反义c-fos寡脱氧核苷酸对hCG诱导大鼠黄体细胞孕酮和雌二醇生成的影响

采用离体孵育大鼠黄体细胞的方法,观察了反义c-fos寡脱氧核苷酸(反义c-fos ODN)对hCG诱导的黄体细胞孕酮(P)和雌二醇(E     

血小板活化因子对大鼠黄体细胞孕酮分泌及血管内皮生长因子表达的作用

本文旨在研究血小板活化因子(platelet-activating factor,PAF)对大鼠黄体细胞孕酮分泌及血管内皮生长因子(vascularendothelial growth factor,VEGF)mRNA表达的作用.将未成年(25～28 d)Sprague-Dawley雌性大鼠颈部皮下注射50 IU孕马血清促性腺激素(pregnant mare serum gonadotrophin,PMSG),48 h后注射25 IU人绒毛膜促性腺激素(human chorionicgonadotrophin.hCG)诱导卵泡发育和黄体生成,第6天(hCG注射日为第1天)收集卵巢黄体细胞,体外培养24 h后,不加或加入不同剂量(0.1 μg/mL、1 μg/mL、10 μg/mL)PAF,37℃、5%CO2培养箱内培养24 h.用放射免疫方法测定培养液中孕酮的含量,流式细胞仪和RT-PCR方法检测黄体细胞凋亡以及VEGF mRNA的表达.结果显示,PAF促进黄体细胞孕酮分泌,1 μg/mL PAF作用最强(P<0.05);PAF促进黄体细胞凋亡无明显剂量依赖性,但10 μg/mL PAF显著促进大鼠黄体细胞凋亡(P<0.05):PAF刺激黄体细胞VEGF mRNA表达,1 μg/mL PAF效果最显著(P<0.01).结果提示,PAF可通过调节黄体细胞孕酮的分泌和VEGF mRNA的表达来促进黄体形成.     

抑制素α亚基片段P33对大鼠离体培养黄体细胞凋亡的影响

我室先前的工作表明,抑制素α亚基片段Ｐ３３显著抑制离体培养大鼠黄体细胞的孕酮分泌,整体实验显示Ｐ３３促进黄体功能萎缩和细胞凋亡。本实验进一步在细胞水平探讨Ｐ３３促进黄体细胞凋亡的作用机制。应用ＤＮＡ电泳检测技术、ＤＮＡ荧光（ＡＯＥＢＰＩ）染色和流式细胞分析方法观察了Ｐ３３对ＰＭＳＧｈＣＧ假孕大鼠胶原酶ＤＮＡ酶分散的黄体细胞的自发凋亡的影响。结果三种方法一致显示,Ｐ３３（１μｇ／ｍｌ）促进黄体细胞的自发凋亡。阻断酪氨酸蛋白激酶活性（ｇｅｎｉｓｔｅｉｎ５０μｇ）则抑制Ｐ３３诱导的黄体凋亡;而阻断ＲＮＡ和蛋白质合成（Ｃｙｘ,５０μｇ／ｍｌ;ＡｃｔＤ,５０μｇ／ｍｌ）均不抑制Ｐ３３促进的黄体细胞凋亡。结果表明,Ｐ３３促进培养大鼠黄体细胞的自发凋亡,其作用机制可能与ＴＰＫ途径有关。本实验为抑制素α亚单位或其相关衍生物可能是卵巢局部调节因子之一的假说提供了又一证据。     

c-erbB_2对大鼠黄体细胞hCG诱导的孕酮分泌的影响

采用离体细胞体外孵育法 ,研究反义c erbB2 寡脱氧核苷酸 (antisensec erbB2 ODN)对大鼠黄体细胞hCG诱导的孕酮分泌的影响 ,及其与外源性cAMP和Ca2 以及蛋白抑制剂放线菌酮 (CYX)之间的关系。结果表明 ,反义c erbB2 以剂量相关方式抑制黄体细胞hCG诱导的孕酮的产生 ,同时使c erbB2 蛋白染色阳性的黄体细胞百分数下降 ,无义tatODN没有相应的作用。10 -4 mol/L的二丁酰cAMP能明显反转反义c erbB2 ODN对孕酮产生和c erbB2 表达的抑制作用 ,钙离子通道阻断剂维拉帕米和蛋白抑制剂CYX对此抑制作用有协同效应。该实验说明c erbB2 参于hCG诱导黄体细胞生孕酮作用     

