RUI: Na+/H+ Exchange in Fish Acid-Base and Ion Regulation

鱼的鳃完成了许多与陆生动物的肺、肝和肾相同的功能。鳃是气体交换、代谢废物排泄、盐平衡和pH调节的场所。在细胞水平上，某些蛋白质被认为负责钠（Na+）和酸（H+）等盐的运动。这些离子在鳃间的转移反过来可能有助于这些动物的酸碱和离子调节。在过去的70年里，人们提出了各种细胞系统来驱动淡水和海洋鱼类物种的这些交换。Na+/H+交换蛋白（NHE）已经在许多哺乳动物和非哺乳动物系统中被描述。这些蛋白被认为具有多种功能，包括细胞体积调节、pH平衡和Na+摄取。几种不同类型的NHEs在哺乳动物中具有特征，其中NHE-2和3被认为是特异性运输上皮组织，如肾和肠。在这项持续的研究中，作者将重点放在了鱼鳃中类似Na+/H+蛋白的存在和功能上。利用RT-PCR和3′RACE，他们最近证明了在海洋长角鱼（Myoxocephalus octodecimspinosus）、宽盐mummichog （Fundulus heteroclitus）、蓝鳃鱼（Raja erinacea和Squalus acanthias）和颅类动物（Myxine glutinosa）的鳃中存在与NHE同源的mRNA转录物。当谷氨酰胺酸中毒时，定量PCR显示鳃中NHE mRNA的增加与体内测量的净酸外排平行。利用针对哺乳动物NHE-1和NHE-3亚型的抗体，也确定了这些物种中存在鳃NHE。研究人员提出，这些海洋物种酸中毒后的净H+排泄是由鳃Na+/H+交换驱动的，其方式类似于哺乳动物运输上皮（如肾近端小管）。他们的研究目标有三个方面：1)确定Na+/H+反转运蛋白mRNA和蛋白在海鱼鳃组织中的存在和异构体分布；2)描述NHE反转运蛋白在鳃上皮中的特定细胞分布；3)描述系统性酸中毒或外部盐度变化对鳃NHE表达的影响。为了实现这些目标，他们建议结合分子、生理和免疫学技术来研究鳃离子的转运机制。他们计划设计鱼类特异性单克隆和/或多克隆抗体，以关联酸中毒或外部盐度改变后的体内和细胞变化。这些蛋白的细胞分布将通过免疫组织化学定位和共聚焦激光显微镜显示。RT-PCR, 3‘和5’ RACE和定量PCR将用于检测和扩增目前未知的NHEs区域。作者已经克隆了鱼类NHE亚型的第一个全长序列，该亚型与哺乳动物的特化上皮NHE-2最密切相关。该研究将为鳃酸碱和离子调控的生理学提供新的见解，并可能有助于更好地理解其他脊椎动物NHE亚型的分子结构和保守调控域。这个为期四年的项目将包括本科生和理科硕士学生在家乡机构（佐治亚南方大学）和海洋野外站（Mount Desert Island生物实验室）进行研究。

The gills of fish accomplish many of the same functions as the lungs, liver and kidneys in terrestrial animals. Gills are the site for gas exchange, metabolic waste excretion, salt balance, and pH adjustments. At the cellular level, certain proteins are thought to be responsible for the movement of salts like sodium (Na+) and acid (H+). The transfers of these ions across the gills may in turn assist in acid-base and ion regulation in these animals. Over the past 70 years, a variety of cellular systems have been proposed to drive these exchanges in freshwater and marine fish species. Na+/H+ exchange proteins (NHE) have been described in a number of mammalian and non-mammalian systems. These proteins are thought to function in several roles including cell volume regulation, pH balance, and Na+ uptake. Several different types of NHEs have been characterized in mammals with NHE-2 and 3 thought to be specific to transporting epithelial tissues such as kidney and intestine. In this continuing study, the authors have focused on the presence and function of similar Na+/H+ proteins in fish gills. Using RT-PCR and 3' RACE they have recently demonstrated the presence of mRNA transcripts homologous to NHE in the gills of the marine long-horned sculpin (Myoxocephalus octodecimspinosus), the euryhaline mummichog (Fundulus heteroclitus), elasmobranchs (Raja erinacea and Squalus acanthias) and a craniate (Myxine glutinosa). When M. glutinosa was made acidotic, quantitative PCR revealed that mRNA for NHE in the gills increases in a fashion parallel to net acid efflux measured in vivo. The presence of gill NHE in these species was also established using antibodies for the mammalian NHE-1 and NHE-3 isoforms. The investigators have proposed that net H+ excretion following acidosis in these marine species is driven by gill Na+/H+ exchange in a fashion similar to mammalian transporting epithelia such as the renal proximal tubule. The objectives of their research are three-fold: 1) to determine the presence and isoform distribution of Na+/H+ antiporter mRNA and protein expression in the gill tissue of marine fish; 2) to describe the specific cellular distribution of the NHE antiporters in the gill epithelium; and 3) to characterize the effects of systemic acidosis or variations in external salinity on gill NHE expression. To accomplish these goals, they propose to study gill ion transport mechanisms using a combination of molecular, physiological, and immunological techniques. They plan to design fish-specific monoclonal and/or polyclonal antibodies to correlate in vivo and cellular changes following acidosis or alterations in the external salinity. The cellular distribution of these proteins will be shown using immunohistochemical localization and confocal laser microscopy. RT-PCR, 3' and 5' RACE and quantitative PCR will be used to detect and amplify regions of currently unknown NHEs. The authors have already cloned the first full-length sequence for a fish NHE isoform which is most closely related to the specialized epithelial NHE-2 in mammals. This study will provide new insight into the physiology of gill acid-base and ion regulation, and may lead to a greater understanding of the molecular structure and conserved regulatory domains of other vertebrate NHE isoforms. This four-year project will involve both undergraduate and Masters of Science students doing research at the home institution (Georgia Southern University) and at a marine field station (The Mount Desert Island Biological Laboratory).