β-Adrenoceptor-mediated Relaxation of Urinary Bladder Muscle in β2-Adrenoceptor Knockout Mice

Abstract

Background and objective: In order to characterize the β-adrenoceptor (AR) subtypes involved in agonist-stimulated relaxation of murine urinary bladder we studied the effects of (-)-isoprenaline and CL 316,243 on tonic contraction and spontaneous contractions in detrusor strips of wild-type (WT) and β2-AR knockout (β2-AR KO) mice.

Materials and methods: Urinary bladders were isolated from male WT and β2-AR KO mice. β-AR subtype expression was determined with quantitative real-time PCR. Intact muscle strips pre-contracted with KCl (40 mM) were exposed to cumulatively increasing concentrations of (-)-isoprenaline or β3-AR agonist CL 316,243 in the presence and absence of the subtype-selective β-AR blockers CGP 20712A (β1-ARs), ICI 118,551 (β2-ARs), and L748,337 (β3-ARs).

Results: Quantitative real-time PCR confirmed lack of β2-AR expression in bladder tissue from β2-AR KO mice. In isolated detrusor strips, pre-contraction with KCl increased basal tone and enhanced spontaneous activity significantly more in β2-AR KO than in WT. (-)-Isoprenaline relaxed tonic tension and attenuated spontaneous activity with similar potency, but the concentrations required were two orders of magnitude higher in β2-AR KO than WT. The concentration-response curves (CRCs) for relaxation were not affected by CGP 20712A (300 nM), but were shifted to the right by ICI 118,551 (50 nM) and L748,337 (10 μM). The -logEC50 values for (-)-isoprenaline in WT and β2-AR KO tissue were 7.98 and 6.00, respectively, suggesting a large receptor reserve of β2-AR. (-)-CL 316,243 relaxed detrusor and attenuated spontaneous contractions from WT and β2-AR KO mice with a potency corresponding to the drug's affinity for β3-AR. L743,337 shifted the CRCs to the right.

Conclusion: Our findings in β2-AR KO mice suggest that there is a large receptor reserve for β2-AR in WT mice so that this β-AR subtype will mediate relaxation of tone and attenuation of spontaneous activity under physiological conditions. Nevertheless, upon removal of this reserve, β3-AR can also mediate murine detrusor relaxation.

Keywords: CL 316,243; detrusor muscle; isoprenaline; mucosa; relaxation; β2-adrenoceptor knockout; β3-adrenoceptors.

FIGURE 1

Expression of β-AR subtypes in urinary bladders und lungs of WT and β₂-AR knockout mice. (A) Agarose gel electrophoresis of qualitative PCR of cDNA obtained from urinary bladder detrusor samples (left panel) and lung samples (right panel) of WT and β₂-AR KO mice for β₁-AR, β₂-AR, β₃-AR and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (B) mRNA expression of β₁-AR, β₂-AR, and β₃-AR normalized to GAPDH in detrusor (WT, n = 3; β₂-AR KO, n = 5). n represent numbers of mice measured in triplicates. Mean values ± SD.

FIGURE 2

Relaxation of KCl-precontracted intact detrusor strips from WT and β₂-AR KO mice. (A) Original tracings of force of contraction (in mN) from two intact detrusor strips precontracted with KCl (40 mM) and subsequently relaxed with cumulatively increasing concentrations of (-)-isoprenaline. Forskolin (10 μM) was added at the end of each experiment. (B) Baseline amplitude (upper panel) and baseline integral (lower panel) of spontaneous activity before, and amplitudes and integrals after addition of KCl. (C) CRCs for the relaxing effects of (-)-isoprenaline on KCl-induced tone in detrusor strips from WT (open symbols) and β₂-AR KO (closed symbols). Since tonic contraction was completely relaxed with 10 μM forskolin, the CRCs were normalized to the response with forskolin (= 100%). (D) CRCs for spontaneous activity expressed as force-time integral in percent of pre-agonist control for WT (open symbols) and β₂-AR KO (closed symbols). A quantitative analysis of the data is shown in Table 1.

FIGURE 3

Effects β-AR blockers on detrusor tone and time-integral of spontaneous activity after exposure to 40 mM KCl (CGP, 300 nM CGP 20712A; ICI, 50 nM ICI 118,551; L748,337, 100 nM L748,337). (A,B) Peak increase (F_peak) and steady-state increase in tone (F_ss), respectively, after exposure to 40 mM KCl from WT and β₂-AR KO preparations. Please note the difference in scale. (C,D) Time-integral of spontaneous contractions from WT and β₂-AR KO preparations; baseline time-integral before (C) and time-integral after (D) exposure to 40 mM KCl. Mean ± SD from number of experiments as indicated. ^∗denotes P < 0.05 compared with control.

FIGURE 4

Effects of various β-AR subtype selective blockers on CRCs for (-)-isoprenaline-induced relaxation of KCl-induced tone (A–C) and spontaneous contractions (D–F) in detrusor strips from WT (open symbols) and β₂-AR KO mice (closed symbols). CRCs were performed in the presence of the β₁-AR-selective blocker CGP 20712A (A,D), the β₂-AR-selective blocker ICI 118,551 (B,E), the β₃-AR-selective blocker L748,337 (C,F). The respective controls (WT, dashed gray lines; β₂-AR KO, continuous gray lines) were taken from the same experiments as in Figure 2. Mean values ± SD from n experiments as indicated in parenthesis (number of strips/number of animals).

FIGURE 5

Effects of the β₃-AR-selective blocker L748,337, 1 μM (A), 3 μM (B), and 10 μM (C), on CRCs for (-)-isoprenaline-induced relaxation of KCl-precontracted detrusor strips from WT mice. The control is taken from the same experiments as in Figure 2. Mean values ± SD from n experiments as indicated in parenthesis (number of strips/number of animals). (D) Schild plot for determining the pA₂ value of L748,337 (see Materials and Methods for further details).

FIGURE 6

Relaxation in response to the β₃-AR-selective agonist CL 316.243 of KCl-pre-contracted detrusor strips from wild type (WT) and β₂-AR KO mice. (A) Original tracings of force of contraction (in mN) from two detrusor strips precontracted with KCl (40 mM) and subsequently relaxed with cumulatively increasing concentrations of CL 316,243. At the end of each experiment complete relaxation was induced with forskolin (10 μM) as indicated. (B) Amplitude (upper panel) and integral (lower panel) of spontaneous activity before and after addition of KCl. (C,D) CRCs for the CL 316,243 effects on tone and spontaneous activity in WT and β₂-AR KO, respectively. Similar lay-out as in Figure 2.

FIGURE 7

Effects of various concentrations of the β₃-AR-selective blocker L748,337 on CRCs for the tone relaxing effects of CL 316,243 in KCl-precontracted detrusor strips from WT (A) and β₂-AR KO mice (B). Control data (WT, dashed gray lines; β₂-AR KO, continuous gray lines) were taken from Figure 2. (C) Schild plot for determining the pA₂ value of L748,337 in β₂-AR KO (see Materials and Methods for further details). Please note that no Schild plot could be obtained for data from WT. Mean values ± SD from n experiments as indicated in parenthesis (number of strips/number of animals).

FIGURE 8

Concentration-response curves for the relaxing effects of forskolin on KCl-induced contractions in detrusor strips from WT and β₂-AR KO mice. (A) Forskolin effects on tone; effects on spontaneous contractions (B). Mean values ± SD from n experiments as indicated in parenthesis (number of strips/number of animals).