title: Distribution and characterisation of [3H]alpha,beta-methylene ATP binding sites in the rat.
abstract: Radioligand binding studies have been performed to study the distribution of the binding sites for the P2x purinoceptor selective agonist radioligand, [3H]alpha,beta-methylene ATP ([3H]alpha beta-meATP), in membranes prepared from various peripheral organs and several brain regions of the rat. In agreement with previous studies in the rat vas deferens, [3H]alpha beta-meATP labelled two populations of sites. One site exhibited high affinity for the ligand (Kd = 0.7 nM; Bmax = 1012 fmol.mg-1 protein) while the other site exhibited lower affinity (Kd = 70.8 nM) and higher capacity (Bmax) = 7470 fmol.mg-1 protein). In competition studies, using a low concentration of radioligand (1 nM), the high affinity alpha beta-meATP binding sites in vas deferens membranes could be preferentially labelled (84-91%). Under these conditions, the P2x purinoceptor agonists, alpha beta-meATP and beta, gamma-methylene ATP, had the highest affinity with pIC50 values of 8.3 and 7.3 respectively. The P2y purinoceptor agonist, 2-methyl-thio-ATP (2-me-S-ATP), had lower affinity (pIC50 = 6.7), while uridine triphosphate, adenosine diphosphate and adenosine, agonists at the P2u, P2t and P1 purinoceptors, respectively, possessed low affinity (pIC50 values < 5.6). In addition, the P2 purinoceptor antagonists, cibacron blue and suramin, inhibited binding over the same concentration range at which they behave as functional antagonists at the P2x purinoceptor. High and low affinity binding sites for [3H]alpha beta-meATP were also identified in a range of other peripheral tissues (spleen, heart and liver) and in several brain regions (striatum, cerebral cortex, hippocampus). In the spleen, heart, cerebral cortex and liver the Kd values at both the high affinity binding sites (Kd = 1-1.2 nM) and the low affinity binding sites (Kd = 98-158 nM) were similar to the respective Kd values at the high and low affinity binding sites in the vas deferens. In competition studies performed using a low concentration of radioligand (1 nM) these sites exhibited a similar pharmacological profile to that seen in the vas deferens. Detailed analysis of competition curves to several of the ATP analogues in each of the tissues revealed that the binding profile of the radioligand was complex since several compounds, and in particular ATP and 2-me-S-ATP, identified a lower proportion of sites with high affinity than did alpha beta-meATP.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Glaxo Institute of Applied Pharmacology, Department of Pharmacology, University of Cambridge, UK.
authors.forename: A D
authors.initials: AD
authors.lastname: Michel
authors.forename: P P
authors.initials: PP
authors.lastname: Humphrey
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D066298:In Vitro Techniques; D008297:Male; D008670:Metals / Q000494:pharmacology; D009130:Muscle, Smooth / Q000187:drug effects / Q000378:metabolism; D058919:Purinergic P2 Receptor Antagonists; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D018048:Receptors, Purinergic P2 / Q000187:drug effects / Q000378:metabolism*; D013696:Temperature; D014649:Vas Deferens / Q000187:drug effects / Q000378:metabolism
chemical_list: D008670:Metals; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; D000255:Adenosine Triphosphate; C002630:alpha,beta-methyleneadenosine 5'-triphosphate