Do β3-adrenergic receptors play a role in guinea pig detrusor smooth muscle excitability and contractility?

Abstract

In many species, β3-adrenergic receptors (β3-ARs) have been reported to play a primary role in pharmacologically induced detrusor smooth muscle (DSM) relaxation. However, their role in guinea pig DSM remains controversial. The aim of this study was to investigate whether β3-ARs are expressed in guinea pig DSM and to evaluate how BRL37344 and L-755,507, two selective β3-AR agonists, modulate guinea pig DSM excitability and contractility. We used a combined experimental approach including RT-PCR, patch-clamp electrophysiology, and isometric DSM tension recordings. β3-AR mRNA message was detected in freshly isolated guinea pig DSM single cells. BRL37344 but not L-755,507 caused a slight decrease in DSM spontaneous phasic contraction amplitude and frequency in a concentration-dependent manner. In the presence of atropine (1 μM), only the spontaneous phasic contractions frequency was inhibited by BRL37344 at higher concentrations. Both BRL37344 and L-755,507 significantly decreased DSM carbachol-induced phasic and tonic contractions in a concentration-dependent manner. However, only BRL37344 inhibitory effect was partially antagonized by SR59230A (10 μM), a β3-AR antagonist. In the presence of atropine, BRL37344 and L-755,507 had no inhibitory effect on electrical field stimulation-induced contractions. Patch-clamp experiments showed that BRL37344 (100 μM) did not affect the DSM cell resting membrane potential and K(+) conductance. Although β3-ARs are expressed at the mRNA level, they play a minor to no role in guinea pig DSM spontaneous contractility without affecting cell excitability. However, BRL37344 and L-755,507 have pronounced inhibitory effects on guinea pig DSM carbachol-induced contractions. The study outlines important DSM β3-ARs species differences.

Fig. 1.

RT-PCR detection of β3-adrenergic receptor (β3-AR) mRNA in detrusor smooth muscle (DSM) whole tissue and isolated DSM single cells. The expected size of the product was 387 bp. RT (+), reverse transcription; RT (−), no reverse transcription. Guinea pig ileal tissue was used as a positive control. Illustrated gel image is a selected representation of at least 5 independent RT-PCR experiments based on RNA extracted from 4 animals.

Fig. 2.

Effects of BRL37344 and L-755,507 on the spontaneous phasic and tonic contractions of guinea pig DSM isolated strips. A–B: original recordings of DSM spontaneous phasic contractions illustrating BRL37344 inhibitory effect (1 nM-100 μM) in the absence (A) or presence (B) of SR59230A (10 μM). C–G: concentration-response curves for BRL37344 (1 nM-100 μM) inhibitory effects on the spontaneous phasic contraction amplitude (C), muscle force integral (D), phasic contraction frequency (E), phasic contraction duration (F), and muscle tone (G) in the presence or absence of SR59230A (10 μM). Concentration-response curves for L-755,507 (1 nM-100 μM) showed no inhibitory effect on the spontaneous phasic and tonic contractions of DSM isolated strips (C–G). Spontaneous contractions were taken to be 100% (n = 7, N = 7 for BRL37344; n = 12, N = 6 for L-755,507; and n = 8, N = 4 for SR59230A + BRL37344; *P < 0.05 for the SR59230A effect). Tetrodotoxin (TTX; 1 μM) was present throughout the experiments.

Fig. 3.

Application of a single concentration of BRL37344 (100 μM) decreased the guinea pig DSM spontaneous phasic contraction amplitude, muscle force integral, and phasic contraction frequency in the absence of atropine. A, C: original recording of DSM contractions illustrating BRL37344 inhibitory effect in the absence (A) or presence of 1 μM atropine (C). B, D: summary data showing BRL37344 (100 μM) inhibitory effect in the absence (B; n = 14, N = 5, ***P < 0.005) or presence of 1 μM atropine (D; n = 9, N = 5, ***P < 0.005). Spontaneous contractions were taken to be 100%. TTX (1 μM) was present throughout the experiments.

Fig. 4.

Original DSM contraction recordings illustrating BRL37344 (1 nM-100 μM) and L-755,507 (1 nM-100 μM) concentration-dependent inhibitory effects on carbachol-induced phasic and tonic contractions of DSM isolated strips in the absence (A, B) or presence (C, D) of SR59230A (10 μM). TTX (1 μM) was present throughout the experiments.

