ADENOSINE RECEPTORS AND ION TRANSPORT IN EPITHELIA

这项研究的长期目标是确定细胞内 和分子基础的调节氯分泌上皮细胞 腺苷受体 了解自育激素调节因子 通过腺苷转运需要（1）识别主要的 分子结构和相应的结构活性谱 上皮细胞中存在的特异性腺苷受体亚型，（2） 识别特定的离子通道， 腺苷受体的效应物，和（3）代谢的整合 以及腺苷转运到受体功能的调节方案中。 G蛋白偶联受体分子生物学的进展现在提供了 腺苷受体系统性鉴定的基础 亚型 我们将联合收割机结合独特的腺苷的分子特征 具有离子电生理特征的受体亚型 通道（C1和K）和腺苷释放细胞膜的测量 为了获得氯化物反馈调节的全面画面， 腺苷分泌。 我们建议的研究重点是高度 专门的，同质的，分泌NaCl的鲨鱼直肠腺， 独特的腺苷受体亚型-A2 e和A1 e受体-双重 调节氯化物分泌。 通过利用这种模式的优势， 我们建议系统提供分子和功能表征 这些受体。 具体目标是：（1）克隆，测序， 在卵母细胞和受体缺陷细胞中功能性地表达 A2 e和A1 e腺苷受体亚型调节NaC 1转运 直肠腺;（2）识别离子通道（C1和K）， 与这些受体亚型偶联，并表征 （3）确定极化运输， 腺苷穿过直肠腺细胞的基底膜和顶膜 并表征新识别的腺苷ATP的顶端受体， UTP。 实验将采用基于PCR的克隆和测序 策略，测量Isc和极化腺苷释放， 原代培养物的单层和使用培养的细胞的膜片钳研究 直肠腺细胞 在建议研究的每个阶段，我们会 将分子结构与受体的功能测定相关联。

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.