Studies using an in vitro model of urothelial barrier dysfunction support Panx1 role in IC/BPS

Objectives: Damage to the protective urothelial barrier and consequent exposure of bladder tissues to high urinary K⁺ has been implicated in the emergence of urinary and pelvic pain symptoms in patients with interstitial cystitis/bladder pain syndrome (IC/BPS) by activating suburothelial sensory fibers and inducing release of signaling molecules, such as ATP and pro-inflammatory mediators. Ongoing studies in our laboratory with animal models have demonstrated that absence of Pannexin 1 (Panx1), a mechanosensitive channel involved in urothelial ATP release, attenuates the development of urinary IC/BPS symptoms. Our goal was to determine if Panx1 channels, which can be activated by high extracellular K⁺, participate in mechanism of K⁺ induced amplification of ATP release and signaling in IC/BPS.

Methods: We used immortalized mouse urothelial cells plated in 35 mm dishes. Panx1 activation by bladder distension, as observed during the voiding cycle, was mimicked by exposing the cells to hypotonic shock (25% and 50%). Panx1 function was then assessed by quantifying cellular ATP release and Yo-Pro-1 (10 µM) dye uptake at 5, 15, 30 min post-stimulation in absence and presence of high K⁺ (10 mM) and Panx1 channel blocker (BB-FCF; 10 µM). Amounts of ATP released were quantified using the Luciferin-luciferase assay method and normalized by non-stimulated baseline levels. Dye-uptake was quantified by fluorescence microscopy. Data was compared pairwise using the Mann-Whitney test.

Results: Mechanical stimulation imposed by 25% and 50% hypotonic shock induced significant ATP release from urothelial cells in amounts that were 3- and 4.5-fold higher than those measured from non-stimulated controls (p < 0.05). Similarly, exposure to high extracellular K⁺ also induced significant ATP release from these cells (4.5-fold compared to controls; p< 0.05). These responses were blunted in the presence of the Panx1 channel blocker BB-FCF. Consistent with these findings, Yo-Pro dye uptake by urothelial cells was significantly higher when cells were mechanically stimulated in the presence than in the absence of high K⁺ (compared to hypo 25%, p< 0.0012; hypo 50%, p< 0.0001, n=20 cells per field of view, 10 fields per dish, 1 dish per experimental condition).

Conclusions: Activation of urothelial Panx1 channels by urinary K⁺ may provide one of the mechanisms whereby breach of the urothelial barrier would result in increased ATP release and signaling, which would contribute to emergence of urinary IC/BPS symptoms. These findings provide basis for future preclinical studies evaluating the potential use of Panx1 channel blockers to manage IC/PBS.