Evidence that most high-affinity ATP binding sites on aortic endothelial cells and membranes do not correspond to P2 receptors

It has recently been demonstrated that two types of ATP receptors, the P2y and P2u receptors, are coexpressed on bovine aortic endothelial cells. The aim of the present study was to characterize directly P2y and P2u subtypes on intact bovine aortic endothelial cells and on membranes prepared from these cells using adenosine 5′-0-(3-thio[35S]triphosphate) ([35S]ATPγS). [α-32P]ATP and [α-32P]UTP as radioligands. [35S]ATPγS binding to bovine aortic endothelial cell membranes was saturable and apparently involved a single class of high-affinity binding sites (Kd: 14 ± 11 nM; Bmax 1.6 ± 0.7 pmol/mg protein; mean ± S.D.). A similar class of high-affinity binding sites was identified with [α-32P]ATP (Kd: 14 ± 9 nM; Bmax: 1.7 ± 1.1 pmol/mg protein; mean ± S.D.). Competition experiments showed that only one third of these sites bound 2-methylthio-ATP (2-MeSATP) with high affinity (Ki: 21 ± 5 and 14 ± 10 nM, mean ± S.D., for [35S]ATPγS and [α-32P]ATP, respectively) and might therefore represent the P2y receptors. UTP did not compete with [35S]ATPγS or [α-32P]ATP for binding at the remaining sites, indicating that they are not the P2u receptors. No high-affinity UTP binding sites could be detected using [α-32P]UTP. [35S]ATPγS binding to intact bovine aortic endothelial cells was competed by ATPγS (Kd: 1.0 ± 0.5 gmM; mean ± S.D.), but not by 2-MeSATP and UTP, indicating that these binding sites are neither the P2y nor the P2u receptors.