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 microM for ATP, 2.5 microM for UTP and 14 microM for adenosine-5'-O-(3-thio)triphosphate (ATPgammaS). EC50 values for 2-methylthioATP, ADP, adenosine-5'-O-(2-thio)diphosphate (ADPbetaS) and AMP were 0.5 microM, 3.5 microM, 15 microM and 4.7 microM 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 microM pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), whereas [Ca2+]c responses to UTP were unaffected by this treatment. 5. ECV304 cells responded to the diadenosine polyphosphate Ap3A with rises in [Ca2+]c. Apparent responses to Ap4A, Ap5A and Ap6A, 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 P2Y2 receptor insensitive to the diadenosine polyphosphates and a P2Y1 receptor sensitive to Ap3A. In addition, ECV304 cells respond to AMP with increases in [Ca2+]c via an as yet uncharacterized receptor.
