Addition and kinetic characterization of mitochondrial calcium in myocardial tissue culture

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Abstract
Ca exchange of myocardial cells in culture was studied with the on-line scintillator disk technique under 2 conditions of perfusion: with and without a permeant anion capable of proton donation. Cells perfused without the proton-donating anion (HEPES [N-2-hydroxyethylpiperazine-N''-2-ethanesulfonic acid] buffer and Cl salts) demonstrated a single rapid component of Ca exchange previously demonstrated to be localized at the sarcolemmal-glycocalyx complex of the cell. Substitution of 10 mM NaH2PO4 for HEPES in the perfusate (at unchanged pH) produced an immediate linear increase in Ca uptake that more than tripled exchangeable cellular Ca uptake in 60 min. The presence of PO4 slowed the efflux of the accumulated Ca significantly. Introduction of inhibitors of mitochondrial respiration (warfarin sodium or antimycin A, 10-5 M) completely prevented the increased Ca uptake induced by PO4. Substitution of a nonproton-donating anion, NO3, for PO4 failed to induce increased Ca uptake. The component of Ca induced by PO4 washed out slowly in the presence of PO4 (rate constant = 0.018/min t1/2 = 39 min) and was not displaceable by La. The characteristics of the Ca compartment strongly indicate that it is localized to the mitochondria. The H2PO4- is visualized to exchange for matrix OH ions in the mitochondria, a reaction equivalent to entry of H3PO4 with loss of a proton to the alkaline matrix. The resulting excess anion provides the milieu for Ca accumulation as the phosphate salt. Beat-to-beat movements of Ca apparently can cycle between cell surface and interior without accumulation in intracellular organelles. If mitochondria are provided with a proton-donating anion, they are capable of accumulating large amounts of Ca in a kinetically identifiable, slowly exchangeable compartment. This adds greatly to the Ca-buffering capacity of the cell, which could be of major importance to cell survival under conditions of high Ca influx.

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