Antidiuretic hormone response in the amphibian urinary bladder: time course of cytochalasin-induced vacuole formation, an ultrastructural study employing ruthenium red.

Cytochalasin is known to inhibit the antidiuretic hormone-induced hydro-osmotic response (bulk water flow) in the amphibian urinary bladder without altering hormone-stimulated diffusional water permeability or short-circuit current. In addition, histological studies have shown that the mold metabolite induces the formation of large intracellular vacuoles or lakes in the epithelial cells. We report here a transmission electron microscopic time-course study which indicates that during the early phases of the ADH response cytochalasin causes the formation of numerous multivesicular bodies or aggregates derived from individual basolateral pinocytotic vesicles. Because of their apparent hypertonic nature, the vesicles, as well as the vesicular aggregates, accumulate water during hormone-stimulated hydro-osmotic flow. As a result, the multivesicular bodies dilate and fuse to form the large intracellular lakes characteristic of cytochalasin treatment in the presence of both an applied osmotic gradient and vasopressin. In the presence of mucosal ruthenium red, the luminal glycocalyx was heavily stained with this tracer. At no time, however, even in the presence of hormone, was there any evidence for the uptake of this dye at the apical epithelial border. In the presence of serosal ruthenium red, the lateral intercellular spaces, basolateral pinocytotic vesicles, basal lamina, and collagen, as well as other subepithelial structures, were ruthenium positive. With cytochalasin D, vasopressin, and serosal ruthenium red, both the pinocytotic vesicles and the multivesicular bodies demonstrated an apparent membrane associated ruthenium positive coat. The tracer data indicates that the basolateral pinocytotic vesicles, increased by the presence of hormone, are indeed endocytotic in nature. The mucopolysaccharide coat associated with these structures may be involved in ionic and/or fluid transport.