There are five muscarinic receptor subtypes expressed in the human heart. The main subtype is the M2-muscarinic receptor. We hypothesized that overexpression of the M2-muscarinic receptor should augment any contractile effects that are M2-muscarinic receptor-mediated. Therefore, we generated a transgenic mouse with overexpression of the human M2-muscarinic receptor under the control of the heart-specific α-myosin heavy chain promoter (M2-TG). We performed contraction experiments with electrically stimulated (1 Hz) left atrial preparations (LA) and spontaneously beating right atrial preparations (RA) from adult M2-TG or from adult wild-type littermate mice (WT). We confirmed the expression of the human M2-muscarinic receptor in the mouse heart by reverse transcription polymerase chain reaction (RT-PCR) and radioligand binding experiments at cardiac membranes and tissue sections. We did not detect differences in hematoxylin/eosin staining or Masson/Goldner staining between M2-TG and WT. We noticed that carbachol (10 nM-10 µM cumulatively applied) alone or in the presence of 1 µM isoprenaline reduced the force of contraction (FOC) to a similar extent in LA from M2-TG and WT. The beating rate in RA was similarly decreased by carbachol alone or by carbachol in the presence of 1 µM isoprenaline in M2-TG and WT. Overall, the number of RA that displayed absolute arrhythmias was higher in atria from M2-TG compared to atria from WT. No arrhythmias were noted in LA from M2-TG or WT. Stimulation of human M2-muscarinic receptors induced absolute atrial arrhythmias more often in RA from M2-TG than in RA from WT. Overexpressed M2-muscarinic receptors were silent to the force and beating rate.

• Klíčová slova
• Arrhythmia, Human M2-muscarinic receptors,
• Publikační typ
• časopisecké články 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

Muscarinic receptors (mAChRs) are typical members of the G protein-coupled receptor (GPCR) family and exist in five subtypes from M1 to M5. Muscarinic receptor subtypes do not sufficiently differ in affinity to orthosteric antagonists or agonists; therefore, the analysis of receptor subtypes is complicated, and misinterpretations can occur. Usually, when researchers mainly specialized in CNS and peripheral functions aim to study mAChR involvement in behavior, learning, spinal locomotor networks, biological rhythms, cardiovascular physiology, bronchoconstriction, gastrointestinal tract functions, schizophrenia, and Parkinson's disease, they use orthosteric ligands and they do not use allosteric ligands. Moreover, they usually rely on manufacturers' claims that could be misleading. This review aimed to call the attention of researchers not deeply focused on mAChR pharmacology to this fact. Importantly, limited selective binding is not only a property of mAChRs but is a general attribute of most neurotransmitter receptors. In this review, we want to give an overview of the most common off-targets for established mAChR ligands. In this context, an important point is a mention the tremendous knowledge gap on off-targets for novel compounds compared to very well-established ligands. Therefore, we will summarize reported affinities and give an outline of strategies to investigate the subtype's function, thereby avoiding ambiguous results. Despite that, the multitargeting nature of drugs acting also on mAChR could be an advantage when treating such diseases as schizophrenia. Antipsychotics are a perfect example of a multitargeting advantage in treatment. A promising strategy is the use of allosteric ligands, although some of these ligands have also been shown to exhibit limited selectivity. Another new direction in the development of muscarinic selective ligands is functionally selective and biased agonists. The possible selective ligands, usually allosteric, will also be listed. To overcome the limited selectivity of orthosteric ligands, the recommended process is to carefully examine the presence of respective subtypes in specific tissues via knockout studies, carefully apply "specific" agonists/antagonists at appropriate concentrations and then calculate the probability of a specific subtype involvement in specific functions. This could help interested researchers aiming to study the central nervous system functions mediated by the muscarinic receptor.

• Klíčová slova
• allosteric, multitarget, muscarinic agonist, muscarinic antagonist, muscarinic receptors, orthosteric,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

UNLABELLED: Autoradiography helps to determine the distribution and density of muscarinic receptor (MR) binding sites in the brain. However, it relies on the selectivity of radioligands toward their target. 3H-Pirenzepine is commonly believed to label predominantly M1MR, 3H-AFDX-384 is considered as M2MR selective ligand. Here we performed series of autoradiographies with 3H-AFDX-384 (2 nM), and 3H-pirenzepine (5 nM) in WT, M1KO, M2KO, and M4KO mice to address the ligand selectivity. Labeling with 3H-pirenzepine using M1KO, M2KO, and M4KO brain sections showed the high selectivity toward M1MR. Selectivity of 3H-AFDX-384 toward M2MR varies among brain regions and depends on individual MR subtype proportion. All binding sites in the medulla oblongata and pons, correspond to M2MR. In caudate putamen, nucleus accumbens and olfactory tubercle, 77.7, 74.2, and 74.6% of 3H-AFDX-384 binding sites, respectively, are represented by M4MR and M2MR constitute only a minor portion. In cortex and hippocampus, 3H-AFDX-384 labels almost similar amounts of M2MR and M4MR alongside significant amounts of non-M2/non-M4MR. In cortex, the proportion of 3H-AFDX-384 binding sites attributable to M2MR can be increased by blocking M4MR with MT3 toxin without affecting non-M4MR. PD102807, which is considered as a highly selective M4MR antagonist failed to improve the discrimination of M2MR. Autoradiography with 3H-QNB showed genotype specific loss of binding sites. IN CONCLUSION: while 3H-pirenzepine showed the high selectivity toward M1MR, 3H-AFDX-384 binding sites represent different populations of MR subtypes in a brain-region-specific manner. This finding has to be taken into account when interpreting the binding data.

