The role of the urothelial mucosa in bladder mechanosensation.

This thesis investigates the role of the urothelium in bladder mechanosensation with focus on the cholinergic signalling pathway. The urothelium can be seen as a first responder to physiological changes in the bladder. It releases a host of mediators including ACh, ATP and NO that play a coordinated role in the stimulation of signalling cascades, which then lead to the onset of afferent nerve activity and detrusor muscle contraction, and finally trigger the micturition reflex. However, the underlying signalling pathways remain elusive. Understanding the signalling pathways involved in the mechanosensation of the urinary bladder will improve the understanding of bladder function, both in health and pathology and ultimately lead to novel treatment options available to patients of lower urinary tract conditions such as overactive bladder syndrome.Mediator release experiments in the whole, isolated murine bladder depicted that ACh is released in a mechanosensitive manner from the urothelium. Distension of as little as 5 mm Hg stimulated a significantly greater release of ACh compared to resting level. However implementation of rising pressure levels showed that the amount of ACh decreased in a negative correlation to the applied pressure. A stretch dependent choline uptake appears to be a convincing explanation for these results. Spontaneous contractions were also measured in the whole, isolated murine bladder and showed a positive correlation to increasing distension levels. Blocking different components of ACh release and choline uptake in the urothelium showed that interfering at any point in the cholinergic pathway does trigger the same response in the muscle, leading to an extremely contracted detrusor muscle, even at low pressure levels. It has been hypothesized in previous studies, that the balance of inhibitory and excitatory mediators released from the urothelium modulates afferent nerve activity and therefore bladder contractility. This balance might be altered when blocking components of the cholinergic pathway. In the present thesis it was also shown, that application of BoNT/A, a novel treatment option for lower urinary tract symptoms, alters this balance by decreasing the excitatory mediator ATP and by increasing the inhibitory mediator NO. How exactly these mediators are balanced and which imbalances occur in the onset of lower urinary tract symptoms will be the topic of future research.New insights on the physiological changes occurring in the naturally aged murine bladder have been shown in this thesis. A significantly higher sensitivity of the aged detrusor in the contractile response to purinergic and muscarinic stimulation was observed. Furthermore, urothelial release of ATP was increased while release of ACh was decreased in the aged bladder. Urothelial NO release was not affected by age and Substance P could not be shown to be released by the urothelium of adult or aged murine bladders. Moreover an increased purinergic receptor sensitivity of aged urothelial cells was shown, which is probably facilitated via the purinergic P2X3 receptor. Further characterisations of the studied pathways are now required to fully validate the data, most suitably in human tissue, as it is not clear if the same pathways are affected by age in the human bladder.Spontaneous contractions and carbachol-induced contractions were measured in male and female porcine bladder strips isolated from the dome, body and trigone region. The amplitude and frequency of these contractions were analysed. After cholinergic stimulation the amplitude of denuded tissue strips was higher compared to the intact counterparts in all bladder regions and in both genders. The existence of an urothelial derived inhibitory factor had been proposed before and would explain these results. Functional differences were also observed between the three bladder regions, particularly the female trigone seems to act differently compared to the bladder dome and body. Gender differences were not observed when comparing spontaneous activity of male and female bladder strips. However, after cholinergic stimulation, female tissue strips from the trigone region showed significantly higher amplitude and frequency in the contractile response. Gender differences and regional disparities should be considered when comparing the findings of detrusor contractility studies. In further experiments it should be examined if mediator release and receptor distribution of the urothelium differs between the different regions of the bladder and between the genders.In summary it can be stated that the urothelium plays an important role in signalling processes and in detrusor contractility of the porcine and murine urinary bladder. Further research is required to fully understand the signalling pathways in the different bladder cells and their interactions