Fig. 5.

Concentration-response curves for BRL37344 (1 nM-100 μM) and L-755,507 (1 nM-100 μM) inhibitory effects on carbachol-induced phasic contraction amplitude (A), muscle force integral (B), phasic contraction frequency (C), phasic contraction duration (D), and muscle tone (E) in the presence or absence of SR59230A (10 μM). Carbachol-induced contractions were taken to be 100% (n = 14, N = 6 for BRL37344; n = 25, N = 6 for BRL37344 + SR59230A; **P < 0.01, ***P < 0.005 for the SR59230A effect) and (n = 12, N = 3 for L-755,507; n = 7, N = 3 for L-755,507 + SR59230A; P > 0.05 for the SR59230A effect). TTX (1 μM) was present throughout the experiments.

Fig. 6.

L-755,507 (100 μM) significantly reduced carbachol-induced phasic and tonic contractions of DSM isolated strips. A: original DSM contraction recording illustrating L-755,507 (100 μM) inhibitory effect in the absence of SR59230A (10 μM). B: summary data showing that L-755,507 (100 μM) significantly reduced the amplitude, muscle force integral, phasic contraction duration, and muscle tone (n = 12, N = 3, *P < 0.05, **P < 0.01, ***P < 0.005). TTX (1 μM) was present throughout the experiments.

Fig. 7.

BRL37344 (100 μM) significantly decreases the amplitude of the electrical field stimulation (EFS)-induced contractions of guinea pig DSM isolated strips in the absence or presence of SR59230A (10 μM). Original recordings illustrating EFS-induced (0.5–50 Hz) contractions of DSM isolated strips in response to 100 μM BRL37344 (A), 10 μM SR59230A and 100 μM BRL37344 combined (C), and 10 μM SR59230A (E). Frequency-response curves for the EFS-induced (0.5–50 Hz) contractions in response to 100 μM BRL37344 (B), 10 μM SR59230A and 100 μM BRL37344 combined (D), 10 μM SR59230A (F; n = 10, N = 4 for B; n = 11, N = 5 for D; n = 9, N = 6, P > 0.05 for F; *P < 0.05, **P < 0.01, ***P < 0.005). EFS-induced contraction amplitude at 50 Hz under control conditions was taken to be 100%.

Fig. 8.

In the presence of suramin (10 μM) and α,β-methylene-ATP (10 μM), BRL37344 (100 μM) decreased the amplitude of the EFS-induced (0.5–50 Hz) contractions in DSM isolated strips. A, C: original EFS-induced contraction recordings showing BRL37344 (100 μM) inhibitory effect in the absence (A) or presence (C) of SR59230A (10 μM). B, D: frequency-response curves showing BRL37344 inhibitory effect on EFS-induced contraction amplitude in the absence (B; n = 10, N = 5, **P < 0.01, ***P < 0.005) or presence (D) of 10 μM SR59230A (n = 10, N = 5, **P < 0.01, ***P < 0.005). EFS-induced contraction amplitude at 50 Hz under control conditions was taken to be 100%.

Fig. 9.

In the presence of atropine (1 μM), BRL37344 (100 μM) does not affect the amplitude of the EFS-induced (0.5–50 Hz) contractions of DSM isolated strips. A, C: original recordings illustrating the lack of BRL37344 (100 μM) effect on EFS-induced contractions in the absence (A) or presence (C) of SR59230A (10 μM). B, D: frequency-response curves showing the lack of BRL37344 effect on the EFS-induced contraction amplitude in the absence (B; n = 13, N = 6, P > 0.05) or presence (D) of 10 μM SR59230A (n = 12, N = 5, P > 0.05).

Fig. 10.

L-755,507 (100 μM) does not affect the amplitude of the EFS-induced (0.5–50 Hz) contractions of DSM isolated strips. A, C: original recordings illustrating a lack of L-755,507 (100 μM) effect on EFS-induced contractions in the absence (A) or presence (C) of atropine (1 μM). B, D: frequency-response curves showing the lack of inhibitory effect on EFS-induced contractions by L-755,507 in the absence (B; n = 9, N = 4, P > 0.05) or presence (D) of 1 μM atropine (n = 6, N = 4, P > 0.05).