ATP, a partial agonist for the P2Z receptor of human lymphocytes

1 Although extracellular adenosine 5'-triphosphate (ATP) is the natural ligand for the P2Z receptor of human lymphocytes it is less potent than 3'-O-(4-benzoylbenzoyl)-ATP (BzATP) in opening the associated ion channel, which conducts a range of permeants including Ba2+ and ethidium(+). We have quantified the influx of ethidium(+) into lymphocytes produced by BzATP, ATP, 2-methylthio-ATP (2MeSATP) and ATP gamma S, studied competition between ATP and BzATP and investigated the effects of KN-62, a new and potent inhibitor of the P2Z receptor. 2 BzATP and ATP stimulated ethidium(+) influx with EC50 values of 15.4 +/- 1.4 mu M (n=5) and 85.6 +/- 8.8 mu M (n=5), respectively. The maximal response to ATP was only 69.8 +/- 1.9% of that for BzATP. Hill analysis gave n(H) of 3.17 +/- 0.24 (n=3) and 2.09 +/- 0.45 (n=4) for BzATP and ATP, suggesting greater positive cooperativity for BzATP than for ATP in opening the P2Z receptor-operated ion channel. 3 A rank order of agonist potency of BzATP>ATP = 2MeSATP>ATP gamma S was observed for agonist-stimulated ethidium(+) influx, while maximal influxes followed a rank order of BzATP>ATP>2MeSATP>ATP gamma S. 4 Preincubation with 30-50 mu M oxidized ATP (ox-ATP), an irreversible P2Z inhibitor, reduced the maximal response but did not change the steepness of the Ba2+ influx-response curve produced by BzATP (n(H) 3.2 and 2.9 for 30 and 50 mu M ox-ATP, respectively (n=2)). 5 ATP (300-1000 mu M) added simultaneously with 30 mu M BzATP (EC90) inhibited both ethidium(+) and Ba2+ fluxes to a maximum of 30-40% relative to the values observed with BzATP alone. Moreover, ATP (300 mu M) shifted the concentration-response curve to the right for BzATP-stimulated Ba2+ influx, confirming competition between ATP and BzATP. 6. KN-62, a new and powerful inhibitor of the lymphocyte P2Z receptor, showed less potency in antagonizing BzATP-mediated fluxes than ATP-induced fluxes when maximal concentrations of both agonists (BzATP, 50 mu M; ATP, 500 mu M) were used. 7 These data suggest that the natural ligand, ATP, is a partial agonist for the P2Z receptor while BzATP is a more efficacious agonist. Moreover the competitive studies show that only a single class of P2-receptor (P2Z class) is expressed on human leukaemic lymphocytes.