RUI: Functional characterization of Na+/H+ exchangers in marine and freshwater fishes

点击翻译按钮获取中文摘要

The fish gill carries out the roles of lungs, liver and kidneys in land-dwelling animals. This organ provides the site for gas exchange, metabolic waste excretion, salt balance, and acid-base adjustments. A specific protein in the gill cells is thought to exchange sodium for hydrogen across the membrane of the cells (Na+/H+ exchange; NHE) and in combination with other protein transporters, allows the fish to regulate the levels of acid (pH) in their blood and tissues. NHE has 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 NHE, each coded by different genes, have been described in mammals. Apically located NHE2 and NHE3 are thought to be specific to transporting epithelial tissues such as kidneys and intestine. In this study, the investigators have focused on the function of the NHE3 antiporter in fish gill and the role this protein plays in acid-base and ion regulation in marine and freshwater fishes.Using molecular biology approaches the investigators have cloned the full length NHE3 transcript in the gills of the marine long-horned sculpin (Myoxocephalus octodecimspinosus). The sculpin NHE3 shows high homology to mammalian NHE3 orthologs. The sequence also includes a variable minisatellite repeat within the coding region. The function (or effect) of this insert is unknown. Northern blots of mRNA for the NHE3 indicated that the 4.5 kb transcript is present in the gills and the level of transcription increases 0.5-2 hours following acidosis. In situ hybridization of gill sections with an NHE3 anti-sense probe identified branchial cells which are thought to also express both NHE2 and Na+/K+-ATPase. Immunological detection of gill NHE3 using a fish specific antibody recognizes a protein on the apical surface of specific gill cells (putative chloride cells) in freshwater adapted Fundulus heteroclitus and the crucian carp (Carassius carassius). The investigators have proposed that net H+ excretion in these species is driven by gill Na+/H+ exchange and that NHE3 plays a predominant role, even in some freshwater species. The aims of this study are three-fold: 1) to determine the presence and cellular distribution of Na+/H+ antiporter ortholog NHE3 expression in gill tissue of a stenohaline marine, freshwater and a euryhaline teleost; 2) to characterize the effects of systemic acidosis, or variations in external salinity on gill NHE3 expression and distribution; 3) to perform a functional analysis of fish NHE2 and NHE3 transfected and expressed in cell culture. To accomplish these goals, they plan to study gill transfer mechanisms using a combination of molecular, physiological, and immunological techniques. They will design fish specific probes and antibodies and correlate in vivo and cellular changes (both mRNA and expressed protein) following acidosis and alterations in the external salinity. The authors will also functionally express the fish NHE in a transfected mammalian fibroblast cell line and begin a study of the kinetics in vitro. This project will provide new insight into the function and physiology of gill acid-base and ion regulation and may lead to a greater understanding of the molecular structure and conserved regulatory domains of vertebrate NHE isoforms. This four year project will involve high school, undergraduate, and Masters of Science students doing research at the home institution (Georgia Southern University), a marine field station (The Mount Desert Island Biological Laboratory) during the summer months, and shorter training visits to Johns Hopkins University.