Activation of the P2Z/P2X₇ receptor in human lymphocytes produces a delayed permeability lesion: Involvement of phospholipase D

Leukemic lymphocytes possess a cytolytic P2Z/P2X₇ receptor which, when activated by extracellular ATP, opens a Ca²⁺ - and Ba²⁺ - permeable ion channel. This ATP-stimulated influx of divalent cations has been shown to activate an intracellular phospholipase D (PLD) which hydrolyzes membrane phosphatidylcholine. Lymphocytes that were exposed to ATP for 20 min at 37°C, washed, and then incubated without ATP for 2 h showed an increased uptake of propidium²⁺, a dye widely used to measure cytotoxicity. The potent P2Z/P2X₇ receptor inhibitor, KN-62, which is known to prevent the channel opening when added with ATP, did not block development of the permeability lesion when added 15 min before dye addition. The activity of lymphocyte PLD was stimulated fourfold by ATP and a proportion of this increased activity persisted for several hours after removal of ATP. Loading lymphocytes with intracellular choline⁺ by prior incubation of cells with ATP in isotonic choline chloride abolished both ATP-stimulated PLD activity and the ATP-induced permeability lesion. Addition of PLD but not phospholipase C to the extracellular medium increased lymphocyte permeability to propidium²⁺ and this effect was not observed in a choline medium. The cytolytic effect of exogenous PLD together with the inhibitory effect of choline, a product of the PLD reaction, suggests that sustained activation of intracellular PLD may be involved in the ATP-initiated cytolytic pathway.