Rodríguez-López A M, Martínez-Salgado C, Eleno N, Arévalo M, López-Novoa J M
Instituto Reina Sofía de Investigación Nefrológica, Departamento de Fisiología y Farmacología, Universidad de Salamanca, España.
Cell Physiol Biochem. 1999;9(6):285-96. doi: 10.1159/000016323.
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.
本研究的目的是探讨毒胡萝卜素诱导大鼠肾小球系膜细胞凋亡的机制以及一氧化氮（NO）在此过程中的可能作用。在内质网Ca(2+)-ATP酶的已知特异性抑制剂10(-6) M毒胡萝卜素存在且无生长因子的培养基中培养系膜细胞单层12小时后，通过碘化丙啶染色（对照条件下为9%，处理后为23%）或末端脱氧核苷酸转移酶介导的dUTP生物素缺口末端标记法（TUNEL；对照条件下为5%，处理后为17%）评估，发现高比例的细胞呈现典型的凋亡核特征。当细胞在含有10%胎牛血清（FCS）的培养基中或无钙培养基中培养时，毒胡萝卜素诱导的凋亡率非常低。在大鼠系膜细胞中，毒胡萝卜素处理降低了BCL-2蛋白和bcl-2 mRNA的表达，而未改变BAX蛋白或bax mRNA的水平。当系膜细胞在无FCS的情况下与毒胡萝卜素一起孵育时，我们检测到亚硝酸盐生成显著增加（3.78±0.96对1.76±0.44微摩尔/孔）。此外，用NO合成抑制剂L-NAME（10(-4) M）处理可使凋亡细胞数量显著减少（9%），而用NO供体SIN-1（10(-5) M）孵育则可使凋亡率显著增加（29%）。对巨噬细胞型诱导型NO合酶（iNOS）的蛋白质免疫印迹和Northern印迹分析表明，毒胡萝卜素处理可诱导iNOS蛋白和iNOS mRNA的表达。用L-NAME处理可防止毒胡萝卜素诱导的BCL-2减少，而用SIN-1孵育则增强了毒胡萝卜素对BCL-2的作用。iNOS和TUNEL的免疫组织化学双重标记显示，发生凋亡的细胞对iNOS呈阳性。总之，我们的结果表明，毒胡萝卜素能够通过胞质游离钙增加介导的机制提高大鼠系膜细胞的凋亡率。iNOS表达增加以及由此导致的NO生成增加似乎参与了这一效应。
