Pharmacological investigation of porcine bladder function and sensory activity.

Overactive bladder (OAB) is a common condition of the lower urinary tract (LUT) that is characterised by urinary urgency, with or without incontinence, accompanied by frequency and nocturia as a result of involuntary urinary bladder contractions. OAB affects millions of people worldwide and has a significant impact on quality of life. A significant amount of research has been undertaken, and is still on-going, investigating the underlying mechanisms of bladder dysfunction and the development of new therapeutic agents for the treatment of LUT conditions.It is now clear that the simplistic ideology of the bladder acting as a passive reservoir is far more complicated, and involves complex mechanisms between the different components of the bladder wall.The recently emerging role of the bladder urothelium and sub-urothelium in regulating bladder functions has laid the foundation for a hypothesis supporting a mechanosensory basis for OAB. The urothelium expresses several different types of receptors and ion channels, and upon stimulation releases mediators such as ATP, nitric oxide and acetylcholine. These mediators act on receptors in the urothelium and in the underlying nerves and muscle to mediate bladder activity, and although these mechanisms are still not completely understood there is evidence to suggest that in pathological conditions, these pathways may be altered. The aim of this project was to investigate the mechanisms, structures and transmitters involved in these mechano-sensory pathways in the bladder wall using in vitro pharmacology, histology, immunohistochemistry and mediator assaysSpontaneous phasic contractions (PCs) developed in tissue strips isolated from the dome, body and trigone regions of the pig bladder, and the amplitude and frequency of these PCs was measured. Functional and structural differences were observed between the different bladder regions, particularly in the trigone versus the dome and body. Removal of the mucosa significantly increased the development time of PCs in the dome and body but not in the trigone. Mucosa removal also significantly decreased the frequency of contractions in the dome and body but not in the trigone. Since increased PCs of the bladder are associated with OAB, the mechanisms regulating these contractions were explored. Phasic contractions amplitude could be increased by low concentrations of the muscarinic receptor agonist carbamylcholine (carbachol - CCh), and functional differences were observed in the trigone compared to the dome and body regions. The muscarinic receptor subtype involved in mediating the effect of CCh was also investigated, but from the results obtained it was not possible to determine which subtype was responsible.Research into the role of interstitial cells (ICC) in the bladder has grown in the past decade since this specialised class of cells was discovered in the urinary bladder. It is postulated that these cells may be involved as mediators of mechanosensory function in the bladder. The expression of c-Kit, a tyrosine kinase receptor, on ICC allowed for the functional role of these cells in PCs of the bladder to be investigated, and to attempt to determine their localization in the different bladder layers using immunohistochemistry. Imatinib, a tyrosine kinase receptor inhibitor, had no effect on the amplitude or frequency of PCs in tissue strips from the different bladder regions, suggesting that c-Kit positive ICC may not be involved in PCs in the pig bladder. A population of cells staining positive for vimentin, but lacking specific c-Kit staining, was identified but cannot be conclusively identified as ICC.Investigation and measurement of ATP release using a luciferase assay kit in response to stretch was also performed in the dome and body regions of the bladder. ATP release was found to increase with stretch. The presence of a heterogeneous population of cells in the bladder including detrusor smooth muscle cells, urothelial cells and ICC makes it difficult to determine the exact source of ATP, although literature suggests the urothelium to be the dominant source.In summary, the data presented in this thesis support a role for the urothelium and suburothelium the complex physiological processes of the pig urinary bladder. Further research is required into the integrated physiology of the bladder wall, and this may contribute to the discovery of new targets for pharmacological agents to treat lower urinary tract conditions.