Interactions of agonists with M2 and M4 muscarinic receptor subtypes mediating cyclic AMP inhibition.

In this study the similarities and differences between the M2 and M4 subtypes in their recognition of agonists were explored. A CHO-K1 cell line transfected with the human m2 receptor was used as a homogeneous M2 tissue for comparison with two putative M4 systems (rat striatum and the N1E-115 mouse neuroblastoma cell line). The equilibrium binding dissociation constants and intrinsic efficacies for seven muscarinic agonists were determined for their stimulation of cyclic AMP inhibition via the M2 and M4 receptors. Partial receptor occlusion with propylbenzilylcholine mustard was used to determine binding constants for the more efficacious drugs and the reference agonist oxotremorine-M. The binding dissociation constants and relative efficacies for other agonists were then determined in reference to oxotremorine-M by a null method. For the M2 receptor the agonist binding dissociation constants ranged in potency from oxotremorine (1.5 microM) to bethanechol (171 microM), whereas relative efficacies varied from that of muscarine (relative efficacy = 0.9) to the value for McN-A343 (relative efficacy = 0.04). In general, most agonists bound with similar potencies to M2 and M4 receptors (Kd values within a factor of 2-3). However, oxotremorine bound to the N1E-115 and striatal M4 receptors about 3-fold and 10-fold less potently, respectively, than it did to the M2 receptor. Another exception was pilocarpine, which bound to the N1E-115 receptor (1.9 microM) with 8-fold and 12-fold higher potency than to the CHO-K1 M2 receptor and the striatal M4 receptor, respectively. Despite the low affinity of bethanechol for the M2 receptor, it was an efficacious agonist (maximal response equivalent to that of oxotremorine-M; relative efficacy = 0.6) at this subtype, whereas it was a partial agonist (60%) with lesser efficacy in the clonal M4 system. In contrast, McN-A343 and arecoline were significantly more efficacious at the two M4 receptors than they were at the M2 receptor. The M4 system in the rat striatum displayed some similarity to the N1E-115 M4 system, with regard to the efficacy ranking for certain agonists (arecoline greater than bethanechol greater than McN-A343 greater than or equal to pilocarpine). This rank order was different from the ranking of these four agonists in the M2 system, indicating that these two M4 receptors are more similar to each other in efficacy ranking than they are to the M2 receptor. However, the rat striatal and N1E-115 M4 receptor differed in their binding of oxotremorine and pilocarpine, indicating that these two M4 systems were not pharmacologically identical.(ABSTRACT TRUNCATED AT 400 WORDS)

Autoradiographic analyses of agonist binding to muscarinic receptor subtypes.

The binding of four muscarinic receptor agonists to regions of rat brain was examined through quantitative autoradiographic techniques. Oxotremorine, arecoline, pilocarpine and bethanechol were chosen based on their different potencies and efficacies in muscarinic second messenger systems. Overall, the order of potency for inhibition of [3H]-l-quinuclidinyl benzilate ([3H]-l-QNB) binding to rat brain slices was oxotremorine greater than pilocarpine = arecoline much greater than bethanechol. Regional assays of agonist potency indicated that all agonists were more selective for brainstem and thalamic regions than for hippocampal and cortical regions. The high selectivity of agonists for areas such as the paraventricular thalamus and the superior colliculus, which also display low affinity for pirenzepine, suggests that muscarinic agonists bind with higher affinity to M2 receptors. Of the four agonists examined, pilocarpine displayed the lowest selectivity for M2 receptors in that IC50 values for pilocarpine were only 3-fold higher in the hippocampal and striatal regions (e.g. CA3: 40.6 +/- 9.4 microM) than in thalamic and brainstem regions (e.g. paraventricular thalamus: 14.9 +/- 6.2 microM). Oxotremorine was 8-fold more potent in the brainstem and thalamus, while arecoline and bethanechol were, respectively, 19- and 100-fold more selective for brainstem and thalamic receptors. Scatchard analyses revealed heterogeneous binding profiles for some agonists within single brain regions, suggesting that multiple agonist sites exist even within regions of predominantly M1 or M2 receptors. For example, arecoline displayed curved Scatchard plots within the external layers of the cerebral cortex, layer CA1 of the hippocampus (predominantly M1 subtype), and the paraventricular thalamus (predominantly M2 subtype). The ability of agonists to recognize multiple sites within a single region may reflect the ability to recognize receptors coupled or uncoupled to second messenger systems through G-proteins.

Effects of four H2 histamine antagonists on bethanechol-stimulated acid and pepsin secretion in the dog.

In five gastric fistula dogs, each of four H2-receptor antagonists was given (at doses roughly equal to the dose inhibiting histamine stimulation at 50%) as background infusion to graded doses of i.v. bethanechol. We measured acid and pepsin secretion and gastrin release. All four compounds noncompetitively inhibited acid secretion, reducing maximum acid outputs by 50 to 70%. Two compounds, tiotidine and cimetidine, shifted the bethanechol dose-response for pepsin secretion about 30% to the right at midpoint without reducing maximum output significantly, whereas the other two, ranitidine and metiamide, did not alter the dose-response. Dose-dependent gastrin release was unaffected by cimetidine, an imidazole compound, and augmented by tiotidine and ranitidine, the two nonimidazole compounds. Actions on pepsin and gastrin are thus not related to H2-receptor effects. The uniform effect of the four antagonists on acid secretion indicates an essential interaction between cholinergic and histamine effects on the parietal cell, although we could not distinguish between prereceptor, receptor and postreceptor sites for such interaction.

Modulation of pancreatic muscarinic receptors by weaning.

We have investigated the effects of early and delayed weaning on the development of the rat pancreatic muscarinic receptors. Weaning after 12, 14 and 16 complete days resulted in significantly increased concentrations of muscarinic receptors. Milk feeding, as the exclusive source of food, from day 12 to 23, 25 or 28, was associated with slight non significant decreases in receptor concentration. In both instances, early or delayed weaning, the apparent KDs of (3H-QNB binding were not affected. It is suggested that early solid food intake can modulate rapidly the pancreatic muscarinic receptor population while exclusive milk feeding does not seem to influence the dynamic of the muscarinic receptor population.