Data are reviewed indicating that allosteric modulators can enhance the affinities of muscarinic receptors for their antagonists and agonists, that the enhancement of the affinity for agonists is relevant functionally, and that the allosterically induced conformational change also affects the interaction between the receptors and the G proteins.

• MeSH
• agonisté muskarinových receptorů farmakologie   MeSH
• alosterická regulace MeSH
• antagonisté muskarinových receptorů farmakologie   MeSH
• konformace proteinů MeSH
• proteiny vázající GTP metabolismus   MeSH
• receptory muskarinové účinky léků   metabolismus   MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• agonisté muskarinových receptorů MeSH
• antagonisté muskarinových receptorů MeSH
• proteiny vázající GTP MeSH
• receptory muskarinové MeSH

Five subtypes of muscarinic acetylcholine receptors have been identified in mammalian tissues, but the selectivity of ligands that are active at these receptors is low. It is possible, however, that selective compounds may be developed by targeting their allosteric site(s). Important new insights into the mechanism of allosteric control of muscarinic receptors have been obtained recently in investigations of the allosteric effects of neuromuscular blockers, and competition between ligands for the allosteric binding site has now been demonstrated. It is now apparent that the binding site for most allosteric ligands is close to the binding site for acetylcholine but that it is located at a more extracellular position. Stanislav Tucek and Jan Proska discuss the pharmacological implications of ligand interaction at these two sites and the therapeutic possibilities.

• MeSH
• alosterická regulace MeSH
• lidé MeSH
• ligandy MeSH
• receptory muskarinové chemie   účinky léků   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Názvy látek
• ligandy MeSH
• receptory muskarinové MeSH

It is well known that allosteric modulators of muscarinic acetylcholine receptors can both diminish and increase the affinity of receptors for their antagonists. We investigated whether the allosteric modulators can also increase the affinity of receptors for their agonists. Twelve agonists and five allosteric modulators were tested in experiments on membranes of CHO cells that had been stably transfected with genes for the M1-M4 receptor subtypes. Allosterically induced changes in the affinities for agonists were computed from changes in the ability of a fixed concentration of each agonist to compete with [3H]N-methylscopolamine for the binding to the receptors in the absence and the presence of varying concentrations of allosteric modulators. The effects of allosteric modulators varied greatly depending on the agonists and the subtypes of receptors. The affinity for acetylcholine was augmented by (-)-eburnamonine on the M2 and M4 receptors and by brucine on the M1 and M3 receptors. Brucine also enhanced the affinities for carbachol, bethanechol, furmethide, methylfurmethide, pilocarpine, 3-(3-pentylthio-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1- methylpyridine (pentylthio-TZTP), oxotremorine-M, and McN-A-343 on the M1, M3, and M4 receptors, for pentylthio-TZTP on the M2 receptors, and for arecoline on the M3 receptors. (-)-Eburnamonine enhanced the affinities for carbachol, bethanechol, furmethide, methylfurmethide, pentylthio-TZTP, pilocarpine, oxotremorine and oxotremorine-M on the M2 receptors and for pilocarpine on the M4 receptors. Vincamine, strychnine, and alcuronium displayed fewer positive allosteric interactions with the agonists, but each allosteric modulator displayed positive cooperativity with at least one agonist on at least one muscarinic receptor subtype. The highest degrees of positive cooperativity were observed between (-)-eburnamonine and pilocarpine and (-)-eburnamonine and oxotremorine-M on the M2 receptors (25- and 7-fold increases in affinity, respectively) and between brucine and pentylthio-TZTP on the M2 and brucine and carbachol on the M1 receptors (8-fold increases in affinity). The discovery that it is possible to increase the affinity of muscarinic receptors for their agonists by allosteric modulators offers a new way to subtype-specific pharmacological enhancement of transmission at cholinergic (muscarinic) synapses.

