INTERNALIZATION IN VIVO OF CHOLERA TOXIN IN THE SMALL INTESTINAL EPITHELIUM OF THE RAT

Abstract

Internalization in vivo of cholera toxin in the small intestinal epithelium of the rat. Acta path. microbiol. immunol. s a n d . Sect. A. 92: 15-21, 1984.The binding and internalization of cholera toxin (CT) into the intestinal epithelium were studied in vivo in rats. The distribution of CT was ascertained using immunofluorescence and by immunoenzymeelectron microscopy, with horse-radish peroxidase anti-CT antibodies as the conjugate. The toxin was rapidly bound and internalized into both epithelial and goblet cells; CT was evenly distributed on the microvilli at the bases of which it appeared in invaginations (coated pits). Though not found in nuclei, CT appeared intracellularly in coated vesicles, and dissolved in the cytoplasm where it was enriched at the terminal web. The basolateral membrane, except for the tight junctions, was outlined with (JT; some staining also appeared in the basement membrane. in fibroblasts, macrophages and in the bloodvessel walls in the submucosa. The lysosomatotrophic agent chloroquine simultaneously inhibited CTinduced fluid secretion and intracellular distribution of C T in the cytoplasmic matrix, but not in the vesicles. The inhibitor of CT-action on adenylate cyclase, chlorpromazine, did not affect the cellular distribution of CT. Our results suggest that CT mainly is internalized by endocytosis into the intestinal epithelium. The toxin is probably released from vesicles into the cytoplasm via secondary lysosomes.

CYTOSKELETAL DISARRANGEMENT IN RAT INTESTINAL EPITHELIUM AFTER IN VIVO EXPOSURE TO SECRETAGOGUES

“…and cytochalasin B causes an immediate secretion presumably by disruption of microfilaments. W e have previously shown that cytoplasmatic CT in epithelial cells is concentrated to the terminal web region (9) supporting the idea that CT might interact directly with cytoskeletal components. However, cholera secretion seems largely to depend on CT-induced activation of adenylate cyclase located in the basolateral membrane (22).…”

“…Liglir and rlocirotz tnicroscop. Sections for light and electron microscopic examination were prepared as previously described (9). Multiple sections from several blocks from each loop were carefully analysed in order to evaluate the significance of the structural changes observed.…”

“…Chloroquine inhibited CT-induced secretion even more strongly than colchicine. W e have previously shown that in rats chloroquine inhibits the endocytosis of receptor-bound CT into epithelial cells (9).…”

“…Recent evidence from experiments in rats ( 9 ) suggest that CT is endocytized into the intestinal epithelial cells and released into the cytoplasm before activation of adenylate cyclase; the cytoplasmically dissolved CT is concentrated to the terminal web region. CT and LT interact with cytoskeletal components in model cells ( I I , 19).…”

“…Three inhibitors to enterotoxic secretion colchicine ( 1 6). chloroquine (9) and chlorpromazine ( 15) were also tested for possible effects on the cytoskeletal organization.…”