Properties of mitochondria and digitonin-treated mitoplasts. A, mitochondria (20 mg/ml) were incubated for 20 min with the indicated amounts of digitonin. The suspensions were then diluted and pelleted by centrifugation as described under “Experimental Procedures.” For each preparation, MAO activity (■) and Δψ_m (□) were determined. The activity was calculated as percent of the starting untreated mitochondria. Error bars represent means ±S.D. of four experiments. B, transmission electron microscopy of mitochondrial preparations without treatment with digitonin (panel a) and after treatment with 0.04 (panel b), 0.09 (panel c), and 0.12 mg (panel d) of digitonin/mg of protein. Scale bars = 1 μm in the main panels and 0.2 μm in the insets. C, Western blot analysis of untreated mitochondria (lane 1) and of preparations obtained after treatment of mitochondria with the indicated amounts of digitonin (lanes 2–4). AIF, apoptosis-inducing factor; mit., mitochondrial; sub., subunit. D, the incubation medium contained 250 mm sucrose, 10 mm Tris/MOPS, 5 mm succinate, 1 mm P_i, 10 μm EGTA/Tris, 0.5 μm calcium green-5N, 0.5 μg/ml oligomycin, and 2 μm rotenone, pH 7.4, at 25 °C. The calcium retention capacity was measured in mitochondria (1 mg/ml; trace a) or mitoplasts prepared with 0.09 mg of digitonin/mg of protein (1 mg/ml; trace a′) by the sequential addition of a train of 10 μm Ca²⁺ pulses at 1-min intervals (arrows).

Effect of the irradiation time in mitochondria and mitoplasts loaded with HP. Mitochondria (■) and mitoplasts (□) at 0.5 mg/ml were loaded with 2 μm HP for 1 min at 25 °C in standard medium before irradiation at 40 W/m² for the indicated times; permeabilization was then studied as the light scattering change at 540 nm following the addition of 60 μm (mitochondria) or 30 μm (mitoplasts) Ca²⁺. The permeabilization rates were normalized to that of the control (non-irradiated organelles). Error bars represent the mean ± S.D. of four experiments.

Effect of DP, HP, and PP on light-dependent activation of the permeability transition. Mitochondria (closed symbols) or mitoplasts (open symbols) at 0.5 mg/ml were incubated for 1 min at 25 °C with concentrations of each porphyrin (PP, circles; DP, squares; HP, triangles) giving a loading of 1.2 nmol of porphyrin/mg of protein (for details, see Table 1 and “Experimental Procedures”). Preparations were then supplemented with 5 μm Ca²⁺ (a concentration not sufficient to induce PTP opening per se) and irradiated for the indicated times at a fluence rate of 40 W/m². The PT was followed as the change in 90° light scattering at 540 nm. Error bars represent the mean ± S.D. of three experiments.

Effect of TSPO ligands on PTP-dependent Ca²⁺ release in mitochondria and mitoplasts. Mitochondria or mitoplasts (1 mg/ml) were incubated as described for Fig. 2D, and experiments were performed as shown in Fig. 7 by adding the stated amounts of FGIN1-27 (squares), Ro 5-4864 (triangles), or PK11195 (circles) after ruthenium red to mitochondria (closed symbols) or mitoplasts (open symbols). The rate of Ca²⁺ efflux was normalized to that obtained after the addition of 0.5 μm carbonyl cyanide p-trifluoromethoxyphenylhydrazone.

Structure of porphyrins. The chemical structures of HP, PP, DP, and CP are shown.

Effect of PhAsO and Cu(OP)₂ on light scattering in mitochondria and mitoplasts. The experimental conditions were as described for Fig. 2C, except that calcium green-5N was omitted. Light scattering was measured at 540 nm in mitochondria (1 mg/ml; A and B) or mitoplasts (1 mg/ml; A′ and B′). A and A′, 5 μm Ca²⁺ and 5 μm PhAsO were added where indicated (traces a); 1 μm CsA was added before (traces b); and 20 μm N-ethylmaleimide (NEM) was added where indicated (traces c). B and B′, 5 μm Ca²⁺ and 3 μm Cu(OP)₂ were added where indicated (traces a); 1 μm CsA was added before the organelles (traces b); and 20 μm N-ethylmaleimide was added where indicated (traces c).

Effect of DP, HP, PP, and CP on light-dependent inactivation of the permeability transition. Mitochondria (A) and mitoplasts (A′) at 0.5 mg/ml were incubated for 1 min with concentrations of each porphyrin giving a loading of 0.2–1.5 nmol of porphyrin/mg of protein at 25 °C (for details, see Table 1 and “Experimental Procedures”). Preparations were irradiated for 45 s at a fluence rate of 40 W/m² and then supplemented with 60 μm (A) or 30 μm (A′) Ca²⁺ to trigger the PT, which was followed as the changes in 90° light scattering at 540 nm. The permeabilization rate was normalized to that of the control (in the absence of porphyrins). Error bars represent the mean ± S.D. of three experiments.

Effect of uncoupler and FGIN1-27 on PTP-dependent Ca²⁺ release in mitochondria and mitoplasts. Mitochondria (1 mg/ml; A) or mitoplasts (1 mg/ml; A′) were incubated as described for Fig. 2D. In traces a and d, the medium was supplemented with 1 μm CsA. Where indicated, Ca²⁺ was added, followed by 0.1 μm ruthenium red (RR) and, at the asterisk, by 0.5 μm carbonyl cyanide p-trifluoromethoxyphenylhydrazone (traces a and b) or 40 μm FGIN1-27 (traces c and d).