title: [3H]A-317491, a novel high-affinity non-nucleotide antagonist that specifically labels human P2X2/3 and P2X3 receptors.
abstract: A-317491 is a potent and selective antagonist of P2X3 and P2X(2/3) receptors. In the present studies, the ability of [3H]A-317491 to label recombinant human P2X(2/3) and P2X(3) receptors was characterized. Using membranes prepared from 1321N1 cells expressing P2X(2/3) receptors, [3H]A-317491 specifically labeled high-affinity (Kd = 0.9 nM) recognition sites. High-affinity [3H]A-317491 binding was not detected in membrane preparations from native 1321N1 cells or cells expressing homomeric P2X1, P2X2, or P2X3 receptors. Specific [3H]A-317491 P2X3 receptors could only be reliably detected following treatment of intact P2X3 receptor-expressing cells with apyrase (1 U/ml) both before and during membrane preparation. Under these conditions, [3H]A-317491 also labeled high-affinity (Kd = 9 nM) binding sites. Lower affinity binding components (Kd values of 87-790 nM) were detected in both assays using higher ligand concentrations that likely represent nonfunctional recognition sites. [3H]A-317491 binding to both P2X(2/3) and P2X3 receptors was reversible, and ligand kinetic studies provided similar estimates of the high-affinity binding constants. Potent P2X3 receptor agonists 2-methylthio-ATP, 2,3-O-(4-benzoylbenzoyl)-ATP, and alpha,beta-methylene adenosine triphosphate also potently inhibited specific [3H]A-317491 binding to both P2X(2/3) and P2X3 receptors. The pharmacological profile for P2X receptor antagonists to inhibit [3H]A-317491 binding to P2X(2/3) and P2X3 receptors was highly correlated (r = 0.98, P < 0.05), and a similar rank order of potency was observed for blockade of P2X(2/3) receptor-mediated calcium influx. These data demonstrate that [3H]A-317491 is the first useful radioligand for the specific labeling of P2X3-containing channels.
authors.affiliation: Neuroscience Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, Illinois, USA. michael.jarvis@abbott.com
authors.forename: Michael F
authors.initials: MF
authors.lastname: Jarvis
authors.forename: Bruce
authors.initials: B
authors.lastname: Bianchi
authors.forename: John T
authors.initials: JT
authors.lastname: Uchic
authors.forename: Jayne
authors.initials: J
authors.lastname: Cartmell
authors.forename: Chih-Hung
authors.initials: CH
authors.lastname: Lee
authors.forename: Michael
authors.initials: M
authors.lastname: Williams
authors.forename: Connie
authors.initials: C
authors.lastname: Faltynek
mesh_terms: D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects; D006801:Humans; D010636:Phenols / Q000494:pharmacology*; D011083:Polycyclic Compounds / Q000494:pharmacology*; D058919:Purinergic P2 Receptor Antagonists; D011869:Radioligand Assay; D018048:Receptors, Purinergic P2 / Q000235:genetics; D058476:Receptors, Purinergic P2X2; D058477:Receptors, Purinergic P2X3; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors; D014316:Tritium; D014407:Tumor Cells, Cultured
chemical_list: C470346:A-317491; C546401:P2RX2 protein, human; C546407:P2RX3 protein, human; D010636:Phenols; D011083:Polycyclic Compounds; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; D058476:Receptors, Purinergic P2X2; D058477:Receptors, Purinergic P2X3; D011994:Recombinant Proteins; D014316:Tritium; D002118:Calcium