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First published online January 5, 2005
Journal of Experimental Biology 208, 345-354 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01382

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Regulation of branchial V-H⁺-ATPase, Na⁺/K⁺-ATPase and NHE2 in response to acid and base infusions in the Pacific spiny dogfish (Squalus acanthias)

Martin Tresguerres^*, Fumi Katoh, Heather Fenton, Edyta Jasinska and Greg G. Goss

Dept of Biological Sciences, University of Alberta, Edmonton, Alberta T5G 2E9, Canada and Bamfield Marine Research Centre, Bamfield, BC V0R 1B0, Canada

View larger version (17K): [in a new window]   Fig. 1. Blood parameters of fish infused intravenously with either 125 mmol l-1 HCl (495±79 µmol kg-1 h-1), 250 mmol l-1 NaHCO3 (981±235 µmol kg-1 h-1) or 500 mmol l-1 NaCl (1832± 128 µmol kg-1 h-1) (mean ± S.E.M., N=4). (A) Arterial blood pH. (B) Total [CO2] in plasma from arterial blood samples. *P<0.05 compared to the control value (NaCl) of the respective time (RM-ANOVA, one-way ANOVA, Dunnet's post test).

View larger version (18K): [in a new window]   Fig. 2. Na+/K+-ATPase in the membrane fraction of the gills of sham-operated, NaCl-, acid- and base-infused fish. (A) Representative quantitative immunoblotting against the -subunit of the Na+/K+-ATPase: a distinct band of 102.8 kDa was obtained. (B) Fluorometric analysis revealed that Na+/K+-ATPase abundance in acid-infused fish increased to 315±88% of sham-operated fish (100±22%; N=4). (C) Na+/K+-ATPase activity. Values are mean ± S.E.M., N=4. No significant differences were found among treatments. *P<0.05 (one-way ANOVA, Dunnet's post test).

View larger version (138K): [in a new window]   Fig. 3. Representative images of Na+/K+-ATPase immunostaining in gills from sham-operated (A), acid-infused (B), base-infused (C) and NaCl-infused (D) fish. The sections were from equivalent regions in the gill filament, near the trailing edge. Note the greater number of labeled cells in the lamellae in B and C. Scale bar, 10 µm.

View larger version (58K): [in a new window]   Fig. 4. High magnification micrographs showing the Na+/K+-ATPase subcellular localization in control (A), acid-(B) and base-infused (C) fish. Note that the immunostaining is basolateral in all cases. Scale bar, 10 µm.

View larger version (21K): [in a new window]   Fig. 5. V-H+-ATPase in the membrane fraction of gills of sham-operated, NaCl-, acid- and base-infused fish. (A) Representative quantitative immunoblotting against the A-subunit of the V-H+-ATPase: a distinct band of 77 kDa was obtained. (B) Fluorometric analysis revealed that the abundance of the A-subunit of the V-H+-ATPase in base-infused fish increased to 300±81% of sham-operated fish (100±28%) (N=4). (C) V-H+-ATPase activity increased in concert with the increase in abundance noted in B (N=4). Values are mean ± S.E.M. *P<0.05 (one-way ANOVA, Dunnet's post test).

View larger version (152K): [in a new window]   Fig. 6. Representative images of H+-ATPase immunostaining in gills from sham-operated (A), acid-infused (B), base-infused (C) and NaCl-infused (D) fish. The sections were from equivalent regions in the gill filament, near the trailing edge. Scale bar, 10 µm.

View larger version (58K): [in a new window]   Fig. 7. High magnification light micrographs showing the V-H+-ATPase subcellular localization in control (A), acid-(B) and base-infused (C) fish. Note the distinct immunostaining at the basolateral region and the absence of staining on the apical membrane (arrowhead) in C. Scale bar, 10 µm.

View larger version (107K): [in a new window]   Fig. 8. Immunohistochemistry of consecutive sections from the trailing edge region of gills from sham-operated fish (A), acid-infused fish (B), and base-infused fish (C). (Ai,Bi,Ci) Na+/K+-ATPase immunoreactivity; (Aii,Bii,Cii) V-H+-ATPase immunoreactivity; (Aiii,Biii,Ciii) diagrams of the approximate location of each immunostained cell. Cells that labeled positive for Na+/K+-ATPase only are black, those labeled for V-H+-ATPase only are white, and cells that labeled positive for both transporters are gray. Scale bar, 10 µm.

View larger version (16K): [in a new window]   Fig. 9. Quantitative immunoblotting of the membrane fraction of gills of sham-operated, NaCl-, acid- and base-infused fish. (A) representative immunoblot incubated with anti-NHE2 antibody, showing a distinct band at 80 kDa. (B) Fluorometric analysis showing that the abundance of NHE2 in acid infused fish was 213±5% of sham-operated fish (100±21%). N=4; *P<0.05 (one-way ANOVA, Dunnet's post test).

View larger version (100K): [in a new window]   Fig. 10. (A) A transmission electron micrograph of a mitochondria-rich cell located on the lamella of a base-infused fish. (B) A detail of the basolateral infoldings. Scale bars, 10 µm (A); 2 µm (B).