polymorphism to regulation of the NOS3 isoform. We observed that in the Glu/Asp and Asp/Asp mutant genotypes, the amount of NOS3 associated with Cav-1 was significantly lower. Additionally, we have shown, using a theoretical computational model, that mutation of an amino acid at position 298 might affect the protein-protein interactions and localization of the NOS3 protein. These alterations might also affect the protein function and explain the enhanced disease risk associated with the presence of Glu298Asp polymorphism in the NOS3 protein.     

Acidic hydrolysis as a mechanism for the cleavage of the Glu(298)-->Asp variant of human endothelial nitric-oxide synthase

The 894G-->T polymorphism within exon 7 of the human endothelial nitric-oxide synthase (eNOS) gene codes for glutamate or aspartate, respectively, at residue 298 and has been associated with several diseases of cardiovascular origin. A recent report indicates that Asp(298)-eNOS (E298D) is cleaved intracellularly to 100- and 35-kDa fragments, suggesting a mechanism for reduced endothelial function. Here we have documented the precise cleavage site of the E298D variant as a unique aspartyl-prolyl (Asp(298)--Pro(299)) bond not seen in wild-type eNOS (Glu(298)). We show that E298D-eNOS, as isolated from cells and in vitro, is susceptible to acidic hydrolysis, and the 100-kDa fragment can be generated ex vivo by increasing temperature at low pH. Importantly, cleavage of E298D was eliminated using a sample buffer system designed to limit acidic hydrolysis of Asp--Pro bonds. These results argue against intracellular processing of E298D-eNOS and suggest that previously described fragmentation of E298D could be a product of sample preparation. We also found that eNOS turnover, NO production, and the susceptibility to cellular stress were not different in cells expressing WT versus E298D-eNOS. Finally, enzyme activities were identical for the respective recombinant enzymes. Thus, intracellular cleavage mechanisms are unlikely to account for associations between the exon 7 polymorphism and cardiovascular diseases.     

The Asp298 allele of endothelial nitric oxide synthase is a risk factor for myocardial infarction among patients with type 2 diabetes mellitus

Background

To establish an efficient prophylaxis of coronary artery disease reliable risk stratification is crucial, especially in the high risk population of patients suffering from diabetes mellitus. This prospective study determined the predictive value of coronary calcifications for future cardiovascular events in asymptomatic patients with diabetes mellitus.

Methods

We included 716 patients suffering from diabetes mellitus (430 men, 286 women, age 55.2 ± 15.2 years) in this study. On study entry all patients were asymptomatic and had no history of coronary artery disease. In addition, all patients showed no signs of coronary artery disease in ECG, stress ECG or echocardiography. Coronary calcifications were determined with the Imatron C 150 XP electron beam computed tomograph. For quantification of coronary calcifications we calculated the Agatston score. After a mean observation period of 8.1 ± 1.1 years patients were contacted and the event rate of cardiac death (CD) and myocardial infarction (MI) was determined.

Results

During the observation period 40 patients suffered from MI, 36 patients died from acute CD. The initial Agatston score in patients that suffered from MI or died from CD (475 ± 208) was significantly higher compared to those without cardiac events (236 ± 199, p < 0.01). An Agatston score above 400 was associated with a significantly higher annualised event rate for cardiovascular events (5.6% versus 0.7%, p < 0.01). No cardiac events were observed in patients with exclusion of coronary calcifications. Compared to the Framingham risk score and the UKPDS score the Agatston score showed a significantly higher diagnostic accuracy in the prediction of MI with an area under the ROC curve of 0.77 versus 0.68, and 0.71, respectively, p < 0.01.

Conclusion

By determination of coronary calcifications patients at risk for future MI and CD could be identified within an asymptomatic high risk group of patients suffering from diabetes mellitus. On the other hand future events could be excluded in patients without coronary calcifications.     

Hydrocortisone modulates the effect of estradiol on endothelial nitric oxide synthase expression in human endothelial cells.

