We investigated the influence of membrane cholesterol content on preferential and non-preferential signaling through the M(2) muscarinic acetylcholine receptor expressed in CHO cells. Cholesterol depletion by 39% significantly decreased the affinity of M(2) receptors for [(3)H]-N-methylscopolamine ([(3)H]-NMS) binding and increased B(max) in intact cells and membranes. Membranes displayed two-affinity agonist binding sites for carbachol and cholesterol depletion doubled the fraction of high-affinity binding sites. In intact cells it also reduced the rate of agonist-induced receptor internalization and changed the profile of agonist binding from a single site to two affinity states. Cholesterol enrichment by 137% had no effects on carbachol E(max) of cAMP synthesis inhibition and on cAMP synthesis stimulation and inositolphosphates (IP) accumulation at higher agonist concentrations (non-preferred pathways). On the other hand, cholesterol depletion significantly increased E(max) of cAMP synthesis inhibition or stimulation without change in potency, and decreased E(max) of IP accumulation. Noteworthy, modifications of membrane cholesterol had no effect on membrane permeability, oxidative activity, protein content, or relative expression of G(s), G(i/o), and G(q/11) alpha subunits. These results demonstrate distinct changes of M(2) receptor signaling through both preferential and non-preferential G-proteins consequent to membrane cholesterol depletion that occur at the level of receptor/G-protein/effector protein interactions in the cell membrane. The significant decrease of IP accumulation by cholesterol depletion was also observed in cells expressing M(3) receptors and by both cholesterol depletion and enrichment in cells expressing M(1) receptors indicating relevance of reduced G(q/11) signaling for the pathogenesis of Alzheimer's disease.

• MeSH
• Acetylcholine analogs & derivatives   MeSH
• Muscarinic Antagonists pharmacology   MeSH
• beta-Cyclodextrins pharmacology   MeSH
• Cell Membrane drug effects   metabolism   MeSH
• CHO Cells MeSH
• Cholesterol metabolism   MeSH
• Cricetulus MeSH
• Carbachol analogs & derivatives   metabolism   pharmacology   MeSH
• Cricetinae MeSH
• Humans MeSH
• N-Methylscopolamine metabolism   pharmacology   MeSH
• GTP-Binding Proteins metabolism   MeSH
• Receptor, Muscarinic M2 metabolism   MeSH
• Second Messenger Systems * drug effects   MeSH
• Animals MeSH
• Check Tag
• Cricetinae MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Acetylcholine MeSH
• Muscarinic Antagonists MeSH
• beta-Cyclodextrins MeSH
• Cholesterol MeSH
• Carbachol MeSH
• methyl-beta-cyclodextrin MeSH Browser
• N-Methylscopolamine MeSH
• GTP-Binding Proteins MeSH
• Receptor, Muscarinic M2 MeSH

Allosteric enhancement of the affinity of muscarinic receptors for their ligands offers a new way to influence cholinergic neurotransmission. The structure of the allosteric binding domain(s) and the features of agonists, antagonists and modulators which determine the occurrence of either positive or negative cooperativity require clarification. We tested interactions between allosteric modulators alcuronium, strychnine and brucine and eight antagonists at muscarinic receptors expressed in CHO cells. In experiments with unlabeled antagonists, all three modulators enhanced the affinity for 4-diphenylacetoxy-N-dimethylpiperidinium (4-DAMP) at the M2 receptors, and strychnine did so also at the M4 receptors. Positive interactions were also observed between alcuronium and L-hyoscyamine (M2) and scopolamine (M2), between strychnine and butylscopolamine (M4), L-hyoscyamine (M2 and M4) and scopolamine (M4), and between brucine and scopolamine (M2). Positive effects of alcuronium, strychnine and brucine on the affinity of the M2 receptors for 4-DAMP have been confirmed by direct measurements of the binding of [3H]-4-DAMP. A comparison of molecular models of several antagonists which are esters revealed that antagonists in which the distance between the N and the carboxyl C atoms corresponds to five chemical bonds are more likely to display positive cooperativity with alcuronium at the M2 receptors than the antagonists in which the N-carboxyl C distance corresponds to four chemical bonds.

• MeSH
• Alcuronium pharmacology   MeSH
• Allosteric Regulation MeSH
• Muscarinic Antagonists chemistry   pharmacology   MeSH
• CHO Cells MeSH
• Cricetinae MeSH
• N-Methylscopolamine metabolism   MeSH
• Piperidines pharmacology   MeSH
• Radioligand Assay MeSH
• Strychnine analogs & derivatives   pharmacology   MeSH
• Tritium MeSH
• Animals MeSH
• Check Tag
• Cricetinae MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 4-diphenylacetoxy-1,1-dimethylpiperidinium MeSH Browser
• Alcuronium MeSH
• Muscarinic Antagonists MeSH
• brucine MeSH Browser
• N-Methylscopolamine MeSH
• Piperidines MeSH
• Strychnine MeSH
• Tritium MeSH

