β3-Adrenoceptor (β3-AR) agonists have been widely used to treat overactive bladder syndrome (OAB), and vibegron is a newly approved β3-AR agonist. It has been reported that β3-AR agonists including vibegron can inhibit non-voiding contractions (NVCs) of the hypertrophied bladder in rats with bladder outlet obstruction (BOO) [1]. Moreover, bladder myogenic microcontractions, which may be similar to NVCs, possibly link to the single-unit mechanosensitive afferent activities (SAAs) of Aδ-fiber, and mirabegron, another another β3-AR agonist, inhibit the afferent activities through the suppression of the microcontractions in normal rats [2]. In addition, SAAs of Aδ- and C-fibers were intermittently enhanced by propagation of bladder myogenic microcontractions in BOO rats [3]. In the present study, we investigated the effects of vibegron on bladder function, specifically on NVCs in cystometry (CMG) and bladder mechanosensitive SAAs. These functions are associated with myogenic microcontractions in a male rat model of BOO. Additionally, an immunohistochemical assay can be used to determine the site of β3-ARs expression in the bladder and whether their expressions relate to substance P (SP), a sensory neuropeptide.

Male Wistar rats were used (10-12 weeks old). The proximal urethra was tightly ligated with a steel rod (1.1 mm in diameter), then the rod was removed. Ten days after the surgical procedure, CMG and SAA measurements were taken under two distinct conditions.
Single CMG measurements were performed under a conscious-restrained condition. A PE-50 catheter with a cuff was implanted into the bladder three days before the measurements. Saline was instilled into the bladder at a rate of 6 mL/h until micturition occurred, and voided urine was collected. CMG recordings were repeated three times both before and after the intravenous (i.v.) administration of vehicle or vibegron (3 mg/kg). The following parameters were averaged and analyzed: basal pressure, threshold pressure, maximum voiding pressure, mean voided volume, residual volume, bladder capacity, and the mean amplitude and the number of NVCs. NVCs were bladder contractions without micturition, which were characterized by amplitudes greater than 2 cm H2O observed for 3 min preceding micturition.
SAAs measurements were performed under a urethane-anesthetized condition (1.0 g/kg, intraperitoneally). Bilateral L6 dorsal roots were transected via a laminectomy. The fine filaments were dissected from the left L6 dorsal roots and placed across a bipolar electrode for monitoring SAAs. Nerve fibers primarily originating from the bladder were identified by electrical stimulation of the left pelvic nerve and by bladder distension. Nerves with conduction velocities (CV) more than 2.5 m/s were designated as Aδ-fibers and those with CV less than 2.5 m/s as C-fibers. Saline instilled until the intravesical pressure reached 30 cmH2O. The bladder was kept under an isovolumetric condition, allowed to stabilize for 5 min, and then vehicle or vibegron (3 mg/kg) was administered i.v. and recording was performed for 5 min. Mean bladder pressure, number of microcontractions and SAAs were analyzed.
In the separates rats, the expression of β3-adrenoceptor and substance P (SP), a sensory neuropeptide, in the bladder was further evaluated following immunohistochemical procedures.

In the CMG measurements, all numeric values of CMG parameters were unchanged before and after vehicle administration. However, the bladder capacity and number of NVCs were significantly increased and decreased, respectively, following vibegron administration (Table 1 middle line). For comparison between vehicle and vibegron groups, data were converted into relative values after each administration (% of each before administration). In this comparison, the number of NVCs in the vibegron group was significantly decreased compared with that in the vehicle group (Table 1 bottom line).
In the SAAs measurements, in the vehicle group, 22 afferent fibers were detected (Aδ-fibers: n = 11, CV: 6.32 ± 1.49 m/s; C-fibers: n = 11, CV: 1.99 ± 0.10 m/s). In the vibegron group, 19 afferent fibers were detected (Aδ-fibers: n = 10, CV: 4.04 ± 0.69 m/s; C-fibers: n = 9, CV: 1.91 ± 0.19 m/s). Values of mean intravesical pressure and amplitude of microcontractions after vehicle administration were significantly decreased, suggesting the suppression of the intravesical pressure time-dependently under an isovolumetric condition. In contrast, the number of microcontractions and mean firing rates of Aδ-fibers and C-fibers were unchanged before and after vehicle administration, whereas the number of microcontractions and firing rates of Aδ- and C-fibers were significantly decreased following vibegron administration (Figure 1AB). For comparison between vehicle and vibegron groups, data were converted as relative values after each administration (% of each before administration), same as CMG parameters. In this comparison, mean intravesical pressure, the number of microcontractions, and the firing rates of Aδ- and C-fibers in the vibegron group were significantly decreased compared with those in the vehicle group.
Immunohistochemical evaluation was conducted to investigate the expression change of β3-AR in BOO rats. We confirmed that this BOO rat showed hypertrophic smooth muscle regions compared with those in the sham rat. Immunoreactivity (IR) of β3-AR in both sham and BOO rats was stronger in the urothelium- and suburothelium-layers rather than in smooth muscle regions, which were not changed between groups. Moreover, the IR of SP in sham and BOO rats was stronger in the suburothelium layer. These expressions were unlikely different between groups. Merged plates for β3-AR and SP depicting co-expression (yellow) were similarly shown in the suburothelium layer in both groups (Figures 1C).

Vibegron decreased NVCs and microcontractions, thus suggesting that vibegron can inhibit bladder myogenic contractile activities in BOO rats. Additionally, the present SAAs measurements showed an inhibitory effect of vibegron on the SAAs of Aδ- and C-fibers. Immunohistochemical assays confirmed the co-expression of SP and β3-AR in the suburothelium layer at a small portion, an expression similar between sham and BOO rats. This finding also shows that β3-ARs contribute to the sensory bladder function.

These findings are the first to suggest that vibegron can inhibit the mechanosensitive afferent transduction via Aδ- and C-fibers, at least partly by suppressing bladder myogenic contractile activities on OAB associated with BOO.

Maruyama I, Yamamoto S, Tsuchioka K, Yamazaki T. Effects of vibegron, a novel beta3-adrenoceptor agonist, and its combination with imidafenacin or silodosin in a rat with partial bladder outlet obstruction. Eur J Pharmacol. 2020; 878: 173096.
Aizawa N, Homma Y, Igawa Y. Effects of mirabegron, a novel beta3-adrenoceptor agonist, on primary bladder afferent activity and bladder microcontractions in rats compared with the effects of oxybutynin. Eur Urol. 2012; 62: 1165-73.
Aizawa N, Ichihara K, Fukuhara H, et al. Characteristics of the mechanosensitive bladder afferent activities in relation with microcontractions in male rats with bladder outlet obstruction. Sci Rep. 2017; 7: 7646.

Continence 2S2 (2022) 100211
DOI: 10.1016/j.cont.2022.100211