Early mitochondrial hyperpolarization and intracellular alkalinization in lactacystin-induced apoptosis of retinal pigment epithelial cells

We investigated the induction and underlying mechanism of apoptosis in retinal pigment epithelial cells by the inhibition of proteasome activity using lactacystin. Rat retinal pigment epithelial cell line retinal pigment epithelial (RPE)-J was used in this study. Apoptosis was evaluated by light and electron microscopies, DNA electrophoresis, and terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. The apoptosis-related proteins were localized in the cells by immunofluorescent microscopy, and the changes of their protein contents and the enzyme activation were monitored by Western blot. Mitochondrial membrane potential was quantified by measuring J aggregate (5,5', 6,6'-tetrachloro-1,1', 3,3'-tetraethylbenzimidazol carbocyanine iodide) fluorescence. To measure changes in intracellular pH, cells were loaded with 2', 7'-bis(carboxyethyl)5( 6')-carboxyfluorescein and assayed by flow cytometry. To elucidate the type of transport system involving intracellular pH regulation, several transporter inhibitors were used, and their effect on pH and membrane potential was assayed as described above. Lactacystin treatment significantly induced apoptosis in RPE-J cells. During the RPE cell apoptosis, 1) cytochrome c and Smac/DIABLO were released into cytosol from mitochondria, 2) translocation of apoptosis-inducing factor to the nucleus was evident, 3) Bax protein seemed to translocate to mitochondria, 4) procaspase-3 and poly(ADP-ribose) polymerase were cleaved, and 5) nuclear condensation and DNA fragmentation were clearly observed. Noticeably, a transient increase of mitochondrial membrane potential was coincidentally detected with the intracellular alkalinization after lactacystin administration. Furthermore, the lactacystin-induced early alkalinization was inhibited by 4-acetamido-4'-isothiocyanostilbene2,2 '-disulfonate, an inhibitor of Cl-/HCO3- anion exchanger, which also prevented early mitochondrial hyperpolarization and apoptosis. Lactacystin-induced apoptosis in RPE-J cells is closely associated with an early mitochondrial hyperpolarization induced by intracellular alkalinization.