Characterization of intestinal adenosine A2b receptor

描述(由申请人提供)： 肠炎性疾病急性发作的特点是中性粒细胞穿过肠上皮进入肠腔，形成“隐窝脓肿”。腺苷是在活动性肠道炎症过程中，中性粒细胞来源的5‘AMP在上皮性5’端核苷酸酶的作用下转化为腺苷而产生的。腺苷通过A2B受体发挥作用，不仅介导电源性氯的分泌，还诱导促炎细胞因子白介素6(IL-6)的顶端极化分泌。IL-6反过来在中性粒细胞中诱导钙离子流动，从而向中性粒细胞提供旁分泌信号。在R03的应用中，我们建议调查A2B受体在协调肠道炎症反应的腹泻和炎症成分中发挥中心作用的假设。如果是这样的话，对A2B受体的药理操作可能对肠道炎症有治疗作用。该方案的总体目标是确定肠道A2B受体的特征，检测腺苷诱导的IL-6分泌的调节，并确定IL-6对中性粒细胞功能的影响。具体目的1：我们将使用表达天然A2B受体的模型肠道细胞系T84和过表达GFP-A2B的Caco 2-BBE细胞来研究：i)A2B受体通过区域特异性生物素化和免疫荧光被募集到根尖和基底膜；II)A2B受体与E3KARP相互作用的分子机制将通过缺失构建和定点突变来研究；III)A2B受体募集在受体信号转导、功能和脱敏中的作用。特定目的2：我们将研究：i)利用IL-6启动子构建的带有各种核因子结合位点缺失和点突变的腺苷介导的IL-6诱导机制。

DESCRIPTION (provided by applicant): Acute flares of inflammatory diseases of the intestine are characterized by the migration of neutrophils across the intestinal epithelium into the lumen to form 'crypt abscess'. Adenosine is generated during active intestinal inflammation by the conversion of neutrophil-derived 5'AMP into adenosine by the epithelial 5'ectonucleotidase. Adenosine, acting via the A2b receptor, not only mediates electrogenic chloride secretion but also induces an apically polarized secretion of the pro-inflammatory cytokine, interleukin-6 (IL-6). IL-6, in turn, induces a calcium flux in neutrophils, thus providing a paracrine signal to neutrophils. In this R03 application, we propose to investigate the hypothesis that the A2b receptors play a central role in orchestrating both the diarrheal and inflammatory components of the intestinal inflammatory response. If so, pharmacological manipulation of the A2b receptor may be therapeutic for intestinal inflammation. The overall goals of this proposal are to characterize the intestinal A2b receptor, to examine the regulation of adenosine-induced IL-6 secretion, and to determine the effect of IL-6 on neutrophil function. Specific aim 1 : We will use model intestinal cell line, T84, expressing native A2b receptor and Caco2-BBE (which do not express A2b receptor) overexpressing GFP-A2b to study: i) the recruitment of the A2b receptor to apical vs basolateral membrane using domain-specific biotinylation and immunoflourescence ii) the molecular mechanism of interaction of A2b receptor with E3KARP will be studied using deletion constructs and site-directed mutagenesis iii) the role of A2b receptor recruitment in receptor signaling and function and desensitization. Specific aim 2: We will investigate: i) the mechanism of adenosine-mediated IL-6 induction using IL-6 promoter constructs with deletion and point mutations of the various nuclear factor binding sites,.Specific aim 3: We will study: i) the effect of IL-6 on degranulation of neutrophils.