胰岛素样生长因子Ⅱ对大鼠离体培养黄体细胞孕酮生成的影响

本实验观察了胰岛素样生长因子Ⅱ（ＩＧＦ－Ⅱ）对大鼠离体培养黄体细胞孕酮生长的影响,并对其作用机制进行了探讨。结果显示,ＩＧＦ－Ⅱ能显著地促进大鼠离体培养黄体细胞孕酮生成并呈剂量－效应关系,同时还能促进＾３Ｈ－亮氨酸掺入黄体细胞蛋白质的合成,促进＾３Ｈ－胸腺嘧啶掺入ＤＮＡ的合成,而上述效应分别被放线菌酮（ＣＹＸ）和放线菌素Ｄ所抑制。此外,ＩＧＦ－Ⅱ对大鼠离体黄体细胞内泊性ｃＡＭＰ和ｈＣＧ诱导的ｃＡＭ     

鞘内注射神经激肽-1受体激动剂Sar-SP增强大鼠脊髓一氧化氮合酶表达和一氧化氮生成

探讨P物质(substance P,SP)对脊髓一氧化氮合酶(nitric oxide synthase,NOS)表达和一氧化氮(nitric oxide,NO)生成的影响。实验用热甩尾法测定大鼠痛阈的变化,分别应用NADPH-d组织化学法和硝酸还原法测定大鼠脊髓内NOS表达和NO生成的变化。结果显示,鞘内注射神经激肽-1受体(neurokinin-1 receptor,NK-1)激动剂[Sar^9,Met(O2)^11]-substance P(Sar-SP)可使大鼠痛阈降低,脊髓后角浅层和中央管周围灰质内NOS表达增强,脊髓腰膨大部位NO生成增多;预先鞘内注射非选择性NK-1受体拮抗剂[D—Arg^1,D-Trp^7,9,Leu^11]-substance P(spantide)可抑制上述变化。结果表明,SP可促进脊髓内NOS表达和NO生成。     

GABA影响大鼠卵巢黄体细胞孕酮的生成

实验用离体培养方法观察ＧＡＢＡ对大鼠黄体细胞孕酮及羟自由基（．ＯＨ）生成的影响。结果表明：ＧＡＢＡ抑制黄体细胞孕酮的生成,同时也促进黄体细胞．ＯＨ的生成。ＧＡＢＡ对孕酮的抑制作用可能与腺苷酸环化酶系统及ＧＡＢＡＡ型受体有关,而与蛋白质合成无关。     

Apelin对大鼠离体肺动脉环的舒张作用及与一氧化氮的关系

目的：探讨新的小分子活性肽Apelin对大鼠离体肺动脉环的舒张作用及与一氧化氮（NO）途径的关系,并比较低氧大鼠的肺动脉环对Apelin的舒张反应与正常大鼠的差异。方法：36只大鼠随机分为正常组与低氧组;采用离体血管环灌流法,检测Apelin对去甲肾上腺素（NE）预收缩的大鼠离体肺主动脉环的舒张效应,观察去内皮或用一氧化氮合酶抑制剂（L-NAME）、可溶性鸟苷酸环化酶（sGC）抑制剂（ODQ）孵育后该舒张率的变化。结果：①在正常组大鼠肺动脉环,Apelin（0.01~100 nmol/L）具有浓度依赖性的舒张效应。去除内皮后,Apelin对NE预先收缩的肺血管舒张效应明显减弱（P〈0.01）。L-NAME或ODQ预孵育后,Apelin的舒张效应均明显减弱（P均〈0.01）。②低氧组大鼠的肺动脉环对Apelin的舒张反应明显低于正常组大鼠,在最大浓度100 nmol/L时,Apelin的效应低60.45%（P〈0.01）,而两组EC50相比差异无显著性（P〉0.05）。结论：Apelin具有内皮依赖性的舒张肺动脉环的作用,该效应与NO-sGC-cGMP信号途径有关;低氧大鼠的离体肺动脉环对Apelin的舒张反应减弱。     

细胞内外钙浓度变化不影响酪氨酸抗hCG致孕酮生成作用

实验取经ＰＭＳＧ－ｈＣＧ处理的未成年雌性大鼠卵巢,用胶原酶－ＤＮＡ酶消化,制得黄体细胞悬浮液,预孵育１ｈ后加入各种处理因素,继续孵育２ｈ,用放射免疫方法测孵育液中孕酮的量。结果：孵育液中含有高钙或高钾或加入Ａ２３１８７时均可增加黄体细胞基础及ｈＣＧ诱导的孕酮生成量。相反,减少钙的浓度或加入ＥＧＡＴ或戊脉胺,孕酮生成量则明显减少。酪氨酸抑制ｈＣＧ刺激的孕酮生成,但对高钙、高钾和Ａ２３１８７增加孕酮的作用没有影响,并对上述三者分别与ｈＣＧ同时作用所致孕酮生成增加也没有影响。提示：大鼠黄体细胞孕酮生成依赖于细胞内外的钙;细胞内外钙浓度的变化不影响酪氨酸抗ｈＣＧ致孕酮生成作用;钙与ｈＣＧ使孕酮增加的作用可能是通过不同机制。     

