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     

生殖周期中小鼠子宫NOS定位及血清NO水平变化

目的　系统研究 3种一氧化氮合酶 (nitricoxidesynthase ,NOS)在生殖周期中小鼠子宫分布变化 ,探讨内源性一氧化氮 (nitricoxide,NO)在生殖周期中可能的生理作用。　方法　免疫组织化学LSAB法 ,比色法。　结果　神经型一氧化氮合酶 (nNOS ,NOS1)在生殖周期小鼠子宫有较为稳定的表达 ,大量的阳性细胞主要分布在子宫内膜上皮 ,内膜基质 ,血管内皮 ,腺上皮及肌层 ;内皮型一氧化氮合酶 (eNOS ,NOS3)在妊娠中期小鼠子宫表达明显增强 ,蜕膜上皮 ,腺上皮和血管内皮呈强阳性标记 ;诱导型一氧化氮合酶 (iNOS ,NOS2 )在妊娠早期表达最强 ,妊娠中、晚期表达稍下降。阳性标记主要分布在肌层 ,血管内皮 ,腺上皮 ,内膜上皮及内膜基质 ;然而动情期小鼠子宫未见iNOS阳性标记。此外 ,还测定了生殖周期小鼠血清NO水平变化。　结论　 3种NOS同工酶在生殖周期小鼠子宫可能既协作又分工 ,由其产生的内源性NO对雌鼠动情、胚胎植入、分娩前子宫静息状态的维持、分娩始动以及产后子宫修复等生理过程可能均有调节作用。     

生后不同发育阶段大鼠卵巢内eNOS和iNOS的表达

目的　观察eNOS和iNOS在卵巢中的表达,并探讨其意义。 方法　采用免疫组化和图像分析系统检测大鼠生后各发育阶段卵巢中eNOS和iNOS的定位分布和表达。 结果　0 d大鼠卵巢卵母细胞eNOS和iNOS均呈弱阳性反应;4 d后,eNOS和iNOS在正常卵母细胞中均呈强阳性,退化卵母细胞中反应非常弱;卵泡细胞与膜细胞中的iNOS在30 d前均呈强阳性表达,30 d后,生长卵泡中表达降低,闭锁卵泡中较正常生长卵泡表达增强,eNOS的表达稳定;240 d 后卵巢内间质成分和闭锁卵泡增多,间质中的iNOS表达增强。360d 后大鼠卵巢逐渐纤维化,iNOS染色强于eNOS。 结论　大鼠不同发育时期卵巢中,eNOS的表达相对稳定;iNOS的表达以性成熟后育龄期最低,随着年龄增加及卵巢功能的衰退,其表达逐渐增高。     

Fmr1基因敲除小鼠生后不同发育阶段的附睾iNOS的变化

目的对不同周龄的Fmr1基因敲除和野生型雄性小鼠附睾组织诱导型一氧化氮合酶(iNOS)的表达进行分析比较,探讨Fmr1基因敲除小鼠的附睾组织iNOS表达的差异,为脆性综合征的研究提供背景资料。方法本研究采用不同周龄(4、6、8、10周)的Fmr1(fragile X mental retardation 1)基因敲除型(knockout,KO)和野生型(wild-type,WT)各6只,先采用聚合酶链式反应(PCR)技术对KO小鼠和WT小鼠进行基因型鉴定,之后所有小鼠麻醉取附睾组织、石蜡包埋切片进行免疫组织化学染色技术对KO小鼠和WT小鼠附睾iNOS的表达进行检测并作对比分析。结果 iNOS在4周小鼠附睾阳性表达,在6、8和10周小鼠呈强阳性表达,且KO小鼠附睾的阳性表达均弱于WT小鼠。结论 Fmr1基因敲除小鼠在缺失Fmr1蛋白(fragile X retardation-1 protein,FMRP)后的附睾iNOS的表达降低。     

一氧化氮合酶(NOS)在膀胱移行细胞癌中的表达及其意义

目的检测膀胱移行细胞癌中一氧化氮合酶(nitric oxide synthase,NOS)的表达,并分析其表达与肿瘤病理特性的关系.方法采用免疫组织化学技术检测35例膀胱移行细胞癌标本、12例癌旁粘膜标本及8例正常膀胱粘膜标本中一氧化氮合酶三种亚型的表达情况.结果 35例肿瘤标本中nNOS、iNOS、eNOS阳性表达率分别为74.3%、85.7%、42.9%,膀胱移行细胞癌中iNOS表达较正常膀胱粘膜增高.但移行细胞癌、癌旁粘膜、正常粘膜三组间nNOS及eNOS表达无差别.nNOS、iNOS、eNOS表达与膀胱移行细胞癌分期分级可能无相关性.结论 iNOS在膀胱移行细胞癌中表达增高,可能参与膀胱移行细胞癌的发生发展.     

