Mitchell Kathryn J, Tsuboi Takashi, Rutter Guy A
Henry Wellcome Laboratories of Integrated Cell Signaling and Department of Biochemistry, School of Medical Sciences, University Walk, University of Bristol, Bristol, U.K.
Diabetes. 2004 Feb;53(2):393-400. doi: 10.2337/diabetes.53.2.393.
Changes in intracellular Ca(2+) concentration play a key role in the regulation of insulin secretion by glucose and other secretagogues. Here, we explore the importance of the secretory pathway Ca(2+)-ATPase, plasma membrane-related Ca(2+)-ATPase-1 (PMR1; human orthologue ATP2C1) in intracellular Ca(2+) homeostasis in pancreatic islet beta-cells. Endogenous PMR1 mRNA and protein were detected in both isolated rat islets and beta-cell-derived lines (MIN6 and INS1). Subcellular fractionation of the cell lines revealed PMR1 immunoreactivity in both microsomal and dense-core secretory vesicle-enriched fractions. Correspondingly, depletion of cellular PMR1 with small interfering RNAs inhibited Ca(2+) uptake into the endoplasmic reticulum and secretory vesicles by approximately 20%, as assessed using organelle-targeted aequorins in permeabilized INS1 cells. In intact cells, PMR1 depletion markedly enhanced flux though L-type Ca(2+) channels and augmented glucose-stimulated, but not basal, insulin secretion. Whereas average cytosolic [Ca(2+)] increases in response to 30.0 mmol/l glucose were unaffected by PMR1 depletion, [Ca(2+)] oscillation shape, duration, and decay rate in response to glucose plus tetraethylammonium were modified in PMR1-depleted single cells, imaged using fluo-3-acetoxymethylester. PMR1 thus plays an important role, which is at least partially nonoverlapping with that of sarco(endo-)plasmic reticulum Ca(2+)-ATPases, in the control of beta-cell Ca(2+) homeostasis and insulin secretion.
细胞内钙离子浓度的变化在葡萄糖及其他促分泌素对胰岛素分泌的调节中起关键作用。在此，我们探讨分泌途径钙离子 -ATP酶，即与质膜相关的钙离子 -ATP酶-1（PMR1；人类同源物ATP2C1）在胰岛β细胞内钙离子稳态中的重要性。在分离的大鼠胰岛和β细胞系（MIN6和INS1）中均检测到内源性PMR1 mRNA和蛋白质。对细胞系进行亚细胞分级分离显示，在富含微粒体和致密核心分泌囊泡的级分中均有PMR1免疫反应性。相应地，使用小干扰RNA耗尽细胞内的PMR1，通过在通透的INS1细胞中使用细胞器靶向水母发光蛋白评估，内质网和分泌囊泡对钙离子的摄取被抑制了约20%。在完整细胞中，耗尽PMR1显著增强了通过L型钙离子通道的通量，并增强了葡萄糖刺激的胰岛素分泌，但对基础胰岛素分泌无影响。虽然对30.0 mmol/l葡萄糖刺激的平均胞质钙离子浓度升高不受PMR1耗尽的影响，但在使用fluo-3-乙酰氧甲酯成像的PMR1耗尽的单细胞中，对葡萄糖加四乙铵反应的钙离子振荡形状、持续时间和衰减率发生了改变。因此，PMR1在β细胞钙离子稳态和胰岛素分泌的控制中发挥重要作用，这至少部分与肌质（内质）网钙离子 -ATP酶的作用不重叠。
