Chloromethyltetramethylrosamine (Mitotracker Orange) induces the mitochondrial permeability transition and inhibits respiratory complex I. Implications for the mechanism of cytochrome c release.
We have investigated the interactions with isolated mitochondria and intact cells of chloromethyltetramethylrosamine (CMTMRos), a probe (Mitotracker Orange) that is increasingly used to monitor the mitochondrial membrane potential (Deltapsi(m)) in situ. CMTMRos binds to isolated mitochondria and undergoes a large fluorescence quenching. Most of the binding is energy-independent and can be substantially reduced by sulfhydryl reagents. A smaller fraction of the probe is able to redistribute across the inner membrane in response to a membrane potential, with further fluorescence quenching. Within minutes, however, this energy-dependent fluorescence quenching spontaneously reverts to the same level obtained by treating mitochondria with the uncoupler carbonylcyanide-p-trifluoromethoxyphenyl hydrazone. We show that this event depends on inhibition of the mitochondrial respiratory chain at complex I and on induction of the permeability transition pore by CMTMRos, with concomitant depolarization, swelling, and release of cytochrome c. After staining cells with CMTMRos, depolarization of mitochondria in situ with protonophores is accompanied by changes of CMTMRos fluorescence that range between small and undetectable, depending on the probe concentration. A lasting decrease of cellular CMTMRos fluorescence associated with mitochondria only results from treatment with thiol reagents, suggesting that CMTMRos binding to mitochondria in living cells largely occurs at SH groups via the probe chloromethyl moiety irrespective of the magnitude of Deltapsi(m). Induction of the permeability transition precludes the use of CMTMRos as a reliable probe of Deltapsi(m) in situ and demands a reassessment of the conclusion that cytochrome c release can occur without membrane depolarization and/or onset of the permeability transition.
Scorrano
L
L
Consiglio Nazionale delle Ricerche Unit for the Study of Biomembranes and the Departments of Biomedical Sciences, University of Padova, Viale Giuseppe Colombo 3, I-35121 Padova, Italy.
Petronilli
V
V
Colonna
R
R
Di Lisa
F
F
Bernardi
P
P
D000818:Animals; D001152:Arsenicals / Q000494:pharmacology; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002460:Cell Line; D003574:Cytochrome c Group / Q000378:metabolism; D042967:Electron Transport Complex I; D005033:Ethylmaleimide / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology*; D008564:Membrane Potentials / Q000187:drug effects; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000187:drug effects*; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors*; D010539:Permeability / Q000187:drug effects; D051381:Rats; D017208:Rats, Wistar; D012235:Rhodamines / Q000494:pharmacology; D014966:Xanthenes / Q000494:pharmacology
D001152:Arsenicals; D003574:Cytochrome c Group; D005456:Fluorescent Dyes; D012235:Rhodamines; D014966:Xanthenes; C121372:mitotracker orange; C029341:oxophenylarsine; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D009247:NADH, NADPH Oxidoreductases; D042967:Electron Transport Complex I; D005033:Ethylmaleimide