Department of Physiology and Neuroscience, NYU School of Medicine, NY 10016, USA.
Cell Calcium. 2010 Jun;47(6):507-13. doi: 10.1016/j.ceca.2010.04.004. Epub 2010 May 26.
Quick cytosolic calcium clearance is essential for the effective modulation of various cellular functions. An excess of cytosolic calcium after influx is largely removed via ATP-dependent mechanisms located in the plasma membrane and the endoplasmic reticulum. Therefore, calcium clearance depends critically on the adequate supply of ATP, which may come from either glycolysis or mitochondria, or both. However, it presently remains unknown the degree to which individual ATP generating pathways - glycolysis and mitochondria power ATP-dependent calcium as well as other vital ion clearance mechanisms in neurons. In this study, we explored the relationship between the energy generating pathways and ion clearance mechanisms in neurons by characterizing the effects of glycolytic and mitochondrial inhibitors of ATP synthesis on calcium clearance kinetics in the soma, dendrites and spines. Stimulation of cultured cerebellar granule cells by brief pulses of 60mM potassium ACSF, and electrical stimulation of purkinje cells in acutely prepared slices led to a transient calcium influx, whose clearance was largely mediated by the plasma membrane Ca(2+)-ATPase pump. Inhibition of glycolysis by deoxyglucose or iodoacetic acid resulted in a marked slowing in calcium clearance in the soma, dendrites, and spines with the spines affected the most. However, inhibition of the mitochondrial citric acid cycle with fluoroacetate and arsenite, or mitochondrial ATP synthase with oligomycin did not produce any immediate effects on calcium clearance kinetics in any of those neuronal regions. Although cytosolic calcium clearance was not affected by the inhibition of mitochondria, the magnitude of the calcium clearance delay induced by glycolytic inhibitors in different neuronal compartments was related to their mitochondrial density. Conversely, the endoplasmic reticulum Ca(2+)-ATPase pump activity is fuelled by both glycolytic and mitochondrial ATP, as evidenced by a minimal change in the endoplasmic reticulum calcium contents in cells treated with either deoxyglucose supplemented with lactate or arsenite. Taken together, these data suggest that calcium clearance in cerebellar granule and purkinje cells relies on the plasma membrane Ca(2+)-ATPase, and is powered by glycolysis.
快速的细胞质钙离子清除对于有效调节各种细胞功能至关重要。流入后的细胞质钙离子过多，主要通过位于质膜和内质网上的 ATP 依赖性机制去除。因此，钙离子清除严重依赖于 ATP 的充分供应，ATP 可以来自糖酵解或线粒体，或者两者都有。然而，目前尚不清楚单个 ATP 生成途径（糖酵解和线粒体）在多大程度上为神经元中的 ATP 依赖性钙以及其他重要离子清除机制提供动力。在这项研究中，我们通过研究糖酵解和线粒体抑制剂对 ATP 合成的影响，来研究能量生成途径与神经元离子清除机制之间的关系。用 60mM 钾 ACSF 短暂脉冲刺激培养的小脑颗粒细胞，以及急性切片中浦肯野细胞的电刺激导致短暂的钙离子内流，其清除主要由质膜 Ca(2+)-ATP 酶泵介导。用脱氧葡萄糖或碘乙酸抑制糖酵解导致胞体、树突和棘突中的钙清除明显减慢，其中棘突受影响最大。然而，用氟乙酸和亚砷酸盐抑制柠檬酸循环或用寡霉素抑制线粒体 ATP 合酶，在这些神经元区域的任何一个中都不会对钙清除动力学产生即时影响。虽然细胞质钙离子清除不受线粒体抑制的影响，但不同神经元区室中糖酵解抑制剂引起的钙离子清除延迟的幅度与它们的线粒体密度有关。相反，内质网 Ca(2+)-ATP 酶泵的活性由糖酵解和线粒体 ATP 共同提供，这可以从用补充有乳酸的脱氧葡萄糖或亚砷酸盐处理的细胞中内质网钙离子含量的微小变化中得到证明。总之，这些数据表明小脑颗粒细胞和浦肯野细胞中的钙离子清除依赖于质膜 Ca(2+)-ATP 酶，并且由糖酵解提供动力。
