Functional and molecular biological evidence for a possible beta3-adrenoceptor in the human detrusor muscle

Abstract

The possible existence of a beta3-adrenergic receptor (beta3-AR) in the human detrusor muscle was investigated by in vitro functional studies and analysis of mRNA expression. Isoprenaline, noradrenaline and adrenaline each produced a concentration-dependent relaxation of the human detrusor. The rank order for their relaxing potencies was isoprenaline (pD2 6.37+/-0.07) > or = noradrenaline (pD2 6.07+/-0.12) > or = adrenaline (pD2 5.88< or =0.11). Neither dobutamine (beta1- and beta2-AR agonist) nor procaterol (beta2-AR agonist) produced any significant relaxation at concentrations up to 10(-5) M. BRL37344A, CL316243 and CGP-12177A (beta3-AR agonists), relaxed the preparations significantly at concentrations higher than 10(-6) M. The pD2 values for BRL37344A, CL316243 and CGP-12177A were 6.42+/-0.25, 5.53+/-0.09 and 5.74+/-0.14, respectively. CGP-20712A (10(-7) - 10(-5) M), a beta1-AR antagonist, did not affect the isoprenaline-induced relaxation. On the other hand, ICI-118,551, a beta2-AR antagonist, produced a rightward parallel shift of the concentration-relaxation curve for isoprenaline only at the highest concentration used (10(-5) > M) and its pKB value was 5.71+/-0.19. Moreover, SR58894A (10(-7) - 10(-5) M), a beta3-AR antagonist, caused a rightward shift of the concentration-relaxation curve for isoprenaline in a concentration-dependent manner. The pA2 value and slope obtained from Schild plots were 6.24+/-0.20 and 0.68+/-0.31. The beta1-, beta2- and beta3-AR mRNAs were all positively expressed in detrusor smooth muscle preparations in a reverse transcription polymerase chain reaction assay. In conclusion, the present results provide the first evidence for the existence of the beta3-AR subtype in the human detrusor. They also suggest that the relaxation induced by adrenergic stimulation of the human detrusor is mediated mainly through beta3-AR activation.

Figures

Figure 1

Representative recording of the effect of isoprenaline on resting tension in a human detrusor preparation.

Figure 2

Effects of isoprenaline, noradrenaline, adrenaline, dobutamine, and procaterol on resting tension in human detrusor preparations. All experiments were performed in the presence of 10−6 M phentolamine. Each point represents the mean±s.e.mean of 6–29 experiments. Data are expressed as a percentage of the maximal relaxation induced by 10−5 M forskolin.

Figure 3

Effects of isoprenaline, BRL37344A, CL316243 and CGP-12177A on resting tension in human detrusor preparations. All experiments were performed in the presence of 10−6 M phentolamine. Each point represents the mean±s.e.mean of 7–29 experiments. Data are expressed as a percentage of the maximal relaxation induced by 10−5 M forskolin.

Figure 4

Effects of CGP-20712A (a) and ICI-118,551 (b) on the isoprenaline-induced relaxation of human detrusor muscle preparations. (a) Concentration-response relationships for isoprenaline, either alone or in the presence of CGP-20712A 10−7 M, 3×10−7 M, 10−6 M, 3×10−6 M or 10−5 M. Each point represents the mean±s.e.mean (n=5–6). (b) Concentration-response relationships for isoprenaline, either alone or in the presence of ICI-118,551 10−7 M, 3×10−7 M, 10−6 M, 3×10−6 M, or 10−5 M. Each point represents the mean±s.e.mean (n=6–7). All experiments were carried out in the presence of 10−6 M phentolamine. Data are expressed as a percentage of the maximal relaxation induced by 10−5 M forskolin.

Figure 5

Effect of SR58894A on the isoprenaline-induced relaxation of human detrusor muscle preparations. All experiments were carried out in the presence of CGP-20712A (10−7 M), ICI-118,551 (10−7 M) and phentolamine (10−6 M). (a) Concentration-response relationships for isoprenaline, either alone or in the presence of SR58894A 10−7 M, 3×10−7 M, 10−6 M, 3×10−6 M or 10−5 M. Each point represents the mean±s.e.mean (n=7–12). Data are expressed as a percentage of the maximal relaxation induced by 10−5 M forskolin. (b) Schild plot for the inhibition of the isoprenaline-induced relaxation produced by SR58894A.

Figure 6

Detection of β1-, β2- and β3-AR mRNA in human detrusor tissue by RT-PCR. Detrusor preparations were obtained from three different patients (#1, #2 and #3). PCR amplification for β1- (lanes 2–4), β2- (lanes 5–7), β3-AR (lanes 8–10) and G3PDH (lanes 11–13) were carried out for 35, 35, 35 and 24 cycles, respectively. Expected size of PCR-products for β1-, β2-, β3- AR and G3PDH were 265, 329, 314 and 452, respectively. Lanes 1 and 14 show 100 bp DNA ladder (Gibco-BRL).