Muscarinic receptor subtypes controlling the cationic current in guinea-pig ileal smooth muscle

Abstract

1. The effects of muscarinic antagonists on cationic current evoked by activating muscarinic receptors with the stable agonist carbachol were studied by use of patch-clamp recording techniques in guinea-pig single ileal smooth muscle cells.

2. Ascending concentrations of carbachol (3–300 μM) activated the cationic conductance in a concentration-dependent manner with conductance at a maximally effective carbachol concentration (G_max) of 27.4±1.4 nS and a mean −log EC₅₀ of 5.12±0.03 (mean±s.e.mean) (n=114).

3. Muscarinic antagonists with higher affinity for the M₂ receptor, methoctramine, himbacine and tripitramine, produced a parallel shift of the carbachol concentration-effect curve to the right in a concentration-dependent manner with pA₂ values of 8.1, 8.0 and 9.1, respectively.

4. All M₃ selective muscarinic antagonists tested, 4-DAMP, p-F-HHSiD and zamifenacin, reduced the maximal response in a concentration-dependent and non-competitive manner. This effect could be observed even at concentrations which did not produce any increase in the EC₅₀ for carbachol. At higher concentrations M₃ antagonists shifted the agonist curve to the right, increasing the EC₅₀, and depressed the maximum conductance response. Atropine, a non-selective antagonist, produced both reduction in G_max (M₃ effect) and significant increase in the EC₅₀ (M₂ effect) in the same concentration range.

5. The depression of the conductance by 4-DAMP, zamifenacin and atropine could not be explained by channel block as cationic current evoked by adding GTPγS to the pipette (without application of carbachol) was unaffected.

6. The results support the hypothesis that carbachol activates M₂ muscarinic receptors so initiating the opening of cationic channels which cause depolarization; this effect is potentiated by an unknown mechanism when carbachol activates M₃ receptors. As an increasing fraction of M₃ receptors are blocked by an antagonist, the effects on cationic current of an increasing proportion of activated M₂ receptors are disabled.

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