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First published online October 7, 2008
Journal of Experimental Biology 211, 3226-3236 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.020396
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The effects of CO2 and external buffering on ammonia excretion and Rhesus glycoprotein mRNA expression in rainbow trout

C. Michele Nawata* and Chris M. Wood

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


Figure 1
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  		Fig. 1. (A) Predicted amino acid sequence alignment of Rhbg1, Rhbg2a and Rhbg2c. Lines above the sequences represent putative transmembrane domains and asterisks indicate predicted N-linked glycosylation sites. The alternative COOH-terminal end of Rhbg2c is underscored. Shading highlights amino acid differences between Rhbg1 and Rhbg2. (B) 5'-Untranslated region (UTR) of the longest Rhbg2 variant. Two upstream open reading frames (ORFs) are underscored. (C) Schematic representation of the mRNA variants identified in Rhbg2. Shading and stippling identifies regions of identity between variants. White background represents the 5'-UTR, black background represents the ORF, and grey background represents the 3'-UTR. Four variants (v1, 2, 3, 4) were detected in the 5'-UTRs, three of which were detected in Rhbg2a.

 

Figure 2
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  		Fig. 2. Tissue distribution of Rhbg2 mRNA in rainbow trout exposed to control conditions (100% air for 12 h) as determined by reverse transcriptase PCR. Elongation factor (EF-1{alpha}) was used as an internal control. NTC, no template control.

 

Figure 3
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  		Fig. 3. Relative abundance of 5'-UTR variants (v1, 2, 3, 4) in the gill under control conditions (12 h exposure to 100% air) and skin during control conditions and after 12 h of exposure to hypercapnia (1% CO2 in air) in 10 mmol l–1 Hepes-buffered water. Expression data were normalized to ng total RNA concentration. Different letters indicate significant differences in mRNA abundance between variants in the skin under control conditions. Data are means ± s.e.m. (N=6).

 

Figure 4
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  		Fig. 4. The effect of 12 h exposure of rainbow trout to: hypercapnia (1% CO2 in air) in unbuffered water, hypercapnia in 10 mmol l–1 Hepes-buffered water, and normocapnia (100% air) in Hepes-buffered water on the net ammonia flux (JAmm). Control fish were exposed to normocapnia for 12 h in unbuffered water. The negative values indicate excretion into the water. Crosses represent values significantly different from the 3 h control value. Asterisks represent values significantly different from the 12 h control value. Control values are significantly different from each other (P<0.05). Data are means ± s.e.m. (N=6–10).

 

Figure 5
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  		Fig. 5. Plasma total ammonia after 12 h of exposure of rainbow trout to 1% CO2 in air (CO2), 1% CO2 in air with 10 mmol l–1 Hepes-buffered water (CO2 + Hepes), 100% air with 10 mmol l–1 Hepes-buffered water (Air+Hepes) and high environmental ammonia (HEA; 1.5 mmol NH4HCO3). Control fish were exposed to 100% air for 12 h in unbuffered water. Asterisks represent plasma values significantly different from the control value (P<0.05). Data are means ± s.e.m. (N=6–10).

 

Figure 6
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  		Fig. 6. Gill Rh mRNA expression in rainbow trout after 12 h of exposure to 1% CO2 in air (CO2), 1% CO2 in air with 10 mmol l–1 Hepes-buffered water (CO2+Hepes), 100% air with 10 mmol l–1 Hepes-buffered water (Air+Hepes) and high environmental ammonia (HEA; 1.5 mmol NH4HCO3). Control fish were exposed to 100% air for 12 h in unbuffered water. Expression data were normalized to ng total RNA concentration with the control value set to one. Asterisks indicate significant difference from the control (P<0.05). Data are means ± s.e.m. (N=6).

 

Figure 7
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  		Fig. 7. Skin Rh mRNA expression in rainbow trout after 12 h of exposure to 1% CO2 in air (CO2), 1% CO2 in air with 10 mmol l–1 Hepes buffered-water (CO2+Hepes), 100% air with 10 mmol l–1 Hepes-buffered water (Air+Hepes) and high environmental ammonia (HEA; 1.5 mmol NH4HCO3). Control fish were exposed to 100% air for 12 h in unbuffered water. Expression data were normalized to ng total RNA concentration with the control value set to one. Asterisks indicate significant difference from the control (P<0.05). Data are means ± s.e.m. (N=6).

 

Figure 8
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  		Fig. 8. Erythrocyte Rh mRNA expression in the rainbow trout exposed for 12 h to 1% CO2 in air (CO2), 1% CO2 in air with 10 mmol l–1 Hepes buffered-water (CO2+Hepes) and 100% air with 10 mmol l–1 Hepes-buffered water (Air+Hepes). Control fish were exposed to 100% air for 12 h in unbuffered water. Expression data were normalized to ng total RNA concentration with the control value set to one. Asterisks indicate significant difference from the control (P<0.05). Data are means ± s.e.m. (N=6).

 

Figure 9
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  		Fig. 9. The effect of 12 h exposure to 1% CO2 in air (CO2), 1% CO2 in air with 10 mmol l–1 Hepes buffered-water (CO2+Hepes), and 100% air with 10 mmol l–1 Hepes-buffered water (Air+Hepes) on the mRNA expression of carbonic anhydrase (CA2), H+-ATPase, NHE2 and Na+/K+-ATPase {alpha}-1a (NKA) in the gills of rainbow trout. Control fish were exposed to 100% air for 12 h in unbuffered water. Expression data were normalized to ng total RNA concentration with the control value set to one. Significant differences from the control are indicated by an asterisk (P<0.05). Data are means ± s.e.m. (N=6).

 

Figure 10
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  		Fig. 10. Plasma cortisol levels after 12 h of exposure to 1% CO2 in air (CO2), 1% CO2 in air with 10 mmol l–1 Hepes-buffered water (CO2+Hepes), 100% air with 10 mmol l–1 Hepes-buffered water (Air+Hepes) and high environmental ammonia (HEA; 1.5 mmol NH4HCO3). Control fish were exposed to 100% air for 12 h in unbuffered water. Asterisk indicates significant difference from the control value (P<0.05). Data are means ± s.e.m. (N=6–10).

 

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© The Company of Biologists Ltd 2008