RENAL PROXIMAL TUBULE TRANSPORT--HORMONAL REGULATION

本申请中提出的研究旨在检查G 调节肾脏转运的蛋白质依赖机制 近端小管(PT)。氯化钠和碳酸氢钠的转运是相互调节的 通过像血管紧张素II(AngII)和一种激动剂这样的激素来增加它， 而甲状旁腺素则会降低它。这些荷尔蒙的受体是通过 G蛋白转化为酶或离子通道，从而产生其生物效应。 由G蛋白(效应物)调节的酶和离子通道可能 包括腺酰环化酶(AC)、磷脂酶A2和C(PLA2和PLC)，以及 钾通道和钙通道。大多数荷尔蒙调节某些激素，但不是全部。 效应器。信号通路的特异性是如何确定的，如何 效应器是受调控的，而G蛋白的具体作用尚不清楚。 血管紧张素转换酶和甲状旁腺素等激素激活了PT中的多个效应器，但 目前尚不清楚一种G蛋白是激活一个还是多个效应器。它是 也不清楚单个G蛋白是与一个还是多个受体偶联。 本申请中提出的研究将解决以下问题：1) 大鼠肾脏切片原位杂交研究 六种G蛋白(Gs、Gil-3、Go和Gz)的寡核苷酸探针将 确定哪些G蛋白在PT中表达。2)Western blotting 肾皮质基底外侧膜(BLMV)和刷状缘膜(BBMV) 蛋白特异性抗血清将确定G蛋白在体内的分布 甲状旁腺的根尖膜和基底侧膜。3)效应器(AC、PLA2、 PLC，以及K和Ca通道)，受G蛋白表达的调节 PT将通过表达突变的、成分活性的G来确定 类PT连续细胞系(OK、BSC-1或永生化)中的蛋白质 原代培养)，并测量潜在效应者的活动。 纯化的、预活化的重组G蛋白将被重组为 测定BLMV、BBMV及钾、钙通道活动。4)G 在BLMV和BBMV中将建立蛋白质-受体偶联 一种生化分析方法，用于鉴定一种G蛋白，这种G蛋白由一种 受体，或通过表达具有异常受体的突变G蛋白 在一个连续的肾细胞系中偶联。甲状旁腺素、血管紧张素转换酶和肾上腺素能 将对激动剂进行研究。5)这些G蛋白依赖的能力 将通过测量建立用于规范运输的信号系统 细胞内Na/H逆向转运体、Na/3HCO3共转运体与Na，KATPase活性 表达活化G蛋白和膜囊泡的研究 预活化的重组G蛋白。这些研究将提供更好的 了解PT-氯化钠和NaHCO3转运是如何调节的，以及如何 确定了G蛋白依赖的信号转导的特异性。

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.