Sustained enhancement of Ca2+ influx by glibenclamide induces apoptosis inRINm5F cells

Cytosolic Ca2+ elevations are known to be involved in triggering apoptosis in many tissues, but the effect of sustained enhancement of Ca2+ influx on apoptosis in beta cells remains unknown. We have found that the viability o f RINm5F cells is decreased dose-dependently by continuous exposure to glib enclamide at concentrations from 10(-7) to 10(-4) M, and that this effect i s partially ameliorated by pretreatment with cycloheximide. Electrophoresis of the cells exposed to glibenclamide revealed ladder-like fragmentation c haracteristic of apoptosis, and which also is suppressed by cycloheximide p retreatment. By using terminal. deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining, we detected increased DNA fragmentation in the nuclei of the cells exposed to glibenclamide, and staining with Hoe chst 33342 and propidium iodide showed a dose-dependent increase in the num ber of cells with the chromatin condensation and fragmentation in their nuc lei that is characteristic of apoptosis. The effects of glibenclamide on ce ll viability and apoptotic cell death were partially inhibited by treatment with Ca2+ channel blocker, and by reducing the extracellular Ca2+ concentr ation during glibenclamide exposure, suggesting that they may be derived fr om increased Ca2+ influx. Furthermore, only the percentage of apoptotic cel ls, and not that of necrotic cells, increased with the increasing intracell ular Ca2+ concentration during glibenclamide exposure. In conclusion, we ha ve demonstrated that the sustained enhancement of Ca2+ influx caused by gli benclamide exposure can induce apoptotic cell death in a pure beta cell lin e. (C) 2000 Academic Press.