APOPTOSIS IN INSULIN-SECRETING CELLS - EVIDENCE FOR THE ROLE OF INTRACELLULAR CA2-ACID METABOLISM( STORES AND ARACHIDONIC)

This study investigated the role of intracellular free Ca2+ concentrat ion ([Ca2+](i)) in apoptosis in MIN6 cells, an insulin secreting cell line, and in mouse islets. Thapsigargin, an inhibitor of sarcoendoplas mic reticulum Ca2+-ATPases (SERCA), caused a time-and concentration-de pendent decrease in the viability of MINE cells and an increase in DNA fragmentation and nuclear chromatin staining changes characteristic o f apoptosis. Two structurally distinct SERCA inhibitors, cyclopiazonic acid and 2,5-di-[t-butyl]-1,4-hydroquinone also caused apoptosis, but agents that increased [Ca2+](i) by other mechanisms did not induce ap optosis in MIN6 cells. Carbachol- or ionomycin-releasible intracellula r Ca2+ stores were completely depleted in cells treated by SERCA inhib itors, but not by other agents that increase [Ca2+](i). The ability of thapsigargin to induce cell death was not affected by blocking Ca2+ i nflux or by clamping [Ca2+](i) with a cytosolic Ca2+ buffer suggesting that the process did not depend on changes in [Ca2+](i) per se. Howev er, application of the lipoxygenase inhibitors 5,8,11-eicosatrienoic a cid and nordihydroguaiaretic acid partially prevented MIN6 cell apopto sis, while exposure of cells to the product of lipoxygenase, 12-hydrox y-[5,8,10,14]-eicosatetraenoic acid, caused apoptosis. In contrast, in hibition of cyclooxygenase with indomethacin did not abolish thapsigar gin-induced apoptosis in MIN6 cells. Our findings indicate that thapsi gargin causes apoptosis in MIN6 cells by depleting intracellular Ca2stores and leading to release of intermediate metabolites of arachidon ic acid metabolism.