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First published online June 11, 2007
Journal of Experimental Biology 210, 2113-2120 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.004101
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Effect of osmotic shrinkage and hormones on the expression of Na+/H+ exchanger-1, Na+/K+/2Cl– cotransporter and Na+/K+-ATPase in gill pavement cells of freshwater adapted Japanese eel, Anguilla japonica

William K. F. Tse1, Doris W. T. Au2 and Chris K. C. Wong1,*

1 Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong
2 Departments of Biology and Chemistry, City University of Hong Kong, Hong Kong


Figure 1
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  		Fig. 1. Hypertonicity-induced cell volume change and the expression of Na+/K+-ATPase, Na+/K+/2Cl– cotransporter (NKCC) and Na+/H+ exchanger-1 (NHE-1) in freshwater gill pavement cells (PVCs). (A) Percoll-purified PVCs were incubated in either normal (317 mOsmol l–1) or hypertonic (500 mOsmol l–1) medium. Blockers for three ion transporters (ouabain, bumetanide or EIPA) were added to the cells before the application of hyperosmotic stress. The change in cell volume was monitored using a Multisizer for a period of 60 min. Note that the three blockers reduced the RVI response in the cells. (B) A primary freshwater PVC culture was established. Over 80% of gill epithelial cells attached after overnight incubation (Bi); the cell suspension was obtained from trypsin digestion. (Bii) Gill epithelial cells cultured for 1 day. (C) Cell lysates were obtained from 6 h hypertonic treatment (HT) of the PVC culture and analysed by western blot (right). The amounts of the respective ion transporter protein were quantified and tabulated in graphical form (left). Significant induction of {alpha} and ß subunits of Na+/K+-ATPase, NKCC and NHE-1 were observed. (D) The gene expression levels for Ostf1 in the control and hypertonic-treated cells. *P<0.05 compared with the control. The results were obtained from more than five independent experiments.

 

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  		Fig. 2. Effect of DEX on the expression of Na+/K+-ATPase, Na+/K+/2Cl– cotransporter (NKCC) and Na+/H+ exchanger-1 (NHE-1) in pavement cells (PVCs) in culture. The cells were exposed to 0.5–2 µmol l–1 DEX for 24 h. (A) Cell lysates were collected for western blot analysis and (B) the amounts of the respective ion transporter proteins were quantified. Note that the three transporters were significantly induced. *P<0.05 compared with the control. The results were obtained from more than four independent experiments.

 

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  		Fig. 3. Effect of prolactin (PRL), thyroid hormone (T3) or insulin-like growth factor-1 (IGF-1) on the expression of Na+/K+-ATPase, Na+/K+/2Cl– cotransporter (NKCC) and Na+/H+ exchanger-1 (NHE-1) in pavement cells (PVCs) in culture. The cells were exposed to the respective hormones for 24 h. Cell lysates were collected for western blot analysis (left) and the amounts of the respective ion transporter proteins were quantified (right). (A) PRL at high dose stimulated the expression of NKCC and NHE-1. (B) IGF-1 elicited a dose-dependent induction of NKCC. (C) T3 had no observable effect to the expression of the ion transporters. *P<0.05 compared with the control. The results were obtained from more than six independent experiments.

 

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  		Fig. 4. Effect of dibutyryl cAMP (dbcAMP) or dibutyryl cGMP (dbcGMP) on the expression of Na+/K+-ATPase, Na+/K+/2Cl– cotransporter (NKCC) and Na+/H+ exchanger-1 (NHE-1) in pavement cells (PVCs) in culture. The cells were exposed to 1–4 mmol l–1 of the drugs for 24 h. (A) Cell lysates were collected for western blot analysis and (B) the amounts of the respective ion transporter proteins were quantified. Dibutyryl cAMP significantly induced the expression of the three ion transporters. The effect of dbcGMP was negligible. *P<0.05 compared with the control. The results were obtained from more than four independent experiments.

 

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© The Company of Biologists Ltd 2007