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Papers In Press, published online ahead of print March 26, 2003
Buck Institute for Age Research, Novato, CA 94945
Corresponding Author: dnicholls{at}buckinstitute.org
Three sequential phases of mitochondrial calcium accumulation can be distinguished: matrix dehydrogenase regulation, buffering of extra-mitochondrial 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 1-5mM as monitored by matrix-loaded fura2-FF, was observed when total matrix calcium was increased from 10 to at least 500nmol calcium/mg 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 10nmol 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.
J. Biol. Chem, 10.1074/jbc.M212661200
Submitted on December 12, 2002
Revised on March 26, 2003
Accepted on March 26, 2003
The relationship between free and total calcium concentrations in the matrix of liver and brain mitochondria
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