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Sample records for muscarinic receptor signal

  1. Direct Interaction of GABAB Receptors with M2 Muscarinic Receptors Enhances Muscarinic Signaling

    PubMed Central

    Boyer, Stephanie B.; Clancy, Sinead M.; Terunuma, Miho; Revilla-Sanchez, Raquel; Thomas, Steven M.; Moss, Stephen J.; Slesinger, Paul A.

    2009-01-01

    Down-regulation of G protein coupled receptors (GPCR) provides an important mechanism for reducing neurotransmitter signaling during sustained stimulation. Chronic stimulation of M2 muscarinic receptors (M2R) causes internalization of M2R and G protein-activated inwardly rectifying potassium (GIRK) channels in neuronal PC12 cells, resulting in loss of function. Here, we show that co-expression of GABAB R2 receptors (GBR2) rescues both surface expression and function of M2R, including M2R-induced activation of GIRKs and inhibition of cAMP production. GBR2 showed significant association with M2R at the plasma membrane but not other GPCRs (M1R, μOR), as detected by FRET measured with TIRF microscopy. Unique regions of the proximal C-terminal domains of GBR2 and M2R mediate specific binding between M2R and GBR2. In the brain, GBR2, but not GBR1, biochemically coprecipitates with M2R and overlaps with M2R expression in cortical neurons. This novel heteromeric association between M2R and GBR2 provides a possible mechanism for altering muscarinic signaling in the brain and represents a previously unrecognized role for GBR2. PMID:20016095

  2. Sphingosine kinase-mediated calcium signaling by muscarinic acetylcholine receptors.

    PubMed

    van Koppen, C J; Meyer zu Heringdorf, D; Alemany, R; Jakobs, K H

    2001-04-27

    Based on the finding that G protein-coupled receptors (GPCRs) can induce Ca2+ mobilization, apparently independent of the phospholipase C (PLC)/inositol-1,4,5-trisphosphate (IP3) pathway, we investigated whether sphingosine kinase, which generates sphingosine-1-phosphate (SPP), is involved in calcium signaling by mAChR and other GPCRs. Inhibition of sphingosine kinase by DL-threo-dihydrosphingosine and N,/N-dimethylsphingosine markedly inhibited [Ca2+]i increases elicited by M2 and M3 mAChRs in HEK-293 cells without affecting PLC activation. Activation of M2 and M3 mAChR rapidly and transiently stimulated production of SPP. Furthermore, microinjection of SPP into HEK-293 cells induced rapid and transient Ca2+ mobilization. Pretreatment of HEK-293 cells with the calcium chelator BAPTA/AM fully blocked mAChR-induced SPP production. On the other hand, incubation of HEK-293 cells with calcium ionophores activated SPP production. Similar findings were obtained for formyl peptide and P2Y2 purinergic receptors in HL-60 cells. On the basis of these studies we propose, that following initial IP3 production by receptor-mediated PLC activation, a local discrete increase in [Ca2+]i induces sphingosine kinase stimulation, which ultimately leads to full calcium mobilization. Thus, sphingosine kinase activation most likely represents an amplification system for calcium signaling by mAChRs and other GPCRs.

  3. Honokiol blocks store operated calcium entry in CHO cells expressing the M3 muscarinic receptor: honokiol and muscarinic signaling

    PubMed Central

    2013-01-01

    Background Honokiol, a cell-permeable phenolic compound derived from the bark of magnolia trees and present in Asian herbal teas, has a unique array of pharmacological actions, including the inhibition of multiple autonomic responses. We determined the effects of honokiol on calcium signaling underlying transmission mediated by human M3 muscarinic receptors expressed in Chinese hamster ovary (CHO) cells. Receptor binding was determined in radiolabelled ligand binding assays; changes in intracellular calcium concentrations were determined using a fura-2 ratiometric imaging protocol; cytotoxicity was determined using a dye reduction assay. Results Honokiol had a potent (EC50 ≈ 5 μmol/l) inhibitory effect on store operated calcium entry (SOCE) that was induced by activation of the M3 receptors. This effect was specific, rapid and partially reversible, and was seen at concentrations not associated with cytotoxicity, inhibition of IP3 receptor-mediated calcium release, depletion of ER calcium stores, or disruption of M3 receptor binding. Conclusions It is likely that an inhibition of SOCE contributes to honokiol disruption of parasympathetic motor functions, as well as many of its beneficial pharmacological properties. PMID:23432810

  4. Muscarinic Receptor Antagonists.

    PubMed

    Matera, Maria Gabriella; Cazzola, Mario

    2017-01-01

    Parasympathetic activity is increased in patients with chronic obstructive pulmonary disease (COPD) and asthma and appears to be the major reversible component of airway obstruction. Therefore, treatment with muscarinic receptor antagonists is an effective bronchodilator therapy in COPD and also in asthmatic patients. In recent years, the accumulating evidence that the cholinergic system controls not only contraction by airway smooth muscle but also the functions of inflammatory cells and airway epithelial cells has suggested that muscarinic receptor antagonists could exert other effects that may be of clinical relevance when we must treat a patient suffering from COPD or asthma. There are currently six muscarinic receptor antagonists licenced for use in the treatment of COPD, the short-acting muscarinic receptor antagonists (SAMAs) ipratropium bromide and oxitropium bromide and the long-acting muscarinic receptor antagonists (LAMAs) aclidinium bromide, tiotropium bromide, glycopyrronium bromide and umeclidinium bromide. Concerns have been raised about possible associations of muscarinic receptor antagonists with cardiovascular safety, but the most advanced compounds seem to have an improved safety profile. Further beneficial effects of SAMAs and LAMAs are seen when added to existing treatments, including LABAs, inhaled corticosteroids and phosphodiesterase 4 inhibitors. The importance of tiotropium bromide in the maintenance treatment of COPD, and likely in asthma, has spurred further research to identify new LAMAs. There are a number of molecules that are being identified, but only few have reached the clinical development.

  5. Pharmacological discrimination between muscarinic receptor signal transduction cascades with bethanechol chloride

    PubMed Central

    Liu, Liwang; Rittenhouse, Ann R

    2003-01-01

    Muscarinic agonist specificity is limited, making it difficult to match receptor subtypes with signal transduction cascades that mediate ion channel modulation. We have characterized the inhibitory effects of two muscarinic agonists, oxotremorine-M (Oxo-M) and bethanechol chloride (BeCh), on Ca2+ currents in neonatal rat superior cervical ganglion neurons. Oxo-M-mediated (10 μM) inhibition occurred via two signaling pathways. The first pathway inhibited whole cell peak currents, consisting primarily of N-type current, but not FPL 64176-induced, long-lasting tail currents, comprised entirely of L-type current. Inhibited currents displayed slowed activation kinetics and voltage dependence, characteristics of membrane-delimited inhibition. Current inhibition was blocked by the selective M2 receptor antagonist, methoctramine (METH; 100 nM), or following pertussis toxin (PTX) pretreatment. Activation of the second pathway inhibited both peak and long-lasting tail currents. This pathway was voltage-independent, PTX-insensitive, but sensitive to internal Ca2+ chelator concentration. Muscarinic toxin 7 (MT-7, 100 nM), an irreversible M1 receptor antagonist, eliminated this inhibition. Oxo-M (100 μM) decreased L- and N-type channel activities in cell-attached patches, indicating that a diffusible second messenger is involved. BeCh (100 μM) also inhibited whole cell currents via the membrane-delimited pathway. Blocking M4 receptors with 100 nM pirenzepine (in the presence of MT-7) had no effect, while antagonizing M2 receptors with METH abolished inhibition. Concentrations of BeCh as high as 3 mM failed to inhibit either peak or long-lasting tail currents following PTX pretreatment. These results indicate that BeCh may be an effective tool for selectively activating M2 receptor stimulation of the membrane-delimited pathway. PMID:12711626

  6. Site of action of the general anesthetic propofol in muscarinic M1 receptor-mediated signal transduction.

    PubMed

    Murasaki, Osamu; Kaibara, Muneshige; Nagase, Yoshihisa; Mitarai, Sayaka; Doi, Yoshiyuki; Sumikawa, Koji; Taniyama, Kohtaro

    2003-12-01

    Although a potential target site of general anesthetics is primarily the GABA A receptor, a chloride ion channel, a previous study suggested that the intravenous general anesthetic propofol attenuates the M1 muscarinic acetylcholine receptor (M1 receptor)-mediated signal transduction. In the present study, we examined the target site of propofol in M1 receptor-mediated signal transduction. Two-electrode voltage-clamp method was used in Xenopus oocytes expressing both M1 receptors and associated G protein alpha subunits (Gqalpha). Propofol inhibited M1 receptor-mediated signal transduction in a dose-dependent manner (IC50 = 50 nM). Injection of guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) into oocytes overexpressing Gqalpha was used to investigate direct effects of propofol on G protein coupled with the M1 receptor. Propofol did not affect activation of Gqalpha-mediated signal transduction with the intracellular injection of GTPgammaS. We also studied effects of propofol on l-[N-methyl-3H]scopolamine methyl chloride ([3H]NMS) binding and M1 receptor-mediated signal transduction in mammalian cells expressing M1 receptor. Propofol inhibited the M1 receptor-mediated signal transduction but did not inhibit binding of [3H]NMS. Effects of propofol on Gs- and Gi/o-coupled signal transduction were investigated, using oocytes expressing the beta2 adrenoceptor (beta2 receptor)/cystic fibrosis transmembrane conductance regulator or oocytes expressing the M2 muscarinic acetylcholine receptor (M2 receptor)/Kir3.1 (a member of G protein-gated inwardly rectifying K(+) channels). Neither beta2 receptor-mediated nor M2 receptor-mediated signal transduction was inhibited by a relatively high concentration of propofol (50 microM). These results indicate that propofol inhibits M1 receptor-mediated signal transduction by selectively disrupting interaction between the receptor and associated G protein.

  7. Muscarinic receptors and ligands in cancer

    PubMed Central

    Shah, Nirish; Khurana, Sandeep; Cheng, Kunrong; Raufman, Jean-Pierre

    2009-01-01

    Emerging evidence indicates that muscarinic receptors and ligands play key roles in regulating cellular proliferation and cancer progression. Both neuronal and nonneuronal acetylcholine production results in neurocrine, paracrine, and autocrine promotion of cell proliferation, apoptosis, migration, and other features critical for cancer cell survival and spread. The present review comprises a focused critical analysis of evidence supporting the role of muscarinic receptors and ligands in cancer. Criteria are proposed to validate the biological importance of muscarinic receptor expression, activation, and postreceptor signaling. Likewise, criteria are proposed to validate the role of nonneuronal acetylcholine production in cancer. Dissecting cellular mechanisms necessary for muscarinic receptor activation as well as those needed for acetylcholine production and release will identify multiple novel targets for cancer therapy. PMID:19036940

  8. Kinetics of M1 muscarinic receptor and G protein signaling to phospholipase C in living cells

    PubMed Central

    Falkenburger, Björn H.; Jensen, Jill B.

    2010-01-01

    G protein–coupled receptors (GPCRs) mediate responses to external stimuli in various cell types. Early events, such as the binding of ligand and G proteins to the receptor, nucleotide exchange (NX), and GTPase activity at the Gα subunit, are common for many different GPCRs. For Gq-coupled M1 muscarinic (acetylcholine) receptors (M1Rs), we recently measured time courses of intermediate steps in the signaling cascade using Förster resonance energy transfer (FRET). The expression of FRET probes changes the density of signaling molecules. To provide a full quantitative description of M1R signaling that includes a simulation of kinetics in native (tsA201) cells, we now determine the density of FRET probes and construct a kinetic model of M1R signaling through Gq to activation of phospholipase C (PLC). Downstream effects on the trace membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) and PIP2-dependent KCNQ2/3 current are considered in our companion paper in this issue (Falkenburger et al. 2010. J. Gen. Physiol. doi:10.1085/jgp.200910345). By calibrating their fluorescence intensity, we found that we selected transfected cells for our experiments with ∼3,000 fluorescently labeled receptors, G proteins, or PLC molecules per µm2 of plasma membrane. Endogenous levels are much lower, 1–40 per µm2. Our kinetic model reproduces the time courses and concentration–response relationships measured by FRET and explains observed delays. It predicts affinities and rate constants that align well with literature values. In native tsA201 cells, much of the delay between ligand binding and PLC activation reflects slow binding of G proteins to receptors. With M1R and Gβ FRET probes overexpressed, 10% of receptors have G proteins bound at rest, rising to 73% in the presence of agonist. In agreement with previous work, the model suggests that binding of PLC to Gαq greatly speeds up NX and GTPase activity, and that PLC is maintained in the active state by cycles of

  9. The GAR-3 muscarinic receptor cooperates with calcium signals to regulate muscle contraction in the Caenorhabditis elegans pharynx.

    PubMed Central

    Steger, Katherine A; Avery, Leon

    2004-01-01

    Muscarinic acetylcholine receptors regulate the activity of neurons and muscle cells through G-protein-coupled cascades. Here, we identify a pathway through which the GAR-3 muscarinic receptor regulates both membrane potential and excitation-contraction coupling in the Caenorhabditis elegans pharyngeal muscle. GAR-3 signaling is enhanced in worms overexpressing gar-3 or lacking GPB-2, a G-protein beta-subunit involved in RGS-mediated inhibition of G(o)alpha- and G(q)alpha-linked pathways. High levels of signaling through GAR-3 inhibit pharyngeal muscle relaxation and impair feeding--but do not block muscle repolarization--when worms are exposed to arecoline, a muscarinic agonist. Loss of gar-3 function results in shortened action potentials and brief muscle contractions in the pharyngeal terminal bulb. High levels of calcium entry through voltage-gated channels also impair terminal bulb relaxation and sensitize worms to the toxic effects of arecoline. Mutation of gar-3 reverses this sensitivity, suggesting that GAR-3 regulates calcium influx or calcium-dependent processes. Because the effects of GAR-3 signaling on membrane depolarization and muscle contraction can be separated, we conclude that GAR-3 regulates multiple calcium-dependent processes in the C. elegans pharyngeal muscle. PMID:15238517

  10. Interaction between muscarinic receptor subtype signal transduction pathways mediating bladder contraction

    PubMed Central

    BRAVERMAN, ALAN S.; TALLARIDA, RONALD J.; RUGGIERI, MICHAEL R.

    2012-01-01

    M3 muscarinic receptors mediate cholinergic-induced contraction in most smooth muscles. However, in the denervated rat bladder, M2 receptors participate in contraction because M3-selective antagonists [para-fluoro-hexahydro-sila-diphenidol (p-F-HHSiD) and 4-DAMP] have low affinities. However, the affinity of the M2-selective antagonist methoctramine in the denervated bladder is consistent with M3 receptor mediating contraction. It is possible that two pathways interact to mediate contraction: one mediated by the M2 receptor and one by the M3 receptor. To determine whether an interaction exists, the inhibitory potencies of combinations of methoctramine and p-F-HHSiD for reversing cholinergic contractions were measured. In normal bladders, all combinations gave additive effects. In denervated bladders, synergistic effects were seen with the 10:1 and 1:1 (methoctramine:p-F-HHSiD wt/wt) combinations. After application of the sarcoplasmic reticulum ATPase inhibitor thapsigargin to normal tissue, the 10:1 and 1:1 ratios became synergistic, mimicking denervated tissue. Thus in normal bladders both M2 and M3 receptors can induce contraction. In the denervated bladder, the M2 and the M3 receptors interact in a facilitatory manner to mediate contraction. PMID:12185001

  11. Signal-transduction pathways that regulate visceral smooth muscle function. III. Coupling of muscarinic receptors to signaling kinases and effector proteins in gastrointestinal smooth muscles.

    PubMed

    Gerthoffer, William T

    2005-05-01

    Stimulation of muscarinic M3 and M2 receptors on gastrointestinal smooth muscle elicits contraction via activation of G proteins that are coupled to a diverse set of downstream signaling pathways and effector proteins. Many studies suggest a canonical excitation-contraction coupling pathway that includes activation of phospholipases, production of inositol 1,4,5-trisphosphate and diacylglycerol, release of calcium from the sarcoplasmic reticulum, activation of L-type calcium channels, and activation of nonselective cation channels. These events lead to elevated intracellular calcium concentration, which activates myosin light chain kinase to phosphorylate and activate myosin II thus causing contraction. In addition, muscarinic receptors are coupled to signaling pathways that modulate the effect of activator calcium. The Rho/Rho kinase pathway inhibits myosin light chain phosphatase, one of the key steps in sensitization of the contractile proteins to calcium. Phosphatidylinositol 3-kinases and Src family tyrosine kinases are also activated by muscarinic agonists. Src family tyrosine kinases regulate L-type calcium and nonselective cation channels. Src activation also leads to activation of ERK and p38 MAPKs. ERK MAPKs phosphorylate caldesmon, an actin filament binding protein. P38 MAPKs activate phospholipases and MAPKAP kinase 2/3, which phosphorylate HSP27. HSP27 may regulate cross-bridge function, actin filament formation, and actin filament attachment to the cell membrane. In addition to the well-known role of M3 muscarinic receptors to regulate myoplasmic calcium levels, the integrated effect of muscarinic activation probably also includes signaling pathways that modulate phospholipases, cyclic nucleotides, contractile protein function, and cytoskeletal protein function.

  12. Ryanodine receptor subtype 2 encodes Ca2+ oscillations activated by acetylcholine via the M2 muscarinic receptor/cADP-ribose signalling pathway in duodenum myocytes.

    PubMed

    Fritz, Nicolas; Macrez, Nathalie; Mironneau, Jean; Jeyakumar, Loice H; Fleischer, Sidney; Morel, Jean-Luc

    2005-05-15

    In this study, we characterized the signalling pathway activated by acetylcholine that encodes Ca2+ oscillations in rat duodenum myocytes. These oscillations were observed in intact myocytes after removal of external Ca2+, in permeabilized cells after abolition of the membrane potential and in the presence of heparin (an inhibitor of inositol 1,4,5-trisphosphate receptors) but were inhibited by ryanodine, indicating that they are dependent on Ca2+ release from intracellular stores through ryanodine receptors. Ca2+ oscillations were selectively inhibited by methoctramine (a M2 muscarinic receptor antagonist). The M2 muscarinic receptor-activated Ca2+ oscillations were inhibited by 8-bromo cyclic adenosine diphosphoribose and inhibitors of adenosine diphosphoribosyl cyclase (ZnCl2 and anti-CD38 antibody). Stimulation of ADP-ribosyl cyclase activity by acetylcholine was evaluated in permeabilized cells by measuring the production of cyclic guanosine diphosphoribose (a fluorescent compound), which resulted from the cyclization of nicotinamide guanine dinucleotide. As duodenum myocytes expressed the three subtypes of ryanodine receptors, an antisense strategy revealed that the ryanodine receptor subtype 2 alone was required to initiate the Ca2+ oscillations induced by acetylcholine and also by cyclic adenosine diphosphoribose and rapamycin (a compound that induced uncoupling between 12/12.6 kDa FK506-binding proteins and ryanodine receptors). Inhibition of cyclic adenosine diphosphoribose-induced Ca2+ oscillations, after rapamycin treatment, confirmed that both compounds interacted with the ryanodine receptor subtype 2. Our findings show for the first time that the M2 muscarinic receptor activation triggered Ca2+ oscillations in duodenum myocytes by activation of the cyclic adenosine diphosphoribose/FK506-binding protein/ryanodine receptor subtype 2 signalling pathway.

  13. Muscarinic acetylcholine receptor activation blocks long-term potentiation at cerebellar parallel fiber-Purkinje cell synapses via cannabinoid signaling.

    PubMed

    Rinaldo, Lorenzo; Hansel, Christian

    2013-07-02

    Muscarinic acetylcholine receptors (mAChRs) are known to modulate synaptic plasticity in various brain areas. A signaling pathway triggered by mAChR activation is the production and release of endocannabinoids that bind to type 1 cannabinoid receptors (CB1R) located on synaptic terminals. Using whole-cell patch-clamp recordings from rat cerebellar slices, we have demonstrated that the muscarinic agonist oxotremorine-m (oxo-m) blocks the induction of presynaptic long-term potentiation (LTP) at parallel fiber (PF)-Purkinje cell synapses in a CB1R-dependent manner. Under control conditions, LTP was induced by delivering 120 PF stimuli at 8 Hz. In contrast, no LTP was observed when oxo-m was present during tetanization. PF-LTP was restored when the CB1R antagonist N-1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide (AM251) was coapplied with oxo-m. Furthermore, the suppressive effect of oxo-m on PF-LTP was abrogated by the GDP analog GDP-β-S (applied intracellularly), the phospholipase C inhibitor U-73122, and the diacylglycerol lipase inhibitor tetrahydrolipstatin (THL), suggesting that cannabinoid synthesis results from the activation of Gq-coupled mAChRs present on Purkinje cells. The oxo-m-mediated suppression of LTP was also prevented in the presence of the M3 receptor antagonist DAU 5884, and was absent in M1/M3 receptor double-KO mice, identifying M3 receptors as primary oxo-m targets. Our findings allow for the possibility that cholinergic signaling in the cerebellum--which may result from long-term depression (LTD)-related disinhibition of cholinergic neurons in the vestibular nuclei--suppresses presynaptic LTP to prevent an up-regulation of transmitter release that opposes the reduction of postsynaptic responsiveness. This modulatory capacity of mAChR signaling could promote the functional penetrance of LTD.

  14. Muscarinic receptor family interacting proteins: role in receptor function.

    PubMed

    Borroto-Escuela, Dasiel O; Correia, Patrícia A; Romero-Fernandez, Wilber; Narvaez, Manuel; Fuxe, Kjell; Ciruela, Francisco; Garriga, Pere

    2011-02-15

    G protein-coupled receptors constitute one of the most important families of membrane receptors through which cells respond to extracellular stimuli. Receptors of this superfamily likely function as signal transduction complexes. The identification and analysis of their components provide new insights into a better understanding of these receptors' function and regulation. We used tandem-affinity purification and mass spectrometry as a systematic approach to characterize multiprotein complexes in the acetylcholine muscarinic receptor subfamily. To overcome the limitations associated with membrane protein receptor solubilization with detergents, we developed a strategy in which receptors are co-expressed with a cytoplasmic minigene construct, encoding the third intracellular loop and the C-terminal tail tagged to the tandem-affinity-cassette of each receptor subtype. Numerous protein complexes were identified, including many new interactions in various signalling pathways. Systematic identification data set together with protein interactions reported in the literature revealed a high degree of connectivity. These allow the proposal, for the first time, of an outline of the muscarinic interactome as a network of protein complexes and a context for a more reasoned and informed approach to drug discovery and muscarinic receptor subtype specificities.

  15. The colon-selective spasmolytic otilonium bromide inhibits muscarinic M3 receptor-coupled calcium signals in isolated human colonic crypts

    PubMed Central

    Lindqvist, Susanne; Hernon, James; Sharp, Paul; Johns, Neil; Addison, Sarah; Watson, Mark; Tighe, Richard; Greer, Shaun; Mackay, Jean; Rhodes, Michael; Lewis, Michael; Stebbings, William; Speakman, Chris; Evangelista, Stefano; Johnson, Ian; Williams, Mark

    2002-01-01

    Otilonium bromide (OB) is a smooth muscle relaxant used in the treatment of irritable bowel syndrome. Otilonium bromide has been shown to interfere with the mobilization of calcium in intestinal smooth muscle, but the effects on other intestinal tissues have not been investigated. We identified the muscarinic receptor subtype coupled to calcium signals in colonic crypt derived from the human colonic epithelium and evaluated the inhibitory effects of OB. Calcium signals were monitored by fluorescence imaging of isolated human colonic crypts and Chinese hamster ovary cells stably expressing the cloned human muscarinic M3 receptor subtype (CHO-M3). Colonic crypt receptor expression was investigated by pharmacological and immunohistochemical techniques. The secretagogue acetylcholine (ACh) stimulated calcium mobilization from intracellular calcium stores at the base of human colonic crypts with an EC50 of 14 μM. The muscarinic receptor antagonists 4-DAMP, AF-DX 384, pirenzepine and methroctamine inhibited the ACh-induced calcium signal with the following respective IC50 (pKb) values: 0.78 nM (9.1), 69 nM (7.2), 128 nM (7.1), and 2510 nM (5.8). Immunohistochemical analyses of muscarinic receptor expression demonstrated the presence of M3 receptor subtype expression at the crypt-base. Otilonium bromide inhibited the generation of ACh-induced calcium signals in a dose dependent manner (IC50=880 nM). In CHO-M3 cells, OB inhibited calcium signals induced by ACh, but not ATP. In addition, OB did not inhibit histamine-induced colonic crypt calcium signals. The present studies have demonstrated that OB inhibited M3 receptor-coupled calcium signals in human colonic crypts and CHO-M3 cells, but not those induced by stimulation of other endogenous receptor types. We propose that the M3 receptor-coupled calcium signalling pathway is directly targeted by OB at the level of the colonic epithelium, suggestive of an anti-secretory action in IBS patients suffering with diarrhoea. PMID

  16. The colon-selective spasmolytic otilonium bromide inhibits muscarinic M(3) receptor-coupled calcium signals in isolated human colonic crypts.

    PubMed

    Lindqvist, Susanne; Hernon, James; Sharp, Paul; Johns, Neil; Addison, Sarah; Watson, Mark; Tighe, Richard; Greer, Shaun; Mackay, Jean; Rhodes, Michael; Lewis, Michael; Stebbings, William; Speakman, Chris; Evangelista, Stefano; Johnson, Ian; Williams, Mark

    2002-12-01

    1. Otilonium bromide (OB) is a smooth muscle relaxant used in the treatment of irritable bowel syndrome. Otilonium bromide has been shown to interfere with the mobilization of calcium in intestinal smooth muscle, but the effects on other intestinal tissues have not been investigated. We identified the muscarinic receptor subtype coupled to calcium signals in colonic crypt derived from the human colonic epithelium and evaluated the inhibitory effects of OB. 2. Calcium signals were monitored by fluorescence imaging of isolated human colonic crypts and Chinese hamster ovary cells stably expressing the cloned human muscarinic M(3) receptor subtype (CHO-M(3)). Colonic crypt receptor expression was investigated by pharmacological and immunohistochemical techniques. 3. The secretagogue acetylcholine (ACh) stimulated calcium mobilization from intracellular calcium stores at the base of human colonic crypts with an EC(50) of 14 micro M. The muscarinic receptor antagonists 4-DAMP, AF-DX 384, pirenzepine and methroctamine inhibited the ACh-induced calcium signal with the following respective IC(50) (pK(b)) values: 0.78 nM (9.1), 69 nM (7.2), 128 nM (7.1), and 2510 nM (5.8). 4. Immunohistochemical analyses of muscarinic receptor expression demonstrated the presence of M(3) receptor subtype expression at the crypt-base. 5. Otilonium bromide inhibited the generation of ACh-induced calcium signals in a dose dependent manner (IC(50)=880 nM). 6. In CHO-M(3) cells, OB inhibited calcium signals induced by ACh, but not ATP. In addition, OB did not inhibit histamine-induced colonic crypt calcium signals. 7. The present studies have demonstrated that OB inhibited M(3) receptor-coupled calcium signals in human colonic crypts and CHO-M(3) cells, but not those induced by stimulation of other endogenous receptor types. We propose that the M(3) receptor-coupled calcium signalling pathway is directly targeted by OB at the level of the colonic epithelium, suggestive of an anti-secretory action

  17. Layer-specific processing of excitatory signals in CA1 interneurons depends on postsynaptic M₂ muscarinic receptors.

    PubMed

    Zheng, Fang; Seeger, Thomas; Nixdorf-Bergweiler, Barbara E; Alzheimer, Christian

    2011-05-02

    The hippocampus receives a diffuse cholinergic innervation which acts on pre- and postsynaptic sites to modulate neurotransmission and excitability of pyramidal cells and interneurons in an intricate fashion. As one missing piece in this puzzle, we explored how muscarinic receptor activation modulates the somatodendritic processing of glutamatergic input in CA1 interneurons. We performed whole-cell recordings from visually identified interneurons of stratum radiatum (SR) and stratum oriens (SO) and examined the effects of the cholinergic agonist carbachol (CCh) on EPSP-like waveforms evoked by brief glutamate pulses onto their proximal dendrites. In SO interneurons, CCh consistently reduced glutamate-induced postsynaptic potentials (GPSPs) in control rat and mice, but not in M₂ muscarinic receptor knockout mice. By contrast, the overwhelming majority of interneurons recorded in SR of control and M₂ receptor-deficient hippocampi exhibited muscarinic enhancement of GPSPs. Interestingly, the non-responding interneurons were strictly confined to the SR subfield closest to the subiculum. Our data suggest that postsynaptic modulation by acetylcholine of excitatory input onto CA1 interneurons occurs in a stratum-specific fashion, which is determined by the absence or presence of M₂ receptors in their (somato-)dendritic compartments. Thus cholinergic projections might be capable of recalibrating synaptic weights in different inhibitory circuits of the CA1 region.

  18. The M2 muscarinic receptors are essential for signaling in the heart left ventricle during restraint stress in mice.

    PubMed

    Tomankova, Hana; Valuskova, Paulina; Varejkova, Eva; Rotkova, Jana; Benes, Jan; Myslivecek, Jaromir

    2015-01-01

    We hypothesized that muscarinic receptors (MRs) in the heart have a role in stress responses and thus investigated changes in MR signaling (gene expression, number of receptors, adenylyl cyclase (AC), phospholipase C (PLC), protein kinase A and C (PKA and PKC) and nitric oxide synthase [NOS]) in the left ventricle, together with telemetric measurement of heart rate (HR) in mice (wild type [WT] and M2 knockout [KO]) during and after one (1R) or seven sessions (7R) of restraint stress (seven mice per group). Stress decreased M2 MR mRNA and cell surface MR in the left ventricle in WT mice. In KO mice, 1R, but not 7R, decreased surface MR. Similarly, AC activity was decreased in WT mice after 1R and 7R, whereas in KO mice, there was no change. PLC activity was also decreased after 1R in WT and KO mice. This is in accord with the concept that cAMP is a key player in HR regulation. No change was found with stress in NOS activity. Amount of AC and PKA protein was not changed, but was altered for PKC isoenzymes (PKCα, β, γ, η and ϵ (increased) in KO mice, and PKCι (increased) in WT mice). KO mice were more susceptible to stress as shown by inability to compensate HR during 120 min following repeated stress. The results imply that not only M2 but also M3 are involved in stress signaling and in allostasis. We conclude that for a normal stress response, the expression of M2 MR to mediate vagal responses is essential.

  19. Muscarinic Receptor Antagonists: Effects on Pulmonary Function

    PubMed Central

    Buels, Kalmia S.

    2014-01-01

    In healthy lungs, muscarinic receptors control smooth muscle tone, mucus secretion, vasodilation, and inflammation. In chronic obstructive pulmonary disease (COPD) and asthma, cholinergic mechanisms contribute to increased bronchoconstriction and mucus secretion that limit airflow. This chapter reviews neuronal and nonneuronal sources of acetylcholine in the lung and the expression and role of M1, M2, and M3 muscarinic receptor subtypes in lung physiology. It also discusses the evidence for and against the role of parasympathetic nerves in asthma, and the current use and therapeutic potential of muscarinic receptor antagonists in COPD and asthma. PMID:22222705

  20. Blocking M2 muscarinic receptor signaling inhibits tumor growth and reverses epithelial-mesenchymal transition (EMT) in non-small cell lung cancer (NSCLC).

    PubMed

    Zhao, Qingnan; Gu, Xiajing; Zhang, Chun; Lu, Qin; Chen, Hongzhuan; Xu, Lu

    2015-01-01

    Lung cancers express non-neuronal, cholinergic autoparacrine loop, which facilitates tumor growth. Interruption of M3 muscarinic cholinergic signaling has been reported to inhibit small cell lung cancer (SCLC) growth. The purpose of this study is to investigate if blocking autoparacrine muscarinic cholinergic signaling could inhibit non-small cell lung cancer (NSCLC) growth and possible underlying mechanisms. Our results showed that PC9 and A549 cells expressed all 5 subtypes of muscarinic receptor (mAChR) and blocking M2 mAChR (M2R) signaling using selective antagonist methoctramine or short hairpin RNA (shRNA) inhibited tumor cell proliferation in vitro and in vivo. Consistent with AChR agonists stimulating p44/42 MAPK (Erk1/2) and Akt phosphorylation, blocking M2R signaling decreased MAPK and Akt phosphorylation, indicating that non-neuronal ACh functions as an autoparacrine growth factor signaling in part through activation of M2R and downstream MAPK and Akt pathways. Importantly, further studies revealed that blocking M2R signaling also reversed epithelial-mesenchymal transition (EMT) in vitro and in vivo, indicating that non-neuronal ACh promotes EMT partially through activation of M2R. These findings demonstrate that M2R plays a role in the growth and progression of NSCLC and suggest M2R antagonists may be an efficacious adjuvant therapy for NSCLC.

  1. Taurolithocholic acid promotes intrahepatic cholangiocarcinoma cell growth via muscarinic acetylcholine receptor and EGFR/ERK1/2 signaling pathway

    PubMed Central

    AMONYINGCHAROEN, SUMET; SURIYO, TAWIT; THIANTANAWAT, APINYA; WATCHARASIT, PIYAJIT; SATAYAVIVAD, JUTAMAAD

    2015-01-01

    Cholangiocarcinoma (CCA) is a malignant cancer of the biliary tract and its occurrence is associated with chronic cholestasis which causes an elevation of bile acids in the liver and bile duct. The present study aimed to investigate the role and mechanistic effect of bile acids on the CCA cell growth. Intrahepatic CCA cell lines, RMCCA-1 and HuCCA-1, were treated with bile acids and their metabolites to determine the growth promoting effect. Cell viability, cell cycle analysis, EdU incorporation assays were conducted. Intracellular signaling proteins were detected by western immunoblotting. Among eleven forms of bile acids and their metabolites, only taurolithocholic acid (TLCA) concentration dependently (1–40 μM) increased the cell viability of RMCCA-1, but not HuCCA-1 cells. The cell cycle analysis showed induction of cells in the S phase and the EdU incorporation assay revealed induction of DNA synthesis in the TLCA-treated RMCCA-1 cells. Moreover, TLCA increased the phosphorylation of EGFR, ERK 1/2 and also increased the expression of cyclin D1 in RMCCA-1 cells. Furthermore, TLCA-induced RMCCA-1 cell growth could be inhibited by atropine, a non-selective muscarinic acetylcholine receptor (mAChR) antagonist, AG 1478, a specific EGFR inhibitor, or U 0126, a specific MEK 1/2 inhibitor. These results suggest that TLCA induces CCA cell growth via mAChR and EGFR/EKR1/2 signaling pathway. Moreover, the functional presence of cholinergic system plays a certain role in TLCA-induced CCA cell growth. PMID:25815516

  2. Blueberry treatment antagonizes C-2 ceramide-induced stress signaling in muscarinic receptor-transfected COS-7 cells.

    PubMed

    Joseph, James A; Bielinski, Donna F; Fisher, Derek R

    2010-03-24

    Previous research has shown that muscarinic receptors (MAChRs) show loss of sensitivity in aging and AD and are selectively sensitive to oxidative stress (OS). Thus, COS-7 cells transfected (tn) with MAChR subtype M1 show > OS sensitivity [as reflected in the ability of the cell to extrude or sequester Ca(2+) following depolarization (recovery) by oxotremorine (oxo) and exposure to dopamine (DA) or amyloid beta (Abeta)] than M3-transfected COS-7 cells. Blueberry (BB) extract pretreatment prevented these deficits. Research has also indicated that C2 ceramide (Cer) has several age-related negative cellular effects (e.g., OS). When these cells were treated with Cer, the significant decrements in the ability of both types of tn cells to initially respond to oxo were antagonized by BB treatment. Present experiments assessed signaling mechanisms involved in BB protection in the presence or absence of DA, Abeta, and/or Cer in this model. Thus, control or BB-treated M1 and M3 tn COS-7 cells were exposed to DA or Abeta(42) in the presence or absence of Cer. Primarily, results showed that the effects of DA or Abeta(42) were to increase stress (e.g., PKCgamma, p38MAPK) and protective signals (e.g., pMAPK). Cer also appeared to raise several of the stress and protective signals in the absence of the other stressors, including PKCgamma, pJNK, pNfkappaB, p53, and p38MAPK, while not significantly altering MAPK, or Akt. pArc was, however, increased by Cer in both types of transfected cells. The protective effects of BB when combined with Cer generally showed greater protection when BB extract was applied prior to Cer, except for one protective signal (pArc) where a greater effect was seen in the M3 cells exposed to Abeta(42.) In the absence of the Abeta(42) or DA, for several of the stress signals (e.g., pNfkappaB, p53), BB lowered their Cer-induced increases in M1- and M3-transfected cells. We are exploring these interactions further, but it is clear that increases in ceramide

  3. Synthesis, Trafficking, and Localization of Muscarinic Acetylcholine Receptors

    PubMed Central

    Nathanson, Neil M.

    2008-01-01

    Muscarinic acetylcholine receptors are members of the G-protein coupled receptor superfamily that are expressed in and regulate the function of neurons, cardiac and smooth muscle, glands, and many other cell types and tissues. The correct trafficking of membrane proteins to the cell surface and their subsequent localization at appropriate sites in polarized cells are required for normal cellular signaling and physiological responses. This review will summarize work on the synthesis and trafficking of muscarinic receptors to the plasma membrane and their localization at the cell surface. PMID:18558434

  4. Effects of toluene exposure on signal transduction: toluene reduced the signaling via stimulation of human muscarinic acetylcholine receptor m2 subtypes in CHO cells.

    PubMed

    Tsuga, Hirofumi; Haga, Tatsuya; Honma, Takeshi

    2002-07-01

    The organic solvent toluene is used widely in industry and is toxic to the central nervous system (CNS). To clarify the mechanisms of CNS toxicity following toluene exposure, especially with respect to the G protein-coupling of receptors, we determined the effects of toluene on the activation of Gi by stimulating human muscarinic acetylcholine receptor m2 subtypes (hm2 receptors) expressed in Chinese hamster ovary (CHO) cells. We first examined whether toluene affects the inhibition of adenylyl cyclase by Gi. The attenuation of forskolin-stimulated cAMP formation by the stimulation of hm2 receptors was reduced in a medium containing toluene. Next, we determined the effects of toluene on carbamylcholine-stimulated [35S]GTPgammaS binding using membrane fractions of CHO cell expressing hm2 receptors. Carbamylcholine-stimulated [35S]GTPgammaS binding activity was markedly reduced when assayed using reaction buffers containing toluene. However, carbamylcholine-stimulated [35S]GTPgammaS binding activity was essentially unchanged following pretreatment of the cells with a toluene-saturated medium prior to membrane isolation. Toluene pretreatment and the toluene itself did not alter the characteristics of the binding of carbamylcholine and [3H]N-methylscopolamine to hm2 receptors. On the contrary of the effect of toluene for [35S]GTPgammaS binding, the effect of toluene for attenuation of forskolin-stimulated cAMP formation by the stimulation of hm2 receptors was irreversible. These observations indicate that toluene acts as an inhibitor of the signal transduction via hm2 receptor stimulation in CHO cells, and at least two mechanisms exist in the inhibition mechanisms by toluene.

  5. Heterogeneity of muscarinic receptor subtypes in cerebral blood vessels

    SciTech Connect

    Garcia-Villalon, A.L.; Krause, D.N.; Ehlert, F.J.; Duckles, S.P. )

    1991-07-01

    The identity and distribution of muscarinic cholinergic receptor subtypes and associated signal transduction mechanisms was characterized for the cerebral circulation using correlated functional and biochemical investigations. Subtypes were distinguished by the relative affinities of a panel of muscarinic antagonists, pirenzepine, AF-DX 116 (11-2-((2-(diethylaminomethyl)- 1-piperidinyl)acetyl)-5,11-dihydro-6H- pyrido(2,3-b)(1,4)benzodiazepine-6-one), hexahydrosiladifenidol, methoctramine, 4-diphenylacetoxy-N-methylpiperidine methobromide, dicyclomine, para-fluoro-hexahydrosiladifenidol and atropine. Muscarinic receptors characterized by inhibition of (3H)quinuclidinylbenzilate binding in membranes of bovine pial arteries were of the M2 subtype. In contrast pharmacological analysis of (3H)-quinuclidinylbenzilate binding in bovine intracerebral microvessels suggests the presence of an M4 subtype. Receptors mediating endothelium-dependent vasodilation in rabbit pial arteries were of the M3 subtype, whereas muscarinic receptors stimulating endothelium-independent phosphoinositide hydrolysis in bovine pial arteries were of the M1 subtype. These findings suggest that characteristics of muscarinic receptors in cerebral blood vessels vary depending on the type of vessel, cellular location and function mediated.

