Methamphetamine (MA) is worldwide known drug with high potential for addiction that causes dopamine, noradrenaline and serotonin release. MA is also able to increase acetylcholine levels in adult rodents. The aim of this study was to map changes in D1-like dopamine receptors (DR), M1 and M2 muscarinic receptors (MR), and the total number of MR (M1-M5 MR) in the CNS of rats exposed to MA prenatally and in adulthood. Rat mothers were exposed to MA (5mg/kg s.c.) or saline during the entire gestation period and their male offspring were administered in adulthood with single MA (1mg/kg) or saline injection. Thus, the animals were divided into 4 groups: prenatally MA-exposed rats treated with saline (MA/S) or MA (MA/MA) in adulthood and prenatally saline-exposed rats treated with saline (S/S) or MA (S/MA) in adulthood. One hour after the acute treatment animals were sacrificed and their brains were removed. The numbers of M1, M2, total MR, and D1-DR were measured by autoradiography. The main effect was detected in the hippocampus with the most affected M1 MR. D1-DR were decreased in motor cortex and substantia nigra. M1MR were decreased in caudate-putamen, dorsal hippocampus, CA1, CA3 and dentate gyrus (DG). M2MR were decreased in DG only. Total number of MR was moreover decreased in dorsal hippocampus, CA1, CA3 and DG. Our results have shown different patterns of changes in DR and MR, suggesting a pilot role of M1 MR in the CNS changes induced by prenatal and adult MA exposure.

• Klíčová slova
• Autoradiography, Dopamine receptors, Methamphetamine, Muscarinic receptors,
• MeSH
• autoradiografie MeSH
• methamfetamin toxicita   MeSH
• mozek účinky léků   růst a vývoj   metabolismus   MeSH
• náhodné rozdělení MeSH
• potkani Wistar MeSH
• receptor muskarinový M1 metabolismus   MeSH
• receptor muskarinový M2 metabolismus   MeSH
• receptory dopaminu D1 metabolismus   MeSH
• těhotenství MeSH
• zpožděný efekt prenatální expozice MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• methamfetamin MeSH
• receptor muskarinový M1 MeSH
• receptor muskarinový M2 MeSH
• receptory dopaminu D1 MeSH

We investigated the role of beta3-adrenoceptors (AR) in cold stress (1 or 7 days in cold) in animals lacking main cardioinhibitive receptors-M2 muscarinic receptors (M(2)KO). There was no change in receptor number in the right ventricles. In the left ventricles, there was decrease in binding to all cardiostimulative receptors (beta1-, and beta2-AR) and increase in cardiodepressive receptors (beta3-AR) in unstressed KO in comparison to WT. The cold stress in WT animals resulted in decrease in binding to beta1- and beta2-AR (to 37%/35% after 1 day in cold and to 27%/28% after 7 days in cold) while beta3-AR were increased (to 216% of control) when 7 days cold was applied. MR were reduced to 46% and 58%, respectively. Gene expression of M2 MR in WT was not changed due to stress, while M3 was changed. The reaction of beta1- and beta2-AR (binding) to cold was similar in KO and WT animals, and beta3-AR in stressed KO animals did not change. Adenylyl cyclase activity was affected by beta3-agonist CL316243 in cold stressed WT animals but CL316243 had almost no effects on adenylyl cyclase activity in stressed KO. Nitric oxide activity (NOS) was not affected by BRL37344 (beta3-agonist) both in WT and KO animals. Similarly, the stress had no effects on NOS activity in WT animals and in KO animals. We conclude that the function of M2 MR is substituted by beta3-AR and that these effects are mediated via adenylyl cyclase rather than NOS.

• MeSH
• adenylátcyklasy metabolismus   MeSH
• beta-3-adrenergní receptory metabolismus   MeSH
• fyziologická adaptace * genetika   MeSH
• fyziologický stres * genetika   MeSH
• katecholaminy biosyntéza   MeSH
• myši MeSH
• nízká teplota * MeSH
• receptor muskarinový M2 nedostatek   genetika   metabolismus   MeSH
• regulace genové exprese MeSH
• srdce patofyziologie   MeSH
• srdeční komory enzymologie   patologie   patofyziologie   MeSH
• synthasa oxidu dusnatého metabolismus   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenylátcyklasy MeSH
• beta-3-adrenergní receptory MeSH
• katecholaminy MeSH
• receptor muskarinový M2 MeSH
• synthasa oxidu dusnatého MeSH

