adenosine-5--o-(3-thiotriphosphate) and Leukemia--Lymphocytic--Chronic--B-Cell

Extracellular ATP is known to trigger apoptosis of thymocytes and lymphocytes through a P2Z receptor at which ATP is a partial agonist, giving only 70% of the maximum response of 3'-O-(4-benzoyl)benzoyl-adenosine 5'-triphosphate (BzATP), a full agonist. This cytolytic receptor and its associated ion channel are Ca2+ (and Ba2+) selective but also pass molecules up to the size of ethidium cation (314 Da). RT-PCR showed identity between lymphocyte P2Z and the hP2X7 gene recently cloned from human monocytes. When human leukemic B lymphocytes were incubated with ATP and 133Ba2+, an immediate influx of isotope occurred. It was augmented by 45% when ATP was added 10 min before isotope. Time-resolved flow cytometry was used to examine kinetics of ethidium uptake in cells incubated with BzATP or the partial agonists ATP, 2-methylthioadenosine 5'-triphosphate, or adenosine 5'-O-(3-thiotriphosphate). Maximally effective concentrations of BzATP (50 microM) induced immediate uptake of ethidium at a rate linear with time. In contrast, a delay was observed (30 s) before ethidium uptake commenced after addition of maximally effective ATP concentrations (500 microM) at 37 degreesC, and the delay was longer at 24 degreesC. ATP addition 2-10 min before ethidium abolished the delay. The delay was longer with other partial agonists and inversely related to maximal flux produced by agonist. A delay was also observed for submaximal BzATP concentrations (10-20 microM). P2Z/P2X7 inhibitors, KN-62 and 5-(N, N-hexamethylene)-amiloride, reduced the rate of agonist-induced ethidium uptake and lengthened the delay. The results support a model in which agonists for P2Z/P2X7 receptor mediate an immediate channel opening allowing passage of small inorganic cations, followed by a slow further permeability increase allowing passage of larger permeant cations like ethidium. The rate of the second step depends on time and temperature and the efficacy and concentration of agonist and is slowed by antagonists, suggesting it depends on the fraction of P2Z/P2X7 channels held in the initial open state.

Topics: Adenosine Triphosphate; B-Lymphocytes; Barium; Calcium; Cell Membrane Permeability; Cells, Cultured; Cloning, Molecular; Humans; Kinetics; Leukemia, Lymphocytic, Chronic, B-Cell; Liver; Macrophages; Monocytes; Purinergic P2 Receptor Agonists; Purinergic P2 Receptor Antagonists; Receptors, Purinergic P2; Receptors, Purinergic P2X7; Reverse Transcriptase Polymerase Chain Reaction; Thionucleotides

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.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adenosine Triphosphate; Affinity Labels; Barium; Dose-Response Relationship, Drug; Enzyme Inhibitors; Ethidium; Flow Cytometry; Humans; In Vitro Techniques; Ion Channels; Leukemia, Lymphocytic, Chronic, B-Cell; Linear Models; Lymphocytes; Purinergic P2 Receptor Agonists; Receptors, Purinergic P2; Receptors, Purinergic P2X7; Structure-Activity Relationship; Thionucleotides