Binding of [35S]adenosine 5'-O-(2-thiodiphosphate) to endothelial cells in culture

We have investigated the binding of [35S]adenosine 5'-O-(2-thiodiphosphate) ([35S]ADP beta S) to intact cultured bovine aortic endothelial cells which have been previously shown to co-express P2y and P2u purinoceptors and to bovine adrenal medulla endothelial cells which solely possess P2u purinoceptors. ADP beta S has been shown to stimulate phospholipase C activity in these cells via the P2y purinoceptor and does not interact with the P2u purinoceptor. We describe a simple equilibrium binding procedure designed for the study of low affinity agonists and compare these results with those obtained by separation of bound and free by filtration. Saturation analysis of equilibrium binding data revealed two sites for ADP beta S binding; one with KD = 3.3 x 10(-8) M, Bmax = 32 pmol/mg protein; and the other with KD = 4.3 x 10(-6) and Bmax = 2155 pmol/mg protein. Use of filtration did not significantly alter the KD of either of these sites, nor the Bmax of the high affinity site, but reduced the Bmax of the low affinity site by more than 95%. The rank order of agonist potency for competing for [35S]ADP beta S binding indicated that most of this was to non-P2y purinoceptor sites as beta,gamma-methylene ATP, a P2x purinoceptor agonist, was more potent than 2-methylthio ATP, a P2y purinoceptor agonist. Binding was also carried out in the presence of beta,gamma-methylene ATP, in an attempt to reduce non-P2y purinoceptor binding and produced similar results. Specific [35S]ADP beta S binding sites were also found in bovine adrenal medulla endothelial cells which do not possess P2y purinoceptors. These results indicate that [35S]ADP beta S was able to bind to endothelial cells from different parts of the vasculature but that the ligand can only be considered suitable for investigation of P2y purinoceptors on mammalian cells when specific conditions are designed to reduce the large amount of non-receptor binding.