title: Beta,gamma-methylene ATP-induced cAMP formation in C6Bu-1 cells: involvement of local metabolism and subsequent stimulation of adenosine A2B receptor.
abstract: The mechanism underlying beta,gamma-methylene ATP (beta,gamma-MeATP)-induced cAMP elevation was investigated in rat glioma C6Bu-1 cells. Beta,gamma-MeATP increased forskolin-stimulated cAMP formation in a manner sensitive to both the P1 antagonist xanthine amine congener (XAC) and the P2 antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). Adenosine deaminase (ADA; 1 U/mL), which abolished the adenosine-induced response, did not eliminate the beta,gamma-MeATP-induced response. However, combination of ADA with alpha,beta-methylene ADP (alpha,beta-MeADP), an ecto-5'-nucleotidase inhibitor, blocked the beta,gamma-MeATP-induced response. AMP, the substrate for ecto-5'-nucleotidase, also induced cAMP formation in a manner sensitive to XAC and alpha,beta-MeADP inhibition. However, the AMP-induced response was not blocked by PPADS. HPLC analyses revealed that adenosine was generated from beta,gamma-MeATP and AMP. In addition, alpha,beta-MeADP inhibited the conversion of beta,gamma-MeATP and AMP to adenosine, whereas PPADS blocked adenosine formation from beta,gamma-MeATP but not from AMP. [3H]Adenosine generated from [3H]AMP was preserved on the cell surface environment even in the presence of ADA. The mRNAs for ecto-phosphodiesterase/pyrophosphatase 1 (EC 3.1.4.1), ecto-5'-nucleotidase (EC 3.1.3.5) and adenosine A2B receptor were detected by RT-PCR. These results suggest that C6Bu-1 cells possess ecto-enzymes converting beta,gamma-MeATP to adenosine, and the locally accumulated adenosine in this mechanism efficiently stimulates A2B receptors in a manner resistant to exogenous ADA.
authors.affiliation: Department of Pharmacology, School of Medicine, Fukushima Medical University, Fukushima, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Ohkubo
authors.forename: K
authors.initials: K
authors.lastname: Kumazawa
authors.forename: K
authors.initials: K
authors.lastname: Sagawa
authors.forename: J
authors.initials: J
authors.lastname: Kimura
authors.forename: I
authors.initials: I
authors.lastname: Matsuoka
mesh_terms: D015720:5'-Nucleotidase / Q000037:antagonists & inhibitors; D000241:Adenosine / Q000096:biosynthesis; D000249:Adenosine Monophosphate / Q000378:metabolism / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000096:biosynthesis*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005110:Extracellular Space / Q000378:metabolism; D009708:Nucleotidases / Q000378:metabolism; D058914:Purinergic Antagonists; D058910:Purinergic P2 Receptor Agonists; D051381:Rats; D043704:Receptor, Adenosine A2B; D018047:Receptors, Purinergic P1 / Q000378:metabolism*; D014407:Tumor Cells, Cultured
chemical_list: D004791:Enzyme Inhibitors; D058914:Purinergic Antagonists; D058910:Purinergic P2 Receptor Agonists; D043704:Receptor, Adenosine A2B; D018047:Receptors, Purinergic P1; D005576:Colforsin; C005147:5'-adenylyl (beta,gamma-methylene)diphosphonate; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D009708:Nucleotidases; D015720:5'-Nucleotidase; D000241:Adenosine; C002630:alpha,beta-methyleneadenosine 5'-triphosphate