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First published online February 4, 2005
Journal of Experimental Biology 208, 749-760 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01440

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Hypotonicity induced K⁺ and anion conductive pathways activation in eel intestinal epithelium

M. G. Lionetto¹, M. E. Giordano¹, F. De Nuccio¹, G. Nicolardi¹, E. K. Hoffmann² and T. Schettino^1,*

¹ Department of Biological and Environmental Sciences and Technologies, University of Lecce, Italy
² Biochemistry Department, August Krogh Institute, 13 Universitetsparken, Copenhagen, Denmark

View larger version (12K): [in a new window]   Fig. 1. Time course of plasma osmolarity of 3 eels transferred at time 0 from seawater to freshwater. Values are means ± S.E.M.

View larger version (73K): [in a new window]   Fig. 2. (A-C). Semithin (0.5 µm) sections of eel intestinal epithelium cut along planes perpendicular to the luminal epithelium surface and stained with 1% Toluidine Blue. (A) Isotonic condition, (B) after 5 min exposure to hypotonic stress, (C) after 45 min exposure to hypotonic stress.

View larger version (11K): [in a new window]   Fig. 3. Epithelium height measured in isosmotic conditions (Ctrl), and after 5 min and 45 min exposure to hypotonic stress (decrease of Ringer osmolarity from 315 mOsm to 175 mOsm). Values are means ± S.E.M. Statistical analysis was performed by one-way ANOVA repeated measures test and Newman-Keuls multiple comparison test. **P<0.01.

View larger version (12K): [in a new window]   Fig. 4. Changes in transepithelial voltage (Vte) and short circuit current (Isc) in response to a hypotonic stress (decrease of Ringer osmolarity from 315 mOsm to 175 mOsm). - sign of Vte refers to the mucosa (grounded); - sign of Isc indicates current flowing from mucosal to serosal side. Vte time course represents the registered trace, while Isc time course was performed by keeping the preparation `short circuited' every 5 min. Representative time course of N=20 trials.

View larger version (9K): [in a new window]   Fig. 5. Transepithelial resistance (Rte) in controls (Ctrl) and after 5, 30 and 60 min exposure to a hypotonic stress (decrease of Ringer osmolarity from 315 mOsm to 175 mOsm). Values are means ± S.E.M. of 10 experiments. The statistical significance of the differences was analysed by one-way ANOVA repeated measures test and Newman-Keuls multiple comparison test. *P<0.05.

View larger version (18K): [in a new window]   Fig. 6. (A) Effect of 10 µmol l-1 bumetanide (added in the mucosal bath) on the Vte response to hypotonic stress expressed as Vte ( values were calculated as the differences between the Vte and Isc values after hypotonic stress application and the value before; values were calculated every 5 min during the time course of the exposure). Time=0 indicated the start of the exposure to hypotonic stress. Data are reported as mean ± S.E.M. of 5 separate experiments. (B) Effect of 1 mmol l-1 ouabain (added in the serosal bath) on the Vte response to hypotonic stress. Values are means ± S.E.M. of 4 experiments. The statistical significance of the differences was tested by paired Student t-test. *P<0.05.

View larger version (22K): [in a new window]   Fig. 7. Effect of 2 mmol l-1 Ba2+, added in the serosal (A) or mucosal (B) baths on the Vte response to hypotonic stress. Data are expressed as mean ± S.E.M. of 5 experiments. Details as in Fig. 6.

View larger version (21K): [in a new window]   Fig. 8. Effect of 0.1 µmol l-1 iberiotoxin on basal Vte in isotonic conditions (A, serosal application; B, mucosal application) and on the Vte response to hypotonic stress (C, serosal application; D, mucosal application). (A,B) Representative time courses (N=4) are shown. (C,D) Values are means ± S.E.M. of 4 experiments. Details as in Figs 4 and 6. s, serosal; m, mucosal. *P<0.05, **P<0.01.

View larger version (23K): [in a new window]   Fig. 9. Effect of 1 µmol l-1 apamin on basal Vte in isotonic conditions (A, serosal application; B, mucosal application) and on the Vte response to hypotonic stress (C, serosal application; D, mucosal application). (A,B) Representative time courses (N=4) are shown. (C,D) Values are means ± S.E.M. of 4 experiments. Details as in Figs 4 and 6. s, serosal; m, mucosal. *P<0.05.

View larger version (21K): [in a new window]   Fig. 10. Effect of 500 µmol l-1 DIDS on basal Vte in isotonic conditions (A, serosal application; B, mucosal application) and on the Vte response to hypotonic stress (C, serosal application; D, mucosal application). (A,B) Representative time courses (N=4) are shown. (C,D) Values are means ± S.E.M. of 5 experiments. Details as in Figs 4 and 6. s, serosal; m, mucosal. *P<0.05.

View larger version (16K): [in a new window]   Fig. 11. Effect of 1 h preincubation with 50 µmol l-1 BAPTA-AM on the Vte response to hypotonic stress. Values are means ± S.E.M. of 4 experiments. Details as in Figs 4 and 6. *P<0.05.

View larger version (18K): [in a new window]   Fig. 12. Effect of 10 µmol l-1 trifluoroperazine on the Vte response to hypotonic stress. Values are means ± S.E.M. of 6 experiments. Details as in Figs 4 and 6. *P<0.05.

View larger version (20K): [in a new window]   Fig. 13. Effect of (A) 1 µmol l-1 thapsigargin preincubation and (B) Ca2+ removal from Ringer solution on the Vte response to hypotonic stress. Values are means ± S.E.M. of 5 experiments. Details as in Figs 4 and 6. *P<0.05.

View larger version (27K): [in a new window]   Fig. 14. Model of ion transport mechanisms activated by hypotonic stress in eel enterocytes.