大鼠脊髓iNOS的过表达参与形成带状疱疹后神经痛

目的:观察诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)在带状疱疹后神经痛(postherpetic neuralgia;PHN)大鼠脊髓的表达变化以及与痛行为的相关性。方法:SD大鼠随机分为水痘带状疱疹病毒感染组(varicella zoster virus,VZV组),假接种组(mock infected,Mock组)和空白对照组(Naive组);每组在接种前和接种后3、5、7、10、14、17、21 d先进行痛觉行为学检测,然后利用免疫组化染色和Western Blot检测脊髓iNOS以及NOS的其它两种同功酶nNOS和eNOS的表达变化;在痛觉阈值最低的时间点,鞘内注入各种NOS抑制剂进行行为药理学检测,同时在脊髓进行iNOS与NeuN,GFAP或OX-42的免疫荧光双重染色。结果:与Mock组和Naive组比较,VZV组在病毒注射后5 d形成了显著的机械性异常疼痛,14 d达到巅峰(P0.05)。VZV组脊髓背角iNOS染色密度和表达量显著增加(P0.05),免疫荧光双重染色显示iNOS由星形胶质细胞生成。iNOS的表达增加与痛觉阈值的降低显著相关(P0.001,r=-0.89)。VZV组脊髓内nNOS和eNOS的表达没有变化。VZV组鞘内给予iNOS抑制剂L-NIL或NO清除剂PTIO能够有效镇痛,而nNOS或eNOS的抑制剂7-NINA,L-NIO则无镇痛作用。结论:脊髓背角iNOS的上调参与了PHN大鼠痛敏状态的形成。     

体外反搏对心肌梗死犬一氧化氮系统的影响

目的:探讨体外反搏对心肌梗死犬一氧化氮(NO)、一氧化氮合酶(NOS)和其基因表达的影响。方法:19只健康杂种犬随机分为对照组、缺血组和缺血+反搏组(反搏组)3组,采用开胸结扎冠状动脉左前降支的方法建立心肌缺血模型,用改良硝酸还原酶法测定心肌缺血前后血清NO含量、以及心肌组织的NO含量和NOS比活性,采用免疫组化方法检测缺血区心肌组织的NOS亚型即诱导型NOS(iNOS)和内皮型NOS(eNOS)的蛋白合成,用原位杂交方法检测构成型NOS(cNOS)信使核糖核酸(mRNA)的基因表达。结果:在冠状动脉结扎前和结扎后60min,3组犬血清NO含量均无明显差异(P＞0.05);结扎后120min和180min时,反搏组犬血清NO含量明显高于缺血组(P＜0.05)。正常组和反搏组犬心肌组织NO含量和NOS比活性均大于缺血组(P＜0.05)。免疫组化结果表明心肌缺血时iNOS蛋白合成增多,而eNOS蛋白合成减少;体外反搏对iNOS有抑制作用,对eNOS有促进作用。此外心肌缺血时cNOSmRNA的表达明显减少,反搏可促进cNOSmRNA的表达。结论:体外反搏促进NO的产生可能是其抗心肌缺血性损伤的重要机制之一。     

雌孕激素与子宫内膜的一氧化氮合酶

NO(NitricOxide) /NOS(NitricOxideSynthase)在雌性生殖生理功能方面如卵泡发育、激素分泌、胚胎着床、维持妊娠、促进分娩及内膜周期性变化、细胞凋亡等方面都具有重要作用。有关子宫内膜NOS的表达及调控机制仍存在异议。三种NOS神经型NOS(neuronalNOS ,nNOS)、内皮型NOS(endothelialNOS ,eNOS)和诱导型NOS(inducibleNOS ,iNOS)在内膜不同部位和时期呈强弱不等的表达 ,生理作用也不尽相同 ,且与雌孕激素对内膜的调节 ,内膜周期性变化关系密切。本文就NOS在子宫内膜的活性表达及其与雌孕激素关系的研究进行综述。     

