Redistribution of cathepsin B activity from the endosomal-lysosomal pathway in chick intestine within 3 min of calcium absorption

Abstract

Earlier work has suggested that calcium-containing lysosomes are involved in 1,25-dihydroxyvitamin D 3 (1,25(OH) 2D 3)-stimulated intestinal absorption of the divalent cation. In the present report immunofluorescent labelling studies on fixed frozen sections of chick intestine were undertaken to determine whether lysosomes could respond to calcium transport conditions in less than 5 min. Tissue prepared from vitamin D-deficient chicks dosed with vehicle or 1.3 nmol of 1,25(OH) 2D 3 15 h prior to use was immunofluorescently labelled for cathepsin B, a lysosomal protease. In the absence of calcium absorption, punctate staining was found in the region below the terminal web, and more diffusely in the cytoplasm. The intensity of staining was noticeably greater in sections from 1,25(OH) 2D 3-treated than control chicks. In sections prepared after 3 min of calcium absorption, cathepsin B staining was localized near the basal and lateral membranes of the epithelial cells. After 30 min of transport, the protease was found in the villus core regardless of vitamin D status; however, immunoreactivity within the epithelial cells of 1,25(OH) 2D 3-treated chick intestine had returned to pretransport intensity, whereas that of controls had not. To further investigate the specificity of the cathepsin B antibody, the intracellular compartmentalization of the protease was determined by biochemical methods. Using dosing procedures and calcium transport times equivalent to those for the immunofluorescent studies mucosae were collected by scraping, homogenized, and subcellular fractions prepared by a combination of differential and Percoll gradient centrifugation. In the absence of calcium transport, cathepsin B-specific activity was enhanced in whole homogenates, endocytic vesicles, and a lysosomal fraction prepared from intestinal epithelium of 1,25(OH) 2D 3-treated chicks, relative to vitamin D-deficient controls. After 3 min of calcium absorption, a profound (~ 4-fold) decrease in endocytic vesicle cathepsin B activity was found regardless of vitamin D status, as well as a similar marked decrease in lysosomes prepared from vitamin D-deficient, but not -treated, chicks. After 30 min of calcium transport, endocytic vesicles prepared from either vitamin D-deficient or 1,25(OH) 2D 3-treated birds had recovered cathepsin B activity to pretransport levels. However, lysosomes prepared from rachitic chicks remained low in protease levels relative to equivalent fractions from dosed chicks. Thus, biochemical analysis of cathepsin B activity in putative endocytic vesicles and lysosomes supports the intracellular redistribution of protease visualized with immunofluorescence microscopy.