Inhibition of ATP-induced cAMP formation by 5'-p-fluorosulfonylbenzoyladenosine in NG108-15 cells.

S Ohkubo, I Matsuoka, J Kimura, H Nakanishi

Index: Naunyn Schmiedebergs Arch. Pharmacol. 358(2) , 153-9, (1998)

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Abstract

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.