Interstitial cystitis (IC) is a chronic voiding disorder characterized by urinary frequency, urinary urgency, pain in the bladder and surrounding pelvic regions, in the absence of any identifiable pathology or bacterial infection. Several lines of evidence suggest that numerous pathophysiological mechanisms trigger the IC, resulting in the urothelial damage, mast cell activation, increased release of ATP, proinflammatory cytokines and accumulation of leukocytes. Pannexin-1, Connexin 43 and P2X7 receptors (P2X7R) are ATP release channels and are implicated in the process of inflammation and cell death [1]. The current study aims to investigate the functional role of P2X7R to mediate the large pore formation in the mouse urinary bladder, and to investigate the role of extracellular ATP to facilitate ininterleukin-1β  release in LPS induced cystitis.

Immunohistochemistry was conducted on mouse bladder tissues and cultured urothelial cells using pannexin-1 (ab124131 Abcam), connexin 43 (C6219 Sigma-Aldrich) and P2X7R (APR-004) antibody. Freshly isolated mouse urothelial cells were cultured in RPMI culture media and then transfected after 3 hrs. with pannexin-1 (500 pmol) siRNA by using Lipofectamine 2000 (Invitrogen). Pannexin-1 expression in siRNA treated and naive urothelial cells was also measured by immunocytochemistry and quantitative RT-PCR after 72 h of cultivation. Pannexin-1 knock out and naive urothelial cells were then treated with LPS (1µg/ml) for 4 h washed and preincubated for 30 min with cell-permeable and irreversible inhibitor of caspase-1 (ac-yvad-cmk, 30 µg/ml), AZ11645373 (selective P2X7 antagonist), 10panx1 (a pannexin-1 mimetic peptide, 100 µM), Peptide 5 (Cx43 inhibitor, 20 µM), and ATP (4mM). Supernatant was collected and interleukin-1β was measured using Quantikine Mouse IL-1 beta/IL-1F2 ELISA Kit (MLB00C).

RT-PCR revealed abundant pannexin-1, connexin 43 and P2X7R mRNA expression in mouse bladder urothelial cells. Pannexin-1, connexin 43 and P2X7R staining was highly prominent in the cytoplasm and at the plasma membrane in primary cultured urothelial cells (Figure 1 A). Pannexin-1 siRNA intervention has reduced the expression and immunoreactivity of pannexin-1 by ~70% compared to the naïve urothelial cells (Figure 1 B). 
ATP induced a significant interleukin-1β release in LPS primed urothelial cells which was blocked by 10panx1 in naïve urothelial cells. However, only a partial inhibition of ATP stimulated interleukin-1β release from naïve urothelial cells was observed in the presence of AZ11645373 and peptide 5. Similarly, in pannexin-1 knock out urothelial cells, ATP stimulated interleukin-1β release was significantly reduced compared to scrambled siRNA treated urothelial cells control. Scrambled siRNA treated urothelial cells didn’t show any difference in ATP induced interleukin-1β release compared to untranslated cells.

In the present study, a functional link has been established for the first time between pannexin-1 and P2X7R to mediate large pore formation and interleukin-1β release in urinary bladder. Our results demonstrated an intense expression of pannexin-1 and P2X7R immunoreactivity throughout the urothelium, including basal, intermediate and terminally differentiated umbrella cells. This may suggest that these channels are involved to mediate the release of various neurotransmitters such as ATP, acetylcholine and several inflammatory mediators in extracellular environment to play an important role in the urinary bladder physiology and pathophysiology. 
In this study, it has been shown that high extracellular concentrations of ATP induced large pore formation via P2X7R activation and mediate interleukin-1β release in mouse bladder urothelial cells. Interestingly, the P2X7R activation-dependent pore forming effect was completely abolished by pannexin-1 depletion and pannexin-1 channel blocker 10Panx1, suggesting cross-talk between the P2X7R and pannexin-1 channels exists in urothelial cells, which may form the base of larger pore formation and cell death.In previous studies, role of pannexin-1 to mediate ATP release during apoptosis, inflammasome activation [2] and increased extracellular ATP in IC patients has already been established [3]. This study has provided a compelling evidence that both the P2X7R and pannexin-1 channel may also play a critical role in the development of inflammatory bladder conditions through pro-inflammatory mediators release.

Here, we have established for the first time that prolonged exposure of LPS primed urothelial cells to high extracellular concentrations of ATP induced a large pore-formation and IL-1β release via the activation of P2X7R which was blocked in the presence of 10Panx1 and in pannexin-1 depleted urothelial cells. This indicates that P2X7R activation-dependent pore-formation and/or the cross-talk between the P2X7R and the pannexin-1 channel in the urinary bladder may play a critical role in the release of pro-inflammatory mediators and hence the development of inflammation. Based on the findings of the present study, it can be concluded that pharmacological interventions with P2X7R and pannexin-1 channel may lead to development of new therapeutics for the clinical management of painful bladder syndrome/interstitial cystitis.

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