Activation of the P2Z/P2X 7 Receptor in Human Lymphocytes Produces a Delayed Permeability Lesion: Involvement of Phospholipase D 1 Kekulu C. Fernando, Caroline E. Gargett and James S. Wiley 2 Sydney University Department of Medicine, The Nepean Hospital, Somerset Street, Penrith, Australia Received June 25, 1998, and in revised form November 13, 1998 Leukemic lymphocytes possess a cytolytic P2Z/P2X 7 receptor which, when activated by extracellular ATP, opens a Ca 2 - and Ba 2 -permeable ion channel. This ATP-stimulated influx of divalent cations has been shown to activate an intracellular phospholipase D (PLD) which hydrolyzes membrane phosphatidylcho- line. 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 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 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 sev- eral 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 phospho- lipase C to the extracellular medium increased lym- phocyte permeability to propidium 2 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, sug- gests that sustained activation of intracellular PLD may be involved in the ATP-initiated cytolytic path- way. © 1999 Academic Press Receptors for extracellular ATP fall into a number of subtypes which include ligand-gated ion channels and G-protein-coupled receptors (1, 2). The ligand-gated ion channels, collectively termed P2X receptors, have been extensively characterized and cloned from vis- ceral smooth muscle and peripheral and central neu- rons where their activation serves in cell– cell commu- nication, usually by depolarizing effector cells (3–5). A separate subtype of ATP receptor, termed the P2Z re- ceptor, has been recognized in cells of hemopoietic or- igin and also functions as a ligand-gated ion channel. Recently, a P2Z receptor was cloned from rat brain and a human monocytic library and identified as a P2X receptor (P2X 7 ), on the basis of sequence homology with the other six members (P2X 1–6 ) of this family of ionotropic ATP receptors (6, 7). Moreover, P2X 7 tran- scripts have been shown by RT-PCR analysis to be present in human peripheral blood lymphocytes, and in this cell type, ATP gives responses which are function- ally similar to hP2X 7 expressed in HEK-293 cells (8 – 10). The P2Z/P2X 7 ion channel show considerable se- lectivity for Ca 2+ over monovalent cations and al- though the fluorescent dye ethidium + is a permeant, the larger propidium 2+ dye (414 Da) does not enter the channel during short-term incubations up to 60 min. Indeed, uptake and staining of cells with propidium 2+ has been widely used as an early measure of cytotox- icity (11, 12). Extracellular ATP has been shown to trigger cell death both in fresh murine thymocytes (13–15) and in cultured murine macrophages (16, 17). Many features of this ATP-mediated cytotoxicity resemble that pro- duced by exposure of these cells to dexamethasone, namely, distinct morphological changes and fragmen- tation of target cell DNA usually associated with apo- ptosis (18). There is considerable evidence that ATP increases cytosolic Ca 2+ in these two cell types, while 1 This work was supported by the National Health and Medical Research Council of Australia and the Cancer Council of NSW. 2 To whom correspondence should be addressed at Sydney Univer- sity Department of Medicine, Nepean Hospital, Penrith, New South Wales 2750, Australia. Fax: (+612)(47)242567. E-mail: wileyj@ medicine.usyd.edu.au. 0003-9861/99 $30.00 197 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved. Archives of Biochemistry and Biophysics Vol. 362, No. 2, February 15, pp. 197–202, 1999 Article ID abbi.1998.1045, available online at http://www.idealibrary.com on