• MeSH
• acetylcholin metabolismus   MeSH
• agonisté muskarinových receptorů metabolismus   MeSH
• alosterická regulace MeSH
• CHO buňky MeSH
• kompetitivní vazba MeSH
• křečci praví MeSH
• ligandy MeSH
• N-methylskopolamin MeSH
• receptory muskarinové klasifikace   metabolismus   MeSH
• skopolaminové deriváty metabolismus   MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Názvy látek
• acetylcholin MeSH
• agonisté muskarinových receptorů MeSH
• ligandy MeSH
• N-methylskopolamin MeSH
• receptory muskarinové MeSH
• skopolaminové deriváty MeSH

It has been shown previously that the third extracellular loop (o3) and its vicinity play a critical role in allosteric modulation at muscarinic acetylcholine receptors (mAChRs) (Ellis et al., 1993; Krejçí and Tuçek, 2001; Buller et al., 2002). In this study interaction of four chemically related substances (strychnine, its dimethoxy derivate brucine, precursor for synthesis of strychnine Wieland-Gumlich aldehyde (WGA), and precursor for synthesis of alcuronium propargyl-WGA) with orthosteric antagonist N-methylscopolamine (NMS) was investigated on the M3 subtype of mAChRs mutated at the o3 loop.

• MeSH
• alosterická regulace MeSH
• asparagin MeSH
• konformace proteinů MeSH
• mutageneze cílená MeSH
• N-methylskopolamin farmakologie   MeSH
• prolin MeSH
• receptory muskarinové chemie   účinky léků   fyziologie   MeSH
• serin MeSH
• strychnin farmakologie   MeSH
• substituce aminokyselin MeSH
• threonin MeSH
• valin MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• asparagin MeSH
• N-methylskopolamin MeSH
• prolin MeSH
• receptory muskarinové MeSH
• serin MeSH
• strychnin MeSH
• threonin MeSH
• valin MeSH

An allosteric modulator is a ligand that binds to an allosteric site on the receptor and changes receptor conformation to produce increase (positive cooperativity) or decrease (negative cooperativity) in the binding or action of an orthosteric agonist (e.g., acetylcholine). Since the identification of gallamine as the first allosteric modulator of muscarinic receptors in 1976, this unique mode of receptor modulation has been intensively studied by many groups. This review summarizes over 30 years of research on the molecular mechanisms of allosteric interactions of drugs with the receptor and for new allosteric modulators of muscarinic receptors with potential therapeutic use. Identification of positive modulators of acetylcholine binding and function that enhance neurotransmission and the discovery of highly selective allosteric modulators are mile-stones on the way to novel therapeutic agents for the treatment of schizophrenia, Alzheimer's disease and other disorders involving impaired cognitive function.

• Klíčová slova
• Alzheimer’s disease, allosteric modulation, muscarinic acetylcholine receptors, schizophrenia,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Muscarinic receptors are known to play important biological roles and are drug targets for several human diseases. In a pilot study, novel muscarinic antagonists were synthesized and used as chemical probes to obtain additional information of the muscarinic pharmacophore. The design of these ligands made use of current orthosteric and allosteric models of drug-receptor interactions together with chemical motifs known to achieve muscarinic receptor selectivity. This approach has led to the discovery of several non-competitive muscarinic ligands that strongly bind at a secondary receptor site. These compounds were found to be non-competitive antagonists that completely abolished carbachol activation in functional assays. Several of these compounds antagonized functional response to carbachol with great potency at M1 and M4 than at the rest of receptor subtypes.

