Activation of the P2Z/P2X(7), receptor in human lymphocytes produces a delayed permeability lesion: Involvement of phospholipase D-1

Leukemic lymphocytes possess a cytolytic P2Z/P2X(7) receptor which, when ac tivated 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 phosphatid ylcholine. Lymphocytes that were exposed to ATP for 20 min at 37 degrees C, washed, and then incubated without ATP for 2 h showed an increased uptake of propidium(2+), a dye widely used to measure cytotoxicity, The potent P2Z /P2X(7) receptor inhibitor, KN-62, which is known to prevent the channel op ening when added with ATP, did not block development of the permeability le sion 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-ind uced permeability lesion. Addition of PLD but not phospholipase C to the ex tracellular medium increased lymphocyte permeability to propidium(2+) and t his effect was not observed in a choline medium. The cytolytic effect of ex ogenous PLD together with the inhibitory effect of choline, a product of th e PLD reaction, suggests that sustained activation of intracellular PLD may be involved in the ATP-initiated cytolytic pathway. (C) 1999 Academic Pres s.