title: 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 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. NG108-15 cells hydrolyzed AMP to adenosine with the K:(M:) value of 18.8+/-2.2 microM 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. 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 ss-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 ss-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.
authors.affiliation: Department of Pharmacology, School of Medicine, Fukushima Medical University, Hikarigaoka-1, Fukushima 960-1295, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Ohkubo
authors.forename: J
authors.initials: J
authors.lastname: Kimura
authors.forename: I
authors.initials: I
authors.lastname: Matsuoka
mesh_terms: D000241:Adenosine / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000378:metabolism / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D000242:Cyclic AMP / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D005994:Glycerophosphates / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis / Q000187:drug effects; D007978:Levamisole / Q000494:pharmacology; D051379:Mice; D010755:Organophosphates / Q000378:metabolism; D058915:Purinergic P1 Receptor Antagonists; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013379:Substrate Specificity; D013997:Time Factors; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000187:drug effects / Q000378:metabolism
chemical_list: D005994:Glycerophosphates; D010755:Organophosphates; D058915:Purinergic P1 Receptor Antagonists; D012333:RNA, Messenger; C077792:pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid; D007978:Levamisole; C005147:5'-adenylyl (beta,gamma-methylene)diphosphonate; D000249:Adenosine Monophosphate; D011732:Pyridoxal Phosphate; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D000469:Alkaline Phosphatase; D000241:Adenosine; C002630:alpha,beta-methyleneadenosine 5'-triphosphate; C031463:beta-glycerophosphoric acid