Ecto-alkaline phosphatase in NG108-15 cells: A key enzyme mediating P1 antagonist-sensitive ATP response

Abstract

We previously demonstrated that extracellular adenine nucleotides induced cyclic AMP elevation through local adenosine production at the membrane surface and subsequent activation of adenosine A2A receptors in NG108-15 cells. Furthermore, the adenosine formation was found to be mediated by an ecto-enzyme distinct from the ecto-5′-nucleotidase (CD73). In this study, we investigated the properties of the ecto-AMP phosphohydrolase activity in NG108-15 cells. NG108-15 cells hydrolyzed AMP to adenosine with the KM value of 18.8±2.2 μM and Vmax of 5.3±1.6 nmol min-1 106 cells-1. This activity was suppressed at pH 6.5, but markedly increased at pH 8.5. The AMP hydrolysis was blocked by levamisole, an alkaline phosphatase (ALP) inhibitor. NG108-15 cells released orthophosphate from 2′- and 3′-AMP as well as from ribose-5-phosphate and β-glycerophosphate, indicating that NG108-15 cells express ecto-ALP. The cyclic AMP accumulation induced by several adenine nucleotides was inhibited by levamisole, p-nitrophenylphosphate and β-glycerophosphate, with a parallel decrease in the extracellular adenosine formation. Reverse transcriptase polymerase chain reaction analysis revealed that NG108-15 cells express mRNA for the tissue-nonspecific isozyme of ALP. These results demonstrate that AMP phosphohydrolase activity in NG108-15 cells is due to ecto-ALP, and suggest that this enzyme plays an essential role for the P1 antagonist-sensitive ATP-induced cyclic AMP accumulation in NG108-15 cells.