白细胞介素—10对血管一氧化氮／一氧化氮合酶系统的影响

为探讨抗炎因子－－白细胞介素－10（IL－10）对大鼠主动脉一氧化氮（NO）／一氧化氮合酶（NOS）系统的影响,应用Griess试剂、^3H－瓜氨酸生成及蛋白免疫印迹杂交等方法,测定IL－10孵育对血管NO释放、NOS活性及表达的影响。结果发现细菌脂多糖（LPS）呈浓度领带性地激活诱导型NOS（iNOS）,促进NO生成。IL－10（10^-10-10^-8g/ml）呈浓度依赖性地上调内皮型NOS（eNOS）蛋白表达及其活性,但对iNOS活性及表达无明显影响,IL－10（10^-9-10^-8g/ml）显著抑制10μg/ml LPS诱导的NO生成和iNOS激活;而高浓度IL－10（10^-7g/ml）则上调iNOS的活性,对eNOS蛋白的表达知活性无明显影响。因此IL－10对NO／NOS系统具有双重影响,一方面可抑制炎症介质诱发的作为炎性物质的iNOS的表达及激活,另一方面可上调内皮源扩血管物质NO的释放。     

Sphingomyelinase causes endothelium-dependent vasorelaxation through endothelial nitric oxide production without cytosolic Ca(2+) elevation

Neutral sphingomyelinase (N-SMase) elevated nitric oxide (NO) production without affecting intracellular Ca(2+) concentration ([Ca(2+)](i)) in endothelial cells in situ on aortic valves, and induced prominent endothelium-dependent relaxation of coronary arteries, which was blocked by N(omega)-monomethyl-L-arginine, a NO synthase (NOS) inhibitor. N-SMase induced translocation of endothelial NOS (eNOS) from plasma membrane caveolae to intracellular region, eNOS phosphorylation on serine 1179, and an increase of ceramide level in endothelial cells. Membrane-permeable ceramide (C(8)-ceramide) mimicked the responses to N-SMase. We propose the involvement of N-SMase and ceramide in Ca(2+)-independent eNOS activation and NO production in endothelial cells in situ, linking to endothelium-dependent vasorelaxation.     

Modulation of CYP3A4 expression by ceramide in human colon carcinoma HT-29 cells

Cytochrome P450 3A4 (CYP3A4) enzyme is responsible for the metabolic activation and inactivation of the majority of clinically used drugs in human liver and intestines. Recent studies have increasingly implicated various inflammatory stimuli to cause changes in the activities and expression levels of CYPs. However, the underlying mechanisms are largely unknown. In the present study, our studies investigated the effects of ceramide on CYP3A4 expression in human colon carcinoma HT-29 cells. Treatment with the cell-permeable ceramide analog C(6)-ceramide to the cells significantly decreased the expression of CYP3A4. By contrast, C(6)-dihydroceramide, a biologically inactive analog of C(6)-ceramide, did not affect CYP3A4 expression. We found that bacterial sphingomyelinase (SMase) and tumor necrosis factor-alpha (TNF), which are known to increase intracellular ceramide levels, also markedly suppressed the synthesis of CYP3A4. To elucidate whether nitric oxide (NO) participates in suppression of CYP3A4 expression by ceramide, the effects of NO modulators were determined. Treatment with N(G)-monomethyl-L-arginine, a competitive inhibitor of inducible nitric oxide synthase (iNOS), was able to protect ceramide-dependent CYP3A4 suppression. In contrast, the addition of S-nitroso-N-acetylpenicillamine, a NO donor, to HT-29 cells reduced CYP3A4 expression. The addition of iNOS antisense oligonucleotide prevented ceramide-mediated induction of iNOS expression and restored CYP3A4 expression. Wortmannin which is known to inhibit phosphatidylinositol 3-kinase (PI3-K) blocked CYP3A4 suppression by ceramide. Taken together, our results demonstrate that ceramide-mediated suppression of CYP3A4 is due to production of NO, which might result from activation of PI3-K.     

