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]ATP gamma S), [alpha-32P]ATP and [alpha-32P]UTP as radioligands. [35S]ATP gamma S 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 [alpha-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]ATP gamma S and [alpha-32P]ATP, respectively) and might therefore represent the P2Y receptors. UTP did not compete with [35S]ATP gamma S or [alpha-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 [alpha-32P]UTP. [35S]ATP gamma S binding to intact bovine aortic endothelial cells was competed by ATP gamma S (Kd: 1.0 +/- 0.5 microM; mean +/- S.D.), but not by 2-MeSATP and UTP, indicating that these binding sites are neither the P2Y nor the P2U receptors.
Motte
S
S
Institute of Interdisciplinary Research, School of Medicine, Brussels, Belgium.
Swillens
S
S
Boeynaems
J M
JM
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000097:blood* / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001011:Aorta / Q000166:cytology / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003570:Cytidine Triphosphate / Q000494:pharmacology; D004727:Endothelium / Q000166:cytology / Q000378:metabolism; D066298:In Vitro Techniques; D007295:Inositol Phosphates / Q000097:blood / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D009994:Osmolar Concentration; D010761:Phosphorus Radioisotopes; D011485:Protein Binding / Q000187:drug effects; D011983:Receptors, Purinergic / Q000097:blood* / Q000378:metabolism; D018048:Receptors, Purinergic P2 / Q000097:blood / Q000378:metabolism; D013462:Sulfur Radioisotopes; D014530:Uridine Diphosphate / Q000494:pharmacology; D014544:Uridine Triphosphate / Q000097:blood / Q000378:metabolism
D007295:Inositol Phosphates; D010761:Phosphorus Radioisotopes; D011983:Receptors, Purinergic; D018048:Receptors, Purinergic P2; D013462:Sulfur Radioisotopes; D014530:Uridine Diphosphate; D000244:Adenosine Diphosphate; D003570:Cytidine Triphosphate; D000255:Adenosine Triphosphate; D014544:Uridine Triphosphate