Acetylcholine receptor. Genes encoding nicotinic receptor subtypes on neurons.
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Animals↗
Biomedical subjects
S W Halvorsen
Publications and source records attributed to S W Halvorsen.
Functional and physical properties of chick atrial and ventricular GTP-binding proteins: relationship to muscarinic acetylcholine receptor-mediated responses.
Activation of muscarinic acetylcholine receptors in chick atria, but not ventricles, causes an increase in K+ permeability. Because of suggestions that this difference in muscarinic receptor-mediated physiological responses may be due to changes in the guanine nucleotide regulatory proteins (termed Go and Gi) associated with the receptor, we compared the functional and biochemical properties of these proteins in atria and ventricles from 8-day chick embryos. The affinity of agonist for the muscarinic receptor in either the absence or presence of guanine nucleotides was the same in membranes from atria and ventricles; similar concentrations of guanyl-5'-yl imidodiphosphate were required to regulate agonist binding in both tissues (EC50 of 2.7 X 10(-8) and 2.0 X 10(-8) M for atria and ventricles, respectively). Forskolin-stimulated adenylate cyclase activity in atria and ventricles was equally sensitive to inhibition by guanyl-5'-yl imidodiphosphate. In addition, the muscarinic agonist carbachol inhibited adenylate cyclase in ventricles with an IC50 similar to that observed in atria, although the magnitude of inhibition was slightly less in ventricles. The physical properties of the alpha subunits of the guanine nucleotide regulatory proteins were examined after covalent modification by islet activating protein. Two [32P]ADP-ribosylated polypeptides were detected by one dimensional gel electrophoresis, with molecular weights equal to those reported for Go and Gi from other tissues. Similar amounts of both proteins were found in atrial and ventricular membranes. Peptide mapping demonstrated that although the 39 and 42 kD proteins had nonidentical peptide maps, atrial and ventricular peptide maps were identical.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenosine Diphosphate Ribose↗
Affinity labeling of neuronal acetylcholine receptor subunits with an alpha-neurotoxin that blocks receptor function.
An alpha-neurotoxin, Bgt 3.1, has previously been shown to recognize the functional acetylcholine receptor (AChR) on chick autonomic neurons, since it specifically blocks receptor function and it binds to a class of sites on the neurons with the pharmacology, kinetics, and affinity expected for the receptor. A monoclonal antibody, mAb 35, to the main immunogenic region of muscle and electric organ AChR alpha subunit cross-reacts with a component on chick autonomic neurons that, from several lines of evidence, also appears to be the functional AChR. The identity of the antibody-binding component has remained in doubt, however, because previous studies indicated that in at least one instance a substantial discrepancy existed between the number of functional AChRs estimated physiologically and the number of putative AChRs detected by mAb 35 on the neurons. The present findings demonstrate that Bgt 3.1 and mAb 35 recognize the same AChRs on the neurons, and provide information about the stoichiometry of binding and the identity of subunits associated with active sites on the receptor. Chick ciliary ganglion neurons examined under a variety of growth and regulatory conditions in culture displayed a constant ratio of about 2:1 for mAb 35 and Bgt 3.1 binding to cell surface sites. Treatment of the cells with mAb 35 induced a substantial decrease in the number of Bgt 3.1 sites and vice versa. AChRs that had been covalently labeled with a photoaffinity derivative of 125I-Bgt 3.1 in situ and then solubilized were specifically immune-precipitated by mAb 35, demonstrating unequivocally that the receptors possessed both Bgt 3.1 and mAb 35 binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Identification of a nicotinic acetylcholine receptor on neurons using an alpha-neurotoxin that blocks receptor function.
An alpha-neurotoxin, Bgt 3.1, that reversibly blocks the ACh response of chick ciliary ganglion neurons has been used to identify 2 classes of high-affinity binding sites on the cells in culture. The first class appears to be the alpha-bungarotoxin binding site on the neurons. The second class of Bgt 3.1 sites is distinct from the alpha-bungarotoxin binding sites and has the properties expected for the functional nicotinic ACh receptor on the cells. Equilibrium binding and kinetic studies indicate a Kd value of 5-6 nM for Bgt 3.1 at the second class of sites. The kinetics and affinity of binding are consistent with those inferred from previous physiological studies for Bgt 3.1 inhibition of receptor function. Bgt 3.1 binding to the sites is completely inhibited by each of the cholinergic ligands ACh, carbachol, nicotine, d-tubocurarine, and trimethaphan, but not by alpha-bungarotoxin. Highest site densities are found in cultures of ciliary and sympathetic ganglion neurons, cell types known to have ganglionic nicotinic ACh receptors. Low levels of sites may be present in cultures of spinal cord and dorsal root ganglion neurons; no binding is found in cultures of skeletal myotubes or cardiac cells when alpha-bungarotoxin is used to block Bgt 3.1 binding to alpha-bungarotoxin sites. These results demonstrate that Bgt 3.1 can be used as a specific probe for the nicotinic ACh receptor on chick autonomic neurons.
