391 Interactions of Chloromethyltetramethylrosamine (Mitotracker Orange) with Isolated Mitochondria and Intact Cells LUCA SCORRANO, a VALERIA PETRONILLI, a RAFFAELE COLONNA, a FABIO DI LISA, b AND PAOLO BERNARDI a,c a Consiglio Nazionale delle Ricerche Unit for the Study of Biomembranes, and the Department of Biomedical Sciences and b Department of Biological Chemistry, University of Padova, I-35121 Padova, Italy Activation of the apoptotic program by cytochrome c and apoptosis-inducing factor (AIF) requires release of these intermembrane proteins into the cytosol, and the mechanism(s) by which this occurs is the subject of intense investigation. One of the most studied targets is the mitochondrial permeability transition (PT). The sudden increase of permeability of the inner membrane to solutes has been extensively stud- ied. 1 A specific issue is whether or not a mitochondrial PT is a requisite for the re- lease of apoptogenic proteins. This is an issue that has generated conflicting results and has become a major controversy in the literature. A specific, PT-independent re- lease mechanism has been proposed based on the finding that cytochrome c release occurs without measurable decrease of the mitochondrial membrane potential (ΔΨ m ). 2–4 This point remains very controversial. Others have reported that cyto- chrome c release matches membrane depolarization, 5 and that it is mediated by a PT. 6,7 This has been indicated as the major determinant of AIF release. 8,9 Many of these studies were performed with the “fixable” probe chloromethyltetramethyl ro- samine (CMTMRos) to monitor changes of ΔΨ m in situ. We have carried out a char- acterization of the interactions of this probe with mitochondria. In the experiments of FIGURE 1, rat liver mitochondria were incubated in an iso- tonic KCl medium and energized with glutamate plus malate. Mitochondrial perme- ability to solutes was measured from the changes of absorbance at 620 nm. 10 Following the addition of a small Ca 2+ load unable to induce changes of membrane permeability per se, EGTA was added to prevent Ca 2+ redistribution among mito- chondria with different stability, and increasing concentrations of CMTMRos were added. Increasing fractions of mitochondria became swollen in a concentration-de- pendent fashion (traces a to d). The swelling induced by the highest concentration of CMTMRos (5 μM) was prevented by cyclosporin A (CsA), indicating that the PT was the underlying cause. The dependence of the fraction of mitochondria having PT c Address for correspondence: Prof. Paolo Bernardi, Dipartimento di Scienze Biomediche Sperimentali, Viale Giuseppe Colombo 3, I-35121 Padova, Italy. Phone: + 39-049-827-6063; fax: +39-049-827-6361. e-mail: bernardi@civ.bio.unipd.it