The Cellular Action of Antidiuretic Hormone
|Title:||The Cellular Action of Antidiuretic Hormone|
|Authors:||Ausiello, M.D., Dennis A.; Brown, Dennis; Verkman, Alan S.; Skorecki, Karl L.|
|Publisher:||Methods in Enzymology|
|Date Published:||January 01, 1990|
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)
They have studied the form and function of VP through freeze-fracture studies. Freeze-fracturing is a method of preparing cells (in this instance, the CD principal cells) for electron-microscopal examination. Researchers freeze the collecting duct cells at -150 degrees Celsius. Then they insert the frozen tissue into a vacuum chamber. Then the cell tissue is cut into thin slices. Researchers then make a platinum carbon replica of the exposed tissue surfaces. Finally, the underlying tissue is removed before the specimen is examined under an electron microscope.
This method applied to VP in the CD cells revealed that specialized membrane domains called IMP clusters appear on the apical membrane of the cells at the same time the VP-induced water flow occurs through the cells. Following VP withdrawal, the apical membranes lose their highly permeable state at the same time the number of IMP cluster leave the apical membrane.
The IMP clusters are absent from the CD cells of mice with nephrogenic diabetes insipidus (NDI). This suggests that IMP clusters contain water channels which increase the permeability of CD cells when VP can influence the cell properly.
Exposing the antibiotic, filipin, to tissues and then preparing the tissue for freeze-fracture examination produces easily visible markers that allow researchers to track VP action on even finer levels. Researchers found IMP clusters located in clathrin-coated pits on the CD apical cell membranes. Clathrin is a protein. It coats pits that form on the apical membrane surface. These clathrin-coated pits were found to be involved in helping bringing the IMP clusters back inside the cells after VP had withdrawn from the cell culture.
These techniques allowed researchers to detect the endpoint of VP action -- the appearance of IMP clusters in the CD cells' apical membrane at the same time the membrane increased its water permeability.
Using tracers such as horseradish peroxidase and fluorescein dextrans, researchers were able to trace the path the IMP clusters took after VP withdrawal. Endocytosis is the process wherein a cell brings something into it. The movement of the IMP clusters from the apical membrane to the inside of the cell cytoplasm is an endocytosic process. In endocytosis, vesicles which have lost their coat of clathrin are called endosomes. Using the tracing techniques, researchers were able to characterize the IMP clusters, during the endocytosic cycles, as endosomes containing water channels (WCs) that moved from the apical membrane to the subapical area of the cell.
Having identified WCs, researchers were able to derive important information on how they functioned in response to VP by directly measuring the effect on CD cell membrane water permeability. New measurement methods enabled researchers to monitor:
- water flow through the cell membranes,
- the variables and processes responsible for the flow, and
- the conditions that enhance and inhibit the flow.
Compiling the information from these measurements suggests to researchers that VP induces the formation of a population of WC-containing endosomes in CD cells, and that the targeting of the WCs is directly influenced by transcellular water flow.
The induction of the WC-containing vesicles to move from the subapical to the apical membrane of the CD principal cells is the last step in a molecular sequence initiated when VP binds with its receptor, the vasopressin-2 receptor, located in the basolateral membranes of the CD cell. The steps of the sequence involving the activation of the enzyme, adenylate cyclase (AdC) became more thoroughly understood when it was investigated by a method called target analysis.
This technique uses radiation to inactivate the molecule under study. Applied to adenylate cyclase, it helped reveal that before AdC is stimulated by VP, the Gs protein to which it is connected, and which is comprised of three parts, is whole. Upon stimulation by VP, one of the parts of the Gs protein (alpha) disassociates from the other two parts (beta and gamma). It appears that this disassociation is required for the full activation of AdC to occur.