  6. The M3-muscarinic receptor regulates learning and memory in a receptor phosphorylation/arrestin-dependent manner.

    PubMed

    Poulin, Benoit; Butcher, Adrian; McWilliams, Phillip; Bourgognon, Julie-Myrtille; Pawlak, Robert; Kong, Kok Choi; Bottrill, Andrew; Mistry, Sharad; Wess, Jürgen; Rosethorne, Elizabeth M; Charlton, Steven J; Tobin, Andrew B

    2010-05-18

    Degeneration of the cholinergic system is considered to be the underlying pathology that results in the cognitive deficit in Alzheimer's disease. This pathology is thought to be linked to a loss of signaling through the cholinergic M(1)-muscarinic receptor subtype. However, recent studies have cast doubt on whether this is the primary receptor mediating cholinergic-hippocampal learning and memory. The current study offers an alternative mechanism involving the M(3)-muscarinic receptor that is expressed in numerous brain regions including the hippocampus. We demonstrate here that M(3)-muscarinic receptor knockout mice show a deficit in fear conditioning learning and memory. The mechanism used by the M(3)-muscarinic receptor in this process involves receptor phosphorylation because a knockin mouse strain expressing a phosphorylation-deficient receptor mutant also shows a deficit in fear conditioning. Consistent with a role for receptor phosphorylation, we demonstrate that the M(3)-muscarinic receptor is phosphorylated in the hippocampus following agonist treatment and following fear conditioning training. Importantly, the phosphorylation-deficient M(3)-muscarinic receptor was coupled normally to G(q/11)-signaling but was uncoupled from phosphorylation-dependent processes such as receptor internalization and arrestin recruitment. It can, therefore, be concluded that M(3)-muscarinic receptor-dependent learning and memory depends, at least in part, on receptor phosphorylation/arrestin signaling. This study opens the potential for biased M(3)-muscarinic receptor ligands that direct phosphorylation/arrestin-dependent (non-G protein) signaling as being beneficial in cognitive disorders.

  7. HIGH AFFINITY ACYLATING ANTAGONISTS FOR MUSCARINIC RECEPTORS

    PubMed Central

    Baumgold, Jesse; Karton, Yishai; Malka, Naftali; Jacobson, Kenneth A.

    2012-01-01

    Summary The muscarinic antagonists pirenzepine and telenzepine were derivitized as alkylamino derivatives at a site on the molecules corresponding to a region of bulk tolerance in receptor binding. The distal primary amino groups were coupled to the cross-linking reagent meta-phenylene diisothiocyanate, resulting in two isothiocyanate derivatives that were found to inhibit muscarinic receptors irreversibly and in a dose-dependent fashion. Preincubation of rat forebrain membranes with an isothiocyanate derivative followed by radioligand binding using [3H]N-methylscopolamine diminished the Bmax value, but did not affect the Kd value. The receptor binding site was not restored upon repeated washing, indicating that irreversible inhibition had occurred. IC50 values for the irreversible inhibition at rat forebrain muscarinic receptors were 0.15 nM and 0.19 nM, for derivatives of pirenzepine and telenzepine, respectively. The isothiocyanate derivative of pirenzepine was non-selective as an irreversible muscarinic inhibitor, and the corresponding derivative prepared from telenzepine was 5-fold selective for forebrain (mainly m1) vs. heart (m2) muscarinic receptors. PMID:1625525

  8. External imaging of cerebral muscarinic acetylcholine receptors

    SciTech Connect

    Eckelman, W.C.; Reba, R.C.; Rzeszotarski, W.J.; Gibson, R.E.; Hill, T.; Holman, B.L.; Budinger, T.; Conklin, J.J.; Eng, R.; Grissom, M.P.

    1984-01-20

    A radioiodinated ligand that binds to muscarinic acetylcholine receptors was shown to distribute in the brain by a receptor-mediated process. With single-photon-emission imaging techniques, radioactivity was detected in the cerebrum but not in the cerebellum, whereas with a flow-limited radiotracer, radioactivity was detected in cerebrum and cerebellum. Single-photon-emission computed tomography showed good definition of the caudate putamen and cortex in man.

  9. External Imaging of Cerebral Muscarinic Acetylcholine Receptors

    NASA Astrophysics Data System (ADS)

    Eckelman, William C.; Reba, Richard C.; Rzeszotarski, Waclaw J.; Gibson, Raymond E.; Hill, Thomas; Holman, B. Leonard; Budinger, Thomas; Conklin, James J.; Eng, Robert; Grissom, Michael P.

    1984-01-01

    A radioiodinated ligand that binds to muscarinic acetylcholine receptors was shown to distribute in the brain by a receptor-mediated process. With single-photon-emission imaging techniques, radioactivity was detected in the cerebrum but not in the cerebellum, whereas with a flow-limited radiotracer, radioactivity was detected in cerebrum and cerebellum. Single-photon-emission computed tomography showed good definition of the caudate putamen and cortex in man.

  10. Homology modeling of human muscarinic acetylcholine receptors.

    PubMed

    Thomas, Trayder; McLean, Kimberley C; McRobb, Fiona M; Manallack, David T; Chalmers, David K; Yuriev, Elizabeth

    2014-01-27

    We have developed homology models of the acetylcholine muscarinic receptors M₁R-M₅R, based on the β₂-adrenergic receptor crystal as the template. This is the first report of homology modeling of all five subtypes of acetylcholine muscarinic receptors with binding sites optimized for ligand binding. The models were evaluated for their ability to discriminate between muscarinic antagonists and decoy compounds using virtual screening using enrichment factors, area under the ROC curve (AUC), and an early enrichment measure, LogAUC. The models produce rational binding modes of docked ligands as well as good enrichment capacity when tested against property-matched decoy libraries, which demonstrates their unbiased predictive ability. To test the relative effects of homology model template selection and the binding site optimization procedure, we generated and evaluated a naïve M₂R model, using the M₃R crystal structure as a template. Our results confirm previous findings that binding site optimization using ligand(s) active at a particular receptor, i.e. including functional knowledge into the model building process, has a more pronounced effect on model quality than target-template sequence similarity. The optimized M₁R-M₅R homology models are made available as part of the Supporting Information to allow researchers to use these structures, compare them to their own results, and thus advance the development of better modeling approaches.

  11. Allosteric binding sites on muscarinic acetylcholine receptors.

    PubMed

    Wess, Jürgen

    2005-12-01

    In this issue of Molecular Pharmacology, Tränkle et al. (p. 1597) present new findings regarding the existence of a second allosteric site on the M2 muscarinic acetylcholine receptor (M2 mAChR). The M2 mAChR is a prototypic class A G protein-coupled receptor (GPCR) that has proven to be a very useful model system to study the molecular mechanisms involved in the binding of allosteric GPCR ligands. Previous studies have identified several allosteric muscarinic ligands, including the acetylcholinesterase inhibitor tacrine and the bis-pyridinium derivative 4,4'-bis-[(2,6-dichloro-benzyloxy-imino)-methyl]-1,1'-propane-1,3-diyl-bis-pyridinium dibromide (Duo3), which, in contrast to conventional allosteric muscarinic ligands, display concentration-effect curves with slope factors >1. By analyzing the interactions of tacrine and Duo3 with other allosteric muscarinic agents predicted to bind to the previously identified ;common' allosteric binding site, Tränkle et al. provide evidence suggesting that two allosteric agents and one orthosteric ligand may be able to bind to the M2 mAChR simultaneously. Moreover, studies with mutant mAChRs indicated that the M2 receptor epitopes involved in the binding of tacrine and Duo3 may not be identical. Molecular modeling and ligand docking studies suggested that the additional allosteric site probably represents a subdomain of the receptor's allosteric binding cleft. Because allosteric binding sites have been found on many other GPCRs and drugs interacting with these sites are thought to have great therapeutic potential, the study by Tränkle et al. should be of considerable general interest.

  12. Identification of a family of muscarinic acetylcholine receptor genes

    SciTech Connect

    Bonner, T.I.; Buckley, N.J.; Young, A.C.; Brann, M.R.

    1987-07-31

    Complementary DNAs for three different muscarinic acetylcholine receptors were isolated from a rat cerebral cortex library, and the cloned receptors were expressed in mammalian cells. Analysis of human and rat genomic clones indicates that there are at least four functional muscarinic receptor genes and that these genes lack introns in the coding sequence. This gene family provides a new basis for evaluating the diversity of muscarinic mechanisms in the nervous system.

  13. Differential regulation of muscarinic M2 and M3 receptor signaling in gastrointestinal smooth muscle by caveolin-1.

    PubMed

    Bhattacharya, Sayak; Mahavadi, Sunila; Al-Shboul, Othman; Rajagopal, Senthilkumar; Grider, John R; Murthy, Karnam S

    2013-08-01

    Caveolae act as scaffolding proteins for several G protein-coupled receptor signaling molecules to regulate their activity. Caveolin-1, the predominant isoform in smooth muscle, drives the formation of caveolae. The precise role of caveolin-1 and caveolae as scaffolds for G protein-coupled receptor signaling and contraction in gastrointestinal muscle is unclear. Thus the aim of this study was to examine the role of caveolin-1 in the regulation of Gq- and Gi-coupled receptor signaling. RT-PCR, Western blot, and radioligand-binding studies demonstrated the selective expression of M2 and M3 receptors in gastric smooth muscle cells. Carbachol (CCh) stimulated phosphatidylinositol (PI) hydrolysis, Rho kinase and zipper-interacting protein (ZIP) kinase activity, induced myosin phosphatase 1 (MYPT1) phosphorylation (at Thr(696)) and 20-kDa myosin light chain (MLC20) phosphorylation (at Ser(19)) and muscle contraction, and inhibited cAMP formation. Stimulation of PI hydrolysis, Rho kinase, and ZIP kinase activity, phosphorylation of MYPT1 and MLC20, and muscle contraction in response to CCh were attenuated by methyl β-cyclodextrin (MβCD) or caveolin-1 small interfering RNA (siRNA). Similar inhibition of PI hydrolysis, Rho kinase, and ZIP kinase activity and muscle contraction in response to CCh and gastric emptying in vivo was obtained in caveolin-1-knockout mice compared with wild-type mice. Agonist-induced internalization of M2, but not M3, receptors was blocked by MβCD or caveolin-1 siRNA. Stimulation of PI hydrolysis, Rho kinase, and ZIP kinase activities in response to other Gq-coupled receptor agonists such as histamine and substance P was also attenuated by MβCD or caveolin-1 siRNA. Taken together, these results suggest that caveolin-1 facilitates signaling by Gq-coupled receptors and contributes to enhanced smooth muscle function.

  14. Muscarinic M1 receptors activate phosphoinositide turnover and Ca2+ mobilisation in rat sympathetic neurones, but this signalling pathway does not mediate M-current inhibition

    PubMed Central

    del Río, Elena; Bevilacqua, Jorge A; Marsh, Stephen J; Halley, Pamela; Caulfield, Malcolm P

    1999-01-01

    The relationship between muscarinic receptor activation, phosphoinositide turnover, calcium mobilisation and M-current inhibition has been studied in rat superior cervical ganglion (SCG) neurones in primary culture. Phosphoinositide-specific phospholipase C (PLC) stimulation was measured by the accumulation of [3H]-cytidine monophosphate phosphatidate (CMP-PA) after incubation with [3H]-cytidine in the presence of Li+. The muscarinic agonist oxotremorine methiodide (oxo-M) stimulated PLC in a dose-dependent manner with an EC50 of approximately 3.5 μm. The concentration-response curve for oxo-M was shifted to the right by a factor of about 10 by pirenzepine (100 nm), suggesting a pKB (—log of the apparent dissociation constant) of 7.9 ± 0.4, while himbacine (1 μm) shifted the curve by a factor of about 13 (pKB∼7.1 ± 0.6). This indicates involvement of the M1 muscarinic receptor in this response. The accumulation of CMP-PA was localised by in situ autoradiography to SCG principal neurones, with no detectable signal in glial cells present in the primary cultures. The ability of oxo-M to release Ca2+ from inositol(1,4,5)trisphosphate (InsP3)-sensitive stores was determined by fura-2 microfluorimetry of SCG neurones voltage clamped in perforated patch mode. Oxo-M failed to evoke intracellular Ca2+ (Cai2+) mobilisation in SCG neurones voltage clamped at −60 mV, but produced a significant Cai2+ rise (67 ± 15 nm, n = 9) in cells voltage clamped at −25 mV. Thapsigargin (0.5–1 μm) caused a 70% inhibition of the oxo-M-induced Cai2+ increase, indicating its intracellular origin, while oxo-M-induced inhibition of M-current in the same cells was unaffected by thapsigargin. Our results do not support the involvement of InsP3-sensitive calcium mobilisation in M-current inhibition. PMID:10517804

  15. TRUSS, TNF-R1, and TRPC Ion Channels Synergistically Reverse Endoplasmic Reticulum Ca2+ Storage Reduction in Response to m1 Muscarinic Acetylcholine Receptor Signaling

    PubMed Central

    Mace, Kimberly E.; Lussier, Marc P.; Boulay, Guylain; Terry-Powers, Jennifer L.; Parfrey, Helen; Perraud, Anne-Laure; Riches, David W.H.

    2015-01-01

    Although most signaling responses initiated by tumor necrosis factor-α (TNF-α) occur in a Ca2+-independent fashion, TNF-α receptor signaling augments Ca2+ entry induced by Gαq/11 G-protein coupled receptors (GPCRs) in endothelial cells and increases transendothelial permeability. The signaling events involved in GPCR-induced Ca2+ influx have been characterized and involve store-operated Ca2+ entry facilitated by the Ca2+ permeable ion channel, transient receptor potential canonical 4 (TRPC4). Little is known about the mechanisms by which TNF-α receptor signaling augments GPCR-induced Ca2+ entry. T NF-α R eceptor U biquitous S ignaling and S caffolding protein (TRUSS) is a tumor necrosis factor receptor-1 (TNF-R1)-associated protein whose gene name is TRPC4-associated protein (TRPC4AP). The goal of our study was to test the hypothesis that TRUSS serves to link TNF-R1 and GPCR-signaling pathways at the level of TRPC4 by: (i) determining if TRUSS and TNF-R1 interact with TRPC4, and (ii) investigating the role of TRUSS, TNF-R1, and TRPC4 in GPCR-induced Ca2+ signaling. Here, we show that TRUSS and TNF-R1 interact with a sub-family of TRPC channels (TRPC1, 4, and 5). In addition, we show that TRUSS and TNF-R1 function together with TRPC4 to elevate endoplasmic reticulum Ca2+ filling in the context of reduced endoplasmic reticulum Ca2+ storage initiated by G-protein coupled m1 muscarinic acetylcholine receptor (m1AchR) signaling. Together, these findings suggest that TNF-R1, TRUSS, and TRPC4 augment Ca2+ loading of endoplasmic reticulum Ca2+ stores in the context of m1AchR stimulation and provide new insights into the mechanisms that connect TNF-R1 to GPCR-induced Ca2+ signaling. PMID:20458742

  16. Type 3 Muscarinic Receptors Contribute to Clearance of Citrobacter rodentium

    USDA-ARS?s Scientific Manuscript database

    Although the alpha 7 nicotinic receptor exerts anti-inflammatory effects on immune cells, the role of muscarinic receptors in mucosal homeostasis, response to enteric pathogens, and modulation of immune cell function is undefined. The contribution of type 3 muscarinic receptor (M3R) to mucosal homeo...

  17. Binding of quinolizidine alkaloids to nicotinic and muscarinic acetylcholine receptors.

    PubMed

    Schmeller, T; Sauerwein, M; Sporer, F; Wink, M; Müller, W E

    1994-09-01

    Fourteen quinolizidine alkaloids, isolated from Lupinus albus, L. mutabilis, and Anagyris foetida, were analyzed for their affinity for nicotinic and/or muscarinic acetylcholine receptors. Of the compounds tested, the alpha-pyridones, N-methylcytisine and cytisine, showed the highest affinities at the nicotinic receptor, while several quinolizidine alkaloid types were especially active at the muscarinic receptor.

  18. Muscarinic acetylcholine receptor-interacting proteins (mAChRIPs): targeting the receptorsome.

    PubMed

    Borroto-Escuela, D O; Agnati, Luigi F; Fuxe, Kjell; Ciruela, F

    2012-01-01

    Muscarinic acetylcholine receptors comprise a large family of G protein-coupled receptors that are involved in the regulation of many important functions of the central and peripheral nervous system. To achieve such a large range of physiological effects, these receptors interact with a large array of accessory proteins including scaffold molecules, ion channels and enzymes that operate as molecular transducers of muscarinic function in addition to the canonical heterotrimeric G proteins. Interestingly, as demonstrated for others G protein-coupled receptors, this type of receptor is also able to oligomerise, a fact that has been shown to play a critical role in their subcellular distribution, trafficking, and fine tuning of cholinergic signalling. On the other hand, the specificity of these receptor interactions may be largely determined by the occurrence of precise protein-interacting motifs, posttranslational modifications, and the differential tissue distribution and stoichiometry of the receptor-interacting proteins. Thus, the exhaustive cataloguing and documentation of muscarinic acetylcholine receptor-interacting proteins and the grasp of their specific function will explain key physiological differences in muscarinic-mediated cholinergic transmission. Overall, a better comprehension of the muscarinic receptor interactome will have a significant impact on the cholinergic pharmacology and thus provide previously unrealised opportunities to achieve greater specificity in muscarinic-related drug discovery and diagnostics.

  19. TRPC Channels Mediate a Muscarinic Receptor-Induced Afterdepolarization in Cerebral Cortex

    PubMed Central

    Yan, Hai-Dun; Villalobos, Claudio; Andrade, Rodrigo

    2009-01-01

    Activation of muscarinic cholinergic receptors on pyramidal cells of the cerebral cortex induces the appearance of a slow afterdepolarization that can sustain autonomous spiking after a brief excitatory stimulus. Accordingly, this phenomenon has been hypothesized to allow for the transient storage of memory traces in neuronal networks. Here we investigated the molecular basis underlying the muscarinic receptor-induced afterdepolarization using molecular biological and electrophysiological strategies. We find that the ability of muscarinic receptors to induce the inward aftercurrent underlying the slow afterdepolarization is inhibited by expression of a Gαq-11 dominant negative and is also markedly reduced in a phospholipase C β1 (PLCβ1) knock-out mouse. Furthermore, we show, using a genetically encoded biosensor, that activation of muscarinic receptor induces the breakdown of phosphatidylinositol 4,5-bisphosphate in pyramidal cells. These results indicate that the Gαq-11/PLCβ1 cascade plays a key role in the ability of muscarinic receptors to signal the inward aftercurrent. We have shown previously that the muscarinic afterdepolarization is mediated by a calcium-activated nonselective cation current, suggesting the possible involvement of TRPC channels. We find that expression of a TRPC dominant negative inhibits, and overexpression of wild-type TRPC5 or TRPC6 enhances, the amplitude of the muscarinic receptor-induced inward aftercurrent. Furthermore, we find that coexpression of TRPC5 and T-type calcium channels is sufficient to reconstitute a muscarinic receptor-activated inward aftercurrent in human embryonic kidney HEK-293 cells. These results indicate that TRPC channels mediate the muscarinic receptor-induced slow afterdepolarization seen in pyramidal cells of the cerebral cortex and suggest a possible role for TRPC channels in mnemonic processes. PMID:19675237

  20. Subtype Differences in Pre-Coupling of Muscarinic Acetylcholine Receptors

    PubMed Central

    Jakubík, Jan; Janíčková, Helena; Randáková, Alena; El-Fakahany, Esam E.; Doležal, Vladimír

    2011-01-01

    Based on the kinetics of interaction between a receptor and G-protein, a myriad of possibilities may result. Two extreme cases are represented by: 1/Collision coupling, where an agonist binds to the free receptor and then the agonist-receptor complex “collides” with the free G-protein. 2/Pre-coupling, where stable receptor/G-protein complexes exist in the absence of agonist. Pre-coupling plays an important role in the kinetics of signal transduction. Odd-numbered muscarinic acetylcholine receptors preferentially couple to Gq/11, while even-numbered receptors prefer coupling to Gi/o. We analyzed the coupling status of the various subtypes of muscarinic receptors with preferential and non-preferential G-proteins. The magnitude of receptor-G-protein coupling was determined by the proportion of receptors existing in the agonist high-affinity binding conformation. Antibodies directed against the C-terminus of the α-subunits of the individual G-proteins were used to interfere with receptor-G-protein coupling. Effects of mutations and expression level on receptor-G-protein coupling were also investigated. Tested agonists displayed biphasic competition curves with the antagonist [3H]-N-methylscopolamine. Antibodies directed against the C-terminus of the α-subunits of the preferential G-protein decreased the proportion of high-affinity sites, and mutations at the receptor-G-protein interface abolished agonist high-affinity binding. In contrast, mutations that prevent receptor activation had no effect. Expression level of preferential G-proteins had no effect on pre-coupling to non-preferential G-proteins. Our data show that all subtypes of muscarinic receptors pre-couple with their preferential classes of G-proteins, but only M1 and M3 receptors also pre-couple with non-preferential Gi/o G-proteins. Pre-coupling is not dependent on agonist efficacy nor on receptor activation. The ultimate mode of coupling is therefore dictated by a combination of the receptor subtype

  1. Evidence of paired M2 muscarinic receptors

    SciTech Connect

    Potter, L.T.; Ballesteros, L.A.; Bichajian, L.H.; Ferrendelli, C.A.; Fisher, A.; Hanchett, H.E.; Zhang, R. )

    1991-02-01

    Binding assays involving various antagonists, including N-(3H) methylscopolamine, (3H)quinuclidinyl benzilate, AFDX-116, pirenzepine, and propylbenzilylcholine mustard, disclosed only a single population of M2 muscarinic receptors in membranes from the rat brainstem (medulla, pons, and colliculi). However, competition curves between N-(3H)methylscopolamine and various agonists, including oxotremorine, cis-dioxolane, and acetylethylcholine mustard, showed approximately equal numbers of guanine nucleotide-sensitive high affinity (H) sites and guanine nucleotide-insensitive low affinity (L) sites. This 50% H phenomenon persisted in different buffers, at different temperatures, after the number of receptors was halved (and, thus, the remaining receptor to guanine nucleotide-binding protein ratio was doubled), after membrane solubilization with digitonin, and when rabbit cardiac membranes were used instead of rat brainstem membranes. Preferential occupation of H sites with acetylethylcholine mustard, and of L sites with quinuclidinyl benzilate or either mustard, yielded residual free receptor populations showing predominantly L and H sites, respectively. Low concentrations of (3H)-oxotremorine-M labeled only H sites, and the Bmax for these sites was 49% of the Bmax found with (3H)quinuclidinyl benzilate plus guanine nucleotide. These and other results are most consistent with the idea that H and L receptor sites exist on separate but dimeric receptor molecules and with the hypothesis that only the H receptors cycle between high and low affinity, depending upon interactions between this receptor molecule and a guanine nucleotide-binding protein.

  2. Amyloid beta-peptide disrupts carbachol-induced muscarinic cholinergic signal transduction in cortical neurons.

    PubMed Central

    Kelly, J F; Furukawa, K; Barger, S W; Rengen, M R; Mark, R J; Blanc, E M; Roth, G S; Mattson, M P

    1996-01-01

    Cholinergic pathways serve important functions in learning and memory processes, and deficits in cholinergic transmission occur in Alzheimer disease (AD). A subset of muscarinic cholinergic receptors are linked to G-proteins that activate phospholipase C, resulting in the liberation of inositol trisphosphate and Ca2+ release from intracellular stores. We now report that amyloid beta-peptide (Abeta), which forms plaques in the brain in AD, impairs muscarinic receptor activation of G proteins in cultured rat cortical neurons. Exposure of rodent fetal cortical neurons to Abeta25-35 and Abeta1-40 resulted in a concentration and time-dependent attenuation of carbachol-induced GTPase activity without affecting muscarinic receptor ligand binding parameters. Downstream events in the signal transduction cascade were similarly attenuated by Abeta. Carbachol-induced accumulation of inositol phosphates (IP, IP2, IP3, and IP4) was decreased and calcium imaging studies revealed that carbachol-induced release of calcium was severely impaired in neurons pretreated with Abeta. Muscarinic cholinergic signal transduction was disrupted with subtoxic levels of exposure to AP. The effects of Abeta on carbachol-induced GTPase activity and calcium release were attenuated by antioxidants, implicating free radicals in the mechanism whereby Abeta induced uncoupling of muscarinic receptors. These data demonstrate that Abeta disrupts muscarinic receptor coupling to G proteins that mediate induction of phosphoinositide accumulation and calcium release, findings that implicate Abeta in the impairment of cholinergic transmission that occurs in AD. PMID:8692890

  3. Muscarinic receptor-stimulated phosphatidylinositol turnover in the rat corpus striatum: role of muscarinic receptor subtypes and regulation

    SciTech Connect

    Monsma, F.J.

    1987-01-01

    The coupling between the M1 and M2 muscarinic receptor subtypes and phosphatidylinositol (Pl) hydrolysis has been examined in the corpus striatum and cerebral cortex of the rat brain. Receptor binding by the various muscarinic ligands was assessed using a preparation of intact brain cell aggregates, under similar conditions as the assay of Pl hydrolysis. In striatal cell aggregates, (/sup 3/H)-quinuclidinyl benzilate ((/sup 3/H)-QNB) bound to a single class of muscarinic sites with high affinity, inhibition of (/sup 3/H)-QNB binding by muscarinic receptor ligands which exhibit selectivity for subtypes of the muscarinic receptor revealed the presence of both the M1 and M2 subtypes in approximately equal numbers.

  4. Regulation and ontogeny of subtypes of muscarinic receptors and muscarinic receptor-mediated

    SciTech Connect

    Lee, W.

    1989-01-01

    The densities of total and M1 muscarinic receptors were measured using the muscarinic receptor antagonists {sup 3}H-quinuclidinyl benzilate and {sup 3}H-pirenzepine, respectively. Thus, the difference between the density of {sup 3}H-quinuclidinyl benzilate and {sup 3}H-pirenzepine binding sites represents the density of M2 sites. In addition, there is no observable change in either acetylcholine-stimulated phosphoinositide breakdown (suggested to be an M1 receptor-mediated response) or in carbachol-mediated inhibition of cyclic AMP accumulation (suggested to be an M2 receptor-mediated response) in slices of cortex+dorsal hippocampus following chronic atropine administration. In other experiments, it has been shown that the M1 and M2 receptors in rat cortex have different ontogenetic profiles. The M2 receptor is present at adult levels at birth, while the M1 receptor develops slowly from low levels at postnatal week 1 to adult levels at postnatal week 3. The expression of acetylcholine-stimulated phosphoinositide breakdown parallels the development of M1 receptors, while the development of carbachol-mediated inhibition of cyclic AMP accumulation occurs abruptly between weeks 2 and 3 postnatally.

  5. Muscarinic receptor binding and muscarinic receptor-mediated inhibition of adenylate cyclase in rat brain myelin

    SciTech Connect

    Larocca, J.N.; Ledeen, R.W.; Dvorkin, B.; Makman, M.H.

    1987-12-01

    High-affinity muscarinic cholinergic receptors were detected in myelin purified from rat brain stem with use of the radioligands /sup 3/H-N-methylscopolamine (/sup 3/H-NMS), /sup 3/H-quinuclidinyl benzilate (/sup 3/H-QNB), and /sup 3/H-pirenzepine. /sup 3/H-NMS binding was also present in myelin isolated from corpus callosum. In contrast, several other receptor types, including alpha 1- and alpha 2-adrenergic receptors, present in the starting brain stem, were not detected in myelin. Based on Bmax values from Scatchard analyses, /sup 3/H-pirenzepine, a putative M1 selective ligand, bound to about 25% of the sites in myelin labeled by /sup 3/H-NMS, a nonselective ligand that binds to both M1 and M2 receptor subtypes. Agonist affinity for /sup 3/H-NMS binding sites in myelin was markedly decreased by Gpp(NH)p, indicating that a major portion of these receptors may be linked to a second messenger system via a guanine-nucleotide regulatory protein. Purified myelin also contained adenylate cyclase activity; this activity was stimulated several fold by forskolin and to small but significant extents by prostaglandin E1 and the beta-adrenergic agonist isoproterenol. Myelin adenylate cyclase activity was inhibited by carbachol and other muscarinic agonists; this inhibition was blocked by the antagonist atropine. Levels in myelin of muscarinic receptors were 20-25% and those of forskolin-stimulated adenylate cyclase 10% of the values for total particulate fraction of whole brain stem. These levels in myelin are appreciably greater than would be predicted on the basis of contamination. Also, additional receptors and adenylate cyclase, added by mixing nonmyelin tissue with whole brain stem, were quantitatively removed during the purification procedure.

  6. Muscarinic receptor pharmacology and circuitry for the modulation of cognition.

    PubMed

    Bubser, Michael; Byun, Nellie; Wood, Michael R; Jones, Carrie K

    2012-01-01

    The muscarinic cholinergic system constitutes an important part of the neuronal circuitry that modulates normal cognition. Muscarinic receptor antagonists are well known to produce or exacerbate impairments in attention, learning, and memory. Conversely, both direct-acting muscarinic receptor agonists and indirect-acting muscarinic cholinergic agonists, such as acetylcholinesterase inhibitors, have shown cognition-enhancing properties, including improvements in normal cognitive function, reversal of cognitive deficits induced by muscarinic receptor antagonists, and attenuation of cognitive deficits in psychiatric and neurological disorders, such as Alzheimer's disease and schizophrenia. However, until recently, the lack of small molecule ligands that antagonize or activate specific muscarinic acetylcholine receptor (mAChR) subtypes with high selectivity has been a major obstacle in defining the relative contributions of individual mAChRs to different aspects of cognitive function and for the development of novel therapeutic agents. These limitations may be potentially overcome by the recent discovery of novel mAChR subtype-selective compounds, notably allosteric agonists and positive allosteric modulators, which exhibit greater selectivity for individual mAChR subtypes than previous mAChR orthosteric agonists. In preclinical studies, these novel ligands have shown promising efficacy in several models for the enhancement of cognition. In this chapter, we will review the muscarinic cholinergic circuitry and pharmacology of mAChR agonists and antagonists relevant to the modulation of different aspects of cognition in animals and clinical populations.

  7. Ethanol inhibits neuritogenesis induced by astrocyte muscarinic receptors.

    PubMed

    Guizzetti, Marina; Moore, Nadia H; Giordano, Gennaro; VanDeMark, Kathryn L; Costa, Lucio G

    2010-09-01

    In utero alcohol exposure can lead to fetal alcohol spectrum disorders, characterized by cognitive and behavioral deficits. In vivo and in vitro studies have shown that ethanol alters neuronal development. We have recently shown that stimulation of M(3) muscarinic receptors in astrocytes increases the synthesis and release of fibronectin, laminin, and plasminogen activator inhibitor-1, causing neurite outgrowth in hippocampal neurons. As M(3) muscarinic receptor signaling in astroglial cells is strongly inhibited by ethanol, we hypothesized that ethanol may also inhibit neuritogenesis in hippocampal neurons induced by carbachol-stimulated astrocytes. In the present study, we report that the effect of carbachol-stimulated astrocytes on hippocampal neuron neurite outgrowth was inhibited in a concentration-dependent manner (25-100 mM) by ethanol. This effect was because of the inhibition of the release of fibronectin, laminin, and plasminogen activator inhibitor-1. Similar effects on neuritogenesis and on the release of astrocyte extracellular proteins were observed after the incubation of astrocytes with carbachol in the presence of 1-butanol, another short-chain alcohol, which like ethanol is a competitive substrate for phospholipase D, but not by tert-butanol, its analog that is not a substrate for this enzyme. This study identifies a potential novel mechanism involved in the developmental effects of ethanol mediated by the interaction of ethanol with cell signaling in astrocytes, leading to an impairment in neuron-astrocyte communication.

  8. Co-incident signalling between mu-opioid and M3 muscarinic receptors at the level of Ca2+ release from intracellular stores: lack of evidence for Ins(1,4,5)P3 receptor sensitization.

    PubMed Central

    Samways, Damien S K; Li, Wen-hong; Conway, Stuart J; Holmes, Andrew B; Bootman, Martin D; Henderson, Graeme

    2003-01-01

    Activation of G(i)/G(o)-coupled opioid receptors increases [Ca2+]i (intracellular free-Ca2+ concentration), but only if there is concomitant G(q)-coupled receptor activation. This G(i)/G(o)-coupled receptor-mediated [Ca2+]i increase does not appear to result from further production of Ins P3 [Ins(1,4,5) P3] in SH-SY5Y cells. In the present study, fast-scanning confocal microscopy revealed that activation of mu-opioid receptors alone by 1 muM DAMGO ([L-Ala, NMe-Phe, Gly-ol]-enkephalin) did not stimulate the Ins P3-dependent elementary Ca2+-signalling events (Ca2+ puffs), whereas DAMGO did evoke Ca2+ puffs when applied during concomitant activation of M3 muscarinic receptors with 1 muM carbachol. We next determined whether mu-opioid receptor activation might increase [Ca2+]i by sensitizing the Ins P3 receptor to Ins P3. DAMGO did not potentiate the amplitude of the [Ca2+]i increase evoked by flash photolysis of the caged Ins P3 receptor agonist, caged 2,3-isopropylidene-Ins P3, whereas the Ins P3 receptor sensitizing agent, thimerosal (10 muM), did potentiate this response. DAMGO also did not prolong the rate of decay of the increase in [Ca2+]i evoked by flash photolysis of caged 2,3-isopropylidene-Ins P3. Furthermore, DAMGO did not increase [Ca2+]i in the presence of the cell-membrane-permeable Ins P3 receptor agonist, Ins P3 hexakis(butyryloxymethyl) ester. Therefore it appears that mu-opioid receptors do not increase [Ca2+]i through either Ins P3 receptor sensitization, enhancing the releasable pool of Ca2+ or inhibition of Ca2+ removal from the cytoplasm. PMID:12880387

  9. Characterization of PCS1055, a novel muscarinic M4 receptor antagonist.

    PubMed

    Croy, Carrie H; Chan, Wai Y; Castetter, Andrea M; Watt, Marla L; Quets, Anne T; Felder, Christian C

    2016-07-05

    Identification of synthetic ligands selective for muscarinic receptor subtypes has been challenging due to the high sequence identity and structural homology among the five muscarinic acetylcholine receptors. Here, we report the pharmacological characterization of PCS1055, a novel muscarinic M4 receptor antagonist. PCS1055 inhibited radioligand [(3)H]-NMS binding to the M4 receptor with a Ki=6.5nM. Though the potency of PCS1055 is lower than that of pan-muscarinic antagonist atropine, it has better subtype selectivity over previously reported M4-selective reagents such as the muscarinic-peptide toxins (Karlsson et al., 1994; Santiago and Potter, 2001a) at the M1 subtype, and benzoxazine ligand PD102807 at the M3-subtype (Bohme et al., 2002). A detailed head-to-head comparison study using [(3)H]-NMS competitive binding assays characterizes the selectivity profiles of PCS1055 to that of other potent muscarinic-antagonist compounds PD102807, tropicamide, AF-DX-384, pirenzapine, and atropine. In addition to binding studies, the subtype specificity of PCS1055 is also demonstrated by functional receptor activation as readout by GTP-γ-[(35)S] binding. These GTP-γ-[(35)S] binding studies showed that PCS1055 exhibited 255-, 69.1-, 342- and >1000-fold greater inhibition of Oxo-M activity at the M4 versus the M1-, M2(-), M3-or M5 receptor subtypes, respectively. Schild analyses indicates that PCS1055 acts as a competitive antagonist to muscarinic M4 receptor, and confirms the affinity of the ligand to be low nanomolar, Kb=5.72nM. Therefore, PCS1055 represents a new M4-preferring antagonist that may be useful in elucidating the roles of M4 receptor signaling. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Current excitement from insect muscarinic receptors.

    PubMed

    Trimmer, B A

    1995-02-01

    Recent electrophysiological, pharmacological and molecular studies suggest that muscarinic ACh receptors (mAChRs) in insects are related to, but distinct from, their mammalian counterparts. Insect mAChRs perform two primary roles that are distinguished by their locations. Presynaptic mAChRs, present on sensory terminals, inhibit transmitter release, thereby reducing the effectiveness of specific afferent inputs. In contrast, postsynaptic mAChRs depolarize and increase the excitability of motoneurons and interneurons, thereby acting as dynamic-gain controls. This postsynaptic modulation is achieved in different ways in specific neurons but generally results from the activation of persistent inward and outward currents. At the level of neural processing, these distinct roles enable insect mAChRs to regulate the transfer of sensory information, and modulate the contributions of central neurons to central pattern generators and reflexes. Because these phenomena can be studied in identified neurons, a combination of physiological and molecular studies of mAChRs in insects should help to elucidate some of their behavioral roles. Furthermore, such studies could lead to the identification of general mechanisms of functional plasticity in neuronal networks.

  11. Acetylcholine acts through M3 muscarinic receptor to activate the EGFR signaling and promotes gastric cancer cell proliferation

    PubMed Central

    Yu, Huangfei; Xia, Hongwei; Tang, Qiulin; Xu, Huanji; Wei, Guoqing; Chen, Ying; Dai, Xinyu; Gong, Qiyong; Bi, Feng

    2017-01-01

    Acetylcholine (ACh), known as a neurotransmitter, regulates the functions of numerous fundamental central and peripheral nervous system. Recently, emerging evidences indicate that ACh also plays an important role in tumorigenesis. However, little is known about the role of ACh in gastric cancer. Here, we reported that ACh could be auto-synthesized and released from MKN45 and BGC823 gastric cancer cells. Exogenous ACh promoted cell proliferation in a does-dependent manner. The M3R antagonist 4-DAMP, but not M1R antagonist trihexyphenidyl and M2/4 R antagonist AFDX-116, could reverse the ACh-induced cell proliferation. Moreover, ACh, via M3R, activated the EGFR signaling to induce the phosphorylation of ERK1/2 and AKT, and blocking EGFR pathway by specific inhibitor AG1478 suppressed the ACh induced cell proliferation. Furthermore, the M3R antagonist 4-DAMP and darifenacin could markedly inhibit gastric tumor formation in vivo. 4-DAMP could also significantly enhance the cytotoxic activity of 5-Fu against the MKN45 and BGC823 cells, and induce the expression of apoptosis-related proteins such as Bax and Caspase-3. Together, these findings indicated that the autocrine ACh could act through M3R and the EGFR signaling to promote gastric cancer cells proliferation, targeting M3R or EGFR may provide us a potential therapeutic strategy for gastric cancer treatment. PMID:28102288

  12. Role of Parasympathetic Nerves and Muscarinic Receptors in Allergy and Asthma

    PubMed Central

    Scott, Gregory D.; Fryer, Allison D.

    2014-01-01

    Parasympathetic nerves control the symptoms and inflammation of allergic diseases primarily by signaling through peripheral muscarinic receptors. Parasympathetic signaling targets classic effector tissues such as airway smooth muscle and secretory glands and mediates acute symptoms of allergic disease such as airway narrowing and increased mucus secretion. In addition, parasympathetic signaling modulates inflammatory cells and non-neuronal resident cell types such as fibroblasts and smooth muscle contributing to chronic allergic inflammation and tissue remodeling. Importantly, muscarinic antagonists are experiencing a rebirth for the treatment of asthma and may be useful for treating other allergic diseases. PMID:22767057

  13. Comparison of muscarinic receptor selectivity of solifenacin and oxybutynin in the bladder and submandibular gland of muscarinic receptor knockout mice.

    PubMed

    Ito, Yoshihiko; Oyunzul, Luvsandorj; Yoshida, Akira; Fujino, Tomomi; Noguchi, Yukiko; Yuyama, Hironori; Ohtake, Akiyoshi; Suzuki, Masanori; Sasamata, Masao; Matsui, Minoru; Yamada, Shizuo

    2009-08-01

    Solifenacin is a novel selective antagonist of M(3) muscarinic receptor developed for the treatment of overactive bladder. The current study was undertaken to characterize in vivo muscarinic receptor subtype selectivity of solifenacin in the bladder and submandibular gland by using muscarinic receptor subtype knockout (KO) mice. Muscarinic receptors in the bladder and submandibular gland of wild type, M(2)R KO and M(3)R KO mice under in vitro and after oral administration of solifenacin and oxybutynin were measured by radioligand binding assay using [N-methyl-(3)H]scopolamine ([(3)H]NMS). There was little difference between the bladder and submandibular gland of M(2)R KO mice in the receptor binding activities of oxybutynin and solifenacin in vitro, suggesting equal affinity for residual (predominantly M(3) subtype) muscarinic receptors in both tissues. In contrast, compared with oral oxybutynin, oral administration of solifenacin exerted a significantly greater activity to bind muscarinic receptors in the bladder of M(2)R KO mice, while exhibiting a significantly less activity to bind those in the submandibular gland. In the bladder and submandibular gland of M(3)R KO mice, the binding activity of solifenacin and oxybutynin showed no significant difference. Plasma concentrations of solifenacin and oxybutynin after oral administration differed little among wild type, M(2)R KO and M(3)R KO mice. The results indicate that oral solifenacin, unlike oral oxybutynin, may selectively bind to the muscarinic M(3) subtype in the bladder compared with such receptors in the submandibular gland in vivo. Oral solifenacin may be advantageous for the treatment of overactive bladder, in terms of high affinity for M(3) receptors in the bladder.

  14. Characterization of muscarinic receptor subtypes in human tissues

    SciTech Connect

    Giraldo, E.; Martos, F.; Gomez, A.; Garcia, A.; Vigano, M.A.; Ladinsky, H.; Sanchez de La Cuesta, F.

    1988-01-01

    The affinities of selective, pirenzepine and AF-DX 116, and classical, N-methylscopolamine and atropine, muscarinic cholinergic receptor antagonists were investigated in displacement binding experiments with (/sup 3/H)Pirenzepine and (/sup 3/H)N-methylscopolamine in membranes from human autoptic tissues (forebrain, cerebellum, atria, ventricle and submaxillary salivary glands). Affinity estimates of N-methylscopolamine and atropine indicated a non-selective profile. Pirenzepine showed differentiation between the M/sub 1/ neuronal receptor of the forebrain and the receptors in other tissues while AF-DX 116 clearly discriminated between muscarinic receptors of heart and glands. The results in human tissues confirm the previously described selectivity profiles of pirenzepine and AF-DX 116 in rat tissues. These findings thus reveal the presence also in man of three distinct muscarinic receptor subtypes: the neuronal M/sub 1/, the cardiac M/sub 2/ and the glandular M/sub 3/.

