尿酸抑制人脐静脉内皮细胞合成一氧化氮

目的 研究尿酸(UA)对人脐静脉内皮细胞(HUVEC)表达内皮型一氧化氮合酶(eNOS)及分泌一氧化氮(NO)的影响.方法 不同浓度UA(0、0.5、1、1.5及2 mg/L)及50 mg/L ox-LDL(阳性对照)分别作用HUVEC 24、48及72 h,用real-time PCR法测定HUVEC eNOS mRNA;Western blot法检测细胞eNOS蛋白;酶法检测上清液NO的含量.结果 UA 0.5 mg/L组eNOS mRNA表达水平明显高于对照组(P＜0.05);随着UA浓度升高(1、1.5及2 mg/L组),及其作用时间延长,HUVEC eNOS mRNA及蛋白表达水平及上清液NO分泌最相比对照均明显下降(72 h N02-/N03-2 mg/L组与对照组分别为0.52±0.18与1.00±0.10,P＜0.05),且趋势与ox-LDL组相同.结论 0.5 mg/L以上浓度的UA呈浓度及时间依赖性抑制HUVEC eNOS表达及NO合成,提示高浓度的UA可能损伤血管内皮功能.     

Protective role of endothelial nitric oxide synthase

Nitric oxide is a versatile molecule, with its actions ranging from haemodynamic regulation to anti-proliferative effects on vascular smooth muscle cells. Nitric oxide is produced by the nitric oxide synthases, endothelial NOS (eNOS), neural NOS (nNOS), and inducible NOS (iNOS). Constitutively expressed eNOS produces low concentrations of NO, which is necessary for a good endothelial function and integrity. Endothelial derived NO is often seen as a protective agent in a variety of diseases.This review will focus on the potential protective role of eNOS. We will discuss recent data derived from studies in eNOS knockout mice and other experimental models. Furthermore, the role of eNOS in human diseases is described and possible therapeutic intervention strategies will be discussed.     

人内皮型一氧化氮合酶cDNA在COS-7细胞中的表达

本研究从人脐静脉血管内皮细胞中提取总RNA，采用逆转录PCR(RT-PCR)方法，扩增出人内皮型一氧化氮合酶(heNOS)cDNA，总长度为3731bp。将其克隆入pUCm—T载体质粒，序列分析表明，克隆所得片段含有完整开放阅读框架，与GenBank中heNOScDNA序列同源性达99．93％，在此基础上发现有若干核酸多态性。将该基因片段亚克隆到真核表达载体pcDNA3．0上，用脂质体转染法将pcDNA3．0／heNOS转染到COS-7细胞株，RT—PCR、Western blot分别检测到外源性eNOS基因在mRNA水平和蛋白质水平的表达，分析表明所表达的heNOS蛋白质分子量为145ku；L-^14C-精氨酸掺入同位素法检测证实所表达的eNOS蛋白具有生物学活性，能将精氨酸氧化为瓜氨酸。     

溶血磷脂酰胆碱对内皮细胞一氧化氮的生成的影响

目的：探讨溶血磷脂酰胆碱（Lysophosphatidylcholine,Lyso-PC)对培养的人脐静脉内皮细胞（human umbilical vein endothelial cells,HUVECs)一氧化氮（nitric oxide,NO)的生成的影响。方法：采用NO酶法测定内皮细胞NO生成的变化。结果：内皮细胞NO的生成对Lyso-PC具有时间和剂量依赖性,结论：Lyso-PC可能通过抑制内皮细胞NO的生成而导致动脉粥样硬化损伤的形成。     

低糖诱导人脐静脉内皮细胞一氧化氮与活性氧变化的研究

目的观察低浓度葡萄糖对人脐静脉内皮细胞一氧化氮（NO）与活性氧（ROS）变化的影响。方法以体外培养人脐静脉内皮细胞株HUVEC-12为研究对象,将实验分为正常对照组（5.5mmol/L葡萄糖）、低糖组（2.8mmol/L葡萄糖）、无糖组（0mmol/L葡萄糖）。分别用2.8、0mmol/L葡萄糖干预HUVEC-12细胞,经过1、2、4、12h后,硝酸还原酶法测定NO产量,化学比色法测定一氧化氮合成酶（NOS）活性,Dihydroethidium荧光探针法测定细胞内ROS水平。结果与正常对照组相比,低糖组与无糖组NO水平降低（P〈0.01）,NOS活性下降（P〈0.01）,细胞内ROS水平升高（P〈0.01）,均呈剂量依赖关系。同一浓度组内随着时间的延长,内皮细胞NO水平、NOS活性进一步降低（P〈0.01）,ROS水平进一步升高（P〈0.05）,各指标都具有浓度和时间依赖性。结论低糖可导致内皮细胞功能障碍,NO水平降低,NOS活性下降,其机制可能与低糖导致内皮细胞氧化应激损伤有关。     

