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

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Abstract
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. Omisson of Mg2+ from a calcium‐free incubation buffer caused a shift to the left of the ATP concentration‐action curve. 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. 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. A similar shift to the left of the concentrations‐action curves was also observed with both UTP and 2MeSATP. 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.