Quantitation of Ca²⁺-induced MPT induction in sensitive and resistant cells.

(A) Illustration of method of detection. Curves were generated using a Ca²⁺-sensitive electrode. Two million cells were resuspended in a buffer containing 0.15 mM KCl, 5 mM KH₂PO₄, 1 mM MgSO₄, 5 mM succinate, and 5 mM Tris, pH 7.4 (see “Materials and methods”). After 2 minutes, cells were permeabilized with 0.005% digitonin and 5 μM rotenone was added to keep mitochondrial pyridine nucleotides in a reduced state. Ca²⁺ addition, in 20 nM increments, resulted in a steep increase in Ca²⁺, followed by restoration of the initial level due to Ca²⁺ accumulation in mitochondria, at which point 20 nM Ca²⁺ was again added. The Ca²⁺ capacity was calculated by adding the total amount of Ca²⁺ that could be taken up in mitochondria before MPT induction occurred, and mitochondrial Ca²⁺ was released. (B) Ca²⁺ capacity in sensitive and resistant cells treated with diluent (open bars) or 1 μM CdA (gray bars) for 12 hours. MPT was measured as described in panel A, and the amount of Ca²⁺ required for MPT induction is expressed graphically. Data shown are representative of 3 separate experiments.