Ecto-alkaline phosphatase in NG108-15 cells : a key enzyme mediating P1 antagonist-sensitive ATP response.
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.
Ohkubo
S
S
Department of Pharmacology, School of Medicine, Fukushima Medical University, Hikarigaoka-1, Fukushima 960-1295, Japan.
Kimura
J
J
Matsuoka
I
I
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
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