M(2) and M(3) muscarinic receptor-mediated contractions in longitudinal smooth muscle of the ileum studied with receptor knockout mice

Abstract

Isometric contractile responses to carbachol were studied in ileal longitudinal smooth muscle strips from wild-type mice and mice genetically lacking M(2) or M(3) muscarinic receptors, in order to characterize the mechanisms involved in M(2) and M(3) receptor-mediated contractile responses. Single applications of carbachol (0.1-100 microM) produced concentration-dependent contractions in preparations from M(2)-knockout (KO) and M(3)-KO mice, mediated via M(3) and M(2) receptors, respectively, as judged by the sensitivity of contractile responses to blockade by the M(2)-preferring antagonist methoctramine (300 nM) or the M(3)-preferring antagonist 4-DAMP (30 nM). The M(2)-mediated contractions were mimicked in shape by submaximal stimulation with high K(+) concentrations (up to 35 mM), almost abolished by voltage-dependent Ca(2+) channel (VDCC) antagonists or depolarization with 140 mM K(+) medium, and greatly reduced by pertussis toxin (PTX) treatment. The M(3)-mediated contractions were only partially inhibited by VDCC antagonists or 140 mM K(+)-depolarization medium, and remained unaffected by PTX treatment. The contractions observed during high K(+) depolarization consisted of different components, either sensitive or insensitive to extracellular Ca(2+). The carbachol contractions observed with wild-type preparations consisted of PTX-sensitive and -insensitive components. The PTX-sensitive component was functionally significant only at low carbachol concentrations. The results suggest that the M(2) receptor, through PTX-sensitive mechanisms, induces ileal contractions that depend on voltage-dependent Ca(2+) entry, especially associated with action potential discharge, and that the M(3) receptor, through PTX-insensitive mechanisms, induces contractions that depend on voltage-dependent and -independent Ca(2+) entry and intracellular Ca(2+) release. In intact tissues coexpressing M(2) and M(3) receptors, M(2) receptor activity appears functionally relevant only when fractional receptor occupation is relatively small.

Figure 1

Contractions to carbachol in ileal smooth muscle strips from M₂-KO (a), M₃-KO (b), and M₂/M₃-double KO mice (c). In each strip, increasing, single concentrations of carbachol were applied for 1 min, as indicated (for details, see Methods). As a standard, a K⁺-induced contraction was obtained by the addition of 70 mM KCl (70 K). In (c), prostaglandin F2α (PGF2α, 0.3 μM) was applied as indicated.

Figure 2

Averaged concentration–response curves for the tension increases caused by carbachol in ileal smooth muscle strips from M₂-KO and M₃-KO mice and their respective WT controls (a). (b) Competitive antagonisms by the M₂-preferring antagonist, methoctramine (300 nM), and the M₃-preferring antagonist, 4-DAMP (30 nM), of carbachol-induced contractions in strips from the M₂-KO and M₃-KO groups. The peak tension generated by carbachol was measured from the mean peak level of pre-existing spontaneous contractions and was expressed as % of the 70 mM K⁺ contraction response in the same strip. In (b), the carbachol curves for the M₂-KO and M₃-KO groups without antagonist treatment (same as in a) are represented by the dotted curves. Each point represents the mean±s.e.m. of three to twelve measurements.

Figure 3

Shape of tension responses to carbachol in the M₃-KO group (a) and to application of high K⁺ in the M₂/M₃-double KO group (b). Agents were applied for 3 min at different concentrations as indicated. In (b), the 70 mM K⁺ contraction response is cut off at its top by one calibration size (0.2 g).

Figure 4

Effects of nicardipine on carbachol contractions in ileal smooth muscle strips from the M₃-KO (a) and M₂-KO groups (b). Carbachol was applied for 3 min at the indicated concentrations, in the absence or presence of nicardipine (3 μM). In (a), the contraction response to 70 mM K⁺ is cut off at its top by one calibration size (0.2 g). The arrows in (b) indicate an initial rapid rising phase (see text for details). (c) Superimposed traces of responses in the absence or presence of nicardipine (data from panel b). Note that the carbachol contractions in (a) are much more sensitive to nicardipine than those in (b).

Figure 5

Effects of isotonic 140 mM K⁺ medium and removal of extracellular calcium (‘Ca²⁺-free') on carbachol contractions in strips from the M₃-KO (a) and M₂-KO groups (b). Carbachol was applied for 3 min at the indicated concentrations. The upward deflections seen at the beginning of ‘Ca^2+-free' are artifacts by changing the bath solution (140 mM K⁺ medium) to Ca²⁺-free medium (140 mM K⁺ medium plus 0.5 mM EGTA). See text for details.

Figure 6

Contractile effects of carbachol on ileal smooth muscle strips from the M₂-KO (a) and M₃-KO groups (b) pretreated with PTX. Increasing concentrations of carbachol and 3 μM PGF2α were applied as indicated. (c) Averaged concentration–response curves for the tension increases caused by carbachol. Each point represents the mean±s.e.m. (n=6 for the PTX-treated M₂-KO group; n=8 for the PTX-treated M₃-KO group). For comparison, the carbachol curves for the M₂-KO and M₃-KO groups without PTX treatment (taken from Figure 2) are represented by the dotted curves.

Figure 7

Contractile effects of carbachol on ileal smooth muscle strips from WT M₃ mice in the absence (a) or after treatment with PTX (b). (c) Averaged concentration–response curves for the tension increases caused by carbachol. Each point represents the mean±s.e.m. (n=8 for the control preparations; n=5 for the PTX-treated preparations). ^*Significantly different (P<0.05) from the corresponding control value. (d) Plot depicting the relationship between carbachol concentration and the PTX-sensitive component of the carbachol-mediated contractions. The percentage of PTX-sensitive component (P) was estimated from the data (mean relative amplitudes) shown in (c) using the following formula: P=100 (T_Cont−T_PTX)/T_Cont, where T_Cont is the mean relative amplitude for the control preparations and T_PTX is the corresponding value for samples treated with PTX. Note that the PTX-sensitive component continuously decreases with increasing carbachol concentrations.