Antidiuretic Hormone Modulates Membrane Phosphoproteins in Toad Urinary Bladder and Retrieved Water Channel Containing Apical Membrane Vesicles

Title: Antidiuretic Hormone Modulates Membrane Phosphoproteins in Toad Urinary Bladder and Retrieved Water Channel Containing Apical Membrane Vesicles
Author: Harris, H. William
Publisher: Journal of the American Society of Nephrology
Date Published: March 01, 1991
Reference Number: 278
Antidiuretic hormone (ADH) dramatically increases the water permeability of toad urinary bladder by insertion of unique highly selective water channels into the apical membranes of granular cells. Before ADH stimulation, water channels are stored in high concentrations in the limiting membranes of large cytoplasmic vesicles called aggrephores. ADH stimulation causes aggrephore fusion with the granular cell apical membrane and increases water permeability. Transepithelial osmotic water flow causes a rapid attenuation of the ADH-elicited increase in water permeability through a process called flux inhibition. Flux inhibition is due to retrieval of ADH water channels by apical membrane endocytosis. When phosphoproteins of intact bladders are labeled with (32P)orthophosphate, the 32P content of 34-, 28-, and 17-kDa proteins is increased by ADH stimulation. When flux inhibition occurs, the 32P-labelling of a 15.5-kDa protein is reduced to approximately one half its original value (Konieczkowski M, Rudolph SA, J Pharmacol Exp Ther 1985;234:515). These observations have been confirmed, and these studies have been extended, by using a combination of subcellular fractionation and membrane protein chemistry techniques. All four of these phosphoproteins are present in membrane fractions of granular cells. Analysis of membrane proteins by a combination of Triton X-114 partitioning and an alkaline stripping technique reveals that the 28- and 17-kDa species are integral membrane proteins of unknown function. In contrast, the 32P-labeled 15.5-kDa protein is a peripheral membrane protein. It is attached to the cytoplasmic (outer) surface of highly water-permeable vesicles retrieved during flux inhibition.
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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 antidiuretic hormone (ADH) binds with its receptors on the granular cells of the toad urinary bladder, it induces a process that increases the water permeability of the apical membrane of the granular cell. It does this by instigating a molecular process that induces a protein called a water channel (WC) to insert itself into the apical membrane of the granular cell. (If you picture the granular cell as an upright rectangle, the top of the rectangle is the apical membrane.) The WC sits within a vesicle called an aggrephore. The aggrephore is located inside the cell beneath the apical membrane. It is the aggrephore that responds to ADH by traveling to and fusing with the apical membrane. Upon fusion, the WC is inserted in the apical membrane. Once inserted into the apical membrane, the WC dramatically increases the amount of water that flows through the membrane.

As the water flows across the cell membrane through the WCs, bits of the apical membrane are taken inside the cell and the apical membrane is restored to its normal state of low water permeability. The bits of apical membrane contain vesicles which contain WCs. Thus, it is the retrieval of WCs from the apical membrane back to inside the cell that returns the apical membrane to its normal, low water permeable state.

Harris wanted to discover in more detail the structure and function of some of the proteins of the toad bladder and see if and how they related to the process of WC retrieval from the apical membrane. He knew that three phosphoproteins, each with different molecular mass (a 34 kilodalton (kDa) protein, a 28 kDa protein and a 17 kDa) increased in phosphate content when stimulated with ADH. And he knew a 15.5 kDa phosphoprotein was reduced to about half its original phosphate content when the WCs are retrieved from the apical membrane when the increased water flow across the apical membrane occurs. (This process is called flux inhibition because it reduces the permeability of the apical membrane and thus reduces the water flow across it.)

Harris confirmed that all four of these phosphoproteins are present in the membrane of granular cells. He found that the 28 and 17 kDa proteins were integral membranes proteins. That is, they were embedded in the cell membranes. The author could not, however, determine their function, but he did determine that these two phosphoproteins were not components of retrieved apical membrane proteins.

In contrast, the 34 kDa and the 15.5 kDa proteins are peripheral membrane proteins, which are merely attached to - not embedded in - the membranes. The 15.5 kDa protein is attached to the outside surface of retrieved apical membrane vesicles containing WCs. Harris speculates that this protein may play a role in the retrieval of vesicles from the apical membrane in response to ADH-elicited water flow during flux inhibition.