葛根素预处理上调内皮型一氧化氮合酶的表达减轻人脐静脉内皮细胞缺氧/复氧损伤

 目的:探讨葛根素(puerarin,PUE)预处理对缺氧/复氧(hypoxia reoxygenation,H/R)损伤的人脐静脉内皮细胞(HUVECs)的保护作用。方法:HUVECs随机分为正常对照(control)组、H/R组、单纯PUE组和PUE+H/R组(1.0×10^-3 mol/L PUE预处理24 h后进行H/R)。采用Western blot方法检测内皮型一氧化氮合酶(eNOS)蛋白的表达,化学比色法检测组成型一氧化氮合酶(cNOS)的活性,TUNEL法检测细胞凋亡。此外,在PUE预处理前使用ERK蛋白激酶抑制剂U0126(1.0×10^-5 mol/L)或PI3K/Akt蛋白激酶抑制剂LY294002(5.0×10^-5mol/L)处理细胞1 h,再进行H/R。结果:与control组相比,H/R组的eNOS蛋白表达水平降低(P<0.05),PUE预处理上调eNOS蛋白的表达水平(P<0.05),该上调作用均可被U0126及LY294002抑制(P<0.05);与control组相比,H/R组的cNOS活力下降(P<0.05),PUE预处理组的cNOS活力增加(P<0.05);与control组相比,H/R组细胞凋亡指数显著增大(P<0.01),PUE预处理组的细胞凋亡指数减小(P<0.01)。结论:H/R可使HUVECs受损伤,eNOS蛋白表达及活性降低,细胞凋亡增加。PUE预处理可通过ERK1/2信号通路和PI3K/Akt信号通路上调HUVECs的eNOS蛋白表达,增强eNOS活性,减少细胞凋亡,发挥内皮细胞保护作用。     

Endothelial nitric oxide synthase gene polymorphism in women with idiopathic recurrent miscarriage

BACKGROUND: Lack of endothelium-derived nitric oxide is associated with vasospasm and vascular infarction. We investigated the relationship between idiopathic recurrent miscarriage and a polymorphism of the gene encoding endothelial nitric oxide synthase (NOS3). METHOD: In a prospective case-control study, 105 women with idiopathic recurrent miscarriage and 91 healthy controls were investigated. We used the polymerase chain reaction to identify the different alleles of a 27 base pair tandem repeat polymorphism in intron 4 of the NOS3 gene. RESULTS: The wild type B allele was identified on 329 out of 392 chromosomes (frequency 0.84). The polymorphic A allele was present on 63 chromosomes (frequency 0.16). The genotype frequencies were as follows: 68% (B/B), 31% (A/B) and.5% (A/A). The distribution of genotype frequencies was significantly different between the study and control groups for allele A/B heterozygotes (NOS3(A/B)) (36.7 versus 23.8%, P = 0.03, OR 1.6, 95% CI 1.1--3.8). Only one individual was homozygous for the A allele (NOS3(A/A)). She was in the study group. Between women with primary and secondary recurrent miscarriages, no statistically significant difference between the distribution of NOS3(A/B) genotypes (28 versus 34%) was observed. CONCLUSIONS: These data support a role for the NOS3 gene as a genetic determinant of the risk of idiopathic recurrent miscarriage.     

血流切应力调控内皮型一氧化氮合酶的分子机制

血流切应力（flowshear stress，rss）是生理或病理条件下调节血管内皮细胞产生一氧化氮的最重要的刺激因素。FSS对内皮型一氧化氮合酶（endothelial nitric oxide synthase，eNOS）的调控包括基因转录的调节、转录后的调节和翻译后的调节。eNOS基因转录以及转录后mRNA的稳定性能被FSS诱导加强。FSS通过游离钙离子浓度、磷酸化、eNOS相关蛋白以及细胞内易位等途径调节eNOS的催化活性。此外，FSS还能调控eNOS催化反应的辅助因子。     

Expression of nitric oxide synthase and effect of substrate manipulation of the nitric oxide pathway in mouse ovarian follicles

