Adrenergic and Purinergic Regulation of Target Cells

描述(由申请人提供)：本申请是评估肾上腺素能受体和P2Y嘌呤能受体信号转导方面的修订建议。我们最近的初步数据包括在MDCK-D1细胞中鉴定和克隆了五种不同的P2Y受体，并发现了ATP释放作为一种自分泌/旁分泌机制的关键作用，以建立这些细胞中信号转导的基础水平。拟议的研究将集中在MDCK-D1细胞中的研究，旨在确定关于P2Y受体的核苷酸释放和自分泌/旁分泌信号的信息，G蛋白偶联受体/G蛋白和效应分子的化学计量学和区隔，激动剂促进的肾上腺素能和P2受体的调节，以及在使用小鼠基因敲除的平行研究中，P2Y受体和肾上腺素能受体在肾功能调节中的作用。这些研究将检验几个假说，包括：1)核苷酸释放是多个信号转导途径设定点的关键因素；2)P2Y受体、A1-AR和(B2-AR)在极化的MDCK细胞中具有不同的靶点，并创建分隔的信号微域；以及3)肾上腺素能和P2Y受体将显示出“交叉调节”的证据。后者可能通过肾脏和其他细胞中的交感节后神经元参与反应的调节。在其他实验中，我们将尝试通过对某些受体敲除的小鼠的研究来确定肾上腺素能和P2受体在体内的功能活性。总体而言，这些数据应该为肾上腺素能和P2Y受体对上皮细胞，特别是肾上皮细胞的调控提供新的见解，并应该有助于确定这些受体的细胞和肾脏生理作用。此外，这些发现可能对心血管、肾脏和其他涉及交感神经系统的疾病或细胞损伤导致核苷酸释放的疾病具有病理生理学和治疗意义。

DESCRIPTION (provided by applicant): This application is a revised proposal to assess aspects of adrenergic receptor and P2Y purinergic receptor signalling. Our recent, preliminary data for this application include the identification and cloning of five different P2Y receptors in MDCK-D1 cells and discovery of a key role of ATP release as an autocrine/paracrine mechanism for establishing basal levels of signal transduction in these cells. The proposed studies will focus on studies in MDCK-D1 cells, designed to define information regarding nucleotide release and autocrine/paracrine signalling by P2Y-receptors, stoichiometry and compartmentation of G protein-coupled receptors/G protein and effector molecules, agonist-promoted regulation of adrenergic and P2-receptors, and in parallel studies using murine knockouts, the role of P2Y receptors and adrenergic receptors in regulation of renal function. The studies will test several hypotheses, including the possibilities that: 1) nucleotide release is a critical factor for the set point of multiple signal transduction pathways; 2) P2Y receptors, A1-AR and (B2-AR differentially target in polarized MDCK cells, and create compartmentalized signalling microdomains; and 3) adrenergic and P2Y receptors will show evidence of"cross regulation." The latter may contribute to modulation of response via sympathetic postganglionic neurons in the kidney and other cells. In additional experiments, we will attempt to define the functional activity of adrenergic and P2 receptors in vivo by the studies of mice with knockouts of certain receptors. Overall, the data should provide new insights into regulation of epithelial cells, in particular renal epithelial cells, by adrenergic and P2Y receptors and should help define the cell and renal physiological role of those receptors. In addition, the findings may have pathophysiologic and therapeutic implications for diseases such as cardiovascular, renal, and other disorders that involve the sympathetic nervous system or in which cell injury leads to release of nucleotides.