The muscarinic receptor of chick embryo cells: correlation between ligand binding and calcium mobilization.
In this report we characterize muscarinic cholinergic receptor on embryonic cells. We established dose-response curves 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. We determined ED50 values for the agonists acetylcholine and carbachol as 3.4 X 10(-6) and 2.7 X 10(-5) M, respectively. Pilocarpine and oxotremorine were found to act as reversible competitive antagonists with inhibition constants (Kl) of 5.0 X 10(-6) and 1.4 X 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 X 10(-4) M. Its antagonistic component is expressed by an inhibition constant of 1.9 X 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 "two 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 fitted KD values of the binding studies when a "one binding-site model" was applied. Combination of dose-response and binding data showed close proportionality between receptor occupancy and calcium mobilization. No spare receptors were present.
Oettling
G
G
Schmidt
H
H
Drews
U
U
D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D018723:Bethanechol; D001626:Bethanechol Compounds / Q000494:pharmacology; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D002642:Chick Embryo; D004305:Dose-Response Relationship, Drug; D010095:Oxotremorine / Q000494:pharmacology; D010862:Pilocarpine / Q000494:pharmacology; D011813:Quinuclidinyl Benzilate / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000502:physiology*; D013050:Spectrometry, Fluorescence
D001626:Bethanechol Compounds; D011976:Receptors, Muscarinic; D018723:Bethanechol; D010862:Pilocarpine; D010095:Oxotremorine; D011813:Quinuclidinyl Benzilate; D002217:Carbachol; D000109:Acetylcholine; D002118:Calcium