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

Abstract

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...

Non-Selectivity of the Monoclonal Antibody M35 for Subtypes of Muscarinic Acetylcholine Receptors

“…Evidently, the epitope for M35 is preserved on all mAChR subtypes, as well as on the embryonic mAChR type [17], which is biochemically different from the adult types of mAChRs [28]. The epitope for M35 must, therefore, be localized on a part of these receptors that is highly homologous.…”

Identification and Functional Characterization of the Muscarinic Receptor M3 in the Human Keratinocyte Cell Line HaCaT

“…For calculation of the dose-response curves ( fi g. 5 ) we quantifi ed the calcium reaction as fi rst described by Oettling et al [1985] for the chlorotetracycline fl uorescence. In order to estimate the dose-response curves for acetylcholine and carbachol we analyzed the mobilization of intracellular calcium in a concentration range between 10 n M and 4 m M .…”

Membrane-bound Ca2+ distribution visualized by chlorotetracycline fluorescence during morphogenesis of soredia in a lichen

“…On the other hand, similar morphogenetic functions have been ascribed to the 'embryonic' cholinergic system (Drews, 1975) and Ca 2+ distribution here detected closely parallels ChE localization during the development of soredia (Raineri & Modenesi, 1986). In ChE-active animal cells, a rise in free cytosolic Ca 2+ is triggered by ligand binding to a muscarinic ACh receptor (Schmidt et al, 1984;Oettling et al, 1985). The earliest event seems to be the hydrolysis of membrane phosphatidyl inositol-4, 5-bisphosphate into diacylglycerol and inositol-1, 4, 5-trisphosphate, the latter acting as a second messenger to cause Ca 2+ release from intracellular stores (Berridge & Irvine, 1984).…”

“…In the chick limb bud, where such a cholinergic regulation has been investigated in depth, ligand binding to a muscarinic acetylcholine (ACh) receptor is associated with a rise in free cytoplasmic Ca 2+ from intracellular stores (Schmidt et al, 1984;Oettling et al, 1985).…”