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

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 mu M) 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 M-2 receptor, methoctramine, himbacine and tripitramine, produced a parallel shift of the carbachol concentration-effect curve to the right in a concentration-dependent manner with pA(2) values of 8.1, 8.0 and 9.1, respectively. 4 All M-3 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 M-3 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) (M-3 effect) and significant increase in the EC50 (M-2 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 GTP gamma S to the pipette (without application of carbachol) was unaffected. 6 The results support the hypothesis that carbachol activates M-2 muscarinic receptors so initiating the opening of cationic channels which cause depolarization; this effect is potentiated by an unknown mechanism when carbachol activates M-3 receptors. As an increasing fraction of M-3 receptors are blocked by an antagonist, the effects on cationic current of an increasing proportion of activated M-2 receptors are disabled.