Kolachala Vasantha L, Obertone Tracy Sitler, Wang Lixin, Merlin Didier, Sitaraman Shanthi V
Division of Digestive Diseases, Department of Medicine, Emory University, Room 201-F, 615 Michael Street, Whitehead Research Building, Atlanta, GA 30322, USA.
Biochim Biophys Acta. 2006 Jul;1760(7):1102-8. doi: 10.1016/j.bbagen.2006.03.010. Epub 2006 Apr 4.
Adenosine 2b receptor (A2bR), a G-protein coupled receptor positively coupled to adenylate cyclase, mediates key events such as chloride, IL-6 and fibronectin secretion in intestinal epithelial cells and is upregulated during intestinal inflammation. In order to gain insight into the overall mechanism of A2bR activation, in this study, we sought to characterize the AC isoform associated with A2bR signaling. The colonic epithelial cell line T84, expressing only the A2b subtype of adenosine receptor, and Chinese hamster ovary (CHO) cells, were used in these studies. cAMP was measured by luminometric assay and AC isoform expression was determined by Western blot, RT-PCR, isoform-specific stealth RNAi and Quantigene. T84 and CHO cells express all nine known AC isoforms. In order to characterize which AC isoform(s) are associated with A2bR, we used the differential inhibition of specific AC isoforms by calcium and nitric oxide. Pretreatment of cells with carbachol or nitric oxide donors such as S-Nitroso-N-acetylpencillamine (SNAP) and PAPANANOATE inhibited A2bR mediated increase in cAMP. Further, overexpression of AC-5 or AC-6 potentiated A2bR-mediated increases in cAMP levels. Finally, transfection with AC isoform-specific RNAi demonstrated that AC-6 but not AC-5 RNAi inhibited adenosine-induced cAMP levels. Taken together, these results suggest that A2bR mediates signaling through AC-6 isoform. Since pro-inflammatory cytokines such as interferon-gamma (IFN-gamma) modulate the expression of specific AC isoforms in the intestinal epithelia, our observation may have therapeutic implications for intestinal inflammation or diarrhea wherein aA2bR is upregulated.
腺苷2b受体（A2bR）是一种与腺苷酸环化酶正性偶联的G蛋白偶联受体，介导肠道上皮细胞中诸如氯离子、白细胞介素-6和纤连蛋白分泌等关键事件，并且在肠道炎症期间上调。为了深入了解A2bR激活的整体机制，在本研究中，我们试图鉴定与A2bR信号传导相关的腺苷酸环化酶（AC）同工型。这些研究中使用了仅表达腺苷受体A2b亚型的结肠上皮细胞系T84和中国仓鼠卵巢（CHO）细胞。通过发光测定法测量环磷酸腺苷（cAMP），并通过蛋白质免疫印迹法、逆转录-聚合酶链反应（RT-PCR）、同工型特异性隐形RNA干扰（RNAi）和基因表达定量分析（Quantigene）确定AC同工型的表达。T84和CHO细胞表达所有九种已知的AC同工型。为了鉴定哪些AC同工型与A2bR相关，我们利用钙和一氧化氮对特定AC同工型的差异性抑制作用。用卡巴胆碱或一氧化氮供体如S-亚硝基-N-乙酰青霉胺（SNAP）和帕潘诺酸预处理细胞可抑制A2bR介导的cAMP增加。此外，AC-5或AC-6的过表达增强了A2bR介导的cAMP水平升高。最后，用AC同工型特异性RNAi转染表明，AC-6 RNAi而非AC-5 RNAi抑制腺苷诱导的cAMP水平。综上所述，这些结果表明A2bR通过AC-6同工型介导信号传导。由于促炎细胞因子如γ干扰素（IFN-γ）调节肠道上皮中特定AC同工型的表达，我们的观察结果可能对肠道炎症或腹泻具有治疗意义，其中A2bR上调。
