Login to Access Video or Poster Abstract: MP42-15
Sources of Funding: BBSRC BB/P004695/1; NIA 1R01AG049321-01A1

Introduction

Recognition of the urothelium as a new sensory structure has significantly advanced our understanding of bladder function. Key to further progress is the identification of novel pathological regulators in this tissue. Oxidative stress is a fundamental pathological mediator; ROS generating enzyme NADPH oxidase (Nox enzyme) has attracted intense interest recently as it is the only enzyme that produces ROS as its sole function and can be targeted without compromising normal biochemical oxidation. Our recent pilot study provided initial evidence for the presence of such system in bladder urothelium and its potential functional significance. This study aimed firstly to define the importance of urothelial superoxide production in the body and secondly to dissect the enzymatic sources of superoxide production in the bladder.

Methods

C57BL/6J mice were euthanized. Bladder and other types of tissue were isolated. Lucigenin-enhanced chemiluminescence quantified superoxide production in live tissue. Western blot determined Nox subtype expressions.

Results

Superoxide production in bladder mucosa (RLU/mg tissue: 536.8±104.8, mean±SEM) was many folds as high as those in detrusor muscle (21.8±3.8, n=15, p<0.01), aorta (67.2±26.6, n=7, p<0.05), brain (9.4±1.7, n=6, p<0.01), kidney (84.3±23.0, n=6, p<0.05), ventricle (21.8±3.8, n=6, p<0.01) and liver (80.8±12.9, n=7, p<0.05). NADPH oxidase inhibitor diphenyleneiodonium (DPI, 20µM)reduced superoxide production to 10.8±3.4 % of control (n=6, p<0.01) in bladder mucosa and to 30.8±8.4% of control (n=6, p<0.01) in detrusor. Mitochondria de-coupler FCCP (10µM) suppressed superoxide production to 51.8±10.5 % of control (n=6; p<0.01) in bladder mucosa and to 59.8±10.4 % of control (n=6, p<0.05) in detrusor. Xanthine oxidase inhibitor oxypurinol (100µM) produced no significant effect in bladder mucosa (87.9±17.4 % of control, n=6, p>0.05) but a small inhibition in detrusor (78.0±6.6% of control). Western blot showed specific bands for Nox1, Nox2 and Nox4 but no Nox3 expression in bladder mucosa and detrusor with significantly higher expression in bladder mucosa (p<0.05, n=4-6)._x000D_

Conclusions

These data demonstrate for the first time that the urothelium is the most active tissue for superoxide production in the body. Nox enzymes are the main enzymatic source for superoxide in bladder. The main Nox subtypes are Nox1, Nox2 and Nox4, mainly located in the urothelium. Exceptionally high levels of Nox-driven superoxide explain why bladder urothelium is prone to oxidative stress, inflammation and sensory dysfunction.

Funding

BBSRC BB/P004695/1; NIA 1R01AG049321-01A1