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 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.