Cyclosporin A Rescues Thymocytes from Apoptosis Induced by Very Low Concentrations of Thapsigargin: Effects on Mitochondrial Function

Raising intracellular calcium levels can induce apoptosis or programmed cell death in many cells. While early rises in intracellular calcium are not universally associated with apoptotic cell death, calcium clearly plays a key role in many of the biochemical events which occur during apoptosis. In this paper we have determined intracellular calcium rises induced by 2, 10, and 100 nMthapsigargin 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 nMcompared to 2 nMthapsigargin is 2.1 ± 0.4 (n= 6). Both 2 and 10 nMthapsigargin cause apoptosis at 24 h as shown by DNA fragmentation and morphology when examined by electron microscopy. Cyclosporin A (CsA) inhibits apoptosis caused by 2 nMthapsigargin but not that caused by 10 nMthapsigargin. Electron microscopy of thymocytes treated with 2 nMthapsigargin at 24 h shows intact mitochondria although with altered morphology. There is no loss of ATP or decrease in the ATP/ADP ratio in these cells over 12 h. Mitochondria in cells treated with 10 nMthapsigargin, 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 ATP/ADP ratio. Examination of the effects of PMA, an activator of the plasma membrane calcium ATPase pump, on cells treated with 10 nMthapsigargin suggests that two pools of calcium may be responsible for the differential 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 nMthapsigargin is unaffected by CsA. The MMP is also reduced in cells treated with 2 nMthapsigargin but this is restored by CsA. Cells are also rescued from apoptosis caused by 2 nMthapsigargin by incubation with FK506. This immunosuppressive agent has no effect on the membrane permeability transition induced in isolated mitochondria. These results suggest that very low rises in intracellular calcium in thymocytes cause activation-induced cell death inhibited by CsA and FK506 and are without effect on ATP levels and therefore do not involve irreversible mitochondrial damage. Exceeding these calcium levels by only twofold results in apoptosis accompanied by reduced ATP levels and mitochondrial damage, although apoptotic cell death in this instance is unaffected by the classic inhibitor of mitochondrial permeability transition, CsA.