Login to Access Video or Poster Abstract: MP82-13
Sources of Funding: Einstein-Montefiore FPMRS Fellowship Program

Introduction

Pannexin 1 (Panx1) channels play essential roles in urothelial mechanotransduction and signaling by providing a mechanosensitive conduit for ATP release in response to bladder distension. It has been proposed that urothelial ATP signaling to bladder sensory fibers relays the degree of bladder fullness and modulates detrusor activity, which are essential for proper micturition. Panx1 channels have been implicated in neurogenic bladder and bladder overactivity. However, little is known about the mechanisms that regulate Panx1 in the bladder. Our goal was to investigate the extent to which mechanical stimulation and bladder overdistension, as occurs with polyuria in diabetes, regulates Panx1 expression and function.

Methods

Studies were performed with a) mouse urothelial cell cultures and b) 10 week old C57BL/6 mice treated for 2 and 4 weeks with 5% sucrose in the drinking water (diuresis mouse model), and non-treated age-match control mice. Urothelial cells cultured on custom made stretchable silicone culture chambers were submitted to uniaxial stretch for 0, 2 and 5 hours (30 sec cycle duration; maximum strain ~10%) and then immediately harvested and processed for quantification of Panx1 mRNA by real-time quantitative PCR (qPCR). Voided urine samples from sucrose-fed and control mice were collected for quantification of ATP levels using the luciferin-luciferase assay. Bladders were then isolated, the urothelium dissected and processed for qPCR analysis of Panx1 mRNA levels.

Results

Prolonged cyclic mechanical stimulation (5hrs) resulted in significant reduction of Panx1 expression in urothelial cells when compared to non-stimulated cells or to shorter (2hrs) stimulation (5 hrs: 0.66 ± 0.07*; 2 hrs: 1.02 ± 0.05; 0 hrs: 1.01 ± 0.05; mRNA norm. to mean 0 hrs levels; N = 6, *P<0.01). Panx1 expression in the bladder urothelium of mice after 2 and 4 weeks of sucrose-induced diuresis was significantly lower than in control mice (2 wks: 0.88 ± 0.05*; 4 wks: 0.74 ± 0.07*; Ctrl: 1.00 ± 0.03; mRNA norm. to mean Ctrl value; N = 4, *P<0.01). Urine ATP levels (nM) in 2 and 4 weeks diuresis mice were also significantly lower when compared to those in control mice (2 wks: 7.1 ± 0.6*; 4 wks: 6.3 ± 0.5*; Ctrl: 38.7 ± 1.8; N = 4, *P<0.0001), consistent with observed Panx1 downregulation.

Conclusions

Prolonged mechanical loading and overdistension reduce urothelial Panx1 expression and ATP release. This impairs proper urothelial ATP signaling and bladder mechanosensory activation, and may thereby contribute to development of detrusor underactivity.

Funding

Einstein-Montefiore FPMRS Fellowship Program