CYCLOSPORINE-A RESCUES THYMOCYTES FROM APOPTOSIS INDUCED BY VERY-LOW CONCENTRATIONS OF THAPSIGARGIN - EFFECTS ON MITOCHONDRIAL-FUNCTION

Raising intracellular calcium levels can induce apoptosis or programme d cell death in many cells, While early rises in intracellular calcium are not universally associated with apoptotic cell death, calcium cle arly plays a key role in many of the biochemical events which occur du ring apoptosis, In this paper we have determined intracellular calcium rises induced by 2, 10, and 100 nM thapsigargin in mouse thymocytes, These concentrations cause increases in cytosolic calcium of 100-250, 400-600, and >1000 nM, respectively, These rises are sustained for at least 85 min and the ratio between the maximum rise caused by 10 nM co mpared to 2 nM thapsigargin is 2.1 +/- 0.4 (n = 6), Both 2 and 10 nM t hapsigargin cause apoptosis at 24 h as shown by DNA fragmentation and morphology when examined by electron microscopy, Cyclosporin A (CsA) i nhibits apoptosis caused by 2 nM thapsigargin but not that caused by 1 0 nM thapsigargin, Electron microscopy of thymocytes treated with 2 nM thapsigargin at 24 h shows intact mitochondria although with altered morphology, There is no loss of ATP or decrease in the ATP/ADP ratio i n these cells over 12 h, Mitochondria in cells treated with 10 nM thap sigargin, however, are swollen by 6 h and many are lost by 24 h, These cells show greatly diminished ATP content by 12 h and a decrease in A TP/ADP ratio, Examination of the effects of PMA, an activator of the p lasma membrane calcium ATPase pump, on cells treated with 10 nM thapsi gargin suggests that two pools of calcium may be responsible for the d ifferential effects of the two calcium levels in the cells, Probing of the mitochondrial membrane potential (MMP) by rhodamine 123 staining of live cells shows that the collapse of the MMP caused by 10 nM thaps igargin is unaffected by CsA, The MMP is also reduced in cells treated with 2 nM thapsigargin but this is restored by CsA, Cells are also re scued from apoptosis caused by 2 nM thapsigargin by incubation with FK 506, This immunosuppressive agent has no effect on the membrane permea bility transition induced in isolated mitochondria, These results sugg est that very low rises in intracellular calcium in thymocytes cause a ctivation-induced cell death inhibited by CsA and FK506 and are withou t effect on ATP levels and therefore do not involve irreversible mitoc hondrial damage, Exceeding these calcium levels by only two-fold resul ts in apoptosis accompanied by reduced ATP levels and mitochondrial da mage, although apoptotic cell death in this instance is unaffected by the classic inhibitor of mitochondrial permeability transition, CsA. ( C) 1996 Academic Press, Inc.