Expression of purinergic P2Y receptor subtypes by INS-1 insulinoma β-cells:: A molecular and binding characterization
Expression of purinergic P2Y receptor subtypes by INS-1 insulinoma β-cells:: A molecular and binding characterization
复制标题
DOI：
10.1016/j.ejphar.2007.04.012
复制
发表时间：
2007-07-30
影响因子：
5
通讯作者：
Vignon, Jacques
中科院分区：
文献类型：
--
作者：
Lugo-Garcia, Laura;Filhol, Romain;Vignon, Jacques
嘌呤能P2 Y受体激动剂可增强葡萄糖诱导的胰腺β细胞胰岛素分泌，从而为2型糖尿病的治疗提供了新的机会。然而，很少有人知道哪些嘌呤能P2 Y受体亚型在这些细胞中表达。INS-1 β细胞系用作胰腺β细胞的模型，表达其大部分特性。因此，我们通过真实的时间聚合酶链反应和蛋白质印迹法研究了不同分子亚型在该细胞系中的表达。我们还对原型嘌呤能P2 Y激动剂腺苷-5 '-O-（1-[（35）S]硫代三磷酸）（ATP-α-[S-35]）与细胞膜匀浆的结合进行了表征。分子生物学分析证实存在五种不同的嘌呤能P2 Y受体亚型（P2 Y 1、P2 Y（2）、P2 Y（4）、P2 Y（6）和P2 Y（12）），它们以相似的水平表达。Western印迹分析允许检测相应的蛋白质。结合试验证明了高（40%）和低（60%）亲和力组分上的特异性ATP-α-[S-35]相互作用。两个位点的ATP-α-[S-35]药理学特征分析允许将高亲和力结合位点分类为嘌呤能P2 Y受体亚型的代表和P2 Y（4）和/或P2 Y（6）受体亚型的低亲和力结合位点。ATP-α-S和腺苷-5 '-O-（2-硫代二磷酸）（ADP-β-S）在高亲和力和低亲和力结合位点上表现出相反的选择性。尽管嘌呤能P2 Y（1）受体或P2 Y（1）样亚型通常被认为与葡萄糖诱导的胰岛素释放的调节有关，但目前的数据表明β细胞表达嘌呤能P2 Y受体亚型的复杂谱，其功能意义仍有待充分阐明。(C)2007 Elsevier B. V.保留所有权利。
Purinergic P2Y-receptor agonists amplify glucose-induced insulin secretion from pancreatic beta-cells, thus offering new opportunities for the treatment of type 2 diabetes. However, little is known about which subtypes of purinergic P2Y receptors are expressed in these cells. The INS-1 beta-cell line is used as a model of pancreatic beta-cells, expressing most of their properties. Therefore, we investigated the expression of different molecular subtypes in this cell line by means of real time Polymerase Chain Reaction and Western blot. We also performed a characterization of the binding of a prototypic purinergic P2Y agonist, Adenosine-5'-O-(1-[(35) S]thiotri phosphate) (ATP-alpha-[S-35]), to cell membrane homogenates. The molecular analysis evidenced the presence of five different purinergic P2Y receptor subtypes (P2Y1, P2Y(2), P2Y(4), P2Y(6) and P2Y(12)), which were expressed at similar levels. The Western blot analysis allowed detecting corresponding proteins. The binding assay demonstrated a specific ATP-alpha-[S-35] interaction on high (40%) and low (60%) affinity components. The analysis of ATP-alpha-[S-35] pharmacological profile on both sites permitted to classify the high affinity binding site as representative of the purinergic P2Y, receptor subtype and the low affinity binding site of the P2Y(4) and/or P2Y(6) receptor subtypes. ATP-alpha-S and Adenosine-5'-O-(2-thiodiphosphate) (ADP-beta-S) exhibited opposite selectivity on high and low affinity binding sites. Although purinergic P2Y(1) receptor, or a P2Y(1)-like subtype, has been generally considered as that implicated in the modulation of glucose-induced insulin release, the present data show that the beta-cell expresses a complex profile of purinergic P2Y receptor subtypes, the functional implication of which remains to be fully elucidated. (C) 2007 Elsevier B.V. All rights reserved.