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 Ba ²⁺ 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. 2. BzATP and ATP stimulated ethidium ⁺ influx with EC ₅₀ 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 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 > ATPγS was observed for agonist-stimulated ethidium ⁺ influx, while maximal influxes followed a rank order of BzATP > ATP > 2MeSATP > ATPγS. 4. 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 Ba ²⁺ influx-response curve produced by BzATP (n(H) 3.2 and 2.9 for 30 and 50 μM ox-ATP, respectively (n = 2)). 5. ATP (300-1000 μM) added simultaneously with 30 μM BzATP (EC ₉₀) inhibited both ethidium ⁺ and Ba ²⁺ 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 Ba ²⁺ 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 μM; ATP, 500 μM) 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.