一氧化氮合酶在坐骨神经夹伤后腓肠肌中的表达变化

目的:探讨外周神经损伤后同侧腓肠肌中3种一氧化氮合酶(nitric oxide synthase,NOS)的表达变化及定位。方法:采用H-E染色及Masson三色染色法分析大鼠坐骨神经夹伤后同侧腓肠肌的病理变化,NADPH-黄递酶组织化学研究其总NOS的改变,并利用Western印迹法、免疫荧光双标法,对3种NOS表达变化及定位进行分析。结果:神经夹伤后相应腓肠肌发生了明显的病理变化且总NOS发生改变,3种NOS变化不尽相同,其表达高峰均约在4周左右。nNOS与神经丝标记物NF-200有共定位,iNOS、eNOS则分别在巨噬细胞、血管内皮细胞中有表达。结论:3种NOS在坐骨神经夹伤后相应腓肠肌中表达变化不同,可能对肌肉损伤及再生修复发挥不同作用。     

兔股动脉结扎诱导动脉生成过程中iNOS和eNOS的表达

目的:研究兔后肢动脉生成过程中诱导型一氧化氮合酶(iNOS)和内皮型一氧化氮合酶(eNOS)表达特征.方法:将兔一侧股动脉结扎,另一侧设为对照组.1周后动物被处死.应用免疫荧光组织化学技术检测侧支血管中iNOS及eNOS的表达.用Leica激光共聚焦显微镜观察并拍照.图片用silicongraphicsoctane进行处理.结果:在正常小动脉血管中iNOS的表达很低,在生长的侧支血管iNOS的表达显著上调,是正常小动脉血管的2.8倍.其表达在血管壁的各层可见.正常小动脉血管eNOS的表达呈现一定基础水平,在生长的侧支血管中eNOS的表达呈强阳性,集中在内皮细胞,是正常小动脉血管的2.2倍.结论:侧支血管发育过程中iNOS的表达上调,上调的iNOS和eNOS可能通过生成NO,调节内皮细胞的增殖、移动及炎症的形成,从而对侧支血管的生长发挥重要作用.     

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.     

mRNA and protein expression and activities of nitric oxide synthases in the lumbar spinal cord of neonatal rats after sciatic nerve transection and melatonin administration

Sciatic axotomy in 2-day-old rats (P2) causes lumbar motoneuron loss, which could be associated with nitric oxide (NO) production. NO may be produced by three isoforms of synthase (NOS): neuronal (nNOS), endothelial (eNOS) and inducible (iNOS). We investigated NOS expression and NO synthesis in the lumbar enlargement of rats after sciatic nerve transection at P2 and treatment with the antioxidant melatonin (sc; 1 mg/kg). At time points ranging from P2 to P7, expression of each isoform was assessed by RT-PCR and immunohistochemistry; catalytic rates of calcium-dependent (nNOS, eNOS) and independent (iNOS) NOS were measured by the conversion of [3H]L-arginine to [3H]L-citrulline. All NOS isoforms were expressed and active in unlesioned animals. nNOS and iNOS were detected in some small cells in the parenchyma. Only endothelial cells were positive for eNOS. No NOS isoform was detected in motoneurons. Axotomy did not change these immunohistochemical findings, nNOS and iNOS mRNA expression and calcium-independent activity at all survival times. However, sciatic nerve transection reduced eNOS mRNA levels at P7 and increased calcium-dependent activity at 1 and 6 h. Melatonin did not alter NOS expression. Despite having no action on NOS activity in unlesioned controls the neurohormone enhanced calcium-dependent activity at 1 and 72 h and reduced calcium-independent catalysis at 72 h in lesioned rats. These results suggest that NOS isoforms are constitutive in the neonatal lumbar enlargement and are not overexpressed after sciatic axotomy. Changes in NO synthesis induced by axotomy and melatonin administration in the current model are discussed considering some beneficial and deleterious effects that NO may have.     

