Study on the origin of apical tubules in ileal absorptive cells of suckling rats using concanavalin-A as a membrane-bound tracer.

Abstract

The ileal absorptive cells of suckling rats exhibit high levels of endocytic activity being engaged in nonselective uptake of macromolecules from the intestinal lumen. The apical cytoplasm usually contains an extensive network of small, membrane-limited tubules (apical tubules: AT), in addition to newly formed endocytic vesicles and large endocytic vacuoles. To determine whether the AT are directly involved in the endocytic process by carrying the tracer into the cell, we have analysed movements of the apical cell membrane of the ileal absorptive cells by using a membrane-bound tracer (horseradish peroxidase-labelled concanavalin-A: Con-A HRP). The ileal absorptive cells were exposed in vitro to Con-A HRP for 10 min at 4 degrees C, incubated for different times in Con-A free medium at 37 degrees C, and prepared for electron microscopy. After 1 min incubation at 37 degrees C, invaginations of the apical cell membrane, including coated pits, and endocytic vesicles were labelled with HRP-reaction product, whereas the AT and large endocytic vacuoles were negative. After 2.5 min, almost all the large endocytic vacuoles were labelled with reaction product, which was seen in their vacuolar lumen and along the luminal surface of their limiting membrane. A few AT with reaction product were seen in the apical cytoplasm; they were in frequent connection with the reaction-positive large endocytic vacuoles. With increasing incubation time, the number of the labelled AT increased. Thus, after 15 min at 37 degrees C, the apical cytoplasm was fully occupied by the reaction-positive AT. The ends of these AT were often continuous with small spherical coated vesicles. No reaction product was detected in the Golgi complex at any time after incubation. These observations indicate that the AT located in the apical cytoplasm probably originate by budding off from the large endocytic vacuoles, rather than being involved in the process of endocytosis.