Keyvanloo Shahrestanaki Mohammad, Aghaei Mahmoud
Department of Clinical Biochemistry, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, I.R. Iran.
Res Pharm Sci. 2019 Mar 8;14(2):107-114. doi: 10.4103/1735-5362.253357. eCollection 2019 Apr.
Diabetes incidence showed ascending trends in recent years indicating urgent need for new therapeutic agents. Extracellular adenosine signaling showed promising results. However, role of its A3 receptor in pancreatic β-cells proliferation and insulin secretion is not well established. Thus, we aimed to determine its main signaling mediators in MIN6 insulinoma cell line. A3 adenosine receptor (A3AR) expression was confirmed using RT-PCR. Receptor functionality was evaluated by measurements of cAMP, using ELISA kit, and intracellular Ca levels, using Fura 2/AM probe in response to the specific A3AR agonist (Cl- IBMECA). Insulin ELISA kit was used to measure insulin release. Herein, we mentioned that MIN6 cells express active form of A3AR, which decreased cAMP levels with the half maximal effective concentration (EC50) value of 5.61. [Ca]i Levels transiently (approximately 120 sec) increased in response to the agonist. Cl-IBMECA increase insulin secretion at 0.01-1 μM, but showed an inhibitory effects at higher concentrations (1-10 μM). Altogether, we found that in MIN6 cells, A3AR, possibly through Ca mediated signaling pathways, potentiated glucose-induced insulin secretion.
近年来，糖尿病发病率呈上升趋势，这表明迫切需要新的治疗药物。细胞外腺苷信号传导显示出有前景的结果。然而，其A3受体在胰腺β细胞增殖和胰岛素分泌中的作用尚未完全明确。因此，我们旨在确定其在MIN6胰岛素瘤细胞系中的主要信号传导介质。使用逆转录聚合酶链反应（RT-PCR）确认A3腺苷受体（A3AR）的表达。通过使用酶联免疫吸附测定（ELISA）试剂盒测量环磷酸腺苷（cAMP）以及使用Fura 2/AM探针测量细胞内钙水平来评估受体功能，以响应特异性A3AR激动剂（Cl-IB-MECA）。使用胰岛素ELISA试剂盒测量胰岛素释放。在此，我们提到MIN6细胞表达活性形式的A3AR，其降低cAMP水平，半数最大效应浓度（EC50）值为5.61。细胞内钙（[Ca]i）水平在响应激动剂时短暂（约120秒）升高。Cl-IB-MECA在0.01-1μM时增加胰岛素分泌，但在较高浓度（1-10μM）时显示出抑制作用。总之，我们发现在MIN6细胞中，A3AR可能通过钙介导的信号通路增强葡萄糖诱导的胰岛素分泌。
