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First published online August 8, 2008
Journal of Experimental Biology 211, 2584-2599 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.018663

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Evidence for an apical Na–Cl cotransporter involved in ion uptake in a teleost fish

Junya Hiroi^1,2,*, Shigeki Yasumasu², Stephen D. McCormick^3,4, Pung-Pung Hwang⁵ and Toyoji Kaneko⁶

¹ Department of Anatomy, St Marianna University School of Medicine, Miyamae-ku, Kawasaki 216-8511, Japan
² Department of Materials and Life Sciences, Faculty of Science and Technology, Sophia University, Chiyoda-ku, Tokyo 102-8554, Japan
³ USGS, Conte Anadromous Fish Research Center, Turners Falls, MA 01376, USA
⁴ Department of Biology, University of Massachusetts, Amherst, MA 01003, USA
⁵ Institute of Cellular and Organismic Biology, Academia Sinica, Nankang, Taipei 11529, Taiwan, Republic of China
⁶ Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan

^* Author for correspondence (e-mail: j-hiroi{at}marianna-u.ac.jp)

Accepted 2 June 2008

Cation–chloride cotransporters, such as the Na⁺/K⁺/2Cl^– cotransporter (NKCC) and Na⁺/Cl^– cotransporter (NCC), are localized to the apical or basolateral plasma membranes of epithelial cells and are involved in active ion absorption or secretion. The objectives of this study were to clone and identify `freshwater-type' and `seawater-type' cation–chloride cotransporters of euryhaline Mozambique tilapia (Oreochromis mossambicus) and to determine their intracellular localization patterns within mitochondria-rich cells (MRCs). From tilapia gills, we cloned four full-length cDNAs homologous to human cation–chloride cotransporters and designated them as tilapia NKCC1a, NKCC1b, NKCC2 and NCC. Out of the four candidates, the mRNA encoding NKCC1a was highly expressed in the yolk-sac membrane and gills (sites of the MRC localization) of seawater-acclimatized fish, whereas the mRNA encoding NCC was exclusively expressed in the yolk-sac membrane and gills of freshwater-acclimatized fish. We then generated antibodies specific for tilapia NKCC1a and NCC and conducted whole-mount immunofluorescence staining for NKCC1a and NCC, together with Na⁺/K⁺-ATPase, cystic fibrosis transmembrane conductance regulator (CFTR) and Na⁺/H⁺ exchanger 3 (NHE3), on the yolk-sac membrane of tilapia embryos acclimatized to freshwater or seawater. The simultaneous quintuple-color immunofluorescence staining allowed us to classify MRCs clearly into four types: types I, II, III and IV. The NKCC1a immunoreactivity was localized to the basolateral membrane of seawater-specific type-IV MRCs, whereas the NCC immunoreactivity was restricted to the apical membrane of freshwater-specific type-II MRCs. Taking account of these data at the level of both mRNA and protein, we deduce that NKCC1a is the seawater-type cotransporter involved in ion secretion by type-IV MRCs and that NCC is the freshwater-type cotransporter involved in ion absorption by type-II MRCs. We propose a novel ion-uptake model by MRCs in freshwater that incorporates apically located NCC. We also reevaluate a traditional ion-uptake model incorporating NHE3; the mRNA was highly expressed in freshwater, and the immunoreactivity was found at the apical membrane of other freshwater-specific MRCs.

Key words: cation–chloride cotransporter, Na⁺/K⁺/2Cl^– cotransporter, Na⁺/Cl^– cotransporter, ion transport, mitochondria-rich cell, chloride cell, tilapia

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