Associate Scientist Lewis Katz School of Medicine at Temple University
Introduction: Generation of reactive oxygen species (ROS) by the enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) is a highly regulated process that constitutes one of the main redox signaling components. Excessive ROS production may cause oxidative damage to tissue and organ. Although, the role of redox signaling in different pathological processes has been of intense interest, its importance in bladder pathology remains unexplored. This aim was to use ex vivo muscle strip contractility studies to further explore the physiological role of ROS/Nox in regulating bladder function in humans.
Methods: Mucosa-denuded bladder muscle strips were obtained from human organ transplant donors; three males and two females were mounted in muscle baths. The maximal contractile responses to 120 mM potassium chloride were measured. Trains of electrical field stimulation (EFS) of 1 ms pulse duration, 12 V, 8 Hz at 90 s intervals were applied to each strip for about 20 minutes. Then, subsets of strips were incubated with ROS/Nox agonists or antagonists for 20 minutes in continued trains of EFS. Next, same subsets of strips that were treated with antagonists were retreated with agonists and their responses were detected. All responses are expressed in milli Newtons (mN) or as percentages of control.
Results: In male and female human bladders, exogenous application of ROS (hydrogen peroxide; H2O2), at the physiological concentration of 100µM, enhanced EFS-induced smooth muscle strip contractions. Apocynin (100µM), the inhibitor of ROS generating enzyme Nox, attenuated intrinsic muscle strip activity. However, treatment with H2O2 brings the EFS-induced muscle activity back to control level. Application of a key Nox activator and a pro-inflammatory peptide angiotensin II (1µM) augmented EFS-induced smooth muscle contractions. Treatment with AT1 receptor specific antagonist ZD7155 (1µM) inhibited EFS-induced activity, while retreatment with angiotensin II after ZD7155 blockade restores muscle activity to control level.
Conclusions: The enhancement of EFS-evoked contractions by H2O2 and the inhibition of these contractions by the Nox inhibitor apocynin demonstrates the functional relevance of ROS in regulating human bladder smooth muscle activity and suggests that endogenous Nox-derived ROS regulates smooth muscle function. The augmentation of contractions by angiotensin II suggests that activation of Nox via a receptor by its ligand can also enhance smooth muscle activity and that the effect of angiotensin II is mediated by AT1, which was further supported by the inhibitory effect of the selective antagonist ZD7155. Collectively, these data provide evidence for the functional significance of Nox-derived ROS in human bladder and that ROS can modulate bladder function without exogenous stimuli. Since, inflammation is an important mechanism associated with oxidative damage, the effects of angiotensin II on bladder smooth muscle function may have significant pathologic implications.