INTRACELLULAR CA2+ SIGNALS ACTIVATE APOPTOSIS IN THYMOCYTES - STUDIES USING THE CA2+-ATPASE INHIBITOR THAPSIGARGIN

The endoplasmic reticular Ca2+-ATPase inhibitor, thapsigargin, was used to study the role of an increase in cytosolic free calcium concentration ([Ca2+](i)) as a signal for the activation of thymocyte apoptosis. Treatment of rat thymocytes with thapsigargin resulted in an early sustained increase in [Ca2+](i) followed by extensive DNA fragmentation. Agarose gel electrophoresis revealed that the pattern of DNA fragments was typical of endonuclease-mediated internucleosomal cleavage. In addition, confocal,microscopy studies showed the formation of apoptotic nuclei in thapsigargin-treated thymocytes. The concentrations of thapsigargin required to induce DNA fragmentation and [Ca2+](i) increase in thymocytes were identical and so were the kinetics of thapsigargin-induced DNA fragmentation and formation of apoptotic nuclei. The lowest concentration of