FIGURE 7.

Az-LPAF-induced mitochondrial dysfunction is enhanced by the pro-apoptotic protein Bid. A, Bid and Az-LPAF synergize to depolarize mitochondria. Isolated rat liver mitochondria were loaded with TMRM, the organelles were provided with glutamate and malate, and changes to the transmembrane potential were recorded as described in the legend to Fig. 2. Recombinant murine Bid (2 μg) was added, or not, as shown by the second arrow, and 2 min later 1 μm Az-LPAF was introduced into the cuvette. CCCP was added at the final arrow to achieve complete depolarization (n = 3). B, mitochondria from Bid^−/− animals were less sensitive to Az-LPAF than mitochondria from the parental strain. Mitochondria isolated from Bid^−/− or BL6 murine livers were loaded with TMRM and changes in transmembrane potential with or without the addition of 5 μm Az-LPAF (second arrow) were recorded as in the proceeding panel (n = 3). C, recombinant Bid restores sensitivity of Bid^−/− mitochondria to Az-LPAF. Mitochondria isolated from the livers of Bid null mice were incubated with 2 μg of recombinant Bid for 5 min before they were loaded with TMRM, energized with glutamate and malate (first arrow), treated or not with 5 μm Az-LPAF (second arrow), and finally depolarized with CCCP (third arrow) (n = 3).