  15. Analgesic Effects Mediated by Muscarinic Receptors: Mechanisms and Pharmacological Approaches.

    PubMed

    De Angelis, Federica; Tata, Ada Maria

    2016-01-01

    Chronic pain represents a research field on great clinical relevance and social impactful. It is associated to a variety of pathological events causing un altered excitability of peripheral nerves derived by tissue damage depending on physical, biological and chemical injury. In the last years much attention has been paid in the identification of novel molecules involved in mediating pain sensation useful as therapeutic tools for the development of new analgesic drugs. Muscarinic receptors are widely distributed both in the central and peripheral nervous system. It is known that muscarinic agonists cause analgesic effects via spinal and supraspinal mechanisms. Considering that the analgesia induced by cholinergic agonists is comparable to that observed with morphine, the identification of receptor subtypes involved and the identification of the muscarinic ligands capable of selectively activate these receptors, is of considerable interest for potential therapeutic application. In the present review we describe the role of muscarinic receptors in mediating central and peripheral pain and the mechanisms downstream these receptors responsible of the modulation of nociceptive stimuli. Moreover the therapeutic perspectives and the identification of potential drugs binding muscarinic receptors involved in pain modulation will also be discussed.

  16. Cholinergic impact on neuroplasticity drives muscarinic M1 receptor mediated differentiation into neurons.

    PubMed

    Benninghoff, Jens; Rauh, Werner; Brantl, Victor; Schloesser, Robert J; Moessner, Rainald; Möller, Hans-Jürgen; Rujescu, Dan

    2013-04-01

    Increasing evidence indicates that canonical neurotransmitters act as regulatory signals during neuroplasticity. Here, we report that muscarinic cholinergic neurotransmission stimulates differentiation of adult neural stem cells in vitro. Adult neural stem cells (ANSC) dissociated from the adult mouse hippocampus were expanded in culture with basic fibroblast growth factor (BFGF) and epidermal growth factor (EGF). Carbachol (CCh), an analog of acetylcholine (ACh) significantly enhanced de novo differentiation into neurons on bFGF- and EGF-deprived stem cells as shown by the percentage of TUJ1 positive cells. By contrast, pirenzepine (PIR), a muscarinic M1 receptor antagonist, reduced the generation of neurons. Activation of cholinergic signaling drives the de novo differentiation of uncommitted stem cells into neurons. These effects appear to be predominantly mediated via the muscarinic M1 receptor subtype.

  17. Muscarinic receptor agonists stimulate matrix metalloproteinase 1-dependent invasion of human colon cancer cells

    SciTech Connect

    Raufman, Jean-Pierre; Cheng, Kunrong; Saxena, Neeraj; Chahdi, Ahmed; Belo, Angelica; Khurana, Sandeep; Xie, Guofeng

    2011-11-18

    Highlights: Black-Right-Pointing-Pointer Muscarinic receptor agonists stimulated robust human colon cancer cell invasion. Black-Right-Pointing-Pointer Anti-matrix metalloproteinase1 antibody pre-treatment blocks cell invasion. Black-Right-Pointing-Pointer Bile acids stimulate MMP1 expression, cell migration and MMP1-dependent invasion. -- Abstract: Mammalian matrix metalloproteinases (MMPs) which degrade extracellular matrix facilitate colon cancer cell invasion into the bloodstream and extra-colonic tissues; in particular, MMP1 expression correlates strongly with advanced colon cancer stage, hematogenous metastasis and poor prognosis. Likewise, muscarinic receptor signaling plays an important role in colon cancer; muscarinic receptors are over-expressed in colon cancer compared to normal colon epithelial cells. Muscarinic receptor activation stimulates proliferation, migration and invasion of human colon cancer cells. In mouse intestinal neoplasia models genetic ablation of muscarinic receptors attenuates carcinogenesis. In the present work, we sought to link these observations by showing that MMP1 expression and activation plays a mechanistic role in muscarinic receptor agonist-induced colon cancer cell invasion. We show that acetylcholine, which robustly increases MMP1 expression, stimulates invasion of HT29 and H508 human colon cancer cells into human umbilical vein endothelial cell monolayers - this was abolished by pre-incubation with atropine, a non-selective muscarinic receptor inhibitor, and by pre-incubation with anti-MMP1 neutralizing antibody. Similar results were obtained using a Matrigel chamber assay and deoxycholyltaurine (DCT), an amidated dihydroxy bile acid associated with colon neoplasia in animal models and humans, and previously shown to interact functionally with muscarinic receptors. DCT treatment of human colon cancer cells resulted in time-dependent, 10-fold increased MMP1 expression, and DCT-induced cell invasion was also blocked by pre

  18. Binding of tropane alkaloids to nicotinic and muscarinic acetylcholine receptors.

    PubMed

    Schmeller, T; Sporer, F; Sauerwein, M; Wink, M

    1995-07-01

    Fourteen tropane and related alkaloids were analyzed for their affinity for nicotinic and/or muscarinic acetylcholine receptors. The biogenetic intermediates littorine, 6 beta-hydroxyhyoscyamine, 7 beta-hydroxyhyoscyamine exhibit similar affinities at the muscarinic receptor as scopolamine and atropine. The quarternary derivatives N-methylatropine and N-methylscopolamine show the highest binding with IC50 values of less than 100 pM and 300 pM, respectively. The tropane alkaloids (including cocaine) also bind to the nicotinic acetylcholine receptor, albeit with much lower affinities.

  19. Biochemical and immunological studies of the Muscarinic acetylcholine receptor

    SciTech Connect

    Gainer, M.W.

    1985-01-01

    Muscarinic acetylcholine receptors were solubilized from bovine brain membranes with 3(3-cholamidopropyl)dimethylammonio)propanesulfonate (CHAPS). A combination of 10 mM CHAPS and 1 M NaCl solubilized 15-40% of the specific receptor binding sites from these membranes. The solubilized receptors displayed high affinity binding of the muscarinic antagonist, (/sup 3/H)quinuclidinyl benzilate with a K/sub D/ = 300 pM. In addition, the solubilized and retained guanyl nucleotide regulation of agonist binding characteristic of membrane bound receptors. Gel filtration experiments showed that solubilized receptors from cortex and cerebellum had different elution profiles. Analysis by sucrose density gradient centrifugation showed that receptors in the lower molecular weight peak sedimented with a coefficient of 5S. Receptors in the larger molecular weight peak sedimented to the bottom of the gradient. Attempts to purify receptors by chromatography on propylbenzilycholine Sepharose were unsuccessful. The technique used to attach the ligand to the solid support, however, was used to synthesize a PrBCM-BSA conjugate and the conjugate used as an antigen in the production of anti-ligand antibodies. Two anti-PrBCM monoclonal antibodies were isolated that recognize muscarinic but not nicotinic cholinergic ligands. The abilities of the antibodies to recognize other muscarinic ligands indicated the antibodies recognized a portion of PrBCM involved in binding to the receptor. Construction of an antibody affinity resin resulted in the purification of this fragment a minimum of 170 fold.

  20. Molecular alteration of a muscarinic acetylcholine receptor system during synaptogenesis

    SciTech Connect

    Large, T.H.; Cho, N.J.; De Mello, F.G.; Klein, W.L.

    1985-07-25

    Biochemical properties of the muscarinic acetylcholine receptor system of the avian retina were found to change during the period when synapses form in ovo. Comparison of ligand binding to membranes obtained before and after synaptogenesis showed a significant increase in the affinity, but not proportion, of the high affinity agonist-binding state. There was no change in receptor sensitivity to antagonists during this period. Pirenzepine binding, which can discriminate muscarinic receptor subtypes, showed the presence of a single population of low affinity sites (M2) before and after synaptogenesis. The change in agonist binding was not due to the late development of receptor function. However, detergent-solubilization of membranes eliminated differences in agonist binding between receptors from embryos and hatched chicks, suggesting a developmental change in interactions of the receptor with functionally related membrane components. A possible basis for altered interactions was obtained from isoelectric point data showing that the muscarinic receptor population underwent a transition from a predominantly low pI form (4.25) in 13 day embryos to a predominantly high pI form (4.50) in newly hatched chicks. The possibility that biochemical changes in the muscarinic receptor play a role in differentiation of the system by controlling receptor position on the surface of nerve cells is discussed.

  1. Characterization of muscarinic cholinergic receptor subtypes in human peripheral lung

    SciTech Connect

    Bloom, J.W.; Halonen, M.; Yamamura, H.I.

    1988-02-01

    The authors have characterized the muscarinic cholinergic receptor subtypes in human peripheral lung membranes using the selective muscarinic antagonist (/sup 3/H)pirenzepine ((/sup 3/H)PZ) and the classical muscarinic antagonist (/sup 3/H)(-)-quinuclidinyl benzilate. High-affinity binding with pharmacologic specificity was demonstrated for both radioligands. The high affinity Kd for (/sup 3/H)PZ binding determined from saturation isotherms was 5.6 nM, and the Kd for (/sup 3/H)(-)-quinuclidinyl benzilate binding was 14.3 pM. Approximately 62% of the total muscarinic binding sites in human peripheral lung bind (/sup 3/H)PZ with high affinity. There was no significant effect of the guanine nucleotide, guanyl-5'-yl imidodiphosphate, on the inhibition of (/sup 3/H)(-)-quinyclidinyl benzilate binding by the muscarinic agonist carbachol in peripheral lung membranes. If the muscarinic receptor with high affinity for PZ has an important role in bronchoconstriction, its characterization could result in the development of more selective bronchodilators.

  2. Acetylcholine stimulates cortical precursor cell proliferation in vitro via muscarinic receptor activation and MAP kinase phosphorylation.

    PubMed

    Ma, W; Maric, D; Li, B S; Hu, Q; Andreadis, J D; Grant, G M; Liu, Q Y; Shaffer, K M; Chang, Y H; Zhang, L; Pancrazio, J J; Pant, H C; Stenger, D A; Barker, J L

    2000-04-01

    Increasing evidence has shown that some neurotransmitters act as growth-regulatory signals during brain development. Here we report a role for the classical neurotransmitter acetylcholine (ACh) to stimulate proliferation of neural stem cells and stem cell-derived progenitor cells during neural cell lineage progression in vitro. Neuroepithelial cells in the ventricular zone of the embryonic rat cortex were found to express the m2 subtype of the muscarinic receptor. Neural precursor cells dissociated from the embryonic rat cortical neuroepithelium were expanded in culture with basic fibroblast growth factor (bFGF). reverse transcriptase-polymerase chain reaction (RT-PCR) revealed the presence of m2, m3 and m4 muscarinic receptor subtype transcripts, while immunocytochemistry demonstrated m2 protein. ACh and carbachol induced an increase in cytosolic Ca2+ and membrane currents in proliferating (BrdU+) cells, both of which were abolished by atropine. Exposure of bFGF-deprived precursor cells to muscarinic agonists not only increased both cell number and DNA synthesis, but also enhanced differentiation of neurons. These effects were blocked by atropine, indicating the involvement of muscarinic ACh receptors. The growth-stimulating effects were also antagonized by a panel of inhibitors of second messengers, including 1,2-bis-(O-aminophenoxy)-ethane-N,N,N', N'-tetraacetic acid (BAPTA-AM) to chelate cytosolic Ca2+, EGTA to complex extracellular Ca2+, pertussis toxin, which uncouples certain G-proteins, the protein kinase C inhibitor H7 and the mitogen-activated protein kinase (MAPK) inhibitor PD98059. Muscarinic agonists activated MAPK, which was significantly inhibited by atropine and the same panel of inhibitors. Thus, muscarinic receptors expressed by neural precursors transduce a growth-regulatory signal during neurogenesis via pathways involving pertussis toxin-sensitive G-proteins, Ca2+ signalling, protein kinase C activation, MAPK phosphorylation and DNA synthesis.

  3. Non-Neuronal Functions of the M2 Muscarinic Acetylcholine Receptor

    PubMed Central

    Ockenga, Wymke; Kühne, Sina; Bocksberger, Simone; Banning, Antje; Tikkanen, Ritva

    2013-01-01

    Acetylcholine is an important neurotransmitter whose effects are mediated by two classes of receptors. The nicotinic acetylcholine receptors are ion channels, whereas the muscarinic receptors belong to the large family of G protein coupled seven transmembrane helix receptors. Beyond its function in neuronal systems, it has become evident that acetylcholine also plays an important role in non-neuronal cells such as epithelial and immune cells. Furthermore, many cell types in the periphery are capable of synthesizing acetylcholine and express at least some of the receptors. In this review, we summarize the non-neuronal functions of the muscarinic acetylcholine receptors, especially those of the M2 muscarinic receptor in epithelial cells. We will review the mechanisms of signaling by the M2 receptor but also the cellular trafficking and ARF6 mediated endocytosis of this receptor, which play an important role in the regulation of signaling events. In addition, we provide an overview of the M2 receptor in human pathological conditions such as autoimmune diseases and cancer. PMID:24705159

  4. Muscarinic receptors of the albino rabbit ciliary process.

    PubMed

    Mallorga, P; Babilon, R W; Buisson, S; Sugrue, M F

    1989-04-01

    Muscarinic receptor binding sites were identified in membranes prepared from albino rabbit ciliary processes, using the muscarinic antagonist [3H]L-quinuclidinyl benzylate as the radioligand. Analysis of saturation binding experiments demonstrated that [3H]L-quinuclidinyl benzylate bound to an apparent homogeneous population of binding sites with a Kd value of 6.4 pm and a Bmax value of 155 fmol mg-1 protein. Seventy percent (70%) of binding sites showed high affinity for pirenzepine, i.e. belonged to the M1 subtype. In contrast, AF-DX 116 was unable to discriminate between subtypes of muscarinic binding sites in this tissue. Carbachol caused a dose-dependent increase in phosphatidylinositol turnover (EC50 = 154 microM) in ciliary processes. A maximum stimulation of 652% of basal activity was obtained following a 45 min incubation with 10 mM carbachol. The potency of muscarinic antagonists to block the carbachol-induced response was comparable to that found for M1 receptors in other tissues. Oxotremorine and pilocarpine behaved like partial agonists in this assay. The carbachol-induced increase in phosphatidylinositol turnover was also observed in a suspension of epithelial cells from ciliary processes and it was blocked by atropine; thus, indicating the presence of muscarinic receptors functionally coupled to phosphatidylinositol turnover in these cells.

  5. Muscarinic and dopaminergic receptor subtypes on striatal cholinergic interneurons

    SciTech Connect

    Dawson, V.L.; Dawson, T.M.; Wamsley, J.K. )

    1990-12-01

    Unilateral stereotaxic injection of small amounts of the cholinotoxin, AF64A, caused minimal nonselective tissue damage and resulted in a significant loss of the presynaptic cholinergic markers (3H)hemicholinium-3 (45% reduction) and choline acetyltransferase (27% reduction). No significant change from control was observed in tyrosine hydroxylase or tryptophan hydroxylase activity; presynaptic neuronal markers for dopamine- and serotonin-containing neurons, respectively. The AF64A lesion resulted in a significant reduction of dopamine D2 receptors as evidenced by a decrease in (3H)sulpiride binding (42% reduction) and decrease of muscarinic non-M1 receptors as shown by a reduction in (3H)QNB binding in the presence of 100 nM pirenzepine (36% reduction). Saturation studies revealed that the change in (3H)sulpiride and (3H)QNB binding was due to a change in Bmax not Kd. Intrastriatal injection of AF64A failed to alter dopamine D1 or muscarinic M1 receptors labeled with (3H)SCH23390 and (3H)pirenzepine, respectively. In addition, no change in (3H)forskolin-labeled adenylate cyclase was observed. These results demonstrate that a subpopulation of muscarinic receptors (non-M1) are presynaptic on cholinergic interneurons (hence, autoreceptors), and a subpopulation of dopamine D2 receptors are postsynaptic on cholinergic interneurons. Furthermore, dopamine D1, muscarinic M1 and (3H)forskolin-labeled adenylate cyclase are not localized to striatal cholinergic interneurons.

  6. Muscarinic receptor-mediated excitation of rat intracardiac ganglion neurons.

    PubMed

    Hirayama, Michiko; Ogata, Masanori; Kawamata, Tomoyuki; Ishibashi, Hitoshi

    2015-08-01

    Modulation of the membrane excitability of rat parasympathetic intracardiac ganglion neurons by muscarinic receptors was studied using an amphotericin B-perforated patch-clamp recording configuration. Activation of muscarinic receptors by oxotremorine-M (OxoM) depolarized the membrane, accompanied by repetitive action potentials. OxoM evoked inward currents under voltage-clamp conditions at a holding potential of -60 mV. Removal of extracellular Ca(2+) markedly increased the OxoM-induced current (IOxoM). The inward IOxoM in the absence of extracellular Ca(2+) was fully inhibited by removal of extracellular Na(+), indicating the involvement of non-selective cation channels. The IOxoM was inhibited by organic cation channel antagonists including SKF-96365 and ML-204. The IOxoM was antagonized by muscarinic receptor antagonists with the following potency: 4-DAMP > pirenzepine = darifenacin > methoctramine. Muscarinic toxin 7 (MT-7), a highly selective inhibitor for M1 receptor, produced partial inhibition of the IOxoM. In the presence of MT-7, concentration-inhibition curve of the M3-preferring antagonist darifenacin was shifted to the left. These results suggest the contribution of M1 and M3 receptors to the OxoM response. The IOxoM was inhibited by U-73122, a phospholipase C inhibitor. The membrane-permeable IP3 receptor blocker xestospongin C also inhibited the IOxoM. Furthermore, pretreatment with thapsigargin and BAPTA-AM inhibited the IOxoM, while KN-62, a blocker of Ca(2+)/calmodulin-dependent protein kinase II, had no effect. These results suggest that the activation mechanism involves a PLC pathway, release of Ca(2+) from intracellular Ca(2+) stores and calmodulin. The cation channels activated by muscarinic receptors may play an important role in neuronal membrane depolarization in rat intracardiac ganglion neurons.

  7. Functional characterization of muscarinic receptors in murine airways.

    PubMed Central

    Garssen, J.; Van Loveren, H.; Gierveld, C. M.; Van der Vliet, H.; Nijkamp, F. P.

    1993-01-01

    1. The effects of muscarinic receptor antagonists considered to be selective for M1 receptors (pirenzepine; PZ), M2 receptors (AFDX-116), and for M3 receptors (4-diphenyl acetoxy N-methyl-piperidine (4-DAMP)) were used to investigate the existence of muscarinic receptors subtypes in murine airways. Atropine was used as a nonselective antagonist. The effects of these antagonists were studied upon tracheal contractions induced either by EFS (electric field stimulation) or by application of an exogenous cholinoceptor agonist (arecoline). 2. The muscarinic receptor antagonists tested inhibited arecoline-induced tracheal contractions with the following rank order of potency: 4-DAMP = atropine > pirenzepine = AFDX-116. The rank order of potency of the muscarinic antagonists used in inhibiting EFS-induced tracheal contractions was: 4-DAMP = atropine > PZ > AFDX-116. The pA2 values for these antagonists were similar when compared to the pA2 values determined in guinea-pig and bovine airway smooth muscle. 3. In addition to in vitro studies, the effects of inhalation of the different muscarinic antagonists on lung function parameters in vivo were investigated. Inhalation of 4-DAMP induced a decrease in airway resistance and an increase in lung compliance. In contrast, inhalation of AFDX-116 induced an increase in airway resistance and almost no change in lung compliance. Apart from some minor effects of atropine on airway resistance, atropine, PZ, and pilocarpine failed to induce changes in lung mechanics as determined by in vivo lung function measurements. 4. The results provide evidence for the existence of M3 receptors on murine tracheae that are involved in the contraction of tracheal smooth muscle.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 6 Figure 7 PMID:8495246

  8. Pirenzepine Promotes the Dimerization of Muscarinic M1 Receptors through a Three-step Binding Process*

    PubMed Central

    Ilien, Brigitte; Glasser, Nicole; Clamme, Jean-Pierre; Didier, Pascal; Piemont, Etienne; Chinnappan, Raja; Daval, Sandrine B.; Galzi, Jean-Luc; Mely, Yves

    2009-01-01

    Ligand binding to G protein-coupled receptors is a complex process that involves sequential receptor conformational changes, ligand translocation, and possibly ligand-induced receptor oligomerization. Binding events at muscarinic acetylcholine receptors are usually interpreted from radioligand binding studies in terms of two-step ligand-induced receptor isomerization. We report here, using a combination of fluorescence approaches, on the molecular mechanisms for Bodipy-pirenzepine binding to enhanced green fluorescent protein (EGFP)-fused muscarinic M1 receptors in living cells. Real time monitoring, under steady-state conditions, of the strong fluorescence energy transfer signal elicited by this interaction permitted a fine kinetic description of the binding process. Time-resolved fluorescence measurements allowed us to identify discrete EGFP lifetime species and to follow their redistribution upon ligand binding. Fluorescence correlation spectroscopy, with EGFP brightness analysis, showed that EGFP-fused muscarinic M1 receptors predominate as monomers in the absence of ligand and dimerize upon pirenzepine binding. Finally, all these experimental data could be quantitatively reconciled into a three-step mechanism, with four identified receptor conformational states. Fast ligand binding to a peripheral receptor site initiates a sequence of conformational changes that allows the ligand to access to inner regions of the protein and drives ligand-receptor complexes toward a high affinity dimeric state. PMID:19451648

  9. Pirenzepine promotes the dimerization of muscarinic M1 receptors through a three-step binding process.

    PubMed

    Ilien, Brigitte; Glasser, Nicole; Clamme, Jean-Pierre; Didier, Pascal; Piemont, Etienne; Chinnappan, Raja; Daval, Sandrine B; Galzi, Jean-Luc; Mely, Yves

    2009-07-17

    Ligand binding to G protein-coupled receptors is a complex process that involves sequential receptor conformational changes, ligand translocation, and possibly ligand-induced receptor oligomerization. Binding events at muscarinic acetylcholine receptors are usually interpreted from radioligand binding studies in terms of two-step ligand-induced receptor isomerization. We report here, using a combination of fluorescence approaches, on the molecular mechanisms for Bodipy-pirenzepine binding to enhanced green fluorescent protein (EGFP)-fused muscarinic M1 receptors in living cells. Real time monitoring, under steady-state conditions, of the strong fluorescence energy transfer signal elicited by this interaction permitted a fine kinetic description of the binding process. Time-resolved fluorescence measurements allowed us to identify discrete EGFP lifetime species and to follow their redistribution upon ligand binding. Fluorescence correlation spectroscopy, with EGFP brightness analysis, showed that EGFP-fused muscarinic M1 receptors predominate as monomers in the absence of ligand and dimerize upon pirenzepine binding. Finally, all these experimental data could be quantitatively reconciled into a three-step mechanism, with four identified receptor conformational states. Fast ligand binding to a peripheral receptor site initiates a sequence of conformational changes that allows the ligand to access to inner regions of the protein and drives ligand-receptor complexes toward a high affinity dimeric state.

  10. Differential activation of nitric oxide synthase through muscarinic acetylcholine receptors in rat salivary glands.

    PubMed

    Leirós, C P; Rosignoli, F; Genaro, A M; Sales, M E; Sterin-Borda, L; Santiago BordaE

    2000-03-15

    Muscarinic receptors play an important role in secretory and vasodilator responses in rat salivary glands. Nitric oxide synthase (NOS) appears to be one of the multiple effectors coupled to muscarinic receptors in both submandibular and sublingual glands although some differences have been found depending on the gland studied. First, submandibular glands had a lower basal activity of nitric oxide synthase than sublingual glands and the concentration-response curve for carbachol was bell-shaped in the former but not in sublingual glands. Second, cGMP levels displayed a similar profile to that observed for NOS activity in both glands. Third, protein kinase C also coupled to muscarinic receptor activation in the glands might have a regulatory effect on nitric oxide production since its activity was higher in basal conditions in submandibular than sublingual glands and it also increased in the presence of the agonist at a concentration that inhibited NOS activity in submandibular glands. The effects appear to be partly related to the expression of a minor population of M(1) receptors in submandibular glands absent in sublingual as determined in binding and signaling experiments with the muscarinic receptor antagonist pirenzepine.

  11. Cardiac Muscarinic Receptor Overexpression in Sudden Infant Death Syndrome

    PubMed Central

    Livolsi, Angelo; Niederhoffer, Nathalie; Dali-Youcef, Nassim; Rambaud, Caroline; Olexa, Catherine; Mokni, Walid; Gies, Jean-Pierre; Bousquet, Pascal

    2010-01-01

    Background Sudden infant death syndrome (SIDS) remains the leading cause of death among infants less than 1 year of age. Disturbed expression of some neurotransmitters and their receptors has been shown in the central nervous system of SIDS victims but no biological abnormality of the peripheral vago-cardiac system has been demonstrated to date. The present study aimed to seek vago-cardiac abnormalities in SIDS victims. The cardiac level of expression of muscarinic receptors, as well as acetylcholinesterase enzyme activity were investigated. Methodology/Principal Findings Left ventricular samples and blood samples were obtained from autopsies of SIDS and children deceased from non cardiac causes. Binding experiments performed with [3H]NMS, a selective muscarinic ligand, in cardiac membrane preparations showed that the density of cardiac muscarinic receptors was increased as shown by a more than doubled Bmax value in SIDS (n = 9 SIDS versus 8 controls). On average, the erythrocyte acetylcholinesterase enzyme activity was also significantly increased (n = 9 SIDS versus 11 controls). Conclusions In the present study, it has been shown for the first time that cardiac muscarinic receptor overexpression is associated with SIDS. The increase of acetylcholinesterase enzyme activity appears as a possible regulatory mechanism. PMID:20209124

  12. Perirhinal Cortex Muscarinic Receptor Blockade Impairs Taste Recognition Memory Formation

    ERIC Educational Resources Information Center

    Gutierrez, Ranier; De la Cruz, Vanesa; Rodriguez-Ortiz, Carlos J.; Bermudez-Rattoni, Federico

    2004-01-01

    The relevance of perirhinal cortical cholinergic and glutamatergic neurotransmission for taste recognition memory and learned taste aversion was assessed by microinfusions of muscarinic (scopolamine), NMDA (AP-5), and AMPA (NBQX) receptor antagonists. Infusions of scopolamine, but not AP5 or NBQX, prevented the consolidation of taste recognition…

  13. Muscarinic receptors in perirhinal cortex control trace conditioning.

    PubMed

    Bang, Sun Jung; Brown, Thomas H

    2009-04-08

    Trace conditioning requires that a transient representation of the conditional stimulus (CS) persists during the time interval between the CS offset and the onset of the unconditional stimulus. According to one hypothesis, this transient CS representation is supported by endogenous activity in "persistent-firing" neurons of perirhinal cortex (PR). By definition, persistent-firing neurons discharge for tens of seconds or minutes after the termination of the original spike-initiating stimulus. This continued spiking does not depend on recurrent circuit activity and can be reliably and completely blocked by muscarinic receptor antagonists. The present study evaluated the role of PR muscarinic receptors in trace fear conditioning. Before conditioning, rats received bilateral intra-PR infusions with either saline or scopolamine, a nonselective muscarinic receptor antagonist. Scopolamine infusions profoundly impaired trace conditioning but had no effect on delay conditioning or context conditioning. The results encourage a more general understanding of muscarinic receptors in PR and they motivate additional tests of the emerging theory that persistent-firing neurons support aspects of transient memory.

  14. Muscarinic receptor heterogeneity in follicle-enclosed Xenopus oocytes

    PubMed Central

    Arellano, Rogelio O; Garay, Edith; Miledi, Ricardo

    1999-01-01

    Ionic current responses elicited by acetylcholine (ACh) in follicle-enclosed Xenopus oocytes (follicles) were studied using the two-electrode voltage-clamp technique. ACh generated a fast chloride current (Fin) and inhibited K+ currents gated by cAMP (IK,cAMP) following receptor activation by adenosine, follicle-stimulating hormone or noradrenaline. These previously described cholinergic responses were confirmed to be of the muscarinic type, and were independently generated among follicles from different frogs.Inhibition of IK,cAMP was about 100 times more sensitive to ACh than Fin activation; the half-maximal effective concentrations (EC50) were 6.6 ± 0.4 and 784 ± 4 nm, respectively.Both responses were blocked by several muscarinic receptor antagonists. Using the respective EC50 concentrations of ACh as standard, the antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide blocked the two effects with very different potencies. Fin was blocked with a half-maximal inhibitory concentration (IC50) of 2.4 ± 0.07 nm, whilst the IC50 for IK,cAMP inhibition was 5.9 ± 0.2 μm.Oxotremorine, a muscarinic agonist, preferentially stimulated IK,cAMP inhibition (EC50= 15.8 ± 1.4 μm), whilst Fin was only weakly activated. In contrast, oxotremorine inhibited Fin generated by ACh with an IC50 of 2.3 ± 0.7 μm.Fin elicited via purinergic receptor stimulation was not affected by oxotremorine, indicating that the inhibition produced was specific to the muscarinic receptor, and suggesting that muscarinic actions do not exert a strong effect on follicular cell-oocyte coupling.Using reverse transcription-PCR, transcripts of a previously cloned muscarinic receptor from Xenopus (XlmR) were amplified from the RNA of both the isolated follicular cells and the oocyte. The pharmacological and molecular characteristics suggest that XlmR is involved in IK,cAMP inhibition.In conclusion, follicular cells possess two different muscarinic receptors, one resembling the M2 (or M4) subtype

  15. A model of the human M2 muscarinic acetylcholine receptor

    NASA Astrophysics Data System (ADS)

    Jöhren, Kirstin; Höltje, Hans-Dieter

    2002-11-01

    The M2 muscarinic acetylcholine receptor belongs to the family of rhodopsin like G-Protein Coupled Receptors. This subtype of muscarinic receptors is of special interest because it bears, aside from an orthosteric binding site, also an allosteric binding site. Based on the X-ray structure of bovine rhodopsin a complete homology model of the human M2 receptor was developed. For the orthosteric binding site point mutations and binding studies with different agonists and antagonists are available. This knowledge was utilized for an initial verification of the M2 model. Allosteric modulation of activity is mediated by structurally different ligands such as gallamine, caracurine V salts or W84 (a hexamethonium-derivative). Caracurine V derivatives with different affinities to M2 were docked using GRID-fields. Subsequent molecular dynamics simulations yielded different binding energies based on diverse electrostatic and lipophilic interactions. The calculated affinities are in good agreement to experimentally determined affinities.

  16. Structural determinants at the M2 muscarinic receptor modulate the RGS4-GIRK response to pilocarpine by impairment of the receptor voltage sensitivity.

    PubMed

    Chen, I-Shan; Furutani, Kazuharu; Kurachi, Yoshihisa

    2017-07-21

    Membrane potential controls the response of the M2 muscarinic receptor to its ligands. Membrane hyperpolarization increases response to the full agonist acetylcholine (ACh) while decreasing response to the partial agonist pilocarpine. We previously have demonstrated that the regulator of G-protein signaling (RGS) 4 protein discriminates between the voltage-dependent responses of ACh and pilocarpine; however, the underlying mechanism remains unclear. Here we show that RGS4 is involved in the voltage-dependent behavior of the M2 muscarinic receptor-mediated signaling in response to pilocarpine. Additionally we revealed structural determinants on the M2 muscarinic receptor underlying the voltage-dependent response. By electrophysiological recording in Xenopus oocytes expressing M2 muscarinic receptor and G-protein-gated inwardly rectifying K(+) channels, we quantified voltage-dependent desensitization of pilocarpine-induced current in the presence or absence of RGS4. Hyperpolarization-induced desensitization of the current required for RGS4, also depended on pilocarpine concentration. Mutations of charged residues in the aspartic acid-arginine-tyrosine motif of the M2 muscarinic receptor, but not intracellular loop 3, significantly impaired the voltage-dependence of RGS4 function. Thus, our results demonstrated that voltage-dependence of RGS4 modulation is derived from the M2 muscarinic receptor. These results provide novel insights into how membrane potential impacts G-protein signaling by modulating GPCR communication with downstream effectors.

  17. Repeated effects of asenapine on adrenergic and cholinergic muscarinic receptors.

    PubMed

    Choi, Yong Kee; Wong, Erik H F; Henry, Brian; Shahid, Mohammed; Tarazi, Frank I

    2010-04-01

    Adrenergic (alpha1 and alpha2) and cholinergic muscarinic (M1-M5) receptor binding in rat forebrain was quantified after 4 wk of twice-daily subcutaneous administration of asenapine or vehicle. Asenapine (0.03, 0.1, and 0.3 mg/kg) produced increases in [3H]prazosin binding to alpha1-adrenergic receptors in the medial prefrontal cortex (mPFC: 30%, 39%, 57%) and dorsolateral frontal cortex (DFC: 27%, 37%, 53%) and increased [3H]RX821002 binding to alpha2-adrenergic receptors in mPFC (36%, 43%, 50%) and DFC (41%, 44%, 52%). Despite showing no appreciable affinity for muscarinic receptors, asenapine produced regionally selective increases in binding of [3H]QNB to M1-M5 receptors in mPFC (26%, 31%, 43%), DFC (27%, 34%, 41%), and hippocampal CA1 (40%, 44%, 42%) and CA3 (25%, 52%, 48%) regions. These regionally selective effects of asenapine on adrenergic and cholinergic muscarinic receptor subtypes may contribute to its beneficial clinical effects in the treatment of schizophrenia and bipolar disorder.

  18. Characterization of muscarinic receptor subtypes in primary cultures of cerebellar granule cells using specific muscarinic receptor antagonists

    SciTech Connect

    McLeskey, S.W.

    1989-01-01

    In cerebellar granule cell cultures, two muscarinic receptor mediated responses were observed: inhibition of adenylate cyclase (M-AC) and stimulation of phosphoinositide hydrolysis (M-PI). These responses were antagonized by three purported specific muscarinic antagonists: pirenzipine and (-)QNX (specific for M-PI) and methoctramine (specific for M-AC). However, the specificity for the three antagonists in blocking these responses is not comparable to the specificity observed in binding studies on these cells or to that quoted in the literature. Two peaks of molecular sizes were found in these cells corresponding to the two molecular sizes of muscarinic receptive proteins reported in the literature. Muscarinic receptive proteins were alkylated with {sup 3}H-propylbenzilylcholine mustard followed by sodium dodecylsulfate polyacrylamide gel electrophoresis. Pirenzipine and (-)QNX were able to block alkylation of the high molecular size peak, which corresponds to the receptive protein m{sub 3} reported in the literature. Methoctramine was able to block alkylation of a portion of the lower molecular size peak, possibly corresponding to the m{sub 2} and/or m{sub 4} receptive proteins reported in the literature. Studies attempting to show the presence of receptor reserve for either of the two biochemical responses present in these cells by alkylation of the receptive protein with nonradiolabeled propylbenzilylcholine mustard (PBCM) were confounded by specificity of this agent for the lower molecular weight peak of muscarinic receptive protein. Thus the muscarinic receptive proteins coupled to M-AC were alkylated preferentially over the ones coupled to M-PI.

  19. Alkylating derivative of oxotremorine interacts irreversibly with the muscarinic receptor

    SciTech Connect

    Ehlert, F.J.; Jenden, D.J.; Ringdahl, B.

    1984-03-05

    A 2-chloroethylamine derivative of oxotremorine was studied in pharmacological experiments and muscarinic receptor binding assays. The compound, N-(4-(2-chloroethylmethylamino)-2-butynyl)-2-pyrrolidone (BM 123), forms an aziridinium ion in aqueous solution at neutral pH that stimulates contractions of guinea pig ileum with a potency similar to that of oxotremorine. Following the initial stimulation, there is a long lasting period of lack of sensitivity of the guinea pig ileum to muscarinic agonists. BM 123 also produces muscarinic effects in vivo. When homogenates of the rat cerebral cortex were incubated with BM 123 and assayed subsequently in muscarinic receptor binding assays, a loss of binding capacity for the muscarinic antagonist, (/sup 3/H)N-methylscopolamine ((/sup 3/H)NMS), was noted without a change in affinity. Similar observations were made in (/sup 3/H)1-3-quinuclidinyl benzilate ((/sup 3/H)-QNB) binding assays on the forebrains of mice that had been injected with BM 123 24 hr earlier. The loss in receptor capacity for both (/sup 3/H)NMS and (/sup 3/H)-QNB was prevented by atropine treatment. Kinetic studies of the interaction of BM 123 with homogenates of the rat cerebral cortex in vitro showed that the half-time for the loss of (/sup 3/H)-QNB binding sites increased from 10 to 45 min as the concentration of BM 123 decreased from 10 to 1 ..mu..M. In contrast to the aziridinium ion, the parent 2-chloroethylamine compound and the alcoholic hydrolysis product were largely devoid of pharmacological and binding activity.

  20. Muscarinic Receptors and Their Antagonists in COPD: Anti-Inflammatory and Antiremodeling Effects

    PubMed Central

    Karakiulakis, George; Roth, Michael

    2012-01-01

    Muscarinic receptors are expressed by most cell types and mediate cellular signaling of their natural ligand acetylcholine. Thereby, they control numerous central and peripheral physiological organ responses to neuronal activity. In the human lung, muscarinic receptors are predominantly expressed by smooth muscle cells, epithelial cells, and fibroblasts. Antimuscarinic agents are used for the treatment of chronic obstructive pulmonary disease and to a lesser extent for asthma. They are primarily used as bronchodilators, but it is now accepted that they are also associated with anti-inflammatory, antiproliferative, and antiremodeling effects. Remodeling of the small airways is a major pathology in COPD and impairs lung function through changes of the extracellular matrix. Glycosaminoglycans, particularly hyaluronic acid, and matrix metalloproteases are among extracellular matrix molecules that have been associated with tissue inflammation and remodeling in lung diseases, including chronic obstructive pulmonary disease and asthma. Since muscarinic receptors have been shown to influence the homeostasis of glycosaminoglycans and matrix metalloproteases, these molecules may be proved valuable endpoint targets in clinical studies for the pharmacological exploitation of the anti-inflammatory and antiremodeling effects of muscarinic inhibitors in the treatment of chronic obstructive pulmonary disease and asthma. PMID:23226927

  1. Muscarinic receptors and their antagonists in COPD: anti-inflammatory and antiremodeling effects.

    PubMed

    Karakiulakis, George; Roth, Michael

    2012-01-01

    Muscarinic receptors are expressed by most cell types and mediate cellular signaling of their natural ligand acetylcholine. Thereby, they control numerous central and peripheral physiological organ responses to neuronal activity. In the human lung, muscarinic receptors are predominantly expressed by smooth muscle cells, epithelial cells, and fibroblasts. Antimuscarinic agents are used for the treatment of chronic obstructive pulmonary disease and to a lesser extent for asthma. They are primarily used as bronchodilators, but it is now accepted that they are also associated with anti-inflammatory, antiproliferative, and antiremodeling effects. Remodeling of the small airways is a major pathology in COPD and impairs lung function through changes of the extracellular matrix. Glycosaminoglycans, particularly hyaluronic acid, and matrix metalloproteases are among extracellular matrix molecules that have been associated with tissue inflammation and remodeling in lung diseases, including chronic obstructive pulmonary disease and asthma. Since muscarinic receptors have been shown to influence the homeostasis of glycosaminoglycans and matrix metalloproteases, these molecules may be proved valuable endpoint targets in clinical studies for the pharmacological exploitation of the anti-inflammatory and antiremodeling effects of muscarinic inhibitors in the treatment of chronic obstructive pulmonary disease and asthma.

  2. Structural analysis of beta-adrenergic and muscarinic cholinergic receptors

    SciTech Connect

    Kerlavage, A.R.; Fraser, C.M.; Venter, J.C.

    1987-05-01

    The authors have recently cloned the gene encoding the human brain beta-adrenergic receptor. Beta-adrenergic and muscarinic cholinergic receptors have also been cloned from other tissues. In order to correlate the primary structures of these receptors with their function, they have undertaken detailed mapping of their functionally important sites. Purified guinea pig lung beta receptor was radioiodinated and digested with trypsin. The resultant peptides were resolved by reverse phase HPLC into nine peaks containing /sup 125/I, corresponding exactly with the predicted number of tyrosine containing peptides in the beta receptor. Hamster lung beta receptor was labeled with (/sup 125/I)-iodocyanopindolol diazarine ((/sup 125/I)CYPD) and partially purified by SDS-PAGE. The (/sup 125/I)CYPD-labeled receptor was extracted from the gel, digested with either trypsin or CNBr and the digests were resolved by reverse phase HPLC. The tryptic digest contained one (/sup 125/I)CYPD-labeled peak and the CNBr digest contained two. Rat brain muscarinic receptor was specifically labeled with (/sup 3/H)-propylbenzilyl-choline mustard ((/sup 3/H)PrBCM) and partially purified by SDS-PABE. The (/sup 3/H)PrBCM-labeled receptor was extracted from the gel and digested with CNBr. The resultant HPLC profile revealed a single (/sup 3/H)PrBCM-labeled peak. These data yield information on the location of functional sites within the primary sequences of these receptors.

  3. Muscarinic acetylcholine receptor X-ray structures: potential implications for drug development.

    PubMed

    Kruse, Andrew C; Hu, Jianxin; Kobilka, Brian K; Wess, Jürgen

    2014-06-01

    Muscarinic acetylcholine receptor antagonists are widely used as bronchodilating drugs in pulmonary medicine. The therapeutic efficacy of these agents depends on the blockade of M3 muscarinic receptors expressed on airway smooth muscle cells. All muscarinic antagonists currently used as bronchodilating agents show high affinity for all five muscarinic receptor subtypes, thus increasing the likelihood of unwanted side effects. Recent X-ray crystallographic studies have provided detailed structural information about the nature of the orthosteric muscarinic binding site (the conventional acetylcholine binding site) and an 'outer' receptor cavity that can bind allosteric (non-orthosteric) drugs. These new findings should guide the development of selective M3 receptor blockers that have little or no effect on other muscarinic receptor subtypes.

