In patients suffering from overactive bladder (OAB) the detrusor is in a hypersensitive state. The exact nature of this hypersensitive state can vary, especially when comparing OAB patients with an otherwise healthy bladder to those with a concomitant inflammation. Mirabegron, a selective β3-agonist, was recently approved for the treatment of OAB in combination with an antimuscarinic drug. Early reports on mirabegron suggested that part of the relaxatory effect was exerted via release of nitric oxide (NO). However, this remains to be shown in conclusive studies. 

Numerous studies utilize an approach in which relaxation of bladder smooth muscle is studied in pre-contracted tissue. However, one needs to be aware that this can be a difficult approach since the pre-contraction tends to fade during the time period that the relaxatory agent is exerting its effect. In the current study, we chose to study the indirect relaxatory influence that activation of β-adrenoceptors exerts on muscarinic receptor-induced contraction.    

The aim of the current study was to determine how β-adrenoceptor subtypes (β1-3) contribute to bladder relaxation and if this is influenced by nitric oxide. Further, it was wondered if this is altered in a state of inflammation, similar to treatment with antimuscarinics which have been shown to be less useful in patients with concomitant cystitis.

A total of 36 adult male Sprague-Dawley rats were used in the current study. Each rat was pre-treated with either cyclophosphamide (CYP; 100 mg/kg i.p.) to induce experimental cystitis or saline (4 mL/kg i.p.), serving as controls. Sixty hours later, the rats were sacrificed and their bladders were excised. From each bladder, two bladder strips (2 x 6 mm) were cut and mounted in an organ bath setup. After an equilibration period, methacholine (1*10-7 – 3*10-4 M) was applied and the contractile force was measured in the absence and presence of increasing concentrations of a selective β-adrenoceptor agonist (5*10-8 M – 5*10-6 M); either dobutamine (β1), isoprenaline (β1/2) or ZD7114 (β3). Further, each series of measurements was performed in the absence or presence of the NO synthase inhibitor L-NNA (1*10-4 M). All data analysis and statistical comparisons were performed using GraphPad Prism 7.03 (GraphPad Software Inc., San Diego, USA). Two-way ANOVA followed by Tukey’s correction for multiple comparisons was used to compare the obtained dose-response curves and one-way ANOVA followed by Tukey’s correction was used to determine statistical significance between logEC50-values.

The presence of a β-adrenoceptor (5*10-8 M – 5*10-6 M) agonist significantly attenuated methacholine-induced bladder contraction, regardless of the selectivity profile of the agonist (β1, β1/2 or β3), causing a right-shift of the concentration-response curve in both healthy and inflamed tissue (p < 0.05 for all comparisons between logEC50 values for methacholine in the absence of a β-adrenoceptor agonist to logEC50 values for methacholine in the presence of a β-adrenoceptor agonist; n = 6 in each group). Inhibition of NO synthase did not significantly shift the methacholine-induced contraction-response curves in the presence of a β-adrenoceptor agonist (p-values remained <0.05 for all comparisons between logEC50 values for methacholine in the absence of a β-adrenoceptor agonist to logEC50 values for methacholine in the presence of a β-adrenoceptor agonist; n = 6 in each group).    

Concentration-dependency could not be seen for any of the β-adrenoceptor agonists in the studied range (5*10-8 – 5*10-6 M) with the exception of isoprenaline in healthy tissue in the absence of L-NNA in which the presence of a high (5*10-6 M) concentration caused a significantly greater right-shift of the methacholine-induced contraction curve than a low (5*10-8 M) concentration (logEC50: -5.283 ± 0.151 and -4.883 ± 0.078, respectively; p = 0.045 for the comparison of logEC50 for methacholine in the presence of a low (5*10-8 M) vs high (5*10-6 M) concentration of isoprenaline; n = 6).

The current data shows that the presence of a β-agonist, regardless of its subtype selectivity (β1, β1/2 or β3), attenuates muscarinic receptor-induced detrusor contraction. Somewhat surprisingly, this attenuation does not seem to be dependent on release of nitric oxide and remains constant in a state of inflammation. It should be noted that the current study does not distinguish between possible sites of action of β-adrenoceptor-induced relaxation, i.e. via β-adrenoceptors located on the detrusor or via β-adrenoceptors in other parts of the bladder, for instance the urothelium.

Activation of β-adrenoceptors attenuates muscarinic receptor-induced bladder contraction. Further, indirect β-adrenoceptor-induced relaxation seems to be independent of nitric oxide, both in the healthy and inflamed bladder. This suggests that treatment against OAB with a β3-adrenoceptor agonist is suitable also for patients with concomitant cystitis.