Parys J B, De Smedt H, Borghgraef R
Biochim Biophys Acta. 1986 Aug 29;888(1):70-81. doi: 10.1016/0167-4889(86)90072-8.
ATP-dependent calcium uptake was measured in membrane vesicles prepared from the renal epithelial LLC-PK1 established cell line. The relative contribution of the nonmitochondrial versus the mitochondrial calcium uptake is larger in LLC-PK1 cell homogenates than in homogenates from renal cortex. Two types of calcium pump, characterized by the formation of calcium-dependent phosphointermediates of 135 kDa and 115 kDa, were found in membrane fractions from LLC-PK1 cells. The 135 kDa calcium pump was also detected by 125I-labelled calmodulin overlay. Although the subcellular localization in LLC-PK1 cell membranes could not be unambiguously determined, it is conceivable that the 135 kDa and the 115 kDa molecules represent the plasma membrane calcium pump and the endoplasmic reticulum calcium pump respectively, in agreement with what was found for renal cortex preparations. Extravesicular sodium partially inhibits ATP-driven calcium uptake in a plasma-membrane-enriched fraction of the LLC-PK1 cells. The effect is potentiated by a vesicle inside-negative membrane potential. Although the effect is less pronounced than in renal cortex basal-lateral membranes, this observation suggests that an Na+-Ca2+ exchange mechanism is also present in LLC-PK1 cells. ATP-dependent calcium uptake in nonmitochondrial intracellular stores was investigated, using saponin-permeabilized cells. Permeabilized LLC-PK1 cells lowered the free calcium concentration in the medium to less than 0.4 microM. More than 60% of the accumulated calcium can be released by addition of inositol 1,4,5-trisphosphate. Our data indicate that the LLC-PK1 cell line can be successfully used as model system for the study of renal calcium handling.
在由肾上皮LLC-PK1建立细胞系制备的膜囊泡中测量了ATP依赖性钙摄取。与肾皮质匀浆相比，LLC-PK1细胞匀浆中非线粒体与线粒体钙摄取的相对贡献更大。在LLC-PK1细胞膜组分中发现了两种类型的钙泵，其特征是形成了135 kDa和115 kDa的钙依赖性磷酸中间产物。135 kDa的钙泵也通过125I标记的钙调蛋白覆盖检测到。尽管无法明确确定LLC-PK1细胞膜中的亚细胞定位，但可以想象135 kDa和115 kDa分子分别代表质膜钙泵和内质网钙泵，这与肾皮质制剂的发现一致。细胞外钠部分抑制LLC-PK1细胞富含质膜的组分中ATP驱动的钙摄取。囊泡内负膜电位可增强这种作用。尽管这种作用不如在肾皮质基底外侧膜中明显，但这一观察结果表明LLC-PK1细胞中也存在Na+-Ca2+交换机制。使用皂素通透细胞研究了非线粒体细胞内储存中ATP依赖性钙摄取。通透的LLC-PK1细胞将培养基中的游离钙浓度降低到小于0.4 microM。添加肌醇1,4,5-三磷酸可释放超过60%的积累钙。我们的数据表明，LLC-PK1细胞系可成功用作研究肾钙处理的模型系统。
