Ohkubo S, Kumazawa K, Sagawa K, Kimura J, Matsuoka I
Department of Pharmacology, School of Medicine, Fukushima Medical University, Fukushima, Japan.
J Neurochem. 2001 Feb;76(3):872-80. doi: 10.1046/j.1471-4159.2001.00098.x.
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.
在大鼠胶质瘤C6Bu-1细胞中研究了β,γ-亚甲基ATP（β,γ-MeATP）诱导细胞内cAMP升高的机制。β,γ-MeATP以对P1拮抗剂黄嘌呤胺类似物（XAC）和P2拮抗剂磷酸吡哆醛-6-偶氮苯基-2',4'-二磺酸（PPADS）均敏感的方式增加了福斯可林刺激的cAMP生成。腺苷脱氨酶（ADA；1 U/mL）可消除腺苷诱导的反应，但并未消除β,γ-MeATP诱导的反应。然而，ADA与ecto-5'-核苷酸酶抑制剂α,β-亚甲基ADP（α,β-MeADP）联合使用可阻断β,γ-MeATP诱导的反应。ecto-5'-核苷酸酶的底物AMP也以对XAC和α,β-MeADP抑制敏感的方式诱导cAMP生成。然而，PPADS不能阻断AMP诱导的反应。高效液相色谱分析显示，腺苷是由β,γ-MeATP和AMP生成的。此外，α,β-MeADP抑制β,γ-MeATP和AMP向腺苷的转化，而PPADS阻断β,γ-MeATP生成腺苷，但不阻断AMP生成腺苷。即使存在ADA，由[3H]AMP生成的[3H]腺苷仍保留在细胞表面环境中。通过逆转录聚合酶链反应（RT-PCR）检测到了ecto-磷酸二酯酶/焦磷酸酶1（EC 3.1.4.1）、ecto-5'-核苷酸酶（EC 3.1.3.5）和腺苷A2B受体的mRNA。这些结果表明，C6Bu-1细胞具有将β,γ-MeATP转化为腺苷的ecto-酶，并且该机制中局部积累的腺苷以对外源ADA有抗性的方式有效刺激A2B受体。
