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Expression of ER is a constitutional feature of the connective tissue and smooth muscle cells of the anal continence organ. Estrogen receptors and PR are not detectable in the striated muscle fibers of the external anal sphincter in either sex. The presence of ER in the stroma and smooth muscles of the anal canal suggests that these tissues are targets for estrogen. This constitutes a theoretical basis for the beneficial effects of estrogen and progestin replacement on anal continence in postmenopausal women.

In this report we characterize muscarinic cholinergic receptor on embryonic cells. We established dose-response curves by fluorometric measurement of Ca 2+ mobilization in cell suspensions of whole chick embryos stage 23•24. Ca 2+ mobilization was quantitated by standardization of chlorotetracycline (CTC) fluorescence changes after stimulation with muscarinic agonists. We determined EDso values for the agonists acetylcholine and carbachol as 3.4 x 10 -6 and 2.7 × 10 -5 M, respectively. Pilocarpine and oxotremorine were found to act as reversible competitive antagonists with inhibition constants (K0 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 EDso 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 Ca 2÷ 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.Undifferentiated cells of the chick limb bud possess a muscarinic cholinergic receptor (1) which is assumed to be part of an embryonic cholinergic system that is expressed in undifferentiated cells during distinct phases of morphogenesis (2, 3). In a preceding publication, we described intracellular Ca 2÷ mobilization upon stimulation of the receptor (4). Ca 2* mobilization was measured in a spectrofluorometric assay with chlorotetracycline (CTC) 1 (5, 6). On addition of muscarinic agonists, the cells responded with a fluorescence decrease. The reaction was blocked by muscarinic antagonists. Nicotinic drugs were ineffective.For further characterization of the muscarinic receptor on embryonic cells, dose-response curves have to be established.Abbreviations used in this paper: B~, total concentration of specific binding sites; CTC, chlorotetracycline; cv, coefficient of variation; ED~o, the agonist concentration that yields 50% of maximal effect; MSE, mean square error; QNB, quinuclidinylbenzilate. Intracellular Ca 2+ mobilization is a biological effect that can be used for this purpose. In the present study, to quantify CTC fluorescence changes, we standardized the fluorometric measurements and improved data processing. The dose-response curves have to be related to receptor occupancy. Since ligand affinities in homogenate are different from those in cell suspensions, our binding data from homogenate (1) cannot be used for discussion of dose-response curves. In particula...

Cell suspensions of chick limb buds (stage 23/24) were loaded with the fluorescent Ca chelator chlorotetracycline. Fluorescence was monitored in a spectrofluorometer. Stimulation with acetylcholine induced a fluorescence decrease, indicating intracellular Ca mobilization. The fluorescence decrease triggered by acetylcholine was inhibited by muscarinic but not by nicotinic antagonists, indicating that a muscarinic acetylcholine receptor is involved. The muscarinic receptor in the chick limb bud has a characteristic pharmacological profile: acetylcholine, carbachol and acetyl-β-methylcholine functioned as full agonists triggering maximal fluorescence decrease. Bethanechol was less effective, producing only one-third of the maximum response. Pilocarpine and oxotremorine, two classical agonists in other systems, were ineffective and functioned as antagonists. In the chick limb bud, cholinesterase, choline acetyltransferase and the presence of a muscarinic receptor have been demonstrated in previous studies. The present experiments show that stimulation of the embryonic muscarinic receptor leads to intracellular Ca mobilization.