Effect of an osmotic gradient on antidiuretic hormone-induced endocytosis and hydroosmosis in the toad urinary bladder

Abstract

The luminal (apical) border of the epithelium of the bladder in the well-hydrated toad is relatively impermeable, so the bladder usually stores hyposmotic urine. When antidiuretic hormone (ADH) increases apical membrane osmotic permeability dramatically, water is resorbed from hyposmotic mucosal solution; in the presence of hyposmotic or isosmotic mucosal solutions, ADH concomitantly induces exocytosis at the apical border of granule-rich (G) cells. Then ADH induces endocytosis at this border. We describe how an osmotic gradient affects ADH-induced endocytosis and hydroosmosis in vitro. We can assess ADH-induced endocytosis in gradient and no-gradient bladders by applying a double-marker technique that distinguishes among endocytosis, completed internalization of previously surface-attached membrane, and surface invagination by comparing the number of horseradish peroxidase (HRP) uptake bodies (endocytosis) with the number of ruthenium red (RR)-delineated bodies (surface invaginations). With this approach we find that gradient bladders have approximately six times more ADH-induced endocytosis than no-gradient bladders during 45-60 min of ADH stimulation. Furthermore, at 60 min approximately 50% of the HRP-containing structures in no-gradient bladders remain surface connected compared with approximately 1% in gradient bladders. In parallel physiological studies, no-gradient bladders reach and maintain higher induced osmotic permeabilities than gradient bladders. These findings support the hypothesis that endocytosis plays an active role in reestablishing impermeable apical surface characteristics in toad bladder.

Current understanding of the cellular biology and molecular structure of the antidiuretic hormone-stimulated water transport pathway.

“…A combination of morphological (20) and tissue capacitance (21) measurements has demonstrated that ADH stimulation causes a 10-30% increase in apical membrane surface area with a time course paralleling the rise in tissue Pf. In a similar fashion, termination ofADH stimulation results in a prompt decrease in tissue Pfand concomitant retrieval ofgranular cell apical membrane in the form ofendocytic vesicles (6,18,20,22) containing particle aggregates (23).…”

“…This conclusion is based on data showing that (a) there is no luminal acidification of WCV in vitro after addition of ATP (26,27,34), (b) measurements of these vesicles in situ using the 342,4-dinitroanilino)-3'-amino-n-methyldipropylamine (DAMP) method show their lumens are not acidic (6), and (c) immunoblots of purified WCV proteins with anti-proton pump antisera reveal these vesicles do not contain the major subunits of the H' ATPase necessary for activity (35). Factors that modulate retrieval of water channels include ADH removal (6,18,20), the magnitude of a transepithelial osmotic gradient (22,27,36), and cytoplasmic dilution or cell swelling (37). Water channel vesicle endocytosis has recently been associated with changes in the phosphorylation state ofa vesicle-associated 1 5.5-kD protein (38).…”

Transepithelial water flow regulates apical membrane retrieval in antidiuretic hormone-stimulated toad urinary bladder.

“…Previous studies have demonstrated that granular cell uptake of fluid phase markers from the mucosal bathing solution during continuous ADH stimulation is greatly enhanced by an osmotic gradient (25,26). In addition, freeze-fracture electron microscopic studies have shown that the number of particle aggregates and aggrephore fusion events in the granular cell membrane is decreased by a transepithelial osmotic gradient during both ADH stimulation (20,21) and the period immediately after hormone removal (21)(22)(23).…”

“…The tracers are found largely in tubular vesicles and multivesicular bodies (25). However, other reports (4,21) disagree as to the importance of apical membrane retrieval during continuous ADH stimulation in the presence of an osmotic gradient.…”

“…Recent studies have shown that the presence of an osmotic gradient markedly increases granular cell uptake of enzymatic (25,26) and fluorescent (27) tracers during ADH stimulation and ADH removal. The tracers are found largely in tubular vesicles and multivesicular bodies (25).…”