RESUMO
First, we present a summary of the evidence for our model of the molecular mechanism of the permeability transition (MPT). Our proposal is that the MPT occurs as a result of the binding of mitochondrial cyclophilin (CyP-D) to the adenine nucleotide translocase (ANT) in the inner mitochondrial membrane. This binding is enhanced by thiol modification of the ANT caused by oxidative stress or other thiol reagents. CyP-D binding enhances the ability of the ANT to undergo a conformational change triggered by Ca2+. Binding of ADP or ATP to a matrix site of the ANT antagonises this effect of Ca2+; modification of other ANT thiol groups inhibits ADP binding and sensitises the MPT to [Ca2+]. Increased membrane potential changes the ANT conformation to enhance ATP binding and hence inhibit the MPT. Our most recent data shows that a fusion protein of CyP-D and glutathione-S-transferase immobilised to Sepharose specifically binds the ANT from Triton-solubilised inner mitochondrial membranes in a cyclosporin A (CsA) sensitive manner. Second we summarise the evidence for the MPT being a major factor in the transition from reversible to irreversible injury during reperfusion of a heart following a period of ischaemia. We describe how in the perfused heart [3H]deoxyglucose entrapment within mitochondria can be used to measure the opening of MPT pore in situ. During ischaemia pore opening does not occur, but significant opening does occur during reperfusion, and recovery of the heart is dependent on subsequent pore closure. Pore opening is inhibited by the presence in the perfusion medium of pyruvate and the anaesthetic propofol which both protect the heart from reperfusion injury. Third we discuss how the MPT may be involved in determining whether cell death occurs by necrosis (extensive pore opening and ATP depletion) or apoptosis (transient pore opening with maintenance of ATP).
Assuntos
Canais de Cálcio/metabolismo , Mitocôndrias Cardíacas/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Apoptose , Cálcio/antagonistas & inibidores , Cálcio/metabolismo , Morte Celular/fisiologia , Desoxiglucose/metabolismo , Sequestradores de Radicais Livres/farmacologia , Coração/fisiopatologia , Humanos , Mitocôndrias Cardíacas/ultraestrutura , Translocases Mitocondriais de ADP e ATP/metabolismo , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Necrose , Peptidilprolil Isomerase/metabolismo , Perfusão , Permeabilidade , Propofol/farmacologiaRESUMO
Human cyclophilin-3 (hCyp-3) cDNA was used to screen a rat skeletal muscle cDNA library which led to the isolation of a full-length cDNA encoding the rat homologue. The derived amino acid sequence showed 89.5% identity with the human sequence, with major differences being restricted to the mitochondrial targeting sequence. The N-terminal sequence of the purified rat liver mitochondrial cyclophilin (CyP-D) corresponded to that derived from the cDNA following 30 amino acids of targeting sequence. This confirms that hCyp-3 encodes mitochondrial matrix CyP (CyP-D), which plays a crucial role in the mitochondrial permeability transition. CyP-D mRNA of a single sized (1.5 kb) was shown by Northern blotting to be present in liver, heart, skeletal muscle and brain.
Assuntos
Isomerases de Aminoácido/genética , Proteínas de Transporte/genética , Ciclofilinas , Mitocôndrias/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Compartimento Celular , Clonagem Molecular , Peptidil-Prolil Isomerase F , DNA Complementar/genética , Humanos , Mitocôndrias/enzimologia , Mitocôndrias Hepáticas/enzimologia , Mitocôndrias Hepáticas/genética , Mitocôndrias Musculares/enzimologia , Mitocôndrias Musculares/genética , Dados de Sequência Molecular , Músculo Esquelético , Precursores de Proteínas/genética , Sinais Direcionadores de Proteínas/genética , RNA Mensageiro/genética , Ratos , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
Stimulation of the mitochondrial permeability transition (MPT) in de-energized mitochondria by phenylarsine oxide (PheArs) is greater than that by diamide and t-butylhydroperoxide (TBH), yet the increase in CyP binding to the inner mitochondrial membrane (Connern, C. P. and Halestrap, A. P. (1994) Biochem. J. 302, 321-324) is less. From a range of nucleotides tested only ADP, deoxy-ADP, and ATP inhibited the MPT. ADP inhibition involved two sites with Ki values of about 1 and 25 microM which were independent of [Ca2+] and CyP binding. Carboxyatractyloside (CAT) abolished the high affinity site. Following pretreatment of mitochondria with TBH or diamide, the Ki for ADP increased to 50-100 microM, whereas pretreatment with PheArs or eosin maleimide increased the Ki to >500 microM; only one inhibitory site was observed in both cases. Eosin maleimide is known to attack Cys159 of the adenine nucleotide translocase (ANT) in a CAT-sensitive manner (Majima, E., Shinohara, Y., Yamaguchi, N., Hong, Y. M., and Terada, H. (1994) Biochemistry 33, 9530-9536), and here we demonstrate CAT-sensitive binding of the ANT to a PheArs affinity column. In adenine nucleotide-depleted mitochondria, no stimulation of the MPT by uncoupler was observed in the presence or absence of thiol reagents, suggesting that membrane potential may inhibit the MPT by increasing adenine nucleotide binding through an effect on the ANT conformation. We conclude that CsA and ADP inhibit pore opening in distinct ways, CsA by displacing bound CyP and ADP by binding to the ANT. Both mechanisms act to decrease the Ca2+ sensitivity of the pore. Thiol reagents and oxidative stress may modify two thiol groups on the ANT and thus stimulate pore opening by both means.
