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

1 We previously demonstrated that extracellular adenine nucleotides induced cyclic AMP elevation through local adenosine production at the membrane surface and subsequent activation of adenosine A(2A) 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. 2 NG108-15 cells hydrolyzed AMP to adenosine with the K-M value of 18.8 +/- 2.2 muM and V-max of 5.3 +/- 1.6 nmol min(-1) 10(6) cells(-1). This activity was suppressed at pH 6.5, but markedly increased at pH 8.5. 3 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 beta -glycerophosphate, indicating that NG108-15 cells express ecto-ALP. 4 The cyclic AMP accumulation induced by several adenine nucleotides was inhibited by levamisole, p-nitrophenylphosphate and beta -glycerophosphate, with a parallel decrease in the extracellular adenosine formation. 5 Reverse transcriptase polymerase chain reaction analysis revealed that NG108-15 cells express mRNA for the tissue-nonspecific isozyme of ALP. 6 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.