BACKGROUND AND PURPOSE: Conventional determination of agonist efficacy at G-protein coupled receptors is measured by stimulation of guanosine-5'-γ-thiotriphosphate (GTPγS) binding. We analysed the role of guanosine diphosphate (GDP) in the process of activation of the M₂ muscarinic acetylcholine receptor and provide evidence that negative cooperativity between agonist and GDP binding is an alternative measure of agonist efficacy. EXPERIMENTAL APPROACH: Filtration and scintillation proximity assays measured equilibrium binding as well as binding kinetics of [³⁵S]GTPγS and [³H]GDP to a mixture of G-proteins as well as individual classes of G-proteins upon binding of structurally different agonists to the M₂ muscarinic acetylcholine receptor. KEY RESULTS: Agonists displayed biphasic competition curves with the antagonist [³H]-N-methylscopolamine. GTPγS (1 µM) changed the competition curves to monophasic with low affinity and 50 µM GDP produced a similar effect. Depletion of membrane-bound GDP increased the proportion of agonist high-affinity sites. Carbachol accelerated the dissociation of [³H]GDP from membranes. The inverse agonist N-methylscopolamine slowed GDP dissociation and GTPγS binding without changing affinity for GDP. Carbachol affected both GDP association with and dissociation from G(i/o) G-proteins but only its dissociation from G(s/olf) G-proteins. CONCLUSIONS AND IMPLICATIONS: These findings suggest the existence of a low-affinity agonist-receptor conformation complexed with GDP-liganded G-protein. Also the negative cooperativity between GDP and agonist binding at the receptor/G-protein complex determines agonist efficacy. GDP binding reveals differences in action of agonists versus inverse agonists as well as differences in activation of G(i/o) versus G(s/olf) G-proteins that are not identified by conventional GTPγS binding.

• MeSH
• agonisté muskarinových receptorů metabolismus   MeSH
• alosterická regulace MeSH
• antagonisté muskarinových receptorů metabolismus   MeSH
• CHO buňky MeSH
• Cricetulus MeSH
• guanosin 5'-O-(3-thiotrifosfát) metabolismus   MeSH
• guanosindifosfát metabolismus   MeSH
• kinetika MeSH
• křečci praví MeSH
• lidé MeSH
• N-methylskopolamin metabolismus   MeSH
• proteiny vázající GTP klasifikace   metabolismus   MeSH
• radioligandová zkouška MeSH
• receptor muskarinový M2 genetika   metabolismus   MeSH
• rekombinantní proteiny genetika   metabolismus   MeSH
• transfekce MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• agonisté muskarinových receptorů MeSH
• antagonisté muskarinových receptorů MeSH
• guanosin 5'-O-(3-thiotrifosfát) MeSH
• guanosindifosfát MeSH
• N-methylskopolamin MeSH
• proteiny vázající GTP MeSH
• receptor muskarinový M2 MeSH
• rekombinantní proteiny MeSH

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
• acetylcholin analogy a deriváty   MeSH
• antagonisté muskarinových receptorů farmakologie   MeSH
• beta-cyklodextriny farmakologie   MeSH
• buněčná membrána účinky léků   metabolismus   MeSH
• CHO buňky MeSH
• cholesterol metabolismus   MeSH
• Cricetulus MeSH
• karbachol analogy a deriváty   metabolismus   farmakologie   MeSH
• křečci praví MeSH
• lidé MeSH
• N-methylskopolamin metabolismus   farmakologie   MeSH
• proteiny vázající GTP metabolismus   MeSH
• receptor muskarinový M2 metabolismus   MeSH
• systémy druhého messengeru * účinky léků   MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• acetylcholin MeSH
• antagonisté muskarinových receptorů MeSH
• beta-cyklodextriny MeSH
• cholesterol MeSH
• karbachol MeSH
• methyl-beta-cyclodextrin MeSH Prohlížeč
• N-methylskopolamin MeSH
• proteiny vázající GTP MeSH
• receptor muskarinový M2 MeSH