• Klíčová slova
• 3H-AFDX-384, 3H-QNB, 3H-pirenzepine, M1 muscarinic receptor, M2 muscarinic receptor, M4 muscarinic receptor, autoradiography,
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Pharmaceuticals with targets in the cholinergic transmission have been used for decades and are still fundamental treatments in many diseases and conditions today. Both the transmission and the effects of the somatomotoric and the parasympathetic nervous systems may be targeted by such treatments. Irrespective of the knowledge that the effects of neuronal signalling in the nervous systems may include a number of different receptor subtypes of both the nicotinic and the muscarinic receptors, this complexity is generally overlooked when assessing the mechanisms of action of pharmaceuticals. METHODS: We have search of bibliographic databases for peer-reviewed research literature focused on the cholinergic system. Also, we have taken advantage of our expertise in this field to deduce the conclusions of this study. RESULTS: Presently, the life cycle of acetylcholine, muscarinic receptors and their effects are reviewed in the major organ systems of the body. Neuronal and non-neuronal sources of acetylcholine are elucidated. Examples of pharmaceuticals, in particular cholinesterase inhibitors, affecting these systems are discussed. The review focuses on salivary glands, the respiratory tract and the lower urinary tract, since the complexity of the interplay of different muscarinic receptor subtypes is of significance for physiological, pharmacological and toxicological effects in these organs. CONCLUSION: Most pharmaceuticals targeting muscarinic receptors are employed at such large doses that no selectivity can be expected. However, some differences in the adverse effect profile of muscarinic antagonists may still be explained by the variation of expression of muscarinic receptor subtypes in different organs. However, a complex pattern of interactions between muscarinic receptor subtypes occurs and needs to be considered when searching for selective pharmaceuticals. In the development of new entities for the treatment of for instance pesticide intoxication, the muscarinic receptor selectivity needs to be considered. Reactivators generally have a muscarinic M2 receptor acting profile. Such a blockade may engrave the situation since it may enlarge the effect of the muscarinic M3 receptor effect. This may explain why respiratory arrest is the major cause for deaths by esterase blocking.

• Klíčová slova
• Acetylcholine, acetylcholinesterase, muscarinic receptor subtypes, pharmacotherapy,
• MeSH
• cholinesterasové inhibitory farmakologie   MeSH
• interakce mezi receptory a ligandy účinky léků   MeSH
• lidé MeSH
• receptory muskarinové účinky léků   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• cholinesterasové inhibitory MeSH
• receptory muskarinové MeSH

Muscarinic receptors (MR) are main cardioinhibitory receptors. We investigated the changes in gene expression, receptor number, echocardiography, muscarinic/adrenergic agonist/antagonist changes in heart rate (HR) and HR biorhythm in M(2) KO mice (mice lacking the main cardioinhibitory receptors) in the left ventricle (LV) and right ventricle (RV). We hypothesize that the disruption of M(2) MR, key players in parasympathetic bradycardia, would change the number of receptors with antagonistic effects on the heart (β(1)- and β(2)-adrenoceptors, BAR), while the function of the heart would be changed only marginally. We have found changes in LV, but not in RV: decrease in M(3) MR, β(1)- and β(2)-adrenoceptor gene expressions that were accompanied by a decrease in MR and BAR receptor binding. No changes were found both in LV systolic and diastolic function as assessed by echocardiography (e.g., similar LV end-systolic and end-diastolic diameter, fractional shortening, mitral flow characteristics, and maximal velocity in LV outflow tract). We have found only marginal changes in specific HR biorhythm parameters. The effects of isoprenaline and propranolol on HR were similar in WT and KO (but with lesser extent). Atropine was not able to increase HR in KO animals. Carbachol decreased the HR in WT but increased HR in KO, suggesting the presence of cardiostimulatory MR. Therefore, we can conclude that although the main cardioinhibitory receptors are not present in the heart, the function is not much affected. As possible mechanisms of almost normal cardiac function, the decreases of both β(1)- and β(2)-adrenoceptor gene expression and receptor binding should be considered.

• MeSH
• atropin farmakologie   MeSH
• beta-1-adrenergní receptory metabolismus   MeSH
• beta-2-adrenergní receptory metabolismus   MeSH
• bradykardie patofyziologie   MeSH
• funkce levé komory srdeční fyziologie   MeSH
• isoprenalin farmakologie   MeSH
• karbachol farmakologie   MeSH
• myši knockoutované MeSH
• myši MeSH
• propranolol farmakologie   MeSH
• receptor muskarinový M2 genetika   MeSH
• regulace genové exprese * MeSH
• srdeční frekvence fyziologie   MeSH
• srdeční komory metabolismus   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• atropin MeSH
• beta-1-adrenergní receptory MeSH
• beta-2-adrenergní receptory MeSH
• isoprenalin MeSH
• karbachol MeSH
• propranolol MeSH
• receptor muskarinový M2 MeSH