Animals↗
Ontogenesis of physiological responsiveness and guanine nucleotide sensitivity of cardiac muscarinic receptors during chick embryonic development.
Atria isolated from 4-day chick embryos were much less responsive to the negative chronotropic effect of muscarinic agonists than were atria from 5- or 8-day embryos, even though the density of muscarinic acetylcholine receptors (mAChR) was similar at all these ages. The mAChR in hearts from 4-day embryos were also significantly less susceptible to regulation of receptor number by in vivo agonist treatment and required a 2-5-fold greater dose of the muscarinic agonist carbachol to achieve a decrease in receptor number equivalent to that observed in 5- or 8-day embryonic hearts. When 4-day atrial membranes were assayed in physiological buffers, agonist binding to the mAChR was not regulated by GTP unless a sulfhydryl reducing agent was present. Receptors from 5- and 8-day embryos did not require addition of a sulfhydryl reducing agent in order to see guanine nucleotide effects on agonist binding. Even in the presence of a sulfhydryl reducing agent, carbachol binding to the mAChR in 4-day membranes was much less sensitive to guanyl-5'-yl imidodiphosphate (GppNHp) than binding to mAChR in 5- or 8-day membranes. In addition, forskolin-activated adenylate cyclase activity was much less sensitive to inhibition by GppNHp in membranes from 4-day atria than from 5- and 8-day atria. The GTP-binding component (NI) which couples the mAChR to inhibition of adenylate cyclase activity was examined by covalent modification with pertussis toxin.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenylate Cyclase Toxin↗
Partial agonist activity of oxotremorine at muscarinic acetylcholine receptors in the embryonic chick heart.
Previous work has demonstrated that prolonged treatment of chick embryos in vivo with muscarinic agonists leads to a decrease in the number of muscarinic acetylcholine binding sites in the heart and a decrease in the sensitivity of isolated atria to the negative chronotropic response to muscarinic stimulation. We show here that treatment with the agonist carbachol leads to greater decreases in receptor number than does treatment with oxotremorine. Simultaneous treatment with oxotremorine and carbachol resulted in partial blockage in the maximal decrease in receptor number seen after treatment with carbachol alone. After treatment of embryos in vivo with carbachol to decrease the reserve of spare receptors in the atria, oxotremorine could no longer elicit a significant negative chronotropic response under conditions in which carbachol was still effective. These results demonstrate that oxotremorine is a partial agonist at the muscarinic acetylcholine receptor in the embryonic chick heart.
Animals↗
Development and regulation of cardiac muscarinic acetylcholine receptor number, function and guanyl nucleotide sensitivity.
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Adenylyl Cyclases↗
In vivo regulation of muscarinic acetylcholine receptor number and function in embryonic chick heart.
We have used the chick embryonic heart to study the regulation of the muscarinic acetylcholine receptor in vivo. Sustained activation of the muscarinic receptor in vivo with the cholinergic agonist carbachol decreases muscarinic receptor number as much as 87% as measured by the specific binding of the potent muscarinic receptor in antagonist [3H]quinuclidinyl benzilate. The decrease in receptor number is both dose and time dependent. After carbachol-induced down regulation, the receptor number recovers to control levels when further receptor activation is blocked by the muscarinic receptor antagonist atropine. When receptor number is decreased 50% in vivo, isolated atria require a 12-fold greater concentration of carbachol than controls to arrest spontaneous beating in an organ bath. Analysis of the binding of carbachol to the muscarinic receptor indicates that this shift in the dose response of atria is accompanied by a change in the relative fraction of the high and low affinity forms of the muscarinic receptor with no change in their respective affinities for carbachol. In addition, this analysis suggests that the low agonist affinity form of the cardiac muscarinic receptor is the physiologically active form.
Animals↗