Regulation of a Novel Intestinal NHE Isoform (NHE8)

描述（申请人提供）：胃肠道在成熟过程中经历了显著的形态和功能变化，以满足日益增长的营养运输需求。胃肠道的主要功能是运输营养物质和液体，以维持足够的电解质稳态。1岁婴儿的胃肠道每天处理大约3升液体，导致每天只有50克粪便排出，这表明胃肠道在生命早期具有复杂的运输能力。我们之前的研究表明，肠道钠/氢交换剂（NHE2和NHE3）的表达在生命早期较低，并随着成熟而显著增加。此外，敲除NHE2和NHE3小鼠模型的表型在液体和电解质平衡中显示出最小的扰动，表明在这一成熟时期存在另一个NHE。我们的研究表明，NHE8在胃肠道上皮细胞的顶膜中表达，在生命早期处于高水平，并在成年后下降。这种定位与NHE8在肾上皮顶端表达的发现相似。因此，本研究旨在验证NHE8是一种独特的、新颖的NHE，在哺乳和断奶发育期间的电解质稳态中起重要作用。我们计划探索五个具体目标，以确定NHE8的生理作用及其在生理和病理条件下的调节。特异性Aim 1旨在表征NHE8的功能和药理学特性。我们的第二个特异性目标是测定NHE8在大鼠肠道和NHE2/3敲除小鼠中沿头尾轴和隐窝绒毛轴的表达。我们还将寻求进一步加强我们的假设，即NHE8在肠上皮细胞的刷缘膜上表达。在Specific Aim 3中，我们计划研究基础条件下EGF和糖皮质激素对NHE8调控的转录机制。我们的初步数据表明NHE8基因的表达受这些生理效应物的调控。EGF和糖皮质激素对胃肠道的功能成熟很重要，并已被证明调节肠道NHEs。此外，已知的肠道NHEs在炎症期间受到调节，我们计划继续我们的初步观察，即NHE8在内毒素血症（例如LPS管理）和TNFa暴露期间受到调节。因此，我们的Specific Aim 4旨在确定TNFa对NHES蛋白和基因表达的急性和慢性影响的机制。特异性Aim 5旨在确定第二信使（环核苷酸和细胞内钙）对NHES的急性调节所涉及的机制。总的来说，拟议的研究可能会对我们理解早期控制电中性NaCI吸收的分子机制及其与肠道内稳态各种扰动的关系产生重大影响。

DESCRIPTION (provided by applicant): The gastrointestinal tract undergoes significant morphological and functional changes during maturation to meet the increasing demands for nutrient transport. The major function of the gastrointestinal tract is to transport nutrients and fluids to maintain adequate electrolyte homeostasis. The gastrointestinal tract of a 1- year-old infant handles approximately 3 liters of fluid per day, resulting in only 50 grams of daily stool output, indicating the complex transport capacity of the gastrointestinal tract during early life. Our previous studies have shown that expression of the intestinal sodium/hydrogen exchangers (NHE2 and NHE3) is low during early life, and increases significantly with maturation. Moreover, the phenotypes of knockout mouse models of NHE2 and NHE3 show minimal perturbations in fluid and electrolyte balance, indicating the presence of another NHE(s) during this period of maturation. Our study demonstrates that NHE8 is expressed in the apical membrane of epithelial cells in the gastrointestinal tract, at high levels during early life, and decreasing into adulthood. This localization is similar to the finding that NHE8 is expressed apically in the renal epithelium. Therefore, the proposed studies are designed to test the hypothesis that NHE8 represents a unique, novel NHE, which is important in electrolyte homeostasis during the suckling and weaning periods of development. We plan to explore five specific aims to determine the physiological role of NHE8 and its regulation in physiological and pathological conditions. Specific Aim 1 is designed to characterize functional and pharmacological properties of NHE8. Our second Specific Aim is designed to determine NHE8 expression along the cephalo-caudal and crypt-villus axes in the rat intestine and in NHE2/3 knock-out mice. We will also seek to further strengthen our supposition that NHE8 is expressed on the brush-border membrane of intestinal epithelial cells. In Specific Aim 3, we plan to investigate the transcriptional mechanisms of NHE8 regulation under basal conditions and as regulated by EGF and glucocorticoids. Our preliminary data show that NHE8 gene expression is regulated by these physiological effectors. EGF and glucocorticoids are important for functional maturation of the gastrointestinal tract, and have been shown to regulate intestinal NHEs. Furthermore, known intestinal NHEs are regulated during inflammation and we plan to follow up on our preliminary observation that NHE8 is regulated during endotoxemia (e.g. LPS administration) and by TNFa exposure. Therefore our Specific Aim 4 is designed to determine the mechanism responsible for acute and chronic effects of TNFa on NHES protein and gene expression. Specific Aim 5 is designed to define the mechanisms involved in the acute regulation of NHES by second messengers (cyclic nucleotides and intracellular calcium). Overall, the proposed studies are likely to have a significant impact on our understanding of the molecular mechanisms controlling electroneutral NaCI absorption in early life and its relationship to various perturbations of intestinal homeostasis.