CLONING & EXPRESSION OF EPITHELIAL NA+/H+ EXCHANGERS

大多数腹泻疾病是通过抑制绒毛细胞刷毛而发生的。 边界Na+/H+交换。我们克隆了两只50%完全相同的兔回肠 绒毛细胞Na+/H+交换器：NHE-1和NHE-2。NHe-1为基准面 Na+/H+交换器在回肠绒毛细胞基底外侧的定位 免疫印迹和免疫细胞化学研究中的膜，其标记 对阿米洛利抑制的敏感性，以及它被有丝分裂原激活。NHE-2 很可能是兔回肠绒毛细胞Na+/H+的顶端异构体 NHE-2在兔肾和回肠中大量表达，它是 对阿米洛利不太敏感。 这项计划的总体目标是克隆人牙根尖上皮 Na+/H+交换器，检测其在人体肠道中的表达，以及其在肠道中的表达。 已知的抑制刷状边界Na+/H+的第二信使的调节 交换。我们将首先证明兔NHE-2是根尖上皮细胞 异构体在兔根尖膜上的定位 回肠在免疫印迹和免疫细胞化学研究中，通过显示 NHE-2仅在中性的NaCI吸收上皮细胞中表达，并通过 阿米洛利的NHE-2‘S K1及其内pH设定值的测定我们会 用NHE-2筛选人肾皮质和空肠组织中的c DNA文库 试图克隆人类心尖部交换器。全长克隆将是 Na~+/H~+交换缺陷型中国仓鼠肺成纤维细胞的表达 行PS120以演示其编码为功能的Na+/H+交换器， 允许成纤维细胞进行阿米洛利敏感的Na+/H+交换。 使用由人NHE-2构建的探针以定量方法 逆转录-聚合酶链式反应分析 胃肠道组织活检组织中的人NHE-2信使RNA 正常成人和心尖Na~+/H~+儿童空肠的肌束 交换缺乏型先天性慢性分泌性腹泻。我们的最终目标 将通过确定人类NHE-2的第二信使调节来定义 如果在PS120成纤维细胞中稳定表达的人NHE-2能够被调控 通过已知的刷状边界Na+？H+交换的抑制剂，如cAMP，蛋白质 我们将确定这种抑制是否是 伴随着交换器的磷酸化。到时候我们会的 确定哪些是功能上假定的蛋白激酶共识 位于Nhe-2‘S C端胞质尾部的序列 这些激酶共识的单向连续缺失的影响 序列对第二信使调节人类NHE-2的能力有影响。

Most diarrheal diseases occur through inhibition of villus cell brush border Na+/H+ exchange. We have cloned two 50% identical rabbit ileal villus cell Na+/H+ exchangers: NHE-1 and NHE-2. NHE-1 is the basolateral Na+/H+ exchanger by its localization to the ileal villus cell basolateral membrane in immunoblotting and immunocytochemical studies, its marked sensitivity to amiloride inhibition, and its activation by mitogens. NHE-2 is most likely the apical isoform of the rabbit ileal villus cell Na+/H+ exchangers; NHE-2 is greatly expressed in rabbit kidney and ileum and it is less sensitive to amiloride. The overall GOALS of this proposal is to clone the human apical epithelial Na+/H+ exchanger, examine its expression in the human gut and its regulation by second messengers known to inhibit brush border Na+/H+ exchange. We will first prove that rabbit NHE-2 is the apical epithelial isoform by demonstrating its localization to the apical membrane of rabbit ileum in immunoblotting and immunocytochemical studies, by showing that NHE-2 is solely expressed in neutral NaC1 absorbing epithelia and by determining NHE-2's K1 of amiloride and its internal pH set point. We will use NHE-2 to screen human kidney cortex and jejunum cDNA libraries in an attempt to clone the human apical exchanger. A full length clone will be expressed in Na+/H+ exchange deficient Chinese Hamster Lung fibroblast cell line PS120 to demonstrate that it codes for a functional Na+/H+ exchanger, allowing the fibroblasts to perform amiloride sensitive Na+/H+ exchange. Using a probe constructed from human NHE-2 in a quantitative method of Reverse Transcription-PCR analysis we will characterize the expression of human NHE-2 messenger RNA in tissue biopsies from the gastrointestinal tract of normal adults and from the jejunum of children with apical Na+/H+ exchange deficient congenital chronic secretory diarrhea. Our final goal will be to define second messenger regulation of human NHE-2 by determining if human NHE-2, as stably expressed in PS120 fibroblasts, can be regulated by known inhibitors of brush border Na+?H+ exchange, such as cAMP, protein kinase C, and Ca++/CaM kinase II. We will determine if such inhibition is accompanied by the phosphorylation of the exchanger. We will then determine which are the functional putative protein kinase consensus sequences located on NHE-2's C-terminus cytoplasmic tail, by examining the effects that unidirectional serial deletions of these kinase consensus sequences have on the ability of second messengers to regulate human NHE-2.