Characterization of the P2 receptors on the human umbilical vein endothelial cell line ECV304

1 To characterize the P2 receptors present on the human umbilical vein endothelial-derived cell line, ECV304, cytosolic Ca2+, ([Ca2+](c)), responses were recorded in single cells and in cell suspensions to a series of nucleotides and nucleotide agonists. 2 Concentration response curves were obtained in fura-2-loaded ECV304 cell suspensions, with EC50 values of 4.2 mu M for ATP, 2.5 mu M for UTP and 14 mu M for adenosine-5'-O-(3-thio)triphosphate (ATP gamma S). EC50 values for 2-methylthioATP, ADP, adenosine-5'-O-(2-thio)diphosphate (ADP beta S) and AMP were 0.5 mu M, 3.5 mu M, 15 mu M and 4.7 mu M respectively, but maximal [Ca2+](c) responses were less than those produced by a maximal addition of ATP/UTP. ECV304 cells were unresponsive to UDP and beta,gamma,methyleneATP. 3 Cross-desensitization studies on ECV304 cells suggested that ATP and UTP recognized the same receptor. However, ADP recognized a receptor distinct from the UTP-sensitive receptor and AMP recognized a third distinct receptor. 4 ECV304 [Ca2+](c) responses to 2-methylthioATP were inhibited in the presence of 30 mu M pyridoxalphosphate-6-azophenyl-2',4'-dis acid (PPADS), whereas [Ca2+](c) responses to UTP were unaffected by this treatment. 5 ECV304 cells responded to the diadenosine polyphosphate Ap,A with rises in [Ca2+](c). Apparent responses to Ap(4)A, Ap(5)A and Ap(6)A, were shown to be due to a minor nucleotide contaminant that could be removed by pre-treatment of the diadenosine samples with either alkaline phosphatase or apyrase. 6 ECV304 cells display a pharmacology consistent with the presence of at least two P2 receptors; a P2Y(2) receptor insensitive to the diadenosine polyphosphates and a P2Y(1) receptor sensitive to Ap(3)A. In addition, ECV304 cells respond to AMP with increases in [Ca2+](c) via an as yet uncharacterized receptor.