RENAL PROXIMAL TUBULE TRANSPORT--HORMONAL REGULATION

本申请中提出的研究旨在检查G 蛋白质依赖性机制，通过该机制调节肾脏中的转运 近端小管（PT）。NaCl和NaHCO 3的运输受盐的调节 血管紧张素II（AngII）和一种增加它的激动剂， 以及降低其水平的甲状旁腺激素。这些激素的受体通过 G蛋白与产生其生物效应的酶或离子通道。 G蛋白调节的酶和离子通道（效应器）可能 包括腺苷酸环化酶（AC）、磷脂酶A2和C（PLA 2和PLC）， K和Ca通道。大多数激素调节一些，但不是所有这些 效应器信号通路的特异性是如何确定的， 效应子的调节，并通过哪些G蛋白是不知道的细节。 激素如AngII和PTH激活PT中的多种效应物，但 目前尚不清楚一种G蛋白是否激活一种或多种效应物。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这些问题：1） 大鼠肾组织原位杂交研究 针对六种G蛋白（Gs、Gil-3、Go和Gz）的寡核苷酸探针将 确定哪些G蛋白在PT中表达。2)的蛋白质印迹 肾皮质基底外侧膜（BLMV）和刷状缘膜（BBMV），G 蛋白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，PLA 2， PLC，以及K和Ca通道），由G蛋白调节，G蛋白表达在细胞中。 PT将通过表达突变的组成型活性G 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），并测量潜在效应物的活性。 纯化的预活化重组G蛋白将重构为 测量BLMV和BBMV以及K和Ca通道活性。4)G 蛋白质-受体偶联将在BLMV和BBMV中建立， 生物化学测定法，其鉴定由G蛋白激活的G蛋白， 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偶联。PTH、AngII和肾上腺素能 将研究激动剂。5)这些G蛋白依赖的 将通过测量来建立调节运输的信号系统， 细胞Na/H逆向转运体、Na/3 HCO 3协同转运体和Na，KATP酶活性 表达活化的G蛋白和含有 预活化的重组G蛋白这些研究将提供更好的 了解PT NaCl和NaHCO 3运输是如何调节的，以及如何 测定对G蛋白依赖性信号传导的特异性。

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.