BACKGROUND: Nitric oxide (NO) is a cell messenger with multiple actions in different biological systems, implicated in the control of follicle and oocyte function. NO is formed from L-arginine by isoforms of nitric oxide synthase (NOS) via NG-hydroxy-L-arginine, with L-citrulline as a byproduct. This study aimed to show how modulation of NO by manipulating NOS substrates would affect mouse follicle growth and ovulation in vitro, where vascular effects of NO are attenuated. METHODS: Immunohistochemistry [endothelial (eNOS) and inducible (iNOS)] and in situ hybridization (iNOS) were applied on mouse ovaries. Cultured follicles were also stained for iNOS by immunohistochemistry. For follicles cultured in the presence or absence of L-arginine, the ability of L-citrulline or NG-hydroxy-L-arginine to substitute for L-arginine was assessed in terms of follicle growth and ovulation. RESULTS: iNOS and eNOS were localized in oocytes and theca, with some staining in granulosa. iNOS mRNA occurred predominantly in granulosa and oocyte. Omission of L-arginine significantly reduced follicle survival and ovulation. Partial compensation for L-arginine withdrawal was achieved with L-citrulline and NG-hydroxy-L- arginine. Specific abnormalities of follicle growth were noted. CONCLUSIONS: NOS is present in mouse follicles, and its action is necessary at a local level for normal follicle development in vitro. Reduced growth, persistent basement membranes and reduced ovulation were associated with in vitro disruption of NO.     

Genetic association between endothelial nitric oxide synthase and Alzheimer disease

Evidence suggests that vascular and inflammatory factors may be important in the etiology of Alzheimer disease (AD). The Glu/Glu genotype at the Glu298Asp variant of the endothelial nitric oxide synthase (NOS3) gene has been tested for association with AD in several Caucasian and Asian populations, with conflicting results. We tested the Glu298Asp variant for association in African American and Caucasian AD patients, unaffected siblings, and unrelated controls from the MIRAGE Study. To explore whether the inconsistent results in previous studies might be due to linkage disequilibrium with a polymorphism or haplotype not previously tested, we genotyped 10 additional NOS3 single nucleotide polymorphisms (SNPs) spanning 25.3 kb. Finally, we compiled results of previous studies of Glu298Asp using meta-analysis, to determine whether the aggregate studies support an association between Glu298Asp and AD. We found that the Glu298 allele was associated with higher risk of AD in the MIRAGE African American (p = 0.002) but not Caucasian (p = 0.9) groups. None of the additional SNPs were associated with AD in the Caucasians, whereas two showed evidence for association in the African Americans. The meta-analysis showed a small effect of the Glu298Asp GG genotype on AD risk across all studies (summary odds ratio = 1.15, 95% confidence interval: 0.97-1.35) and significant heterogeneity of this association among studies (p = 0.02).     

Gene polymorphisms of endothelial nitric oxide synthase and angiotensin-converting enzyme in patients with asthma

BACKGROUND: Nitric oxide, including that produced by endothelial constitutive nitric oxide synthase (ecNOS), may regulate vascular and airway tone in the lungs and may influence various aspects of airway homeostasis. Angiotensin-converting enzyme (ACE) is expressed at high levels in the lungs and plays a role in the metabolism of angiotensin II, bradykinin, and substance P, all of which are potentially involved in the pathogenesis of asthma. An insertion-deletion polymorphism of the ACE gene has been shown to be associated with enzyme activity levels of ACE. To examine the possible involvement of the ecNOS and/or ACE genes as the genetic basis of bronchial asthma, we investigated whether there was any association between bronchial asthma and polymorphisms of the ecNOS and/or ACE genes. METHODS: A total of 310 patients with bronchial asthma and 121 healthy subjects took part in this study. The ecNOS and ACE genotypes were determined in all subjects by polymerase chain reaction. RESULTS: 1) The distribution of one genotype (bb) of ecNOS was significantly higher in the asthma group than in the control population. 2) The ACE genotype distribution was not significantly different between the control and the asthma groups. 3) In asthmatic patients, the ACE and ecNOS genotype distribution did not differ significantly among groups of patients with different severities of asthma. CONCLUSIONS: These results suggest that polymorphisms of the ecNOS gene, but not the ACE gene, may be associated with the development of asthma. However, the severity of asthma may not be influenced by polymorphisms of the ecNOS and ACE genes.     

