Mechanical forces affect many cellular functions. How cells respond to mechanical stimuli and how the stimuli are transduced into cellular signals are important questions in cell biology. To this end, the effects of hydrostatic pressure (filling) on bladder uroepithelium were examined by exposing isolated pieces of uroepithelium to hydrostatic pressure in modified Ussing chambers, thus mimicking the filling process of rabbit urinary bladders. Filling had profound effects on both ion and membrane transport in the uroepithelium. Membrane distention activated mechanosensitive ion channels, stimulating Na+ absorption across the umbrella cells via epithelial Na+ channels, K+ secretion via nonselective cation channels, and Cl- secretion via Cl- channels. In addition to elevated ion transport, pressure also stimulated discoidal vesicle exocytosis in umbrella cells, resulting in ~55% increase in apical surface area of the umbrella cells. Exocytosis in these cells also increased the amount of uroplakin III at the apical surface by 67%, and caused release of secretory proteins into the bladder lumen. One pressure-sensing mechanism that could regulate umbrella cell exocytosis is ATP and purinergic receptor signaling. Significant amounts of ATP were released from the uroepithelium during filling. However, when the serosal surface of the uroepithelium was treated with the ATPase apyrase or hexokinase, or incubated with the purinergic receptor antagonist PPADS, pressure-activated exocytosis was blocked. More importantly, filling-induced exocytosis was blocked when P2X2 or P2X3 receptors were absent from mice bladders. The exocytic events were mediated by Ca2+, cAMP, and PKA-dependent mechanisms. In addition to exocytosis, hydrostatic pressure induced endocytosis of 100% of a biotinylated membrane pool within 5 minutes of stimulation. The endocytosed membrane was delivered to lysosome and degraded by a leupeptin-sensitive pathway. The endocytic event could be activated by the purinergic receptor agonist ATP?S, indicating that filling-induced endocytosis may depend on purinergic receptor signaling as well. These results have shed light on how hydrostatic pressure regulates ion and membrane transport in uroepithelial cells and may provide insights to how other mechanosensitive cell types respond to external forces.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-02062004-163352 |
| Date | 16 February 2004 |
| Creators | Wang, Edward Chi Yu |
| Contributors | Dr. Rebecca Hughey, Ph.D., Dr. Thomas Kleyman, M.D., Dr. Gerard Apodaca, Ph.D., Dr. Lori Birder, Dr. John Johnson, M.D. |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-02062004-163352/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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