Immunolocalization of H+-ATPase in the gill epithelia of rainbow trout

The localization of proton pumps (H+-ATPase) in gill epithelia of rainbow trout [Oncorhynchus mykiss (Walbaum)] was elucidated by immunofluorescence microscopy, using rabbit polyclonal antibodies against the 70 kDa subunit of H+-ATPase purified from clathrin-coated vesicles of bovine brain. In the gill epithelia of freshwater trout, the immunostaining was uniformly distributed along the lamellae and generally concentrated in apical regions. It is concluded, therefore, that H+-ATPase is located in the apex of both chloride cells and epithelial cells of freshwater fish. Hypercapnic treatment resulted in a non-polarized and restrictive distribution of H+-ATPase in the chloride cell. No fluorescent staining was observed in the gill epithelium of seawater-adapted rainbow trout, except in some unidentified anucleate surface material. The presence of the 70 kDa subunit in fish gill epithelia was confirmed by Western blot. These results support the proposed role of a proton pump in sodium uptake in freshwater fish and demonstrate that the H+-ATPase in fish gills is of the vacuolar type, antigenically similar to the H+-ATPase in mammalian brain and kidney.