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首页> 外文期刊>Cellular Physiology and Biochemistry >Nitric oxide is involved in apoptosis induced by thapsigargin in rat mesangial cells.
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Nitric oxide is involved in apoptosis induced by thapsigargin in rat mesangial cells.

机译：一氧化氮参与毒胡萝卜素诱导的大鼠系膜细胞凋亡。

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The purpose of this study was to examine the mechanisms of thapsigargin-induced apoptosis in rat glomerular mesangial cells and the possible involvement of nitric oxide (NO) in this process. In mesangial cell monolayers incubated for 12 h in a medium without growth factors and with 10(-6) M thapsigargin, a known specific inhibitor of endoplasmic reticulum Ca(2+)-ATPase, a high percentage of cells showed typical nuclear features of apoptosis, assessed either by staining with propidium iodide (23 vs. 9% in control conditions) or by terminal desoxynucleotidyl transferase-mediated dUTP biotin nick end labelling (TUNEL; 17 vs. 5% in control conditions). When cells were maintained in a medium containing 10% fetal calf serum (FCS) or in a free-calcium medium, the thapsigargin-induced apoptosis rate was very low. In rat mesangial cells treatment with thapsigargin decreased the expression of BCL-2 protein and bcl-2 mRNA, whereas it did not alter the levels of BAX protein or bax mRNA. When mesangial cells were incubated with thapsigargin in the absence of FCS, we detected a significant increase in nitrite production (3.78 +/- 0.96 vs. 1.76 +/- 0.44 micromol/well). Furthermore, the treatment with the NO synthesis inhibitor L-NAME (10(-4) M) induced a significant decrease in the number of apoptotic cells (9%), whereas incubation with the NO donor SIN-1 (10(-5) M) induced a marked increase in the rate of apoptosis (29%). Western and Northern blot analysis of macrophage-type inducible NO synthase (iNOS) demonstrated that thapsigargin treatment induces the expression of the iNOS protein and iNOS mRNA. Treatment with L-NAME prevented the thapsigargin-induced BCL-2 decrease, whereas incubation with SIN-1 potentiated the effect of thapsigargin on BCL-2. Double labelling by immunohistochemistry for iNOS and TUNEL revealed that the same cells that suffered apoptosis were positive for iNOS. In summary, our results indicate that thapsigargin is able to enhance the apoptosis rate of rat mesangial cells by a mechanism that is mediated by an increase in cytosolic free calcium. Increased iNOS expression, and hence increased NO production, seems to be involved in this effect. Copyright 1999 S. Karger AG, Basel
机译：这项研究的目的是检查thapsigargin诱导的大鼠肾小球系膜细胞凋亡的机制，以及一氧化氮（NO）可能参与这一过程。在没有生长因子和10（-6）M thapsigargin（一种已知的内质网Ca（2 +）-ATPase特异性抑制剂）的培养基中孵育12 h的系膜细胞单层细胞中，高百分比的细胞显示出典型的细胞凋亡核特征可以通过碘化丙啶染色（在对照条件下为23％，相对于9％）或通过末端脱氧核苷酸转移酶介导的dUTP生物素缺口末端标记（TUNEL;在对照条件下为17：5％）来评估。当将细胞保存在含有10％胎牛血清（FCS）的培养基中或游离钙培养基中时，毒胡萝卜素诱导的细胞凋亡率非常低。在大鼠肾小球系膜细胞中，thapsigargin处理可降低BCL-2蛋白和bcl-2 mRNA的表达，而不会改变BAX蛋白或bax mRNA的水平。当在不存在FCS的情况下将肾小球系膜细胞与毒胡萝卜素一起孵育时，我们检测到亚硝酸盐产量显着增加（3.78 +/- 0.96对1.76 +/- 0.44 micromol /孔）。此外，用NO合成抑制剂L-NAME（10（-4）M）处理可诱导凋亡细胞数量显着减少（9％），而与NO供体SIN-1（10（-5）孵育） M）诱导凋亡率显着增加（29％）。巨噬细胞型诱导型一氧化氮合酶（iNOS）的蛋白质印迹和Northern印迹分析表明，毒胡萝卜素处理诱导了iNOS蛋白和iNOS mRNA的表达。 L-NAME处理可防止毒胡萝卜素诱导的BCL-2减少，而SIN-1孵育则可增强毒胡萝卜素对BCL-2的作用。通过免疫组织化学对iNOS和TUNEL的双重标记显示，遭受凋亡的相同细胞对iNOS呈阳性。总之，我们的结果表明，thapsigargin能够通过细胞溶质游离钙增加介导的机制来提高大鼠系膜细胞的凋亡率。 iNOS表达增加，因此NO产生增加，似乎与这种作用有关。版权所有1999 S.Karger AG，巴塞尔

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