Augmented endothelial nitric oxide synthase (eNOS) protein expression in human pregnant myometrium: possible involvement of eNOS promoter activation by estrogen via both estrogen receptor (ER)alpha and ERbeta

The aim of the present study was to investigate the possible contribution of estrogen to pregnancy-associated modulation of nitric oxide production in the human myometrium during pregnancy. Both endothelial nitric oxide synthase (eNOS) and inducible NOS (iNOS) proteins were clearly expressed in the non-pregnant myometrium and were elevated in the first trimester of pregnancy. Oral contraceptive pills augmented eNOS, but not iNOS, protein expression in the non-pregnant human myometrium. In cultured human myometrial cells, estrogen receptor (ER)alpha and ERbeta expression was extremely low. Therefore, we used either ERalpha or ERbeta expression vector to investigate the effect of 17beta-estradiol treatment on eNOS promoter activity using eNOS promoter/luciferase vector in cultured human myometrial cells. 17beta-estradiol treatment significantly augmented eNOS promoter activity in cells co-transfected with either ERalpha or ERbeta, and this augmentation was dose-dependently suppressed by ICI 182780, an estrogen antagonist. These data suggest the possibility that both ERalpha and ERbeta are involved in the estrogen-associated regulation of eNOS gene expression in the human myometrium.     

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.     

Hyperoxia reduces basal release of nitric oxide and contracts porcine coronary arteries

Aim: The purpose of the present study was to investigate whether changes in nitric oxide (NO) concentration is involved in hyperoxia‐induced vasoconstriction in porcine conduit coronary arteries. Methods: The effect of hyperoxia on NO release and vasoconstriction was evaluated by tension recording, microsensor measurements, and immunoblotting in porcine conduit coronary arteries contracted with U46619 or 5‐hydroxytryptamine. Results: In endothelium‐intact segments exchanging 20% O₂, 5% CO₂, 75% N₂ (normoxia) for 95% O₂, 5% CO₂ (hyperoxia) increased contraction. In segments without endothelium hyperoxia‐evoked contraction was abolished, but restored by an encircling donor segment with endothelium. An inhibitor of NOS, asymmetric dimethylarginine (ADMA, 300 μm ), reduced hyperoxic contraction and basal NO concentration by, respectively, 38 ± 12% and 46 ± 3% (P < 0.05, n = 9). A NO donor, S‐nitroso‐N‐acetylpenicillamine (SNAP), increased NO concentration and evoked relaxation to the same levels in normoxic and hyperoxic conditions. β‐actin and endothelial NO synthase (eNOS) protein expression was similar in normoxic and hyperoxic arterial segments. Phosphorylation of eNOS was unaltered in normoxia vs. hyperoxia, but phosphorylation of eNOS‐Ser¹¹⁷⁷ was increased and phosphorylation of eNOS‐Thr⁴⁹⁵ decreased by U46619. Blockers of ATP‐sensitive, voltage‐dependent and calcium‐activated K⁺ channels did not change hyperoxic contraction. However, high extracellular K⁺ concentration or a second and third exposure to hyperoxia decreased contraction. Conclusion: The present study provides direct evidence that hyperoxia reduces basal release of NO leading to depletable endothelium‐dependent vasoconstriction in porcine coronary arteries independent of changes in eNOS phosphorylation.     

Immunohistochemical localization of inducible and endothelial constitutive nitric oxide synthase in neoplastic and autoimmune thyroid disorders

AIM: To investigate the distribution of nitric oxide synthase in tissues derived from patients with autoimmune or neoplastic disorders of the thyroid gland in order to test whether the expression of inducible nitric oxide synthase or endothelial constitutive nitric oxide synthase (two subtypes of EC 1.14.13.39) may be related to the inflammatory activity or degree of neoplasia. EXPERIMENTAL DESIGN: The expression of nitric oxide synthases was examined by immunohistochemistry in tissues from patients with either Hashimoto's thyroiditis (n=6), hyperplastic glands (Graves' disease) (n=7), adenomas (n=8), multinodular goitres (n=7), papillary carcinomas (n=4) or follicular carcinomas (n=5). RESULTS: Expression of inducible nitric oxide synthase was found in 22 of the tissues and was not specific for any of the examined thyroid disorders. Expression of endothelial constitutive nitric oxide synthase was found in some of the epithelial cells in all the tissues. There was no correlation between the intensity and distribution of the immunostaining and the thyroid disorders. CONCLUSION: Demonstration of nitric oxide synthase cannot be used for diagnostic purposes. The expression of endothelial constitutive nitric oxide synthase in all tissues indicates that the enzyme may be of importance for the function or growth of the thyroid epithelial cells.     

