Reduced mitochondrial volume contributes but cannot fully explain the increase in matrix free calcium after addition of ADP

Abstract

In an earlier study we showed a transient doubling of mitochondrial matrix (m) [Ca2+] in response to added ADP and proposed that greater buffering of Ca2+ by ATP than by ADP is largely responsible for the increase. Previous studies have described mitochondrial volume contraction in response to ADP. Thus we explored if reduced mitochondrial volume with ADP also contributes to the apparent increase in m[Ca2+]. Guinea pig heart mitochondria were isolated by differential centrifugation. Mitochondria were suspended in respiration media, including 1 mM EGTA, 1 mM MgCl2, 140 mM KCl, and 5 mM K2HPO4. Mitochondria were energized with pyruvic acid followed by 250 μM ADP. Mitochondrial volume was measured by light scattering at a wavelength of 520 nm. Alamethicin (5 μg/ml) was added at the end to attain maximal matrix volume. Changes in matrix volume after addition of ADP were calculated as a percentage of maximal volume change. We showed a matrix contraction in response to ADP of approximately 5% of the maximal change in matrix volume. We conclude that although matrix contraction can contribute to the increase in [Ca2+] after addition of ADP, it explains only a small proportion of the rise in [Ca2+] during phosphorylation of ADP, suggesting differential Ca2+ buffering of ADP and ATP remains a major factor.