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Immunochemical analysis of the vacuolar proton-ATPase B-subunit in the gills of a euryhaline stingray (Dasyatis sabina): effects of salinity and relation to Na⁺/K⁺-ATPase

Peter M. Piermarini^* and David H. Evans

University of Florida, Department of Zoology, Box 118525, 223 Bartram Hall, Gainesville, FL 32611, USA

View larger version (70K): [in a new window]   Fig. 1. Representative immunoblot for V-H+-ATPase B-subunit in gill membrane enrichments from freshwater (lane 1), seawater-acclimated (lane 2) and seawater (lane 3) Atlantic stingrays. The positions of molecular mass markers (kDa) are shown. The antibody recognized a 60.5±1.08 kDa protein in gills (arrow). Note the reduction in band intensity from lanes 1 to 3.

View larger version (9K): [in a new window]   Fig. 2. Relative intensity of 60.5 kDa band representing the B subunit of V-H+-ATPase in the gills of freshwater (FW), seawater-acclimated (SWA) and seawater (SW) stingrays. N=5 for all groups. Although statistical differences were detected with a non-parametric test (Kruskal–Wallis ANOVA), values are shown as means + 1 S.E.M. for clarity. Lower case letters indicate statistical categorization of groups as determined by a Kruskal–Wallis post-hoc test (P <0.05).

View larger version (101K): [in a new window]   Fig. 3. Representative photomicrographs of immunostaining for V-H+-ATPase in longitudinal sections of gill filaments from freshwater (A), seawater-acclimated (B) and seawater (C) Atlantic stingrays (x400). Scale bars, 100 µm. V-H+-ATPase-rich cells occurred on lamellae (finger-like projections) and/or interlamellar regions (basal to and between lamellae). Note the differences in abundance and distribution (lamella versus interlamellar region) of V-H+-ATPase-rich cells among groups.

View larger version (14K): [in a new window]   Fig. 4. V-H+-ATPase-rich cell numbers in the gills of freshwater (FW), seawater-acclimated (SWA) and seawater (SW) Atlantic stingrays. (A) ‘Sum’ number of V-H+-ATPase-rich cells (per 100 µm of lamella + per interlamellar region); (B) number of V-H+-ATPase-rich cells per 100 µm of lamella; (C) number of V-H+-ATPase-rich cells per interlamellar region. Values are means + 1 S.E.M. Lower case letters indicate statistical categorization of the means as determined by a Student–Newman–Keuls post-hoc test (P<0.05). Note that both mean and S.E.M. for the number of V-H+-ATPase-rich cells per 100 µm of lamella = 0 in SW stingrays.

View larger version (83K): [in a new window]   Fig. 5. Representative high-magnification (x1000) photomicrographs of V-H+-ATPase-rich cells in gills from freshwater (A), seawater-acclimated (B), and seawater (C) Atlantic stingrays. Scale bars, 50 µm. Arrows indicate cells that best demonstrate the qualitative differences in staining observed among the groups. In freshwater stingrays, localization of V-H+-ATPase was diffuse throughout the cytoplasm and discrete along the basolateral plasma membrane of relatively large cells. In seawater-acclimated and seawater stingrayss V-H+-ATPase staining appeared to be stronger in the cytoplasm and less discrete along the basolateral membrane, relative to freshwater individuals. In all groups, no distinct staining was observed on the apical plasma membrane of V-H+-ATPase-rich cells.

View larger version (89K): [in a new window]   Fig. 6. Representative photomicrographs of double labeling for V-H+-ATPase-rich (brown) and Na+/K+-ATPase-rich (blue) in longitudinal sections of gill filaments from freshwater (A), seawater-acclimated (B), and seawater (C) Atlantic stingrays (x400). Scale bars, 100 µm. Note that regardless of salinity there were separate brown and blue cells, indicating the two transporters occur in distinct cell types.

View larger version (19K): [in a new window]   Fig. 7. Hypothetical model of NaCl and acid–base transport in the gills of the Atlantic stingray. Results from this and an earlier study (Piermarini and Evans, 2000) suggest that V-H+-ATPase and Na+/K+-ATPase occur on the basolateral cell membrane of distinct mitochondrion-rich cell types. We hypothesize that the V-H+-ATPase-rich cells act as base excreting cells via an apical Cl-/HCO3- exchanger that would also result in Cl- uptake. In contrast, we hypothesize that Na+/K+-ATPase-rich cells act as acid-excreting cells via an apical NHE that would also result in Na+ uptake. This model can explain NaCl and acid–base transport in both freshwater and seawater stingrays. For example, in freshwater animals, the gradient for NaCl entry into the cells is unfavorable, therefore ATPases would be required to establish electrochemical gradients to drive Na+/H+ and Cl-/HCO3- exchange, which is supported by the greater expression and number of ATPase-rich cells found in freshwater stingray gills. In seawater animals, the gradient for NaCl entry into the cells is favorable, therefore ATPases would not be as important for driving Na+/H+ and Cl-/HCO3- exchange, which is supported by the lower overall expression and number of ATPase-rich cells found in seawater-acclimated and seawater stingrays. In addition, the qualitative differences in V-H+-ATPase staining that we observed (see Fig. 5) may suggest that trafficking of cytoplasmic vesicles containing V-H+-ATPase to the basolateral membrane is enhanced in freshwater stingrays, which would enhance active proton transport across this membrane. ? indicates that the presence of the transporter needs to be demonstrated in the Atlantic stingray.