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1 Department of Zoology, University of Florida, Gainesville, FL, USA
2 Department of Biological Sciences, Tokyo Institute of Technology, Yokohama, Japan
3 Department of Physiology and Biophysics and Department of Pharmacology, Case Western Reserve University, Cleveland, OH, USA
4 Department of Biology, Georgia Southern University, Statesboro, GA, USA
* To whom correspondence should be addressed. E-mail: kchoe{at}zoo.ufl.edu.
In mammals, the Na+/H+ exchanger 3 (NHE3) is expressed with Na+/K+-ATPase in renal proximal tubules where it secretes H+ and absorbs Na+ to maintain blood pH and volume. In elasmobranchs (sharks, skates, and stingrays), the gills are the dominant site of pH and osmoregulation. This study was conducted to determine if epithelial NHE homologues exist in elasmobranchs, and if so, to localize their expression in gills and determine if their expression is altered by environmental salinity or hypercapnia. Degenerate primers and RT-PCR were used to deduce partial sequences of mammalian NHE2 and NHE3 homologues from the gills of the euryhaline Atlantic stingray (Dasyatis sabina). Real-time PCR was then used to demonstrate that mRNA expression of the NHE3 homologue increased when stingrays were transferred to low salinities, but not during hypercapnia. Expression of the NHE2 homologue did not change with either treatment. Rapid amplification of cDNA was then used to deduce the complete sequence of a putative NHE3. The 2744 base pair cDNA includes a coding region for a 2511 amino acid protein that is 70% identical to human NHE3 (SLC9A3). Antisera generated against the carboxyl tail of the putative stingray NHE3 labeled the apical membranes of Na+/K+-ATPase-rich epithelial cells, and acclimation to fresh water caused a redistribution of labeling in the gills. This study provides the first NHE3 cloned from an elasmobranch and is the first to demonstrate an increase in gill NHE3 expression during acclimation to low salinities, suggesting that NHE3 can absorb Na+ from ion-poor environments.
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