  4. Muscarinic receptor size on smooth muscle cells and membranes

    SciTech Connect

    Collins, S.M.; Jung, C.Y.; Grover, A.K.

    1986-08-01

    The loss of (/sup 3/H)quinuclidinyl benzilate ((/sup 3/H)QNB) binding following high-energy radiation was used to compare the muscarinic receptor size on single smooth muscle cells isolated by collagenase digestion from the canine stomach and on plasma membranes derived from intact gastric smooth muscle without exposure to exogenous proteolysis. Radiation inactivation of galactose oxidase (68 kdaltons), yeast alcohol dehydrogenase (160 kdaltons), and pyruvate kinase (224 kdaltons) activities were used as molecular-weight standards. Radiation inactivation of (/sup 3/H)QNB binding to rat brain membranes, which gave a target size of 86 kdaltons, served as an additional control. In isolated smooth muscle cells, the calculated size of the muscarinic receptor was 80 +/- 8 kdaltons. In contrast, in a smooth muscle enriched plasma membrane preparation, muscarinic receptor size was significantly smaller at 45 +/- 3 kdaltons. Larger molecular sizes were obtained either in the presence of protease inhibitors (62 +/- 4 kdaltons) or by using a crude membrane preparation of gastric smooth muscle 86 +/- 7 kdaltons).

  5. Action of AF64A on rat brain muscarinic receptors

    SciTech Connect

    Eva, C.; Costa, E.

    1986-03-01

    ICV administration of compound AF64A (ethylcholine mustard aziridium ion) induces a long-term selective cholinergic hypofunction; however, it does not modify the characteristics of muscarinic receptors. In brain muscarinic receptor activation can either stimulate phosphoinositide turnover or inhibit adenylate cyclase. ICV infusion of AF64A (5 nmol/side/2.5 ..mu..l) reduced the hippocampal ACh content 10 or 30 days after the treatment to 75% of the control values. Under these conditions neither in the striatum nor in the frontal cortex ACh levels were decreased. The carbachol dose-dependent stimulation in hippocampal slices differed from that observed in control rats. The carbachol efficacy was increased but its potency was unchanged by AF64A. In contrast, ICV administration of AF64A failed to alter the oxotremorine efficacy or potency in inhibiting the forskolin stimulated adenylate cyclase in rat hippocampal membranes. These results suggest the two transducer systems coupled to muscarinic receptors may be differentially regulatable by cholinergic input.

  6. Role of dopamine receptor and muscarinic acetylcholine receptor blockade in the antiapomorphine action of neuroleptics

    SciTech Connect

    Zharkovskii, A.M.; Langel, Yu.L.; Chereshka, K.S.; Zharkovskaya, T.A.

    1987-08-01

    The authors analyze the role of dopamine and muscarinic acetylcholine receptor blocking components in the antistereotypic action of neuroleptics with different chemical structure. To determine dopamine-blocking activity in vitro, binding of /sup 3/H-spiperone with membranes of the rat striatum was measured. To study the blocking action of the substances on muscarinic acetylcholine receptors, binding of /sup 3/H-quinuclidinyl benzylate with brain membranes was chosen.

  7. Muscarinic acetylcholine receptors in the nucleus accumbens core and shell contribute to cocaine priming-induced reinstatement of drug seeking

    PubMed Central

    Yee, Judy; Famous, Katie R.; Hopkins, Thomas J.; McMullen, Michael C.; Pierce, R. Christopher; Schmidt, Heath D.

    2011-01-01

    Muscarinic acetylcholine receptors in the nucleus accumbens play an important role in mediating the reinforcing effects of cocaine. However, there is a paucity of data regarding the role of accumbal muscarinic acetylcholine receptors in the reinstatement of cocaine-seeking behavior. The goal of these experiments was to assess the role of muscarinic acetylcholine receptors in the nucleus accumbens core and shell in cocaine and sucrose priming-induced reinstatement. Rats were initially trained to self-administer cocaine or sucrose on a fixed-ratio schedule of reinforcement. Lever-pressing behavior was then extinguished and followed by a subsequent reinstatement phase during which operant responding was induced by either a systemic injection of cocaine in cocaine-experienced rats or non-contingent delivery of sucrose pellets in subjects with a history of sucrose self-administration. Results indicated that systemic administration of the muscarinic acetylcholine receptor antagonist scopolamine (5.0 mg/kg, i.p.) dose-dependently attenuated cocaine, but not sucrose, reinstatement. Furthermore, administration of scopolamine (36.0 μg) directly into the nucleus accumbens shell or core attenuated cocaine-priming induced reinstatement. In contrast, infusion of scopolamine (36.0 μg) directly into the accumbens core, but not shell, attenuated sucrose reinstatement, which suggests that muscarinic acetylcholine receptors in these two subregions of the nucleus accumbens have differential roles in sucrose seeking. Taken together, these results indicate that cocaine-priming induced reinstatement is mediated, in part, by increased signaling through muscarinic acetylcholine receptors in the shell subregion of the nucleus accumbens. Muscarinic acetylcholine receptors in the core of the accumbens, in contrast, appear to play a more general (i.e. not cocaine specific) role in motivated behaviors. PMID:21034738

  8. Effects of Anticholinesterase Exposure on Transport and Distribution of High vs. Low Affinity Muscarinic Cholinergic Receptors.

    DTIC Science & Technology

    1983-11-11

    atypical antagonist pirenzepine . Thus, it will be possible to localize several different muscarinic cholinergic receptor subtypes in animals chronically... pirenzepine . These results indicate the presence of two distinct muscarinic receptor subtypes (M1 and M2 ) in the brain. The data suggest that these two...defined by direct labeling for receptor autoradiography. The discovery of the atypical muscarinic antagonist pirenzepine has prompted the definition of

  9. Muscarinic receptors in the bladder: from basic research to therapeutics

    PubMed Central

    Hegde, Sharath S

    2006-01-01

    Muscarinic receptor antagonists (antimuscarinics) serve as the cornerstone in the pharmacological management of overactive bladder (OAB) by relieving the symptoms of urgency, frequency and incontinence. These drugs operate primarily by antagonizing post-junctional excitatory muscarinic receptors (M2/M3) in the detrusor. The combination of pharmacological and gene knockout studies has greatly advanced our understanding of the functional role of muscarinic receptors in the bladder. M3 receptors produce direct smooth muscle contraction by a mechanism that relies on entry of extracellular calcium through L-type channels and activation of a rho kinase. M2 receptors, which predominate in number, appear to facilitate M3-mediated contractions. M2 receptors can also produce bladder contractions indirectly by reversing cAMP-dependent β-adrenoceptor-mediated relaxation, although the physiological role of β-adrenoceptors in detrusor relaxation is controversial. Emerging evidence suggests that muscarinic receptors in the urothelium/suburothelium can modulate the release of certain factors, which in turn may affect bladder function at the efferent or afferent axis. Currently, oxybutynin, tolterodine, darifenacin, solifenacin and trospium are the five major antimuscarinics approved for the treatment of OAB. Comparative clinical studies have shown that oxybutynin and solifenacin may be marginally more effective than tolterodine, although the latter seems to be better tolerated. Pharmacokinetic–pharmacodynamic analyses using plasma concentrations of ‘total drug' indicate that, at therapeutic doses, the clinical efficacy of darifenacin and solifenacin may be driven primarily by selective M3 receptor occupation, whereas the pharmacodynamic effects of pan-selective molecules (such as tolterodine, trospium) may potentially involve multiple receptors, including M2 and M3. Furthermore, high M3 receptor occupation is the likely explanation for the greater propensity of darifenacin

  10. Selectivity of oxomemazine for the M1 muscarinic receptors.

    PubMed

    Lee, S W; Woo, C W; Kim, J G

    1994-12-01

    The binding characteristics of pirenzepine and oxomemazine to muscarinic receptor were studied to evaluate the selectivity of oxomemazine for the muscarinic receptor subtypes in rat cerebral microsomes. Equilibrium dissociation constant (KD) of (-)-[3H]quinuclidinyl benzilate([3H]QNB) determined from saturation isotherms was 64 pM. Analysis of the pirenzepine inhibition curve of [3H]QNB binding to cerebral microsome indicated the presence of two receptor subtypes with high (Ki = 16 nM, M1 receptor) and low (Ki = 400 nM, M3 receptor) affinity for pirenzepine. Oxomemazine also identified two receptor subtypes with about 20-fold difference in the affinity for high (Ki = 84 nM, OH receptor) and low (Ki = 1.65 microM, OL receptor) affinity sites. The percentage populations of M1 and M3 receptors to the total receptors were 61:39, and those of OH and OL receptors 39:61, respectively. Both pirenzepine and oxomemazine increased the KD value for [3H]QNB without affecting the binding site concentrations and Hill coefficient for the [3H]QNB binding. Oxomemazine had a 10-fold higher affinity at M1 receptors than at M3 receptors, and pirenzepine a 8-fold higher affinity at OH receptors than at OL receptors. Analysis of the shallow competition binding curves of oxomemazine for M1 receptors and pirenzepine for OL receptors yielded that 69% of M1 receptors were of OH receptors and the remaining 31% of OL receptors, and that 29% of OL receptors were of M1 receptors and 71% of M3 receptors. However, M3 for oxomemazine and OH for pirenzepine were composed of a uniform population. These results suggest that oxomemazine could be classified as a selective drug for M1 receptors and also demonstrate that rat cerebral microsomes contain three different subtypes of M1, M3 and the other site which is different from M1, M2 and M3 receptors.

  11. Selective antagonism of muscarinic receptors is neuroprotective in peripheral neuropathy.

    PubMed

    Calcutt, Nigel A; Smith, Darrell R; Frizzi, Katie; Sabbir, Mohammad Golam; Chowdhury, Subir K Roy; Mixcoatl-Zecuatl, Teresa; Saleh, Ali; Muttalib, Nabeel; Van der Ploeg, Randy; Ochoa, Joseline; Gopaul, Allison; Tessler, Lori; Wess, Jürgen; Jolivalt, Corinne G; Fernyhough, Paul

    2017-02-01

    Sensory neurons have the capacity to produce, release, and respond to acetylcholine (ACh), but the functional role of cholinergic systems in adult mammalian peripheral sensory nerves has not been established. Here, we have reported that neurite outgrowth from adult sensory neurons that were maintained under subsaturating neurotrophic factor conditions operates under cholinergic constraint that is mediated by muscarinic receptor-dependent regulation of mitochondrial function via AMPK. Sensory neurons from mice lacking the muscarinic ACh type 1 receptor (M1R) exhibited enhanced neurite outgrowth, confirming the role of M1R in tonic suppression of axonal plasticity. M1R-deficient mice made diabetic with streptozotocin were protected from physiological and structural indices of sensory neuropathy. Pharmacological blockade of M1R using specific or selective antagonists, pirenzepine, VU0255035, or muscarinic toxin 7 (MT7) activated AMPK and overcame diabetes-induced mitochondrial dysfunction in vitro and in vivo. These antimuscarinic drugs prevented or reversed indices of peripheral neuropathy, such as depletion of sensory nerve terminals, thermal hypoalgesia, and nerve conduction slowing in diverse rodent models of diabetes. Pirenzepine and MT7 also prevented peripheral neuropathy induced by the chemotherapeutic agents dichloroacetate and paclitaxel or HIV envelope protein gp120. As a variety of antimuscarinic drugs are approved for clinical use against other conditions, prompt translation of this therapeutic approach to clinical trials is feasible.

  12. Interaction of a radiolabeled agonist with cardiac muscarinic cholinergic receptors

    SciTech Connect

    Harden, T.K.; Meeker, R.B.; Martin, M.W.

    1983-12-01

    The interaction of a radiolabeled muscarinic cholinergic receptor agonist, (methyl-/sup 3/H)oxotremorine acetate ((/sup 3/H)OXO), with a washed membrane preparation derived from rat heart, has been studied. In binding assays at 4 degrees C, the rate constants for association and dissociation of (/sup 3/H)OXO were 2 X 10(7) M-1 min-1 and 5 X 10(-3) min-1, respectively, Saturation binding isotherms indicated that binding was to a single population of sites with a Kd of approximately 300 pM. The density of (/sup 3/H)OXO binding sites (90-100 fmol/mg of protein) was approximately 75% of that determined for the radiolabeled receptor antagonist (/sup 3/H)quinuclidinyl benzilate. Both muscarinic receptor agonists and antagonists inhibited the binding of (/sup 3/H)OXO with high affinity and Hill slopes of approximately one. Guanine nucleotides completely inhibited the binding of (/sup 3/H)OXO. This effect was on the maximum binding (Bmax) of (/sup 3/H)OXO with no change occurring in the Kd; the order of potency for five nucleotides was guanosine 5'-O-(3-thio-triphosphate) greater than 5'-guanylylimidodiphosphate greater than GTP greater than or equal to guanosine/diphosphate greater than GMP. The (/sup 3/H)OXO-induced interaction of muscarinic receptors with a guanine nucleotide binding protein was stable to solubilization. That is, membrane receptors that were prelabeled with (/sup 3/H)OXO could be solubilized with digitonin, and the addition of guanine nucleotides to the soluble, (/sup 3/H)OXO-labeled complex resulted in dissociation of (/sup 3/H)OXO from the receptor. Pretreatment of membranes with relatively low concentrations of N-ethylmaleimide inhibited (/sup 3/H)OXO binding by 85% with no change in the Kd of (/sup 3/H)OXO, and with no effect on (/sup 3/H)quinuclidinyl benzilate binding.

  13. Muscarinic receptor subtype selectivity of novel heterocyclic QNB analogues

    SciTech Connect

    Baumgold, J.; Cohen, V.I.; Paek, R.; Reba, R.C. )

    1991-01-01

    In an effort at synthesizing centrally-active subtype-selective antimuscarinic agents, the authors derivatized QNB (quinuclidinyl benzilate), a potent muscarinic antagonist, by replacing one of the phenyl groups with less lipophilic heterocyclic moieties. The displacement of ({sup 3}H)-N-methyl scopolamine binding by these novel compounds to membranes from cells expressing ml - m4 receptor subtypes was determined. Most of the novel 4-bromo-QNB analogues were potent and slightly selective for ml receptors. The 2-thienyl derivative was the most potent, exhibiting a 2-fold greater potency than BrQNB at ml receptors, and a 4-fold greater potency than BrQNB at ml receptors, and a 4-fold greater potency at m2 receptors. This compound was also considerably less lipophilic than BrQNB as determined from its retention time on C18 reverse phase HPLC. This compound may therefore be useful both for pharmacological studies and as a candidate for a radioiodinated SPECT imaging agent for ml muscarinic receptors in human brain.

  14. Muscarinic receptor subtypes in cilia-driven transport and airway epithelial development

    PubMed Central

    Klein, Maike K.; Haberberger, Rainer V.; Hartmann, Petra; Faulhammer, Petra; Lips, Katrin S.; Krain, Benjamin; Wess, Jürgen; Kummer, Wolfgang; König, Peter

    2014-01-01

    Ciliary beating of airway epithelial cells drives the removal of mucus and particles from the airways. Mucociliary transport and possibly airway epithelial development are governed by muscarinic acetylcholine receptors but the precise roles of the subtypes involved are unknown. This issue was addressed by determining cilia-driven particle transport, ciliary beat frequency, and the composition and ultrastructural morphology of the tracheal epithelium in M1–M5 muscarinic receptor gene-deficient mice. Knockout of M3 muscarinic receptors prevented an increase in particle transport speed and ciliary beat frequency in response to muscarine. Furthermore, the ATP response after application of muscarine was blunted. Pretreatment with atropine before application of muscarine restored the response to ATP. Additional knockout of the M2 receptor in these mice partially restored the muscarine effect most likely through the M1 receptor and normalized the ATP response. M1, M4, and M5 receptor deficient mice exhibited normal responses to muscarine. None of the investigated mutant mouse strains had any impairment of epithelial cellular structure or composition. In conclusion, M3 receptors stimulate whereas M2 receptors inhibit cilia-driven particle transport. The M1 receptor increases cilia-driven particle transport if the M3 and M2 receptor are missing. None of the receptors is necessary for epithelial development. PMID:19213795

  15. The effects of sigma (σ1) receptor-selective ligands on muscarinic receptor antagonist-induced cognitive deficits in mice

    PubMed Central

    Malik, Maninder; Rangel-Barajas, Claudia; Sumien, Nathalie; Su, Chang; Singh, Meharvan; Chen, Zhenglan; Huang, Ren-Qi; Meunier, Johann; Maurice, Tangui; Mach, Robert H; Luedtke, Robert R

    2015-01-01

    Background and Purpose Cognitive deficits in patients with Alzheimer's disease, Parkinson's disease, traumatic brain injury and stroke often involve alterations in cholinergic signalling. Currently available therapeutic drugs provide only symptomatic relief. Therefore, novel therapeutic strategies are needed to retard and/or arrest the progressive loss of memory. Experimental Approach Scopolamine-induced memory impairment provides a rapid and reversible phenotypic screening paradigm for cognition enhancement drug discovery. Male C57BL/6J mice given scopolamine (1 mg·kg−1) were used to evaluate the ability of LS-1–137, a novel sigma (σ1) receptor-selective agonist, to improve the cognitive deficits associated with muscarinic antagonist administration. Key Results LS-1–137 is a high-affinity (Ki = 3.2 nM) σ1 receptor agonist that is 80-fold selective for σ1, compared with σ2 receptors. LS-1–137 binds with low affinity at D2-like (D2, D3 and D4) dopamine and muscarinic receptors. LS-1–137 was found to partially reverse the learning deficits associated with scopolamine administration using a water maze test and an active avoidance task. LS-1–137 treatment was also found to trigger the release of brain-derived neurotrophic factor from rat astrocytes. Conclusions and Implications The σ1 receptor-selective compound LS-1–137 may represent a novel candidate cognitive enhancer for the treatment of muscarinic receptor-dependent cognitive deficits. PMID:25573298

  16. Muscarinic M3 receptor subtype gene expression in the human heart.

    PubMed

    Hellgren, I; Mustafa, A; Riazi, M; Suliman, I; Sylvén, C; Adem, A

    2000-01-20

    The heart is an important target organ for cholinergic function. In this study, muscarinic receptor subtype(s) in the human heart were determined using reverse transcription-polymerase chain reaction. Our results demonstrated muscarinic receptor M2 and M3 subtype RNA in left/right atria/ventricles of donor hearts. Receptor autoradiography analysis using selective muscarinic ligands indicated an absence of M1 receptor subtype in the human heart. The level of muscarinic receptor binding in atria was two to three times greater than in ventricles. Our results suggest that muscarinic receptors in the human heart are of the M2 and M3 subtypes. This is the first report of M3 receptors in the human myocardium.

  17. The binding of some antidepressant drugs to brain muscarinic acetylcholine receptors.

    PubMed Central

    Golds, P. R.; Przyslo, F. R.; Strange, P. G.

    1980-01-01

    1 The binding of some antidepressant drugs, including some new drugs of atypical structure (flupenthixol, iprindole, maprotiline, mianserin, nomifensine, tofenacine and viloxazine) to muscarinic acetylcholine receptors in the brain has been studied by displacement of [3H]-atropine. 2 Many of the drugs are potent muscarinic antagonists. 3 Some correlation can be made between the affinity for binding to the muscarinic acetylcholine receptor and the incidence of anticholinergic side effects in clinical usage. PMID:7052344

  18. Down regulation of the muscarinic cholinergic receptor of the rat prostate following castration

    SciTech Connect

    Shapiro, E.; Miller, A.R.; Lepor, H.

    1985-07-01

    Prostatic secretion is dependent upon the integrity of the endocrine and autonomic nervous systems and is dramatically influenced by muscarinic cholinergic analogs. In this study, the authors have used radioligand receptor binding methods on whole tissue homogenates and slide mounted tissue sections of rat prostate to determine whether androgens regulate the density of muscarinic cholinergic receptors in the prostate. The muscarinic cholinergic receptor binding affinities (Kd) of (/sup 3/H) N-methylscopolamine in prostatic homogenates obtained from intact, castrate, and castrate rats receiving testosterone replacement (castrate + T) were similar (0.07 to 0.10 nM). The muscarinic cholinergic receptor binding capacity decreased 73 per cent following castration. Testosterone administration restored the density of muscarinic cholinergic receptors in castrate rats to intact levels. In order to ensure that the loss of receptor density was not due to a decrease in the epithelial: stromal cell ratio, the number of muscarinic cholinergic receptors per unit area of epithelium was determined in the 3 treatment groups using autoradiography on slide mounted tissue sections. The density of muscarinic cholinergic receptors in a unit area of epithelium was decreased 91 per cent following castration. Testosterone administration restored the density of muscarinic cholinergic receptors in the castrate rats to intact levels. The modulation of neurotransmitter receptors by steroid hormones may be a mechanism by which sex steroids regulate biological responsiveness of target tissues.

  19. Characterization and photoaffinity labeling of the muscarinic acetylcholine receptor

    SciTech Connect

    Cremo, C.R.

    1983-01-01

    The muscarinic acetylcholine receptor, identified by tritiated L-quinuclidinyl benzilate (L-(/sup 3/H)QNB) binding, was solubilized from porcine atrial membranes using a 5:1 (w/w) ratio of digitonin and cholate. Specific binding activities of the solubilized receptor solutions usually exceeded 1.0 nmol L-(/sup 3/H)QNB sites per gram of protein, representing 75-98% total site recovery and a two- to three-fold enrichment over untreated atrial membranes. Two rapid assays for measuring the binding activities of detergent extracts were devised and compared with equilibrium dialysis. All three methods gave similar results. The equilibrium dissociation constant of the solubilized receptor for L-(/sup 3/H)QNB as determined by the three methods varied from 230 to 450 pM depending on the method and temperature. The interaction of alkyl quanidines and decahydrohistrionicotoxin with the membrane-bound and solubilized muscarinic acetylcholine receptor (mAcChR) from porcine atria was described. Alkyl guanidines with alkyl chain lengths from one to ten carbons displaced (/sup 3/H)L-quinuclidinyl bensilate ((/sup 3/H)L-QNB) competitively from a single class of sites for the membrane-bound mAcChR. From a plot of -1n K/sub i/ versus alkyl carbon chain number, a value of -(473 +/- 30) cal/mol was estimated as the energetic contribution per methylene group to the total binding energy. The synthesis and properties of a radiolabeled muscarinic antagonist photoaffinity probe, (/sup 3/H) p-azidoatropine methyl iodide were reported.

  20. Muscarinic Receptor Agonists and Antagonists: Effects on Cancer

    PubMed Central

    2012-01-01

    Many epithelial and endothelial cells express a cholinergic autocrine loop in which acetylcholine acts as a growth factor to stimulate cell growth. Cancers derived from these tissues similarly express a cholinergic autocrine loop and ACh secreted by the cancer or neighboring cells interacts with M3 muscarinic receptors expressed on the cancer cells to stimulate tumor growth. Primary proliferative pathways involve MAPK and Akt activation. The ability of muscarinic agonists to stimulate, and M3 antagonists to inhibit tumor growth has clearly been demonstrated for lung and colon cancer. The ability of muscarinic agonists to stimulate growth has been shown for melanoma, pancreatic, breast, ovarian, prostate and brain cancers, suggesting that M3 antagonists will also inhibit growth of these tumors as well. As yet no clinical trials have proven the efficacy of M3 antagonists as cancer therapeutics, though the widespread clinical use and low toxicity of M3 antagonists support the potential role of these drugs as adjuvants to current cancer therapies. PMID:22222710

  1. Vasoactive Intestinal Polypeptide and Muscarinic Receptors: Supersensitivity Induced by Long-Term Atropine Treatment

    NASA Astrophysics Data System (ADS)

    Hedlund, Britta; Abens, Janis; Bartfai, Tamas

    1983-04-01

    Long-term treatment of rats with atropine induced large increases in the numbers of muscarinic receptors and receptors for vasoactive intestinal polypeptide in the salivary glands. Since receptors for vasoactive intestinal polypeptide coexist with muscarinic receptors on the same neurons in this preparation, the results suggest that a drug that alters the sensitivity of one receptor may also affect the sensitivity of the receptor for a costored transmitter and in this way contribute to the therapeutic or side effects of the drug.

  2. Agonists with supraphysiological efficacy at the muscarinic M2 ACh receptor

    PubMed Central

    Schrage, R; Seemann, WK; Klöckner, J; Dallanoce, C; Racké, K; Kostenis, E; De Amici, M; Holzgrabe, U; Mohr, K

    2013-01-01

    Background and Purpose Artificial agonists may have higher efficacy for receptor activation than the physiological agonist. Until now, such ‘superagonism’ has rarely been reported for GPCRs. Iperoxo is an extremely potent muscarinic receptor agonist. We hypothesized that iperoxo is a ‘superagonist’. Experimental Approach Signalling of iperoxo and newly synthesized structural analogues was compared with that of ACh at label-free M2 muscarinic receptors applying whole cell dynamic mass redistribution, measurement of G-protein activation, evaluation of cell surface agonist binding and computation of operational efficacies. Key Results In CHO-hM2 cells, iperoxo significantly exceeds ACh in Gi/Gs signalling competence. In the orthosteric loss-of-function mutant M2-Y1043.33A, the maximum effect of iperoxo is hardly compromised in contrast to ACh. ‘Superagonism’ is preserved in the physiological cellular context of MRC-5 human lung fibroblasts. Structure–signalling relationships including iperoxo derivatives with either modified positively charged head group or altered tail suggest that ‘superagonism’ of iperoxo is mechanistically based on parallel activation of the receptor protein via two orthosteric interaction points. Conclusion and Implications Supraphysiological agonist efficacy at muscarinic M2 ACh receptors is demonstrated for the first time. In addition, a possible underlying molecular mechanism of GPCR ‘superagonism’ is provided. We suggest that iperoxo-like orthosteric GPCR activation is a new avenue towards a novel class of receptor activators. Linked Article This article is commented on by Langmead and Christopoulos, pp. 353–356 of this issue. To view this commentary visit http://dx.doi.org/10.1111/bph.12142 PMID:23062057

  3. Muscarinic receptor subtypes involved in regulation of colonic motility in mice: functional studies using muscarinic receptor-deficient mice.

    PubMed

    Kondo, Takaji; Nakajima, Miwa; Teraoka, Hiroki; Unno, Toshihiro; Komori, Sei-ichi; Yamada, Masahisa; Kitazawa, Takio

    2011-11-16

    Although muscarinic M(2) and M(3) receptors are known to be important for regulation of gastric and small intestinal motility, muscarinic receptor subtypes regulating colonic function remain to be investigated. The aim of this study was to characterize muscarinic receptors involved in regulation of colonic contractility. M(2) and/or M(3) receptor knockout (KO) and wild-type mice were used in in vivo (defecation, colonic propulsion) and in vitro (contraction) experiments. Amount of feces was significantly decreased in M(3)R-KO and M(2)/M(3)R-KO mice but not in M(2)R-KO mice. Ranking of colonic propulsion was wild-type=M(2)R-KO>M(3)R-KO>M(2)/M(3)R-KO. In vitro, the amplitude of migrating motor complexes in M(2)R-KO, M(3)R-KO and M(2)/M(3)R-KO mice was significantly lower than that in wild-type mice. Carbachol caused concentration-dependent contraction of the proximal colon and distal colon from wild-type mice. In M(2)R-KO mice, the concentration-contraction curves shifted to the right and downward. In contrast, carbachol caused non-sustained contraction and relaxation in M(3)R-KO mice depending on its concentration. Carbachol did not cause contraction but instead caused relaxation of colonic strips from M(2)/M(3)R-KO mice. 4-[[[(3-chlorophenyl)amino]carbonyl]oxy]-N,N,N-trimethyl-2-butyn-1-aminium chloride (McN-A-343) caused a non-sustained contraction of colonic strips from wild-type mice, and this contraction was changed to a sustained contraction by tetrodotoxin, pirenzepine and L-nitroarginine methylester (L-NAME). In the colon of M(2)/M(3)R-KO mice, McN-A-343 caused only relaxation, which was decreased by tetrodotoxin, pirenzepine and L-NAME. In conclusion, M(1), M(2) and M(3) receptors regulate colonic motility of the mouse. M(2) and M(3) receptors mediate cholinergic contraction, but M(1) receptors on inhibitory nitrergic nerves counteract muscarinic contraction.

  4. Widespread decreases in cortical muscarinic receptors in a subset of people with schizophrenia.

    PubMed

    Gibbons, Andrew Stuart; Scarr, Elizabeth; Boer, Simone; Money, Tammie; Jeon, Won-Je; Felder, Chris; Dean, Brian

    2013-02-01

    These studies were undertaken to investigate the selectivity of cortical muscarinic receptor radioligand binding in muscarinic M(1) and M(4) receptor knockout mice and to determine whether a marked decrease in [(3)H]pirenzepine binding in Brodmann's area (BA) 9 from a subset of people with schizophrenia was predictive of decreased muscarinic receptors in other central nervous system (CNS) regions. Our data show that, under the conditions used, [(3)H]pirenzepine binding was highly selective for the muscarinic M(1) receptor whereas both [(3)H]AF-DX 386 and [(3)H]4DAMP had less discriminatory power. In addition, the data suggest that a marked decrease in [(3)H]pirenzepine binding in BA 9 from a subset of people with schizophrenia is predictive of decreases in muscarinic receptors in other CNS regions. However, there were some region-specific decreases in muscarinic receptors in tissue from people with schizophrenia who were outside this subset. These data add to a growing body of evidence suggesting there are widespread decreases in muscarinic receptors in the CNS of some subjects with schizophrenia, as demonstrated by neuroimaging. Our data have implications for understanding the potential clinical utility of drugs directed at the orthosteric and allosteric sites of muscarinic receptors to treat schizophrenia.

  5. Does pirenzepine distinguish between 'subtypes' of muscarinic receptors?

    PubMed Central

    Szelenyi, I.

    1982-01-01

    Pharmacological studies with pirenzepine were carried out on the isolated ileum and atrium of the guinea-pig and on the acid secretion from the isolated stomach of the mouse. Pirenzepine inhibited the bethanechol-evoked changes in all three organs in a dose-dependent manner. The slopes of the Schild-plots confirmed the competitive nature of the antagonism by pirenzepine. The estimated pA2-values were very similar. Based on these data, it might be concluded that pirenzepine is an anticholinoceptor compound without specific affinity for gastric muscarinic receptors. PMID:6897522

  6. Muscarinic receptor occupancy by biperiden in living human brain.

    PubMed

    Sudo, Y; Suhara, T; Suzuki, K; Okubo, Y; Yoshikawa, K; Uchida, S; Sassa, T; Okauchi, T; Sasaki, Y; Matsushita, M

    1999-01-01

    Anticholinergic drug is often used to treat extrapyramidal symptoms. We measured muscarinic cholinergic receptor (mAchR) occupancy by the oral administration of biperiden in eight healthy subjects using positron emission tomography (PET) and [11C]N-methyl-4-piperidylbenzilate (NMPB). After the baseline scan each subject underwent one or two post-dose PET scans. mAchR occupancy was 10-45% in the frontal cortex three hours after the oral administration of 4 mg of biperiden. The occupancy correlated with the plasma concentration of biperiden in a curvilinear manner.

  7. Discovery of new muscarinic acetylcholine receptor antagonists from Scopolia tangutica

    PubMed Central

    Du, Nana; Liu, Yanfang; Zhang, Xiuli; Wang, Jixia; Zhao, Jianqiang; He, Jian; Zhou, Han; Mei, Lijuan; Liang, Xinmiao

    2017-01-01

    Scopolia tangutica (S. tangutica) is a traditional Chinese medicinal plant used for antispasmodics, anesthesia, analgesia and sedation. Its pharmacological activities are mostly associated with the antagonistic activity at muscarinic acetylcholine receptors (mAchRs) of several known alkaloids such as atropine and scopolamine. With our recent identification of four hydroxycinnamic acid amides from S. tangutica, we hypothesized that this plant may contain previously unidentified alkaloids that may also contribute to its in vivo effect. Herein, we used a bioassay-guided multi-dimension separation strategy to discover novel mAchR antagonists from S. tangutica. The core of this approach is to use label-free cell phenotypic assay to first identify active fractions, and then to guide purification of active ligands. Besides four tropanes and six cinnamic acid amides that have been previously isolated from S. tangutica, we recently identified two new tropanes, one new cinnamic acid amide, and nine other compounds. Six tropane compounds purified from S. tangutica for the first time were confirmed to be competitive antagonists of muscarinic receptor 3 (M3), including the two new ones 8 and 12 with IC50 values of 1.97 μM and 4.47 μM, respectively. Furthermore, the cinnamic acid amide 17 displayed 15-fold selectivity for M1 over M3 receptors. These findings will be useful in designing lead compounds for mAchRs and elucidating mechanisms of action of S. tangutica. PMID:28387362

  8. Revisiting the endocytosis of the m2 muscarinic acetylcholine receptor.

    PubMed

    Ockenga, Wymke; Tikkanen, Ritva

    2015-05-12

    The agonist-induced endocytosis of the muscarinic acetylcholine receptor M2 is different from that of the other members of the muscarinic receptor family. The uptake of the M2 receptor involves the adapter proteins of the β-arrestin family and the small GTPase ADP-ribosylation factor 6. However, it has remained inconclusive if M2 endocytosis is dependent on clathrin or the large GTPase dynamin. We here show by means of knocking down the clathrin heavy chain that M2 uptake upon agonist stimulation requires clathrin. The expression of various dominant-negative dynamin-2 mutants and the use of chemical inhibitors of dynamin function revealed that dynamin expression and membrane localization as such appear to be necessary for M2 endocytosis, whereas dynamin GTPase activity is not required for this process. Based on the data from the present and from previous studies, we propose that M2 endocytosis takes place by means of an atypical clathrin-mediated pathway that may involve a specific subset of clathrin-coated pits/vesicles.

  9. Revisiting the Endocytosis of the M2 Muscarinic Acetylcholine Receptor

    PubMed Central

    Ockenga, Wymke; Tikkanen, Ritva

    2015-01-01

    The agonist-induced endocytosis of the muscarinic acetylcholine receptor M2 is different from that of the other members of the muscarinic receptor family. The uptake of the M2 receptor involves the adapter proteins of the β-arrestin family and the small GTPase ADP-ribosylation factor 6. However, it has remained inconclusive if M2 endocytosis is dependent on clathrin or the large GTPase dynamin. We here show by means of knocking down the clathrin heavy chain that M2 uptake upon agonist stimulation requires clathrin. The expression of various dominant-negative dynamin-2 mutants and the use of chemical inhibitors of dynamin function revealed that dynamin expression and membrane localization as such appear to be necessary for M2 endocytosis, whereas dynamin GTPase activity is not required for this process. Based on the data from the present and from previous studies, we propose that M2 endocytosis takes place by means of an atypical clathrin-mediated pathway that may involve a specific subset of clathrin-coated pits/vesicles. PMID:25985102

  10. Autoradiographic visualization of muscarinic receptors in human bronchi

    SciTech Connect

    van Koppen, C.J.; Blankesteijn, W.M.; Klaassen, A.B.; Rodrigues de Miranda, J.F.; Beld, A.J.; van Ginneken, C.A.

    1988-02-01

    To visualize muscarinic receptors in human bronchi, the stripping film method was used which permits direct autoradiographic localization of tissue labeling. Cryostate sections of human bronchi were fixed in 0.5% glutaraldehyde in Krebs-Ringer buffer, pH 7.0 for 30 min at 0/sup 0/C, washed in Krebs-Ringer buffer for 20 min at 0/sup 0/C and incubated with (-)-(/sup 3/H)Quinuclidinyl benzilate ((-)-(/sup 3/H)QNB) for 90 min at 37/sup 0/C. Specific (-)-(/sup 3/H)QNB binding to tissue sections was saturable (receptor density of 0.14 +/- 0.03 fmol/tissue section) and of high affinity (Kd of 40 +/- 9 pM). For autoradiography, labeled tissue sections were covered with stripping film and exposed for 5 months. Muscarinic receptors in human bronchi were located predominantly in submucosal glands and parasympathetic ganglia. There was less labeling in smooth muscle cells and nerve bundles. Epithelium and blood vessels located within the bronchial wall were devoid of specific labeling.

  11. Modulation of M(2) muscarinic receptor-receptor interaction by immunoglobulin G antibodies from Chagas' disease patients.

    PubMed

    Beltrame, S P; Auger, S R; Bilder, C R; Waldner, C I; Goin, J C

    2011-05-01

    Circulating immunoglobulin (Ig)G antibodies against M(2) muscarinic acetylcholine receptors (M(2) mAChR) have been implicated in Chagas' disease (ChD) pathophysiology. These antibodies bind to and activate their target receptor, displaying agonist-like activity through an unclear mechanism. This study tested the ability of serum anti-M(2) mAChR antibodies from chronic ChD patients to modulate M(2) muscarinic receptor-receptor interaction by bioluminescence resonance energy transfer (BRET). Human embryonic kidney (HEK) 293 cells co-expressing fusion proteins M(2) mAChR-Renilla luciferase (RLuc) and M(2) mAChR-yellow fluorescent protein (YFP) were exposed to the serum IgG fraction from ChD patients, and BRET between RLuc and YFP was assessed by luminometry. Unlike serum IgG from healthy subjects and conventional muscarinic ligands, ChD IgG promoted a time- and concentration-dependent increase in the BRET signal. This effect neither required cellular integrity nor occurred as a consequence of receptor activation. Enhancement of M(2) receptor-receptor interaction by ChD IgG was receptor subtype-specific and mediated by the recognition of the second extracellular loop of the M(2) mAChR. The monovalent Fab fragment derived from ChD IgG was unable to reproduce the effect of the native immunoglobulin. However, addition of ChD Fab in the presence of anti-human Fab IgG restored BRET-enhancing activity. These data suggest that the modulatory effect of ChD IgG on M(2) receptor-receptor interaction results from receptor cross-linking by bivalent antibodies.

  12. Inhibition of adenylate cyclase attenuates muscarinic Ca²(+) signaling by a PKA-independent mechanism in rat carotid body Type I cells.

    PubMed

    Thompson, Carrie M; Wyatt, Christopher N

    2011-01-31

    Carotid body (CB) Type I cells respond to hypoxia by releasing excitatory and inhibitory neurotransmitters. This mechanism leads to increased firing of the carotid sinus nerve (CSN) which alters breathing to maintain blood gases within the physiological range. Acetylcholine targets both muscarinic and nicotinic receptors in the rat CB, acting postsynaptically on CSN and presynaptically on Type I cells. Muscarinic Ca²(+) signaling is inhibited by the activation of G(i)-coupled receptors including histamine H3 receptors. Here inhibition of adenylate cyclase with SQ22536 mimicked H3 receptor activation. Using Ca²(+) imaging techniques it was observed that inhibition of muscarinic Ca²(+) signaling was independent of protein kinase A (PKA) as PKA inhibitors H89 and KT5720 were without effect on the muscarinic Ca²(+) response. By contrast the Epac (exchange protein activated by cAMP) inhibitor brefeldin A inhibited muscarinic Ca²(+) signaling whereas the Epac activator 8-pCPT-2'-O-Me-cAMP-AM potentiated Ca²(+) signaling. Thus in Type I cells inhibition of adenylate cyclase inhibited muscarinic Ca²(+) signaling via a PKA-independent pathway that may rely upon modulation of Epac.

  13. Ozone-induced loss of neuronal M2 muscarinic receptor function is prevented by cyclophosphamide.

    PubMed

    Gambone, L M; Elbon, C L; Fryer, A D

    1994-09-01

    We tested the hypothesis that inflammatory cells mediate the loss of neuronal M2 muscarinic receptors in the lung after ozone exposure. Pathogen-free guinea pigs treated with cyclophosphamide (30 mg.kg-1.day-1 i.p. for 7 days) before exposure to ozone were compared with untreated ozone-exposed animals. This dose of cyclophosphamide significantly reduced leukocytes in peripheral blood and bronchoalveolar lavage fluid. Twenty-four hours after ozone, muscarinic receptor function was tested in anesthetized animals. In air-exposed guinea pigs, vagally induced bronchoconstriction was attenuated by the muscarinic agonist pilocarpine (0.1-100 micrograms/kg i.v.) and potentiated by the selective M2 antagonist gallamine (0.1-10 mg/kg i.v.), indicating that the neuronal M2 muscarinic receptors were functioning. These responses were significantly reduced after ozone, indicating loss of neuronal M2 muscarinic receptor function. However, in those animals treated with cyclophosphamide, M2 muscarinic receptor function was not altered by ozone. These data suggest that ozone-induced loss of neuronal muscarinic receptor function is mediated via inflammatory cells and that the link between ozone-induced hyperresponsiveness and inflammation may be the neuronal M2 muscarinic receptor.

