Estimates of antagonist affinities at P2X 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 pKB 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 [3H]alpha,beta-meATP binding sites in rat vas deferens membranes with pKi 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 pKB and pKi 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 [3H]alpha,beta-meATP binding sites can be identified by iso-PPADS, 26.4% of these having low affinity (pKi of 4.4 +/- 0.2), and 73.6% having high affinity (pKi of 6.5 +/- 0.02) for iso-PPADS. The pKi of 6.5 obtained at the high affinity sites identified by iso-PPADS was close to the equivalent pKB value of 6.6 from functional studies. These studies therefore show a good agreement between pKB and pKi estimates for several antagonists, and suggest that the high affinity binding sites labelled with [3H]alpha,beta-meATP in rat vas deferens represents binding to functional P2X purinoceptors.
Glaxo Institute of Applied Pharmacology, Department of Pharmacology, University of Cambridge, UK.
B S
BS
Khakh
A
A
Michel
P P
PP
Humphrey
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D003224:Congo Red / Q000378:metabolism / Q000494:pharmacology; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors; D008297:Male; D009119:Muscle Contraction / Q000187:drug effects; D009282:Naphthalenesulfonates / Q000378:metabolism / Q000494:pharmacology; D058914:Purinergic Antagonists; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D011983:Receptors, Purinergic / Q000378:metabolism; D013498:Suramin / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D014227:Triazines / Q000378:metabolism / Q000494:pharmacology; D014343:Trypan Blue / Q000378:metabolism / Q000494:pharmacology; D014649:Vas Deferens / Q000187:drug effects / Q000378:metabolism*
C091798:GR 200282; D009282:Naphthalenesulfonates; D058914:Purinergic Antagonists; D011983:Receptors, Purinergic; D014227:Triazines; C077792:pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid; D003224:Congo Red; C012842:Cibacron Blue F 3GA; D011732:Pyridoxal Phosphate; D013498:Suramin; D000255:Adenosine Triphosphate; D005982:Glutathione Transferase; D014343:Trypan Blue