DISTRIBUTION AND CHARACTERIZATION OF [H-3] ALPHA,BETA-METHYLENE ATP BINDING-SITES IN THE RAT

Radioligand binding studies have been performed to study the distribut ion of the binding sites for the P2x purinoceptor selective agonist ra dioligand, [H-3]alpha,beta-methylene ATP ([H-3]alphabeta-meATP), in me mbranes prepared from various peripheral organs and several brain regi ons of the rat. In agreement with previous studies in the rat vas defe rens, [H-3]alphabeta-meATP labelled two populations of sites. One site exhibited high affinity for the ligand (K(d) = 0.7 nM; B(max) = 1012 fmol. mg-1 protein) while the other site exhibited lower affinity (K(d ) = 70.8 nM) and higher capacity (B..) = 7470 fmol. mg-1 protein). In competition studies, using a low concentration of radioligand (1 nM), the high affinity alphabeta-meATP binding sites in vas deferens membra nes could be preferentially labelled (84 - 91 %). Under these conditio ns, the P2x purinoceptor agonists, alphabeta-meATP and beta,gamma-meth ylene ATP, had the highest affinity with pIC50 values of 8.3 and 7.3 r espectively. The P2y purinoceptor agonist, 2-methyl-thio-ATP (2-me-S-A TP), had lower affinity (pIC50 = 6.7), while uridine triphosphate, ade nosine diphosphate and adenosine, agonists at the P2u, P2t and P1 puri noceptors, respectively, possessed low affinity (pIC50 values < 5.6). In addition, the P2 purinoceptor antagonists, cibacron blue and surami n, inhibited binding over the same concentration range at which they b ehave as functional antagonists at the P2x purinoceptor. High and low affinity binding sites for [H-3]alphabeta-MeATP were also identified i n a range of other peripheral tissues (spleen, heart and liver) and in several brain regions (striatum, cerebral cortex, hippocampus). In th e spleen, heart, cerebral cortex and liver the K(d) Values at both the high affinity binding sites (K(d) = 1 - 1.2 nM) and the low affinity binding sites (K(d) = 98 - 158 nM) were similar to the respective K(d) values at the high and low affinity binding sites in the vas deferens . In competition studies performed using a low concentration of radiol igand (1 nM) these sites exhibited a similar pharmacological profile t o that seen in the vas deferens. Detailed analysis of competition curv es to several of the ATP analogues in each of the tissues revealed tha t the binding profile of the radioligand was complex since several com pounds, and in particular ATP and 2-me-S-ATP, identified a lower propo rtion of sites with high affinity than did alphabeta-meATP. The simple st interpretation of these data is that there are both high and low af finity sites for [H-3]alphabeta-meATP in all tissues, but that the hig h affinity sites display heterogeneity with respect to various purinoc eptor agonists. We conclude that this study demonstrates the presence of high affinity binding sites for [H-3]alphabeta-meATP in a range of peripheral tissues and in several brain regions. These sites display a pharmacological profile which suggests that they reflect binding to a P2x purinoceptor. The apparent heterogeneity of these high affinity s ites may be a consequence of agonist-induced conformational changes wh ich we speculate may be linked with desensitisation states of the rece ptor.