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The cultured branchial epithelium of the rainbow trout as a model for diffusive fluxes of ammonia across the fish gill

Scott P. Kelly and Chris M. Wood*

Department of Biology, McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada L8S 4K1



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  		Fig. 1. (A) Transepithelial resistance (TER) of cultured epithelia before (symmetrical conditions, L-15 apical/L-15 basolateral) and after (asymmetrical conditions, fresh water apical/L-15 basolateral) the addition of apical fresh water at three different basolateral concentrations of total ammonia (TAmm) in series 1. Values are means +1 S.E.M. (N=10 per group). All increases in TER were highly significant (P<0.001), but there were no significant differences (P>0.05) associated with different TAmm levels. (B) Ammonia flux rates (JAmm) from basolateral-to-apical compartments under asymmetrical conditions over a 3 h flux period at the three different basolateral TAmm levels in the same preparations. An asterisk denotes a statistically significant difference (P<0.05) from the 123 µmol l–1 group and a double dagger denotes a statistically significant difference (P<0.05) from both other groups. (C) Relationships between JAmm and TER in individual cultured epithelia under asymmetrical conditions in each of the three groups in B. The regression equations are as follows. At 123 µmol l–1, filled circles, JAmm=–0.103TER+6.593, r=0.33, N=10, P>0.05; at 400 µmol l–1, open circles, JAmm=–0.176TER+10.495, r=0.49, N=10, P>0.05; at 1055 µmol l–1, open squares, JAmm=–0.924TER+22.483, r=0.90, N=10, P<0.0001.

 


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  		Fig. 2. The effects of varying basolateral pH (pHBl) at total ammonia (TAmm)=650 µmol l–1 on ammonia flux rates (JAmm, over 3 h) across cultured branchial epithelia under asymmetrical (fresh water apical/L-15 basolateral) conditions in series 2. Values are means +1 S.E.M. (N=3 per group). The overall effect of pHBl was significant (P<0.05), although none of the individual differences was significant.

 


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  		Fig. 3. (A) A comparison of ammonia flux rates (JAmm, over 6 h) between epithelia exposed to either symmetrical (L-15 apical/L-15 basolateral; filled columns) or asymmetrical (fresh water apical/L-15 basolateral; open columns) conditions at varying basolateral pH (pHBl), total ammonia (TAmm)=650 µmol l–1, in series 3. The overall effects of pH and of symmetrical versus asymmetrical conditions were significant (P<0.05). (B) Mean pH gradients and (C) mean PNH3 gradients associated with these treatments. Values are means +1 S.E.M. (N=6 for all symmetrical groups and N=3 for all asymmetrical groups). Significant differences between specific groups within a treatment (pHBl effect) are denoted by different letters; significant differences between specific groups held under either symmetrical or asymmetrical conditions are denoted by an asterisk. 1 nmHg=0.133 Pa.

 


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  		Fig. 4. (A) A comparison of ammonia flux rates (JAmm, over 6 h) between epithelia exposed under symmetrical conditions (L-15 apical/L-15 basolateral) to either basolateral manipulations (basolateral-to-apical fluxes; open columns, data from Fig. 3) or apical manipulations (apical-to-basolateral fluxes; filled columns) of pH at total ammonia (TAmm)=650 µmol l–1 in series 3. The overall effects of pH and of direction were significant (P<0.05), although there were no significant differences with respect to direction at the same pH values. (B) Mean pH gradients and (C) mean PNH3 gradients associated with these treatments. In B and C, there were no significant differences (P>0.05) attributable to direction, although the overall effects of pH were highly significant (P<0.0001). Values are means +1 S.E.M. (N=6 per group for all basolateral manipulations and N=5–6 per group for all apical manipulations). Significant differences between specific groups within a treatment (pHBl effect) are denoted by different letters. 1 nmHg=0.133 Pa.

 


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  		Fig. 5. Relationships between ammonia flux rates (JAmm, over 6 h) and average PNH3 gradients in individual epithelial preparations of series 3 under (A) asymmetrical conditions with basolateral-to-apical fluxes, (B) symmetrical conditions with basolateral-to-apical fluxes and (C) symmetrical conditions with apical-to-basolateral fluxes. The regression equations are as follows: (A) JAmm=+0.0242PNH3+23.94, r=0.53, N=18, P<0.05; (B) JAmm=+0.0174PNH3+5.52, r=0.74, N=31, P<0.05; (C) JAmm=+0.0313PNH3+30.95, r=0.28, N=27, P>0.05. 1 nmHg=0.133 Pa. FW, fresh water; L-15, L15 medium.

 


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  		Fig. 6. Relationships between ammonia flux rates (JAmm, over 6 h) and average net driving force for NH4+ (DF) in individual epithelial preparations of series 3 under (A) asymmetrical conditions with basolateral-to-apical fluxes, (B) symmetrical conditions with basolateral-to-apical fluxes and (C) symmetrical conditions with apical-to-basolateral fluxes. The regression equations are as follows: (A) JAmm=–1.4415DF+111.40, r=–0.69, N=18, P<0.05; (B) JAmm=–0.4616DF+21.37, r=–0.28, N=33, P>0.05; (C) JAmm=–0.2370DF+34.40, r=–0.03, N=29, P>0.05. FW, fresh water; L-15, L15 medium.

 


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  		Fig. 7. Relationships between the apparent basolateral-to-apical flux rates of NH4+ (as calculated from ammonia flux rates, JAmm, the relevant PNH3 gradient and the regression slopes from Fig. 5A,B as described in the text) and the transepithelial conductance (C, inverse of transepithelial resistance) in individual cultured epithelial preparations of series 3 under symmetrical and asymmetrical conditions. The regression equations are as follows: asymmetrical conditions, NH4+ flux=85.05C–4.61, r=0.68, N=18, P<0.05; symmetrical conditions, NH4+ flux=0.03C+6.21, r=0.01, N=29, P>0.05. 1 nmHg=0.133 Pa. FW, fresh water; L-15, L15 medium.

 

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© The Company of Biologists Ltd 2001