Chalmers Susan, Nicholls David G
Buck Institute for Age Research, Novato, California 94945, USA.
J Biol Chem. 2003 May 23;278(21):19062-70. doi: 10.1074/jbc.M212661200. Epub 2003 Mar 26.
Three sequential phases of mitochondrial calcium accumulation can be distinguished: matrix dehydrogenase regulation, buffering of extramitochondrial free calcium, and finally activation of the permeability transition. Relationships between these phases, free and total matrix calcium concentration, and phosphate concentration are investigated in rat liver and brain mitochondria. Slow, continuous calcium infusion is employed to avoid transient bioenergetic consequences of bolus additions. Liver and brain mitochondria undergo permeability transitions at precise matrix calcium loads that are independent of infusion rate. Cytochrome c release precedes the permeability transition. Cyclosporin A enhances the loading capacity in the presence or absence of acetoacetate. A remarkably constant free matrix calcium concentration, in the range 1-5 microM as monitored by matrix-loaded fura2-FF, was observed when total matrix calcium was increased from 10 to at least 500 nmol of calcium/mg of protein. Increasing phosphate decreased both the free matrix calcium and the matrix calcium-loading capacity. Thus the permeability transition is not triggered by a critical matrix free calcium concentration. The rate of hydrogen peroxide detection by Amplex Red decreased during calcium infusion arguing against a role for oxidative stress in permeability pore activation in this model. A transition between a variable and buffered matrix free calcium concentration occurred at 10 nmol of total matrix calcium/mg protein. The solubility product of amorphous Ca3(PO4)2 is consistent with the observed matrix free calcium concentration, and the matrix pH is proposed to play the major role in maintaining the low matrix free calcium concentration.
基质脱氢酶调节、线粒体外游离钙的缓冲，以及最终通透性转变的激活。在大鼠肝脏和脑线粒体中研究了这些阶段、游离和总基质钙浓度以及磷酸盐浓度之间的关系。采用缓慢、持续的钙输注以避免推注添加钙带来的短暂生物能量学后果。肝脏和脑线粒体在精确的基质钙负荷下发生通透性转变，且该负荷与输注速率无关。细胞色素c释放先于通透性转变。无论有无乙酰乙酸，环孢素A均可提高钙负荷能力。当总基质钙从10增加到至少500 nmol钙/毫克蛋白质时，通过基质加载的fura2-FF监测到，游离基质钙浓度在1-5 microM范围内显著恒定。增加磷酸盐会降低游离基质钙和基质钙负荷能力。因此，通透性转变不是由临界的游离基质钙浓度触发的。在钙输注过程中，Amplex Red检测到的过氧化氢速率下降，这表明在该模型中氧化应激在通透性孔激活中不起作用。在总基质钙为10 nmol/毫克蛋白质时，游离基质钙浓度发生了从可变到缓冲的转变。无定形Ca3(PO4)2的溶度积与观察到的游离基质钙浓度一致，并且提出基质pH在维持低游离基质钙浓度中起主要作用。
