Figure 4.
Comparison of the effects of diazoxide and phorbol esters on Ca-induced MPT in nonenergized mitochondria. In each image, the upper trace shows TMRM fluorescence and the lower trace shows matrix swelling. Fresh, isolated, nonenergized mitochondria (1–3 h on ice) were subjected to a small Ca load (10 μM) sufficient to dissipate Δψm and cause matrix swelling. Ala, alamethicin. (A) With 300 μM 5-HD added to diazoxide, full Δψm recovery required arachidonic acid + TMPD, EGTA, and CsA, indicating MPT had occurred. (B) With 50 μM diazoxide present, energization with complex IV substrates (IV) led to full Δψm recovery. (C–E) Treatment with 400 nM PMA (D), but not the inactive phorbol ester 4α-PMA (E), reproduced the effects of diazoxide. The effects of PMA were blocked by 300 μM 5-HD (C). (F) Data summary showing the degree of matrix swelling after Ca loading (relative to alamethicin, solid bars) and the extent of Δψm recovery after the addition of complex IV substrates (TMPD) for the various interventions in A–E.