Ohkubo S, Matsuoka I, Kimura J, Nakanishi H
Department of Pharmacology, School of Medicine, Fukushima Medical University, Hikarigaoka, Japan.
Naunyn Schmiedebergs Arch Pharmacol. 1998 Aug;358(2):153-9. doi: 10.1007/pl00005237.
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.
已知三磷酸腺苷（ATP）通过一种新型嘌呤受体增加NG108 - 15细胞内的环磷酸腺苷（cAMP）水平，且这种反应会被P1嘌呤受体拮抗剂甲基黄嘌呤抑制。在本研究中，我们检测了ATP结合蛋白的亲和配体5'-对氟磺酰苯甲酰腺苷（FSBA）对NG108 - 15细胞中由腺苷酸环化酶（AC）偶联嘌呤受体激活介导的cAMP形成的影响。使用抗琥珀酰 - cAMP抗血清通过放射免疫分析（RIA）测定cAMP水平。FSBA（100微摩尔）使细胞内cAMP增加约2.6倍。然而，用腺苷脱氨酶预处理可消除FSBA诱导的cAMP形成，这表明FSBA的分解产物腺苷参与了FSBA诱导的cAMP形成。相反，在腺苷脱氨酶存在下用FSBA预处理细胞可抑制ATP和β,γ - 亚甲基 - ATP（β,γ - MeATP）诱导的cAMP形成，而不影响前列腺素E1（PGE1）诱导的反应。FSBA对ATP诱导的cAMP形成的抑制作用呈浓度依赖性，半数最大抑制浓度（IC50）约为3微摩尔。FSBA的抑制作用不受百日咳毒素（PTX）处理的影响。用FSBA（10微摩尔）预处理使对β,γ - MeATP的最大反应降低60%，但不影响对5'-N - 乙基甲酰胺基腺苷的反应。FSBA（100微摩尔）的抑制作用在预处理期间呈时间依赖性增加，且部分抵抗洗脱。在FSBA预处理期间同时添加β,γ - MeATP可保护其抑制作用，表明FSBA和ATP类似物与同一受体位点相互作用。用FSBA预处理不影响ATP、尿苷三磷酸（UTP）或苯甲酰苯甲酸ATP诱导的细胞内钙离子浓度（[Ca2+]i）升高。这些结果表明，FSBA通过选择性修饰AC偶联嘌呤受体来抑制ATP或相关激动剂在NG108 - 15细胞中诱导的cAMP积累。