Mammal heart tissue has long been assumed to be the exclusive domain of the M(2) subtype of muscarinic receptor, but data supporting the presence of other subtypes also exist. We have tested the hypothesis that muscarinic receptors other than the M(2) subtype are present in the heart as minor populations. We used several approaches: a set of competition binding experiments with pirenzepine, AFDX-116, 4-DAMP, PD 102807, p-F-HHSiD, AQ-RA 741, DAU 5884, methoctramine and tripinamide, blockage of M(1) muscarinic receptors using MT7 toxin, subtype-specific immunoprecipitation experiments and determination of phospholipase C activity. We also attempted to block M(1)-M(4) receptors using co-treatment with MT7 and AQ-RA 741. Our results show that only the M(2) subtype is present in the atria. In the ventricles, however, we were able to determine that 20% (on average) of the muscarinic receptors were subtypes other than M(2), with the majority of these belonging to the M(1) subtype. We were also able to detect a marginal fraction (6 +/- 2%) of receptors that, based on other findings, belong mainly to the M(5) muscarinic receptors. Co-treatment with MT7 and AQ-RA 741 was not a suitable tool for blocking of M(1)-M(4) receptors and can not therefore be used as a method for M(5) muscarinic receptor detection in substitution to crude venom. These results provide further evidence of the expression of the M(1) muscarinic receptor subtype in the rat heart and also show that the heart contains at least one other, albeit minor, muscarinic receptor population, which most likely belongs to the M(5) muscarinic receptors but not to that of the M(3) receptors.

• MeSH
• exprese genu MeSH
• fosfolipasy typu C metabolismus   MeSH
• kompetitivní vazba MeSH
• krysa rodu Rattus MeSH
• potkani Wistar MeSH
• receptor muskarinový M1 účinky léků   metabolismus   MeSH
• receptor muskarinový M2 účinky léků   metabolismus   MeSH
• receptor muskarinový M3 účinky léků   metabolismus   MeSH
• receptor muskarinový M5 účinky léků   metabolismus   MeSH
• receptory muskarinové účinky léků   metabolismus   MeSH
• srdeční komory účinky léků   metabolismus   MeSH
• srdeční síně účinky léků   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfolipasy typu C MeSH
• receptor muskarinový M1 MeSH
• receptor muskarinový M2 MeSH
• receptor muskarinový M3 MeSH
• receptor muskarinový M5 MeSH
• receptory muskarinové MeSH

Xanomeline is a functionally selective M(1)/M(4) muscarinic acetylcholine receptor agonist that nevertheless binds with high affinity to all five subtypes of muscarinic receptors. A novel mode of interaction of this ligand with the muscarinic M(1) receptors characterized by persistent binding and receptor activation after extensive washout has been shown previously. In the present study, using human M(1) and M(2) receptors expressed in Chinese hamster ovary cells and [(3)H]N-methylscopolamine as a tracer, we show that persistent binding of xanomeline also occurs at the M(2) receptor with similar affinity as at the M(1) receptor (K(I) = 294 and 296 nM, respectively). However, kinetics of formation of xanomeline wash-resistant binding to M(2) receptors was markedly slower than to M(1) receptors. Xanomeline was a potent fast-acting full agonist in stimulating guanosine 5'-O-(3-[(35)S]thio)triphosphate binding at M(1) receptors, whereas at M(2) receptors it behaved as a potent partial agonist (40% of carbachol maximal response) only upon preincubation for 1 h. Development of xanomeline agonistic effects at the M(2) receptor was slower than its ability to attenuate carbachol responses. We also demonstrate that xanomeline discriminates better between G protein subtypes at M(1) than at M(2) receptors. Our data support the notion that xanomeline interacts with multiple sites on the muscarinic receptor, resulting in divergent conformations that exhibit differential effects on ligand binding and receptor activation. These conformations are both time- and concentration-dependent and vary between the M(1) and the M(2) receptor.

• MeSH
• agonisté muskarinových receptorů farmakologie   MeSH
• CHO buňky MeSH
• guanosin 5'-O-(3-thiotrifosfát) metabolismus   MeSH
• karbachol farmakologie   MeSH
• kinetika MeSH
• křečci praví MeSH
• N-methylskopolamin metabolismus   MeSH
• proteiny vázající GTP fyziologie   MeSH
• pyridiny metabolismus   farmakologie   MeSH
• receptor muskarinový M1 účinky léků   metabolismus   MeSH
• receptor muskarinový M2 účinky léků   metabolismus   MeSH
• thiadiazoly metabolismus   farmakologie   MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• agonisté muskarinových receptorů MeSH
• guanosin 5'-O-(3-thiotrifosfát) MeSH
• karbachol MeSH
• N-methylskopolamin MeSH
• proteiny vázající GTP MeSH
• pyridiny MeSH
• receptor muskarinový M1 MeSH
• receptor muskarinový M2 MeSH
• thiadiazoly MeSH
• xanomeline MeSH Prohlížeč