Cell-specific nitric oxide synthase-isoenzyme expression and regulation in response to endotoxin in intact rat lungs

Nitric oxide (NO) produced by NO synthase (NOS) serves as a ubiquitous mediator molecule involved in many physiologic lung functions, including regulation of vascular and bronchial tone, immunocompetence, and neuronal signaling. On the other hand, excessive and inappropriate NO synthesis in inflammation and sepsis has been implicated in vascular abnormalities and cell injury. At least three different NOS isoforms (neuronal/brain [bNOS], inducible [iNOS], and endothelial [eNOS]) have been described, which are all expressed in normal lung tissue. We investigated the cell-specific expression of bNOS, iNOS, and eNOS in perfused control rat lungs and lungs undergoing stimulation with endotoxin in the presence and absence of plasma constituents. Lung immunohistochemistry and quantitative evaluation of staining intensity showed endotoxin-induced increase in iNOS expression in particular in bronchial epithelial cells, cells of the bronchus-associated lymphoid tissue (BALT), alveolar macrophages, and vascular smooth muscle cells in a time- and dose-dependent fashion. In endothelial cells, which did not express iNOS at baseline, newly induced iNOS was found in response to endotoxin. In contrast, expression of eNOS was markedly suppressed under endotoxin challenge, particularly in bronchial epithelium, BALT, and alveolar macrophages but also in vascular smooth muscle cells and endothelial cells. eNOS expression in bronchial smooth muscle cells was not altered. In contrast to iNOS and eNOS, cellular expression of bNOS in epithelial cells, nerve fibers, BALT, and endothelial cells did not change in response to endotoxin. All changes in NOS regulation were found to be independent of plasma constituents. We conclude that endotoxin exerts a profound impact on the cell-specific NOS regulation in a large number of lung cell types. Prominent features include de novo synthesis or up-regulation of iNOS, in contrast to down-regulation of eNOS, which may well contribute to vascular abnormalities, inflammatory sequelae, and loss of physiologic functions in septic lung failure.     

Inducible and endothelial nitric oxide synthase expression during development of transplant arteriosclerosis in rat aortic grafts.

In the vascular system, distinct isoforms of nitric oxide synthase (NOS) generate nitric oxide (NO), which acts as a biological messenger. Its role in the development of transplant arteriosclerosis (TA) is still unclear. To investigate whether NO is involved in TA, we studied the expression of NOS isoforms, inducible NOS (iNOS) and endothelial NOS (eNOS), by immunohistochemistry and in situ hybridization during the first two post-transplantation months and their relation with cold ischemia (1 to 24 hours) and reperfusion injury using an aortic transplantation model in the rat. We found an increased iNOS expression in the intima and adventitia and a decreased expression in the media, whereas eNOS expression was not significantly altered during the development of TA. Co-localization studies suggested that iNOS-positive cells were vascular smooth muscle cells, monocyte-derived macrophages, and endothelial cells. Prolonged ischemic storage time resulted in an increase in eNOS expression in the neointima. In situ hybridization showed iNOS mRNA expression by vascular cells in the neointima and media. NO produced by iNOS and eNOS may be involved, at least in part, in the pathogenesis of TA in aortic grafts. Additional studies are needed to confirm the modulatory mechanism of NO during the development of TA.     

Protective effects of Big-leaf mulberry and physiological roles of nitric oxide synthases in the testis of mice following water immersion and restraint stress

Big-leaf mulberry is a new hybrid plant from the application of cell engineering technology, but its effect in stress-induced testicular dysfunction is unknown. Nitric oxide (NO) is a tiny, highly reactive lipophilic molecule produced by nitric oxide synthases (NOS). Three isoforms of NOS (neuronal NOS, inducible NOS and endothelial NOS) have been identified. Our aim was to investigate the effect of water immersion and restraint stress (WIRS) on NOS in the testis, and the effect of Big-leaf mulberry to protect against WIRS. The activity and expression of NOS, and total antioxidant capacity (T-AOC) in the mouse testis of different treatment groups (non-WIRS, 3 h-WIRS, WIRS-recovery) were examined. Histological analysis of WIRS-induced testicular damage and immunohistochemical staining of NOS were also analyzed. Results demonstrated that WIRS-exposed mice produced several injuries and showed an increased iNOS and eNOS mRNA expression in testes, whereas pretreatment with Big-leaf mulberry down-regulated iNOS and eNOS mRNA expressions and up-regulated T-AOC activities. Immunohistochemical studies showed that both iNOS and eNOS were localized in germ cells, spermatozoa and blood vessels in addition to Leydig cells and Sertoli cells, but nNOS was not present in these areas. In conclusion, our results suggested that Big-leaf mulberry exerted a protective effect on WIRS-induced testicular dysfunction, and iNOS and eNOS appeared to exert an important action in mouse testes exposed to WIRS.     