  14. Binding characteristics of the muscarinic receptor subtype in rabbit pancreas

    SciTech Connect

    van Zwam, A.J.; Willems, P.H.; Rodrigues de Miranda, J.F.; de Pont, J.J.; van Ginneken, C.A. )

    1990-01-01

    The muscarinic receptor in the rabbit pancreas was characterized with the use of the labeled ligand ({sup 3}H)-(-)-quinuclidinyl-benzylate (({sup 3}H)-(-)-QNB). Specific binding of ({sup 3}H)-(-)-QNB to pancreatic acini was found to be reversible and of high affinity, with an equilibrium dissociation constant (KD) of 68 pmol/l and a receptor density (RT) of 170 fmol/mg protein. Agonist binding behaviour was investigated by displacement of ({sup 3}H)-(-)-QNB binding by eight agonists like arecoline, arecadine-propargylester (APE) and carbachol, yielding only low affinity binding sites. The inhibition of ({sup 3}H)-(-)-QNB binding by the selective antagonists pirenzepine, hexahydrosiladifenidol (HHSiD) and (11-(2-(diethyl-amino)-methyl-1-piperidinyl)acetyl)-5,11-dihydro-6H-pyr ido (2,3-b) (1,4) benzodiazepin-6-one (AF-DX 116) confirmed the M3 nature of the rabbit pancreatic receptor.

  15. Effects of selective activation of M1 and M4 muscarinic receptors on object recognition memory performance in rats.

    PubMed

    Galloway, Claire R; Lebois, Evan P; Shagarabi, Shezza L; Hernandez, Norma A; Manns, Joseph R

    2014-01-01

    Acetylcholine signaling through muscarinic receptors has been shown to benefit memory performance in some conditions, but pan-muscarinic activation also frequently leads to peripheral side effects. Drug therapies that selectively target M1 or M4 muscarinic receptors could potentially improve memory while minimizing side effects mediated by the other muscarinic receptor subtypes. The ability of three recently developed drugs that selectively activate M1 or M4 receptors to improve recognition memory was tested by giving Long-Evans rats subcutaneous injections of three different doses of the M1 agonist VU0364572, the M1 positive allosteric modulator BQCA or the M4 positive allosteric modulator VU0152100 before performing an object recognition memory task. VU0364572 at 0.1 mg/kg, BQCA at 1.0 mg/kg and VU0152100 at 3.0 and 30.0 mg/kg improved the memory performance of rats that performed poorly at baseline, yet the improvements in memory performance were the most statistically robust for VU0152100 at 3.0 mg/kg. The results suggested that selective M1 and M4 receptor activation each improved memory but that the likelihood of obtaining behavioral efficacy at a given dose might vary between subjects even in healthy groups depending on baseline performance. These results also highlighted the potential of drug therapies that selectively target M1 or M4 receptors to improve memory performance in individuals with impaired memory.

  16. Evidence for cross-talk between M2 and M3 muscarinic acetylcholine receptors in the regulation of second messenger and extracellular signal-regulated kinase signalling pathways in Chinese hamster ovary cells

    PubMed Central

    Hornigold, David C; Mistry, Rajendra; Raymond, Pamela D; Blank, Jonathan L; John Challiss, R A

    2003-01-01

    We have examined possible mechanisms of cross-talk between the Gq/11-linked M3 muscarinic acetylcholine (mACh) receptor and the Gi/o-linked M2 mACh receptor by stable receptor coexpression in Chinese hamster ovary (CHO) cells. A number of second messenger (cyclic AMP, Ins(1,4,5)P3) and mitogen-activated protein kinase (ERK and JNK) responses stimulated by the mACh receptor agonist methacholine were examined in CHO-m2m3 cells and compared to those stimulated in CHO-m2 and CHO-m3 cell-lines, expressing comparable levels of M2 or M3 mACh receptors. Based on comparisons between cell-lines and pertussis toxin (PTx) pretreatment to eliminate receptor-Gi/o coupling, evidence was obtained for (i) an M2 mACh receptor-mediated contribution to the predominantly M3 mACh receptor-mediated Ins(1,4,5)P3 response and (ii) a facilitation of the inhibitory effect of M2 mACh receptor on forskolin-stimulated cyclic AMP accumulation by M3 mACh receptor coactivation at low agonist concentrations (MCh 10−9–10−6 M). The most profound cross-talk effects were observed with respect to ERK activation. Thus, while MCh stimulated ERK activation in both CHO-m2 and CHO-m3 cells (pEC50 values: 5.64±0.09 and 5.57±0.16, respectively), the concentration–effect relation was approx 50-fold left-shifted in CHO-m2m3 cells (pEC50: 7.17±0.07). In addition, the ERK response was greater and more sustained in CHO-m2m3 cells. In contrast, only minor differences were seen in the time-courses and concentration-dependencies of JNK activation in CHO-m3 and CHO-m2m3 cells. Costimulation of endogenous P2Y2 purinoceptors also caused an approx 10-fold left-shift in the MCh-stimulated ERK response in CHO-m2 cells, suggesting that the Gq/11/Gi/o interaction to affect ERK activation is not specific to muscarinic receptors. PTx pretreatment of cells had unexpected effects on ERK activation by MCh in both CHO-m2m3 and CHO-m3 cells. Thus, in CHO-m3 cells PTx pretreatment caused a marked left-shift in the MCh

  17. Activation and dynamic network of the M2 muscarinic receptor

    PubMed Central

    Miao, Yinglong; Nichols, Sara E.; Gasper, Paul M.; Metzger, Vincent T.; McCammon, J. Andrew

    2013-01-01

    G-protein-coupled receptors (GPCRs) mediate cellular responses to various hormones and neurotransmitters and are important targets for treating a wide spectrum of diseases. Although significant advances have been made in structural studies of GPCRs, details of their activation mechanism remain unclear. The X-ray crystal structure of the M2 muscarinic receptor, a key GPCR that regulates human heart rate and contractile forces of cardiomyocytes, was determined recently in an inactive antagonist-bound state. Here, activation of the M2 receptor is directly observed via accelerated molecular dynamics simulation, in contrast to previous microsecond-timescale conventional molecular dynamics simulations in which the receptor remained inactive. Receptor activation is characterized by formation of a Tyr2065.58–Tyr4407.53 hydrogen bond and ∼6-Å outward tilting of the cytoplasmic end of transmembrane α-helix 6, preceded by relocation of Trp4006.48 toward Phe1955.47 and Val1995.51 and flipping of Tyr4307.43 away from the ligand-binding cavity. Network analysis reveals that communication in the intracellular domains is greatly weakened during activation of the receptor. Together with the finding that residue motions in the ligand-binding and G-protein-coupling sites of the apo receptor are correlated, this result highlights a dynamic network for allosteric regulation of the M2 receptor activation. PMID:23781107

  18. Modified expression of peripheral blood lymphocyte muscarinic cholinergic receptors in asthmatic children.

    PubMed

    Cherubini, Emanuela; Tabbì, Luca; Scozzi, Davide; Mariotta, Salvatore; Galli, Elena; Carello, Rossella; Avitabile, Simona; Tayebati, Seyed Koshrow; Amenta, Francesco; De Vitis, Claudia; Mancini, Rita; Ricci, Alberto

    2015-07-15

    Lymphocytes possess an independent cholinergic system. We assessed the expression of muscarinic cholinergic receptors in lymphocytes from 49 asthmatic children and 10 age matched controls using Western blot. We demonstrated that CD4+ and CD8+ T cells expressed M2 and M4 muscarinic receptors which density were significantly increased in asthmatic children in comparison with controls. M2 and M4 receptor increase was strictly related with IgE and fraction of exhaled nitric oxide (FeNO) measurements and with impairment in objective measurements of airway obstruction. Increased lymphocyte muscarinic cholinergic receptor expression may concur with lung cholinergic dysfunction and with inflammatory molecular framework in asthma.

  19. Effects of ovarian hormones on beta-adrenergic and muscarinic receptors in rat heart

    SciTech Connect

    Klangkalya, B.; Chan, A.

    1988-01-01

    The in vitro and in vivo effects of estrogen and progesterone on muscarinic and ..beta..-adrenergic receptors of cardiac tissue were studied in ovariectomized (OVX) rats. The binding assay for muscarinic receptors was performed under a nonequilibrium condition; whereas the binding assay for ..beta..-adrenergic receptors, under an equilibrium condition. Estrogenic compounds and progesterone were found to have no effect on the binding of the radioligand, (/sup 3/H)-dihydroalprenolol, to ..beta..-adrenergic receptors in vitro. However, progestins but not estrogenic compounds inhibited the binding of the radioligand, (/sup 3/H)-quinuclidinyl benzilate, to muscarinic receptors in vitro, with progesterone as the most potent inhibitor. Progesterone was found to decrease the apparent affinity of muscarinic receptors for (/sup 3/H)(-)QNB in vitro. Daily treatment of OVX rats with estradiol benzoate or progesterone for 4 days had no effect on the muscarinic or ..beta..-adrenergic receptors with respect to the binding affinity and receptor density. However, administrations of these hormones together for 4 days caused an increase in the receptor density of muscarinic receptors without a significant effect on their apparent binding affinity; also these hormones induced a decrease in the binding affinity and an increase in the receptor density of ..beta..-adrenergic receptors.

  20. Identification of muscarinic receptor subtypes involved in catecholamine secretion in adrenal medullary chromaffin cells by genetic deletion

    PubMed Central

    Harada, Keita; Matsuoka, Hidetada; Miyata, Hironori; Matsui, Minoru; Inoue, Masumi

    2015-01-01

    Background and Purpose Activation of muscarinic receptors results in catecholamine secretion in adrenal chromaffin cells in many mammals, and muscarinic receptors partly mediate synaptic transmission from the splanchnic nerve, at least in guinea pigs. To elucidate the physiological functions of muscarinic receptors in chromaffin cells, it is necessary to identify the muscarinic receptor subtypes involved in excitation. Experimental Approach To identify muscarinic receptors, pharmacological tools and strains of mice where one or several muscarinic receptor subtypes were genetically deleted were used. Cellular responses to muscarinic stimulation in isolated chromaffin cells were studied with the patch clamp technique and amperometry. Key Results Muscarinic M1, M4 and M5 receptors were immunologically detected in mouse chromaffin cells, and these receptors disappeared after the appropriate gene deletion. Mouse cells secreted catecholamines in response to muscarinic agonists, angiotensin II and a decrease in external pH. Genetic deletion of M1, but not M3, M4 or M5, receptors in mice abolished secretion in response to muscarine, but not to other stimuli. The muscarine-induced secretion was suppressed by MT7, a snake peptide toxin specific for M1 receptors. Similarly, muscarine failed to induce an inward current in the presence of MT7 in mouse and rat chromaffin cells. The binding affinity of VU0255035 for the inhibition of muscarine-induced currents agreed with that for the M1 receptor. Conclusions and Implications Based upon the effects of genetic deletion of muscarinic receptors and MT7, it is concluded that the M1 receptor alone is responsible for muscarine-induced catecholamine secretion. PMID:25393049

  1. Muscarinic receptor subtypes in human and rat colon smooth muscle.

    PubMed

    Gómez, A; Martos, F; Bellido, I; Marquez, E; Garcia, A J; Pavia, J; Sanchez de la Cuesta, F

    1992-06-09

    Muscarinic receptor subtypes in human and rat colon smooth muscle homogenates were characterized with [3H]N-methylscopolamine ([3H]NMS) by ligand binding studies. [3H]NMS saturation experiments show the existence of a homogeneous population of non-interacting binding sites with similar affinity (KD values of 1.38 +/- 0.20 nM in human colon smooth muscle and 1.48 +/- 0.47 nM in rat colon smooth muscle) and with Hill slopes close to unity in both samples of tissue. However, a significant (P less than 0.01) increase in muscarinic receptor density (Bmax) is found in human colon (29.9 +/- 2.9 fmol/mg protein) compared with rat colon (17.2 +/- 1.5 fmol/mg protein). Inhibition of [3H]NMS binding by non-labelled compounds shows the following order in human colon: atropine greater than AF-DX 116 greater than pirenzepine. Whereas in rat colon the rank order obtained is atropine greater than pirenzepine greater than AF-DX 116. Atropine and pirenzepine bind to a homogeneous population of binding sites, although pirenzepine shows higher affinity to bind to the sites present in rat colon (Ki = 1.08 +/- 0.08 microM) than those in human colon (Ki = 1.74 +/- 0.02 microM) (P less than 0.05). Similarly, IC50 values obtained in AF-DX 116 competition experiments were significantly different (P less than 0.01) in human colon (IC50 = 1.69 +/- 0.37 microM) than in rat colon (IC50 = 3.78 +/- 0.75 microM). Unlike atropine and pirenzepine, the inhibition of [3H]NMS binding by AF-DX 116 did not yield a simple mass-action binding curve (nH less than 1, P less than 0.01) suggesting the presence of more than one subtype of muscarinic receptor in both species. Computer analysis of these curves with a two binding site model suggests the presence of two populations of receptor. The apparent Ki1 value for the high affinity binding site is 0.49 +/- 0.07 microM for human colon smooth muscle and 0.33 +/- 0.05 microM for rat colon smooth muscle. The apparent Ki2 for the low affinity binding site is 8

  2. Effects of novel muscarinic M3 receptor ligand C1213 in pulmonary arterial hypertension models.

    PubMed

    Ahmed, Mohamed; VanPatten, Sonya; Lakshminrusimha, Satyan; Patel, Hardik; Coleman, Thomas R; Al-Abed, Yousef

    2016-12-01

    Pulmonary hypertension (PH) is a complex disease comprising a pathologic remodeling and thickening of the pulmonary vessels causing an after load on the right heart ventricle that can result in ventricular failure. Triggered by oxidative stress, episodes of hypoxia, and other undetermined causes, PH is associated with poor outcomes and a high rate of morbidity. In the neonate, this disease has a similar etiology but is further complicated by the transition to breathing after birth, which requires a reduction in vascular resistance. Persistent pulmonary hypertension of the newborn (PPHN) is one form of PH that is frequently unresponsive to current therapies including inhaled nitric oxide (due to lack of proper absorption and diffusion), and other therapeutics targeting signaling mediators in vascular endothelium and smooth muscle. The need for novel agents, which target distinct pathways in pulmonary hypertension, remains. Herein, we investigated the therapeutic effects of novel muscarinic receptor ligand C1213 in models of PH We demonstrated that via M3 muscarinic receptors, C1213 induced activating- eNOS phosphorylation (serine-1177), which is known to lead to nitric oxide (NO) production in endothelial cells. Using signaling pathway inhibitors, we discovered that AKT and calcium signaling contributed to eNOS phosphorylation induced by C1213. As expected for an eNOS-stimulating agent, in ex vivo and in vivo models, C1213 triggered pulmonary vasodilation and induced both pulmonary artery and systemic blood pressure reductions demonstrating its potential value in PH and PPHN In brief, this proof-of-concept study provides evidence that an M3 muscarinic receptor functionally selective ligand stimulates downstream pathways leading to antihypertensive effects using in vitro, ex vivo, and in vivo models of PH.

  3. The location of muscarinic type 2 receptors within the synaptic circuitry of the cat lateral posterior nucleus.

    PubMed

    Carden, W B; Bickford, M E

    1999-12-10

    The ultrastructural distribution of the muscarinic type 2 acetylcholine receptor (M2) was examined in the lateral division of the lateral posterior (LP) nucleus of the cat thalamus, using immunocytochemistry. Postembedding immunocytochemical staining for gamma-aminobutyric acid (GABA) further characterized M2 stained profiles. M2 receptors were predominately found on small caliber (presumably distal) dendritic arbors of thalamocortical cells and interneurons in the lateral LP nucleus. While glomeruli were not abundant in the lateral LP nucleus, occasionally they contained dendritic terminals of interneurons (F2 profiles) stained for M2 receptors. Some GABAergic terminals throughout the neuropil also stained for M2 receptors. The location of M2 receptors correlates well with the cholinergic innervation of the lateral LP nucleus and suggests that muscarinic modulation of visual signals differs in the lateral LP nucleus as compared with the lateral geniculate and pulvinar nuclei.

  4. The importance of muscarinic receptors in domestic animal diseases and therapy: Current and future perspectives.

    PubMed

    Abraham, Getu

    2016-02-01

    This review provides an overview of the early and current literature including contributions that highlight the parasympathetic cholinergic receptor systems in domestic animal tissues. Muscarinic acetylcholine receptors (mAChRs) belong to the subfamily of G protein-coupled receptors and regulate many fundamental functions of the central and peripheral nervous systems and have been subject to research over at least 40 years. Nonetheless, there are few studies specifying mAChRs in domestic animal tissues. This review focuses on the pharmacology of muscarinic acetylcholine receptor (mAChR) system and its pathological as well as the therapeutic importance in organ systems of domestic animals. Illustration and discussion of recent advances in distribution, function, biochemistry and pharmacology of mAChRs are followed by summaries of the involvement of this family of receptors in cardiovascular, respiratory, neurological, gastrointestinal (GI) and urological diseases as well as in anaesthesia and toxicology. Specific functions of mAChRs are described in detail including subtype characterization, smooth muscle functions, signal transduction and regulation. Due to their wide tissue distribution, mAChRs have shown promise as targets for the treatment of some animal diseases such as equine recurrent airway obstruction, glaucoma, abnormalities of gastric acid secretion and GI disturbances including colic. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. The role of muscarinic cholinergic signaling in cost-benefit decision making

    NASA Astrophysics Data System (ADS)

    Fobbs, Wambura

    Animals regularly face decisions that affect both their immediate success and long term survival. Such decisions typically involve some form of cost-benefit analysis and engage a number of high level cognitive processes, including learning, memory and motivational influences. While decision making has been a focus of study for over a century, it's only in the last 20 years that researchers have begun to identify functional neural circuits that subserve different forms of cost-benefit decision making. Even though the cholinergic system is both functionally and anatomically positioned to modulate cost-benefit decision circuits, the contribution of the cholinergic system to decision making has been little studied. In this thesis, I investigated the cognitive and neural contribution of muscarinic cholinergic signaling to cost-benefit decision making. I, first, re-examined the effects of systemic administration of 0.3 mg/kg atropine on delay and probability discounting tasks and found that blockade of muscarinic acetylcholine receptors by atropine induced suboptimal choices (impulsive and risky) in both tasks. Since the effect on delay discounting was restricted to the No Cue version of the delay discounting task, I concluded that muscarinic cholinergic signaling mediates both forms of cost-benefit decision making and is selectively engaged when decisions require valuation of reward options whose costs are not externally signified. Second, I assessed the impact of inactivating the nucleus basalis (NBM) on both forms decision making and the effect of injecting atropine locally into the orbitofrontal cortex (OFC), basolateral amygdala (BLA), or nucleus accumbens (NAc) core during the No Cue version of the delay discounting task. I discovered that although NBM inactivation failed to affect delay discounting, it induced risk aversion in the probability discounting task; and blockade of intra- NAc core, but not intra-OFC or intra-BLA, muscarinic cholinergic signaling lead to

  6. A Novel Voltage Sensor in the Orthosteric Binding Site of the M2 Muscarinic Receptor.

    PubMed

    Barchad-Avitzur, Ofra; Priest, Michael F; Dekel, Noa; Bezanilla, Francisco; Parnas, Hanna; Ben-Chaim, Yair

    2016-10-04

    G protein-coupled receptors (GPCRs) mediate many signal transduction processes in the body. The discovery that these receptors are voltage-sensitive has changed our understanding of their behavior. The M2 muscarinic acetylcholine receptor (M2R) was found to exhibit depolarization-induced charge movement-associated currents, implying that this prototypical GPCR possesses a voltage sensor. However, the typical domain that serves as a voltage sensor in voltage-gated channels is not present in GPCRs, making the search for the voltage sensor in the latter challenging. Here, we examine the M2R and describe a voltage sensor that is comprised of tyrosine residues. This voltage sensor is crucial for the voltage dependence of agonist binding to the receptor. The tyrosine-based voltage sensor discovered here constitutes a noncanonical by which membrane proteins may sense voltage. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  7. Blocking muscarinic receptors in the olfactory bulb impairs performance on an olfactory short-term memory task

    PubMed Central

    Devore, Sasha; Manella, Laura C.; Linster, Christiane

    2012-01-01

    Cholinergic inputs to cortical processing networks have long been associated with attentional and top-down processing. Experimental and theoretical studies suggest that cholinergic inputs to the main olfactory bulb (OB) can modulate both neural and behavioral odor discrimination. Previous experiments from our laboratory and others demonstrate that blockade of nicotinic receptors directly impairs olfactory discrimination, whereas blockade of muscarinic receptors only measurably impairs olfactory perception when task demands are made more challenging, such as when very low-concentration odors are used or rats are required to maintain sensory memory over long durations. To further investigate the role of muscarinic signaling in the OB, we developed an olfactory delayed match-to-sample task using a digging-based behavioral paradigm. We find that rats are able to maintain robust short-term odor memory for 10–100 s. To investigate the role of muscarinic signaling in task performance, we bilaterally infused scopolamine into the OB. We find that high dosages of scopolamine (38 mM) impair performance on the task across all delays tested, including the baseline condition with no delay, whereas lower dosages (7.6 mM and 22.8 mM) had no measureable effects. These results indicate that general execution of the match-to-sample task, even with no delay, is at least partially dependent on muscarinic signaling in the OB. PMID:22973212

  8. RGS4 regulates partial agonism of the M2 muscarinic receptor-activated K+ currents

    PubMed Central

    Chen, I-Shan; Furutani, Kazuharu; Inanobe, Atsushi; Kurachi, Yoshihisa

    2014-01-01

    Partial agonists are used clinically to avoid overstimulation of receptor-mediated signalling, as they produce a submaximal response even at 100% receptor occupancy. The submaximal efficacy of partial agonists is due to conformational change of the agonist–receptor complex, which reduces effector activation. In addition to signalling activators, several regulators help control intracellular signal transductions. However, it remains unclear whether these signalling regulators contribute to partial agonism. Here we show that regulator of G-protein signalling (RGS) 4 is a determinant for partial agonism of the M2 muscarinic receptor (M2R). In rat atrial myocytes, pilocarpine evoked smaller G-protein-gated K+ inwardly rectifying (KG) currents than those evoked by ACh. In a Xenopus oocyte expression system, pilocarpine acted as a partial agonist in the presence of RGS4 as it did in atrial myocytes, while it acted like a full agonist in the absence of RGS4. Functional couplings within the agonist–receptor complex/G-protein/RGS4 system controlled the efficacy of pilocarpine relative to ACh. The pilocarpine–M2R complex suppressed G-protein-mediated activation of KG currents via RGS4. Our results demonstrate that partial agonism of M2R is regulated by the RGS4-mediated inhibition of G-protein signalling. This finding helps us to understand the molecular components and mechanism underlying the partial agonism of M2R-mediated physiological responses. PMID:24421355

  9. Muscarinic M1 receptor and cannabinoid CB1 receptor do not modulate paraoxon-induced seizures

    PubMed Central

    Kow, Rebecca L; Cheng, Eugene M; Jiang, Kelly; Le, Joshua H; Stella, Nephi; Nathanson, Neil M

    2015-01-01

    One of the major signs of severe organophosphate poisoning is seizures. Previous studies have shown that both muscarinic agonist- and organophosphate-induced seizures require activation of muscarinic acetylcholine receptors in the central nervous system. Seizures induced by the muscarinic agonist pilocarpine require the M1 receptor and are modulated by cannabinoid CB1 receptors. In this study, we determined whether M1 and CB1 receptors also regulated seizures induced by the organophosphate paraoxon. We found no differences in seizures induced by paraoxon in wild-type (WT) and M1 knockout (KO) mice, indicating that in contrast to pilocarpine seizures, M1 receptors are not required for paraoxon seizures. Furthermore, we found that pilocarpine administration resulted in seizure-independent activation of ERK in the hippocampus in a M1 receptor-dependent manner, while paraoxon did not induce seizure-independent activation of ERK in the mouse hippocampus. This shows that pilocarpine and paraoxon activated M1 receptors in the hippocampus to different extents. There were no differences in seizures induced by paraoxon in WT and CB1 KO mice, and neither CB1 agonist nor antagonist administration had significant effects on paraoxon seizures, indicating that, in contrast to pilocarpine seizures, paraoxon seizures are not modulated by CB1 receptors. These results demonstrate that there are fundamental molecular differences in the regulation of seizures induced by pilocarpine and paraoxon. PMID:25692018

  10. Muscarinic ACh Receptors Contribute to Aversive Olfactory Learning in Drosophila

    PubMed Central

    Silva, Bryon; Molina-Fernández, Claudia; Ugalde, María Beatriz; Tognarelli, Eduardo I.; Angel, Cristian; Campusano, Jorge M.

    2015-01-01

    The most studied form of associative learning in Drosophila consists in pairing an odorant, the conditioned stimulus (CS), with an unconditioned stimulus (US). The timely arrival of the CS and US information to a specific Drosophila brain association region, the mushroom bodies (MB), can induce new olfactory memories. Thus, the MB is considered a coincidence detector. It has been shown that olfactory information is conveyed to the MB through cholinergic inputs that activate acetylcholine (ACh) receptors, while the US is encoded by biogenic amine (BA) systems. In recent years, we have advanced our understanding on the specific neural BA pathways and receptors involved in olfactory learning and memory. However, little information exists on the contribution of cholinergic receptors to this process. Here we evaluate for the first time the proposition that, as in mammals, muscarinic ACh receptors (mAChRs) contribute to memory formation in Drosophila. Our results show that pharmacological and genetic blockade of mAChRs in MB disrupts olfactory aversive memory in larvae. This effect is not explained by an alteration in the ability of animals to respond to odorants or to execute motor programs. These results show that mAChRs in MB contribute to generating olfactory memories in Drosophila. PMID:26380118

  11. Muscarinic receptor subtypes in neuronal and non-neuronal cholinergic function.

    PubMed

    Eglen, R M

    2006-07-01

    1 Muscarinic M1-M5 receptors mediate the metabotropic actions of acetylcholine in the nervous system. A growing body of data indicate they also mediate autocrine functions of the molecule. The availability of novel and selective muscarinic agonists and antagonists, as well as in vivo gene disruption techniques, has clarified the roles of muscarinic receptors in mediating both functions of acetylcholine. 2 Selective M1 agonists or mixed M1 agonists/M2 antagonists may provide an approach to the treatment of cognitive disorders, while M3 antagonism, or mixed M2/M3 antagonists, are approved for the treatment of contractility disorders including overactive bladder and chronic obstructive pulmonary disease. Preclinical data suggest that selective agonism of the M4 receptor will provide novel anti-nociceptive agents, while therapeutics-based upon agonism or antagonism of the muscarinic M5 receptor have yet to be reported. 3 The autocrine functions of muscarinic receptors broadly fall into two areas - control of cell growth or proliferation and mediation of the release of chemical mediators from epithelial cells, ultimately causing muscle relaxation. The former particularly are involved in embryological development, oncogenesis, keratinocyte function and immune responsiveness. The latter regulate contractility of smooth muscle in the vasculature, airways and urinary bladder. 4 Most attention has focused on muscarinic M1 or M3 receptors which mediate lymphocyte immunoresponsiveness, cell migration and release of smooth muscle relaxant factors. Muscarinic M4 receptors are implicated in the regulation of keratinocyte adhesion and M2 receptors in stem cell proliferation and development. Little data are available concerning the M5 receptor, partly due to the difficulties in defining the subtype pharmacologically. 5 The autocrine functions of acetylcholine, like those in the nervous system, involve activation of several muscarinic receptor subtypes. Consequently, the role of

  12. Scopolamine administration modulates muscarinic, nicotinic and NMDA receptor systems.

    PubMed

    Falsafi, Soheil Keihan; Deli, Alev; Höger, Harald; Pollak, Arnold; Lubec, Gert

    2012-01-01

    Studies on the effect of scopolamine on memory are abundant but so far only regulation of the muscarinic receptor (M1) has been reported. We hypothesized that levels of other cholinergic brain receptors as the nicotinic receptors and the N-methyl-D-aspartate (NMDA) receptor, known to be involved in memory formation, would be modified by scopolamine administration.C57BL/6J mice were used for the experiments and divided into four groups. Two groups were given scopolamine 1 mg/kg i.p. (the first group was trained and the second group untrained) in the multiple T-maze (MTM), a paradigm for evaluation of spatial memory. Likewise, vehicle-treated mice were trained or untrained thus serving as controls. Hippocampal levels of M1, nicotinic receptor alpha 4 (Nic4) and 7 (Nic7) and subunit NR1containing complexes were determined by immunoblotting on blue native gel electrophoresis.Vehicle-treated trained mice learned the task and showed memory retrieval on day 8, while scopolamine-treatment led to significant impairment of performance in the MTM. At the day of retrieval, hippocampal levels for M1, Nic7 and NR1 were higher in the scopolamine treated groups than in vehicle-treated groups.The concerted action, i.e. the pattern of four brain receptor complexes regulated by the anticholinergic compound scopolamine, is shown. Insight into probable action mechanisms of scopolamine at the brain receptor complex level in the hippocampus is provided. Scopolamine treatment is a standard approach to test cognitive enhancers and other psychoactive compounds in pharmacological studies and therefore knowledge on mechanisms is of pivotal interest.

  13. Structure and dynamics of the M3 muscarinic acetylcholine receptor

    SciTech Connect

    Kruse, Andrew C.; Hu, Jianxin; Pan, Albert C.; Arlow, Daniel H.; Rosenbaum, Daniel M.; Rosemond, Erica; Green, Hillary F.; Liu, Tong; Chae, Pil Seok; Dror, Ron O.; Shaw, David E.; Weis, William I.; Wess, Jürgen; Kobilka, Brian K.

    2012-03-01

    Acetylcholine, the first neurotransmitter to be identified, exerts many of its physiological actions via activation of a family of G-protein-coupled receptors (GPCRs) known as muscarinic acetylcholine receptors (mAChRs). Although the five mAChR subtypes (M1-M5) share a high degree of sequence homology, they show pronounced differences in G-protein coupling preference and the physiological responses they mediate. Unfortunately, despite decades of effort, no therapeutic agents endowed with clear mAChR subtype selectivity have been developed to exploit these differences. We describe here the structure of the G{sub q/11}-coupled M3 mAChR ('M3 receptor', from rat) bound to the bronchodilator drug tiotropium and identify the binding mode for this clinically important drug. This structure, together with that of the G{sub i/o}-coupled M2 receptor, offers possibilities for the design of mAChR subtype-selective ligands. Importantly, the M3 receptor structure allows a structural comparison between two members of a mammalian GPCR subfamily displaying different G-protein coupling selectivities. Furthermore, molecular dynamics simulations suggest that tiotropium binds transiently to an allosteric site en route to the binding pocket of both receptors. These simulations offer a structural view of an allosteric binding mode for an orthosteric GPCR ligand and provide additional opportunities for the design of ligands with different affinities or binding kinetics for different mAChR subtypes. Our findings not only offer insights into the structure and function of one of the most important GPCR families, but may also facilitate the design of improved therapeutics targeting these critical receptors.

  14. Quantitative in vivo receptor binding. I. Theory and application to the muscarinic cholinergic receptor

    SciTech Connect

    Frey, K.A.; Ehrenkaufer, R.L.; Beaucage, S.; Agranoff, B.W.

    1985-02-01

    A novel approach to in vivo receptor binding experiments is presented which allows direct quantitation of binding site densities. The method is based on an equilibrium model of tracer uptake and is designed to produce a static distribution proportional to receptor density and to minimize possible confounding influences of regional blood flow, blood-brain barrier permeability, and nonspecific binding. This technique was applied to the measurement of regional muscarinic cholinergic receptor densities in rat brain using (/sup 3/H)scopolamine. Specific in vivo binding of scopolamine demonstrated saturability, a pharmacologic profile, and regional densities which are consistent with interaction of the tracer with the muscarinic receptor. Estimates of receptor density obtained with the in vivo method and in vitro measurements in homogenates were highly correlated. Furthermore, reduction in striatal muscarinic receptors following ibotenic acid lesions resulted in a significant decrease in tracer uptake in vivo, indicating that the correlation between scopolamine distribution and receptor density may be used to demonstrate pathologic conditions. We propose that the general method presented here is directly applicable to investigation of high affinity binding sites for a variety of radioligands.

  15. Muscarinic receptors activity in the perirhinal cortex and hippocampus has differential involvement in the formation of recognition memory.

    PubMed

    Balderas, Israela; Morin, Jean-Pascal; Rodriguez-Ortiz, Carlos J; Bermudez-Rattoni, Federico

    2012-05-01

    In this work we probed the effects of post-trial infusions of the muscarinic receptor antagonist scopolamine on object recognition memory formation. Scopolamine was infused bilaterally immediately after the sample phase in the perirhinal cortex or dorsal hippocampus and animals were tested for short-term (90 min) or long-term (24 h) memory. Results showed that scopolamine impaired short-term memory when injected in either the perirhinal cortex or hippocampus. Nevertheless, scopolamine disrupted long-term memory when administrated in the perirhinal cortex but not when applied in the hippocampus. Long-term memory was unaffected when scopolamine was infused 160 min after the sample phase or 90 min before test phase. Our data indicate that short-term recognition memory requires muscarinic receptors signaling in both the perirhinal cortex and hippocampus, whereas long-term recognition memory depends on muscarinic receptors in the perirhinal cortex but not hippocampus. These results support a differential involvement of muscarinic activity in these two medial temporal lobe structures in the formation of recognition memory. Copyright © 2012 Elsevier Inc. All rights reserved.

  16. Coupling of G Proteins to Reconstituted Monomers and Tetramers of the M2 Muscarinic Receptor*

    PubMed Central

    Redka, Dar'ya S.; Morizumi, Takefumi; Elmslie, Gwendolynne; Paranthaman, Pranavan; Shivnaraine, Rabindra V.; Ellis, John; Ernst, Oliver P.; Wells, James W.

    2014-01-01

    G protein-coupled receptors can be reconstituted as monomers in nanodiscs and as tetramers in liposomes. When reconstituted with G proteins, both forms enable an allosteric interaction between agonists and guanylyl nucleotides. Both forms, therefore, are candidates for the complex that controls signaling at the level of the receptor. To identify the biologically relevant form, reconstituted monomers and tetramers of the purified M2 muscarinic receptor were compared with muscarinic receptors in sarcolemmal membranes for the effect of guanosine 5′-[β,γ-imido]triphosphate (GMP-PNP) on the inhibition of N-[3H]methylscopolamine by the agonist oxotremorine-M. With monomers, a stepwise increase in the concentration of GMP-PNP effected a lateral, rightward shift in the semilogarithmic binding profile (i.e. a progressive decrease in the apparent affinity of oxotremorine-M). With tetramers and receptors in sarcolemmal membranes, GMP-PNP effected a vertical, upward shift (i.e. an apparent redistribution of sites from a state of high affinity to one of low affinity with no change in affinity per se). The data were analyzed in terms of a mechanistic scheme based on a ligand-regulated equilibrium between uncoupled and G protein-coupled receptors (the “ternary complex model”). The model predicts a rightward shift in the presence of GMP-PNP and could not account for the effects at tetramers in vesicles or receptors in sarcolemmal membranes. Monomers present a special case of the model in which agonists and guanylyl nucleotides interact within a complex that is both constitutive and stable. The results favor oligomers of the M2 receptor over monomers as the biologically relevant state for coupling to G proteins. PMID:25023280

  17. Vitamin C Deficiency Reduces Muscarinic Receptor Coronary Artery Vasoconstriction and Plasma Tetrahydrobiopterin Concentration in Guinea Pigs

    PubMed Central

    Skovsted, Gry Freja; Tveden-Nyborg, Pernille; Lindblad, Maiken Marie; Hansen, Stine Normann

    2017-01-01

    Vitamin C (vitC) deficiency is associated with increased cardiovascular disease risk, but its specific interplay with arteriolar function is unclear. This study investigates the effect of vitC deficiency in guinea pigs on plasma biopterin status and the vasomotor responses in coronary arteries exposed to vasoconstrictor/-dilator agents. Dunkin Hartley female guinea pigs (n = 32) were randomized to high (1500 mg/kg diet) or low (0 to 50 mg/kg diet) vitC for 10–12 weeks. At euthanasia, coronary artery segments were dissected and mounted in a wire-myograph. Vasomotor responses to potassium, carbachol, sodium nitroprusside (SNP), U46619, sarafotoxin 6c (S6c) and endothelin-1 (ET-1) were recorded. Plasma vitC and tetrahydrobiopterin were measured by HPLC. Plasma vitC status reflected the diets with deficient animals displaying reduced tetrahydrobiopterin. Vasoconstrictor responses to carbachol were significantly decreased in vitC deficient coronary arteries independent of their general vasoconstrictor/vasodilator capacity (p < 0.001). Moreover, in vitC deficient animals, carbachol-induced vasodilator responses correlated with coronary artery diameter (p < 0.001). Inhibition of cyclooxygenases with indomethacin increased carbachol-induced vasoconstriction, suggesting an augmented carbachol-induced release of vasodilator prostanoids. Atropine abolished carbachol-induced vasomotion, supporting a specific muscarinic receptor effect. Arterial responses to SNP, potassium, S6c, U46619 and ET-1 were unaffected by vitC status. The study shows that vitC deficiency decreases tetrahydrobiopterin concentrations and muscarinic receptor mediated contraction in coronary arteries. This attenuated vasoconstrictor response may be linked to altered production of vasoactive arachidonic acid metabolites and reduced muscarinic receptor expression/signaling. PMID:28671625

  18. Muscarinic receptors, leukotriene B4 production and neutrophilic inflammation in COPD patients.

    PubMed

    Profita, M; Giorgi, R Di; Sala, A; Bonanno, A; Riccobono, L; Mirabella, F; Gjomarkaj, M; Bonsignore, G; Bousquet, J; Vignola, A M

    2005-11-01

    Acetylcholine (ACh) plays an important role in smooth muscle contraction and in the development of airway narrowing; preliminary evidences led us to hypothesize that ACh might also play a role in the development of airways inflammation in chronic obstructive pulmonary disease (COPD). We evaluated the concentrations of leukotriene B4 (LTB4) in induced sputum, and the expression of Ach M1, M2, and M3 receptors in sputum cells (SC) obtained from 16 patients with COPD, 11 smokers, and 14 control subjects. The SC were also treated with ACh and the production of LTB4 assessed in the presence or absence of a muscarinic antagonist (oxitropium). In blood monocytes, we evaluated LTB4 release and activation of the extracellular signal-regulated kinases (ERK) pathway after treatment with Ach. The LTB4 concentrations were higher in COPD than in controls (P < 0.01) and correlated with the number of neutrophil (P < 0.01). The M3 receptors expression was increased in COPD subjects when compared to smokers and control (P < 0.05 and 0.0001, respectively), while M2 expression resulted decreased (P < 0.05 and 0.01). The ACh-induced LTB(4) production was observed in peripheral blood monocytes, and was sensitive to ERK inhibition. Similarly, ACh significantly increased neutrophil chemotactic activity and LTB4 released from SC of COPD patients only, and these effects were blocked by pretreatment with the inhibitor of ERK pathway PD98059. The results obtained show that muscarinic receptors may be involved in airway inflammation in COPD subjects through ACh-induced, ERK1/2-dependent LTB4 release. Muscarinic antagonism may contribute to reduce neutrophil infiltration and activation in COPD.

  19. Genetic deletion of muscarinic M4 receptors is anxiolytic in the shock-probe burying model.

    PubMed

    Degroot, Aldemar; Nomikos, George G

    2006-02-15

    We used muscarinic M2 and M4 receptor knockout (KO) mice to further explore the role of the cholinergic system in anxiety. Using the shock-probe burying model we were able to both assess anxiety and cognition. In this paradigm, an anxiolytic response is reflected by decreased burying behavior. In addition, retention latency depicts long-term memory performance. Whereas muscarinic M2 receptor KO mice did not differ behaviorally from wild-type mice, muscarinic M4 receptor KO mice showed increased anxiolysis, but normal long-term memory compared to wild-type mice. Therefore, muscarinic M4 receptors are of particular significance in anxiety modulation that seems dissociated from changes in long-term memory.

  20. Muscarinic receptors modulate the intrinsic excitability of infralimbic neurons and consolidation of fear extinction.

    PubMed

    Santini, Edwin; Sepulveda-Orengo, Marian; Porter, James T

    2012-08-01

    There is considerable interest in identifying pharmacological compounds that could be used to facilitate fear extinction. Recently, we showed that the modulation of M-type K(+) channels regulates the intrinsic excitability of infralimbic (IL) neurons and fear expression. As muscarinic acetylcholine receptors inhibit M-type K(+) channels, cholinergic inputs to IL may have an important role in controlling IL excitability and, thereby, fear expression and extinction. To test this model, we combined whole-cell patch-clamp electrophysiology and auditory fear conditioning. In prefrontal brain slices, muscarine enhanced the intrinsic excitability of IL neurons by reducing the M-current and the slow afterhyperpolarization, resulting in an increased number of spikes with shorter inter-spike intervals. Next, we examined the role of endogenous activation of muscarinic receptors in fear extinction. Systemic injected scopolamine (Scop) (muscarinic receptor antagonist) before or immediately after extinction training impaired recall of extinction 24-h later, suggesting that muscarinic receptors are critically involved in consolidation of extinction memory. Similarly, infusion of Scop into IL before extinction training also impaired recall of extinction 24-h later. Finally, we demonstrated that systemic injections of the muscarinic agonist, cevimeline (Cev), given before or immediately after extinction training facilitated recall of extinction the following day. Taken together, these findings suggest that cholinergic inputs to IL have a critical role in modulating consolidation of fear extinction and that muscarinic agonists such as Cev might be useful for facilitating extinction memory in patients suffering from anxiety disorders.

  1. Alterations of muscarinic receptor subtypes in pathways relating to memory: Effects of lesions and transplants

    SciTech Connect

    Dawson, V.L.

