Evidence that most high-affinity ATP binding sites on aortic endothelial cells and membranes do not correspond to P-2 receptors

It has recently been demonstrated that two types of ATP receptors, the P-2Y and P-2U receptors, are coexpressed on bovine aortic endothelial cells. The aim of the present study was to characterize directly P-2Y and P-2U subtypes on intact bovine aortic endothelial cells and on membranes prepared from these cells using adenosine 5'-0-(3-thio[S-35]triphosphate) ([S-35]ATP gamma S), [alpha-P-32]ATP and [alpha-P-32]UTP as radioligands. [S-35]ATP gamma S binding to bovine aortic endothelial cell membranes was saturable and apparently involved a single class of high-affinity binding sites (K-d: 14 +/- 11 nM; B-max: 1.6 +/- 0.7 pmol/mg protein; mean +/- S.D.). A similar class of high-affinity binding sites was identified with [alpha-P-32]ATP (K-d: 14 +/- 9 nM; B-max: 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 (K-i: 21 +/- 5 and 14 +/- 10 nM, mean +/- S.D., for [S-35]ATP gamma S and [alpha-P-32]ATP, respectively) and might therefore represent the P-2Y receptors. UTP did not compete with [S-35]ATP gamma S or [alpha-P-32]ATP for binding at the remaining sites, indicating that they are not the P-2U receptors. No high-affinity UTP binding sites could be detected using [alpha-P-32]UTP. [S-35]ATP gamma S binding to intact bovine aortic endothelial cells was competed by ATP gamma S (K-d: 1.0 +/- 0.5 mu M; mean +/- S.D.), but not by 2-MeSATP and UTP, indicating that these binding sites are neither the P-2Y nor the P-2U receptors.