Cytoplasmic Dilution Induces Antidiuretic Hormone Water Channel Retrieval in Toad Urinary Bladder

Title: Cytoplasmic Dilution Induces Antidiuretic Hormone Water Channel Retrieval in Toad Urinary Bladder
Authors: Botelho, MD, Barbara; Zeidel, Mark L.; Strange, Kevin; Harris, H. William
Publisher: American Journal of Physiology
Date Published: July 01, 1992
Reference Number: 274
Antidiuretic hormone (ADH) increases the osmotic water permeability (Pf) of the toad urinary bladder by insertion of water channels into the apical cell membrane. Transepithelial water flow (Jv) reduces Pf by inducing endocytosis of apical water channels despite continuous ADH stimulation. This phenomenon is termed flux inhibition. We wished to determine whether cytoplasmic dilution or transcellular Jv causes flux inhibition because both have been proposed previously as a primary regulatory mechanism for this process. Apical membrane endocytosis was quantified by monitoring the uptake of the fluid phase marker fluorescein isothiocyanate dextran (FITC-dextran). FITC-dextran fluorescence was monitored in Triton X-100 extracts of epithelial cells as the ratio of total tissue fluorescence compared with background fluorescence. The background was defined as cellular autofluorescence and nonspecific tissue staining due to the presence of small amounts of free fluorescein contaminating the FITC-dextran. FITC-dextran uptake measured under symmetric isotonic (220 mosmol/kgH2O) conditions in either the absence (1.0 +/- 0.4 SD; n = 14) or presence (1.3 +/- 0.3; n = 4) of ADH was not statistically different from that of background. In contrast, flux inhibition induced by a 180 mosmol/kgH2O apical-to-basolateral osmotic gradient increased FITC-dextran uptake to 3.4 +/- 1.3 (n = 7). FITC-dextran uptake was identical in bladders exposed to symmetric hypotonic (150 mosmol/kgH2O) solutions during ADH (3.6 +/- 0.9; n = 6) or adenosine 3',5'-cyclic monophosphate (3.1 +/- 0.4 fold; n = 3) stimulation.

This translation by the NDI Foundation is to assist the lay reader. To provide a clear, accessible interpretation of the original article, we eliminated or simplified some technical detail and complicated scientific language. We concentrated our translation on those aspects of the article dealing directly with NDI. The NDI Foundation thanks the researchers for their work toward understanding and more effectively treating this disorder.
© Copyright NDI Foundation 2007 (JC)

When a cell membrane is called permeable it means that solutions, e.g. body water, can pass through it. Different cell membranes have different degrees of permeability, and sometimes different portions of the same cell membrane will have different degrees of permeability. For example, in the granular cells of the toad urinary bladder, the apical membrane is far less water permeable than the basolateral membranes. (If you picture the granular cell as an upright rectangle, and the perimeter of the rectangle as the cell's membrane, the bottom and sides are the basolateral membrane and the top is the apical membrane.)

Osmosis is the process by which a solution such as body water can move through the membranes. Osmosis is the passage of the solvent part of a solution through a membrane. (A solution is comprised of solvent, e.g. the pure water of salt water, and solutes, e.g. the particles of salt in salt water.) In normal osmosis, the direction of the solvent flow is always from the solution with the lesser solute concentration to the solution with the greater solute concentration. So, for osmosis to happen there must be two solutions of unequal solute concentrations, one on each side of a permeable membrane. And the membrane must be able to let solvent through and not let most of the solutes through.

Antidiuretic hormone (ADH), when it binds to receptors on the granular cell basolateral membrane of the toad urinary bladder, induces a great increase in the apical membrane's degree of water permeability (Pf). It does this by initiating a molecular sequence that induces water channels (WCs) to insert themselves into the apical membranes. WCs are proteins that act as channels through which water can flow. Most WCs only allow water to flow through them. They sit inside little sacs called WC vesicles (WCVs) located within the cell. When signaled by ADH, the WCVs travel to the apical membrane and fuse with it. At this point the WCs are inserted. This allows the solvent aspect (e.g. free body water) of the toad's urine to flow across the cells that make up the epithelium (the tissue lining of the toad bladder) and move into other parts of the toad's interior. The direction of this transepithelial water flow (Jv) is from the urine to the fluid on the other side of the granular cell because the urine has a lower concentration of solutes than the fluid.

However, this transepitheleal water flow does not go on forever. There is a process that reduces the flow of water across the granular cell apical membrane by reducing the apical membrane's Pf. The ADH-stimulated insertion of WCVs into the apical membrane increases the membrane's Pf. The increased Pf results in increased transepithelial Jv (provided there is an osmotic gradient, i.e. two solutions of unequal solute concentration on either side of the membrane.) However, within minutes after the transepithelial Jv is initiated, there is a reduction of Pf. This phenomena is called flux inhibition and it reduces Pf in proportion to the increase in transepithelial Jv.

During flux inhibition there is a simultaneous retrieval of WCVs from the apical membrane. This causes a decrease in apical membrane Pf, which results in a decrease in transepithelial Jv. Researchers determined that the decrease in membrane Pf occurs because the WCVs are removed from the apical membrane. Research revealed that the retrieval of WCVs is responsible for flux inhibition, but what signaled WCV retrieval?

Harris, et al, investigated the mechanisms that regulated the removal of WCVs during flux inhibition. It had been proposed that either transepithelial Jv or cytoplasmic dilution cause flux inhibition by signaling the retrieval of WCVs from the apical membrane. (Cytoplasmic dilution is a state where the protoplasm in the cell - all the stuff except the nucleus - is diluted because of an increase of water in the cell.) The authors devised a series of experiments to determine if either of these processes was indeed the cause.

The authors were able to observe and quantify the movement of WCVs from the apical membrane to the interior of granular cells by staining them with a marker called FITC-dextran. They then subjected their granular cell cultures to various hormonal and osmotic conditions which would either inhibit or encourage transepithelial Jv.

They demonstrated that when the granular cells were stimulated by ADH while being exposed to osmotic conditions that resulted in a complete absence of transepithelial Jv, there was both a reduction in the membrane Pf and a retrieval of WCVs identical to what occurs after the membrane is exposed to osmotic conditions that cause a large transepithelial Jv. In other words, WCV retrieval could take place independent of transepithelial Jv.

This demonstrated that the removal of WCVs and their movement back within the cell can occur independently of transcellular Jv and that cytoplasmic dilution alone is sufficient to activate retrieval of ADH water channels. Because identical declines in transepithelial Pf occur in conjunction with identical quantities of apical membrane retrieval under both cytoplasmic dilution minus transepithelial Jv and flux inhibition caused by transepithelial Jv, it is not necessary to postulate the existence of a Jv-sensing mechanism that regulates WC retrieval. This strongly suggests that cytoplasmic dilution is the mechanism that induces antidiuretic hormone WC retrieval in toad urinary bladder.