Inhibition of Ca2(+)-induced large-amplitude swelling of liver and heart mitochondria by cyclosporin is probably caused by the inhibitor binding to mitochondrial-matrix peptidyl-prolyl cis-trans isomerase and preventing it interacting with the adenine nucleotide translocase.
1. Isolated rat liver and heart mitochondria incubated in 150 mM-KSCN or sucrose medium in the presence of respiratory-chain inhibitors showed a large increase in swelling when exposed to 250 microM-Ca2+. Swelling was inhibited by bongkrekic acid and cyclosporin A in both media and by ADP in KSCN medium; the effect of ADP was reversed by carboxyatractyloside. These results demonstrate that this is a suitable technique with which to study the opening of the Ca2(+)-induced non-specific pore of the mitochondrial inner membrane and implicate the adenine nucleotide carrier in this process. 2. Titration of the rate of swelling with increasing concentrations of cyclosporin showed the number of cyclosporin-binding sites (+/- S.E.M.) in liver and heart mitochondria to be respectively 113.7 +/- 5.0 (n = 9) and 124.3 +/- 11.2 (n = 10) pmol/mg of protein, with a Ki of about 5 nM. 3. Liver and heart mitochondrial-matrix fractions were prepared free of membrane and cytosolic contamination and shown to contain cyclosporin-sensitive peptidyl-prolyl cis-trans isomerase (cyclophilin) activity. Titration of isomerase activity with cyclosporin gave values (+/- S.E.M.) of 110.6 +/- 10.1 (n = 5) and 165.4 +/- 15.0 (n = 3) pmol of enzyme/mg of liver and heart mitochondrial protein respectively, with a Ki of 2.5 nM. The similarity of these results to those from the swelling experiments suggest that the isomerase may be involved in the Ca2(+)-induced swelling. 4. The rapid light-scattering change induced in energized heart mitochondria exposed to submicromolar Ca2+ [Halestrap (1987) Biochem. J. 244, 159-164] was inhibited by ADP and bongkrekic acid, the former effect being reversed by carboxyatractyloside. These results suggest an interaction of Ca2+ with the adenine nucleotide carrier when the 'c' conformation. 5. A model is proposed in which mitochondrial peptidyl-prolyl cis-trans isomerase interacts with the adenine nucleotide carrier in the presence of Ca2+ to cause non-specific pore opening. The model also explains the involvement of the adenine nucleotide translocase in the PPi-mediated cyclosporin-insensitive increase in K+ permeability described in the preceding paper [Davidson & Halestrap (1990) Biochem. J. 268, 147-152]. 6. The physiological and pathological implications of the model are discussed in relation to reperfusion injury and cyclosporin toxicity.
Halestrap
A P
AP
Department of Biochemistry, School of Medical Sciences, University of Bristol, U.K.
Davidson
A M
AM
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000591:Amino Acid Isomerases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D001865:Bongkrekic Acid / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology*; D002463:Cell Membrane Permeability; D003524:Cyclosporins / Q000378:metabolism / Q000494:pharmacology*; D007700:Kinetics; D008027:Light; D008929:Mitochondria, Heart / Q000187:drug effects / Q000502:physiology*; D008930:Mitochondria, Liver / Q000187:drug effects / Q000502:physiology*; D000226:Mitochondrial ADP, ATP Translocases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008933:Mitochondrial Swelling / Q000187:drug effects*; D009713:Nucleotidyltransferases / Q000378:metabolism*; D019696:Peptidylprolyl Isomerase; D051381:Rats; D012542:Scattering, Radiation; D013395:Sucrose; D013861:Thiocyanates
D003524:Cyclosporins; D013861:Thiocyanates; D001865:Bongkrekic Acid; D013395:Sucrose; D000244:Adenosine Diphosphate; D000226:Mitochondrial ADP, ATP Translocases; D009713:Nucleotidyltransferases; D000591:Amino Acid Isomerases; D019696:Peptidylprolyl Isomerase; D002118:Calcium; C009941:potassium thiocyanate