Effect of adenovirus-mediated gene transfer of nitric oxide synthase on vascular reactivity of rat isolated pulmonary arteries

Aerosol gene transfer of endothelial nitric oxide synthase (eNOS) and inducible NOS (iNOS) to rat lungs increased NOS expression and activity, and prevented hypoxic pulmonary vasoconstriction (HPV) in vivo. Hereby, we examined the effect of eNOS and iNOS aerosol gene transfer on the endothelium-dependent relaxation (EDR) and on acute HPV in isolated rat pulmonary arteries. Changes in isometric forces were recorded in organ baths for large conduit arteries (diameter 1.8±0.1 mm) and in a wire myograph for small resistance arteries (258±35 μm). Male Wistar rats were randomly aerosolized with adenovirus (Ad) encoding β-galactosidase (control), eNOS, or iNOS. Four days later, exhaled nitric oxide was measured, NOS expression within rat lungs was evaluated by quantitative real-time polymerase chain reaction and immunohistochemistry, vasoconstricting agonist and acetylcholine concentration response curves were generated, and the time course of HPV was recorded. Human eNOS and murine iNOS were expressed within rat lung tissue mostly in parenchyma and endothelial cells. Large arteries isolated from Ad-i, eNOS-aerosolized rats developed lower agonist-induced tension than those of control rats. The first and second contractions of the HPV were smaller in the Ad-i, eNOS-aerosolized rats. Contractions were modestly, but significantly and inversely, related to exhaled NO. Agonist- and hypoxia-induced contractions were even more reduced after eNOS aerosolization. There was no significant effect on EDR and no notable difference between small and large vessels. We conclude that adenovirus (Ad)-mediated NOS gene transfer can counteract both pharmacologically and hypoxia-induced increases in pulmonary vascular tone in isolated rat pulmonary arteries. eNOS seems as efficient as iNOS in regulating pulmonary vascular tone.     

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.     

Involvement of endogenous nitric oxide in the regulation of K+ channel activity in cultured human proximal tubule cells

Nitric oxide (NO) modulates the activity of an inwardly rectifying K(+) channel in cultured human proximal tubule cells. In this study, we investigated which NO synthase (NOS) isoform(s) was involved in the endogenous production of NO and hence the regulation of channel activity. The patch-clamp experiments using the cell-attached mode showed that a nonselective NOS inhibitor, N(omega)-nitro-L-arginine methyl ester (L-NAME; 100 microM), suppressed channel activity, whereas a NOS substrate, L-arginine (500 microM), stimulated it. A neuronal NOS (nNOS)/inducible NOS (iNOS)-selective inhibitor, 1-(alpha,alpha,alpha-trifluoro-o-tolyl)-imidazole (TRIM; 100 microM), suppressed channel activity to the same extent as L-NAME. TRIM also blocked the stimulatory effect of L-arginine. In contrast, an NO donor, sodium nitroprusside (10 microM) or 8-bromoguanosine 3',5'-cyclic monophosphate (100 microM) stimulated channel activity even in the presence of TRIM. RT-PCR revealed that iNOS mRNA alone was expressed in most of the cultures, i.e., 34 out of 40. In the other 6 cases, endothelial NOS (eNOS) and iNOS mRNA were simultaneously expressed. This finding was confirmed at the protein level by Western blotting. Indeed, in the patch-clamp experiments TRIM sometimes failed to suppress the channel activity, but the following addition of L-NAME suppressed it. However, since the suppressive effect of TRIM was usually similar to that of L-NAME, the involvement of eNOS in K(+) channel regulation would be relatively low. These results suggest that iNOS plays a pivotal role in the endogenous production of NO under the basal condition, which is involved in the activity of the inwardly rectifying K(+) channel in cultured human proximal tubule cells.