title: ATP, a partial agonist for the P2Z receptor of human lymphocytes.
abstract: 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 ATPgammaS, 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 microM (n=5) and 85.6+/-8.8 microM (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. 3. A rank order of agonist potency of BzATP>ATP=2MeSATP>ATPgammaS was observed for agonist-stimulated ethidium+ influx, while maximal influxes followed a rank order of BzATP>ATP>2MeSATP>ATPgammaS. 4. Preincubation with 30-50 microM 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 microM ox-ATP, respectively (n=2)). 5. ATP (300-1000 microM) added simultaneously with 30 microM 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 microM) 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 microM; ATP, 500 microM) 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.
authors.affiliation: Department of Haematology, Austin Campus, Austin and Repatriation Medical Centre, Heidelberg, Vic, Australia.
authors.forename: C E
authors.initials: CE
authors.lastname: Gargett
authors.forename: J E
authors.initials: JE
authors.lastname: Cornish
authors.forename: J S
authors.initials: JS
authors.lastname: Wiley
mesh_terms: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology*; D000345:Affinity Labels; D001464:Barium / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004996:Ethidium / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D066298:In Vitro Techniques; D007473:Ion Channels / Q000187:drug effects; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000097:blood; D016014:Linear Models; D008214:Lymphocytes / Q000187:drug effects* / Q000378:metabolism; D058910:Purinergic P2 Receptor Agonists; D018048:Receptors, Purinergic P2 / Q000097:blood; D058486:Receptors, Purinergic P2X7; D013329:Structure-Activity Relationship; D013873:Thionucleotides / Q000494:pharmacology
chemical_list: D000345:Affinity Labels; D004791:Enzyme Inhibitors; D007473:Ion Channels; C546468:P2RX7 protein, human; D058910:Purinergic P2 Receptor Agonists; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; D013873:Thionucleotides; D001464:Barium; C022571:adenosine 5'-O-(3-thiotriphosphate); C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; C063302:KN 62; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000255:Adenosine Triphosphate; D004996:Ethidium; C008501:2-methylthio-ATP