Mice deficient in endothelial nitric oxide synthase exhibit a selective deficit in hippocampal long-term potentiation

Long-term potentiation, a persistent increase in synaptic efficacy, may require a retrograde signal originating in the postsynaptic cell that induces an increase in presynaptic neurotransmitter release. We have constructed a mouse homozygous for a targeted null mutation in the endothelial isoform of nitric oxide synthase and report that long-term potentiation in the CA1 region of these mice is entirely absent under weak stimulation conditions. Application of a membrane-permeant guanosine-3′,5′-cyclic monophosphate analogue during tetanus fails to compensate for this deficit, suggesting that nitric oxide produced by endothelial nitric oxide synthase may affect long-term potentiation through a cascade that does not include guanylyl cyclase. We also report that strong tetanic stimulation can induce robust long-term potentiation in these mice which is not blocked by pharmacological inhibitors of nitric oxide synthase. Furthermore, mice lacking endothelial nitric oxide synthase show no shift in the frequency–response curve for the induction of long-term potentiation. Basal synaptic transmission, paired-pulse facilitation and the electrical properties of CA1 cells in these mice were similar to controls.

These results support a selective role for endothelial nitric oxide synthase in long-term potentiation, but also demonstrate that nitric oxide synthase is not involved in this process under all conditions.     

Ovary and ovulation: Cell-specific localization of nitric oxide synthases (NOS) in the rat ovary during follicular development, ovulation and luteal formation

Nitric oxide (NO) has emerged as one of several important intraovarianregulatory factors. In particular, NO has been implicated inthe processes of ovulation and atresia-related apoptosis. Theaim of the present study was to investigate the presence anddistribution of the NO-generating nitric oxide synthase (NOS)enzymes in the ovary during follicular development, ovulationand luteal formation of the equine chorionic gonadotrophin (ECG)/humanchorionic gonado-trophin (HCG)-primed rat NADPH diaphorase activitywas used as a histochemical marker for NOS within the ovary.Diaphorase reactivity was most abundant in the stroma (S) ofthe ovary and in the theca (T) layer of the follicle. In luteinizedovaries, weaker diaphorase reactivity was present within thecorpora lutea (CL). Two different isoforms of NOS, the constitutivelyexpressed endothelial NOS (eNOS) and the inducible isoform ofNOS (iNOS), were immunolocalized in ovaries of immature ratsand in ECG/HCG-primed rats during the periovulatory period fromHCG injection until 2 days after ovulation. In addition, ovarianconcentrations of eNOS and iNOS were quantified by immunoblotting.Immunoblotting with a monoclonal anti-eNOS antibody demonstratedthe presence of eNOS mainly in the residual ovary (ROV) duringthe periovulatory period. In luteinized ovaries, higher concentrationsof eNOS were seen in CL, while those in the ROV at this stagewere lower than in the periovulatory ovary. Immature ovariescontained diminutive amounts of eNOS, detectable mostly in theROV compartment. In contrast, iNOS was barely detectable duringfollicular development to the preovulatory stage. A slight elevationof iNOS was observed in the granulosa cells at 6 h after theHCG injection. The levels of iNOS during the luteal phase werealso low. Immunohistochemical analysis using polyclonal eNOSand iNOS antibodies revealed the localization of these two isoformsprimarily in the S and the T of the periovulatory ovary. Inluteinized ovaries, positive immunoreactivity was also seenwithin the CL. With a monoclonal antibody against eNOS, intenseimmunoreactivity was observed in the S, T and within CL. Therewas a particularly strong staining in blood vessels. These datademonstrate the presence of an intraovarian NO-generating system.The localization of this system to the S, T and CL suggestsa role for NO in the ovulatory process and in the regulationof CL function.     

L-精氨酸和一氧化氮抑制剂对大鼠血管钙化的影响

观察给予一氧化氮合酶(NOS)的抑制剂左旋硝基精氨酸(L-NNA)或底物L-精氨酸(L-Arg)对血管钙化的影响。利用维生素D3和尼古丁制备大鼠血管钙化模型，测定大鼠尾动脉压，血管钙含量、碱性磷酸酶活性及^45Ca沉积；并检测血管组织的L-Arg转运，NOS活性及NO2^-和cGMP含量。发现钙化大鼠血压略升高；动脉钙含量、ALP活性及^45Ca沉积明显增加，von Kossa染色可以看到明显的钙化颗粒；钙化血管组织NOS活性升高；NO和cGMP含量均减少。给予L-NNA或L-Arg后，与单纯钙化组相比，L-NNA干预组的L-Arg转运、NOS活性及NO和cGMP的含量均降低；而L-Arg干预组上述指标的变化正相反。同时，L-NNA干预组的钙化程度比钙化组加重，而L-Arg干预组的钙化程度比钙化组减轻；提示血管钙化时NO—NOS—cGMP途径发生紊乱，干预NO—NOS—cGMP途径可以影响钙化的进程。