IMMUNOLOCALIZATION OF H-ATPASE IN THE GILL EPITHELIA OF RAINBOW-TROUT()

The localization of proton pumps (H+-ATPase) in gill epithelia of rain bow trout [Oncorhynchus mykiss (Walbaum)] was elucidated by immunofluo rescence microscopy, using rabbit polyclonal antibodies against the 70 kDa subunit of H+-ATPase purified from clathrin-coated vesicles of bov ine brain. In the gill epithelia of freshwater trout, the immunostaini ng was uniformly distributed along the lamellae and generally concentr ated in apical regions. It is concluded, therefore, that H+-ATPase is located in the apex of both chloride cells and epithelial cells of fre shwater fish. Hypercapnic treatment resulted in a non-polarized and re strictive distribution of H+-ATPase in the chloride cell. No fluoresce nt staining was observed in the gill epithelium of seawater-adapted ra inbow trout, except in some unidentified anucleate surface material. T he presence of the 70kDa subunit in fish gill epithelia was confirmed by Western blot. These results support the proposed role of a proton p ump in sodium uptake in freshwater fish and demonstrate that the H+-AT Pase in fish gills is of the vacuolar type, antigenically similar to t he H+-ATPase in mammalian brain and kidney.