Duque I, García-Escribano C, Rodríguez-Puyol M, Díez-Marqués M L, López-Novoa J M, Arribas I, Hernando L, Rodríguez-Puyol D
University Hospital, Madrid, Spain.
Am J Physiol. 1992 Sep;263(3 Pt 2):F466-73. doi: 10.1152/ajprenal.1992.263.3.F466.
The effects of reactive oxygen species (ROS) on cultured rat mesangial cells were studied by measuring planar cell surface area (PCSA) after incubation with xanthine plus xanthine oxidase (XXO), in the presence of superoxide dismutase (SOD; 5 micrograms/ml) or catalase (CAT; 20 micrograms/ml), or after incubation with H2O2. Myosin light chain (MLC) phosphorylation was assessed in cells prelabeled with o-[32P]phosphoric acid and incubated with H2O2, after protein separation with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A possible intermediate role for platelet-activating factor (PAF) was analyzed by preincubation of the cells with a PAF antagonist BN 52021 (BN, 5 x 10(-5) M) and by measuring PAF-specific [3H]acetate incorporation and immunoassayable PAF. XXO significantly decreased PCSA (14%), an effect abolished by CAT but not by SOD. H2O2 induced a similar effect, in a dose-dependent and time-dependent manner. MLC phosphorylation increased by 81 +/- 15% after H2O2 incubation, and this effect was blocked by BN. BN also completely blocked the effect of H2O2 on PCSA. PAF-specific [3H]acetate incorporation increased in the presence of H2O2 (from 6,886 +/- 2,030 to 58,703 +/- 16,063 counts.min-1.mg-1) as well as the immunoassayable PAF production by cells (from 0.90 +/- 0.19 to 6.71 +/- 2.27 ng/mg). These results suggest that ROS, particularly H2O2, could modulate the surface area of mesangial cells, modifying the ultrafiltration coefficient, thus explaining the decrease in glomerular filtration rate in those pathological situations characterized by an increased ROS synthesis. PAF could be involved in the genesis of these effects.
通过在黄嘌呤加黄嘌呤氧化酶（XXO）孵育后，在超氧化物歧化酶（SOD；5微克/毫升）或过氧化氢酶（CAT；20微克/毫升）存在的情况下，或在与过氧化氢（H2O2）孵育后测量平面细胞表面积（PCSA），研究了活性氧（ROS）对培养的大鼠系膜细胞的影响。在用o-[32P]磷酸预标记并与H2O2孵育的细胞中，在通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳进行蛋白质分离后，评估肌球蛋白轻链（MLC）磷酸化。通过用血小板活化因子（PAF）拮抗剂BN 52021（BN，5×10（-5）M）预孵育细胞并测量PAF特异性[3H]乙酸掺入和可免疫测定的PAF，分析了PAF可能的中间作用。XXO显著降低PCSA（14%），CAT可消除此效应，但SOD不能。H2O2以剂量依赖性和时间依赖性方式诱导类似效应。H2O2孵育后MLC磷酸化增加81±15%，且该效应被BN阻断。BN也完全阻断了H2O2对PCSA的影响。在H2O2存在下，PAF特异性[3H]乙酸掺入增加（从6,886±2,030增加到58,703±16,063计数·分钟-1·毫克-1），细胞产生的可免疫测定的PAF也增加（从0.90±0.19增加到6.71±2.27纳克/毫克）。这些结果表明，ROS，特别是H2O2，可能调节系膜细胞的表面积，改变超滤系数，从而解释了在那些以ROS合成增加为特征的病理情况下肾小球滤过率的降低。PAF可能参与了这些效应的发生。
