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

Abstract

It has recently been demonstrated that two types of ATP receptors, the P 2 y and P 2 u receptors, are coexpressed on bovine aortic endothelial cells. The aim of the present study was to characterize directly P 2 y and P 2 u 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 ( 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 [ α- 32P]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 [ 35S]ATPγS and [ α- 32P]ATP, respectively) and might therefore represent the P 2 y receptors. UTP did not compete with [ 35S]ATPγS or [ α- 32P]ATP for binding at the remaining sites, indicating that they are not the P 2 u 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 ( K d : 1.0 ± 0.5 gmM; mean ± S.D.), but not by 2-MeSATP and UTP, indicating that these binding sites are neither the P 2 y nor the P 2 u receptors.