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First published online January 19, 2006
Journal of Experimental Biology 209, 518-530 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02018

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The role of branchial carbonic anhydrase in acid-base regulation in rainbow trout (Oncorhynchus mykiss)

T. Georgalis, S. F. Perry and K. M. Gilmour^*

Department of Biology and Centre for Advanced Research in Environmental Genomics, University of Ottawa, Ottawa, Ontario, K1N 6N5, Canada

View larger version (146K): [in a new window]   Fig. 1. Trout cytoplasmic carbonic anhydrase (tCAc) mRNA localization in the gills of rainbow trout (Oncorhynchus mykiss) by in situ hybridization. The images present two sets (A,B and C,D) of in situ hybridization results from serial sections of the gill incubated with probe (A,C), no probe (B) or probe in the presence of excess unlabelled probe (D) and are typical of the sections examined. Strong hybridization signals for tCAc are evident in epithelial cells of the lamellae at both low (A) and high (C) magnification. Scale bars, 40 µm (A,B) and 20 µm (C,D).

View larger version (81K): [in a new window]   Fig. 2. Localization of trout cytoplasmic carbonic anhydrase (tCAc) protein in the gills of rainbow trout (Oncorhynchus mykiss) by immunohistochemistry. The images are overlays of three images collected individually for tCAc immunoreactivity (green), 5 immunoreactivity (red) and nuclei visualization (blue). Areas of overlap of tCAc and 5 immunoreactivity are indicated in yellow. Nuclei were visualized using 4',6'-diamidino-2-phenylindole. Medium (A) and high (D) magnification images representative of the sections examined indicate that cells displaying only tCAc immunoreactivity, only 5 immunoreactivity or co-localization of tCAc and 5 immunoreactivities were present in the lamellar epithelia as well as in the interlamellar regions. Omission of primary antibodies eliminated all immunofluorescence (B). Pre-absorption of the tCAc antibody using the peptide against which the antibody was raised eliminated tCAc immunoreactivity without affecting 5 immunoreactivity (C). Scale bars, 20 µm.

View larger version (46K): [in a new window]   Fig. 3. (A) The effect of exposure to hypercarbia (filled bars), for periods ranging from 1 h to 24 h, on trout cytoplasmic carbonic anhydrase (tCAc) mRNA expression in the gills of rainbow trout (Oncorhynchus mykiss) relative to the expression of ß-actin mRNA (relative tCAc mRNA expression), as determined by real-time PCR. Values are means ± 1 s.e.m. and N=6 for each period of exposure to hypercarbia. For statistical analysis, values were compared to corresponding relative tCAc mRNA expression values for fish (N=6 in each case) held under control (normocarbic) conditions for the corresponding period of time; a single group of control fish sampled at 3 h served as the control for 1, 2 and 3 h of exposure to hypercarbia. In each case, mRNA expression in the control group was given a relative value of 1 (open bar), and an asterisk therefore indicates relative tCAc mRNA expression in the hypercarbic fish that was significantly different from 1 (one-sample Student's t-test, P<0.05). (B,C) Representative images of tCAc mRNA localization by in situ hybridization in the gills of (B) control and (C) hypercarbic (24 h at a nominal PWCO2 of 6 Torr; 1 Torr=133.3 Pa) trout. Scale bars, 20 µm.

View larger version (49K): [in a new window]   Fig. 4. The effects of exposure to hypercarbia on the expression of trout cytoplasmic carbonic anhydrase (tCAc) protein in the gills of rainbow trout (Oncorhynchus mykiss). An antibody raised against a peptide sequence specific to tCAc was used to detect tCAc protein expression by (A) western analysis and (B,C) immunohistochemistry in perfused gills from fish held under control conditions or exposed to hypercarbia (nominal PWCO2=6 Torr; 1 Torr=133.3 Pa) for 24 h. In A, expression of a 30 kDa protein was significantly greater (one-tailed Student's t-test, P=0.031) in gills extracted from hypercarbic trout (N=6) than in those extracted from normocarbic fish (N=6). Protein expression from the immunoblots presented on the right was quantified by digital image processing software and depicted in the figure on the left. In B and C, representative overlay images visualizing tCAc immunoreactivity (green), 5 immunoreactivity (red) and nuclei (4',6'-diamidino-2-phenylindole; blue) are presented for gill sections from trout exposed to (B) control conditions and (C) hypercarbia for 24 h. Co-localization of tCAc and 5 is indicated in yellow. Scale bars, 20 µm.

View larger version (19K): [in a new window]   Fig. 5. The effects of exposure to hypercarbia (nominal PWCO2=6 Torr; 1 Torr=133.3 Pa) and acetazolamide treatment (30 mg kg-1) on branchial (A) net excretion of acidic equivalents (JnetH+), (B) titratable net acid flux (JnetTA) and (C) net ammonia excretion (JnetNH3) in rainbow trout (Oncorhynchus mykiss). JnetH+ was calculated as the sum of JnetTA and JnetNH3, signs considered. Data are mean values ± 1 s.e.m.; N=11 for both control and hypercarbia treatment groups. Data were analyzed by two-way RM ANOVA with treatment group (control versus hypercarbic) and sampling time [pre- or post-acetazolamide (Az) injection] as factors; P values are indicated on the figure. An asterisk (*) indicates a significant difference as a result of Az injection within a treatment group, while a dagger () indicates a significant difference between control and hypercarbia treatment groups within a given sampling time. Where significant interactions did not occur, only the P values are indicated on the figure.

View larger version (21K): [in a new window]   Fig. 6. A pH-[HCO3-] diagram for rainbow trout at 13°C. The PCO2 for a given combination of pH and [HCO3-] was calculated using the Henderson-Hasselbalch equation and the appropriate values for pK' and the solubility coefficient CO2 (Boutilier et al., 1984). The buffer line for rainbow trout true plasma was constructed using buffer values derived by Wood et al. (1982). The labelled points represent mean data (±1 s.e.m., N=8) for (1) pre-exposure, (2) pre-acetazolamide injection and (3) post-acetazolamide injection sampling points for trout exposed to hypercarbia (nominal PWCO2 of 6 Torr; 1 Torr=133.3 Pa).