STEADY-STATE BINDING OF [H-3]ATP TO RAT-LIVER PLASMA-MEMBRANES AND COMPETITION BY VARIOUS PURINERGIC AGONISTS AND ANTAGONISTS

Steady-state analysis of nucleotide-binding sites on rat liver plasma membranes was carried out using H-3-labelled ATP as radioligand under complete inhibition of ecto-ATPase activity by excess EDTA. Binding of [H-3]ATP to the membranes is saturable, reversible and apparently inv olves one population of specific binding sites with K-d of about 90 nM and binding capacity (B-max) of 15 pmol/mg protein. A broad spectrum of purinergic agonists and antagonists was examined as potential inhib itors of the measured binding. The displacement studies showed the fol lowing rank order of inhibitory potency for [H-3]ATP-binding sites (pI C(50) values in parentheses:): ATP gamma S (7.49) > 2-MeSATP (7.18) > ATP (6.91) >ADP beta S (6.64) greater than or equal to ADP (6.56) much greater than RB2 (6.14) much greater than suramin (5.40) much greater than Ap(4)A (4.57) = alpha,beta-MeATP (4.19) greater than or equal to beta,gamma-MeATP (3.97). AMP, adenosine, Ap(5)-A, PPADS, beta-glycero phosphate as well as non-adenine nucleoside triphosphates GTP, UTP and CTP did not exert any effect on the measured binding at concentration ranges of 10(-6)-10(-4) M. In order to ascertain whether ATP and its analogues are capable of interacting with the same binding domain, 2-M eSATP and ADP were treated as alternative ligands that could compete w ith unlabelled ATP for its binding sites. A 2-fold increase of K-d val ue for ATP-receptor interaction was observed in the presence of 2-MeSA TP (60 nM) or ADP (250 nM) without any modulation of B-max value, conf irming that inhibitory effects of these compounds are competitive in n ature. These studies demonstrate that ATP and its analogues are able t o interact with a single binding domain on liver plasma membranes, whi ch may be identified as ligand-binding component of P2 purinoceptors o f the P2Y(1) subtype. (C) 1998 Elsevier Science B.V. All rights reserv ed.