Poulsen J C, Caspersen C, Mathiasen D, East J M, Tunwell R E, Lai F A, Maeda N, Mikoshiba K, Treiman M
Department of Medical Physiology, Panum Institute, University of Copenhagen, Denmark.
Biochem J. 1995 May 1;307 ( Pt 3)(Pt 3):749-58. doi: 10.1042/bj3070749.
In chromaffin cells of adrenal medulla, heterogeneity of Ca2+ stores has been suggested with respect to the mechanisms of Ca2+ uptake and release. We have examined Ca(2+)-ATPases responsible for loading of Ca2+ stores in these cells for their sensitivity to thapsigargin, a highly selective inhibitor of the SERCA [sarco(endo)plasmic reticulum calcium ATPase] family of intracellular Ca2+ pumps. Using immunostaining, we studied the distribution of Ca(2+)-ATPases, and of receptors for inositol 1,4,5-trisphosphate (InsP3) and ryanodine, in the density-gradient fractions of microsomes from bovine adrenal medulla. In parallel, we examined distribution profiles of ATP-dependent Ca2+ uptake in the same fractions, along with subcellular markers for plasma membranes and endoplasmic reticulum (ER). Two Ca(2+)-ATPase-like proteins (116 and 100 kDa) were detected, consistent with the presence of SERCA 2b and SERCA 3 isoenzymes of Ca2+ pumps. The distribution of these putative Ca(2+)-ATPase iso-enzymes paralleled that of InsP3 and ryanodine receptors. This distribution of ER Ca(2+)-ATPases, as determined immunologically, was consistent with that of thapsigargin-sensitive, but not of thapsigargin-insensitive, ATP-dependent Ca2+ uptake. In contrast, the distribution profile of the thapsigargin-insensitive Ca2+ uptake was strongly correlated to that of plasma membranes, and co-distributed with plasma membrane Ca(2+)-ATPase detected immunologically. In isolated, permeabilized chromaffin cells, InsP3 and caffeine induced Ca2+ release following an ATP-dependent Ca2+ accumulation to the stores. This accumulation was abolished by thapsigargin. Together, these data strongly indicate that the thapsigargin-sensitive, presumably SERCA-type Ca(2+)-ATPases account for Ca2+ uptake to InsP3-sensitive, as well as to caffeine-sensitive, Ca2+ stores in bovine adrenal chromaffin cells.
在肾上腺髓质的嗜铬细胞中，关于钙离子摄取和释放机制，已有人提出钙离子储存存在异质性。我们研究了负责这些细胞中钙离子储存加载的钙ATP酶对毒胡萝卜素的敏感性，毒胡萝卜素是肌浆网/内质网钙ATP酶（SERCA）家族细胞内钙泵的一种高度选择性抑制剂。利用免疫染色，我们研究了牛肾上腺髓质微粒体密度梯度组分中钙ATP酶、肌醇1,4,5-三磷酸（InsP3）受体和兰尼碱受体的分布。同时，我们检测了相同组分中ATP依赖的钙离子摄取分布情况，以及质膜和内质网（ER）的亚细胞标志物。检测到两种类钙ATP酶蛋白（116 kDa和100 kDa），这与钙离子泵的SERCA 2b和SERCA 3同工酶的存在一致。这些假定的钙ATP酶同工酶的分布与InsP3和兰尼碱受体的分布平行。通过免疫测定确定的内质网钙ATP酶的这种分布与毒胡萝卜素敏感但与毒胡萝卜素不敏感的ATP依赖的钙离子摄取的分布一致。相反，毒胡萝卜素不敏感的钙离子摄取的分布与质膜的分布密切相关，并与免疫检测到的质膜钙ATP酶共分布。在分离的、透化的嗜铬细胞中，InsP3和咖啡因在ATP依赖的钙离子积累到储存库后诱导钙离子释放。这种积累被毒胡萝卜素消除。总之，这些数据有力地表明，毒胡萝卜素敏感的、可能是SERCA型的钙ATP酶负责牛肾上腺嗜铬细胞中对InsP3敏感以及对咖啡因敏感的钙离子储存的钙离子摄取。
