Hypothesis / aims of study
Inflammation and excessive production of reactive oxygen species (ROS) and consequent oxidative damage is a fundamental pathogenic mechanism underlying many inflammatory, sensory and motor dysfunctions of the bladder, in particular, in urothelium-specific pathologies. The sources of ROS from NADPH-oxidase family (Nox) in pathological contributions have generated intense interest for potential pharmacological control of oxidative damage. Almost all ROS-generating enzymes in the body produce ROS as the by-products while performing biochemical oxidations required for physiological activity. Targeting these enzymes will inevitably compromise the normal biochemical oxidation. Nox enzymes, however, produce ROS as their sole function and therefore can be selectively targeted to control excessive ROS with minimal side effect on physiological oxidation. Such system has recently been identified in our pilot studies (1). The pathological significance is further highlighted by the Nox's response to the inflammatory factors with urothelium-specific actions. The bottle neck for progress is the mechanism involved in Nox activation by the inflammatory mediators. This has not been examined in the bladder.
We hypothesize that Nox subtype 2 (Nox2) is the main subtype involved in the inflammatory factor mediated, urothelium-specific activation of Nox activity and protein kinase C, p47phox/22 phox and small GTP-linked Rac protein are the key signaling molecules involved.
The objective of this study is to test the hypothesis by exploration of the mode of action of the prototype inflammatory factor and vaso-active trophic factor angiotensin II, known to underlie many bladder pathologies (2) using molecular and functional approaches.
Study design, materials and methods
Adult C57BL/6J mice (5-10 months) and the knockout mice based on this background were used as the experimental models in compliance with UK and EU regulations, and maintained in pathogen-free and standard feeding conditions. Nox1 (B6.129X1-Nox1tm1 Kkr/J), Nox2 (B6.129S-Cybbtm1Din/J) and Nox4 (B6.129- Nox4tm1Kkr/J) knockout mice and their wild type littermates (The Jackson Laboratory) were employed to specifically dissect molecular mechanisms. The genotyping was performed for each mouse by PCR with specific primers and gel electrophoresis. Mice were euthanized to obtain bladder tissue. Bladder mucosa and smooth muscle were micro-dissected under microscopic guide.
Lucigenin-enhanced chemiluminescence quantified real-time NADPH-stimulated superoxide production in live tissue. The specificity of the superoxide was validated by its sensitivity to the superoxide scavenger Tiron. Tissue preparations were incubated in a HEPES-buffered physiological saline with pharmacological interventions to identify the sub-cellular signaling mechanisms. Data are expressed as mean±SEM. Student’s t-test examined two paired and non-paired normally distributed data sets. ANOVA with post-hoc pair-wise comparison tested the difference between multiple means.
Results
Angiotensin II (AngII, 1µM) significantly stimulated the NADPH- dependent (Nox-derived) superoxide production in the urothelium of the wildtype mice (171±15% of control, n=20, p<0.01). The control experiments showed that Nox-derived superoxide could only be attributed to Nox1, Nox2 and Nox3 subtypes. The stimulatory effect of AngII was lost in Nox2 KO mice (107±13% of control, n=20, p>0.05). In contrast, the positive effect of AngII remained in Nox1 KO mice (148±14% of control, n=19, p<0.01) and Nox4 KO mice (161±22% of control, n=15, p<0.05). Nox2 subtype selective inhibitor GSK2759039, also abolished the excitatory effect of AngII (106±4% of control, n=18, p>0.05). Further sets of experiments with the pathway interventions showed that the stimulatory effect of AngII was also inhibited by PKC inhibitor staurosporine (1µM, 129±17% of control, n=8, p>0.05), the inhibitor of p47phox and p22phox interaction, ebselen (1µM, 96±13% of control, n=8, p>0.05) and the Rac GTPase inhibitor EHT1864 (25µM, 111±10% of control, n=20, p>0.05). The above reversal effects were not observed with the smooth muscle.
Interpretation of results
The loss of the excitatory effect in Nox2 knockout mice suggests involvement of Nox2 subtype in the angiotensin II mediated stimulation of Nox-derive ROS release. The preservation of angiotensin II's positive effect in Nox1 and Nox4 knockout mice excludes the contribution from Nox1 and Nox4 to the angiotensin II induced Nox-dependent ROS release. The ability of Nox2 specific inhibitor GSK2759039 to inhibit the angiotensin II action provides further proof for the Nox2 specific activation. The antagonizing effect of PKC inhibitor staurosporine supports the involvement of PKC phosphorylation in angiotensin II-mediated Nox activation. The inhibitory effect on angiotensin II activation by the inhibitor for p47phox and p22phox interaction suggests an involvement of this protein to protein interaction as the signaling mechanism for angiotensin II action. These two evidences together suggest a pathway of PKC phosphorylation of p47phox and p47phox to p22phox interaction in angiotensin II induced Nox activation. The counteracting effect of Rac protein inhibitor EHT1864 suggests additional activation of Rac GTPase in angiotensin II-augmented ROS release, via recruiting p67phox (3). These signaling molecules are characteristic of Nox2-mediated intracellular pathways for ROS production. A lack of these reversal effects in the smooth muscle demonstrates that these mechanisms are urothelium-specific. As angiotensin II is a key bladder pathology relevant inflammatory mediator, these molecular pathways may provide new insight into the mechanisms of action for other inflammatory factors encountered in the bladder pathologies. These findings also provide a biological basis for selective targeting of Nox2 protein to limit inflammatory Nox activation in the urothelium.