Distinct mixtures of muscarinic receptor subtypes mediate inhibition of noradrenaline release in different mouse peripheral tissues, as studied with receptor knockout mice

Abstract

The muscarinic heteroreceptors modulating noradrenaline release in atria, urinary bladder and vas deferens were previously studied in mice in which the M(2) or the M(4) muscarinic receptor genes had been disrupted. These experiments showed that these tissues possessed both M(2) and non-M(2) heteroreceptors. The analysis was now extended to mice in which either the M(3), both the M(2) and the M(3), or both the M(2) and the M(4) genes had been disrupted (M(3)-knockout, M(2/3)-knockout and M(2/4)-knockout). Tissues were preincubated with (3)H-noradrenaline and then stimulated electrically (20 pulses per 50 Hz). In wild-type atria, carbachol (0.01-100 microM) decreased the electrically evoked tritium overflow by maximally 60-78%. The maximum inhibition of carbachol was reduced to 57% in M(3)-knockout and to 23% in M(2/4)-knockout atria. Strikingly, the effect of carbachol was abolished in M(2/3)-knockout atria. In wild-type bladder, carbachol (0.01-100 microM) reduced the evoked tritium overflow by maximally 57-71%. This effect remained unchanged in the M(3)-knockout, but was abolished in the M(2/4)-knockout bladder. In wild-type vas deferens, carbachol (0.01-100 microM) reduced the evoked tritium overflow by maximally 34-48%. The maximum inhibition of carbachol was reduced to 40% in the M(3)-knockout and to 18% in the M(2/4)-knockout vas deferens. We conclude that the postganglionic sympathetic axons of mouse atria possess M(2) and M(3), those of the urinary bladder M(2) and M(4), and those of the vas deferens M(2), M(3) and M(4) release-inhibiting muscarinic receptors.

Figure 1

Effect of carbachol on the evoked overflow of tritium from atria of M₃-wild-type or M₃-knockout (a), M_2/4-wild-type or M_2/4-knockout (b), and M_2/3-wild-type or M_2/3-knockout (c) mice. After preincubation with ³H-noradrenaline, tissues were superfused and stimulated six times by 20 pulses at 50 Hz (S₁–S₆). Carbachol was added at increasing concentrations (abscissae) before S₂–S₆. Ordinates, evoked overflow of tritium, calculated from S_n/S₁ ratios and expressed as a percentage of the corresponding control (no carbachol). Means±s.e.m. from n=6–14 tissue pieces. Significant differences from corresponding control (no carbachol): ^#P<0.05. Significant differences from corresponding wild type: ^*P<0.05.

Figure 2

Effect of carbachol on the evoked overflow of tritium from urinary bladder of M₃-wild-type or M₃-knockout (a) and M_2/4-wild-type or M_2/4-knockout (b) mice. After preincubation with ³H-noradrenaline, tissues were superfused and stimulated six times by 20 pulses at 50 Hz (S₁–S₆). Carbachol was added at increasing concentrations (abscissae) before S₂–S₆. Ordinates, evoked overflow of tritium, calculated from S_n/S₁ ratios and expressed as a percentage of the corresponding control (no carbachol). Means±s.e.m. from n=9–14 tissue pieces. Significant differences from corresponding control (no carbachol): ^#P<0.05. Significant differences from corresponding wild type: ^*P<0.05.

Figure 3

Effect of carbachol on the evoked overflow of tritium from vas deferens of M₃-wild-type or M₃-knockout (a) and M_2/4-wild-type or M_2/4-knockout (b) mice. After preincubation with ³H-noradrenaline, tissues were superfused and stimulated six times by 20 pulses at 50 Hz (S₁–S₆). Carbachol was added at increasing concentrations (abscissae) before S₂–S₆. Ordinates, evoked overflow of tritium, calculated from S_n/S₁ ratios and expressed as a percentage of the corresponding control (no carbachol). Means±s.e.m. from n=6–15 tissue pieces. Significant differences from corresponding control (no carbachol): ^*P<0.05. Significant differences from corresponding wild type: ^#P<0.05.

Figure 4

Interaction of the muscarinic antagonists methoctramine and pirenzepine with carbachol on the evoked overflow of tritium from vas deferens of M₃-knockout mice. After preincubation with ³H-noradrenaline, tissues were superfused and stimulated six times by 20 pulses at 50 Hz (S₁–S₆). Carbachol was added at increasing concentrations (abscissae) before S₂–S₆. Carbachol was given either alone or combined with the indicated antagonists which were present throughout superfusion. Ordinates, evoked overflow of tritium, calculated from S_n/S₁ ratios and expressed as a percentage of the corresponding control (no carbachol). Means±s.e.m. from n=15–19 tissue pieces. Significant differences from carbachol alone: ^#P<0.05.