INDUCTION OF APOPTOSIS IN CULTURED HUMAN RETINAL-PIGMENT EPITHELIAL-CELLS IS COUNTERACTED BY FLUPIRTINE

Purpose, The aim of the study was to determine whether flupirtine can counteract the induction of apoptosis in cultured retinal pigment epit helium (RPE) cells. Methods. Confluent cultures were subjected to expe rimental ischemia (medium free of serum, glucose, and oxygen) with or without various substances for specific periods. The cells were then e xamined for breakdown of DNA by the TUNEL procedure and agarose gel el ectrophoresis, Moreover cells were processed for the localization of o ncogene proteins (bcl-2, TIAR, ICH-1(L)) associated with apoptosis. Th e effect of flupirtine on reactive oxygen species also was determined. Results. When RPE cells were subjected to ischemia for 72 hours simil ar to 65% of cells remained attached to the coverslips and similar to 65% of their nuclei showed clear fragmentation of DNA by TUNEL. Most o f the cells exhibited a shrunken appearance typical of apoptosis. Frag mentation of the DNA from cells given ischemia for 72 hours was also c onfirmed by agarose gel electrophoresis. Inclusion of flupirtine (flup irtine gluconate, 100 mu M) or 10% fetal calf serum in the medium prev ented ischemia-induced apoptosis occurring after 72 hours. Neither N-m ethyl-D-aspartate (NMDA) (100 mu M) nor deferoxamine (100 mu M) nor th e NMDA antagonists dextromethorphan (100 mu M), memantine (100 mu M), and MK-801 (10 mu M) had a similar effect. NMDA, and to a lesser exten t memantine, induced apoptosis independently. Treatment of RPE cells i n serum-free medium with flupirtine (flupirtine gluconate, 100 mu M) f or 72 hours caused an upregulation of bcl-2 protein. In contrast, the oncogene proteins for TIAR and ICH-1(L) were lower in flupirtine-treat ed cells than in control cells. Flupirtine, like deferoxamine, prevent s iron-ascorbate-induced reactive oxygen species formation in retinal cells, but only flupirtine prevents ischemia-induced apoptosis in RPE cells. Conclusions. The combined data demonstrate that flupirtine is a n effective agent in preventing death by apoptosis. Flupirtine reduces formation of reactive oxygen species in retinal dissociates and cause s changes in various oncogene products in RPE cultures, which may expl ain its action in preventing apoptosis induced by ischemia. The curren t results also suggest that NMDA receptors are not involved in the ind uction of ischemia-induced apoptosis in RPE cells.