ESTIMATES OF ANTAGONIST AFFINITIES AT P-2X PURINOCEPTORS IN RAT VAS-DEFERENS

In functional studies pyridoxalphosphate-6-azophenyl-2',5'-disulphonic acid (iso-PPADS), suramin, GR200282 (4,4'-[carbonyl- bis(imino-3-benzoylimino)]-bis[5-hydroxy-naphthalene-2,7-disulfonic acid] tetrapotassium salt), cibacron blue, trypan blue and congo red, each produced specific antagonism of the contractile responses of isolated rat vas deferens, induced by alpha,beta-methylene ATP (alpha,beta-meATP), with antagonist pK(B) estimates of 6.6 +/- 0.3, 5.5 +/- 0.2, 5.1 +/- 0.3, 5.8 +/- 0.2, 4.7 +/- 0.2 and 4.6 +/- 0.2, respectively. In radioligand binding studies, iso-PPADS, suramin, cibacron blue, GR200282, trypan blue and congo red competed for the high affinity [H-3]alpha,beta-meATP binding sites in rat vas deferens membranes with pK(i) estimates of 5.6 +/- 0.04, 5.5 +/- 0.08, 5.6 +/- 0.15, 5.6 +/- 0.04, 4.3 +/- 0.06 and 4.9 +/- 0.10, respectively. Comparison of pK(B) and pK(i) estimates revealed a good agreement between the two approaches for estimating measures of affinity for the putative antagonists, except in the case of iso-PPADS. However, we found that two populations of [H-3]alpha,beta-meATP binding sites can be identified by iso-PPADS, 26.4% of these having low affinity (pK(i) of 4.4 +/- 0.2), and 73.6% having high affinity (pK(i) of 6.5 +/- 0.02) for iso-PPADS. The pK(i) of 6.5 obtained at the high affinity sites identified by iso-PPADS was close to the equivalent pK(B) value of 6.6 from functional studies. These studies therefore show a good agreement between pK(B) and pK(i) estimates for several antagonists, and suggest that the high affinity binding sites labelled with [H-3]alpha,beta-meATP in rat vas deferens represents binding to functional P-2X purinoceptors.