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

鱼的鳃完成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。 这些离子通过鳃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，已经提出了各种各样的细胞系统来推动淡水和海洋鱼类物种的这些交流。Na+/H+交换蛋白（NHE）已在许多哺乳动物和非哺乳动物系统中被描述。这些蛋白质被认为具有多种功能，包括细胞体积调节、pH平衡和Na+摄取。已经在哺乳动物中表征了几种不同类型的NHE，其中NHE-2和3被认为对转运上皮组织如肾和肠具有特异性。在这项持续的研究中，作者专注于鱼鳃中类似Na+/H+蛋白的存在和功能。 利用RT-PCR和3' RACE，他们最近证明了在海洋长角杜父鱼（Myoxocephalus decimspinosus），广盐性mummichog（底heteroclitus），板鳃类（Raja erinacea和Squalus acanthias）和颅（Myxine spinosa）的鳃中存在与NHE同源的mRNA转录本。当M.将红腹锦鸡制成酸中毒，定量PCR显示鳃中NHE的mRNA以与体内测量的净酸流出平行的方式增加。也使用哺乳动物NHE-1和NHE-3同种型的抗体建立了这些物种中鳃NHE的存在。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的运输上皮细胞，如肾近端小管。他们的研究目标有三个方面：1）确定海水鱼类鳃组织中Na+/H+反向转运蛋白mRNA和蛋白表达的存在和亚型分布; 2）描述鳃上皮中NHE反向转运蛋白的特定细胞分布; 3）表征全身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，他们建议使用分子、生理和免疫学技术相结合的方法来研究鳃离子转运机制。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变化后的体内和细胞变化。这些蛋白质的细胞分布将使用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以及定量PCR将用于检测和扩增目前未知的NHE区域。 作者已经克隆了第一个全长序列的鱼类NHE亚型，这是最密切相关的专门上皮NHE-2在哺乳动物。 这项研究将提供新的洞察鳃酸碱和离子调节的生理学，并可能导致更好地了解其他脊椎动物NHE亚型的分子结构和保守的调节结构域。这个为期四年的项目将涉及本科生和理学硕士学生在家乡机构（格鲁吉亚南方大学）和海洋野外站（山荒岛生物实验室）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).