Anesthesiology Research Laboratories, Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, Wisconsin, USA.
Biophys J. 2010 Aug 9;99(4):997-1006. doi: 10.1016/j.bpj.2010.04.069.
ADP influx and ADP phosphorylation may alter mitochondrial free [Ca2+] (Ca2+) and consequently mitochondrial bioenergetics by several postulated mechanisms. We tested how Ca2+ is affected by H2PO4(-) (P(i)), Mg2+, calcium uniporter activity, matrix volume changes, and the bioenergetic state. We measured Ca2+, membrane potential, redox state, matrix volume, pH(m), and O2 consumption in guinea pig heart mitochondria with or without ruthenium red, carboxyatractyloside, or oligomycin, and at several levels of Mg2+ and P(i). Energized mitochondria showed a dose-dependent increase in Ca2+ after adding CaCl2 equivalent to 20, 114, and 485 nM extramatrix free [Ca2+] (Ca2+); this uptake was attenuated at higher buffer Mg2+. Adding ADP transiently increased Ca2+ up to twofold. The ADP effect on increasing Ca2+ could be partially attributed to matrix contraction, but was little affected by ruthenium red or changes in Mg2+ or P(i). Oligomycin largely reduced the increase in Ca2+ by ADP compared to control, and Ca2+ did not return to baseline. Carboxyatractyloside prevented the ADP-induced Ca2+ increase. Adding CaCl2 had no effect on bioenergetics, except for a small increase in state 2 and state 4 respiration at 485 nM Ca2+. These data suggest that matrix ADP influx and subsequent phosphorylation increase Ca2+ largely due to the interaction of matrix Ca2+ with ATP, ADP, P(i), and cation buffering proteins in the matrix.
ADP 内流和 ADP 磷酸化可能通过几种假设的机制改变线粒体游离 [Ca2+]（[Ca2+]（m）），进而影响线粒体生物能学。我们测试了 H2PO4（-）（Pi）、Mg2+、钙单向转运体活性、基质体积变化以及生物能状态如何影响 [Ca2+]（m）。我们在豚鼠心脏线粒体中测量了 [Ca2+]（m）、膜电位、氧化还原状态、基质体积、pH（m）和 O2 消耗，同时使用了钌红、羧基三叶草苷或寡霉素，并在不同的 Mg2+和 Pi 水平下进行了测量。在加入相当于 20、114 和 485 nM 细胞外游离 [Ca2+]（[Ca2+]（e）的 CaCl2 后，能量充足的线粒体显示出 [Ca2+]（m）的剂量依赖性增加；在更高的缓冲 Mg2+下，这种摄取会减弱。添加 ADP 会使 [Ca2+]（m）短暂增加两倍。ADP 增加 [Ca2+]（m）的作用部分归因于基质收缩，但受钌红或 Mg2+或 Pi 变化的影响很小。与对照相比，寡霉素使 ADP 引起的 [Ca2+]（m）增加减少了很多，并且 [Ca2+]（m）没有回到基线。羧基三叶草苷可防止 ADP 诱导的 [Ca2+]（m）增加。添加 CaCl2 对生物能学没有影响，除了在 485 nM [Ca2+]（e）下，状态 2 和状态 4 呼吸略有增加。这些数据表明，基质 ADP 内流和随后的磷酸化增加了 [Ca2+]（m），主要是由于基质 Ca2+与 ATP、ADP、Pi 和基质中的阳离子缓冲蛋白相互作用所致。
