Fluorescent markers to study membrane retrieval in antidiuretic hormone-treated toad urinary bladder

Abstract

Antidiuretic hormone (ADH) stimulation of toad urinary bladder causes fusion of intracellular vesicles called aggrephores with the apical plasma membrane of granular cells. Aggrephores contain intramembrane particle aggregates whose appearance in the apical membrane is believed to produce a large increase in its water permeability. ADH removal (ADH washout) is thought to cause the retrieval of aggrephores into granular cell cytoplasm. We studied granular cell uptake of dextran and horseradish peroxidase conjugated with fluorescein, rhodamine, or both during ADH washout. Granular cell uptake of fluorescent dextran was dependent on prior exposure to ADH, a linear function of dextran concentration, and increased by a transepithelial osmotic gradient. Immediately after removal of ADH, granular cell fluorescence was finely dispersed and located near the apical surface. Subsequently, it coalesced into larger bodies. This change was most apparent when a single bladder was subjected to two cycles of ADH stimulation and removal using a dextran containing a different fluorophore for each cycle. The ultrastructural correlate for these fluorescent patterns was identified using rhodamine-labeled horseradish peroxidase. Electron microscopy showed that after detachment from the apical membrane, label was initially in tubular-shaped vesicles near the apical surface. Later, these vesicles clustered near multivesicular bodies and transferred their label to these structures. These tubular vesicles closely resemble the morphology of aggrephores visualized by freeze-fracture electron microscopy. We conclude that these fluorescent compounds can be used as markers for the luminal contents of membrane retrieved during ADH washout and allow detailed study of its intracellular processing.

Identification of specific apical membrane polypeptides associated with the antidiuretic hormone-elicited water permeability increase in the toad urinary bladder.

“…Finally, the apical surface of ADH-stimulated bladders was exposed to an isosmotic solution containing lactoperoxidase at 5 mg/ml. Under these conditions there was no endocytosis (26,31), and peroxidase levels were the same as in cells from control bladders never exposed to lactoperoxidase/glucose oxidase (open circle in Fig. 2).…”

“…Retrieval of granular cell apical membrane induced by ADH stimulation and removal has been studied by using a variety of fluid-phase markers, including HRP (6,10,26,31), ferritin (6), colloidal gold (7,10), and fluorescent dextran (31). Fig.…”

“…Removing the ADH from the solution bathing a bladder's basal surface causes the retrieval of vesicles that is stimulated by osmotic water flow (9,30,31). A large proportion of these vesicles is morphologically identical to aggrephores (5,6,8 gradient (no apical membrane retrieval occurs).…”

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

“…Measurements ofuptake of fluorescent dextran or horseradish peroxidase (HRP) by granular cells were performed as previously described (27) using fluorescein-cnjugated dextran (F-dextran) and rhodamine-labeled horseradish peroxidase (R-HRP) at concentrations of20 and 5 mg/ml, respectively. The fluorescent marker was present in the mucosal solution in contact with the apical surface of granular cells during the uptake period as described in the text.…”

“…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).…”