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Vacuolar-type proton pump in the basolateral plasma membrane energizes ion uptake in branchial mitochondria-rich cells of killifish Fundulus heteroclitus, adapted to a low ion environment

Fumi Katoh^*, Susumu Hyodo and Toyoji Kaneko

Ocean Research Institute, University of Tokyo, 1-15-1 Minamidai, Nakano, Tokyo 164-8639, Japan

View larger version (60K): [in a new window]   Fig. 4. Nucleotide sequence of the cDNA encoding the A-subunit of the killifish vacuolar-type proton pump and the deduced amino acid sequence. The antibody was raised against a synthetic peptide corresponding to the highlighted amino acid sequence. The sites of PCR primers are shown in the boxes.

View larger version (93K): [in a new window]   Fig. 1. Confocal laser scanning micrographs of whole-mount preparations of gill filaments in killifish acclimated to defined freshwater with low- (A,D), mid- (B,E) and high- (C,D) NaCl. The gill filaments were stained with FITC-labeled anti-Na+, K+-ATPase. (D-F) Magnified views of a flat region of the afferent-vascular edge, where most branchial mitochondria-rich (MR) cells are distributed. Scale bars, 100 µm (A-C); 50 µm (D-F).

View larger version (175K): [in a new window]   Fig. 2, Scanning electron micrographs of gill filaments in killifish adapted to defined freshwater with low- (A,B), mid- (C,D) and high- (E,F) NaCl. The apical membrane of mitochondria-rich (MR) cells is flat or slightly projecting, and equipped with microvilli (arrowheads) in low- and mid-NaCl groups, whereas the apical membrane of MR cells forms an apical pit (arrows) in the high-NaCl group. pv, pavement cell. Scale bars, 10 µm.

View larger version (144K): [in a new window]   Fig. 3. Transmission electron micrographs of branchial chloride cells (cc) of killifish adapted to defined freshwater with low- (A—C), mid- (D—F) and high- (G—I) NaCl. (B,E,H) Magnified views of apical regions of mitochondria-rich (MR) cells. In low- and mid-NaCl groups, the apical membrane of MR cells is flat or slightly projecting, and equipped with microvilli (asterisks) (B,E). In the high-NaCl group, on the other hand, MR cells form an apical pit (ap) (H). (C,F,I) Magnified views of the cytoplasm of MR cells. Note numerous mitochondria (m) and an extensive tubular system (t) in all experimental groups. pv, pavement cell; ac, accessory cell. Scale bars, 5 µm (A,D,G); 1 µm (B,C,E,F,H,I).

View larger version (33K): [in a new window]   Fig. 5. Western blot analysis for vacuolar-type proton pump (V-ATPase) protein expressed in the gills of killifish adapted to a low-NaCl environment. The membranes were incubated with anti-V-ATPase (lane B) and antibody pre-incubated with the antigen (lane A). Positions of molecular markers (kDa) are indicated on the left side of the figure. Two specific protein bands (arrows) were obtained.

View larger version (108K): [in a new window]   Fig. 6. Double immunofluorescence staining of the vacuolar-type proton pump (V-ATPase: A,C,E,G) and Na+/K+-ATPase (B,D,F,H) in the gill filaments of killifish adapted to freshwater with low-(A,B,G,H), mid- (C,D) and high- (E,F) NaCl. V-ATPase immunoreactivity was detected in Na+/K+-ATPase-immunoreactive mitochondria-rich (MR) cells in fish adapted to a low-NaCl environment (A,B), but the staining was extinguished when antibody was pre-absorbed with the antigen (G). V-ATPase-immunoreactivity was faint in higher NaCl environments (C,E). Scale bar, 50 µm.

View larger version (201K): [in a new window]   Fig. 7. Immuno-electron micrographs of the vacuolar-type proton pump (V-ATPase) in gill chloride cells of killifish adapted to low-NaCl (A,B) and mid-NaCl (C,D). V-ATPase immunoreactivity was detected in the tubular system (arrows) continuous with the basolateral membrane, but not in the apical membrane (asterisks) and mitochondria (m) in mitochondria-rich (MR) cells (A,C). (B,D) Magnified views of the cytoplasm in MR cells. The staining was more intense in the low-NaCl environment than in the mid-NaCl environment. pv, pavement cell. Scale bars, 1 µm (A,C); 0.5 µm (B,D).