    1989-01-01

    Muscarinic cholinergic receptors have been classified pharmacologically into two distinct populations designated muscarinic type-one (M-1) and mscarinic type-two (M-2). The semiquantitative technique of receptor autoradiography was used to examine the anatomical and cellular distribution, and densities of M-1 and M-2 receptors in the rate brain. Muscarinic receptors were labeled with the classical antagonist ({sup 3}H)quinuclidinyl benzilate (QNB). Differentiation of the muscarinic subtypes was accomplished by competition studies of ({sup 3}H)QNB against the relatively selective M-1 antagonist pirenzepine (PZ), and the relatively selective M-2 antagonist, AFDX-116. In addition, M-1 and M-2 receptors were directly labeled with ({sup 3}H)PZ and ({sup 3}H)AFDX-116, respectively. Cholinergic pathways from the large cholinergic neurons in the nucleus basalis magnocellularis (NBM) to the cortex and from the medial septum (MS) to the hippocampus were examined by lesioning with the selective cholinergic neurotoxin, AF64A. Bilateral cerebral cortical infarction was performed in order to analyze potential changes in muscarinic receptor populations in subcortical structures that are sensitive to cortical infarction. Finally, the response of muscarinic receptors to fetal septodiagonal band transplants in the deafferentated hippocampus was examined.

  2. Muscarinic receptor antagonists, from folklore to pharmacology; finding drugs that actually work in asthma and COPD

    PubMed Central

    Moulton, Bart C; Fryer, Allison D

    2011-01-01

    In the lungs, parasympathetic nerves provide the dominant control of airway smooth muscle with release of acetylcholine onto M3 muscarinic receptors. Treatment of airway disease with anticholinergic drugs that block muscarinic receptors began over 2000 years ago. Pharmacologic data all indicated that antimuscarinic drugs should be highly effective in asthma but clinical results were mixed. Thus, with the discovery of effective β-adrenergic receptor agonists the use of muscarinic antagonists declined. Lack of effectiveness of muscarinic antagonists is due to a variety of factors including unwanted side effects (ranging from dry mouth to coma) and the discovery of additional muscarinic receptor subtypes in the lungs with sometimes competing effects. Perhaps the most important problem is ineffective dosing due to poorly understood differences between routes of administration and no effective way of testing whether antagonists block receptors stimulated physiologically by acetylcholine. Newer muscarinic receptor antagonists are being developed that address the problems of side effects and receptor selectivity that appear to be quite promising in the treatment of asthma and chronic obstructive pulmonary disease. LINKED ARTICLES This article is part of a themed issue on Respiratory Pharmacology. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2011.163.issue-1 PMID:21198547

  3. Bovine pancreatic polypeptide as an antagonist of muscarinic cholinergic receptors

    SciTech Connect

    Pan, G.Z.; Lu, L.; Qian, J.; Xue, B.G.

    1987-03-01

    In dispersed acini from rat pancreas, it was found that bovine pancreatic polypeptide (BPP) and its C-fragment hexapeptide amide (PP-6), at concentrations of 0.1 and 30 ..mu..M, respectively, could significantly inhibit amylase secretion stimulated by carbachol, and this inhibition by BPP was dose dependent. /sup 45/Ca outflux induced by carbachol was also inhibited by BPP or PP-6, but they had no effect on cholecystokinin octapeptide- (CCK-8) or A23187-stimulated /sup 45/Ca outflux. BPP was also capable of displacing the specific binding of (/sup 3/H)-quinuclidinyl benzilate to its receptors, and it possessed a higher affinity (K/sub i/35nM) than carbachol (K/sub i/ 1.8 ..mu..M) in binding with M-receptors. It is concluded from this study that BPP acts as an antagonist of muscarinic cholinergic receptors in rat pancreatic acini. In addition, BPP inhibited the potentiation of amylase secretion caused by the combination of carbachol plus secretin or vasoactive intestinal peptide. This may be a possible explanation of the inhibitory effect of BPP on secretin-induced pancreatic enzyme secretion shown in vivo, since pancreatic enzyme secretion stimulated by secretin under experimental conditions may be the result of potentiation of enzyme release produced by the peptide in combination with a cholinergic stimulant.

  4. Structure and Dynamics of the M3 Muscarinic Acetylcholine Receptor

    PubMed Central

    Kruse, Andrew C.; Hu, Jianxin; Pan, Albert C.; Arlow, Daniel H.; Rosenbaum, Daniel M.; Rosemond, Erica; Green, Hillary F.; Liu, Tong; Chae, Pil Seok; Dror, Ron O.; Shaw, David E.; Weis, William I.; Wess, Jurgen; Kobilka, Brian

    2012-01-01

    Acetylcholine (ACh), the first neurotransmitter to be identified1, exerts many of its physiological actions via activation of a family of G protein-coupled receptors (GPCRs) known as muscarinic ACh receptors (mAChRs). Although the five mAChR subtypes (M1-M5) share a high degree of sequence homology, they show pronounced differences in G protein coupling preference and the physiological responses they mediate.2–4 Unfortunately, despite decades of effort, no therapeutic agents endowed with clear mAChR subtype selectivity have been developed to exploit these differences.5–6 We describe here the structure of the Gq/11-coupled M3 mAChR bound to the bronchodilator drug tiotropium and identify the binding mode for this clinically important drug. This structure, together with that of the Gi/o-coupled M2 receptor, offers new possibilities for the design of mAChR subtype-selective ligands. Importantly, the M3 receptor structure allows the first structural comparison between two members of a mammalian GPCR subfamily displaying different G-protein coupling selectivities. Furthermore, molecular dynamics simulations suggest that tiotropium binds transiently to an allosteric site en route to the binding pocket of both receptors. These simulations offer a structural view of an allosteric binding mode for an orthosteric GPCR ligand and raise additional opportunities for the design of ligands with different affinities or binding kinetics for different mAChR subtypes. Our findings not only offer new insights into the structure and function of one of the most important GPCR families, but may also facilitate the design of improved therapeutics targeting these critical receptors. PMID:22358844

  5. Ozone-induced airway hyperresponsiveness and loss of neuronal M2 muscarinic receptor function.

    PubMed

    Schultheis, A H; Bassett, D J; Fryer, A D

    1994-03-01

    The effect of acute ozone exposure on the function of efferent parasympathetic nerves, M3 muscarinic receptors on airway smooth muscle, and inhibitory M2 muscarinic receptors on the parasympathetic nerves was studied. Immediately after exposure to 2.0 ppm ozone for 4 h, guinea pigs became hyperresponsive to electrical stimulation of the vagus nerves. The normal airway response to intravenous cholinergic agonists at this time demonstrates normal M3 receptor function. M2 muscarinic receptors on the nerves, which normally inhibit release of acetylcholine, were dysfunctional after ozone exposure, as demonstrated by the failure of the muscarinic agonist pilocarpine to inhibit, and the failure of the M2 antagonist gallamine to potentiate, vagally mediated bronchoconstriction. Thus, loss of inhibitory M2 muscarinic receptor function after ozone exposure potentiates release of acetylcholine from the vagus nerves, increasing vagally mediated bronchoconstriction. By 14 days, postozone responses to vagal nerve stimulation were not different from those of air-exposed animals and the function of the neuronal M2 muscarinic receptor was normal, confirming that ozone-induced hyperresponsiveness is reversible.

  6. Differential receptor dependencies: expression and significance of muscarinic M1 receptors in the biology of prostate cancer.

    PubMed

    Mannan Baig, Abdul; Khan, Naveed A; Effendi, Vardah; Rana, Zohaib; Ahmad, H R; Abbas, Farhat

    2017-01-01

    Recent reports on acetylcholine muscarinic receptor subtype 3 (CHRM3) have shown its growth-promoting role in prostate cancer. Additional studies report the proliferative effect of the cholinergic agonist carbachol on prostate cancer by its agonistic action on CHRM3. This study shows that the type 1 acetylcholine muscarinic receptor (CHRM1) contributes toward the proliferation and growth of prostate cancer. We used growth and cytotoxic assays, the prostate cancer microarray database and CHRM downstream pathways' homology of CHRM subtypes to uncover multiple signals leading to the growth of prostate cancer. Growth assays showed that pilocarpine stimulates the proliferation of prostate cancer. Moreover, it shows that carbachol exerts an additional agonistic action on nicotinic cholinergic receptor of prostate cancer cells that can be blocked by tubocurarine. With the use of selective CHRM1 antagonists such as pirenzepine and dicyclomine, a considerable inhibition of proliferation of prostate cancer cell lines was observed in dose ranging from 15-60 µg/ml of dicyclomine. The microarray database of prostate cancer shows a dominant expression of CHRM1 in prostate cancer compared with other cholinergic subtypes. The bioinformatics of prostate cancer and CHRM pathways show that the downstream signalling include PIP3-AKT-CaM-mediated growth in LNCaP and PC3 cells. Our study suggests that antagonism of CHRM1 may be a potential therapeutic target against prostate cancer.

  7. Cell type-specific dependence of muscarinic signalling in mouse hippocampal stratum oriens interneurones

    PubMed Central

    Lawrence, J Josh; Statland, Jeffrey M; Grinspan, Zachary M; McBain, Chris J

    2006-01-01

    Cholinergic signalling is critically involved in learning and memory processes in the hippocampus, but the postsynaptic impact of cholinergic modulation on morphologically defined subtypes of hippocampal interneurones remains unclear. We investigated the influence of muscarinic receptor (mAChR) activation on stratum oriens interneurones using whole-cell patch clamp recordings from hippocampal slices in vitro. Upon somatic depolarization, mAChR activation consistently enhanced firing frequency and produced large, sustained afterdepolarizations (ADPs) of stratum oriens–lacunosum moleculare (O-LM) interneurones. In contrast, stratum oriens cell types with axon arborization patterns different from O-LM cells not only lacked large muscarinic ADPs but also appeared to exhibit distinct responses to mAChR activation. The ADP in O-LM cells, mediated by M1/M3 receptors, was associated with inhibition of an M current, inhibition of a slow calcium-activated potassium current, and activation of a calcium-dependent non-selective cationic current (ICAT). An examination of ionic conductances generated by firing revealed that calcium entry through ICAT controls the emergence of the mAChR-mediated ADP. Our results indicate that cholinergic specializations are present within anatomically distinct subpopulations of hippocampal interneurones, suggesting that there may be organizing principles to cholinergic control of GABA release in the hippocampus. PMID:16322052

  8. Muscarinic acetylcholine receptors: location of the ligand binding site

    SciTech Connect

    Hulme, E.; Wheatley, M.; Curtis, C.; Birdsall, N.

    1987-05-01

    The key to understanding the pharmacological specificity of muscarinic acetylcholine receptors (mAChR's) is the location within the receptor sequence of the amino acid residues responsible for ligand binding. To approach this problem, they have purified mAChR's from rat brain to homogeneity by sequential ion-exchange chromatography, affinity chromatography and molecular weight fractionation. Following labelling of the binding site with an alkylating affinity label, /sup 3/H-propylbenzilycholine mustard aziridinium ion (/sup 3/H-PrBCM), the mAChR was digested with a lysine-specific endoproteinase, and a ladder of peptides of increasing molecular weight, each containing the glycosylated N-terminus, isolated by chromatography on wheat-germ agglutinin sepharose. The pattern of labelling showed that a residue in the peptides containing transmembrane helices 2 and/or 3 of the mAChR was alkylated. The linkage was cleaved by 1 M hydroxylamine, showing that /sup 3/H-PrBCM was attached to an acidic residue, whose properties strongly suggested it to be embedded in a hydrophobic intramembrane region of the mAChR. Examination of the cloned sequence of the mAChR reveals several candidate residues, the most likely of which is homologous to an aspartic acid residue thought to protonate the retinal Schiff's base in the congeneric protein rhodopsin.

  9. Muscarinic signaling influences the patterning and phenotype of cholinergic amacrine cells in the developing chick retina

    PubMed Central

    Stanke, Jennifer J; Lehman, Bret; Fischer, Andy J

    2008-01-01

    Background Many studies in the vertebrate retina have characterized the differentiation of amacrine cells as a homogenous class of neurons, but little is known about the genes and factors that regulate the development of distinct types of amacrine cells. Accordingly, the purpose of this study was to characterize the development of the cholinergic amacrine cells and identify factors that influence their development. Cholinergic amacrine cells in the embryonic chick retina were identified by using antibodies to choline acetyltransferase (ChAT). Results We found that as ChAT-immunoreactive cells differentiate they expressed the homeodomain transcription factors Pax6 and Islet1, and the cell-cycle inhibitor p27kip1. As differentiation proceeds, type-II cholinergic cells, displaced to the ganglion cell layer, transiently expressed high levels of cellular retinoic acid binding protein (CRABP) and neurofilament, while type-I cells in the inner nuclear layer did not. Although there is a 1:1 ratio of type-I to type-II cells in vivo, in dissociated cell cultures the type-I cells (ChAT-positive and CRABP-negative) out-numbered the type-II cells (ChAT and CRABP-positive cells) by 2:1. The relative abundance of type-I to type-II cells was not influenced by Sonic Hedgehog (Shh), but was affected by compounds that act at muscarinic acetylcholine receptors. In addition, the abundance and mosaic patterning of type-II cholinergic amacrine cells is disrupted by interfering with muscarinic signaling. Conclusion We conclude that: (1) during development type-I and type-II cholinergic amacrine cells are not homotypic, (2) the phenotypic differences between these subtypes of cells is controlled by the local microenvironment, and (3) appropriate levels of muscarinic signaling between the cholinergic amacrine cells are required for proper mosaic patterning. PMID:18254959

  10. M1 muscarinic receptors increase calcium current and phosphoinositide turnover in guinea-pig ventricular cardiocytes.

    PubMed Central

    Gallo, M P; Alloatti, G; Eva, C; Oberto, A; Levi, R C

    1993-01-01

    1. Physiological and molecular evidence for the presence and functional role of M1 muscarinic cholinergic receptors (mAChRs) in adult guinea-pig ventricular cells is presented. 2. Whole-cell clamp measurements of the L-type calcium current (ICa) in isolated myocytes were performed. Caesium was used to suppress potassium currents. ICa was increased by the muscarinic agonist carbachol in cells pretreated with pertussis toxin which blocked the M2 mAChR-triggered cascade of intracellular signalling, while it was not changed in untreated cells. 3. If the M2-mediated regulation of ICa was blocked by directly saturating the cell with cyclic adenosine monophosphate (cAMP) through the patch pipette, application of carbachol induced a further small increase of the current above the level reached after cAMP perfusion. This increase was more pronounced in cells pretreated with pertussis toxin. 4. The carbachol-induced increase of ICa was blocked by the selective M1 mAChR antagonist pirenzepine. 5. The application of high concentrations of carbachol increased the accumulation of [3H]inositol monophosphate up to 240% above control levels. This increase was reduced by application of pirenzepine. 6. The expression of M1 receptor mRNA in ventricular cardiocytes was shown by reverse transcriptase-polymerase chain reaction. 7. These results suggest that M1 mAChR regulation of ICa can be a component of the paradoxical positive inotropism induced by high concentrations of muscarinic agonists. Images Fig. 11 PMID:8120813

  11. P2×7 purinergic signaling in dilated cardiomyopathy induced by auto-immunity against muscarinic M2 receptors: autoantibody levels, heart functionality and cytokine expression

    PubMed Central

    Martinez, Camila Guerra; Zamith-Miranda, Daniel; da Silva, Marcia Gracindo; Ribeiro, Karla Consort; Brandão, Izaíra Trincani; Silva, Celio Lopes; Diaz, Bruno Lourenço; Bellio, Maria; Persechini, Pedro Muanis; Kurtenbach, Eleonora

    2015-01-01

    Autoantibodies against the M2 receptors (M2AChR) have been associated with Dilated Cardiomyopathy (DCM). In the heart, P2×7 receptors influence electrical conduction, coronary circulation and response to ischemia. They can also trigger pro-inflammatory responses and the development of neurological, cardiac and renal disorders. Here, P2×7−/− mice displayed an increased heart rate and ST segment depression, but similar exercise performance when compared to wild type (WT) animals. After immunization with plasmid containing M2AChR cDNA sequence, WT mice produced anti-M2AChR antibodies, while P2×7−/− mice showed an attenuated production. Despite this, WT and P2×7−/− showed left ventricle cavity enlargement and decreased exercise tolerance. Transfer of serum from M2AChR WT immunized mice to näive recipients led to an alteration in heart shape. P2×7−/− mice displayed a significant increase in the frequency of spleen regulatory T cells population, which is mainly composed by the FoxP3+CD25− subset. M2AChR WT immunized mice showed an increase in IL-1β, IFNγ and IL-17 levels in the heart, while P2×7−/− group produced lower amounts of IL-1β and IL-17 and higher amounts of IFNγ. These results pointed to previously unnoticed roles of P2×7 in cardiovascular and immune systems, and underscored the participation of IL-17 and IFNγ in the progress of autoimmune DCM. PMID:26592184

  12. Muscarinic receptors modulate dendrodendritic inhibitory synapses to sculpt glomerular output.

    PubMed

    Liu, Shaolin; Shao, Zuoyi; Puche, Adam; Wachowiak, Matt; Rothermel, Markus; Shipley, Michael T

    2015-04-08

    Cholinergic [acetylcholine (ACh)] axons from the basal forebrain innervate olfactory bulb glomeruli, the initial site of synaptic integration in the olfactory system. Both nicotinic acetylcholine receptors (nAChRs) and muscarinic acetylcholine receptors (mAChRs) are expressed in glomeruli. The activation of nAChRs directly excites both mitral/tufted cells (MTCs) and external tufted cells (ETCs), the two major excitatory neurons that transmit glomerular output. The functional roles of mAChRs in glomerular circuits are unknown. We show that the restricted glomerular application of ACh causes rapid, brief nAChR-mediated excitation of both MTCs and ETCs in the mouse olfactory bulb. This excitation is followed by mAChR-mediated inhibition, which is blocked by GABAA receptor antagonists, indicating the engagement of periglomerular cells (PGCs) and/or short axon cells (SACs), the two major glomerular inhibitory neurons. Indeed, selective activation of glomerular mAChRs, with ionotropic GluRs and nAChRs blocked, increased IPSCs in MTCs and ETCs, indicating that mAChRs recruit glomerular inhibitory circuits. Selective activation of glomerular mAChRs in the presence of tetrodotoxin increased IPSCs in all glomerular neurons, indicating action potential-independent enhancement of GABA release from PGC and/or SAC dendrodendritic synapses. mAChR-mediated enhancement of GABA release also presynaptically suppressed the first synapse of the olfactory system via GABAB receptors on sensory terminals. Together, these results indicate that cholinergic modulation of glomerular circuits is biphasic, involving an initial excitation of MTC/ETCs mediated by nAChRs followed by inhibition mediated directly by mAChRs on PGCs/SACs. This may phasically enhance the sensitivity of glomerular outputs to odorants, an action that is consistent with recent in vivo findings.

  13. Acetylcholine-induced neuronal differentiation: muscarinic receptor activation regulates EGR-1 and REST expression in neuroblastoma cells.

    PubMed

    Salani, Monica; Anelli, Tonino; Tocco, Gabriella Augusti; Lucarini, Elena; Mozzetta, Chiara; Poiana, Giancarlo; Tata, Ada Maria; Biagioni, Stefano

    2009-02-01

    Neurotransmitters are considered part of the signaling system active in nervous system development and we have previously reported that acetylcholine (ACh) is capable of enhancing neuronal differentiation in cultures of sensory neurons and N18TG2 neuroblastoma cells. To study the mechanism of ACh action, in this study, we demonstrate the ability of choline acetyltransferase-transfected N18TG2 clones (e.g. 2/4 clone) to release ACh. Analysis of muscarinic receptors showed the presence of M1-M4 subtypes and the activation of both IP(3) and cAMP signal transduction pathways. Muscarinic receptor activation increases early growth response factor-1 (EGR-1) levels and treatments with agonists, antagonists, and signal transduction enzyme inhibitors suggest a role for M3 subtype in EGR-1 induction. The role of EGR-1 in the enhancement of differentiation was investigated transfecting in N18TG2 cells a construct for EGR-1. EGR-1 clones show increased neurite extension and a decrease in Repressor Element-1 silencing transcription factor (REST) expression: both these features have also been observed for the 2/4 clone. Transfection of this latter with EGR zinc-finger domain, a dominant negative inhibitor of EGR-1 action, increases REST expression, and decreases fiber outgrowth. The data reported suggest that progression of the clone 2/4 in the developmental program is dependent on ACh release and the ensuing activation of muscarinic receptors, which in turn modulate the level of EGR-1 and REST transcription factors.

  14. Divergence of allosteric effects of rapacuronium on binding and function of muscarinic receptors

    PubMed Central

    2009-01-01

    Background Many neuromuscular blockers act as negative allosteric modulators of muscarinic acetylcholine receptors by decreasing affinity and potency of acetylcholine. The neuromuscular blocker rapacuronium has been shown to have facilitatory effects at muscarinic receptors leading to bronchospasm. We examined the influence of rapacuronium on acetylcholine (ACh) binding to and activation of individual subtypes of muscarinic receptors expressed in Chinese hamster ovary cells to determine its receptor selectivity. Results At equilibrium rapacuronium bound to all subtypes of muscarinic receptors with micromolar affinity (2.7-17 μM) and displayed negative cooperativity with both high- and low-affinity ACh binding states. Rapacuronium accelerated [3H]ACh association with and dissociation from odd-numbered receptor subtypes. With respect to [35S]GTPγS binding rapacuronium alone behaved as an inverse agonist at all subtypes. Rapacuronium concentration-dependently decreased the potency of ACh-induced [35S]GTPγS binding at M2 and M4 receptors. In contrast, 0.1 μM rapacuronium significantly increased ACh potency at M1, M3, and M5 receptors. Kinetic measurements at M3 receptors showed acceleration of the rate of ACh-induced [35S]GTPγS binding by rapacuronium. Conclusions Our data demonstrate a novel dichotomy in rapacuronium effects at odd-numbered muscarinic receptors. Rapacuronium accelerates the rate of ACh binding but decreases its affinity under equilibrium conditions. This results in potentiation of receptor activation at low concentrations of rapacuronium (1 μM) but not at high concentrations (10 μM). These observations highlight the relevance and necessity of performing physiological tests under non-equilibrium conditions in evaluating the functional effects of allosteric modulators at muscarinic receptors. They also provide molecular basis for potentiating M3 receptor-mediated bronchoconstriction. PMID:20038295

  15. Distinct roles of bulbar muscarinic and nicotinic receptors in olfactory discrimination learning.

    PubMed

    Devore, Sasha; de Almeida, Licurgo; Linster, Christiane

    2014-08-20

    The olfactory bulb (OB) and piriform cortex receive dense cholinergic projections from the basal forebrain. Cholinergic modulation within the piriform cortex has long been proposed to serve important functions in olfactory learning and memory. We here investigate how olfactory discrimination learning is regulated by cholinergic modulation of the OB inputs to the piriform cortex. We examined rats' performance on a two-alternative choice odor discrimination task following local, bilateral blockade of cholinergic nicotinic and/or muscarinic receptors in the OB. Results demonstrate that acquisition, but not recall, of novel discrimination problems is impaired following blockade of OB cholinergic receptors, although the relative contribution of muscarinic and nicotinic receptors depends on task difficulty. Blocking muscarinic receptors impairs learning for nearly all odor sets, whereas blocking nicotinic receptors only affects performance for perceptually similar odors. This pattern of behavioral effects is consistent with predictions from a model of cholinergic modulation in the OB and piriform cortex (de Almeida et al., 2013). Model simulations suggest that muscarinic and nicotinic receptors may serve complementary roles in regulating coherence and sparseness of the OB network output, which in turn differentially regulate the strength and overlap in cortical odor representations. Overall, our results suggest that muscarinic receptor blockade results in a bona fide learning impairment that may arise because cortical neurons are activated less often. Behavioral impairment following nicotinic receptor blockade may not be due to the inability of the cortex to learn, but rather arises because the cortex is unable to resolve highly overlapping input patterns.

  16. Stimulation of acid secretion and phosphoinositol production by rat parietal cell muscarinic M sub 2 receptors

    SciTech Connect

    Pfeiffer, A.; Rochlitz, H.; Herz, A.; Paumgartner, G. )

    1988-04-01

    The muscarinic receptor system involved in hydrogen production by enriched rat gastric parietal cells was investigated. Muscarinic receptor density determined by (N-methyl-{sup 3}H)scopolamine binding was 8,100/cell. The receptor appeared to be of the M{sub 2} muscarinic receptor subtype, since it had a low affinity (K{sub d} 189 nM) for the M{sub 1} receptor antagonist pirenzepine compared with atropine. Receptor activation by carbachol rapidly augmented levels of polyphosphoinositides, indicating an activation of phospholipase C. The dose-response relations for the increase in inositol phosphates closely paralleled the binding of carbachol to muscarinic receptors. The inositol phosphate response was antagonized by pirenzepine with a K{sub i} of 177 nM. the stimulation of inositol phosphate levels by carbachol correlated well with the stimulation of ({sup 14}C)aminopyrine uptake, determine as an index of acid secretion. The muscarinic agonists oxotremorine, pilocarpine, and bethanechol elicited partial increases in inositol phosphates at maximal drug concentrations, and these partial increases correlated with their ability to stimulate ({sup 14}C)aminopyrine uptake. These data indicate that inositolpolyphosphates may be a second messenger of M{sub 2} receptors stimulating acid secretion.

  17. Identification of M(1) muscarinic receptor subtype in rat stomach using a tissue segment binding method, and the effects of immobilization stress on the muscarinic receptors.

    PubMed

    Anisuzzaman, Abu Syed Md; Morishima, Shigeru; Suzuki, Fumiko; Tanaka, Takashi; Muramatsu, Ikunobu

    2008-12-03

    Distinct muscarinic acetylcholine receptor subtypes widely distribute in stomach tissues and are involved in many physiological functions. Although mRNA of M(1) subtype was found in gastric mucosa, the M(1) subtype has not been detected by conventional membrane binding assays. In the present study, muscarinic receptor subtypes in the rat stomach were reevaluated by using the tissue segment binding technique recently developed to recognize the inherent/native profiles of receptors without receptor environment perturbation. [(3)H]-N-methylscopolamine (NMS) bound to muscarinic receptors in the intact segments of rat gastric mucosa and muscle layers. The muscarinic receptors in the mucosal segments were composed of M(1), M(2) and M(3) subtypes, among which the M(1) subtype selectively showed high affinity for pirenzepine. However, in the membrane preparations, binding sites with high affinity for pirenzepine could not be detected. In the muscle layer, M(2) and M(3) subtypes, but not M(1), were identified in tissue segment and conventional membrane binding assays. Western blotting analysis recognized the M(1) subtype in the membrane preparations of mucosal but not muscle layers. Chronic immobilization stress increased the M(3) subtype in mucosal and muscle layers and decreased the M(2) subtype in the muscle layer, whereas M(1) and M(2) subtypes in mucosal layer did not change after the stress. The current study shows that M(1) subtype occurs as a pirenzepine-high affinity entity in intact segments of rat gastric mucosa, but that it loses the affinity for pirenzepine upon homogenization. Careful identification of native in vivo muscarinic receptors may further elucidate their functions in stomach.

  18. Autoradiographic visualization of muscarinic receptor subtypes in human and guinea pig lung

    SciTech Connect

    Mak, J.C.; Barnes, P.J. )

    1990-06-01

    Muscarinic receptor subtypes have been localized in human and guinea pig lung sections by an autoradiographic technique, using (3H)(-)quinuclidinyl benzilate (( 3H)QNB) and selective muscarinic antagonists. (3H)QNB was incubated with tissue sections for 90 min at 25 degrees C, and nonspecific binding was determined by incubating adjacent serial sections in the presence of 1 microM atropine. Binding to lung sections had the characterization expected for muscarinic receptors. Autoradiography revealed that muscarinic receptors were widely distributed in human lung, with dense labeling over submucosal glands and airway ganglia, and moderate labeling over nerves in intrapulmonary bronchi and of airway smooth muscle of large and small airways. In addition, alveolar walls were uniformly labeled. In guinea pig lung, labeling of airway smooth muscle was similar, but in contrast to human airways, epithelium was labeled but alveolar walls were not. The muscarinic receptors of human airway smooth muscle from large to small airways were entirely of the M3-subtype, whereas in guinea pig airway smooth muscle, the majority were the M3-subtype with a very small population of the M2-subtype present. In human bronchial submucosal glands, M1- and M3-subtypes appeared to coexist in the proportions of 36 and 64%, respectively. In human alveolar walls the muscarinic receptors were entirely of the M1-subtype, which is absent from the guinea pig lung. No M2-receptors were demonstrated in human lung. The localization of M1-receptors was confirmed by direct labeling with (3H)pirenzepine. With the exception of the alveolar walls in human lung, the localization of muscarinic receptor subtypes on structures in the lung is consistent with known functional studies.

  19. High-affinity binding of (/sup 3/H)acetylcholine to muscarinic cholinergic receptors

    SciTech Connect

    Kellar, K.J.; Martino, A.M.; Hall, D.P. Jr.; Schwartz, R.D.; Taylor, R.L.

    1985-06-01

    High-affinity binding of (/sup 3/H)acetylcholine to muscarinic cholinergic sites in rat CNS and peripheral tissues was measured in the presence of cytisin, which occupies nicotinic cholinergic receptors. The muscarinic sites were characterized with regard to binding kinetics, pharmacology, anatomical distribution, and regulation by guanyl nucleotides. These binding sites have characteristics of high-affinity muscarinic cholinergic receptors with a Kd of approximately 30 nM. Most of the muscarinic agonist and antagonist drugs tested have high affinity for the (/sup 3/H)acetylcholine binding site, but pirenzepine, an antagonist which is selective for M-1 receptors, has relatively low affinity. The ratio of high-affinity (/sup 3/H)acetylcholine binding sites to total muscarinic binding sites labeled by (/sup 3/H)quinuclidinyl benzilate varies from 9 to 90% in different tissues, with the highest ratios in the pons, medulla, and heart atrium. In the presence of guanyl nucleotides, (/sup 3/H) acetylcholine binding is decreased, but the extent of decrease varies from 40 to 90% in different tissues, with the largest decreases being found in the pons, medulla, cerebellum, and heart atrium. The results indicate that (/sup 3/H)acetylcholine binds to high-affinity M-1 and M-2 muscarinic receptors, and they suggest that most M-2 sites have high affinity for acetylcholine but that only a small fraction of M-1 sites have such high affinity.

  20. Muscarinic receptor M3 mediates human gallbladder contraction through voltage-gated Ca2+ channels and Rho kinase.

    PubMed

    Lee, Ming-Che; Yang, Ying-Chin; Chen, Yen-Cheng; Huang, Shih-Che

    2013-02-01

    Muscarinic receptors mediate contraction of the human gallbladder through unclear receptor subtypes. The aim of the present study was to characterize muscarinic acetylcholine receptors mediating contraction of the human gallbladder. Contraction of human gallbladder muscle strips caused by agonists carbachol and muscarine was measured and the inhibition of carbachol-induced contraction by muscarinic receptor antagonists was evaluated. Reverse transcription polymerase chain reaction was performed to determine the existence of muscarinic receptor subtypes. Carbachol and muscarine caused concentration-dependent contraction of gallbladder strips. Four receptor antagonists, including atropine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), methoctramine, and pirenzepine, inhibited the carbachol-induced contraction. The relative inhibitory potency of these receptor antagonists was atropine > 4-DAMP > methoctramine > pirenzepine. The antagonist affinity estimates (pA(2) values) correlated with the known affinities at M(3), M(4), and M(5) muscarinic receptors. In addition, the M(4)-selective antagonist muscarinic toxin 3 did not inhibit and the M(5)-selective positive allosteric modulator VU0238429 did not potentiate carbachol-induced gallbladder contraction. This suggests that M(3) muscarinic receptors mediate the muscarinic response predominantly. The contractile response of carbachol was attenuated by the voltage-gated Ca(2+) channel inhibitor nifedipine and Rho-kinase inhibitor H-1152, but not affected by protein kinase C inhibitor chelerythrine. This implies the involvement of voltage-gated Ca(2+) channel and Rho kinase but not protein kinase C. These results suggest a major role of M(3) muscarinic receptors mediating the human gallbladder contraction through voltage-gated Ca(2+) channels and Rho kinase. M(3)-selective muscarinic receptor antagonists could be of therapeutic importance in the treatment of biliary motility disorders.

  1. M1-muscarinic receptors promote fear memory consolidation via phospholipase C and the M-current.

    PubMed

    Young, Matthew B; Thomas, Steven A

    2014-01-29

    Neuromodulators released during and after a fearful experience promote the consolidation of long-term memory for that experience. Because overconsolidation may contribute to the recurrent and intrusive memories of post-traumatic stress disorder, neuromodulatory receptors provide a potential pharmacological target for prevention. Stimulation of muscarinic receptors promotes memory consolidation in several conditioning paradigms, an effect primarily associated with the M1 receptor (M1R). However, neither inhibiting nor genetically disrupting M1R impairs the consolidation of cued fear memory. Using the M1R agonist cevimeline and antagonist telenzepine, as well as M1R knock-out mice, we show here that M1R, along with β2-adrenergic (β2AR) and D5-dopaminergic (D5R) receptors, regulates the consolidation of cued fear memory by redundantly activating phospholipase C (PLC) in the basolateral amygdala (BLA). We also demonstrate that fear memory consolidation in the BLA is mediated in part by neuromodulatory inhibition of the M-current, which is conducted by KCNQ channels and is known to be inhibited by muscarinic receptors. Manipulating the M-current by administering the KCNQ channel blocker XE991 or the KCNQ channel opener retigabine reverses the effects on consolidation caused by manipulating β2AR, D5R, M1R, and PLC. Finally, we show that cAMP and protein kinase A (cAMP/PKA) signaling relevant to this stage of consolidation is upstream of these neuromodulators and PLC, suggesting an important presynaptic role for cAMP/PKA in consolidation. These results support the idea that neuromodulatory regulation of ion channel activity and neuronal excitability is a critical mechanism for promoting consolidation well after acquisition has occurred.

  2. Cardiac effects of muscarinic receptor antagonists used for voiding dysfunction

    PubMed Central

    Andersson, Karl-Erik; Campeau, Lysanne; Olshansky, Brian

    2011-01-01

    Antimuscarinic agents are the main drugs used to treat patients with the overactive bladder (OAB) syndrome, defined as urgency, with or without urgency incontinence, usually with increased daytime frequency and nocturia. Since the treatment is not curative and since OAB is a chronic disease, treatment may be life-long. Antimuscarinics are generally considered to be ‘safe’ drugs, but among the more serious concerns related to their use is the risk of cardiac adverse effects, particularly increases in heart rate (HR) and QT prolongation and induction of polymorphic ventricular tachycardia (torsade de pointes). An elevated resting HR has been linked to overall increased morbidity and mortality, particularly in patients with cardiovascular diseases. QT prolongation and its consequences are not related to blockade of muscarinic receptors, but rather linked to inhibition of the hERG potassium channel in the heart. However, experience with terodiline, an antimuscarinic drug causing torsade de pointes in patients, has placed the whole drug class under scrutiny. The potential of the different antimuscarinic agents to increase HR and/or prolong the QT time has not been extensively explored for all agents in clinical use. Differences between drugs cannot be excluded, but risk assessments based on available evidence are not possible. PMID:21595741

  3. Muscarinic receptors in amygdala control trace fear conditioning.

    PubMed

    Baysinger, Amber N; Kent, Brianne A; Brown, Thomas H

    2012-01-01

    Intelligent behavior requires transient memory, which entails the ability to retain information over short time periods. A newly-emerging hypothesis posits that endogenous persistent firing (EPF) is the neurophysiological foundation for aspects or types of transient memory. EPF is enabled by the activation of muscarinic acetylcholine receptors (mAChRs) and is triggered by suprathreshold stimulation. EPF occurs in several brain regions, including the lateral amygdala (LA). The present study examined the role of amygdalar mAChRs in trace fear conditioning, a paradigm that requires transient memory. If mAChR-dependent EPF selectively supports transient memory, then blocking amygdalar mAChRs should impair trace conditioning, while sparing delay and context conditioning, which presumably do not rely upon transient memory. To test the EPF hypothesis, LA was bilaterally infused, prior to trace or delay conditioning, with either a mAChR antagonist (scopolamine) or saline. Computerized video analysis quantified the amount of freezing elicited by the cue and by the training context. Scopolamine infusion profoundly reduced freezing in the trace conditioning group but had no significant effect on delay or context conditioning. This pattern of results was uniquely anticipated by the EPF hypothesis. The present findings are discussed in terms of a systems-level theory of how EPF in LA and several other brain regions might help support trace fear conditioning.

  4. Muscarinic Receptors in Amygdala Control Trace Fear Conditioning

    PubMed Central

    Baysinger, Amber N.; Kent, Brianne A.; Brown, Thomas H.

    2012-01-01

    Intelligent behavior requires transient memory, which entails the ability to retain information over short time periods. A newly-emerging hypothesis posits that endogenous persistent firing (EPF) is the neurophysiological foundation for aspects or types of transient memory. EPF is enabled by the activation of muscarinic acetylcholine receptors (mAChRs) and is triggered by suprathreshold stimulation. EPF occurs in several brain regions, including the lateral amygdala (LA). The present study examined the role of amygdalar mAChRs in trace fear conditioning, a paradigm that requires transient memory. If mAChR-dependent EPF selectively supports transient memory, then blocking amygdalar mAChRs should impair trace conditioning, while sparing delay and context conditioning, which presumably do not rely upon transient memory. To test the EPF hypothesis, LA was bilaterally infused, prior to trace or delay conditioning, with either a mAChR antagonist (scopolamine) or saline. Computerized video analysis quantified the amount of freezing elicited by the cue and by the training context. Scopolamine infusion profoundly reduced freezing in the trace conditioning group but had no significant effect on delay or context conditioning. This pattern of results was uniquely anticipated by the EPF hypothesis. The present findings are discussed in terms of a systems-level theory of how EPF in LA and several other brain regions might help support trace fear conditioning. PMID:23029199

  5. Effect of pirenzepine and gallamine on cardiac and pulmonary muscarinic receptors in the rabbit.

    PubMed Central

    Maclagan, J.; Faulkner, D.

    1989-01-01

    1. The effect of muscarinic antagonists considered to be selective for M1 receptors (pirenzepine) and for M2 receptors (gallamine) were studied on bronchoconstriction and bradycardia elicited by stimulation of the vagal nerves and by i.v. acetylcholine (ACh) in anaesthetized rabbits. 2. Pirenzepine was equipotent as an antagonist of ACh-induced responses at postjunctional muscarinic receptors in the heart, lung and blood vessels, whereas gallamine was at least ten times less potent at pulmonary and vascular muscarinic receptors. Thus, gallamine never caused complete inhibition of bronchoconstrictor or hypotensive responses to i.v. ACh, whereas doses of pirenzepine in excess of 1 mumol kg-1 abolished all muscarinic responses. 3. In the lung, both antagonists inhibited bronchoconstriction caused by vagal stimulation and ACh-induced bronchoconstriction to the same extent (pirenzepine, mean ED50 65 +/- 22 and, 130 +/- 28 nmol kg-1 respectively; gallamine, ED50 greater than 10,000 nmol kg-1 for both responses). Enhancement of vagally-induced bronchoconstriction was never observed. 4. In the heart, however, both pirenzepine and gallamine were ten times less potent as antagonists of vagally-induced bradycardia than of ACh-induced bradycardia. This differential blockade was unaltered by propranolol (1 mg kg-1) pretreatment. 5. It is concluded that there is no evidence for M1 or M2 muscarinic receptors in the pulmonary innervation of the rabbit and the potency of the antagonists in abolishing in abolishing vagally-induced bronchoconstriction was consistent with blockade of M3 muscarinic receptors on airway smooth muscle. 6. The results suggest that M2 muscarinic receptors may exert an inhibitory effect on transmission in the parasympathetic nerves innervating the heart in the rabbit.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2758229

  6. Role of the M3 Muscarinic Acetylcholine Receptor Subtype in Murine Ophthalmic Arteries After Endothelial Removal

    PubMed Central

    Gericke, Adrian; Steege, Andreas; Manicam, Caroline; Böhmer, Tobias; Wess, Jürgen; Pfeiffer, Norbert

    2014-01-01

    Purpose. We tested the hypothesis that the M3 muscarinic acetylcholine receptor subtype mediates cholinergic responses in murine ophthalmic arteries after endothelial removal. Methods. Muscarinic receptor gene expression was determined in ophthalmic arteries with intact and with removed endothelium using real-time PCR. To examine the role of the M3 receptor in mediating vascular responses, ophthalmic arteries from M3 receptor-deficient mice (M3R−/−) and respective wild-type controls were studied in vitro. Functional studies were performed in nonpreconstricted arteries with either intact or removed endothelium using video microscopy. Results. In endothelium-intact ophthalmic arteries, mRNA for all five muscarinic receptor subtypes was detected, but M3 receptor mRNA was most abundant. In endothelium-removed ophthalmic arteries, M1, M2, and M3 receptors displayed similar mRNA expression levels, which were higher than those for M4 and M5 receptors. In functional studies, acetylcholine evoked vasoconstriction in endothelium-removed arteries from wild-type mice that was virtually abolished after incubation with the muscarinic receptor blocker atropine, indicative of the involvement of muscarinic receptors. In concentration-response experiments, acetylcholine and carbachol concentration-dependently constricted endothelium-removed ophthalmic arteries from wild-type mice, but produced only negligible responses in arteries from M3R−/− mice. In contrast, acetylcholine concentration-dependently dilated ophthalmic arteries with intact endothelium from wild-type mice, but not from M3R−/− mice. Responses to the nitric oxide donor nitroprusside and to KCl did not differ between ophthalmic arteries from wild-type and M3R−/− mice, neither in endothelium-intact nor in endothelium-removed arteries. Conclusions. These findings provide evidence that in murine ophthalmic arteries the muscarinic M3 receptor subtype mediates cholinergic endothelium-dependent vasodilation

  7. Muscarinic receptors: their distribution and function in body systems, and the implications for treating overactive bladder

    PubMed Central

    Abrams, Paul; Andersson, Karl-Erik; Buccafusco, Jerry J; Chapple, Christopher; de Groat, William Chet; Fryer, Alison D; Kay, Gary; Laties, Alan; Nathanson, Neil M; Pasricha, Pankaj Jay; Wein, Alan J

    2006-01-01

    The effectiveness of antimuscarinic agents in the treatment of the overactive bladder (OAB) syndrome is thought to arise through blockade of bladder muscarinic receptors located on detrusor smooth muscle cells, as well as on nondetrusor structures. Muscarinic M3 receptors are primarily responsible for detrusor contraction. Limited evidence exists to suggest that M2 receptors may have a role in mediating indirect contractions and/or inhibition of detrusor relaxation. In addition, there is evidence that muscarinic receptors located in the urothelium/suburothelium and on afferent nerves may contribute to the pathophysiology of OAB. Blockade of these receptors may also contribute to the clinical efficacy of antimuscarinic agents. Although the role of muscarinic receptors in the bladder, other than M3 receptors, remains unclear, their role in other body systems is becoming increasingly well established, with emerging evidence supporting a wide range of diverse functions. Blockade of these functions by muscarinic receptor antagonists can lead to similarly diverse adverse effects associated with antimuscarinic treatment, with the range of effects observed varying according to the different receptor subtypes affected. This review explores the evolving understanding of muscarinic receptor functions throughout the body, with particular focus on the bladder, gastrointestinal tract, eye, heart, brain and salivary glands, and the implications for drugs used to treat OAB. The key factors that might determine the ideal antimuscarinic drug for treatment of OAB are also discussed. Further research is needed to show whether the M3 selective receptor antagonists have any advantage over less selective drugs, in leading to fewer adverse events. PMID:16751797

  8. Use of intact rat brain cells as a model to study regulation of muscarinic acetylcholine receptors

    SciTech Connect

    Lee, J.H.; El-Fakahany, E.E.

