Rintoul G L, Raymond L A, Baimbridge K G
Department of Physiology, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.
Cell Calcium. 2001 Apr;29(4):277-87. doi: 10.1054/ceca.2000.0190.
Calbindin-D28k (CaBP) is a calcium-binding protein found in specific neuronal populations in the mammalian brain that, as a result of its proposed calcium-buffering action, may protect neurons against potentially harmful increases in intracellular calcium. We have stably transfected HEK 293 cells with recombinant human CaBP in order to determine the influence of this protein upon transient increases in intracellular ionic calcium concentration (Ca(2+)) induced either by transient transfection of the NR1 and NR2A subunits of the N-methyl-D-aspartate (NMDA) receptor and brief exposure to glutamate, photolysis of the caged calcium compound NP-EGTA, or exposure to the Ca(2+)]-ionophore 4-Br-A23187. The presence of CaBP did not significantly reduce the peak Ca(2+)stimulated by glutamate activation of NMDA receptors but significantly prolonged the recovery to baseline values. Flash photolysis of NP-EGTA in control cells resulted in an almost instantaneous increase in Ca(2+)followed by a bi-exponential recovery to baseline values. In cells stably expressing CaBP, the peak Ca(2+)levels were not statistically different from the controls, however, there was a significant prolongation of the initial portion of the slow recovery phase. In cells exposed to 4-Br-A23187, the presence of CaBP significantly reduced the rate of rise of Ca(2+), reduced the peak response, slowed the rate of recovery, and reduced the depolarization of mitochondria. In studies of delayed, Ca(2+)]-dependent cell death, CaBP transfected cells exhibited enhanced survival 24h after a 1-h exposure to 200 microM NMDA. However, necrotic cell death observed after the first 6h was not prevented by the presence of CaBP. These results provide direct evidence for a Ca(2+)-buffering effect of CaBP which serves to limit Ca(2+)entry and the depolarization of mitochondria, thereby protecting cells from death mediated most likely by apoptosis.
钙结合蛋白-D28k（CaBP）是一种在哺乳动物大脑特定神经元群体中发现的钙结合蛋白，由于其推测的钙缓冲作用，可能保护神经元免受细胞内钙潜在有害的增加。我们用重组人CaBP稳定转染了HEK 293细胞，以确定该蛋白对由N-甲基-D-天冬氨酸（NMDA）受体的NR1和NR2A亚基瞬时转染并短暂暴露于谷氨酸、笼形钙化合物NP-EGTA的光解或暴露于Ca(2+)]离子载体4-Br-A23187所诱导的细胞内离子钙浓度（Ca(2+)）瞬时增加的影响。CaBP的存在并未显著降低由NMDA受体谷氨酸激活刺激的Ca(2+)峰值，但显著延长了恢复到基线值的时间。对照细胞中NP-EGTA的闪光光解导致Ca(2+)几乎瞬间增加，随后以双指数方式恢复到基线值。在稳定表达CaBP的细胞中，Ca(2+)峰值水平与对照无统计学差异，然而，缓慢恢复阶段初始部分有显著延长。在暴露于4-Br-A23187的细胞中，CaBP的存在显著降低了Ca(2+)的上升速率，降低了峰值反应，减缓了恢复速率，并降低了线粒体的去极化。在延迟的、Ca(2+)]依赖性细胞死亡研究中，CaBP转染的细胞在暴露于200 microM NMDA 1小时后24小时显示出增强的存活率。然而，CaBP的存在并不能阻止最初6小时后观察到的坏死性细胞死亡。这些结果为CaBP的Ca(2+)缓冲作用提供了直接证据，该作用有助于限制Ca(2+)进入和线粒体去极化，从而保护细胞免受最可能由凋亡介导的死亡。
