beta,gamma-methylene ATP-induced cAMP formation in C6Bu-1 cells: involvement of local metabolism and subsequent stimulation of adenosine A(2B) receptor

The mechanism underlying beta,gamma -methylene ATP (beta,gamma -MeATP)induc ed cAMP elevation was investigated in rat glioma C6Bu-1 cells. beta,gamma - MeATP increased forskolin-stimulated cAMP formation in a manner sensitive t o both the P, antagonist xanthine amine congener (XAC) and the P, antagonis t pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). Adenosine d eaminase (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'-nucl eotidase inhibitor, blocked the beta,gamma -MeATP-induced response. AMP, th e substrate for ecto-5'-nucleotidase, also induced cAMP formation in a mann er sensitive to XAC and alpha,beta -MeADP inhibition. However, the AMP-indu ced response was not blocked by PPADS. HPLC analyses revealed that adenosin e was generated from beta,gamma -MeATP and AMP. In addition, beta,gamma -Me ADP inhibited the conversion of beta,gamma -MeATP and AMP to adenosine, whe reas PPADS blocked adenosine formation from beta,gamma -MeATP but not from AMP. [H-3]Adenosine generated from [3H]AMP was preserved on the cell surfac e environment even in the presence of ADA. The mRNAs for ecto-phosphodieste rase/pyrophosphatase 1 (EC 3.1.4.1), ecto-5'-nucleotidase (EC 3.1.3.5) and adenosine A(2B) receptor were detected by RT-PCR. These results suggest tha t (CBu)-Bu-6-1 cells possess ecto-enzymes converting beta,gamma -MeATP to a denosine, and the locally accumulated adenosine in this mechanism efficient ly stimulates A(2B) receptors in a manner resistant to exogenous ADA.