Pharmacological identification of β-adrenoceptor subtypes mediating isoprenaline-induced relaxation of guinea pig colonic longitudinal smooth muscle

Abstract

Object We aimed to identify the β-adrenoceptor (β-AR) subtypes involved in isoprenaline-induced relaxation of guinea pig colonic longitudinal smooth muscle using pharmacological and biochemical approaches. Methods Longitudinal smooth muscle was prepared from the male guinea pig ascending colon and contracted with histamine prior to comparing the relaxant responses to three catecholamines (isoprenaline, adrenaline, and noradrenaline). The inhibitory effects of subtype-selective β-AR antagonists on isoprenaline-induced relaxation were then investigated. Results The relaxant potencies of the catecholamines were ranked as: isoprenaline > noradrenaline ≈ adrenaline, whereas the rank order was isoprenaline > noradrenaline > adrenaline in the presence of propranolol (a non-selective β-AR antagonist; 3 × 10^-7 M). Atenolol (a selective β₁-AR antagonist; 3 × 10^-7-10^-6 M) acted as a competitive antagonist of isoprenaline-induced relaxation, and the pA₂ value was calculated to be 6.49 (95% confidence interval: 6.34-6.83). The relaxation to isoprenaline was not affected by ICI-118,551 (a selective β₂-AR antagonist) at 10^-9-10^-8 M, but was competitively antagonized by 10^-7-3 × 10^-7 M, with a pA₂ value of 7.41 (95% confidence interval: 7.18-8.02). In the presence of propranolol (3 × 10^-7 M), the relaxant effect of isoprenaline was competitively antagonized by bupranolol (a non-selective β-AR antagonist), with a pA₂ value of 5.90 (95% confidence interval: 5.73-6.35). Conclusion These findings indicated that the β-AR subtypes involved in isoprenaline-induced relaxation of colonic longitudinal guinea pig muscles are β₁-AR and β₃-AR.

Keywords: guinea pig colonic longitudinal smooth muscle; isoprenaline; smooth muscle relaxation; β-adrenoceptor.

Fig. 1.

Representative traces of the contractile responses of isolated segments of the longitudinal smooth muscle of the guinea pig colon to various stimulants over a 3-h period.

Fig. 2.

Analysis of the 3-h contractile responses of isolated segments of guinea pig colonic longitudinal smooth muscle to the indicated compounds. Contractions are expressed as a percentage of the contraction induced by 10⁻⁶ M histamine (100%), which was applied before starting the 3-h recording period; n = 4 for each data point.

Fig. 3.

Reproducibility of the concentration-response curves for isoprenaline-induced relaxation of the isolated guinea pig colonic longitudinal smooth muscle preparations. Concentration-response curves for isoprenaline were obtained in the same preparations. Relaxation is expressed as a percentage of the maximum relaxation induced by 10⁻⁴ M papaverine (100%), which was applied at the end of each experiment; n = 3 for each data point.

Fig. 4.

Relaxant responses to isoprenaline, adrenaline, and noradrenaline in guinea pig colonic longitudinal smooth muscle preparations contracted with histamine (10⁻⁵ M) in the absence (A) and presence (B) of propranolol (3 × 10⁻⁷ M). Relaxation is expressed as a percentage of the maximum relaxation induced by 10⁻⁴ M papaverine (100%), which was applied at the end of each experiment; n = 3 for each data point.

Fig. 5.

Inhibitory effects of propranolol on isoprenaline-induced relaxation of isolated guinea pig colonic longitudinal muscle preparations. A–D: Effects of the indicated concentrations of propranolol on isoprenaline-induced relaxation. A sustained contraction was generated by histamine (10⁻⁵ M) and isoprenaline was then applied to the bath solution cumulatively. Relaxation is expressed as a percentage of the maximum relaxation induced by 10⁻⁴ M papaverine (100%), which was applied at the end of each experiment; n = 3 for each data point. E: Schild plot analyses of the effects of the indicated concentrations of propranolol against isoprenaline; n = 6 for each line.

Fig. 6.

Inhibitory effects of atenolol on isoprenaline-induced relaxation of isolated guinea pig colonic longitudinal muscle preparations. A, B: Effects of the indicated concentrations of atenolol on isoprenaline-induced relaxation of colonic longitudinal muscle preparations contracted with histamine (10⁻⁵ M). Relaxation is expressed as a percentage of the maximum relaxation induced by 10⁻⁴ M papaverine (100%), which was applied at the end of each experiment; n = 3 for each data point. C: Schild plot analysis of the effects of the indicated concentrations of atenolol against isoprenaline; n = 6.

Fig. 7.

Effects of ICI-118,551 on isoprenaline-induced relaxation of isolated guinea pig colonic longitudinal muscle preparations. A–D: Effects of the indicated concentrations of ICI-118,551 on isoprenaline-induced relaxation. Relaxation is expressed as a percentage of the maximum relaxation induced by 10⁻⁴ M papaverine (100%), which was applied at the end of each experiment; n = 4 for each data point. E: Schild plot analysis of the effects of the indicated concentrations of ICI-118,551 against isoprenaline; n = 8.

Fig. 8.

Inhibitory effects of bupranolol on isoprenaline-induced relaxation of isolated guinea pig colonic longitudinal muscle preparations, in the presence of propranolol. The effects of the indicated concentrations of bupranolol on isoprenaline-induced relaxation were investigated in the presence of propranolol (3 × 10⁻⁷ M). Relaxation is expressed as a percentage of the maximum relaxation induced by 10⁻⁴ M papaverine (100%), which was applied at the end of each experiment; n = 4 for each data point. C: Schild plot analysis of the effects of the indicated concentrations of bupranolol against isoprenaline; n = 8.

Fig. 9.

Representative images of agarose gels showing expression of the indicated mRNAs in guinea pig colonic longitudinal smooth muscle (lane 1), atria (lane 2), and ileal longitudinal smooth muscle (lane 3). PCR products were separated by 1.5% agarose gel electrophoresis and cDNA was detected using ethidium bromide. PCR products for β₁-, β₂-, and β₃-ARs were detected as the expected corresponding PCR products of 444, 434, and 383 base pairs, respectively. RT(+): reverse transcription, RT(–): no reverse transcription. These results were representative of four experiments.