Mechanisms mediating cholinergic antral circular smooth muscle contraction in rats

Abstract

Aim: To investigate the pathway (s) mediating rat antral circular smooth muscle contractile responses to the cholinomimetic agent, bethanechol and the subtypes of muscarinic receptors mediating the cholinergic contraction.

Methods: Circular smooth muscle strips from the antrum of Sprague-Dawley rats were mounted in muscle baths in Krebs buffer. Isometric tension was recorded. Cumulative concentration-response curves were obtained for (+)-cis-dioxolane (cD), a nonspecific muscarinic agonist, at 10(-8)-10(-4) mol/L, in the presence of tetrodotoxin (TTX, 10(-7) mol/L). Results were normalized to cross sectional area. A repeat concentration-response curve was obtained after incubation of the muscle for 90 min with antagonists for M1 (pirenzepine), M2 (methoctramine) and M3 (darifenacin) muscarinic receptor subtypes. The sensitivity to PTX was tested by the ip injection of 100 mg/kg of PTX 5 d before the experiment. The antral circular smooth muscles were removed from PTX-treated and non-treated rats as strips and dispersed smooth muscle cells to identify whether PTX-linked pathway mediated the contractility to bethanechol.

Results: A dose-dependent contractile response observed with bethanechol, was not affected by TTX. The pretreatment of rats with pertussis toxin decreased the contraction induced by bethanechol. Lack of calcium as well as the presence of the L-type calcium channel blocker, nifedipine, also inhibited the cholinergic contraction, with a reduction in response from 2.5+/-0.4 g/mm2 to 1.2+/-0.4 g/mm(2) (P<0.05). The dose-response curves were shifted to the right by muscarinic antagonists in the following order of affinity: darifenacin (M(3))>methocramine (M(2)) >pirenzepine (M(1)).

Conclusion: The muscarinic receptors-dependent contraction of rat antral circular smooth muscles was linked to the signal transduction pathway(s) involving pertussis-toxin sensitive GTP-binding proteins and to extracellular calcium via L-type voltage gated calcium channels. The presence of the residual contractile response after the treatment with nifedipine, suggests that an additional pathway could mediate the cholinergic contraction. The involvement of more than one muscarinic receptor (functionally predominant type 3 over type 2) also suggests more than one pathway mediating the cholinergic contraction in rat antrum.

Figure 1

Effect of bethanechol on smooth muscle contraction. Antrum circular smooth muscle strips were incubated with increasing concentrations of bethanechol (10^-4 mol/L to 10^-7 mol/L). The vertical axis represents the developed tension (in grams per mm²). Bethanechol significantly increased circular muscle tension (P < 0.05; paired t-test). Each data point represents mean ± SE, n = 4.

Figure 2

Effect of depletion of Ca²⁺ from medium on the contraction of antral circular smooth muscle strips to bethanechol (10^-4 mol/L). Ca²⁺ depletion caused a significant decrease in the muscle tension; after 5 min incubation (P < 0.06, paired t-test); and after 10 min incubation (P < 0.005, paired t-test) compared to the base contraction induced by bethanechol before Ca²⁺ depletion. Each data point represents mean ± SE, n = 3.

Figure 3

Effect of PTX on antrum circular smooth muscle strips contraction to bethanechol (10^-4 mol/L to 10^-6 mol/L). PTX significantly inhibited the contractile activity of the smooth muscle (P ≤ 0.01; paired t-test) compared to control rats. Each point represents mean ± SE, n = 7; dotted bars represent control animals; solid bars-PTX-pretreated animals.

Figure 4

Effect of PTX on dispersed antral circular smooth muscle myocytes from PTX-pretreated rats contraction to bethanechol (10^-7 mol/L and 10^-8 mol/L). Myocytes from PTX-treated rats contracted less than myocytes from control rats. Each point represents mean ± SE; of the percentage of the control cells diastolic length (no bethanechol). At least 50 myocytes per point were calculated, P < 0.005; n = 7 and 11. Dotted bars represent control animals; solid bars PTX-pretreated animals.

Figure 5

Concentration-response curves for the contractile effect of (+)-cis-Dioxolane alone and in combination with different concentrations of M1 antagonist, pirenzepine (10^-8 mol/L to 10^-5 mol/L) on the rat antral circular smooth muscle strips. Each point represents the mean ± SE, n = 6, 6, 7 and 8 strips. Cumulative concentration-effect curve was constructed for each strip for (+)-cis-Dioxolane, (10^-8 to 3 × 10^-5 mol/L). After washing, strips were equilibrated in either the absence (control) or presence of pirenzepine for 90 min. Subsequently, the second concentration-effect curve for (+)-cis-Dioxolane was constructed for each strip. Pirenzepine caused a significant inhibition of the contractile muscle response to (+)-cis-dioxolane.

Figure 6

Concentration-response curves for the contractile effect of (+)-cis-Dioxolane alone and in combination with different concentrations of M₂ antagonist, methocramine (from 10^-8 mol/L to 10^-5 mol/L) on the rat antral circular smooth muscle strips. Each point represents the mean ± SE; n = 4, 9, 10 and 11 strips. First, cumulative concentration-effect curve was constructed for each strip for (+)-cis-Dioxolane, (10^-8 to 3 × 10^-5 mol/L). After washing, tissues were equilibrated in either the absence (control) or presence of methocramine for a 90 min. Subsequently, the second concentration-effect curve for (+)- cis-Dioxolane was constructed for each strip. Methocramine caused a significant inhibition of the contractile muscle response to (+)-cis-Dioxolane.

Figure 7

Concentration-response curves for the contractile effect of (+)-cis-Dioxolane alone and in combination with different concentrations of M₃ antagonist, darifenacin (from 10^-10 mol/L to 10^-6 mol/L) on the rat antral circular smooth muscle strips. Each point represents the mean ± SE; n = 4, 7, 11, 10 strips. First, cumulative concentration-effect curve was constructed for each strip for (+)-cis-Dioxolane, (10^-8 to 3 × 10^-5 mol/L). After wash, tissues were equilibrated in either the absence (control) or presence of darifenacin for a 90 min. Subsequently, the second concentration-effect curve for (+)-cis-Dioxolane was constructed for each strip. Darifenacin caused a significant inhibition of the contractile muscle response to (+)-cis-Dioxolane.

Figure 8

Comparison of the inhibition of the contractile response to cis-Dioxolane by 3 muscarinic receptor antagonists: M₃- darifenacin, M₂- methocramine, and M₁- pirenzepine. The M₃ antagonist, darifenacin, was the most potent inhibitor of the contraction induced by (+)-cis Dioxolane (10^-6 mol/L). The EC₅₀ values for each were: for darifenacin (M₃) -7.9; for methocramine (M₂) -7.2; and for pirenzepine (M₁) -6.8.