Abstract

We recently demonstrated that extracellular ATP effectively activates adenosine (Ade) A_2B receptors indirectly through a localized rapid conversion to Ade by ectonucleotidases on the membrane surface of C6Bu-1 rat glioma cells. These responses were observed even in the presence of adenosine deaminase (ADA). Here, we demonstrate that such responses indeed occur in A_2B receptor-expressingXenopus laevis oocytes, which possess endogenous ectonucleotidase activity. In oocytes coexpressing the A_2Breceptor and cystic fibrosis transmembrane conductance regulator (CFTR), Ade induced a concentration-dependent increase in a cyclic AMP-activated CFTR current, a response that was inhibited by the P1 antagonist xanthine-amine congener (XAC). A brief application of ATP and β,γ-methylene ATP (β,γ-MeATP) also induced the CFTR current in a manner similar to that seen with Ade. Among several nucleotide agonists, ADP, AMP, and adenosine-5′-O-(3-thio)triphosphate induced the CFTR current. Although adenine nucleotide-induced CFTR currents were inhibited by XAC, they were highly resistant to ADA treatment; 5 U/ml ADA was required for inhibition of adenine nucleotide-induced CFTR current, whereas 1 U/ml ADA was sufficient to abolish the Ade-induced response. In addition, the ecto-5′-nucleotidase inhibitor α,β-methylene ADP markedly inhibited the β,γ-MeATP-induced response but not the Ade-induced one. These results support our hypothesis that adenine nucleotides are rapidly and locally converted into Ade on the membrane surface, resulting in the activation of A_2B receptors.