• Klíčová slova
• G-protein-coupled receptor, N-methylscopolamine, Parkinson's disease, muscarinic acetylcholine receptor, positive allosteric modulator,
• MeSH
• acetylcholinesterasa metabolismus   MeSH
• alosterická regulace MeSH
• antagonisté muskarinových receptorů chemická syntéza   chemie   metabolismus   MeSH
• CHO buňky MeSH
• Cricetulus MeSH
• křečci praví MeSH
• lidé MeSH
• ligandy MeSH
• N-methylskopolamin chemická syntéza   chemie   metabolismus   MeSH
• pilotní projekty MeSH
• protein - isoformy chemie   genetika   metabolismus   MeSH
• pyridiny chemie   MeSH
• racionální návrh léčiv MeSH
• receptory muskarinové chemie   genetika   metabolismus   MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• acetylcholinesterasa MeSH
• antagonisté muskarinových receptorů MeSH
• ligandy MeSH
• N-methylskopolamin MeSH
• protein - isoformy MeSH
• pyridiny MeSH
• receptory muskarinové MeSH

We have investigated allosteric interactions of four closely related strychnine-like substances: Wieland-Gumlich aldehyde (WGA), propargyl Wieland-Gumlich aldehyde, strychnine, and brucine with N-methylscopolamine (NMS) on M(3) subtype of muscarinic receptor genetically modified in the second or the third extracellular loop to corresponding loops of M(2) subtype (M(3)o2 and M(3)o3 chimera). The M(3)o2 chimeric receptor The exhibited no change in either affinity of strychnine, brucine, and WGA or in cooperativity of brucine or WGA, whereas both parameters for propargyl-WGA changed. In contrast, there was a change in affinity of all tested modulators (except for brucine) and in their cooperativity in the M(3)o3 chimera. Directions of affinity changes in both chimeras were always toward values of the donor M(2) subtype, but changes in cooperativity were variable. Compared with the native M(3) receptor, strychnine displayed a slight increase in positive cooperativity and propargyl-WGA a robust decrease in negative cooperativity at M(3)o2 chimera. Similar changes were found in the M(3)o3 chimera. Interestingly, cooperativity of brucine and WGA at the M(3)o3 chimera changed from negative to positive. This is the first evidence of constitution of positive cooperativity of WGA by switching sequences of two parental receptors, both exhibiting negative cooperativity. Gradual replacement of individual amino acids revealed that only three residues (NVT of the o3 loop of the M(2) receptor) are involved in this effect. Data suggest that these amino acids are essential for propagation of a conformation change resulting in positive cooperativity induced by these modulators.

• MeSH
• alosterická regulace genetika   MeSH
• asparagin chemie   genetika   metabolismus   MeSH
• Cercopithecus aethiops MeSH
• COS buňky MeSH
• extracelulární prostor chemie   genetika   MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• mutageneze cílená MeSH
• receptory muskarinové chemie   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• strychnin analogy a deriváty   metabolismus   MeSH
• terciární struktura proteinů genetika   MeSH
• threonin chemie   genetika   metabolismus   MeSH
• valin chemie   genetika   metabolismus   MeSH
• vazba proteinů fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• asparagin MeSH
• receptory muskarinové MeSH
• strychnin MeSH
• threonin MeSH
• valin MeSH

To clarify the involvement of specific domains of muscarinic receptors in the action of allosteric modulators, muscarinic M(3) receptors (on which allosteric interactions are weak) were genetically modified to become more similar to M(2) receptors (on which allosteric interactions are strong) and were expressed in COS-7 cells. Affinity for allosteric modulator gallamine was enhanced 25- to 50-fold by modifications of the third external loop (o3) and the negative effect of gallamine on the affinity for classical antagonist N-[(3)H]methylscopolamine ([(3)H]NMS) was augmented. Affinity for alcuronium became 3-fold higher after the o3 loop of M(3) receptors was made identical with the o3 loop of M(2) receptors, and alcuronium acquired positive influence on the affinity for [(3)H]NMS. This is the first instance of inducing positive cooperativity on muscarinic receptors by genetic manipulation. Transferring whole o2 loop from M(2) to M(3) receptors substantially enhanced affinities for gallamine and alcuronium without augmenting their negative action on [(3)H]NMS binding. In contrast, effects of simply adding two negative charges into the o2 loop of M(3) receptors were small. Removal of Arg from o1 loop abolished the negative effect of gallamine but not of alcuronium on [(3)H]NMS binding at equilibrium. Data point to an important role of o3 loop in the mechanism of the positive and negative cooperativity between [(3)H]NMS and alcuronium and gallamine, respectively, and in the binding of both modulators to M(2) receptors and reveal independence between mutation-induced changes in the affinity for a modulator and in the magnitude and direction of the allosteric effect of the modulator.

