Localization of the vacuolar-type ATPase in swimbladder gas gland cells of the European eel (Anguilla anguilla)

Abstract

SUMMARY The vacuolar ATPase is a multifunctional enzyme that consists of several subunits. Subunit B is part of the catalytic domain of the enzyme and is present in two isoforms in fish as well as in mammals. Possibly, these two isoforms — vatB1 (kidney isoform) and vatB2 (brain isoform) —serve different functions. A localization of the two isoforms was attempted in swimbladder gas gland cells of the European eel Anguilla anguilla by immunohistochemistry. Two antibodies were produced by immunization of rabbits with synthetic peptides. Specificity of the antibodies, on the one hand, an isoform-specific antibody for vatB1 and, on the other hand, an antibody that recognizes both isoforms (vatB1 and vatB2), was confirmed by western blot analysis using recombinant proteins produced in a bacterial expression system. The immunohistochemical localization with the antibody directed against both isoforms of the B subunit revealed a positive staining in apical membranes of swimbladder gas gland cells as well as in the basolateral membranes. Significant staining was observed in vesicles located near the apical membrane. Staining with the vatB1-specific antibody resulted in a similar picture in the apical region of the cells. In contrast to the staining with the first antibody, only a poor signal was observed in the basal region. The nature of the vesicles in the apical region of the gas gland cells was determined by using an antibody directed against surfactant protein D.