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First published online July 6, 2005
Journal of Experimental Biology 208, 2673-2682 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01684
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Aquaporin-3 expressed in the basolateral membrane of gill chloride cells in Mozambique tilapia Oreochromis mossambicus adapted to freshwater and seawater

Soichi Watanabe*, Toyoji Kaneko and Katsumi Aida

Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo, Tokyo 113-8657, Japan



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  		Fig. 1. Sequencing analysis. (A) Alignment of deduced amino acid sequences of AQP3 from Mozambique tilapia, European eel, Japanese dace, Xenopus and human. Asterisks and hyphens indicate identical residues and gaps introduced for alignment, respectively. Two NPA motifs are shaded. The antibody was raised against a synthetic peptide corresponding to the underlined sequence. (B) Hydropathy analysis of the deduced amino acid sequence of tilapia AQP3. Characteristic features of AQP are conserved in tilapia AQP3: putative six transmembrane domains (TM1–6) and five connecting loops (A–E). Consensus NPA motifs are present in loops B and E.

 


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  		Fig. 2. Northern blot analysis of AQP3 in the gill of tilapia adapted to freshwater. An arrowhead indicates the signal detected with the [32P]-labeled tilapia AQP3 cDNA specific probe.

 


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  		Fig. 3. Tissue distribution of AQP3 mRNA, detected by RT-PCR, in freshwater (FW)- and seawater (SW)-adapted tilapia. Total RNAs were reverse-transcribed and amplified by PCR to produce 500 bp bands. NC, negative control; PC, positive control.

 


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  		Fig. 4. Functional expression of tilapia AQP3 in Xenopus oocytes. (A) Representative data of osmotic oocyte volume V changes in control oocytes (control), tilapia AQP3 cRNA-injected oocytes (AQP3), those with 0.3 mmol l–1 HgCl2 treatment (AQP3+Hg) and those with HgCl2 treatment followed by 5 mmol l–1 ß-mercaptoethanol (AQP3+Hg+2ME). (B) Osmotic water permeability (Pf) in different experimental groups. Values are means ± S.E.M. Values marked with different letters are significantly different from one another at P<0.01.

 


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  		Fig. 5. Western blot analysis with an antibody raised against a synthetic peptide corresponding to part of the C-terminal region of tilapia AQP3. After immunoprecipitation with anti-tilapia AQP3, the gill protein sample was separated by SDS-polyacrylamide gel electrophoresis and transferred to a membrane. Immunoreactive bands were detected by the ABC method. Positions of molecular mass markers (kDa) are shown on the left.

 


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  		Fig. 6. Double immunofluorescence microscopy of gills in tilapia adapted to freshwater (FW; A,C,E,G) and seawater (SW; B,D,F,H), stained with anti-tilapia AQP3 (A,B) and anti-Na+/K+-ATPase as a marker of chloride cells (C,D). E and F are merged images of A,C and B,D, respectively. AQP3-immunoreactive cells are observed in both FW and SW gills (A,B), and immunoreactions for AQP3 and Na+/K+-ATPase coincided completely with each other (E,F). No immunoreaction is observed in control sections (G,H). Bar, 50 µm.

 


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  		Fig. 7. Electron-microscopic immunocytochemistry of gill chloride cells in tilapia adapted to freshwater (FW; A,B) and seawater (SW; C,D), incubated with anti-tilapia AQP3 (A,C) and with pre-immune serum as controls (B,D). Intense immunoreaction for AQP3 is observed along the membrane of the tubular system (arrowheads) continuous with the basolateral membrane in chloride cells of FW- and SW-adapted tilapia (A,C), but not in controls (B,D). m, mitochondrion. Bar, 200 nm.

 

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© The Company of Biologists Ltd 2005