HIGH-AFFINITY P-2X-PURINOCEPTOR BINDING-SITES FOR [S-35] ADENOSINE 5'-O-[3-THIOTRIPHOSPHATE] IN RAT VAS-DEFERENS MEMBRANES

1 The binding sites labelled by [S-35]-adenosine 5'-O-[3-thiotriphosph ate]([S-35]-ATP gamma S) at 4 degrees C in rat vas deferens membranes were studied and compared to the sites labelled by [H-3]-alpha,beta-me thylene ATP ([H-3]- alpha beta meATP) to ascertain whether [S-35]-ATP gamma S can be used to label the P-2x purinoceptor. 2 In the presence of 4 mM CaCl2, the binding of 0.2 nM [S-35]-ATP gamma S to vas deferen s membranes was increased 3.4 fold, when compared to studies performed in the absence of calcium. However, binding did not appear to be sole ly to P-2X purinoceptors since [S-35]-ATP gamma S labelled a heterogen eous population of sites and about 72% of the sites possessed high aff inity (pIC(50)=7.5) for guanosine 5'-O-[3-thiotriphosphate] (GTP gamma S). Even in the presence of 1 mu M GTP gamma S, to occlude the sites with high affinity for GTP gamma S, the binding of [S-35]-ATP gamma S was heterogeneous and since there was also evidence of extensive metab olism of ATP in the presence of calcium, the binding of [S-35]-ATP gam ma S under these conditions was not studied further. 3 In the absence of calcium ions, [S-35]-ATP gamma S bound to a single population of si tes (pK(D)=9.23; B-max=4270 fmol mg(-1) protein). Binding reached stea dy state within 3 h (t(1/2)=38 min), was stable for a further 4 h and was readily reversible upon addition of 10 mu M unlabelled ATP gamma S (t(1/2)=45 min). In competition studies the binding of 0.2 nM [S-35]- ATP gamma S was inhibited by a number of P-2X purinoceptor agonists an d antagonists, but not by adenosine receptor agonists, staurosporine ( 1 mu M) or several ATPase inhibitors. The rank order of agonist affini ty estimates (pIC(50) values) in competing for the [S-35]ATP gamma S b inding sites was: ATP (9.01), 2-methylthio-ATP (8.79), ATP gamma S (8. 73), alpha beta meATP (7.57), ADP (7.24), beta,gamma-methylene ATP (7. 18), L-beta,gamma-methylene ATP (5.83), alpha,beta-methylene ADP (4.36 ). 4 Affinity estimates (pIC(50) values) for the P-2X purinoceptor ant agonists, suramin (5.20), pyridoxalphosphate-6-azophenyl-2',4'-disulph onic acid (4.23), pyridoxal 5-phosphate (3.42), cibacron blue (5.70) a nd Evan's blue (5.79) were broadly similar to those obtained at the [H -3]-alpha,beta meATP binding sites in vas deferens. However, ATP, 2-me thylthio-ATP, ATP gamma S and ADP displayed 17-512 fold higher affinit y for the [S-35]-ATP gamma S, than for the [H-3]-alpha beta meATP bind ing sites, whereas alpha beta meATP and L-beta,gamma-methylene ATP dis played 5 and 28 fold, respectively, higher affinity for the [H-3]-alph a beta meATP than for the [S-35]-ATP gamma S binding sites. 5 The diff erences in agonist affinity for the [S-35]-ATP gamma S and [H-3]-alpha beta meATP binding sites probably reflect the fact that the former si tes were labelled in the absence of calcium, while the latter sites we re labelled in its presence. This could differentially affect ionisati on state and/or metabolism of the nucleotides when using the two radio ligands. Since affinity estimates for ATP, 2-methylthio-ATP, ATP gamma S, alpha beta meATP and L-(b)eta,gamma-methylene ATP were different w hen calcium ions were omitted in studies using [H-3]-alpha beta meATP but similar to the affinity estimates obtained at the [S-35]-ATP gamma S binding sites labelled in the absence of calcium, it is likely that [S-35]-ATP gamma S and [H-3]-alpha beta meATP label the same sites in rat vas deferens. 6 We conclude that, in the absence of divalent cati ons, [S-35]-ATP gamma S labels P-2X purinoceptors in rat vas deferens and as such may represent a new, high specific activity, radioligand f or the study of such receptors.