Date of Award
Doctor of Philosophy (PhD)
Laberge , Martine
Webb , Charles K
Bell , Phillip D
Overactive bladder (OAB) is a bladder disorder that is characterized by bladder storage symptoms of urgency with or without urge incontinence, frequency, nocturia, and, as of 2003, affected approximately 16.5% of adults in the United States with an annual treatment cost of over $65 billion. While therapies are available to mitigate the symptoms of OAB, there are no treatments for the cause of OAB, due to the lack of understanding of the etiology of the disorder. Recent research has provided evidence that the bladder urothelium is not just a passive barrier, but is also sensitive to various chemical and mechanical stimuli and responds by releasing neurotransmitters (NO, ATP) to participate in the sensory function of the bladder. Thus, it has been suggested that overactive bladder is caused, at least in part, by altered sensitivity to bladder fullness by urothelial cells (UCs). While previous research has demonstrated that the urothelium responds to tissue stretch induced by elevated pressure, the role of hydrostatic pressure alone in the cellular events was unclear. Therefore, this doctoral thesis sought to decouple the two stimuli and investigate the effects of pressure on UCs, especially the role of select ion channels in UC pressure mechanotransduction. Overall, the objective of this project was to test the hypothesis that UCs sense hydrostatic pressure via activation of membrane-bound ion channels, and that these cells respond by releasing ATP. In order to test this hypothesis, multiple in vitro studies were performed in which UCs were subjected to controlled hydrostatic pressure while monitoring their response to the stimulus.
First, using a custom-made pressure chamber, rat bladder UCs were exposed to sustained hydrostatic pressure (5-20 cmH2O) for up to 30 min. When compared to the control, the supernatant culture media of UCs exposed to hydrostatic pressure (10-15 cmH2O) exhibited a significant increase in ATP. In the absence of extracellular calcium, ATP release due to hydrostatic pressure was attenuated. Pharmacologically blocking transient receptor potential (TRP) channels, stretch-activated channels (SACs), and the epithelial sodium channel (ENaC) all abolished the hydrostatic pressure-evoked ATP release. These results provided evidence for the first time that cultured UCs are sensitive to physiologically relevant levels of hydrostatic pressure and that one or multiple mechanosensitive ion channels play a role in the mechanotransduction of hydrostatic pressure. These findings support the view that not only tissue stretch or tension, but also pressure is an important parameter for the sensing of bladder fullness.
Second, UCs loaded with a fluorescence dye, calcein-AM were exposed to hydrostatic pressure up to 20 cmH2O while fluorescence intensity was measured in real-time. UCs exposed to hydrostatic pressure exhibited a sharp decrease in fluorescence intensity, indicative of a cell volume increase. These observations were confirmed by confocal imaging of primary UCs, which displayed a 7.7% volume increase when exposed to 20 cmH2O. Exposing the cells to Na-free solution and blocking of ENaC during pressure application resulted in a significantly lower change in cell volume. These results provided part of a novel mechanism involving cell swelling for mechanotransduction of hydrostatic pressure and an explanation for the previously reported ENaC-dependent, pressure-evoked ATP release by UCs.
Finally, UCs were exposed to hydrostatic pressure while monitoring the kinetics of ATP release. UCs exposed to pressure (10 cmH2O) exhibited a sharp increase in ATP release that slowly decreased over time, while remaining elevated compared to baseline levels. This response was inhibited by blocking TRPV4 and ENaC. When UCs were exposed to a hypotonic stimulus, a sharp increase in ATP was exhibited followed by an immediate decrease to baseline levels. This increase in ATP was inhibited when blocking TRPV4, but was still present, albeit attenuated compared to the unblocked cells, when blocking ENaC. These results suggest that both ENaC and TRPV4 activation are necessary for ATP release during exposure to hydrostatic pressure. In addition, the osmotic shock results suggest that ENaC is upstream of the cell swelling response, while TRPV4 activation occurs between cell swelling and the ATP release.
In summary, the results of the present research provide evidence that UCs respond to hydrostatic pressure with an increase in cell volume, activation of multiple mechanosensitive ion channels, and elevated ATP release. We have identified a possible mechanism by which UCs detect pressure and are the first to show that UCs respond to hydrostatic pressure in the absence of tissue stretch, which provides a novel contribution to cell mechanobiology that hydrostatic pressure alone is an important parameter involved in bladder mechanotransduction. This knowledge, in turn, will help elucidate the mechanism by which UCs, in normal and pathological cases, sense bladder fullness and ultimately aide in treatment of bladder disorders such as OAB.
Olsen, Shawn, "Investigating the Role of Mechanosensitive Ion Channels in Urothelial Cell Pressure Mechanotransduction" (2012). All Dissertations. 982.