In addition to its role as a physical barrier, the urothelium is considered to play an active role in mechanosensation. A key mechanism is the release of transient mediators that activate purinergic P2 receptors and transient receptor potential (TRP) channels to effect changes in intracellular Ca 2ϩ . Despite the implied importance of these receptors and channels in urothelial tissue homeostasis and dysfunctional bladder disease, little is known about their functional expression by the human urothelium. To evaluate the expression and function of P2X and P2Y receptors and TRP channels, the human ureter and bladder were used to separate urothelial and stromal tissues for RNA isolation and cell culture. RT-PCR using stringently designed primer sets was used to establish which P2 and TRP species were expressed at the transcript level, and selective agonists/antagonists were used to confirm functional expression by monitoring changes in intracellular Ca 2ϩ and in a scratch repair assay. The results confirmed the functional expression of P2Y4 receptors and excluded nonexpressed receptors/channels (P2X 1, P2X3, P2X6, P2Y6, P2Y11, TRPV5, and TRPM8), while a dearth of specific agonists confounded the functional validation of expressed P2X 2, P2X4, P2Y1, P2Y2, TRPV2, TRPV3, TRPV6 and TRPM7 receptors/channels. Although a conventional response was elicited in control stromal-derived cells, the urothelial cell response to well-characterized TRPV1 and TRPV4 agonists/ antagonists revealed unexpected anomalies. In addition, agonists that invoked an increase in intracellular Ca 2ϩ promoted urothelial scratch repair, presumably through the release of ATP. The study raises important questions about the ligand selectivity of receptor/ channel targets expressed by the urothelium. These pathways are important in urothelial tissue homeostasis, and this opens the possibility of selective drug targeting. calcium; purinergic; transient receptor potential channel; urothelium THERE HAS BEEN a growing appreciation that rather than a simple passive barrier, the urothelium plays a more active role in the urinary tract. After physical or other damage, the urothelium will self-repair by switching from a mitotically quiescent to a highly regenerative state More intriguingly, the urothelium has been reported to possess sensory neuronal-like properties and to respond to mechanical and chemical stimulation through the release of transient mediators (4). Various mediators have been implicated, including ATP, nitric oxide, acetylcholine, and substance P (1, 7, 11). These short-lived mediators are considered to actuate suburothelial afferent neurons involved in the regulation of sensory perception and pain, but the urothelium is itself widely reported to express an array of receptors and channels that may respond in an autocrine/paracrine fashion to released mediators. These include purinergic P2X and P2Y (8, 24, 27), transient receptor potential (TRPV1, TRPV2, TRPV4, and TRPM8), acetylcholine (nicotinic and muscarinic), tachykinin, nerve growth factor, endothelin, sphingosine-1-phosphate, and bradykinin (3, 9, 15, 17) receptors. The outcome of such signaling is incompletely understood as it may play a bidirectional feedback role in modulating the neuronal signal and/or effect changes in urothelial homeostasis, such as barrier repair. It has also been suggested that abnormal expression of receptors and/or mediator release by the urothelium may be involved in dysfunctional diseases of the bladder, including idiopathic detrusor instability and interstitial cystitis Despite the literature reporting expression of these channels and receptors by the urothelium, consensus is confounded by contradictions in experimental approaches, including the species, specificity of reagents, and the nature of the tissue preparation (for a review, see Ref. 30). There has been limited characterization of these receptor/mediator signaling pathways in the human urothelium, where functional TRPV1 (10) and an autocrine-activated P2Y receptor pathway (19, 26) have been reported. Ultimately, this conflict and the lack of consensus are hindrances to the development of selective drugs. To attribute expression and function to specific tissue compartments, the present study was designed to define the functional expression of purinergic and transient receptors in the isolated human urothelium and stromal cells in situ and in vitro. A preliminary investigation revealed a lack of specificity of commercially available antibodies. For this reason, our rationalized experimental approach was to identify candidate receptors based on mRNA expression followed by confirmatory functional experiments to measure changes in intracellular Ca 2ϩ using specific agonists/antagonists. Finally, to examine whether receptor activation plays a role in urothelial homeostasis, we examined the effect of receptor activation on human urothelial scratch wound repair in vitro
