Intracellular pH changes of cultured bovine aortic endothelial cells in response to ATP addition

The changes of the intracellular pH (pH_i) of cultured bovine aortic endothelial cells were fluorometrically monitored using 2′,7′-bis(carboxyethyl)carboxyfluorescein (BCECF). A biphasic pH_i change was observed by addition of ATP: an initial acidification followed by an alkalinization of about 0.2 pH unit above the resting level of pH_i 7.23. The alkalinization was dependent on [Na⁺]_o and [H⁺]_o, and was inhibited by 5-(N,N-hexamethylene)amiloride, indicating that the alkalinization is mediated by the Na⁺/H⁺ exchanger. The 50% effective concentration of ATP was about 1.4 μM. ADP similarly induced pH_i changes, whereas AMP and adenosine were inactive. The pH_i changes induced by ATP were dependent on the extracellular Ca²⁺, and the addition of calcium ionophore A23187 induced similar pH_i changes. The results indicate that ATP activates the Na⁺/H⁺ exchanger in cultured bovine aortic endothelial cells and the activation is mediated by the P₂-purinergic receptor and is dependent on the extracellular Ca²⁺.