Despite the wealth of information on the functional and pharmacological properties of the M2 muscarinic receptor, we know relatively little of structure and regulation of the M2 receptor gene. Here, we describe the organisation of the human M2 gene and its promoters. Four exons are present in the 5' untranslated region of the human M2 mRNA distributed over 146 kb on chromosome 7 which produce eight different splice variants in the IMR-32 neuroblastoma cell line. The unexpectedly large size of this gene indicates that transcription initiates much further upstream of the coding region than earlier studies had indicated. We present evidence that there are three distinct human M2 promoters. Analysis of endogenous transcripts revealed that promoter 2 is preferentially used in neuroblastoma cells, whereas promoter 1 in cardiac cells. All promoters are highly conserved across human, mouse, rat and pig. They contain multiple start sites and none possess a TATA-box. In addition, we describe another M2 promoter that is specific for rat. We show that GATA-4 transcription factor binds to two sites within the regulatory regions of the M2 gene using reporter gene assays, electromobility shift assays and mutational analysis.

• MeSH
• buněčné linie MeSH
• chromatin metabolismus   MeSH
• DNA vazebné proteiny metabolismus   MeSH
• exony MeSH
• imunoprecipitace metody   MeSH
• klonování DNA metody   MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• luciferasy metabolismus   MeSH
• messenger RNA genetika   MeSH
• molekulární sekvence - údaje MeSH
• mutageneze fyziologie   MeSH
• myoblasty MeSH
• myši MeSH
• neuroblastom MeSH
• orgánová specificita MeSH
• polymerázová řetězová reakce s reverzní transkripcí metody   MeSH
• promotorové oblasti (genetika) fyziologie   MeSH
• protein vázající TATA box metabolismus   MeSH
• receptor muskarinový M2 genetika   metabolismus   MeSH
• regulace genové exprese fyziologie   MeSH
• reportérové geny fyziologie   MeSH
• retardační test metody   MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení metody   MeSH
• transkripční faktor GATA4 MeSH
• transkripční faktory metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• chromatin MeSH
• DNA vazebné proteiny MeSH
• luciferasy MeSH
• messenger RNA MeSH
• protein vázající TATA box MeSH
• receptor muskarinový M2 MeSH
• transkripční faktor GATA4 MeSH
• transkripční faktory MeSH

Muscarinic acetylcholine receptors mediate transmission of an extracellular signal represented by released acetylcholine to neuronal or effector cells. There are five subtypes of closely homologous muscarinic receptors which are coupled by means of heterotrimeric G-proteins to a variety of signaling pathways resulting in a multitude of target cell effects. Endogenous agonist acetylcholine does not discriminate among individual subtypes and due to the close homology of the orthosteric binding site the same holds true for most of exogenous agonists. In addition to the classical binding site muscarinic receptors have one or more allosteric binding sites at extracellular domains. Binding of allosteric modulators induces conformational changes in the receptor that result in subtype-specific changes in orthosteric binding site affinity for both muscarinic agonists and antagonists. This overview summarizes our recent experimental effort in investigating certain aspects of M2 muscarinic receptor functioning concerning i) the molecular determinants that contribute to the binding of allosteric modulators, ii) G-protein coupling specificity and subsequent cellular responses and iii) possible functional assays that exploit the unique properties of allosteric modulators for characterization of muscarinic receptor subtypes in intact tissue. A detailed knowledge of allosteric properties of muscarinic receptors is required to permit drug design that will modulate signal transmission strength of specific muscarinic receptor subtypes. Furthermore, allosteric modulation of signal transmission strength is determined by cooperativity rather than concentration of allosteric modulator and thus reduces the danger of overdose.

• MeSH
• acetylcholin antagonisté a inhibitory   metabolismus   MeSH
• alosterická regulace * MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• proteiny vázající GTP metabolismus   MeSH
• receptor muskarinový M2 metabolismus   MeSH
• receptor muskarinový M3 metabolismus   MeSH
• receptor muskarinový M4 metabolismus   MeSH
• sekvence aminokyselin MeSH
• signální transdukce MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• acetylcholin MeSH
• proteiny vázající GTP MeSH
• receptor muskarinový M2 MeSH
• receptor muskarinový M3 MeSH
• receptor muskarinový M4 MeSH