Role of bivalent cations and choline in ATP-induced apoptosis of human lymphocytes with P2Z receptors

Abstract

The role of bivalent cations and choline in ATP-induced apoptosis via P2Z purinoceptor was investigated in human leukemic lymphocytes. In vitro exposure of leukemic lymphocytes with P2Z receptors to 1 mmol/L ATP or 0.1 mmol/L benzoylbenzoic ATP (BzATP) for 8 h in the presence of choline, 1 mmol/L Mg2+ or other bivalent cations, and ATP-induced DNA breaks, associated with apoptosis were quantified by TdT assay. We observed that (1) Extracellular Mg2+ or Ca2+ stimulated ATP-induced DNA fragmentation in a dose-dependent manner, and the compatible evidence was provided by the inhibition of ATP-induced DNA fragmentation in the present of EGTA or EDTA; (2) ATP-induced DNA fragmentation was completely inhibited by 1 mmol/L Zn2+; (3) ATP-induced DNA breaks were not affected by Ba2+, Sr2+, Co2+ when they were substituted for extracellular Mg2+ or Ca2+; (4) Choline, an inhibitor of phospholipase D (PLD) stimulated by ATP through P2Z receptor in human lymphocytes, was also a partial inhibitor of ATP-induced DNA fragmentation, and the results were confirmed by flow cytometric analysis (FCA); (5) ATP-induced DNA fragmentation was completely obliterated when the temperature was lower than 10 degrees C. These results suggest that the endonuclease and PLD may be involved in ATP-induced apoptosis in human lymphocytes via P2Z receptor.