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First published online February 27, 2009
Journal of Experimental Biology 212, 878-892 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.021899
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Ammonia transport in cultured gill epithelium of freshwater rainbow trout: the importance of Rhesus glycoproteins and the presence of an apical Na+/NH4+ exchange complex

T. K. N. Tsui1, C. Y. C. Hung1, C. M. Nawata1, J. M. Wilson2, P. A. Wright3 and C. M. Wood1,*

1 Department of Biology, McMaster University, Hamilton, Ontario, Canada L8S 4K1
2 Ecofisiologia CIMAR, 4550-123 Porto, Portugal
3 Department of Integrative Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1


Figure 1
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  		Fig. 1. Relationships between basolateral total ammonia concentration and ammonia efflux rate across asymmetrical DSI in Series 1. Flux rates are portrayed as negative values to represent basolateral-to-apical effluxes. (A) Physiological range of basolateral [ammonia]; data are described by a relationship which combines a Michaelis–Menten saturable component and a linear component (R2=0.993, P<0.0001):

Formula
where JAmm is ammonia efflux rate (nmol cm–2 h–1), [Amm] is basolateral total ammonia concentration (µmol l–1), Jmax is maximum ammonia efflux rate (–3.92±0.75 nmol cm–2 h–1), Km is the affinity constant (66±44 µmol l–1) equal to the [Amm] which supports 50% of Jmax, and C is the slope (–0.0066±0.0004 nmol cm–2 h–1/µmol l–1) of the linear component. The dotted line indicates the linear component which was subtracted to yield the saturable component. The amount of efflux above this line is due to the saturable component. (B) Supra-physiological range of basolateral [ammonia]; data are described by the same equation as for A (R2=0.953, P<0.0002) with the same linear component but with a much lower affinity (Km=4818±1275 µmol l–1) and higher maximum transport capacity (Jmax=–429±47 nmol cm–2 h–1). The dotted line indicates the same linear component as in A which was subtracted to yield the saturable component. The amount of efflux above this line is again due to the saturable component. Data points are means ± s.e.m. (N=4–6).

 

Figure 2
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  		Fig. 2. The effect of 20 h pre-exposure on the ammonia flux across asymmetrical DSI in Series 2. (A) Ammonia efflux (negative); (B) ammonia influx (positive). Amm, 2000 µmol l–1 NH4Cl pre-exposure; Cortisol, 1000 ng ml–1 cortisol pre-exposure; Cort+Amm, 1000 ng ml–1 cortisol and 2000 µmol l–1 NH4Cl pre-exposure. Means not sharing the same letter are significantly different from one another (P<0.05). Data are means ± s.e.m. (N=4 or 5).

 

Figure 3
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  		Fig. 3. The effect of 20 h pre-exposure on the ammonia flux across symmetrical DSI in Series 2. (A) Ammonia efflux (negative); (B) Ammonia influx (positive). Amm, 2000 µmol l–1 NH4Cl pre-exposure; Cortisol, 1000 ng ml–1 cortisol pre-exposure; Cort+Amm, 1000 ng ml–1 cortisol and 2000 µmol l–1 NH4Cl pre-exposure. Means not sharing the same letter are significantly different from one another (P<0.05). Data are means ± s.e.m. (N=4 or 5).

 

Figure 4
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  		Fig. 4. The effect of 20 h pre-exposure on the [3H]PEG-4000 permeability across (A) asymmetrical DSI and (B) symmetrical DSI in Series 2. Amm, 2000 µmol l–1 NH4Cl pre-exposure; Cortisol, 1000 ng ml–1 cortisol pre-exposure; Cort+Amm, 1000 ng ml–1 cortisol and 2000 µmol l–1 NH4Cl pre-exposure. Means not sharing the same letter are significantly different from one another (P<0.05). Data are means ± s.e.m. (N=4 or 5).

 

Figure 5
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  		Fig. 5. The effect of 20 h pre-exposure on the transepithelial resistance (TER) across (A) asymmetrical DSI and (B) symmetrical DSI in Series 2. Amm, 2000 µmol l–1 NH4Cl pre-exposure; Cortisol, 1000 ng ml–1 cortisol pre-exposure; Cort+Amm, 1000 ng ml–1 cortisol and 2000 µmol l–1 NH4Cl pre-exposure. FW, freshwater. Asterisks represent values significantly different from the corresponding control value (P<0.05). Data are means ± s.e.m. (N=4 or 5).