• MeSH
• alkuronium farmakologie   MeSH
• alosterická regulace MeSH
• arginin genetika   MeSH
• glycin genetika   MeSH
• konformace proteinů MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• mutace MeSH
• N-methylskopolamin farmakologie   MeSH
• nikotinoví antagonisté farmakologie   MeSH
• parasympatolytika farmakologie   MeSH
• radioligandová zkouška MeSH
• receptor muskarinový M3 MeSH
• receptory muskarinové chemie   účinky léků   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• triethojodid gallaminia farmakologie   MeSH
• tritium MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• alkuronium MeSH
• arginin MeSH
• glycin MeSH
• N-methylskopolamin MeSH
• nikotinoví antagonisté MeSH
• parasympatolytika MeSH
• receptor muskarinový M3 MeSH
• receptory muskarinové MeSH
• triethojodid gallaminia MeSH
• tritium 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
• alkuronium farmakologie   MeSH
• alosterická regulace MeSH
• antagonisté muskarinových receptorů chemie   farmakologie   MeSH
• CHO buňky MeSH
• křečci praví MeSH
• N-methylskopolamin metabolismus   MeSH
• piperidiny farmakologie   MeSH
• radioligandová zkouška MeSH
• strychnin analogy a deriváty   farmakologie   MeSH
• tritium MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 4-diphenylacetoxy-1,1-dimethylpiperidinium MeSH Prohlížeč
• alkuronium MeSH
• antagonisté muskarinových receptorů MeSH
• brucine MeSH Prohlížeč
• N-methylskopolamin MeSH
• piperidiny MeSH
• strychnin MeSH
• tritium MeSH

Muscarinic acetylcholine receptors are metabotropic G-protein coupled receptors. Muscarinic receptors in the cardiovascular system play a central role in its regulation. Particularly M2 receptors slow down the heart rate by reducing the impulse conductivity through the atrioventricular node. In general, activation of muscarinic receptors has sedative effects on the cardiovascular system, including vasodilation, negative chronotropic and inotropic effects on the heart, and cardioprotective effects, including antifibrillatory effects. First, we review the signaling of individual subtypes of muscarinic receptors and their involvement in the physiology and pathology of the cardiovascular system. Then we review age and disease-related changes in signaling via muscarinic receptors in the cardiovascular system. Finally, we review molecular mechanisms involved in cardioprotection mediated by muscarinic receptors leading to negative chronotropic and inotropic and antifibrillatory effects on heart and vasodilation, like activation of acetylcholine-gated inward-rectifier K+-currents and endothelium-dependent and -independent vasodilation. We relate this knowledge with well-established cardioprotective treatments by vagal stimulation and muscarinic agonists. It is well known that estrogen exerts cardioprotective effects against atherosclerosis and ischemia-reperfusion injury. Recently, some sex hormones and neurosteroids have been shown to allosterically modulate muscarinic receptors. Thus, we outline possible treatment by steroid-based positive allosteric modulators of acetylcholine as a novel pharmacotherapeutic tactic. Keywords: Muscarinic receptors, Muscarinic agonists, Allosteric modulation, Cardiovascular system, Cardioprotection, Steroids.

• MeSH
• agonisté muskarinových receptorů farmakologie   MeSH
• kardiotonika farmakologie   terapeutické užití   MeSH
• lidé MeSH
• receptory muskarinové * metabolismus   MeSH
• vazodilatace fyziologie   účinky léků   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• agonisté muskarinových receptorů MeSH
• kardiotonika MeSH
• receptory muskarinové * MeSH