Oligomycin strengthens the effect of cyclosporin A on mitochondrial permeability transition by inducing phosphate uptake Edmundo Cha´vez * , Jose´ S. Rodrı´guez, Gerardo Garcı´a, Noemı´ Garcı´a, Francisco Correa Departamento de Bioquı´mica, Instituto Nacional de Cardiologı´a, Ignacio Cha ´vez, Me ´xico, D.F. 014080, Me ´xico Received 8 September 2004; revised 27 January 2005; accepted 13 March 2005 Abstract The purpose of this work was to assess the effect of oligomycin on the mitochondrial membrane permeability transition. The antibiotic was found to strengthen cyclosporin A (CSA)-induced protection of non-specific permeability, which is triggered by a matrix Ca 2C load in the absence of ADP. Oligomycin also reinforced the protective effect of CSA on carboxyatractyloside-induced pore opening in the absence of ADP, but failed to do so in mitochondria incubated under anaerobic conditions or after addition of CCCP. Analyzing the efflux of matrix Ca 2C , we found that mitochondrial swelling and the collapse of the transmembrane electric gradient coincided with membrane leakage. The effects of the antibiotic were observed in phosphate-containing media but not in the presence of acetate. Furthermore, N-ethylmaleimide hindered the protective effect of oligomycineCSA. In addition, the matrix phosphate concentration increased concurrently with a diminution in the matrix-free fraction of Ca 2C . We concluded that oligomycin increases phosphate uptake by stimulating the phosphate ÿ /OH ÿ exchange reaction. Ó 2005 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Key words: Mitochondria; Oligomycin; Permeability transition; Calcium; Cyclosporin A; Kidney 1. Introduction Mitochondrial Ca 2C overload results in non-specific permeabilization of the inner membrane, allowing the efflux of molecules from the matrix. The transmembrane pore involved in this leakage is not yet well characterized. However, a growing body of evidence indicates that the adenine nucleotide translocase (ANT) is the entity that switches itself into a high conductance pore (Tikhonova et al., 1994; Ru¨ck et al., 1998; Woodfield et al., 1998; Brustovetsky et al., 2002). The facts that ADP, and to a lesser degree ATP, inhibits pore opening (Haworth and Hunter, 1980), and that carboxyatractyloside opens the pore (Le Quoc and Le Quoc, 1988), constitute part of the evidence implicating this carrier. The transition of mitochondrial permeability from specific to non-specific is involved in pathological and natural processes (heart reperfusion damage and apoptosis, respectively) (Arteaga et al., 1992; Halestrap, 1999; Petronilli et al., 2001). An important characteristic of the permeability transition is its inhibition by cyclosporin A (CSA). This immunosuppressant might close the specific pore by binding to the enzyme cyclophilin, a cis-trans isomerase that interacts with ANT, thus opening the non-specific pore (Tanveer et al., 1996). Ca 2C is required to induce pore opening, but the site to which this cation binds has not been clearly identified. * Corresponding author. Departamento de Bioquı´mica, Instituto Nacional de Cardiologı´a, Ignacio Cha´vez, Juan Badiano No. 1 Col. Seccio´n XVI, Me´xico, D.F. 014080, Me´xico. Fax: C52 55 5573 0926. E-mail address: echavez@mail.ssa.gob.mx (E. Cha´vez). 1065-6995/$ - see front matter Ó 2005 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2005.03.009 www.elsevier.com/locate/cellbi Cell Biology International 29 (2005) 551e558