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

Abstract

Antidiuretic hormone (ADH) increases the water permeability of the toad urinary bladder. The increase occurs in the apical plasma membrane of granular cells that line the urinary surface of the bladder and is produced by the insertion of water permeability units that have been identified by freeze-fracture electron microscopy as intramembrane particle aggregates. Under water-impermeable conditions, particle aggregates reside in intracellular vesicles called "aggrephores." In response to ADH, the aggrephores fuse with the apical plasma membrane and render it water permeable. When ADH is removed, intramembrane particle aggregates and aggrephores are retrieved from the apical membrane, and it returns to a water-impermeable state. To identify proteins involved in the water permeability response, we used lactoperoxidase/glucose oxidase to "MI-label external apical membrane proteins to compare control and ADH-treated bladders. Several polypeptides were consistently labeled in ADH-treated bladders and not in paired controls. After demonstrating that lactoperoxidase behaves as a fluid-phase marker and is sequestered in aggrephore-like vesicles when ADH is withdrawn, we used the technique of Mellman et al. BMl. 86, 712-7221 to label proteins endocytosed when water permeability declines after ADH is withdrawn to test whether the membrane proteins labeled in ADH-treated bladders behaved like particle aggregates. The internalized membranes contained polypeptides of the same molecular weights (55,000, 17,000-14,000, and 7,000) as those labeled on the apical surface of ADH-treated but not control bladders. These polypeptides are evidently involved in the ADH-stimulated water permeability response and may be components of particle aggregates.Antidiuretic hormone (ADH) plays a major role in the control of water balance of many vertebrates. It stimulates the conversion of specific water-impermeable epithelia to a state of high water permeability. For example, the water permeability of the urinary bladder of the toad Bufo marinus increases by >50-fold, allowing water to flow from dilute urine to blood (1). The purpose of this study was to identify membrane proteins involved in the water permeability change that occurs in the toad bladder in response to ADH.The apical plasma membranes of granular cells form >90o of the bladder surface area in contact with urine, and it is these apical membranes that undergo the remarkable change in water permeability (2). Under basal conditions, the apical cytoplasm of granular cells contains tubularshaped vesicles that have been named "aggrephores" because on freeze-fracture electron microscopy they contain distinctive particle aggregates within their limiting membranes (3-5). Aggrephores store and shuttle particle aggregates into and out of the apical plasma membrane in response to changes in the concentration of ADH (6). In response to ADH, aggrephores fuse with the apical plasma membrane and the plasma membrane becomes enriched in particle aggregates (4-8). When ADH is withdrawn, parti...

Discovery of the Aquaporins and Development of the Field

“…Many groups tried examining known channels and carriers for water transport (reviewed in van Os et al 1994). William Harris and colleagues radioactively labeled proteins on the apical surface of toad bladder exposed to antidiuretic hormones (ADH) and compared those proteins to ones labeled without ADH in an attempt to find the protein that was specifically localized on the plasma membrane in response to ADH (Harris et al 1988). Expression cloning was attempted using mRNA from various tissues that were injected into Xenopus laevis oocytes to see if there was an increase in water permeability (Zhang et al 1990).…”

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

“…Fractionation of these labeled proteins by SDS-PAGE has identified bands of 55, 53, 17, 15, and 7 kD as appearing exclusively on the surface of ADH-stimulated water-permeable bladders. Furthermore, sequestration ofa lactoperoxidase membrane iodination mixture within retrieved WCV in intact living cells results in the 125I labeling of the same bands of 55,53,17,15, and 7 kD (53). To further identify the protein bands that are likely parts of the water channel, we have examined the ability of these proteins to interact with lipid using the Triton X-1 14 partitioning analysis.…”

“…We (53) and others (54) have performed detailed comparisons using membrane-impermeant probes that covalently label exposed protein domains. Fractionation of these labeled proteins by SDS-PAGE has identified bands of 55, 53, 17, 15, and 7 kD as appearing exclusively on the surface of ADH-stimulated water-permeable bladders.…”

Aquaporins (Water Channels): Role in Vasopressin-Activated Water Transport

“…The exact sequence of events between cAMP production and the fusion of water channels at the luminal membrane is largely unknown. However, Harris et al (125) have shown that vasopressin induced the insertion of at least 3 proteins of 55, 14-17, and 7 kDa at the apical membrane as detected by 1251 surface labeling. However, whereas one report has detected changes in the iodination patterns (126), another study failed to detect any changes in the apical membrane insertion using similar methods (127).…”

“…The vehicle used to drive these vesicles is a protein known as dynein and is an ATP-dependent process (99)(100). Water vesicles are associated with a number of GTPbinding proteins that appear important for vesicle fusion and trafficking at the apical membrane (125). Fusion at the plasma membrane is likely involving a PLA, enzyme.…”