Acidification of the glandular lumen by a vacuolar-type H⁺-ATPase in the anterior silk gland cell of Bombyx mori

Abstract

An active transport system of vacuolar-type H⁺-ATPase (V-ATPase) was identified and characterized in the anterior silk gland of 5th instar larvae of the silkworm, Bombyx mori. Using fluorescent acridine orange, acidic granules were found in the apical region of cytoplasm. The acidification of these granules was completely and reversibly inhibited under the presence of 0.1 to 1μM bafilomycin A₁. This was observed throughout the larval stage of the 5th instar until the onset of spinning. During the incubation with acridine orange, acidic granules seemed to be transported apically and to be concentrated beneath the apical plasma membrane. These findings imply that V-ATPase is shuttled between an intracellular vesicular compartment and the plasma membrane. The microsomal membrane vesicles from the anterior silk gland cell showed ATP-dependent H⁺-transport activity, which was greatly enhanced by the presence of Cl^-. The immunological blotting test with the antibody against V-ATPase holoenzyme of Manduca sexta midgut showed the presence of subunits A, B, C and G of V-ATPase in the anterior silk gland cell. The V-ATPase was shown to be distributed in the cytoplasmic area of the anterior silk gland cell by immunohistochemistry with the monoclonal antibody against subunit A of Manduca V-ATPase. The rough estimation of luminal pH with pH test papers indicated that the pH value in the 5th instar feeding larvae was around 5.5. On the other hand, that of spinning larvae was around 6.5, suggesting that the acidification by the V-ATPase is off at the spinning stage. These lines of evidence suggest that the V-ATPase of the anterior silk gland participates in the acidification of the glandular lumen during the feeding period of 5th instar.