Kosterin S A, Babich L G, Shlykov S G, Rovenets N A
Department of Biochemical Kinetics, National Academy of Sciences of the Ukraine, Kiev.
Biokhimiia. 1996 Jan;61(1):73-81.
An in-depth study of the effects of Ca(2+)-precipitating anions (oxalate, phosphate), the Ca2+ ionophore A23187 and some highly effective selective inhibitors of the endo(sarco)plasmic reticulum Ca(2+)-pump (tapsigargin, cyclopiasonic acid) on ruthenium red (10 microM)-insensitive, Mg2+,ATP-dependent accumulation of Ca2+ in digitonin-permeabilized myometrium cells of nonpregnant estrogen-treated rats, has been carried out. Oxalate and phosphate stimulated Mg2+,ATP-dependent accumulation of Ca2+ in permeabilized smooth muscle cells. The Ca2+ ionophore A23187 (5 microM) added to the medium containing oxalate (10 mM) or phosphate (20 mM) prevented the energy-dependent accumulation of Ca2+. Tapsigargin (50 nM) and cyclopiasonic acid (10 microM) fully suppressed the Mg2+,ATP-dependent Ca2+ increase measured in the presence of the Ca(2+)-precipitating anions. The kinetic data indicate that the tapsigargin affinity for the energy-dependent Ca2+ transporting system is much higher than that of cyclopiasonic acid: Ki app values are 0.9 nM and 0.6 microM, respectively. Oxalate (10 mM) added to the incubation medium initially containing tapsigargin (50 nM) failed to induce the Mg2+,ATP-dependent accumulation of Ca2+ in permeabilized myocytes. The data obtained provide biochemical evidence for the existence in myometrium cells of a Ca(2+)-pump providing the Mg2+,ATP-dependent accumulation of Ca2+ in the nonmitochondrial Ca2+ depot--the endoplasmic reticulum. This reaction is stimulated by precipitating penetrating anions and suppressed by selective inhibitors.
深入研究了钙离子沉淀阴离子（草酸盐、磷酸盐）、钙离子载体A23187以及一些内质网（肌浆网）钙离子泵的高效选择性抑制剂（毒胡萝卜素、环匹阿尼酸）对未孕雌激素处理大鼠子宫肌层细胞中钌红（10微摩尔）不敏感、镁离子、ATP依赖的钙离子积累的影响。草酸盐和磷酸盐刺激了通透平滑肌细胞中镁离子、ATP依赖的钙离子积累。向含有草酸盐（10毫摩尔）或磷酸盐（20毫摩尔）的培养基中添加钙离子载体A23187（5微摩尔）可阻止钙离子的能量依赖积累。毒胡萝卜素（50纳摩尔）和环匹阿尼酸（10微摩尔）完全抑制了在存在钙离子沉淀阴离子的情况下测得的镁离子、ATP依赖的钙离子增加。动力学数据表明，毒胡萝卜素对能量依赖的钙离子转运系统的亲和力远高于环匹阿尼酸：表观抑制常数（Ki app）值分别为0.9纳摩尔和0.6微摩尔。向最初含有毒胡萝卜素（50纳摩尔）的孵育培养基中添加草酸盐（10毫摩尔）未能诱导通透心肌细胞中镁离子、ATP依赖的钙离子积累。所获得的数据为子宫肌层细胞中存在一种钙离子泵提供了生化证据，该泵在非线粒体钙离子储存库——内质网中提供镁离子、ATP依赖的钙离子积累。该反应受到沉淀穿透性阴离子的刺激，并被选择性抑制剂抑制。
