Evidence that a form of ATP uncomplexed with divalent cations is the ligand of P2y and nucleotide/P2u receptors on aortic endothelial cells

1. The response of bovine aortic endothelial cells to adenosine 5'-triphosphate (ATP) is mediated by both P2y and nucleotide/P2u receptors. In order to determine which form of the nucleotide is the true ligand of these receptors, we have investigated the effects of divalent cations on ATP-, uridine 5'-triphosphate (UTP)- and 2 methylthioadenosine 5'-triphosphate (2MeSATP)-induced inositol phosphate accumulation in these cells. 2. Omisson of Mg2+ from a calcium-free incubation buffer caused a shift to the left of the ATP concentration-action curve. 3. In the presence of EDTA (1 mM), the basal level of inositol trisphosphate (InsP3) was markedly increased and the absolute maximal response to ATP was decreased; however, the response to low concentrations of ATP was enhanced. 4. When the results were plotted in terms of calculated ATP4- concentrations, the concentration-response curves obtained in the presence of 1.25 mM Mg2+ lay closer to the respective curves obtained when Mg2+ was omitted from the medium or when Mg2+ was omitted and EDTA (1 mM) was added. The curves became almost superimposable when the baseline value was subtracted. 5. A similar shift to the left of the concentrations-action curves was also observed with both UTP and 2MeSATP. 6. Our data provide evidence that a form of ATP uncomplexed with divalent cation is the preferential agonist of both the nucleotide/P2u and the P2y receptors expressed on bovine aortic endothelial cells.