Ligands that bind to the allosteric-binding sites on muscarinic acetylcholine receptors alter the conformation of the classical-binding sites of these receptors and either diminish or increase their affinity for muscarinic agonists and classical antagonists. It is not known whether the resulting conformational change also affects the interaction between the receptors and the G proteins. We have now found that the muscarinic receptor allosteric modulators alcuronium, gallamine, and strychnine (acting in the absence of an agonist) alter the synthesis of cAMP in Chinese hamster ovary (CHO) cells expressing the M2 or the M4 subtype of muscarinic receptors in the same direction as the agonist carbachol. In addition, most of their effects on the production of inositol phosphates in CHO cells expressing the M1 or the M3 muscarinic receptor subtypes are also similar to (although much weaker than) those of carbachol. The agonist-like effects of the allosteric modulators are not observed in CHO cells that have not been transfected with the gene for any of the subtypes of muscarinic receptors. The effects of alcuronium on the formation of cAMP and inositol phosphates are not prevented by the classical muscarinic antagonist quinuclidinyl benzilate. These observations demonstrate for the first time that the G protein-mediated functional responses of muscarinic receptors can be evoked not only from their classical, but also from their allosteric, binding sites. This represents a new mechanism of receptor activation.

• MeSH
• Muscarinic Agonists pharmacology   MeSH
• Alcuronium pharmacology   MeSH
• Allosteric Regulation MeSH
• Cyclic AMP metabolism   MeSH
• Muscarinic Antagonists pharmacology   MeSH
• Quinuclidinyl Benzilate pharmacology   MeSH
• CHO Cells MeSH
• Inositol Phosphates physiology   MeSH
• Cricetinae MeSH
• Periodicity MeSH
• GTP-Binding Proteins physiology   MeSH
• Receptors, Muscarinic physiology   MeSH
• Recombinant Proteins MeSH
• Signal Transduction MeSH
• Strychnine pharmacology   MeSH
• Transfection MeSH
• Gallamine Triethiodide pharmacology   MeSH
• Animals MeSH
• Check Tag
• Cricetinae MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Names of Substances
• Muscarinic Agonists MeSH
• Alcuronium MeSH
• Cyclic AMP MeSH
• Muscarinic Antagonists MeSH
• Quinuclidinyl Benzilate MeSH
• Inositol Phosphates MeSH
• GTP-Binding Proteins MeSH
• Receptors, Muscarinic MeSH
• Recombinant Proteins MeSH
• Strychnine MeSH
• Gallamine Triethiodide MeSH

1. By measuring the binding of N-[3H-methyl]-scopolamine ([3H]-NMS) and of unlabelled subtype-specific muscarinic antagonists, two populations of muscarinic binding sites can be distinguished in the membranes of cardiac ventricles taken from 1-day-old chicks. One of them, corresponding to approximately 80% of [3H]-NMS binding sites, has higher affinities for AF-DX116 (pKi = 6.42) and methoctramine (pKi = 7.33); the rate of [3H]NMS dissociation from these sites is fast. The other population, corresponding to approximately 20% of [3H]-NMS binding sites, has lower affinities for AF-DX116 (pKi = 5.00) and methoctramine (pKi = 6.19); the rate of [3H]-NMS dissociation from these sites is slow. Both populations have high affinities for pirenzepine, but the affinity of the former (major) population is lower (pKi = 7.99) than that of the latter (minor) population (pKi = 10.14). 2. Since it has been shown earlier that two mRNAs for muscarinic receptors are expressed in the chick heart, one of them close to the genetically defined m2 and the other to the m4 subtype, we propose that the major population of binding sites with high affinities for AF-DX116 and methoctramine and the lower affinity for pirenzepine represents the M2-like receptors, while the minor population represents the M4-like receptors. 3. It proved possible to obtain isolated samples of either population by selectively protecting the M2-like sites with AF-DX116 and the M4-like sites with pirenzepine, and by inactivating the unprotected sites with benzilylcholine mustard. The properties of the isolated populations corresponded to those derived from the analysis of [3H]-NMS binding to the original mixed population.4 Alcuronium exerted positive allosteric action on the binding of [3H]-NMS both to the M2-like and the M4-like population and severely slowed down [3H]-NMS dissociation from them; its affinity for the M2-like sites was 3-10 times higher.

• MeSH
• Alcuronium pharmacokinetics   MeSH
• Muscarinic Antagonists * MeSH
• Choline analogs & derivatives   MeSH
• Cholinergic Agents MeSH
• Diamines pharmacokinetics   MeSH
• Rats MeSH
• Chickens MeSH
• Ligands MeSH
• RNA, Messenger metabolism   MeSH
• Myocardium metabolism   MeSH
• N-Methylscopolamine MeSH
• Parasympatholytics pharmacology   MeSH
• Pirenzepine analogs & derivatives   pharmacokinetics   MeSH
• Rats, Wistar MeSH
• Radioligand Assay MeSH
• Receptors, Muscarinic metabolism   MeSH
• Scopolamine Derivatives pharmacokinetics   MeSH
• In Vitro Techniques MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Alcuronium MeSH
• Muscarinic Antagonists * MeSH
• benzilylcholine mustard MeSH Browser
• Choline MeSH
• Cholinergic Agents MeSH
• Diamines MeSH
• Ligands MeSH
• RNA, Messenger MeSH
• methoctramine MeSH Browser
• N-Methylscopolamine MeSH
• otenzepad MeSH Browser
• Parasympatholytics MeSH
• Pirenzepine MeSH
• Receptors, Muscarinic MeSH
• Scopolamine Derivatives MeSH