尾加压素对新生大鼠心肌细胞一氧化氮合成的影响

应用半定量逆转录－多聚酶链反应法,观察尾加压素（urotensin Ⅱ,UⅡ）对培养的新生SD大鼠心肌细胞内皮型一氧化氮合酶（endothelial nitric oxide synthase,eNOS)mRNA表达的影响,并测定UⅡ对心肌细胞内一氧化氮合酶（nitric oxide synthase,NOS)活性和一氧化氮（nitric oxide,NO)释放的影响。结果显示：UⅡ抑制培养的新生大鼠心肌细胞eNOS mRNA表达、抑制NOS的活性及NO释放;0.1μmol/L浓度的UⅡ呈时间依赖性抑制心肌细胞NOS的活性及NO生成。上述实验结果提示UⅡ的心血管作用可能与NO合成系统有关。     

Characterization of ceramide-induced apoptotic death in cerebellar granule cells in culture

Sphingomyelin signalling system has been involved in several examples of cell death through apoptosis. We have characterised the effect of exposure to the cell permeable ceramide analogue, C2-ceramide, on cultures of differentiated cerebellar granule cells. C(2)-ceramide was toxic to granule cells in a dose- and time-dependent way at concentrations higher than 10 microM. Ceramide exposure was accompanied by characteristic alterations of cell morphology, namely swollen cell bodies and punctuate appearance and arcuate direction of processes. The final outcome of ceramide exposure was a form of cell death largely apoptotic in nature. Hoechst stain, followed by counts of nuclei with normal appearance and size or with condensed chromatin and reduced size, revealed a large increase of the proportion of shrunken nuclei in treated cultures. In situ visualisation of fragmented DNA through the TUNEL technique, additionally marked cells undergoing apoptosis as a consequence of ceramide treatment. Accordingly, the DNA extracted from cultures exposed to C2-ceramide and subjected to agarose gel electrophoresis showed the peculiar ladder of fragmented low molecular weight DNA. Treatments with inhibitors of two caspases or of nitric oxide synthase were unable to rescue neurons exposed to ceramide, thus suggesting a neurotoxic action not primarily dependent on activation of death proteases or on nitric oxide production.     

Gonadotropin-releasing hormone-agonist inhibits synthesis of nitric oxide and steroidogenesis by luteal cells in the pregnant rat

We have demonstrated that continuous administration of a gonadotropin-releasing hormone agonist (GnRH-Ag) in vivo suppressed progesterone production and induced apoptosis in the corpus luteum (CL) of the pregnant rat. To investigate the mechanism(s) by which progesterone secretion is suppressed and apoptosis is induced in the luteal cells, we studied nitric oxide (NO) as a messenger molecule for GnRH action. Rats were treated individually on Day 8 of pregnancy with 5 microg/day of GnRH-Ag for 4, 8, and 24 h. GnRH-Ag decreased the production of progesterone and pregnenolone 8 and 24 h after the administration. Corresponding with the reduction in these steroid hormones, luteal NO concentrations decreased at 8 and 24 h. Western blotting and immunohistochemical studies of endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), and neuronal nitric oxide synthase (nNOS) in the CL demonstrated that administration of GnRH-Ag was associated with a marked decrease in eNOS and iNOS compared with sham controls at 4 and 8 h, but nNOS did not change throughout the experimental period. We demonstrated, for the first time, the presence of nNOS protein in the CL of the pregnant rat. To determine if this suppressive action of GnRH-Ag is directly on the CL, luteal cells were treated with GnRH-Ag for 4, 8, 12, and 24 h in vitro. Progesterone and NO concentrations in the media decreased at 8 and 12 h after the treatment and recovered at 24 h. Western blots revealed that eNOS and iNOS decreased in luteal cells treated with GnRH-Ag compared with controls at 4 and 8 h. These results demonstrate that suppression of luteal NO synthesis by GnRH-Ag is direct and leads to a decrease in the luteal production and release of progesterone and pregnenolone and thus suggest that GnRH could induce luteolysis in pregnant rats via NO.     

Interaction of caveolin-1, nitric oxide, and nitric oxide synthases in hypoxic human SK-N-MC neuroblastoma cells

