Sustained enhancement of Ca(2+) influx by glibenclamide induces apoptosis in RINm5F cells.
Cytosolic Ca(2+) elevations are known to be involved in triggering apoptosis in many tissues, but the effect of sustained enhancement of Ca(2+) influx on apoptosis in beta cells remains unknown. We have found that the viability of RINm5F cells is decreased dose-dependently by continuous exposure to glibenclamide at concentrations from 10(-7) to 10(-4) M, and that this effect is partially ameliorated by pretreatment with cycloheximide. Electrophoresis of the cells exposed to glibenclamide revealed ladder-like fragmentation characteristic of apoptosis, and which also is suppressed by cycloheximide pretreatment. 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 Hoechst 33342 and propidium iodide showed a dose-dependent increase in the number of cells with the chromatin condensation and fragmentation in their nuclei that is characteristic of apoptosis. The effects of glibenclamide on cell viability and apoptotic cell death were partially inhibited by treatment with Ca(2+) channel blocker, and by reducing the extracellular Ca(2+) concentration during glibenclamide exposure, suggesting that they may be derived from increased Ca(2+) influx. Furthermore, only the percentage of apoptotic cells, and not that of necrotic cells, increased with the increasing intracellular Ca(2+) concentration during glibenclamide exposure. In conclusion, we have demonstrated that the sustained enhancement of Ca(2+) influx caused by glibenclamide exposure can induce apoptotic cell death in a pure beta cell line.
Iwakura
T
T
Department of Metabolism and Clinical Nutrition, Kyoto University, Kyoto, 606-8507, Japan.
Fujimoto
S
S
Kagimoto
S
S
Inada
A
A
Kubota
A
A
Someya
Y
Y
Ihara
Y
Y
Yamada
Y
Y
Seino
Y
Y
D000818:Animals; D017209:Apoptosis; D002118:Calcium / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D005905:Glyburide / Q000494:pharmacology*; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D020287:In Situ Nick-End Labeling; D007340:Insulinoma / Q000378:metabolism; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009336:Necrosis; D010190:Pancreatic Neoplasms / Q000378:metabolism; D051381:Rats; D014407:Tumor Cells, Cultured
D007004:Hypoglycemic Agents; D005905:Glyburide; D002118:Calcium