 

Figure 6
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  		Fig. 6. The effect of 20 h pre-exposure on the transepithelial potential (TEP) across (A) asymmetrical DSI and (B) symmetrical DSI in Series 2. Amm, 2000 µmol l–1 NH4Cl pre-exposure; Cortisol, 1000 ng ml–1 cortisol pre-exposure; Cort+Amm, 1000 ng ml–1 cortisol and 2000 µmol l–1 NH4Cl pre-exposure. Asterisks represent values significantly different from the corresponding control value (P<0.05). Data are means ± s.e.m. (N=4 or 5).

 

Figure 7
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  		Fig. 7. The effect of 20 h pre-exposure on the mRNA expression in asymmetrical DSI, relative to that of elongation factor 1{alpha} (EF-1{alpha}), of (A) Rhbg, (B) Rhcg1, (C) Rhcg2, (D) H+-ATPase, (E) carbonic anhydrase-2 (CA-2), (F) Na+/H+ exchanger-2 (NHE-2) and (G) Na+/K+-ATPase {alpha}1a (NKA) in Series 2. Amm, 2000 µmol l–1 NH4Cl pre-exposure; Cortisol, 1000 ng ml–1 cortisol pre-exposure; Cort+Amm, 1000 ng ml–1 cortisol and 2000 µmol l–1 NH4Cl pre-exposure. Asterisks represent values significantly different from control value (P<0.05). Data are means ± s.e.m. (N=4 or 5).

 

Figure 8
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  		Fig. 8. Effects of different transport inhibitors on ammonia flux across asymmetrical DSI in Series 3. Baf, bafilomycin (1 µmol l–1); Amil, amiloride (100 µmol l–1); Phen, phenamil (10 µmol l–1); HMA, 5-(N,N-hexamethylene)amiloride (10 µmol l–1); Ap, apical solution; Bl, basolateral solution. Asterisks represent values significantly different from control value (P<0.05). Data are means ± s.e.m. (N=4 or 5).

 

Figure 9
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  		Fig. 9. The effect of 20 h low [sodium] pre-exposure on the apical side on (A) ammonia efflux (negative) and (B) [3H]PEG-4000 permeability across asymmetrical DSI in Series 4. Asterisk represents value significantly different from cortisol value (P<0.05). Data are means ± s.e.m. (N=5).

 

Figure 10
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  		Fig. 10. The effect of 20 h low [sodium] pre-exposure on the apical side on the mRNA expression in asymmetrical DSI relative to EF-1{alpha} of (A) Rhbg, (B) Rhcg1, (C) Rhcg2, (D) H+-ATPase and (E) NHE-2 in Series 4. Asterisk represents value significantly different from cortisol value (P<0.05). Data are means ± s.e.m. (N=4 or 5).

 

Figure 11
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  		Fig. 11. The effect of 20 h high [ammonia] pre-exposure on (A) Na+ influx (positive), (B) Na+ efflux (negative), (C) TEP and (D) ammonia efflux (negative) of asymmetrical DSI in Series 5. High [ammonia] pre-exposure was performed on symmetrical DSI for 20 h prior to the switch to apical freshwater. Cortisol (1000 ng ml–1) was added to all DSI. Asterisks represent values significantly different from control value (P<0.05). Data are means ± s.e.m. (N=4).

 

Figure 12
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  		Fig. 12. Correlations between (A) Na+ influx and TEP, (B) ammonia efflux and TEP, and (C) ammonia efflux and Na+ influx in Series 5.

 

Figure 13
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  		Fig. 13. Proposed model of a `Na+/NH4+ exchange complex' in DSI. The roles of various CA isoforms in ammonia excretion require further investigation; therefore they have not been included in the model. Non-ionic NH3 diffusion, shown by the lower arrow, also plays an important role in ammonia efflux.

 

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© The Company of Biologists Ltd 2009