The human bladder and associated urinary tract is lined by a specialised transitional epithelium referred to as the urothelium. The urothelium was initially thought to be exclusively an impermeable barrier, however it has recently been shown to have a sensory role. Transient receptor potential (TRP) channel signalling was implicated in normal bladder function by Birder et al. (1) who showed thatTRPV1 knockout mice displayed increased bladder capacity and a greater frequency of non-voiding contractions. Dysregulation of urothelial cell sensory signalling may contribute to the pathogenesis of dysfunctional syndromes, such as interstitial cystitis and overactive bladder syndrome. Although animal models provide a useful research tool, cross-species variation may limit their relevance to man. The relatively recent development of in vitro culture models of proliferative (2) and differentiated (biomimetic) (3) normal human urothelial (NHU) cells has permitted investigation of TRP signalling pathways in human urothelium for the first time. The aims of this study are: 1. Determine the expression of TRP channels in human urothelium and cultured NHU cells to validate the use of NHU cells as a model for investigating TRP channel signalling. 2. Identify and assess the modulatory effect of other sensory signalling pathways on TRP signalling in NHU cells. Normal human urothelium was obtained at surgery with local research ethics committee approval and patient informed consent. In culture, NHU cells adopted a proliferative phenotype, however addition of 5% adult bovine serum and 2mM calcium to the cell medium induced a stratified epithelium that displayed characteristics of native urothelium with high trans-epithelial resistance and markers of terminal differentiation. TRPV1, TRPV4 and TRPM8, but not TRPA1, transcripts were identified by RT-PCR in cultured NHU cells. Ratiometric laser confocal microscopic techniques were used to detect calcium fluxes in cultured NHU cells in response to the perfusion of specific agonists to reveal the presence of functional receptors. Capsaicin (300μM), indicated the presence of functional TRPV1, while UTP (5μM) demonstrated the presence of purinergic P2Y receptors. Pre-treatment of UTP augmented the calcium flux following capsaicin perfusion and immunohistochemical experiments revealed increased immunolabelling with anti-TRPV1 antibodies following UTP treatment of NHU cells suggesting TRPV1 signalling is modulated by P2Y signalling. In conclusion, proliferative and stratified NHU cells provide a useful tool for the investigation of normal human urothelial cell signalling. TRP channels are functionally expressed and the TRPV1 signalling pathway is modulated by P2Y receptor signalling, the significance of the in normal and deranged human bladder function has yet to be elucidated.