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

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Abstract
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 ATPγS, studied competition between ATP and BzATP and investigated the effects of KN‐62, a new and potent inhibitor of the P2Z receptor. BzATP and ATP stimulated ethidium+ influx with EC50 values of 15.4±1.4 μM (n=5) and 85.6±8.8 μM (n=5), respectively. The maximal response to ATP was only 69.8±1.9% of that for BzATP. Hill analysis gave nH 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. A rank order of agonist potency of BzATP>ATP=2MeSATP>ATPγS was observed for agonist‐stimulated ethidium+ influx, while maximal influxes followed a rank order of BzATP>ATP>2MeSATP>ATPγS. Preincubation with 30–50 μM 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 (nH 3.2 and 2.9 for 30 and 50 μM ox‐ATP, respectively (n=2)). ATP (300–1000 μM) added simultaneously with 30 μM 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 μM) shifted the concentration‐response curve to the right for BzATP‐stimulated Ba2+ influx, confirming competition between ATP and BzATP. 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 μM; ATP, 500 μM) were used. 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. British Journal of Pharmacology (1997) 122, 911–917; doi:10.1038/sj.bjp.0701447