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

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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).