Mechanisms involved in ATP release in the urinary bladder: role of pannexin-1, CALHM1, AND connexins 43 and 45

A prominent role of ATP in bladder sensation via the activation of purinergic receptors on sensory afferent neurons to regulate urinary bladder functions under normal and pathophysiological conditions has been established. Extracellular ATP signalling is altered substantially in bladder diseases, such as overactive bladder and interstitial cystitis. However, cellular pathways responsible for ATP release from the bladder remain largely unknown. Thus, the present thesis aimed to investigate the role of pannexin-1, CALHM1, connexin (Cx) 43 and Cx45 hemichannels in mediating ATP release in the urinary bladder. The expression and localisation of pannexin-1, CALHM1, Cx43 and Cx45 were studied in the porcine bladder, as well as in human urothelial RT4 cells. Double labelling with cell marker antibodies confirmed the expression and localisation of pannexin-1, CALHM1, Cx43 and Cx45 in urothelial, suburothelial and detrusor muscle cells of the porcine bladder, as well as in human urothelial RT4 cells. Inhibition of pannexin-1, CALHM1, Cx43 and Cx45 channels in the presence of their respective inhibitors significantly reduced ATP release from porcine urothelial cells and from human urothelial RT4 cells in response to hypotonic induced stretch. Extracellular Ca2+ depletion induced ATP release was also significantly attenuated by the blockage of the CALHM1, Cx43 and Cx45 channels. Moreover, to study the barrier functions of urothelium, an in vitro model of inflammation was developed in primary cultures of porcine urothelial cells. In this model, TNFα and IL-1β caused a tight junction disruption in urothelial cells, as demonstrated by reduced transepithelial electrical resistance and fragmentation or absence of tight junction protein ZO-1. TNFα and IL-1β induced urothelial barrier damage was prevented by incubation of urothelial cells with the pannexin-1 channel blocker, 10Panx1. This is the first study to show that pannexin-1, CALHM1, Cx43 and Cx45 are functionally expressed in the porcine bladder and in human urothelial RT4 cells, where they mediate ATP release. Furthermore, this study has provided the basis for further investigations targeting the inhibition of pannexin-1 channels to repair the discontinuous urothelium and decrease urothelial permeability for relieving patients with interstitial cystitis.