Identification of the NADPH Oxidase (Nox) Subtype and the Source of Superoxide Production in the Micturition Centre

SIMPLE SUMMARY: Reactive oxygen species (ROS) are chemically active oxygen-containing molecules and overproduction of ROS can cause oxidative damage to cells and tissues in the body. Oxidative damage to brain cells can not only cause lesions to the brain but also lead to disorders in peripheral organs under the control of the corresponding brain centres, such as the urinary bladder. A unique class of enzymes that produce ROS are the special oxidising enzymes called “Nox” enzymes. These are the body’s only enzymes that can be selectively controlled without affecting normal cell activity. Therefore, Nox enzymes are considered to be a drug target. Whether Nox exists in the brain centres that control urination has not been examined. We investigated whether the type 2 Nox enzyme-Nox 2 exists in the brain urination control centre and whether such a Nox enzyme is functional. Our results show that the brain urination control centre has Nox 2 proteins, and the Nox enzyme produces a significant amount of ROS, higher than heart tissue, suggesting the importance of Nox-associated ROS production in physiology and pathology. These findings lay the groundwork for future investigation into Nox 2 and the associated oxidative damage in brain urination control centres and consequent bladder abnormalities. ABSTRACT: Oxidative inflammatory damage to specialised brain centres may lead to dysfunction of their associated peripheral organs, such as the bladder. However, the source of reactive oxygen species (ROS) in specific brain regions that regulate bladder function is poorly understood. Of all ROS-generating enzymes, the NADPH oxidase (Nox) family produces ROS as its sole function and offers an advantage over other enzymes as a drug-targetable molecule to selectively control excessive ROS. We investigated whether the Nox 2 subtype is expressed in the micturition regulatory periaqueductal gray (PAG) and Barrington’s nucleus (pontine micturition centre, PMC) and examined Nox-derived ROS production in these structures. C57BL/6J mice were used; PAG, PMC, cardiac tissue, and aorta were isolated. Western blot determined Nox 2 expression. Lucigenin-enhanced chemiluminescence quantified real-time superoxide production. Western blot experiments demonstrated the presence of Nox 2 in PAG and PMC. There was significant NADPH-dependent superoxide production in both brain tissues, higher than that in cardiac tissue. Superoxide generation in these brain tissues was significantly suppressed by the Nox inhibitor diphenyleneiodonium (DPI) and also reduced by the Nox-2 specific inhibitor GSK2795039, comparable to aorta. These data provide the first evidence for the presence of Nox 2 and Nox-derived ROS production in micturition centres.