Isolation and characterization of specialized regions of toad urinary bladder apical plasma membrane involved in the water permeability response to antidiuretic hormone

Abstract

Antidiuretic hormone (ADH) increases the apical (external facing) membrane water permeability of granular cells that line the toad urinary bladder. In response to ADH, cytoplasmic vesicles called aggrephores fuse with the apical plasma membrane and insert particle aggregates which are visualized by freeze-fracture electron microscopy. Aggrephores contain particle aggregates within their limiting membranes. It is generally accepted that particle aggregates are or are related to water channels. High rates of transepithelial water flow during ADH stimulation and subsequent hormone removal decrease water permeability and cause the endocytosis of apical membrane and aggrephores which retrieve particle aggregates. We loaded the particle aggregate-rich endocytic vesicles with horseradish peroxidase (HRP) during ADH stimulation and removal. Epithelial cells were isolated and homogenized, and a subcellular fraction was enriched for sequestered HRP obtained. The HRP-enriched membrane fraction was subjected to a density shifting maneuver (Courtoy et al., J. Cell Biol. 98:870, 1984), which yielded a purified membrane fraction containing vesicles with entrapped HRP. The density shifted vesicles were composed of approximately 20 proteins including prominent species of 55, 17 and 7 kD. Proteins of these molecular weights appear on the apical surface of ADH-stimulated bladders, but not the apical surface of control bladders. Therefore, we believe these density shifted vesicles contain proteins involved in the ADH-stimulated water permeability response, possibly components of particle aggregates and/or water channels.

Vesicle Recycling and Cell-Specific Function in Kidney Epithelial Cells

“…BROWN vesicles, and identification of specific proteins as candidate water channels; initial steps in this direction have been reported recently for the toad urinary bladder (16). After fixation the tissue was exposed to filipino which disrupts most of the apical membrane.…”

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

“…1). These retrieved vesicles containing either entrapped carboxyfluorescein, fluorescein dextran (F dextran), or horseradish peroxidase (HRP) have been recovered from homogenates ofgranular cells (6,(24)(25)(26)(27)(28) and the renal papillas ofBrattleboro rats (29) and used to measure vesicle membrane Pf and proton permeability (PH+). The Pf of these water channel-containing vesicles (WCV) is extraordinarily high, demonstrating that functional water channels are removed from the apical membrane by vesicle endocytosis.…”

“…We have purified toad bladder WCV using either a density shift protocol (28) or flow cytometry vesicle sorting (55) and characterized their membrane proteins. Purified WCV are composed of -12-15 protein bands as determined by SDS-PAGE (Fig.…”

Very low osmotic water permeability and membrane fluidity in isolated toad bladder granules

“…The gel proteins are identified as components of the osmotically shocked granule membrane: " G 114" are the lipophilic proteins that partition into the detergent phase. " G aq" are the hydrophilic proteins found in the aqueous phase after multiple temperaturedependent extractions shows a 14-160 kDa range of intrinsic proteins, which includes proteins of 17 and 55 kDa, corresponding to two out of the three most prominent proteins reported by Harris et al (1987) in density shifted endosomes from toad bladder.…”