Mirabegron relaxes urethral smooth muscle by a dual mechanism involving β3 -adrenoceptor activation and α1 -adrenoceptor blockade

Abstract

Linked article: This article is commented on by Michel, M. C., pp. 429-430 of this issue. To view this commentary visit http://dx.doi.org/10.1111/bph.13379.

Background and purpose: Mirabegron is the first β3 -adrenoceptor agonist approved for treatment of overactive bladder syndrome. This study aimed to investigate the effects of β3 -adrenoceptor agonist mirabegron in mouse urethra. The possibility that mirabegron also exerts α1 -adrenoceptor antagonism was also tested in rat smooth muscle preparations presenting α1A - (vas deferens and prostate), α1D - (aorta) and α1B -adrenoceptors (spleen).

Experimental approach: Functional assays were carried out in mouse and rat isolated tissues. Competition assays for the specific binding of [(3) H]prazosin to membrane preparations of HEK-293 cells expressing each of the human α1 -adrenoceptors, as well as β-adrenoceptor mRNA expression and cyclic AMP measurements in mouse urethra, were performed.

Key results: Mirabegron produced concentration-dependent urethral relaxations that were shifted to the right by the selective β3 -adrenoceptor antagonist L-748,337 but unaffected by β1 - and β2 -adrenoceptor antagonists (atenolol and ICI-118,551 respectively). Mirabegron-induced relaxations were enhanced by the PDE4 inhibitor rolipram, and the agonist stimulated cAMP synthesis. Mirabegron also produced rightward shifts in urethral contractions induced by the α1 -adrenoceptor agonist phenylephrine. Schild regression analysis revealed that mirabegron behaves as a competitive antagonist of α1 -adrenoceptors in urethra, vas deferens and prostate (α1A -adrenoceptor, pA2 ≅ 5.6) and aorta (α1D -adrenoceptor, pA2 ≅ 5.4) but not in spleen (α1B -adrenoceptor). The affinities estimated for mirabegron in functional assays were consistent with those estimated in radioligand binding with human recombinant α1A - and α1D -adrenoceptors (pKi ≅ 6.0).

Conclusion and implications: The effects of mirabegron in urethral smooth muscle are the result of β3 -adrenoceptor agonism together with α1A and α1D -adrenoceptor antagonism.

Figure 1

CRCs to isoprenaline and mirabegron in mouse isolated urethra smooth muscle. Responses to isoprenaline (A) were evaluated with or without either L‐748,337 (30 μM) alone or in combination with atenolol (10 μM) and ICI‐118,551 (10 μM). Responses to mirabegron were evaluated with or without atenolol (10 μM; B), ICI‐118,551 (10 μM; C) or L‐748,337 (1–30 μM; D). (D) Dotted lines indicate the urethral relaxations at the levels of 25% and 75%. (E) The Schild plot for L‐748,337 at mirabegron's pEC₂₅. (F) CRCs to mirabegron in urethra pre‐contracted with KCl (80 mM), arginine‐vasopressin (60 nM) and endothelin‐1 (100 nM). Relaxations were calculated relative to the maximal changes from the contraction produced by phenylephrine (10 μM) in each urethral ring, which was taken as 100%. Data are presented as mean ± SEM (n = 3–4 different animals for each curve).

Figure 2

Concentration–response curves to mirabegron in mouse isolated urethra smooth muscle and cyclic AMP production. Relaxant responses were measured with or without either rolipram (10 μM; A) or ODQ (10 μM; C). Responses to rolipram (1 μM) were measured in the presence of threshold concentrations of mirabegron (0.1 and 1 μM; B). Cyclic AMP production in mirabegron and isoprenaline‐stimulated urethra in the presence of L‐748,337 is shown in (D). Relaxations were calculated relative to the maximal changes from the contraction produced by phenylephrine (10 μM) in each urethral ring, which was taken as 100%. Data are presented as mean ± SEM. (n = 4–6). ^* P < 0.05, ^** P < 0.01; significantly different from corresponding respective control/basal; anova followed by Tukey's test.

Figure 3

Concentration–response curves to phenylephrine (PE) in mouse isolated urethra with or without different concentrations of mirabegron. Urethral preparations were not pretreated (A) or pretreated (B) with a cocktail of inhibitors containing yohimbine (100 nM), propranolol (100 nM), L‐748,337 (10 μM), 17β‐estradiol (10 μM) and desipramine (100 nM). (C) Schild plots for mirabegron with and without the cocktail of inhibitors. (D) Representative original traces for PE‐induced urethral contractions in a preparation pretreated with the cocktail of inhibitors. Data are presented as mean ± SEM (n = 4–6).

Figure 4

Concentration–response curves to noradrenaline or phenylephrine (PE) in absence and presence of increasing concentrations of mirabegron (10–100 μM) in rat isolated vas deferens (A), prostate (B), aorta (C) and spleen (D) preparations. (E) The Schild plots for mirabegron in vas deferens, prostate and aorta. (F) Competition for the specific binding of [³H]prazosin by mirabegron in membrane preparations from HEK‐293 cells expressing α_1A‐, α‐ and Δ^1–79α_1D‐adrenoceptors. Data are presented as mean ± SEM (n = 4–6).