Abstract
Bladder disorders such as overactive bladders affect millions of people worldwide but their pathogenesis is poorly understood. Recently, the role of the urothelium in bladder function and pathology has generated intense interest, in particular its regulation by inflammatory mediators. Angiotensin II is trophic factor and inflammatory mediator, and is involved in bladder obstruction and hypertrophy. However, its role in urothelial function has never been studied. The aim of this body of research was to identify the presence and localisation of angiotensin receptor type1 within the bladder wall and to elucidate the role of angiotensin II in urothelial and bladder function. In this investigation we also looked at the presence of nicotinamide adenine diphosphate oxidase and its affect in bladder physiology. Guinea-pigs (male Dunkin-Hartley 450-550g) were chosen for their physiological homology to humans and were euthanized with schedule-1 procedure. Immuno-fluorescence was performed on frozen bladder sections with an angiotensin receptor type1, nicotinamide adenine diphosphate oxidase 1 and nicotinamide adenine diphosphate oxidase 2 primary antibodies and an Alexa-586-conugated secondary antibody; Dihydroethidium dye was used as a superoxide dye for imaging studies. Mucosa-intact smooth muscle strips and mucosal strips were isolated from the urinary bladders. The preparations were superfused in a HEPES-buffered Tyrode’s solution for functional measurement. The isometric tension was recorded with a tension-transducer via a bridge-amplifier. The superfusate adjacent to the tissue strip was sampled and adenosine triphosphate release from the tissue was measured using a luciferin-luciferase assay. Immunofluorescence highlighted the novel presentation of angiotensin II type1 receptors in the urothelium of the bladder wall. Application of angiotensin II (200nM and 1µM) significantly increased adenosine triphosphate release from the bladder mucosa and full thickness bladder tissue; angiotensin II type 1 receptor antagonist ZD7155 largely inhibited this effect. Angiotensin II also increased the contractile activity in isolated mucosa and mucosa-intact muscle strips. Experiments into the mechanism of angiotensin II action on adenosine triphosphate release revealed that this was through vesicular release, using Brefeldin A. Further data on nicotinamide adenine diphosphate oxidase and reactive oxygen species, exhibited the presence of nicotinamide adenine diphosphate oxidase 1 and nicotinamide adenine diphosphate oxidase 2 in the urothelium, and a positive effect of hydrogen peroxide on spontaneous bladder activity. Additionally, Apocynin was able to inhibit spontaneous activity. Dihydroethidium stains as well as reactive oxygen species detection assays showed that reactive oxygen species was not only present in the bladder but that angiotensin II is able to increase its activity. These data provide the first evidence that angiotensin II type 1receptor is expressed in the bladder urothelium and angiotensin II stimulates adenosine triphosphate release from the urothelium via angiotensin II type 1 receptors and vesicular transport. Angiotensin II is also able to generate a positive inotropic effect on mucosa-intact smooth muscle, partly through a paracrine effect of the released adenosine triphosphate. Additionally, investigations into the action of reactive oxygen species in the bladder, reveal its presence and regulation by angiotensin II. These findings suggest the significance of angiotensin II in bladder physiology and pathophysiology.