Effect of Butylhydroxytoluene and Related Compounds on Permeability of the Inner Mitochondrial Membrane

Mitochondrial inner membrane contains a latent pore (PTP) that when opened uncouples mitochondrial energy transduction and allows rapid equilibration of low-molecular-weight solutes between the matrix and exterior. Based on sensitivity of the PTP to well-known free radical scavenger butylhydroxytoluene (BHT), it has been proposed that increased steady-state level of oxygen radicals, and subsequent radical attack of proteins and lipids, is a central event in activation of this pore (Novgorodovet al., J. Bioenerg. Biomembr.19, 191–202, 1987; Carbonera and Azzone,Biochim. Biophys. Acta943, 245–255, 1988). Present studies revealed that DBT, a derivative of BHT devoid of radical scavenging activity, exerts an analogous effect on the permeability of the inner membrane. Inhibition of the Ca2+-induced PTP opening is essentially complete at dose range of 50–60 nmol/mg protein with IC50values of about 32 and 23 nmol/mg protein for DBT and BHT, respectively. Electron microscopy and osmotic experiments utilizing polyethylene glycols with different Stokes radii showed that the apparent lack of inhibition seen at high concentrations of these compounds results from cyclosporin A- and Ca2+-insensitive pore formation in the inner membrane. Experiments employing antioxidants with similar structure but dissimilar hydrophobicity provided evidence for localization of the antioxidant binding sites within the hydrophobic zone of the inner membrane or in the matrix space. The data obtained do not refute the notion that oxygen radicals modulate the PTP, but rather indicate that BHT operates independently of its free radical scavenging activity. Overall, the sensitivity to BHT and other antioxidants is not always a reliable criterion for the involvement of free radical reactions in the processes under study.