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

Radioligand binding studies have been performed to study the distribution of the binding sites for the P2x purinoceptor selective agonist radioligand, [H-3]alpha,beta-methylene ATP ([H-3]alphabeta-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, [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 membranes could be preferentially labelled (84 - 91 %). Under these conditions, the P2x purinoceptor agonists, alphabeta-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 [H-3]alphabeta-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 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 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 alphabeta-meATP. The simplest interpretation of these data is that there are both high and low affinity sites for [H-3]alphabeta-meATP in all tissues, but that the high affinity sites display heterogeneity with respect to various purinoceptor 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 sites may be a consequence of agonist-induced conformational changes which we speculate may be linked with desensitisation states of the receptor.