Inhibition of ATP-induced cAMP formation by 5'-p-fluorosulfonylbenzoyladenosine in NG108-15 cells.
ATP is known to increase intracellular cAMP levels in NG108-15 cells via a novel purinoceptor and this response is inhibited by the P1 purinoceptor antagonist methylxanthine. In the present study, we examined the effects of 5'-p-fluorosulfonylbenzoyladenosine (FSBA), an affinity ligand for ATP-binding proteins, on cAMP formation mediated by activation of adenylate cyclase (AC)-linked purinoceptors in NG108-15 cells. cAMP levels were determined by RIA using an anti-succinyl-cAMP antiserum. FSBA (100 microM) increased intracellular cAMP about 2.6-fold. However, FSBA-induced cAMP formation was abolished by pretreatment with adenosine deaminase, suggesting that adenosine, a breakdown product of FSBA, is involved in FSBA-induced cAMP formation. In contrast, pretreatment of cells with FSBA in the presence of adenosine deaminase inhibited cAMP formation induced by ATP and beta,gamma-methylene-ATP (beta,gamma-MeATP), without affecting the prostaglandin E1 (PGE1)-induced response. The inhibitory effect of FSBA on ATP-induced cAMP formation was concentration-dependent with a concentration required for half-maximal inhibition (IC50) of around 3 microM. The inhibitory effect of FSBA was not affected by pertussis toxin (PTX)-treatment. Pretreatment with FSBA (10 microM) depressed the maximal response to beta,gamma-MeATP by 60%, but did not affect the response to 5'-N-ethylcarboxamidoadenosine. The inhibitory effect of FSBA (100 microM) increased time-dependently during pretreatment and partly resisted wash-out. The inhibition by FSBA was protected by simultaneous addition of beta,gamma-MeATP during the FSBA pretreatment, indicating that both FSBA and the ATP analogue interacted with the same receptor site. The pretreatment with FSBA did not affect the increase in [Ca2+]i induced by ATP, UTP or benzoylbenzoic ATP. These results suggest that FSBA inhibits cAMP accumulation induced in NG108-15 cells by ATP or related agonists by selective modification of an AC-linked purinoceptor.
Ohkubo
S
S
Department of Pharmacology, School of Medicine, Fukushima Medical University, Hikarigaoka, Japan.
Matsuoka
I
I
Kimura
J
J
Nakanishi
H
H
D000241:Adenosine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology*; D037361:Adenylate Cyclase Toxin; D000345:Affinity Labels / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D006822:Hybrid Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D037342:Pertussis Toxin; D011485:Protein Binding; D058910:Purinergic P2 Receptor Agonists; D011983:Receptors, Purinergic / Q000378:metabolism / Q000502:physiology; D058486:Receptors, Purinergic P2X7; D058473:Receptors, Purinergic P2Y2; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D014544:Uridine Triphosphate / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D000345:Affinity Labels; D058910:Purinergic P2 Receptor Agonists; D011983:Receptors, Purinergic; D058486:Receptors, Purinergic P2X7; D058473:Receptors, Purinergic P2Y2; D010566:Virulence Factors, Bordetella; C005147:5'-adenylyl (beta,gamma-methylene)diphosphonate; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D037342:Pertussis Toxin; D000241:Adenosine; C002630:alpha,beta-methyleneadenosine 5'-triphosphate; D002118:Calcium; D014544:Uridine Triphosphate