RENAL PROXIMAL TUBULE TRANSPORT--HORMONAL REGULATION

肾近端肾小管运输——激素调节

基本信息

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
项目类别：
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

项目摘要

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.

本申请中提出的研究旨在检查 G 肾脏转运调节的蛋白质依赖性机制近端小管（PT）。 NaCl 和 NaHCO3 运输是相互调节的通过血管紧张素 II (AngII) 等激素和增加血管紧张素 II 的激动剂，并通过 PTH 降低它。这些激素的受体通过以下方式耦合 G 蛋白与酶或离子通道结合，产生其生物效应。 G 蛋白（效应器）调节的酶和离子通道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，但不是全部效应器。信号通路的特异性是如何确定的，如何效应子受到调节，但 G 蛋白的详细情况尚不清楚。 AngII 和 PTH 等激素可激活 PT 中的多个效应器，但是目前尚不清楚一种 G 蛋白是否会激活一个或多个效应器。这是也不清楚单个 G 蛋白是否与一个或多个受体偶联。本申请中提出的研究将解决以下问题：1) 使用大鼠肾脏切片进行原位杂交研究六种 G 蛋白（Gs、Gil-3、Go 和 Gz）的寡核苷酸探针将确定 PT 中表达的 G 蛋白。 2) 蛋白质印迹法肾皮质基底侧膜 (BLMV) 和刷状缘膜 (BBMV) 与 G 蛋白质特异性抗血清将确定 G 蛋白在 PT 的顶膜和基底外侧膜。 3）效应器（AC、PLA2、 PLC、K 和 Ca 通道），受 G 蛋白表达的调节 PT 将通过表达突变型、组成型活性 G 来确定 PT 样连续细胞系（OK、BSC-1 或永生化细胞系）中的蛋白质原代培养物），并测量潜在效应器的活性。纯化的、预激活的重组 G 蛋白将被重构为测量 BLMV 和 BBMV，以及 K 和 Ca 通道活性。 4）G 将使用以下方法在 BLMV 和 BBMV 中建立蛋白质-受体偶联鉴定 G 蛋白被激活的生化分析受体，或通过表达具有异常受体的突变G蛋白连续肾细胞系中的偶联。 PTH、AngII 和肾上腺素能将研究激动剂。 5）这些G蛋白的能力依赖将通过测量建立调节运输的信号系统细胞中的 Na/H 逆向转运蛋白、Na/3HCO3 协同转运蛋白和 Na,KATP 酶活性表达活化的 G 蛋白和膜囊泡，其中含有预激活的重组 G 蛋白。这些研究将提供更好的了解 PT NaCl 和 NaHCO3 运输是如何调节的，以及如何确定 G 蛋白依赖性信号传导的特异性。