Adrenergic and Purinergic Regulation of Target Cells

描述（由申请人提供）：此更新申请寻求继续使用Madin Darby犬肾（MDCK）细胞和敲除小鼠来评估肾上腺素能和P2Y（核苷酸，嘌呤能）受体的分子和功能方面的研究。我们提出的研究将检验与我们在MDCK细胞和最近在小鼠原发性肾收集管（mpkCCD）细胞以及P2Y2敲除小鼠中获得的发现相关的假设，我们最近发现P2Y2敲除小鼠具有耐盐性高血压和多种肾功能异常。具体目的包括研究：1)MDCK和mpkCCD细胞中G蛋白偶联受体/G蛋白/肾上腺素能受体和P2Y受体效应器的raft/caveolar微域的表达和区隔，以及caveolins的sumoylation（我们最近发现的一种新的修饰）的评估；2)受体敲除小鼠肾P2Y和肾上腺素能受体的功能作用。联合使用培养的肾上皮细胞和敲除小鼠提供了一种互补的策略，旨在揭示肾上腺素能和P2Y受体对上皮细胞和肾功能的调节的新见解。这一发现可能对涉及交感神经系统或细胞损伤导致核苷酸释放的心血管、肾脏和其他疾病有启示。

DESCRIPTION (provided by applicant): This renewal application seeks to continue studies that use Madin Darby Canine Kidney (MDCK) cells and knockout mice to assess molecular and functional aspects of adrenergic and P2Y (nucleotide, purinergic) receptors. The proposed studies will test hypotheses related to findings that we have obtained in MDCK cells and more recently in mouse primary kidney collecting duct (mpkCCD) cells as well as in P2Y2 knockout mice, which we have recently shown have salt-resistant hypertension and multiple abnormalities in renal function. The Specific Aims involve studies of: 1) Expression and compartmentation in raft/caveolar microdomains of G protein-coupled receptor/G-proteins/effectors of adrenergic and P2Y receptors in MDCK and mpkCCD cells and assessment of sumoylation (a novel modification that we have recently identified) of caveolins; 2) Functional roles of renal P2Y and adrenergic receptors using receptor-knockout mice. The combined use of cultured renal epithelial cells and knockout mice provides a complementary strategy that is designed to reveal new insights regarding the regulation of epithelial cells and renal function by adrenergic and P2Y receptors. The findings may have implications for cardiovascular, renal and other disorders that involve the sympathetic nervous system or in which cell injury leads to release of nucleotides. PUBLIC HEALTH RELEVANCE: This project will assess 2 important classes of proteins, receptors for adrenaline (and adrenaline-like drugs) and for a major cell constituent, ATP. The receptors, which are located on the surface of cells, recognize those molecules in the extracellular environment but then alter intracellular function. Experiments are proposed in: 1) tissue culture cells to assess the organization of the components involved in information transfer across the cell surface and 2) mice engineered to have loss in receptors for ATP or adrenaline with the goal of understanding how the receptors regulate fluid and salt handling, in particular by the kidney.