The muscarinic receptor of chick embryo cells: correlation between ligand binding and calcium mobilization.

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Abstract
The muscarinic cholinergic receptor on embryonic cells is characterized. Dose-response curves were established by fluorometric measurement of Ca2+ mobilization in cell suspensions of whole chick embryos stage 23/24. Ca2+ mobilization was quantitated by standardization of chlorotetracycline (CTC) fluorescence changes after stimulation with muscarinic agonists. ED50 values were determined for the agonists acetylcholine and carbachol as 3.4 .times. 10-6 and 2.7 .times. 10-5 M, respectively. Pilocarpine and oxotremorine were found to act as reversible competitive antagonists with Ki of 5.0 .times. 10-6 and 1.4 .times. 10-6 M, respectively. Bethanechol, which induced only 23% of the maximal effect obtained by acetylcholine, was a partial agonist with an ED50 of 4.8 .times. 10-4 M. Its antagonistic component is expressed by an inhibition constant of 1.9 .times. 10-4 M. In parallel, binding studies were performed in a competition assay with [3H]-quinuclidinylbenzilate. For the agonists acetylcholine and carbachol, binding parameters were best fitted by a 2 binding-sites model. Comparison with dose-response curves indicated that Ca2+ mobilization was triggered via the high-affinity binding site. The inhibition constants of antagonists derived from the shift of dose-response curves corresponded to the fitter Kd values of the binding studies when a 1 binding-site model was applied. Combination of dose-response and binding data showed close proportionality between receptor occupancy and Ca mobilization. No spare receptors were present.