Prentki M, Janjic D, Biden T J, Blondel B, Wollheim C B
J Biol Chem. 1984 Aug 25;259(16):10118-23.
The regulation of extramicrosomal Ca2+ concentration maintained by suspensions of rat insulinoma microsomes was studied using Ca2+-selective minielectrodes. The Ca2+-transporting activity was MgATP dependent and correlated with the endoplasmic reticulum marker NADPH-cytochrome c reductase. When incubated in a high KCl medium containing Mg2+ and phosphate, the microsomes lowered [Ca2+] within less than 10 min to around 0.2 microM. They had a high Ca2+-sequestering activity since they were able to take up and retain several small Ca2+ additions. No evidence for a Na+/Ca2+ countertransport was obtained. The accumulated Ca2+ was released by the Ca2+ ionophore A23187 or upon transforming ATP into ADP using glucose plus hexokinase. The addition of ADP, at concentrations present in cells, resulted in a dose-dependent and reversible net Ca2+ efflux from the microsomes until a higher [Ca2+] steady state was reached. This was specific for ADP since GDP, UDP, CDP, IDP, and the nonhydrolyzable analogue methylene-ADP as well as AMP and cAMP did not reproduce the effect. Insulin secretory granules were unable to lower medium [Ca2+] or to take up a pulse addition of Ca2+. However, most of the large granular calcium content was released by A23187. The addition of Na+ and lowering or increasing medium pH by 0.2 pH unit did not induce Ca2+ uptake or efflux from the secretory granules. The results indicate that insulinoma endoplasmic reticulum but not insulin secretory granules may play a critical role in the regulation of cytosolic Ca2+. A variation in cellular ADP content following secretagogue addition might modulate Ca2+ fluxes across the endoplasmic reticulum and contribute in raising cytosolic Ca2+.
使用钙离子选择性微电极研究了大鼠胰岛素瘤微粒体悬浮液维持的微粒体外钙离子浓度的调节。钙离子转运活性依赖于MgATP，并与内质网标志物NADPH-细胞色素c还原酶相关。当在含有镁离子和磷酸盐的高钾培养基中孵育时，微粒体在不到10分钟内将[Ca2+]降低至约0.2微摩尔/升。它们具有高钙离子螯合活性，因为它们能够吸收并保留多次少量添加的钙离子。未获得钠离子/钙离子逆向转运的证据。积累的钙离子可通过钙离子载体A23187释放，或在使用葡萄糖加己糖激酶将ATP转化为ADP时释放。添加细胞内存在浓度的ADP会导致微粒体出现剂量依赖性和可逆的净钙离子外流，直至达到更高的[Ca2+]稳态。这对ADP具有特异性，因为GDP、UDP、CDP、IDP以及不可水解的类似物亚甲基-ADP以及AMP和cAMP均未重现该效应。胰岛素分泌颗粒无法降低培养基中的[Ca2+]或吸收脉冲添加的钙离子。然而，大部分大颗粒钙含量可被A23187释放。添加钠离子以及将培养基pH降低或升高0.2个pH单位均未诱导分泌颗粒摄取或释放钙离子。结果表明，胰岛素瘤内质网而非胰岛素分泌颗粒可能在细胞质钙离子调节中起关键作用。添加促分泌剂后细胞内ADP含量的变化可能会调节内质网的钙离子通量，并有助于提高细胞质钙离子浓度。