Neuroblastoma cells are capable of hypoxic adaptation, but the mechanisms involved are not fully understood. We hypothesized that caveolin-1 (cav-1), a plasma membrane signal molecule, might play a role in protecting neuroblastoma cells from oxidative injury by modulating nitric oxide (NO) production. We investigated the alterations of cav-1, cav-2, nitric oxide synthases (NOS), and NO levels in human SK-N-MC neuroblastoma cells exposed to hypoxia with 2% [O2]. The major discoveries include: (i) cav-1 but not cav-2 was up-regulated in the cells exposed to 15 h of hypoxia; (ii) NO donor 1-[N, N-di-(2-aminoethyl) amino] diazen-1-ium-1, 2-diolate up-regulated the expression of cav-1, whereas the non-selective NOS inhibitor N(G)-nitro-L-arginine methyl ester and inducible NOS (iNOS) inhibitor 1400W each abolished the increase in cav-1 expression in the hypoxic SK-N-MC cells. These results suggest that iNOS-induced NO production contributes to the up-regulation of cav-1 in the hypoxic SK-N-MC cells. Furthermore, we studied the roles played by cav-1 in regulating NO, NOS, and apoptotic cell death in the SK-N-MC cells subjected to 15 h of hypoxic treatment. Both cav-1 transfection and cav-1 scaffolding domain peptide abolished the induction of iNOS, reduced the production of NO, and reduced the rates of apoptotic cell death in the hypoxic SK-N-MC cells. These results suggest that increased expression of cav-1 in response to hypoxic stimulation could prevent oxidative injury induced by reactive oxygen species. The interactions of cav-1, NO, and NOS could be an important signal pathway in protecting the neuroblastoma cells from oxidative injury, contributing to the hypoxic tolerance of neuroblastoma cells.     

Ceramide increases steroid hormone production in MA-10 Leydig cells.

Ceramide is known to have major roles in the control of cell proliferation, differentiation, and apoptosis. Recent studies also have shown that ceramide affects steroid production by JEG-3 choriocarcinoma cells, acutely dispersed rat Leydig cells, and ovarian granulosa cells, but the mechanism by which this occurs is unknown. Because ceramide induces apoptosis in many different cell types, we hypothesized that ceramide might affect steroidogenesis and/or induce apoptosis in MA-10 murine Leydig cells. To test this, MA-10 cells were incubated with either the water soluble C2-ceramide, (N-acetyl-sphingosine, 0.01-10 cm); bacterial sphingomyelinase (1-100 mU/ml); or C2-dihydroceramide (N-acetyl-sphinganine, 0.1-10 microM). The data show that N-acetyl-sphingosine significantly increased basal (0.87 +/- 0.2 vs. 0.42 +/- 0.09 ng/mg cell protein, P < 0.01) and human chorionic gonadotropin (hCG) stimulated progesterone (P) synthesis (204 +/- 12 vs. 120 +/- 5 ng/mg cell protein, P < 0.001); as did sphingomyelinase (basal P = 0.83 +/- 0.1 ng/mg cell protein, P < 0.01; hCG stimulated P = 173 +/- 7 ng/mg cell protein, P < 0.001). C2-dihydroceramide also increased basal P synthesis but was less effective than ceramide on a molar basis. Neither sphingomyelinase (100 mU/ml) nor ceramide (10 microM) had any effect on cAMP production or human chorionic gonadotropin binding; and neither induced any signs of apoptosis (FragEL DNA fragmentation assay and electron microscopy). Cells incubated with anti-Fas (300 ng/ml) demonstrated DNA fragmentation, nuclear condensation, and frequent apoptotic bodies, but had no change in P synthesis. These data show that ceramide significantly increases MA-10 Leydig cell P synthesis but does not induce apoptosis. The mechanism by which ceramide increases steroid hormone synthesis remains unknown but does not appear to be linked to the induction of apoptosis in MA-10 cells.     

胰岛素促进血管内皮细胞产生一氧化氮的实验研究

目的：探讨胰岛素对血管内皮细胞增殖、NO产生和NOS基因表达的影响。方法：培养牛主动脉内皮细胞,测定培养上清液中NO氧化产物NO2^－的水平并应用定量RT－PCR技术检测内皮细胞NOS mRNA的表达水平。结果：①胰岛素对大血管内皮细胞无细胞毒作用,也不影响细胞增殖;②在1－15μg/ml浓度范围内,胰岛素加强内皮细胞释放NO,且呈剂量依赖的方式,NOS特异性抑制剂L－NAME可阻抑之;③胰岛素轻度增加NOS mRNA表达水平,但无统计学意义。结论：胰岛素既不影响大血管内皮细胞增殖,也不影响内皮细胞NOS mRNA表达水平,但以剂量依赖的方式加强内皮细胞产生NO,推测其诱导NO产生的机制可能是通过酶活性的诱导,加速NO的合成。     

大鼠脑线粒体NOS及L—Arg转运的生化特性

测定分离纯化的大鼠脑线粒体(mitochondria,Mt)L-精氨酸(L-arginine,L-Arg)/一氧化氮合酶(nitricoxidesynthase,NOS)/NO系统,L-Arg转运和NOS的活性。结果显示正常大鼠脑Mt膜上存在高亲和、低转运、可饱和的L-Arg转运体。最大转运速率Vmax为5.87±0.46nmol/mgpro·min