The interaction between hydrocortisone and estradiol on the regulation of endothelial nitric oxide synthase (eNOS) expression was investigated in human umbilical vein endothelial cells (HUVECs). Following incubation in medium containing dextran-coated-charcoal-stripped serum (DCC-stripped medium) for 4 days, incubation of HUVECs with 0.1 nM estradiol for 24 hr in the absence of hydrocortisone increased levels of eNOS mRNA measured by ribonuclease protection assay above control (0 nM estradiol). 2 microM hydrocortisone applied for 24 hr preceding and during estradiol application inhibited the estradiol-elicited increase in eNOS mRNA levels, reducing mRNA levels from 134% +/- 14% of control to 85% +/- 5% of control. Significant (ANOVA, p<0.01) reductions of estradiol-mediated increases of mRNA levels occurred over a range of hydrocortisone concentrations (10 nM, p<0.05; 2 microM, p<0.05; n=3-12). In the presence of 2 microM hydrocortisone, 10 nM estradiol significantly reduced eNOS mRNA levels to 59% +/- 3% of control. The ability of hydrocortisone to block or reverse the estradiol-mediated increase in eNOS mRNA levels may provide a link between elevated hydrocortisone levels and decreased NO production, potentially contributing to the development of hypertension and cardiovascular disease in vivo and antagonizing cardioprotective effects of estrogens.     

PIN inhibits nitric oxide and superoxide production from purified neuronal nitric oxide synthase

A protein inhibitor of neuronal nitric oxide synthase (nNOS) was identified and designated as PIN. PIN was reported to inhibit nNOS activity in cell lysates through disruption of enzyme dimerization. However, there has been lack of direct characterization of the effect of PIN on NO production from purified nNOS. Furthermore, nNOS also generates superoxide (.O(2)(-)) at low levels of L-arginine. It is unknown whether PIN affects .O(2)(-) generation from nNOS. Therefore, we performed direct measurements of the effects of PIN on NO and .O(2)(-) generation from purified nNOS using electron paramagnetic resonance spin trapping techniques. nNOS was isolated by affinity chromatography and a fusion protein CBP-PIN was used to probe the effect of PIN. While the tag CBP did not affect nNOS activity, CBP-PIN caused a dose-dependent inhibition on both NO and L-citrulline production. In the absence of L-arginine, strong .O(2)(-) generation was observed from nNOS, and this was blocked by CBP-PIN in a dose-dependent manner. With low-temperature polyacrylamide gel electrophoresis, neither CBP nor CBP-PIN was found to affect nNOS dimerization. Thus, these results suggested that PIN not only inhibits NO but also .O(2)(-) production from nNOS, and this is through a mechanism other than decomposition of nNOS dimers.     

Characterization of recombinant human endothelial nitric-oxide synthase purified from the yeast Pichia pastoris

Human endothelial nitric-oxide synthase (eNOS) was expressed in the methylotrophic yeast Pichia pastoris, making use of the highly inducible alcohol oxidase promoter. The recombinant protein constituted approximately 3% of total protein and was largely soluble (>75%). About 1 mg of purified eNOS was obtained from 100-ml yeast cell cultures by affinity chromatography of crude cell supernatants. The purified enzyme had a V(max) of 192 +/- 18 nmol of L-citrulline x mg(-1) x min(-1), had a K(m) for L-arginine of 3.9 +/- 0.2 microM, and showed an absolute requirement for tetrahydrobiopterin (H(4)biopterin). NADPH oxidase activity was 136 +/- 9 and 342 +/- 24 nmol x mg(-1) x min(-1) in the absence and presence of 0.1 mM L-arginine, respectively, and not affected by H(4)biopterin. The protein contained 0.56 +/- 0.06 equivalents of FAD and 0.79 +/- 0.08 equivalents of FMN. On-line gel filtration/inductively coupled plasma mass spectrometry analysis confirmed that both iron (0.80 +/- 0.09 mol/subunit) and zinc (0.43 +/- 0.03 mol/subunit) were bound to the enzyme. Graphite furnace-atomic absorption spectroscopy yielded a value for bound iron of 0.84 +/- 0.04 mol/subunit. The absorbance of the enzyme at 398 nm implied a heme content of 0.85 +/- 0.09 mol/subunit, and the high pressure liquid chromatography heme assay gave an estimate of 0.71 +/- 0.02 mol heme/subunit. Gel permeation chromatography yielded one single peak with a Stokes radius of 6.62 +/- 0.7 nm, indicating that the native protein is dimeric. Upon low temperature gel electrophoresis the untreated protein appeared mainly as a monomer (88 +/- 3%), but pretreatment with H(4)biopterin and L-arginine led to a pronounced shift toward dimers (77 +/- 4%). Thus, in contrast to bovine eNOS (List, B. M., Kl?sch, B., V?lker, C., Gorren, A. C. F., Sessa, W. C., Werner, E. R., Kukovetz, W. R., Schmidt, K., and Mayer, B. (1997) Biochem. J. 323, 159-165; Rodriguez-Crespo, I., Gerber, N. C., and Ortiz de Montellano, P. R. (1996) J. Biol. Chem. 271, 11462-11467), the human eNOS appears to be markedly stabilized by H(4)biopterin.     

