RENAL PROXIMAL TUBULE TRANSPORT--HORMONAL REGULATION

肾近端肾小管运输——激素调节

• 批准号: 3463897
• 负责人: Richard Tyler Miller
• 金额: $ 10.4万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-08-01 至 1994-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3463897
• 关键词: G protein; adenosinetriphosphatase; adenylate cyclase; adrenergic receptor; angiotensin II; antiport; apical membrane; bicarbonates; biological signal transduction; calcium channel; cell line; genetic manipulation; hormone regulation /control mechanism; in situ hybridization; membrane transport proteins; oligonucleotides; parathyroid hormones; phospholipase A2; phospholipase C; potassium channel; renal cortex; renal tubular transport; sodium; western blottings

The studies proposed in this application are designed to examine G protein-dependent mechanisms by which transport is regulated in the renal proximal tubule (PT). NaCl and NaHCO3 transport is reciprocally regulated by hormones like angiotensin II (AngII) and a agonists which increase it, and by PTH which decreases it. Receptors for these hormones are coupled by G proteins to enzymes or ion channels which produce their biologic effects. The enzymes and ion channels regulated by G proteins (effectors) may include adenylyl cyclase (AC), phospholipases A2 and C (PLA2 and PLC), and K and Ca channels. Most hormones regulate some, but not all of these effectors. How the specificity of the signalling pathway is determined, how effectors are regulated, and by which G proteins is not known in detail. Hormones such as AngII and PTH activate multiple effectors in the PT, but it is not clear if one G protein activates one or multiple effectors. It is also not clear if a single G protein couples to one or multiple receptors. The studies proposed in this application will address these questions: 1) In situ hybridization studies in sections of rat kidneys using oligonucleotide probes to six G proteins (Gs, Gil-3, Go, and Gz) will determine which G proteins are expressed in the PT. 2) Western blotting of renal cortical basloateral (BLMV) and brush border membranes (BBMV) with G protein-specific antisera will establish the distribution of G proteins on the apical and basolateral membranes of the PT. 3) The effectors (AC, PLA2, PLC, and K and Ca channels), regulated by the G proteins expressed in the PT will be determined by expressing mutant, constitutively active G proteins in PT-like continuous cell lines (OK, BSC-1, or immortalized primary cultures), and measuring the activity of the potential effectors. Purified, preactivated recombinant G proteins will be reconstituted into BLMV and BBMV, and K and Ca channel activity measured. 4) G protein-receptor coupling will be established in BLMV and BBMV using biochemical assays that identify a G protein that is activated by a receptor, or by expressing a mutant G protein with abnormal receptor coupling in a continuous renal cell line. PTH, AngII, and a adrenergic agonists will be studied. 5) The ability of these G protein-dependent signalling systems to regulate transport will be established by measuring Na/H antiporter, Na/3HCO3 cotransporter, and Na,KATPase activity in cells expressing activated G proteins and membrane vesicles containing preactivated, recombinant G proteins. These studies will provide a better understanding of how PT NaCl and NaHCO3 transport is regulated, and how specificity for G protein-dependent signalling is determined.

本应用程序中提出的研究旨在检查G 在肾脏中调节转运的蛋白质依赖性机制 近端小管（PT）。 NACL和NAHCO3运输经过相互监管 通过血管紧张素II（Angii）和增加其增加的激素，增加了激素， 并通过pth减少它。这些激素的受体与 G蛋白到产生其生物学作用的酶或离子通道。 由G蛋白调节的酶和离子通道（效应子）可能 包括腺苷酸环化酶（AC），磷脂酶A2和C（PLA2和PLC），以及 K和CA通道。大多数激素调节一些，但并非全部 效应子。如何确定信号通路的特异性，如何确定 效应子受到调节，并通过该效应子详细介绍G蛋白。 诸如ANGII和PTH之类的激素激活了PT中的多个效应子，但是 目前尚不清楚一个G蛋白是否激活一个或多个效应子。这是 同样尚不清楚单个G蛋白是否与一个或多个受体伴侣。 本申请中提出的研究将解决以下问题：1） 使用的原位杂交研究在大鼠肾脏的切片中使用 寡核苷酸探针至六种G蛋白（GS，GIL-3，GO和GZ）将 确定哪些G蛋白在PT中表达。 2）蛋白质印迹 带有G 蛋白质特异性抗血清将建立G蛋白的分布 PT的顶端和基底外侧膜。 3）效应子（AC，PLA2， PLC和K和CA通道），由在 PT将通过表达突变体，组成性活性G来确定 PT样连续细胞系中的蛋白质（OK，BSC-1或永生 原始培养物），并测量潜在效应子的活性。 纯化的，预归作的重组G蛋白将被重构为 BLMV和BBMV，以及K和CA通道活性。 4）g 蛋白质受体耦合将在BLMV和BBMV中建立 鉴定被A激活的G蛋白的生化测定 受体，或通过表达具有异常受体的突变G蛋白 在连续的肾细胞系中耦合。 PTH，ANGII和肾上腺素能 激动剂将被研究。 5）这些G蛋白依赖性的能力 通过测量将建立调节运输的信号传导系统 Na/h抗胞菌，Na/3HCO3共转运蛋白和Na，Katpase活性在细胞中 表达活化的G蛋白和膜囊泡 预先活化的重组G蛋白。这些研究将提供更好的 了解如何调节PT NaHCO3运输以及如何调节 确定G蛋白依赖性信号的特异性。