Figure 3.

Effect of the cyclophilin D inhibitor, cyclosporin A, on Ca²⁺ uptake in mitochondria. A, CRC was measured in the absence (−CsA) or presence (+CsA) of 1 μm CsA in a buffer containing glutamate plus malate. Changes in [Ca²⁺]_exm were monitored by the fluorescent Ca²⁺-sensitive dye, Calcium Green-5N. Ca²⁺ solution was subsequently applied at a final concentration of 7.8 μm for untreated mitochondria (−CsA) or for CsA-treated mitochondria (+CsA) at 60 s intervals. The traces marked by a–c were used for analysis in B. Inset, The CRC was determined from three independent experiments (n = 6; *p < 0.05). Data are expressed as mean ± SEM. B, Kinetics of [Ca²⁺]_exm changes. Representative traces of [Ca²⁺]_exm marked by a–c in A were expanded. The traces were normalized by maximal peak amplitude and compared between the absence (−CsA) and presence (+CsA) of CsA. After the repeated addition of Ca²⁺, the recovery time became shorter in the presence of CsA than in the absence of CsA. As shown in C, CsA apparently decreased the recovery time of Ca²⁺. C, Changes in recovery time with sequential additions of Ca²⁺, plotted by the amount of Ca²⁺ added in the absence (−CsA) or presence (+CsA) of CsA. Each circle corresponds to a Ca²⁺ addition, and each trace of [Ca²⁺]_exm is from one experiment. Two-way repeated-measures ANOVA in the first four additions revealed a significant interaction between the number of Ca²⁺ addition and CsA treatment (F = 4.78; df = 1.08; p < 0.05, repeated two-way ANOVA, Greenhouse–Geisser correction). Although there was no statistically significant difference of recovery time after the first addition of Ca²⁺ (p = 0.35, Mann–Whitney U test), recovery time in the presence of CsA (+CsA) became shorter than that in the absence of CsA at the fourth addition of Ca²⁺ (p < 0.05, Mann–Whitney U test). D, Changes in recovery time with the subsequent additions of Ca²⁺. Each circle corresponds to a Ca²⁺ addition and each trace of [Ca²⁺]_exm is from one experiment using mitochondria from wild-type (WT) and mutPOLG Tg (Tg) mice in the absence of CsA.