    1985-08-12

    Intact rat brain cells were dissociated and used to study the regulation of muscarinic acetylcholine receptors upon exposure to muscarinic receptor agonists. Incubation of cells with carbamylcholine resulted in a time-dependent decrease in subsequent (/sup 3/H)N-methylscopolamine specific binding, an effect which reached a steady state after 3 hr at 37/sup 0/C. This effect of carbamylcholine was dependent on the concentration of the agonist in the incubation medium and was due to a reduction in the maximal binding capacity of the receptor with no decrease in the affinity of the remaining receptors. This preparation might be useful in future studies to elucidate the mechanisms underlying the regulation of muscarinic acetylcholine receptors in the central nervous system. 20 references, 3 tables.

  9. Muscarinic inhibition of hippocampal and striatal adenylyl cyclase is mainly due to the M(4) receptor.

    PubMed

    Sánchez, Gonzalo; Colettis, Natalia; Vázquez, Pablo; Cerveñansky, Carlos; Aguirre, Alejandra; Quillfeldt, Jorge A; Jerusalinsky, Diana; Kornisiuk, Edgar

    2009-08-01

    The five muscarinic acetylcholine receptors (M(1)-M(5)) are differentially expressed in the brain. M(2) and M(4) are coupled to inhibition of stimulated adenylyl cyclase, while M(1), M(3) and M(5) are mainly coupled to the phosphoinositide pathway. We studied the muscarinic receptor regulation of adenylyl cyclase activity in the rat hippocampus, compared to the striatum and amygdala. Basal and forskolin-stimulated adenylyl cyclase activity was higher in the striatum but the muscarinic inhibition was much lower. Highly selective muscarinic toxins MT1 and MT2-affinity order M(1) > or = M(4) > others-and MT3-highly selective M(4) antagonist-did not show significant effects on basal or forskolin-stimulated cyclic AMP production but, like scopolamine, counteracted oxotremorine inhibition. Since MTs have negligible affinity for M(2), M(4) would be the main subtype responsible for muscarinic inhibition of forskolin-stimulated enzyme. Dopamine stimulated a small fraction of the enzyme (3.1% in striatum, 1.3% in the hippocampus). Since MT3 fully blocked muscarinic inhibition of dopamine-stimulated enzyme, M(4) receptor would be responsible for this regulation.

  10. Enhanced sensitivity of muscarinic cholinergic receptor associated with dopaminergic receptor subsensitivity after chronic antidepressant treatment

    SciTech Connect

    Koide, T.; Matsushita, H.

    1981-03-09

    The chronic effects of antidepressant treatment on striatal dopaminergic (DA) and muscarinic cholinergic (mACh) receptors of the rat brain have been examined comparatively in this study using /sup 3/H-spiroperidol (/sup 3/H-SPD) and /sup 3/H-quinuclidinyl benzilate (/sup 3/H-QNB) as the respective radioactive ligands. Imipramine and desipramine were used as prototype antidepressants. Although a single administration of imipramine or desipramine did not affect each receptor sensitivity, chronic treatment with each drug caused a supersensitivity of mACh receptor subsequent to DA receptor subsensitivity. Furthermore, it has been suggested that anti-mACh properties of imipramine or desipramine may not necessarily be related to the manifestation of mACh receptor supersensitivity and that sustained DA receptor subsensitivity may play some role in the alterations of mACh receptor sensitivity.

  11. Darifenacin: a novel M3 muscarinic selective receptor antagonist for the treatment of overactive bladder.

    PubMed

    Chapple, Christopher R

    2004-11-01

    Darifenacin is a novel M3 muscarinic selective receptor antagonist for once-daily treatment of overactive bladder (OAB), a highly prevalent, chronic and debilitating disease defined by urinary urgency with or without urge incontinence, usually with increased frequency of micturition and nocturia. In vitro, darifenacin is a potent and specific muscarinic receptor antagonist with muscarinic M3 receptors relative to other muscarinic receptor subtypes. This profile may, therefore, confer clinical efficacy in the treatment of OAB, with a lower propensity for adverse effects and safety issues related to blockade of other muscarinic receptor subtypes. Indeed, consistent with its low relative affinity for M1 and M2 receptors, no effects on cognitive function and heart-rate variability, respectively, have been observed with darifenacin. Subsequent large-scale clinical trials have confirmed that darifenacin (at doses of 7.5 and 15 mg/day) results in central nervous system and cardiac adverse events comparable to placebo, and provides early and meaningful improvement across a range of OAB symptoms including incontinence episodes, urgency and urinary frequency. On the basis of such findings, darifenacin would appear to meet the current need for an effective OAB pharmacotherapy that is efficacious, well-tolerated and, more importantly, minimises the risk of safety-related adverse effects.

  12. Castration decreases amylase release associated with muscarinic acetylcholine receptor downregulation in rat parotid gland.

    PubMed

    Busch, Lucila; Borda, Enri

    2003-05-01

    1 The mechanism and receptor subtypes involved in carbachol-stimulated amylase release and its changes after castration were studied in parotid slices from male rats. 2 Carbachol induced both amylase release and inositol phosphate (IP) accumulation in parotid slices from control and castrated rats, but castration induced a decrease of carbachol maximal effect. The effect of castration was reverted by testosterone replacement. 3 The selective M(1) and M(3) muscarinic receptor antagonists, pirenzepine and 4-diphenylacetoxy-N-methylpiperidine methiodide, respectively, inhibited carbachol-stimulated amylase release and IP accumulation in a dose-dependent manner in parotid slices from control and castrated rats. 4 A diminution of binding sites of muscarinic receptor in parotid membrane from castrated rats was observed. Competition binding assays showed that both, M(1) and M(3) muscarinic receptor subtypes are expressed in membranes of parotid glands from control and castrated rats, M(3) being the greater population. 5 These results suggest that amylase release induced by carbachol in parotid slices is mediated by phosphoinositide accumulation. This mechanism appears to be triggered by the activation of M(1) and M(3) muscarinic receptor subtypes. Castration induced a decrease of the maximal effect of carbachol evoked amylase release and IP accumulation followed by a diminution in the number of parotid gland muscarinic acetylcholine receptors.

  13. Molecular Probes for Muscarinic Receptors: Derivatives of the M1-Antagonist Telenzepine

    PubMed Central

    Karton, Yishai; Baumgold, Jesse; Handen, Jeffrey S.; Jacobson, Kenneth A.

    2012-01-01

    Functionalized congeners of the M1-selective muscarinic antagonist telenzepine (4,9-dihydro-3-methyl-4-[(4-methyl-1-piperazinyl)acetyl]-10H-thieno[3,4–b][1,5]benzodiazepin-10-one) were developed and found to bind to the receptor with affinities (Ki values) in approximately the nanomolar range. The derivatives contain a 10-aminodecyl group, which provides a nucleophilic functionality for further derivatization. The attachment of a spacer chain to the distal piperazinyl nitrogen was based on previous findings of enhanced affinity at muscarinic receptors in an analogous series of alkylamino derivatives of pirenzepine [J. Med. Chem. (1991) 34, 2133–2145]. The telenzepine derivatives contain prosthetic groups for radioiodination, protein cross-linking, photoaffinity labeling, and fluorescent labeling and biotin for avidin complexation. The affinity for muscarinic receptors in rat forebrain (mainly m1 subtype) was determined in competitive binding assays vs [3H]-N-methylscopolamine. A (p-aminophenyl)-acetyl derivative for photoaffinity labeling had a Ki value of 0.29 nM at forebrain muscarinic receptors (16-fold higher affinity than telenzepine). A biotin conjugate displayed a Ki value of 0.60 nM at m2-receptors and a 5-fold selectivity versus forebrain. The high affinity of these derivatives makes them suitable for the characterization of muscarinic receptors in pharmacological and spectroscopic studies, for peptide mapping, and for histochemical studies. PMID:1520727

  14. New pharmacological approaches to the cholinergic system: an overview on muscarinic receptor ligands and cholinesterase inhibitors.

    PubMed

    Greig, Nigel H; Reale, Marcella; Tata, Ada M

    2013-08-01

    The cholinergic system is expressed in neuronal and in non-neuronal tissues. Acetylcholine (ACh), synthesized in and out of the nervous system can locally contribute to modulation of various cell functions (e.g. survival, proliferation). Considering that the cholinergic system and its functions are impaired in a number of disorders, the identification of new pharmacological approaches to regulate cholinergic system components appears of great relevance. The present review focuses on recent pharmacological drugs able to modulate the activity of cholinergic receptors and thereby, cholinergic function, with an emphasis on the muscarinic receptor subtype, and additionally covers the cholinesterases, the main enzymes involved in ACh hydrolysis. The presence and function of muscarinic receptor subtypes both in neuronal and non-neuronal cells has been demonstrated using extensive pharmacological data emerging from studies on transgenic mice. The possible involvement of ACh in different pathologies has been proposed in recent years and is becoming an important area of study. Although the lack of selective muscarinic receptor ligands has for a long time limited the definition of therapeutic treatment based on muscarinic receptors as targets, some muscarinic ligands such as cevimeline (patents US4855290; US5571918) or xanomeline (patent, US5980933) have been developed and used in pre-clinical or in clinical studies for the treatment of nervous system diseases (Alzheimer' and Sjogren's diseases). The present review focuses on the potential implications of muscarinic receptors in different pathologies, including tumors. Moreover, the future use of muscarinic ligands in therapeutic protocols in cancer therapy will be discussed, considering that some muscarinic antagonists currently used in the treatment of genitourinary disease (e.g. darifenacin, patent, US5096890; US6106864) have also been demonstrated to arrest tumor progression in nude mice. The involvement of muscarinic

  15. Grid cell spatial tuning reduced following systemic muscarinic receptor blockade

    PubMed Central

    Newman, Ehren L.; Climer, Jason R.; Hasselmo, Michael E.

    2014-01-01

    Grid cells of the medial entorhinal cortex exhibit a periodic and stable pattern of spatial tuning that may reflect the output of a path integration system. This grid pattern has been hypothesized to serve as a spatial coordinate system for navigation and memory function. The mechanisms underlying the generation of this characteristic tuning pattern remain poorly understood. Systemic administration of the muscarinic antagonist scopolamine flattens the typically positive correlation between running speed and entorhinal theta frequency in rats. The loss of this neural correlate of velocity, an important signal for the calculation of path integration, raises the question of what influence scopolamine has on the grid cell tuning as a read out of the path integration system. To test this, the spatial tuning properties of grid cells were compared before and after systemic administration of scopolamine as rats completed laps on a circle track for food rewards. The results show that the spatial tuning of the grid cells was reduced following scopolamine administration. The tuning of head direction cells, in contrast, was not reduced by scopolamine. This is the first report to demonstrate a link between cholinergic function and grid cell tuning. This work suggests that the loss of tuning in the grid cell network may underlie the navigational disorientation observed in Alzheimer's patients and elderly individuals with reduced cholinergic tone. PMID:24493379

  16. Selective activation of the M1 muscarinic acetylcholine receptor achieved by allosteric potentiation

    PubMed Central

    Ma, Lei; Seager, Matthew A.; Wittmann, Marion; Jacobson, Marlene; Bickel, Denise; Burno, Maryann; Jones, Keith; Graufelds, Valerie Kuzmick; Xu, Guangping; Pearson, Michelle; McCampbell, Alexander; Gaspar, Renee; Shughrue, Paul; Danziger, Andrew; Regan, Christopher; Flick, Rose; Pascarella, Danette; Garson, Susan; Doran, Scott; Kreatsoulas, Constantine; Veng, Lone; Lindsley, Craig W.; Shipe, William; Kuduk, Scott; Sur, Cyrille; Kinney, Gene; Seabrook, Guy R.; Ray, William J.

    2009-01-01

    The forebrain cholinergic system promotes higher brain function in part by signaling through the M1 muscarinic acetylcholine receptor (mAChR). During Alzheimer's disease (AD), these cholinergic neurons degenerate, therefore selectively activating M1 receptors could improve cognitive function in these patients while avoiding unwanted peripheral responses associated with non-selective muscarinic agonists. We describe here benzyl quinolone carboxylic acid (BQCA), a highly selective allosteric potentiator of the M1 mAChR. BQCA reduces the concentration of ACh required to activate M1 up to 129-fold with an inflection point value of 845 nM. No potentiation, agonism, or antagonism activity on other mAChRs is observed up to 100 μM. Furthermore studies in M1−/− mice demonstrates that BQCA requires M1 to promote inositol phosphate turnover in primary neurons and to increase c-fos and arc RNA expression and ERK phosphorylation in the brain. Radioligand-binding assays, molecular modeling, and site-directed mutagenesis experiments indicate that BQCA acts at an allosteric site involving residues Y179 and W400. BQCA reverses scopolamine-induced memory deficits in contextual fear conditioning, increases blood flow to the cerebral cortex, and increases wakefulness while reducing delta sleep. In contrast to M1 allosteric agonists, which do not improve memory in scopolamine-challenged mice in contextual fear conditioning, BQCA induces β-arrestin recruitment to M1, suggesting a role for this signal transduction mechanism in the cholinergic modulation of memory. In summary, BQCA exploits an allosteric potentiation mechanism to provide selectivity for the M1 receptor and represents a promising therapeutic strategy for cognitive disorders. PMID:19717450

  17. Quantitative in vivo receptor binding. III. Tracer kinetic modeling of muscarinic cholinergic receptor binding

    SciTech Connect

    Frey, K.A.; Hichwa, R.D.; Ehrenkaufer, R.L.; Agranoff, B.W.

    1985-10-01

    A tracer kinetic method is developed for the in vivo estimation of high-affinity radioligand binding to central nervous system receptors. Ligand is considered to exist in three brain pools corresponding to free, nonspecifically bound, and specifically bound tracer. These environments, in addition to that of intravascular tracer, are interrelated by a compartmental model of in vivo ligand distribution. A mathematical description of the model is derived, which allows determination of regional blood-brain barrier permeability, nonspecific binding, the rate of receptor-ligand association, and the rate of dissociation of bound ligand, from the time courses of arterial blood and tissue tracer concentrations. The term ''free receptor density'' is introduced to describe the receptor population measured by this method. The technique is applied to the in vivo determination of regional muscarinic acetylcholine receptors in the rat, with the use of (TH)scopolamine. Kinetic estimates of free muscarinic receptor density are in general agreement with binding capacities obtained from previous in vivo and in vitro equilibrium binding studies. In the striatum, however, kinetic estimates of free receptor density are less than those in the neocortex--a reversal of the rank ordering of these regions derived from equilibrium determinations. A simplified model is presented that is applicable to tracers that do not readily dissociate from specific binding sites during the experimental period.

  18. Batrachotoxin Changes the Properties of the Muscarinic Receptor in Rat Brain and Heart: Possible Interaction(s) between Muscarinic Receptors and Sodium Channels

    NASA Astrophysics Data System (ADS)

    Cohen-Armon, Malca; Kloog, Yoel; Henis, Yoav I.; Sokolovsky, Mordechai

    1985-05-01

    The effects of Na+-channel activator batrachotoxin (BTX) on the binding properties of muscarinic receptors in homogenates of rat brain and heart were studied. BTX enhanced the affinity for the binding of the agonists carbamoylcholine and acetylcholine to the muscarinic receptors in brainstem and ventricle, but not in the cerebral cortex. Analysis of the data according to a two-site model for agonist binding indicated that the effect of BTX was to increase the affinity of the agonists to the high-affinity site. Guanyl nucleotides, known to induce interconversion of high-affinity agonist binding sites to the low-affinity state, canceled the effect of BTX on carbamoylcholine and acetylcholine binding. BTX had no effect on the binding of the agonist oxotremorine or on the binding of the antagonist [3H]-N-methyl-4-piperidyl benzilate. The local anesthetics dibucaine and tetracaine antagonized the effect of BTX on the binding of muscarinic agonists at concentrations known to inhibit the activation of Na+ channels by BTX. On the basis of these findings, we propose that in specific tissues the muscarinic receptors may interact with the BTX binding site (Na+ channels).

  19. Physiological and biochemical studies of newly synthesized muscarinic acetylcholine receptors in embryonic chicken heart

    SciTech Connect

    Hunter, D.D.

    1986-01-01

    Exposure of either chicken embryos in ovo or cultured embryonic chicken cardiac cells in vitro to the muscarinic agonist carbachol results in a 70-90% decrease in the number of muscarinic acetylcholine receptors (mAChR) expressed in cardiac cells. Block of agonist-receptor interactions in ovo with the antagonist atropine or removal of the agonist in vitro results in a gradual increase in mAChR number, reaching the control level in 14 hr. Measurements of physiological sensitivity of atria or cultured cells show that, even after the complete recovery of receptor number, the sensitivity to agonist is reduced. The sensitivity of the mAChR-mediated inhibition of adenylate cyclase is also decreased at this time. Newly synthesized mAChR which appear following affinity alkylation in cultured cells are also poorly coupled to the stimulation of /sup 86/Rb/sup +/ efflux, indicating that decreased physiological sensitivity is not due to an unknown effect of long-term agonist exposure on general cellular function, but rather reflects an intrinsic property of newly synthesized mAChR. This increase in sensitivity is also not blocked by cycloheximide. The increase in sensitivity of the mAChR-mediated responses is due neither to a lack of expression of newly synthesized mAChR on the surface nor to reduced agonist affinity of the mAChR. The diminished sensitivity and subsequent maturation observed in cells containing newly synthesized receptors is due either to a small change in mAChR, or to a change in an as-yet-undefined component of the mAChR transduction system; this alteration represents a novel locus for modulation of cholinergic signals in the heart.

  20. Different antagonist binding properties of rat pancreatic and cardiac muscarinic receptors

    SciTech Connect

    Waelbroeck, M.; Camus, J.; Winand, J.; Christophe, J.

    1987-11-09

    The antagonist binding properties of rat pancreatic and cardiac muscarinic receptors were compared. In both tissues pirenzepine (PZ) had a low affinity for muscarinic receptors labelled by (/sup 3/H)N-methylscopolamine ((/sup 3/)NMS) (K/sub D/ values of 140 and 280nM, respectively, in pancreatic and cardiac homogenates). The binding properties of pancreatic and cardiac receptors were, however, markedly different. This was indicated by different affinities for dicyclomine, (11-(/(2-((diethylamino)-methyl)-1-piperidinyl/acetyl)-5, 11-dihydro-6H-pyrido(2,3-b)(1,4) benzodiazepin-6-on)(AFDX-116), 4-diphenylacetoxy-N-methyl-piperidine methobromide (4-DAMP) and hexahydrosiladifenidol (HHSiD). Pancreatic and cardiac muscarinic receptros also showed different (/sup 3/H)NMS association and dissociation rates. These results support the concept of M2 receptor subtypes have different binding kinetic properties. 20 references, 3 figures, 1 table.

  1. Muscarinic acetylcholine receptor-mediated stimulation of retinal ganglion cell photoreceptors.

    PubMed

    Sodhi, Puneet; Hartwick, Andrew T E

    2016-09-01

    Melanopsin-dependent phototransduction in intrinsically photosensitive retinal ganglion cells (ipRGCs) involves a Gq-coupled phospholipase C (PLC) signaling cascade. Acetylcholine, released in the mammalian retina by starburst amacrine cells, can also activate Gq-PLC pathways through certain muscarinic acetylcholine receptors (mAChRs). Using multielectrode array recordings of rat retinas, we demonstrate that robust spiking responses can be evoked in neonatal and adult ipRGCs after bath application of the muscarinic agonist carbachol. The stimulatory action of carbachol on ipRGCs was a direct effect, as confirmed through calcium imaging experiments on isolated ipRGCs in purified cultures. Using flickering (6 Hz) yellow light stimuli at irradiances below the threshold for melanopsin activation, spiking responses could be elicited in ipRGCs that were suppressed by mAChR antagonism. Therefore, this work identified a novel melanopsin-independent pathway for stimulating sustained spiking in ganglion cell photoreceptors. This mAChR-mediated pathway could enhance ipRGC spiking responses in conditions known to evoke retinal acetylcholine release, such as those involving flickering or moving visual stimuli. Furthermore, this work identifies a pharmacological approach for light-independent ipRGC stimulation that could be targeted by mAChR agonists.

  2. Regulation of muscarinic acetylcholine receptors in the 1321N1 human astrocytoma cell line

    SciTech Connect

    Hoover, R.K.

    1989-01-01

    The binding of muscarinic agonists, partial agonists and antagonists to muscarinic receptors of 1321N1 human astrocytoma cells was studied. Binding was studied in both intact cells and cell lysates. Partial agonists and antagonists exhibited similar apparent affinities in intact cell competition binding assays with either the lipophilic radioligand ({sup 3}H)QNB or the hydrophilic radioligand ({sup 3}H)NMS. In contrast, full agonists exhibited markedly lower apparent affinities in intact cells with ({sup 3}H)QNB than with ({sup 3}H)NMS. Treatment of cells with antimycin A to deplete intracellular ATP prevented agonist-induced internalization of muscarinic receptors as assessed by sucrose density gradient assays of receptor subcellular distribution. In ATP-depleted cells, the apparent affinities of full agonists vs ({sup 3}H)QNB were markedly higher. The apparent affinities of partial agonists and of antagonists were unaffected by ATP depletion. In other studies, the effects of the protein kinase C activator phorbol 12-myristate, 13-acetate (PMA) on muscarinic receptor downregulation and internalization in 1321N1 cells were determined. PMA alone did not induce muscarinic receptor downregulation but instead decreased both the rate and final extent of downregulation induced by the agonist carbachol. The specificity of other protein kinase C activators for inhibiting carbachol-induced downregulation indicated involvement of protein kinase C. Furthermore, the protein kinase C inhibitor staurosporine prevented the inhibitory effect of PMA on downregulation. However, staurosporine did not inhibit agonist-induced downregulation.

  3. Affinity labeling of muscarinic receptors in rat cerebral cortex with a photolabile antagonist.

    PubMed Central

    Amitai, G; Avissar, S; Balderman, D; Sokolovsky, M

    1982-01-01

    Highly potent photoaffinity probes for muscarinic binding sites were prepared by the incorporation of an azido group into the benzilic acid moiety in two compound, 3-quinuclidinyl benzilate (3QNB) and N-methyl-4-piperidyl benzilate (4NMPB). Inactivation of muscarinic sites in rat cortex depends on the formation of a reversible complex with the azides prior to their photolytic conversion to the highly reactive nitrenes. During photolysis, radiolabeled azido-4NMPB interacted specifically and with high affinity (Kd = 1.06 nM) with the muscarinic receptors, and the ligand could be covalently incorporated into a macromolecule of about 86,000 Mr, presumably the muscarinic receptor. The incorporation was almost stoichiometric when compared to determination of receptor density by reversible ligands. Atropine (10 microM) afforded specific protection (greater than 83%) of the receptor against inactivation by azido-[3H]4NMPB. This compound and the other ligands described here (i.e., amino-4NMPB, amino-3QNB, and azido-3QNB) represent powerful potential probes for the biochemical isolation and characterization of muscarinic receptors. Images PMID:6952181

  4. Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist.

    PubMed

    Haga, Kazuko; Kruse, Andrew C; Asada, Hidetsugu; Yurugi-Kobayashi, Takami; Shiroishi, Mitsunori; Zhang, Cheng; Weis, William I; Okada, Tetsuji; Kobilka, Brian K; Haga, Tatsuya; Kobayashi, Takuya

    2012-01-25

    The parasympathetic branch of the autonomic nervous system regulates the activity of multiple organ systems. Muscarinic receptors are G-protein-coupled receptors that mediate the response to acetylcholine released from parasympathetic nerves. Their role in the unconscious regulation of organ and central nervous system function makes them potential therapeutic targets for a broad spectrum of diseases. The M2 muscarinic acetylcholine receptor (M2 receptor) is essential for the physiological control of cardiovascular function through activation of G-protein-coupled inwardly rectifying potassium channels, and is of particular interest because of its extensive pharmacological characterization with both orthosteric and allosteric ligands. Here we report the structure of the antagonist-bound human M2 receptor, the first human acetylcholine receptor to be characterized structurally, to our knowledge. The antagonist 3-quinuclidinyl-benzilate binds in the middle of a long aqueous channel extending approximately two-thirds through the membrane. The orthosteric binding pocket is formed by amino acids that are identical in all five muscarinic receptor subtypes, and shares structural homology with other functionally unrelated acetylcholine binding proteins from different species. A layer of tyrosine residues forms an aromatic cap restricting dissociation of the bound ligand. A binding site for allosteric ligands has been mapped to residues at the entrance to the binding pocket near this aromatic cap. The structure of the M2 receptor provides insights into the challenges of developing subtype-selective ligands for muscarinic receptors and their propensity for allosteric regulation.

  5. Neuromodulatory actions of substance P on the muscarinic receptors of the vas deferens of the rat.

    PubMed

    Silva, W I; Miranda, H F; Wolstenholme, W W; Cuevas, N; Ucros, A

    1988-01-01

    The response of post-synaptic neurokinin receptors to SP were not changed by pirenzepine or N-methyl-scopolamine. Atropine led to a slight increase in the EC50 of SP for its post-synaptic neurokinin (NK-A) receptor. In the presence of neostigmine no changes in the Emax and EC50 values of SP for its post- and pre-synaptic receptor site were observed. Only the muscarinic receptor site were observed. Only the muscarinic receptor antagonists, atropine and NMS, elicited statistically significant increases in the Emax of SP at its presynaptic receptor (NK-A). Addition of 7.4-740 nM SP resulted in a decrease in the EC50 and Emax values of ACh for its post-synaptic muscarinic receptor (M1). Conversely, 740 nM SP produced an increase in the EC50 and Emax values of ACh at its pre-synaptic muscarinic receptor (M2). Concentrations of 7.4 and 74 nM SP did not produce statistically significant changes in the Emax of ACh for its pre-synaptic M2 receptor.

  6. Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist

    SciTech Connect

    Haga, Kazuko; Kruse, Andrew C.; Asada, Hidetsugu; Yurugi-Kobayashi, Takami; Shiroishi, Mitsunori; Zhang, Cheng; Weis, William I.; Okada, Tetsuji; Kobilka, Brian K.; Haga, Tatsuya; Kobayashi, Takuya

    2012-03-15

    The parasympathetic branch of the autonomic nervous system regulates the activity of multiple organ systems. Muscarinic receptors are G-protein-coupled receptors that mediate the response to acetylcholine released from parasympathetic nerves. Their role in the unconscious regulation of organ and central nervous system function makes them potential therapeutic targets for a broad spectrum of diseases. The M2 muscarinic acetylcholine receptor (M2 receptor) is essential for the physiological control of cardiovascular function through activation of G-protein-coupled inwardly rectifying potassium channels, and is of particular interest because of its extensive pharmacological characterization with both orthosteric and allosteric ligands. Here we report the structure of the antagonist-bound human M2 receptor, the first human acetylcholine receptor to be characterized structurally, to our knowledge. The antagonist 3-quinuclidinyl-benzilate binds in the middle of a long aqueous channel extending approximately two-thirds through the membrane. The orthosteric binding pocket is formed by amino acids that are identical in all five muscarinic receptor subtypes, and shares structural homology with other functionally unrelated acetylcholine binding proteins from different species. A layer of tyrosine residues forms an aromatic cap restricting dissociation of the bound ligand. A binding site for allosteric ligands has been mapped to residues at the entrance to the binding pocket near this aromatic cap. The structure of the M2 receptor provides insights into the challenges of developing subtype-selective ligands for muscarinic receptors and their propensity for allosteric regulation.

  7. Cholinergic submandibular effects and muscarinic receptor expression in blood vessels of the rat.

    PubMed

    Ryberg, Anders T; Selberg, Hanna; Soukup, Ondrej; Gradin, Kathryn; Tobin, Gunnar

    2008-07-01

    In order to functionally characterise the muscarinic vasodilator responses, effects of cholinergic agonists were studied on isolated preparations of the rat submandibular artery and vein and carotid and jugular vessels. Tentatively, a cholinergic regulatory mechanism having different effects on the arterial and venous vessels would enhance vascular fluid recruitment for the secretory response. In vitro functional findings were correlated to the expression and cellular location of the different receptors that were assessed by immunohistochemistry. In order to find in vivo correlates to the in vitro findings, the influence of muscarinic receptors on permeability was studied on the vasculature of the submandibular gland in anaesthetised rats. Staining for muscarinic M1 receptors occurred in the endothelium, and muscarinic M5 receptors, and possibly M3 also, were detected in the arterial smooth muscle. In venous endothelium, muscarinic M1 and M4 receptors occurred. In the jugular smooth muscle layer, staining for M1, and possibly also for M3, appeared. Muscarinic agonists caused arteries to relax and veins to contract. The nitric oxide synthase inhibitor Nomega-nitro-L-arginine (L-NNA; 10(-4)M) markedly reduced the cholinergic-evoked relaxation of pre-contracted carotid arterial preparations. In the presence of 4-DAMP (10(-7)M), the relaxation to cholinergic agonists was inhibited. Pirenzepine (10(-5)M) did not only inhibit the relaxatory effects, but even reversed the effects, while it in the jugular vein abolished the cholinergic effects. The arterial nitric oxide-dependent response to muscarinic receptor stimulation consisted of two parts -- one sensitive to pirenzepine and 4-DAMP and the other to 4-DAMP only. Inhibition of the former part only, resulted in cholinergic arterial contraction. Also, the submandibular artery and vein responses to muscarinic receptor stimulation show a resemblance with those of the carotid and jugular vessels, i.e. a pronounced arterial

  8. Unraveling a molecular determinant for clathrin-independent internalization of the M2 muscarinic acetylcholine receptor

    PubMed Central

    Wan, Min; Zhang, Wenhua; Tian, Yangli; Xu, Chanjuan; Xu, Tao; Liu, Jianfeng; Zhang, Rongying

    2015-01-01

    Endocytosis and postendocytic sorting of G-protein-coupled receptors (GPCRs) is important for the regulation of both their cell surface density and signaling profile. Unlike the mechanisms of clathrin-dependent endocytosis (CDE), the mechanisms underlying the control of GPCR signaling by clathrin-independent endocytosis (CIE) remain largely unknown. Among the muscarinic acetylcholine receptors (mAChRs), the M4 mAChR undergoes CDE and recycling, whereas the M2 mAChR is internalized through CIE and targeted to lysosomes. Here we investigated the endocytosis and postendocytic trafficking of M2 mAChR based on a comparative analysis of the third cytoplasmic domain in M2 and M4 mAChRs. For the first time, we identified that the sequence 374KKKPPPS380 servers as a sorting signal for the clathrin-independent internalization of M2 mAChR. Switching 374KKKPPPS380 to the i3 loop of the M4 mAChR shifted the receptor into lysosomes through the CIE pathway; and therefore away from CDE and recycling. We also found another previously unidentified sequence that guides CDE of the M2 mAChR, 361VARKIVKMTKQPA373, which is normally masked in the presence of the downstream sequence 374KKKPPPS380. Taken together, our data indicate that endocytosis and postendocytic sorting of GPCRs that undergo CIE could be sequence-dependent. PMID:26094760

  9. Unraveling a molecular determinant for clathrin-independent internalization of the M2 muscarinic acetylcholine receptor.

    PubMed

    Wan, Min; Zhang, Wenhua; Tian, Yangli; Xu, Chanjuan; Xu, Tao; Liu, Jianfeng; Zhang, Rongying

    2015-06-22

    Endocytosis and postendocytic sorting of G-protein-coupled receptors (GPCRs) is important for the regulation of both their cell surface density and signaling profile. Unlike the mechanisms of clathrin-dependent endocytosis (CDE), the mechanisms underlying the control of GPCR signaling by clathrin-independent endocytosis (CIE) remain largely unknown. Among the muscarinic acetylcholine receptors (mAChRs), the M4 mAChR undergoes CDE and recycling, whereas the M2 mAChR is internalized through CIE and targeted to lysosomes. Here we investigated the endocytosis and postendocytic trafficking of M2 mAChR based on a comparative analysis of the third cytoplasmic domain in M2 and M4 mAChRs. For the first time, we identified that the sequence (374)KKKPPPS(380) servers as a sorting signal for the clathrin-independent internalization of M2 mAChR. Switching (374)KKKPPPS(380) to the i3 loop of the M4 mAChR shifted the receptor into lysosomes through the CIE pathway; and therefore away from CDE and recycling. We also found another previously unidentified sequence that guides CDE of the M2 mAChR, (361)VARKIVKMTKQPA(373), which is normally masked in the presence of the downstream sequence (374)KKKPPPS(380). Taken together, our data indicate that endocytosis and postendocytic sorting of GPCRs that undergo CIE could be sequence-dependent.

  10. G-protein mediates voltage regulation of agonist binding to muscarinic receptors: effects on receptor-Na/sup +/ channel interaction

    SciTech Connect

    Cohen-Armon, M.; Garty, H.; Sokolovsky, M.

    1988-01-12

    The authors previous experiments in membranes prepared from rat heart and brain led them to suggest that the binding of agonist to the muscarinic receptors and to the Na/sup +/ channels is a coupled event mediated by guanine nucleotide binding protein(s) (G-protein(s)). These in vitro findings prompted us to employ synaptoneurosomes from brain stem tissue to examine (i) the binding properties of (/sup 3/H) acetylcholine at resting potential and under depolarization conditions in the absence and presence of pertussis toxin; (ii) the binding of (/sup 3/H)batrachotoxin to Na/sup +/ channel(s) in the presence of the muscarinic agonists; and (iii) muscarinically induced /sup 22/Na/sup +/ uptake in the presence and absence of tetrodotoxin, which blocks Na/sup +/ channels. The findings indicate that agonist binding to muscarinic receptors is voltage dependent, that this process is mediated by G-protein(s), and that muscarinic agonists induce opening of Na/sup +/channels. The latter process persists even after pertussis toxin treatment, indicating that it is not likely to be mediated by pertussis toxin sensitive G-protein(s). The system with its three interacting components-receptor, G-protein, and Na/sup +/ channel-is such that at resting potential the muscarinic receptor induces opening of Na/sup +/ channels; this property may provide a possible physiological mechanism for the depolarization stimulus necessary for autoexcitation or repetitive firing in heart or brain tissues.

  11. The binding of (3H)AF-DX 384 to rat ileal smooth muscle muscarinic receptors

    SciTech Connect

    Entzeroth, M.; Mayer, N. )

    1991-01-01

    The tritiated cardioselective muscarinic antagonist AF-DX 384 (5,11-dihydro-11-(2-(-(8-dipropylamino)methyl)-1-piperidinyl-ethyl-amino-carbonyl)-6H-pyrido (2,3-b) (1,4)benzodiazepin-6-one) was used to label muscarinic receptors in the rat ileum. Saturation binding to membrane suspensions revealed a high affinity binding site with a Kd of 9.2 nM. The maximal number of binding sites labeled in this tissue (Bmax) is 237 fmol/mg protein. The association and dissociation kinetics were well represented by single exponential reactions, and the dissociation constant obtained from the ratio of rate constants was in agreement with that derived from saturation experiments. Specific binding was inhibited by muscarinic antagonists with a rank order of potencies of atropine (pKi: 8.80) greater than 4-DAMP (pKi: 8.23) = AF-DX 384 (pKi: 8.20) greater than AF-DX 116 (pKi: 7.09) = hexahydro-sila-difenidol (pKi: 6.97) greater than pirenzepine (pKi: 6.49) and is consistent with the interaction of (3H)AF-DX 384 with muscarinic receptors of the M2 subtype. It can be concluded that (3H)AF-DX 384 can be used to selectively label M2 muscarinic receptors in heterogeneous receptor populations.

  12. Evaluation of 1,2,5-thiadiazoles as modulators of M₁/M₅ muscarinic receptor subtypes.

    PubMed

    Maheshwari, Aditya; Rao, P S S; Messer, William S

    2014-03-15

    Studies have demonstrated the presence of allosteric binding sites on each of the muscarinic acetylcholine receptor (mAChR) subtypes. Since most drugs targeting muscarinic receptors bind to the highly conserved orthosteric binding site, they fail to achieve appreciable subtype selectivity. Targeting non-conserved allosteric sites may provide a new way of enhancing selectivity for individual subtypes of muscarinic receptor. Tetra(ethyleneglycol)(3-methoxy-1,2,5-thiadiazol-4-yl)[3-(1-methyl-1,2,5,6-tetrahydropyrid-3-yl)-1,2,5-thiadiazol-4-yl] ether, CDD-0304 (10), was found to be a M₁/₂/₄ selective muscarinic agonist and might prove useful in treating the symptoms associated with schizophrenia (J. Med. Chem.2003, 46, 4273). It was hypothesized that the observed subtype selectivity demonstrated by 10 may be due to its ability to function as a bitopic ligand (J. Med. Chem.2006, 49, 7518). To further investigate this possibility, a novel series of compounds was synthesized using a 1,2,5-thiadiazole moiety along with varying lengths of a polyethylene glycol linker and terminal groups, for evaluation as potential allosteric modulators of muscarinic receptors. Preliminary biological studies were performed using carbachol to stimulate M₁ and M₅ receptors. No significant agonist activity was observed at either M₁ or M₅ receptors for any of the compounds. Compound 18, 2-(4-methoxy-1,2,5-thiadiazol-3-yloxy)-N,N-dimethylethanamine fumarate (CDD-0361F) was found to block the effects of carbachol at M5 muscarinic receptors.

  13. In vitro functional interactions of acetylcholine esterase inhibitors and muscarinic receptor antagonists in the urinary bladder of the rat.

    PubMed

    Killi, Uday K; Wsol, Vladimir; Soukup, Ondrej; Kuca, Kamil; Winder, Michael; Tobin, Gunnar

    2014-02-01

    Obidoxime, a weak acetylcholine-esterase (AChE) inhibitor, exerts muscarinic receptor antagonism with a significant muscarinic M2 receptor selective profile. The current examinations aimed to determine the functional significance of muscarinic M2 receptors in the state of AChE inhibition, elucidating muscarinic M2 and M3 receptor interaction. In the in vitro examinations, methacholine evoked concentration-dependent bladder contractile and atrial frequency inhibitory responses. Although atropine abolished both, methoctramine (1 μmol/L) only affected the cholinergic response in the atrial preparations. However, in the presence of methoctramine, physostigmine, an AChE inhibitor, increased the basal tension of the bladder strip preparations (+68%), as well as the contractile responses to low concentrations of methacholine (< 5 μmol/L; +90-290%). In contrast to physostigmine, obidoxime alone raised the basal tension (+58%) and the responses to low concentrations of methacholine (< 5 μmol/L; +80-450%). Physostigmine concentration-dependently increased methacholine-evoked responses, similarly to obidoxime at low concentrations. However, at large concentrations (> 5 μmol/L), obidoxime, because of its unselective muscarinic receptor antagonism, inhibited the methacholine bladder responses. In conclusion, the current results show that muscarinic M2 receptors inhibit muscarinic M3 receptor-evoked contractile responses to low concentrations of acetylcholine in the synaptic cleft. The muscarinic M2 and M3 receptor crosstalk could be a counteracting mechanism in the treatment of AChE inhibition when using reactivators, such as obidoxime.

