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WATER AND ELECTROLYTE HOMEOSTASIS

Molecular detection and immunological localization of gill Na⁺/H⁺ exchanger in the dogfish (Squalus acanthias)

¹Department of Biology, Georgia Southern University, Statesboro, Georgia; ²Mount Desert Island Biological Laboratory, Salsbury Cove, Maine; ³Department of Zoology, University of Florida, Gainesville; ⁴Department of Biology, University of Central Florida, Orlando, Florida; and ⁵Department of Biology, Appalachian State University, Boone, North Carolina

Submitted 4 October 2007 ; accepted in final form 12 December 2007

The dogfish (Squalus acanthias) can make rapid adjustments to gill acid-base transfers to compensate for internal acidosis/alkalosis. Branchial Na⁺/H⁺ exchange (NHE) has been postulated as one mechanism driving the excretion of H⁺ following acidosis. We have cloned gill cDNA that includes an open reading frame coding for a 770-residue protein most homologous (71%) to mammalian NHE2. RT-PCR revealed NHE2 transcripts predominantly in gill, stomach, rectal gland, intestine, and kidney. In situ hybridization with an antisense probe against NHE2 in gill sections revealed a strong mRNA signal from a subset of interlamellar and lamellae cells. We developed dogfish-specific polyclonal antibodies against NHE2 that detected a 70-kDa protein in Western blots and immunologically recognized branchial cells having two patterns of protein expression. Cytoplasmic and apical NHE2 immunoreactivity were observed in cells coexpressing basolateral Na⁺-K⁺-ATPase. Other large ovoid cells more generally staining for NHE2 also were strongly positive for basolateral H⁺-ATPase. Gill mRNA levels for NHE2 and H⁺-ATPase did not change following systemic acidosis (as measured by quantitative PCR 2 h after a 1- or 2-meq/kg acid infusion). These data indicate that posttranslational adjustments of NHE2 and other transport systems (e.g., NHE3) following acidosis may be of importance in the short-term pH adjustment and net branchial H⁺ efflux observed in vivo. NHE2 may play multiple roles in the gills, involved with H⁺ efflux from acid-secreting cells, basolateral H⁺ reabsorption for pH_i regulation, and in parallel with H⁺-ATPase for the generation of HCO₃^– in base-secreting cells.

acid-base regulation; branchial; elasmobranch; sodium/hydrogen antiporter

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