In this study, using both pharmacological and genetic approaches, we demonstrate that Panx1 channels and P2X7R are functionally co-expressed in urothelial cells. This functional interplay between P2X7R and Panx1 is clearly observed here in the YoPro uptake, ATP release and ICW spread experiments performed with TRT-HU1 cells. Most notable, however, are our findings that Panx1 channels not only provide the distensioninduced ATP release that can initiate paracrine signaling between urothelial cells, but may also provide ATP release from neighboring non-stimulated cells through P2X7R stimulation. In this scenario, ATP-induced ATP release mediated by activation of the P2X7R-Panx1 complex would not only support the mechanically-initiated intercellular signaling among urothelial cells but provide a Dinaciclib mechanism for mechanosensory amplification. A role for urothelial-derived ATP as the transmitter that communicates bladder distension to the CNS through activation of P2Rs in suburothelial afferent nerve terminals is broadly accepted. However, the actual role played by distension-induced ATP mediated signaling within the urothelium is still largely unknown, but is expected to be important for urothelial function as a syncytium, providing for synchronization and coordination of the urothelial cells. For example, activation of P2Rs has been shown to be essential for increasing the apical surface area of the urothelium during bladder filling. In this regard, ATP release and signaling within the same and between urothelial layers is likely essential to convey the information of bladder distension and provide for proper synchronization of urothelial cell response and adaptation to bladder wall distension. In this study we focused on investigating the role of Panx1 channels and P2X7Rs in urothelial ATP release and signaling. Our immunohistochemical studies, however, indicate that in the rat bladder mucosa Panx1 channels and PX7R are also expressed by spindle-shape cells in the lamina propria that likely correspond to suburothelial myofibroblasts. These cells are in close contact with suburothelial nerves and form a network functionally connected by gap junctions. Isolated suburothelial myofibroblasts have been shown to respond to exogenous ATP with generation of intracellular Ca2+ transients and when mechanically stimulated in intact bladder cross-sections they initiate transmission of ICWs, which spread across the suburothelial network and invade the underlying detrusor layer. These features prompted the proposal that suburothelial myofibroblasts may act as amplifiers in the sensory response to bladder wall distension. Future studies are needed to determine whether Panx1 channels are functionally expressed in suburothelial myofibroblasts. Given the characteristic properties of Panx1 channels and findings presented here for urothelial cells, we can speculate that Panx1 channels and P2X7R may also participate in responses of suburothelial myofibroblasts to mechanical stimulation and in ATP signaling among these cells. Similar to its role discussed here for the urothelium, the P2X7R-Panx1 complex could be a key participant in a mechanism for mechanosensory amplification at the level of the suburothelial layer.