Molecular cloning and expression of a cDNA encoding endothelial cell nitric oxide synthase.

Endothelium-derived relaxing factor (EDRF), identified as nitric oxide (NO), is derived from a guanidino nitrogen of L-arginine via its metabolism by nitric oxide synthase (NOS). Herein, we report the molecular cloning of a cDNA encoding the constitutive calcium-calmodulin (Ca2+/CaM)-regulated nitric oxide synthase (ECNOS). A full-length ECNOS clone was isolated by screening a bovine aortic endothelial cell cDNA library using a fragment of rat brain NOS (bNOS) cDNA. This cDNA has an open reading frame of 3615 nucleotides encoding a 1205-amino acid protein. Membranes prepared from COS cells transfected with the ECNOS cDNA demonstrated NADPH- and Ca2+/CaM- dependent conversion of L-, but not D-, arginine to NO and citrulline that was inhibited by NG-nitro-L-arginine methyl ester. Comparison of the deduced amino acid sequence of ECNOS to the bNOS and macrophage NOS (Mac-NOS) sequences revealed 57 and 50% identity, respectively. In addition, ECNOS contains a unique N-myristylation consensus sequence (not shared by bNOS or Mac-NOS) that may explain its membrane localization.     

Generation of superoxide by purified brain nitric oxide synthase.

Brain nitric oxide synthase (NOS), which utilizes NADPH and calcium/calmodulin as cofactors for metabolizing L-arginine to nitric oxide (NO) and L-citrulline, contains recognition sites for the flavins FAD and FMN. Using a spin-trapping technique combined with electron spin resonance spectroscopy, we report that brain NOS generates superoxide O2-. in a calcium/calmodulin-dependent manner. The "specific inhibitors" of NOS, NG-monomethyl L-arginine (L-NMMA), and NG-nitro-L-arginine methyl ester (L-NAME), have different effects on O2-. generation. For L-NMMA, O2-. production is unaffected, while for L-NAME, inhibition of this free radical is concentration-dependent.     

Alternative polyadenylation sites of human endothelial nitric oxide synthase mRNA

The mRNA 3'-untranslated region (3'-UTR) has been shown to have important roles in the regulation of mRNA function. In this study, we investigated the human endothelial nitric oxide synthase (eNOS) 3'-UTR to evaluate its potential regulatory role. 3'-RACE analysis revealed that the human eNOS mRNA has multiple alternative polyadenylation sites. Apart from the proximal site (418bp downstream of the stop codon), we identified two additional distal sites approximately 770 and 1478bp downstream of the stop codon. In addition, Northern analysis showed that the usage of these sites differed among human tissues. Further, amounts of these eNOS mRNAs were changed during growth of cultured human aortic endothelial cells; mRNAs with long 3'-UTRs decreased more rapidly than total mRNA, as cells approached confluency. Thus, the 3'-UTRs of human eNOS results from alternative polyadenylation sites and differ across tissues and during cell growth.     

人eNOS复制缺陷型重组腺病毒的构建

目的:构建以巨型细胞病毒(CMV)为启动子的heNCS重组腺病毒转移载体.方法和结果:将heNOS cDNA全长亚克隆到穿梭质粒启动子CMV的下游,通过I-Ceu Ⅰ和PI-SceⅠ两个稀有酶切位点将目的基因heNOS与腺病毒质粒DNA(pAdeno-X)进行体外连接,获得重组腺病毒质粒DNA(pAdeno-heNOS),后者经限制性内切酶PacⅠ切割,利用脂质体转染法获得heNOS重组腺病毒.PCR双引物法鉴定是否成功构建heNCS重组腺病毒.结论:PCR双引物检测含有heNOS片段,表明成功构建了heNOS重组腺病毒转移载体AdhCMV-heNOS.     

NO Level and Endothelial NO Synthase Gene Polymorphism （Glu298Asp） in the Patients with Coronary Artery Disease from the Turkish Population

A healthy endothelium plays a core role in cardiovascu-lar control [1]. In the endothelial cell, nitric oxide (NO) issynthesized by the endothelial nitric oxide synthase (eNOS)encoded by a 26-exon gene (NOS 3) located on chromo-some 7 [2]. Besides its regulatory functions on vasomotortone and blood flow, endothelial NO is known to inhibitthe platelet activation and modulate migration and growthof the vascular smooth muscle [3]. Indirect evidence sug-gests that alterations of the NO pathwa…