There is evidence that both vesicular and non-vesicular ATP release mechanisms operate in bladder urothelial cells. Several receptors and channels have been shown to participate in these mechanisms, such as the TRPV1 and TRPV4 channels, Piezo1, acid-sensing ion channel, epithelial Na + channels, muscarinic acetylcholine receptors, bradykinin receptors, PACAP PAC1 receptor and P2Rs. Observation that removal of extracellular Ca2+ augments ATP release from the bladder urothelium, a condition known to enhance P2X7R activation, strongly suggests the participation of this P2R subtype in mechanisms of urothelial ATP release. In addition, in other cell types P2X7R stimulation has been shown to induce ATP release by opening pannexin 1 channels. Panx1 is a member of the gap junction family of proteins that forms large non-junctional channels which allow diffusion of ions and small molecules between the cytosol and extracellular space. Besides being activated by P2X7R and other P2Rs, Panx1 channels are sensitive to voltage, high extracellular and mechanical stimulation. Panx1 is expressed in various cell types and has been shown to participate in key cellular events, such as intercellular signaling, mechanotransduction, and inflammatory responses. The involvement of Panx1 in pathophysiological mechanisms is also becoming increasingly apparent. We have recently shown that Panx1 contributes to Nilotinib development of neurogenic bladder in mice with experimental autoimmune encephalomyelitis, a model of Multiple Sclerosis. Panx1 has also been proposed to participate in mechanisms of bladder overactivity involving P2Y6R activation. However, little is still known of the actual role played by Panx1 channels in the urinary bladder under physiological conditions. Based on the characteristic mechanosensitivity of Panx1 channels and their demonstrated function as conduits for cellular ATP release, and the key role of ATP as an urothelial mechanosignaling molecule, in this study we investigated whether Panx1 channels participate in mechanisms of urothelial mechanotransduction and intercellular signaling. First we immunolocalized Panx1 and P2X7R in the rat bladder mucosa, and determined the effects of intravesical administration of mefloquine on amounts of ATP released in the bladder lumen in response to bladder distension. Then, to specifically demonstrate the functional interaction of Panx1 and P2X7R in the urothelium, we used the TRT-HU1 immortalized human urothelial cell line to measure the effects of pharmacological blockade of Panx1 channels and P2X7R on mechanicallyinduced urothelial ATP release, dye-uptake and transmission of intercellular Ca2+ waves, which is a form of long range cell-cell communication mediated by ATP. Bladders and urothelial cells isolated from mice deficient in Panx1 or P2X7R were also used in ATP release and dye-uptake experiments to support the pharmacological findings obtained from rat bladders and human urothelial cells. Our findings indicate that Panx1 is expressed in the bladder urothelium, that Panx1 channels provide a mechanosensitive conduit for urothelial ATP release and participate in urothelial ATP signaling by functionally.