Figure 3. Chronic ethanol consumption intensifies the mitochondrial permeability transition (MPT) and cytochrome C release from mitochondria of Sod1 ^−/− mice.

For MPT determination, mitochondria (0.5mg/ml) were incubated in respiration buffer at 25° C as described in Materials and Methods. Swelling was monitored for 30 min at 540 nm in mitochondria (○); mitochondria plus respiratory substrates 1 mM glutamate and 1mM malate without calcium (●); with 10 μM CaCl₂ (▲); with 50 μM CaCl₂ (◆), or with 150 μM CaCl₂ (■). 2 μM cyclosporin A was added to mitochondria before incubation with ( ) or without ( ) 150 μM Ca to prevent MPT. Each curve represents the Mean of 3–4 mice

For cytochrome C release to the incubation medium, mitochondria (1 mg/ml) were incubated for 15 min at 25 °C in respiration buffer with the indicated additions. The reaction mixtures were then centrifuged at 9,000 × g for 10 min at 4 °C and supernatant was concentrated by ultrafiltration through Centrikon 10 membranes (Amicon) at 4 °C. The concentrated supernatants were subjected to SDS-PAGE and analyzed by Western blotting using anti-cytochrome c antibody. Results show the cytochrome c present in the concentrated supernatants obtained after mitochondria from the 4 groups were incubated with no addition, or 10 uM calcium, or 150 uM calcium or 150 uM calcium plus 2 uM cyclosporin A.