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First published online January 31, 2006
Journal of Experimental Biology 209, 599-609 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02059

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Microtubule-dependent relocation of branchial V-H⁺-ATPase to the basolateral membrane in the Pacific spiny dogfish (Squalus acanthias): a role in base secretion

Martin Tresguerres^*, Scott K. Parks, Fumi Katoh and Greg G. Goss

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

View larger version (21K): [in a new window]   Fig. 1. 6 h infusions. Blood parameters of colchicine-treated NaCl-infused fish, HCO3--infused fish and colchicine-treated HCO3--infused fish (means ± s.e.m.). (A) Arterial blood pH. (B) Total CO2 in plasma from arterial blood samples. The arrowheads indicate injection of colchicine (15 mg kg-1). *P<0.05 compared with base-infused fish of the respective time (RM-ANOVA, 1-way ANOVA, Dunnet's post test) (N=4).

View larger version (28K): [in a new window]   Fig. 2. 6 h infusions. Quantitative analysis of V-H+-ATPase in gills from colchicine-treated, NaCl-infused (col-NaCl IF), base-infused (BIF) and colchicine-treated, base-infused (col-BIF) fish. (A) Whole-gill homogenates. (B) Membrane fraction. V-H+-ATPase abundance was significantly greater only in the membrane fraction of BIF (N=4). Representative immunoblots are shown above each panel. The lower-case letters indicate difference levels of statistical significance (1-way ANOVA, Bonferroni's post test). Results are means ± s.e.m.

View larger version (95K): [in a new window]   Fig. 3. 6 h infusions. Representative images of V-H+-ATPase immunostaining in gills from (A) colchicine-treated, NaCl-infused, (B) base-infused and (C) colchicine-treated, base-infused fish. Scale bar, 10 µm. (D) The mean number of cells (± s.e.m.; N=4) that labelled for H+-ATPase per interlamellar space (cells IL-1). No significant differences were found between treatments (1-way ANOVA, Bonferroni's post test).

View larger version (113K): [in a new window]   Fig. 4. 6 h infusions. High-magnification (2000x) light micrographs showing H+-ATPase staining in gills from (A) colchicine-treated, NaCl-infused, (B) base-infused and (C) colchicine-treated, base-infused fish. c, cytoplasmic; b, basolateral; i, intermediate staining pattern. Scale bar, 10 µm.

View larger version (22K): [in a new window]   Fig. 5. 24 h infusions. Blood parameters of colchicine-treated NaCl-infused fish (col-NaCl IF), NaHCO3-infused fish (BIF), and colchicine-treated NaHCO3-infused fish (col-BIF) (means ± s.e.m.). (A) Arterial blood pH. (B) Total CO2 in plasma from arterial blood samples. The arrowheads indicate a bolus injection of colchicine (15 mg kg-1). Subsequent arrows indicate injection of half doses of colchicine (7.5 mg kg-1). *P<0.05 compared with base-infused fish of the respective time (RM-ANOVA, 1-way ANOVA, Dunnet's post test) (N=5).

View larger version (22K): [in a new window]   Fig. 6. 24 h infusions. Quantitative analysis of V-H+-ATPase in gills from colchicine-treated, NaCl-infused (col-NaCl IF; N=4), base-infused (BIF; N=5) and colchicine-treated, base-infused fish (col-BIF; N=5). (A) Whole-gill homogenates. (B) Membrane fraction. Representative immunoblots are shown above each panel. The lower-case letters indicate difference levels of statistical significance (1-way ANOVA, Bonferroni's post test). Results are means ± s.e.m.

View larger version (93K): [in a new window]   Fig. 7. 24 h infusions. Representative images of V-H+-ATPase immunostaining in gills from (A) colchicine-treated, NaCl-infused, (B) base-infused and (C) colchicine-treated, base-infused fish. Scale bar: 10 µm. (D) The mean number of cells (± s.e.m.) that labelled for H+-ATPase per interlamellar space (cells IL-1) (N=4 for col-NaCl IF, N=5 for the other two groups). No significant differences were found between treatments (1-way ANOVA, Bonferroni's post test).

View larger version (118K): [in a new window]   Fig. 8. 24 h infusions. High magnification (2000x) light micrographs showing H+-ATPase staining in gills from (A) colchicine-treated, NaCl-infused, (B) base-infused and (C) colchicine-treated, base-infused fish. c, cytoplasmic; b, basolateral; i, intermediate staining pattern. Scale bar, 10 µm.

View larger version (18K): [in a new window]   Fig. 9. H+-ATPase-dependent base secretion in H+-ATPase-rich cells. (A) Basal base secretion. Most of the H+-ATPase is in cytoplasmic vesicles. (i) Continuous recycling with the basolateral membrane. (B) Upregulation of base secretion under blood alkalosis. (ii) Short-term response: microtubule-dependent relocation of the pre-existent cytoplasmic pool to the basolateral membrane. (iii) In the longer term, there is also upregulation of H+-ATPase synthesis. (B) Model for base secretion in H+-ATPase-rich cells. P, Pendrin; CA, carbonic anhydrase; CC, chloride channel; a, apical side; b, basolateral side; N, nucleus.