UCL Discovery

Abstract

We have generated and characterised a clone of chicken DT40 lymphocytes stably transfected with the rat P2X(7) receptor (rP2X(7))Successful transfection was confirmed by Western blotting. Under voltage clamp, P2X(7)-expressing cells responded to ATP and dibenzoyl-ATP (Bz-ATP) (a more potent P2X(7) receptor agonist) with a rapidly activating and sustained inward current. The EC50 values for these agonists were 305 and 15 muM, respectively. Bz-ATP evoked Ca2+ and Mn2+ influx into transfected cells as determined by Fura-2 spectrofluorimetry. Responses to Bz-ATP were inhibited by pre-treatment of cells with oxidised ATP. Treatment of cells with Bz-ATP for up to 24 hr produced time- and concentration-dependent cell death. This was associated with an increase in caspase-3-like activity, exposure of phosphatidylserine on the outside of cell membrane and DNA cleavage, indicating death by apoptosis. Pre-treatment with Z-VAD-fmk, a pan-caspase inhibitor, reduced the DNA fragmentation and phosphatidylserine externalisation, but did not affect overall rates of cell death at 24 hr, implicating caspase-independent mechanisms. The properties of rP2X(7) receptors expressed in DT40 cells are similar to those described for other expression systems. Because DT40 cells lack functionally detectable endogenous P2 receptors and are highly amenable to genetic manipulation, stably transfected DT40 cells provide a novel and potentially useful model system in which to investigate the intracellular signal transduction pathways associated with P2X(7) receptor stimulation, in particular those involved in induction of cell death. (C) 2003 Elsevier Science Inc. All rights reserved.