ADENOSINE RECEPTORS AND ION TRANSPORT IN EPITHELIA

这项拟议研究的长期目标是确定细胞 和调节上皮细胞氯离子分泌的分子基础 腺苷受体。理解眼镜蛇的调节功能 腺苷的转运需要(1)识别原发的 化合物的分子结构和相应的构效关系 上皮细胞中存在特定的腺苷受体亚型，(2) 确定特定的离子通道是最终的 腺苷受体效应物；(3)代谢的整合 以及将腺苷运输到受体功能的调节机制中。 G蛋白偶联受体的分子生物学研究进展 识别腺苷受体的系统方法的基础 子类型。我们将结合独特的腺苷的分子特征 离子的电生理特性与受体亚型 腺苷释放细胞膜的通道(C_1和K)及其测量 全面了解氯离子的反馈规律 腺苷分泌。我们提出的研究重点是高度 特化的，均一的，分泌NaC1的鲨鱼直肠腺 独特的腺苷受体亚型-A2e和A1e受体-双重 调节氯化物的分泌。通过利用该模式的优势 我们建议提供分子和功能表征的系统 这些受体中的一个。具体目标是：(1)克隆、测序和 在卵母细胞和受体缺陷细胞中功能表达的独特性 A2E和A1E腺苷受体亚型调节Nc1转运 直肠腺；(2)确定离子通道(C_1和K) 偶联到这些受体亚型上，并表征 这一规定；和(3)确定极化传输 腺苷穿过直肠腺细胞的基侧膜和顶膜 并表征了新发现的腺苷三磷酸腺苷和三磷酸腺苷的心尖受体 UTP。这些实验将采用基于聚合酶链式反应的克隆和测序 ISC和极化腺苷释放的策略、测量方法 原代培养细胞的单层和膜片钳研究 直肠腺细胞。在建议研究的每一阶段，我们都会 将分子结构与受体的功能分析相关联。

The long term goal of the proposed research is to determine the cellular and molecular basis for regulation of chloride secretion in epithelia by adenosine receptors. Understanding the autacoid regulation f ion transport by adenosine requires (1) identification of the primary molecular structure and corresponding structure activity profiles of specific adenosine receptor subtypes present in epithelia, (2) identification of the specific ion channels that are the ultimate effectors of adenosine receptors, and (3) integration of the metabolism and transport of adenosine into regulatory schemes of receptor function. Advances in molecular biology of G-protein coupled receptors now provide the basis for a systematic approach to identifying adenosine receptor subtypes. We will combine molecular characterization of unique adenosine receptor subtypes with electrophysiologic characterization of ion channels (C1 and K) and measurements of adenosine release cell membranes to obtain a comprehensive picture of the feedback regulation of chloride secretion by adenosine. Our proposed research focuses on the highly specialized, homogeneous, NaC1 secreting shark rectal gland in which unique adenosine receptor subtypes - A2e and A1e receptors - dually regulate chloride secretion. by exploiting advantages of this model system we propose to provide molecular and functional characterization of these receptors. Specific aims are: (1) to clone, sequence, and functionally express in oocytes and receptor deficient cells the unique A2e and A1e adenosine receptor subtypes that regulate NaC1 transport in the rectal gland; (2) to identify the ion channels (C1 and K) that are coupled to these receptor subtypes and to characterize the pathways of this regulation; and (3) to determine the polarized transport of adenosine across basolateral and apical membranes of rectal gland cells and characterize newly recognized apical receptors for adenosine ATP and UTP. The experiments will employ PCR based cloning and sequencing strategies, measurements of Isc and polarized adenosine release in monolayers of primary cultures and patch clamp studies using cultured rectal gland cells. At each stage of the proposed studies we will correlate molecular structure with functional assays for the receptors.