  14. M2muscarinic receptors inhibit cell proliferation and migration in urothelial bladder cancer cells

    PubMed Central

    Pacini, Luca; De Falco, Elena; Di Bari, Maria; Coccia, Andrea; Siciliano, Camilla; Ponti, Donatella; Pastore, Antonio Luigi; Petrozza, Vincenzo; Carbone, Antonio; Tata, Ada Maria; Calogero, Antonella

    2014-01-01

    The role of muscarinic receptors in several diseases including cancer has recently emerged. To evaluate the hypothesis that muscarinic acetylcholine receptors may play a role in bladder cancer as well as in other tumor types, we investigated their expression in bladder tumor specimens. All examined samples expressed the M1, M2 and M3 receptor subtypes. We also found that the level of M2 transcripts, but not those of M1 or M3, significantly increased with the tumor histologic grade. In view of these results, we proceeded to investigate whether the M2 agonist Arecaidine had any effect on in vitro cell growth and migration of T24 cells, a bladder tumor cell line expressing the muscarinic receptors, including the M2 subtype. We observed that Arecaidine significantly reduced T24 and 5637 cell proliferation and migration in a concentration dependent manner. The silencing of M2 receptor by siRNA in T24 and 5637 cell lines showed the inability of Arecaidine (100 μM) to inhibit cell proliferation after 48 hours, whereas the use of M1 and M3 antagonists in T24 appeared not to counteract the Arecaidine effect, suggesting that the inhibition of cell proliferation was directly dependent on M2 receptor activation. These data suggest that M2 muscarinic receptors may play a relevant role in bladder cancer and represent a new attractive therapeutic target. PMID:25482946

  15. Muscarinic cholinergic and alpha/sub 1/ adrenergic receptors in murine atria: phosphatidylinositol breakdown and receptor interaction

    SciTech Connect

    Scherer, R.W.

    1987-01-01

    Upon stimulation of muscarinic cholinergic receptors, there is a decrease in the force of contraction rate of firing in heart, while stimulation of ..cap alpha.. adrenergic receptors causes an increase in the force of contraction with no change in the heart rate. Yet both receptors stimulate the breakdown of phosphatidylinositol (PI). Therefore, the breakdown of PI was examined to determine how the process differed between the two receptor systems. Murine atria, prelabelled with (/sup 3/H)inositol, were stimulated with the muscarinic cholinergic agonists, carbamylcholine (CARB), and oxotremorine (OXO); and with the ..cap alpha.. adrenergic agonists, norepinephrine (NE) and phenylephrine (PE); either singly or in combination. Breakdown of PI was assessed by measurement of individual inositol phosphates by anion exchange chromatography. Binding of CARB to atrial muscarinic receptors was measured by competition with (/sup 3/H)quinuclidinyl benzilate.

  16. Ozone-induced loss of neuronal M{sub 2} muscarinic receptor function is prevented by cyclophosphamide

    SciTech Connect

    Gambone, L.M.; Elbon, C.L.; Fryer, A.D.

    1994-09-01

    The authors tested the hypothesis that inflammatory cells mediate the loss of neuronal M{sub 2} muscarinic receptors in the lung after ozone exposure. Pathogen-free guinea pigs treated with cyclophosphamide (30 mg {center_dot} kg{sup {minus}1} {center_dot} day{sup {minus}1} ip for 7 days) before exposure to ozone were compared with untreated ozone-exposed animals. This dose of cyclophosphamide significantly reduced leukocytes in peripheral blood and bronchoalveolar lavage fluid. Twenty-four hours after ozone, muscarinic receptor function was tested in anesthetized animals. In air-exposed guinea pigs, vagally induced bronchoconstriction was attenuated by the muscarinic agonist pilocarpine (0.1-100 {mu}g/kg iv) and potentiated by the selective M{sub 2} antagonist gallamine (0.1-10 mg/kg iv), indicating that the neuronal M{sub 2} muscarinic receptors were functioning. These responses were significantly reduced after ozone, indicating loss of neuronal M{sub 2} muscarinic receptor function. However, in those animals treated with cyclophosphamide, M{sub 2} muscarinic receptor function was not altered by ozone. These data suggest that ozone-induced loss of neuronal muscarinic receptor function is mediated via inflammatory cells and that the link between ozone-induced hyperresponsiveness and inflammation may be the neuronal M{sub 2} muscarinic receptor. 27 refs., 9 figs.

  17. Presynaptic Muscarinic Acetylcholine Receptors and TrkB Receptor Cooperate in the Elimination of Redundant Motor Nerve Terminals during Development

    PubMed Central

    Nadal, Laura; Garcia, Neus; Hurtado, Erica; Simó, Anna; Tomàs, Marta; Lanuza, Maria A.; Cilleros, Victor; Tomàs, Josep

    2017-01-01

    The development of the nervous system involves the overproduction of synapses but connectivity is refined by Hebbian activity-dependent axonal competition. The newborn skeletal muscle fibers are polyinnervated but, at the end of the competition process, some days later, become innervated by a single axon. We used quantitative confocal imaging of the autofluorescent axons from transgenic B6.Cg-Tg (Thy1-YFP)16 Jrs/J mice to investigate the possible cooperation of the muscarinic autoreceptors (mAChR, M1-, M2- and M4-subtypes) and the tyrosine kinase B (TrkB) receptor in the control of axonal elimination after the mice Levator auris longus (LAL) muscle had been exposed to several selective antagonist of the corresponding receptor pathways in vivo. Our previous results show that M1, M2 and TrkB signaling individually increase axonal loss rate around P9. Here we show that although the M1 and TrkB receptors cooperate and add their respective individual effects to increase axonal elimination rate even more, the effect of the M2 receptor is largely independent of both M1 and TrkB receptors. Thus both, cooperative and non-cooperative signaling mechanisms contribute to developmental synapse elimination. PMID:28228723

  18. Increased function of inhibitory neuronal M2 muscarinic receptors in diabetic rat lungs

    PubMed Central

    Belmonte, Kristen E; Jacoby, David B; Fryer, Allison D

    1997-01-01

    The function of inhibitory neuronal M2 muscarinic receptors in diabetic rat lungs was investigated. Neuronal M2 muscarinic receptors inhibit acetylcholine release from parasympathetic nerves. Thus, stimulation of neuronal M2 muscarinic receptors with muscarinic agonists, such as pilocarpine, inhibits acetylcholine release and vagally induced bronchoconstriction. In contrast, blockade of neuronal M2 muscarinic receptors with selective M2 muscarinic antagonists, such as AF-DX 116, potentiates acetylcholine release and vagally induced bronchoconstriction. Rats were made diabetic by streptozotocin (65 mg kg−1, i.v.). After 7–14 days the rats were anaesthetized with urethane (1.5 g kg−1, i.p.), tracheostomized, vagotomized, ventilated and paralysed with suxamethonium (30 mg kg−1, i.v.). Some 7 day diabetic rats were treated with low doses of long acting (NPH) insulin (2 units day−1, s.c.) for 7 days before experimentation. This dose of insulin was not sufficient to restore normoglycaemia in diabetic rats. Thus, insulin-treated diabetic rats remained hyperglycaemic. Distal electrical stimulation (5–70 Hz, 6 s, 40 V, 0.4 ms) of the vagi caused bronchoconstriction, measured as an increase in inflation pressure and bradycardia. In diabetic rats, vagally induced bronchoconstriction was significantly depressed vs controls. In contrast, bronchoconstriction caused by i.v. acetylcholine was similar in diabetic and control animals. The function of neuronal M2 muscarinic receptors was tested with the muscarinic agonist pilocarpine (0.001–100.0 μg kg−1, i.v.) and the antagonist AF-DX 116 (0.01–3.0 mg kg−1, i.v.). Pilocarpine inhibited vagally-induced bronchoconstriction (30 Hz, 20–40 V, 0.4 ms at 6 s) and AF-DX 116 potentiated vagally-induced bronchoconstriction (20 Hz, 20–40 V, 0.4 ms at 6 s) to a significantly greater degree in diabetic rats compared to controls. Both frequency-dependent vagally

  19. Comparative study of muscarinic acetylcholine receptors of human and rat cortical glial cells

    SciTech Connect

    Demushkin, V.P.; Burbaeva, G.S.; Dzhaliashvili, T.A.; Plyashkevich, Y.G.

    1985-04-01

    The aim of the present investigation was a comparative studyof muscarinic acetylcholine receptors in human and rat glial cells. (/sup 3/H)Quinuclidinyl-benzylate ((/sup 3/H)-QB), atropine, platiphylline, decamethonium, carbamylcholine, tubocurarine, and nicotine were used. The glial cell fraction was obtained from the cerebral cortex of rats weighing 130-140 g and from the frontal pole of the postmortem brain from men aged 60-70 years. The use of the method of radioimmune binding of (/sup 3/H)-QB with human and rat glial cell membranes demonstrated the presence of a muscarinic acetylcholine receptor in the glial cells.

  20. Amplification of the rat m2 muscarinic receptor gene by the polymerase chain reaction: Functional expression of the M sub 2 muscarinic receptor

    SciTech Connect

    Lai, J.; Bloom, J.W.; Yamamura, H.I.; Roeske, W.R. )

    1990-01-01

    A selective amplification of the coding sequence of the rat M{sub 2} muscarinic receptor gene was achieved by the polymerase chain reaction. The error rate of this amplification system under conditions specified was 1 nucleotide substitution in 841 base pairs. In vitro expression of this gene in murine fibroblasts (B82) via the eukaryotic expression vector, pH{beta}APr-1-neo, resulted in high level expression of specific ({sup 3}H)(-)MQNB binding in transfected B82 cell lines. One of these clones, M2LKB2-2, showed a stable expression of ({sup 3}H)(-)MQNB binding with a K{sub d} value of 265 pM and a B{sub max} value of 411{plus minus}50 fmol/10{sup 6} cells. Cardiac selective muscarinic antagonists such as himbacine and AF-DX 116 show high affinities for this binding site in the M2LKB2-2 cells. The rank order of potency of several antagonists in inhibiting ({sup 3}H)(-)MQNB binding in these cells conformed to the characteristics of an M{sub 2} type muscarinic receptor. Carbachol showed a single affinity state for the receptors in the M2LKB2-2 cells with a K{sub i} value of 2.0 {mu}M. This receptor appeared to be inversely coupled to adenylate cyclase via a pertussis toxin sensitive G-protein. Carbachol also had a slight stimulatory effect on the hydrolysis of inositol lipids. The polymerase chain reaction proves highly effective in cloning genes from genomic material, as demonstrated by the first in vitro functional expression of the rat M{sub 2} type muscarinic receptor.

  1. Differential role of insular cortex muscarinic and NMDA receptors in one-trial appetitive taste learning.

    PubMed

    Parkes, Shauna L; De la Cruz, Vanesa; Bermúdez-Rattoni, Federico; Coutureau, Etienne; Ferreira, Guillaume

    2014-12-01

    Our current understanding of the neurobiology of taste learning and memory has been greatly facilitated by the use of a reliable behavioural model, conditioned taste aversion (CTA). This model has revealed that the insular cortex (IC), specifically muscarinic and N-methyl-d-aspartate (NMDA) receptor activation in the IC, is critical for the formation of aversive taste memories. In contrast, current models of appetitive taste learning are less adequate, relying on the use of neophobic tastes (attenuation of neophobia) or on the integration of appetitive and aversive taste memories (latent inhibition of CTA). While these models have implicated IC muscarinic receptors, the involvement of NMDA receptors in the IC remains unclear. Here, we examined the role of both muscarinic and NMDA receptors in appetitive taste learning using a simple paradigm that is independent of neophobic and aversive components. First, we demonstrated that a single exposure to a novel taste, saccharin 0.1%, is sufficient to promote an appetitive taste memory as revealed by an increase in saccharin consumption during the second presentation. This increase was blocked by bilateral infusion in the IC of the muscarinic receptor antagonist, scopolamine. In contrast, infusion of the NMDA receptor antagonist, AP5, did not block appetitive taste learning but did abolish CTA. Therefore, common and distinct molecular substrates within the IC mediate appetitive versus aversive learning about the same taste.

  2. Heterogeneity of binding of muscarinic receptor antagonists in rat brain homogenates

    SciTech Connect

    Lee, J.H.; el-Fakahany, E.E.

    1985-06-01

    The binding properties of (-)-(/sup 3/H)quinuclidinyl benzilate and (/sup 3/H) N-methylscopolamine to muscarinic acetylcholine receptors have been investigated in rat brain homogenates. The binding of both antagonists demonstrated high affinity and saturability. Analysis of the binding data resulted in linear Scatchard plots. However, (-)-(/sup 3/H)quinuclidinyl benzilate showed a significantly higher maximal binding capacity than that of (/sup 3/H)N-methylscopolamine. Displacement of both ligands with several muscarinic receptor antagonists resulted in competition curves in accordance with the law of mass-action for quinuclidinyl benzilate, atropine and scopolamine. A similar profile was found for the quaternary ammonium analogs of atropine and scopolamine when (/sup 3/H)N-methylscopolamine was used to label the receptors. However, when these hydrophilic antagonists were used to displace (-)-(/sup 3/H) quinuclidinyl benzilate binding, they showed interaction with high- and low-affinity binding sites. On the other hand, the nonclassical muscarinic receptor antagonist, pirenzepine, was able to displace both ligands from two binding sites. The present data are discussed in terms of the relationship of this anomalous heterogenity of binding of these hydrophilic muscarinic receptor antagonists and the proposed M1 and M2 receptor subtypes.

  3. Distribution of muscarinic acetylcholine receptor subtypes in the murine small intestine.

    PubMed

    Muise, Eleanor D; Gandotra, Neeru; Tackett, John J; Bamdad, Michaela C; Cowles, Robert A

    2017-01-15

    Serotonin stimulates enterocyte turnover in the small intestine and studies suggest this is mediated by neuronal signaling via a cholinergic pathway. Distribution of the five known muscarinic receptor subtypes (mAChRs) in the small intestine has not been fully studied, and their role in intestinal growth is unknown. We hypothesized that mAChRs have distinct anatomic distributions within the bowel, and that mAChRs present within intestinal crypts mediate the effects of acetylcholine on the small intestinal mucosa. Small intestine from male C57BL/6 mice ages 2, 4, 6, and 8weeks were harvested. RNA was isolated and cDNA synthesized for PCR-amplification of subtype specific mAChRs. Ileum was fixed with Nakane, embedded in epon, and immunofluorescence microscopy performed using polyclonal antibodies specific to each mAChR1-5. All five mAChR subtypes were present in the mouse duodenum, jejunum, and ileum at all ages by RT-PCR. Immunofluorescence microscopy suggested the presence of mAChR1-5 in association with mature enterocytes along the villus and within the myenteric plexus. Only mAChR2 clearly localized to the crypt stem cell compartment, specifically co-localizing with Paneth cells at crypt bases. Muscarinic receptors are widely distributed along the entire alimentary tract. mAChR2 appears to localize to the crypt stem cell compartment, suggesting it is a plausible regulator of stem cell activity. The location of mAChR2 to the crypt makes it a potential therapeutic target for treatment of intestinal disease such as short bowel syndrome. The exact cellular location and action of each mAChR requires further study. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Antibodies to β adrenergic and muscarinic cholinergic receptors in patients with Chronic Fatigue Syndrome.

    PubMed

    Loebel, Madlen; Grabowski, Patricia; Heidecke, Harald; Bauer, Sandra; Hanitsch, Leif G; Wittke, Kirsten; Meisel, Christian; Reinke, Petra; Volk, Hans-Dieter; Fluge, Øystein; Mella, Olav; Scheibenbogen, Carmen

    2016-02-01

    Infection-triggered disease onset, chronic immune activation and autonomic dysregulation in CFS point to an autoimmune disease directed against neurotransmitter receptors. Autoantibodies against G-protein coupled receptors were shown to play a pathogenic role in several autoimmune diseases. Here, serum samples from a patient cohort from Berlin (n=268) and from Bergen with pre- and post-treatment samples from 25 patients treated within the KTS-2 rituximab trial were analysed for IgG against human α and β adrenergic, muscarinic (M) 1-5 acetylcholine, dopamine, serotonin, angiotensin, and endothelin receptors by ELISA and compared to a healthy control cohort (n=108). Antibodies against β2, M3 and M4 receptors were significantly elevated in CFS patients compared to controls. In contrast, levels of antibodies against α adrenergic, dopamine, serotonin, angiotensin, and endothelin receptors were not different between patients and controls. A high correlation was found between levels of autoantibodies and elevated IgG1-3 subclasses, but not with IgG4. Further patients with high β2 antibodies had significantly more frequently activated HLA-DR+ T cells and more frequently thyreoperoxidase and anti-nuclear antibodies. In patients receiving rituximab maintenance treatment achieving prolonged B-cell depletion, elevated β2 and M4 receptor autoantibodies significantly declined in clinical responder, but not in non-responder. We provide evidence that 29.5% of patients with CFS had elevated antibodies against one or more M acetylcholine and β adrenergic receptors which are potential biomarkers for response to B-cell depleting therapy. The association of autoantibodies with immune markers suggests that they activate B and T cells expressing β adrenergic and M acetylcholine receptors. Dysregulation of acetylcholine and adrenergic signalling could also explain various clinical symptoms of CFS.

  5. Role of specific muscarinic receptor subtypes in cholinergic parasympathomimetic responses, in vivo phosphoinositide hydrolysis, and pilocarpine-induced seizure activity.

    PubMed

    Bymaster, Frank P; Carter, Petra A; Yamada, Masahisa; Gomeza, Jesus; Wess, Jürgen; Hamilton, Susan E; Nathanson, Neil M; McKinzie, David L; Felder, Christian C

    2003-04-01

    Muscarinic agonist-induced parasympathomimetic effects, in vivo phosphoinositide hydrolysis and seizures were evaluated in wild-type and muscarinic M1-M5 receptor knockout mice. The muscarinic agonist oxotremorine induced marked hypothermia in all the knockout mice, but the hypothermia was reduced in M2 and to a lesser extent in M3 knockout mice. Oxotremorine-induced tremor was abolished only in the M2 knockout mice. Muscarinic agonist-induced salivation was reduced to the greatest extent in M3 knockout mice, to a lesser degree in M1 and M4 knockout mice, and was not altered in M2 and M5 knockout mice. Pupil diameter under basal conditions was increased only in the M3 knockout mice. Pilocarpine-induced increases in in vivo phosphoinositide hydrolysis were completely absent in hippocampus and cortex of M1 knockout mice, but in vivo phosphoinositide hydrolysis was unaltered in the M2-M5 knockout mice. A high dose of pilocarpine (300 mg/kg) caused seizures and lethality in wild-type and M2-M5 knockout mice, but produced neither effect in the M1 knockout mice. These data demonstrate a major role for M2 and M3 muscarinic receptor subtypes in mediating parasympathomimetic effects. Muscarinic M1 receptors activate phosphoinositide hydrolysis in cortex and hippocampus of mice, consistent with the role of M1 receptors in cognition. Muscarinic M1 receptors appear to be the only muscarinic receptor subtype mediating seizures.

  6. Radioligand binding to muscarinic receptors of bovine aortic endothelial cells.

    PubMed Central

    Brunner, F.; Kukovetz, W. R.

    1991-01-01

    1. Muscarinic receptors on endothelial cells of bovine thoracic aorta were characterized by binding assays in which (-)-[3H]-N-methyl quinuclidinyl benzilate ([3H]-NMeQNB) was used as radioligand. 2. Binding of [3H]-NMeQNB to crude membranes of freshly isolated endothelial cells was atropine-displaceable and of high affinity (KD = 0.48 nM) to a single class of sites (maximum binding capacity: 14 +/- 3 fmol mg-1 protein). Stereospecificity of the binding sites was demonstrated in experiments in which [3H]-NMeQNB binding was inhibited by dexetimide in the nanomolar range (KI = 0.63 nM) and by levetimide, its stereoisomer in the micromolar range (KI = 3.2 microM) (selectivity factor: approximately 5000). 3. Drug competition curves indicated a single class of binding sites for antagonists and the following apparent affinities (KI, nM): methyl atropine: 1.1: 4-diphenylacetoxy N-methyl piperidine methyl bromide (4-DAMP): 3.4; pirenzepine: 16; 11-[2-diethylamino-methyl)-1-piperidinyl- acetyl]-5,11-dihydro-6H-pyrido(2,3-b)1,4-benzodiazepine-6-one (AF-DX 116); 2.500. Competition of acetylcholine with [3H]-NMeQNB was best described by two affinity sites (or states) (KH = 0.82 microM, KL = 1.6 microM). In the presence of guanylimido diphosphate [Gpp(NH)p] (100 microM), acetylcholine affinity (IC50) was slightly, but significantly reduced (factor approximately 4). 4. Binding of [3H]-NMeQNB to freshly harvested intact cells was also atropine-displaceable, stereospecific (selectivity factor: approximately 3500) and of high affinity (KD = 0.35 nM). The maximum binding capacity (9 +/- 2 fmol mg-1 total cell protein) was comparable to that of membranes and corresponded to approximately 900 binding sites per endothelial cell.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2015420

  7. Direct muscarinic and nicotinic receptor-mediated excitation of rat medial vestibular nucleus neurons in vitro

    NASA Technical Reports Server (NTRS)

    Phelan, K. D.; Gallagher, J. P.

    1992-01-01

    We have utilized intracellular recording techniques to investigate the cholinoceptivity of rat medial vestibular nucleus (MVN) neurons in a submerged brain slice preparation. Exogenous application of the mixed cholinergic agonists, acetylcholine (ACh) or carbachol (CCh), produced predominantly membrane depolarization, induction of action potential firing, and decreased input resistance. Application of the selective muscarinic receptor agonist muscarine (MUSC), or the selective nicotinic receptor agonists nicotine (NIC) or 1,1-dimethyl-4-phenylpiperazinium (DMPP) also produced membrane depolarizations. The MUSC-induced depolarization was accompanied by decreased conductance, while an increase in conductance appeared to underlie the NIC- and DMPP-induced depolarizations. The muscarinic and nicotinic receptor mediated depolarizations persisted in tetrodotoxin and/or low Ca2+/high Mg2+ containing media, suggesting direct postsynaptic receptor activation. The MUSC-induced depolarization could be reversibly blocked by the selective muscarinic-receptor antagonist, atropine, while the DMPP-induced depolarization could be reversibly suppressed by the selective ganglionic nicotinic-receptor antagonist, mecamylamine. Some neurons exhibited a transient membrane hyperpolarization during the depolarizing response to CCh or MUSC application. This transient inhibition could be reversibly blocked by the gamma-aminobutyric acid (GABA) antagonist, bicuculline, suggesting that the underlying hyperpolarization results indirectly from the endogenous release of GABA acting at GABA receptors. This study confirms the cholinoceptivity of MVN neurons and establishes that individual MVN cells possess muscarinic as well as nicotinic receptors. The data provide support for a prominent role of cholinergic mechanisms in the direct and indirect regulation of the excitability of MVN neurons.

  8. Muscarinic receptors involved in airway vascular leakage induced by experimental gastro-oesophageal reflux.

    PubMed

    Cui, Yong-Yao; Zhu, Liang; Wang, Hao; Advenier, Charles; Chen, Hong-Zhuan; Devillier, Philippe

    2008-04-23

    Gastro-oesophageal acid reflux may cause airway responses such as cough, bronchoconstriction and inflammation in asthmatic patients. Studies in humans or in animals have suggested that these responses involve cholinergic nerves. The purpose of this study was to investigate the role of the efferent vagal component on airway microvascular leakage induced by instillation of hydrochloric acid (HCl) into the oesophagus of guinea-pigs and the subtype of muscarinic receptors involved. Airway microvascular leakage induced by intra-oesophageal HCl instillation was abolished by bilateral vagotomy or by the nicotinic receptor antagonist, hexamethonium. HCl-induced leakage was inhibited by pretreatment with atropine, a non-specific muscarinic receptor antagonist, and also by pretreatment with either pirenzepine, a muscarinic M(1) receptor antagonist, or 4-DAMP, a muscarinic M(3) receptor antagonist. Pirenzepine was more potent than atropine and 4-DAMP. These antagonists were also studied on airway microvascular leakage or bronchoconstriction induced by intravenous administration of acetylcholine (ACh). Atropine, pirenzepine and 4-DAMP inhibited ACh-induced airway microvascular leakage with similar potencies. In sharp contrast, 4-DAMP and atropine were more potent inhibitors of ACh-induced bronchoconstriction than pirenzepine. Methoctramine, a muscarinic M(2) receptor antagonist, was ineffective in all experimental conditions. These results suggest that airway microvascular leakage caused by HCl intra-oesophageal instillation involves ACh release from vagus nerve terminals and that M(1) and M(3) receptors play a major role in cholinergic-mediated microvascular leakage, whereas M(3) receptors are mainly involved in ACh-induced bronchoconstriction.

  9. Direct muscarinic and nicotinic receptor-mediated excitation of rat medial vestibular nucleus neurons in vitro

    NASA Technical Reports Server (NTRS)

    Phelan, K. D.; Gallagher, J. P.

    1992-01-01

    We have utilized intracellular recording techniques to investigate the cholinoceptivity of rat medial vestibular nucleus (MVN) neurons in a submerged brain slice preparation. Exogenous application of the mixed cholinergic agonists, acetylcholine (ACh) or carbachol (CCh), produced predominantly membrane depolarization, induction of action potential firing, and decreased input resistance. Application of the selective muscarinic receptor agonist muscarine (MUSC), or the selective nicotinic receptor agonists nicotine (NIC) or 1,1-dimethyl-4-phenylpiperazinium (DMPP) also produced membrane depolarizations. The MUSC-induced depolarization was accompanied by decreased conductance, while an increase in conductance appeared to underlie the NIC- and DMPP-induced depolarizations. The muscarinic and nicotinic receptor mediated depolarizations persisted in tetrodotoxin and/or low Ca2+/high Mg2+ containing media, suggesting direct postsynaptic receptor activation. The MUSC-induced depolarization could be reversibly blocked by the selective muscarinic-receptor antagonist, atropine, while the DMPP-induced depolarization could be reversibly suppressed by the selective ganglionic nicotinic-receptor antagonist, mecamylamine. Some neurons exhibited a transient membrane hyperpolarization during the depolarizing response to CCh or MUSC application. This transient inhibition could be reversibly blocked by the gamma-aminobutyric acid (GABA) antagonist, bicuculline, suggesting that the underlying hyperpolarization results indirectly from the endogenous release of GABA acting at GABA receptors. This study confirms the cholinoceptivity of MVN neurons and establishes that individual MVN cells possess muscarinic as well as nicotinic receptors. The data provide support for a prominent role of cholinergic mechanisms in the direct and indirect regulation of the excitability of MVN neurons.

  10. Development of a photoactivatable allosteric ligand for the m1 muscarinic acetylcholine receptor.

    PubMed

    Davie, Briana J; Sexton, Patrick M; Capuano, Ben; Christopoulos, Arthur; Scammells, Peter J

    2014-10-15

    The field of G protein-coupled receptor drug discovery has benefited greatly from the structural and functional insights afforded by photoactivatable ligands. One G protein-coupled receptor subfamily for which photoactivatable ligands have been developed is the muscarinic acetylcholine receptor family, though, to date, all such ligands have been designed to target the orthosteric (endogenous ligand) binding site of these receptors. Herein we report the synthesis and pharmacological investigation of a novel photoaffinity label, MIPS1455 (4), designed to bind irreversibly to an allosteric site of the M1 muscarinic acetylcholine receptor; a target of therapeutic interest for the treatment of cognitive deficits. MIPS1455 may be a valuable molecular tool for further investigating allosteric interactions at this receptor.

  11. Mixed nicotinic-muscarinic properties of the alpha9 nicotinic cholinergic receptor.

    PubMed

    Verbitsky, M; Rothlin, C V; Katz, E; Elgoyhen, A B

    2000-10-01

    The rat alpha9 nicotinic acetylcholine receptor (nAChR) was expressed in Xenopus laevis oocytes and tested for its sensitivity to a wide variety of cholinergic compounds. Acetylcholine (ACh), carbachol, choline and methylcarbachol elicited agonist-evoked currents, giving maximal or near maximal responses. Both the nicotinic agonist suberyldicholine as well as the muscarinic agonists McN-A-343 and methylfurtrethonium behaved as weak partial agonists of the receptor. Most classical cholinergic compounds tested, being either nicotinic (nicotine, epibatidine, cytisine, methyllycaconitine, mecamylamine, dihydro-beta-erythroidine), or muscarinic (muscarine, atropine, gallamine, pilocarpine, bethanechol) agonists and antagonists, blocked the recombinant alpha9 receptor. Block by nicotine, epibatidine, cytisine, methyllycaconitine and atropine was overcome at high ACh concentrations, suggesting a competitive type of block. The present results indicate that alpha9 displays mixed nicotinic-muscarinic features that resemble the ones described for the cholinergic receptor of cochlear outer hair cells (OHCs). We suggest that alpha9 contains the structural determinants responsible for the pharmacological properties of the native receptor.

  12. Quantitative autoradiographic analysis of muscarinic receptor subtypes and their role in representational memory

    SciTech Connect

    Messer, W.S.

    1986-01-01

    Autoradiographic techniques were used to examine the distribution of muscarinic receptors in rat brain slices. Agonist and selective antagonist binding were examined by measuring the ability for unlabeled ligands to inhibit (/sup 3/H)-1-QNB labeling of muscarinic receptors. The distribution of high affinity pirenzepine binding sites (M/sub 1/ subtype) was distinct from the distribution of high affinity carbamylcholine sites, which corresponded to the M/sub 2/ subtype. In a separate assay, the binding profile for pirenzepine was shown to differ from the profile for scopolamine, a classical muscarinic antagonist. Muscarinic antagonists, when injected into the Hippocampus, impaired performance of a representational memory task. Pirenzepine, the M/sub 1/ selective antagonist, produced representational memory deficits. Scopolamine, a less selective muscarinic antagonist, caused increases in running times in some animals which prevented a definitive interpretation of the nature of the impairment. Pirenzepine displayed a higher affinity for the hippocampus and was more effective in producing a selective impairment of representational memory than scopolamine. The data indicated that cholinergic activity in the hippocampus was necessary for representation memory function.

  13. Activation of muscarinic acetylcholine receptors elicits pigment granule dispersion in retinal pigment epithelium isolated from bluegill

    PubMed Central

    González, Alfredo; Crittenden, Elizabeth L; García, Dana M

    2004-01-01

    Background In fish, melanin pigment granules in the retinal pigment epithelium disperse into apical projections as part of the suite of responses the eye makes to bright light conditions. This pigment granule dispersion serves to reduce photobleaching and occurs in response to neurochemicals secreted by the retina. Previous work has shown that acetylcholine may be involved in inducing light-adaptive pigment dispersion. Acetylcholine receptors are of two main types, nicotinic and muscarinic. Muscarinic receptors are in the G-protein coupled receptor superfamily, and five different muscarinic receptors have been molecularly cloned in human. These receptors are coupled to adenylyl cyclase, calcium mobilization and ion channel activation. To determine the receptor pathway involved in eliciting pigment granule migration, we isolated retinal pigment epithelium from bluegill and subjected it to a battery of cholinergic agents. Results The general cholinergic agonist carbachol induces pigment granule dispersion in isolated retinal pigment epithelium. Carbachol-induced pigment granule dispersion is blocked by the muscarinic antagonist atropine, by the M1 antagonist pirenzepine, and by the M3 antagonist 4-DAMP. Pigment granule dispersion was also induced by the M1 agonist 4-[N-(4-chlorophenyl) carbamoyloxy]-4-pent-2-ammonium iodide. In contrast the M2 antagonist AF-DX 116 and the M4 antagonist tropicamide failed to block carbachol-induced dispersion, and the M2 agonist arecaidine but-2-ynyl ester tosylate failed to elicit dispersion. Conclusions Our results suggest that carbachol-mediated pigment granule dispersion occurs through the activation of Modd muscarinic receptors, which in other systems couple to phosphoinositide hydrolysis and elevation of intracellular calcium. This conclusion must be corroborated by molecular studies, but suggests Ca2+-dependent pathways may be involved in light-adaptive pigment dispersion. PMID:15251036

  14. Expression of muscarinic acetylcholine and dopamine receptor mRNAs in rat basal ganglia

    SciTech Connect

    Weiner, D.M. Howard Hughes Medical Inst., Bethesda, MD ); Levey, A.I. Johns Hopkins Univ., Baltimore, MD ); Brann, M.R. )

    1990-09-01

    Within the basal ganglia, acetylcholine and dopamine play a central role in the extrapyramidal control of motor function. The physiologic effects of these neurotransmitters are mediated by a diversity of receptor subtypes, several of which have now been cloned. Muscarinic acetylcholine receptors are encoded by five genes (m1-m5), and of the two known dopamine receptor subtypes (D1 and D2) the D2 receptor gene has been characterized. To gain insight into the physiological roles of each of these receptor subtypes, the authors prepared oligodeoxynucleotide probes to localize receptor subtype mRNAs within the rat striatum and substantia nigra by in situ hybridization histochemistry. Within the striatum, three muscarinic (m1, m2, m4) receptor mRNAs and the D2 receptor mRNA were detected. The m1 mRNA was expressed in most neurons; the m2 mRNA, in neurons which were both very large and rare; and the m4 and D2 mRNAs, in 40-50% of the neurons, one-third of which express both mRNAs. Within the substantia nigra, pars compacta, only the m5 and D2 mRNAs were detected, and most neurons expressed both mRNAs. These data provide anatomical evidence for the identity of the receptor subtypes which mediate the diverse effects of muscarinic and dopaminergic drugs on basal ganglia function.

  15. Desensitization and internalization of the m2 muscarinic acetylcholine receptor are directed by independent mechanisms.

    PubMed

    Pals-Rylaarsdam, R; Xu, Y; Witt-Enderby, P; Benovic, J L; Hosey, M M

    1995-12-01

    The phenomenon of acute desensitization of G-protein-coupled receptors has been associated with several events, including receptor phosphorylation, loss of high affinity agonist binding, receptor:G-protein uncoupling, and receptor internalization. However, the biochemical events underlying these processes are not fully understood, and their contributions to the loss of signaling remain correlative. In addition, the nature of the kinases and the receptor domains which are involved in modulation of activity have only begun to be investigated. In order to directly measure the role of G-protein-coupled receptor kinases (GRKs) in the desensitization of the m2 muscarinic acetylcholine receptor (m2 mAChR), a dominant-negative allele of GRK2 was used to inhibit receptor phosphorylation by endogenous GRK activity in a human embryonic kidney cell line. The dominant-negative GRK2K220R reduced agonist-dependent phosphorylation of the m2 mAChR by approximately 50% and prevented acute desensitization of the receptor as measured by the ability of the m2 mAChR to attenuate adenylyl cyclase activity. In contrast, the agonist-induced internalization of the m2 mAChR was unaffected by the GRK2K220R construct. Further evidence linking receptor phosphorylation to acute receptor desensitization was obtained when two deletions of the third intracellular loop were made which created m2 mAChRs that did not become phosphorylated in an agonist-dependent manner and did not desensitize. However, the mutant mAChRs retained the ability to internalize. These data provide the first direct evidence that GRK-mediated receptor phosphorylation is necessary for m2 mAChR desensitization; the likely sites of in vivo phosphorylation are in the central portion of the third intracellular loop (amino acids 282-323). These results also indicate that internalization of the m2 receptor is not a key event in desensitization and is mediated by mechanisms distinct from GRK phosphorylation of the receptor.

  16. Muscarinic acetylcholine receptor compounds alter net Ca2+ flux and contractility in an invertebrate smooth muscle.

    PubMed

    Devlin, C Leah; Amole, William; Anderson, Shawn; Shea, Kyle

    2003-11-01

    Responses of a holothurian smooth muscle to a range of muscarinic (M(1) to M(5)) acetylcholine receptor (mAChR) agonists and antagonists were surveyed using calcium (Ca(2+))-selective electrodes and a mechanical recording technique. Most of the mAChR agonists and antagonists tested increased both contractility and net Ca(2+) efflux, with M(1)-specific agents like oxotremorine M being the most potent in their action. To investigate the possible sources of Ca(2+) used during mAChR activation, agents that disrupt intracellular Ca(2+) ion sequestration [cyclopiazonic acid (CPA), caffeine, ryanodine], the phosphoinositide signaling pathway [lithium chloride (LiCl)], and L-type Ca(2+) channels (diltiazem and verapamil) were used to challenge contractions induced by oxotremorine M. These contractions were blocked by treatment with CPA, caffeine, LiCl, and by channel blockers, diltiazem and verapamil, but were unaltered by ryanodine. Our data suggest that this smooth muscle had an M(1,3,5)-like receptor that was associated with the phosphoinositide signaling pathway that relied on intracellular Ca(2+) stores, but secondarily used extracellular Ca(2+) via the opening of L-type channels.

  17. [Lesions in the pars compacta substantiae nigra and the subthalamic nucleus modify the density of muscarinic receptors in different nuclei of the basal ganglia].

    PubMed

    Blanco-Lezcano, L; Rocha-Arrieta, L L; Martínez-Martí, L; Alvarez-González, L; Pavón-Fuentes, N; Macías-González, R; Serrano-Sánchez, T; Rosillo-Martí, J C; Coro-Grave de Peralta, Y; Bauza-Calderín, Y; Briones, M

    Several studies that has focused to the dopaminergic transmission in the basal ganglia in parkinsonian condition, but only a few article has taking into account the imbalance between dopaminergic and cholinergic transmission. To evaluate the muscarinic cholinergic receptors density in SNc and PPN in the 6-OHDA model. Were organized five experimental groups in correspondence to the place of the lesion: I. Non treated rats, II. 6-OHDA lesion in SNc, III. 6-OHDA lesion in SNc + quinolinic acid lesion in NST, IV. Sham operated rats, V. Quinolinic acid in STN. Were obtained coronal sections of 20 microm thickness of SNc and PPN from rats and in these sections was evaluated the muscarinic receptors density through autoradiographic technique with [3H]quinuclidinylbenzilate (QNB) (1.23 nM). The muscarinic antagonist atropine (1 microM) was utilized as non-specific union. The density was evaluated in both hemispheres and the density optical was converted in fentomolas/mg of tissue with base to values obtained from tritium standards. Significant diminution of the muscarinic receptors density was found in the SNc ipsilateral to the 6-OHDA lesion from experimental groups II (t=2.76; p<0.05) and III (t=4.06; p<0.05). In the group V, was seen a significant increase of muscarinic receptor density in the SNc ipsilateral to the 6-OHDA lesion. The comparison between experimental groups evidenced significant differences among them (F=13.13; p<0.001) with a significant decrease in the density from SNc of groups II and III and significant increase in the density from SNc of group V in comparison of the others groups. In relation to PPN, muscarinic receptors density from right PPN ipsilateral to the 6-OHDA lesion, shown significant differences (F=3.93; p<0.01) between the experimental groups with a significant increase of this variable in the group II. These results signal a modification of cholinergic activity after 6-OHDA lesion. The changes in the muscarinic receptors populations

  18. Pyridophens: binary pyridostigmine-aprophen prodrugs with differential inhibition of acetylcholinesterase, butyrylcholinesterase, and muscarinic receptors.

    PubMed

    Leader, Haim; Wolfe, Alan David; Chiang, Peter K; Gordon, Richard K

    2002-02-14

    A series of "binary prodrugs" called carbaphens,(1) carbamylated derivatives on one or both of the aromatic rings of the muscarinic receptor antagonist aprophen [(N,N-diethylamino)ethyl 2,2-diphenylpropionate], were synthesized to develop binary prophylactic agents against organophosphorus intoxication. As a group, the carbaphens retained the muscarinic receptor antagonist properties of aprophen but also preferentially inhibited butyrylcholinesterase (BChE) in contrast to acetylcholinesterase (AChE). Therefore, a new series of compounds named pyridophens were designed and synthesized to achieve binary prodrugs to preferentially inhibit AChE over BChE, while still retaining the muscarinic receptor antagonism of aprophen. The pyridophens consist of the basic pyridostigmine skeleton combined with the 2,2-diphenylpropionate portion of aprophen by replacement of the diethylamino group. Three compounds, 9 (a tertiary pyridine), 10 (a quaternary pyridine), and 12 (a tertiary tetrahydropyridine), were found to be effective inhibitors of both BChE and AChE. However, 10, N-methyl-3-[[(dimethylamino)carbonyl]oxy]-2-(2'2'-diphenylpropionoxy-methyl)pyridinium iodide, inhibited AChE selectively over BChE, with a bimolecular rate constant similar to pyridostigmine. In contrast to their potent cholinesterase inhibitory activity, all of the pyridophen analogues were less potent antagonists of the muscarinic receptor than aprophen.

  19. Mixed nicotinic and muscarinic features of cholinergic receptor coupled to secretion in bovine chromaffin cells

    SciTech Connect

    Shirvan, M.H.; Pollard, H.B.; Heldman, E. )

    1991-06-01

    Acetylcholine evokes release from cultured bovine chromaffin cells by a mechanism that is believed to be classically nicotinic. However, the authors found that the full muscarinic agonist oxotremorine-M (Oxo-M) induced a robust catecholamine (CA) secretion. By contrast, muscarine, pilocarpine, bethanechol, and McN-A-343 did not elicit any secretory response. Desensitization of the response to nicotine by Oxo-M and desensitization of the response to Oxo-M by nicotine suggest that both nicotine and Oxo-M were acting at the same receptor. Additional experiments supporting this conclusion show that nicotine-induced secretion and Oxo-M-induced secretion were similarly blocked by various muscarinic and nicotinic antagonists. Moreover, secretion induced by nicotine and Oxo-M were Ca{sup 2+} dependent, and both agonists induced {sup 45}Ca{sup 2+} uptake. Equilibrium binding studies showed that ({sup 3}H)Oxo-M bound to chromaffin cell membranes with a K{sub d} value of 3.08 {times} 10{sup {minus}8}M and a Hill coefficient of 1.00, suggesting one binding site for this ligand. Nicotine inhibited Oxo-M binding in a noncompetitive manner, suggesting that both ligands bind at two different sites on the same receptor. They propose that the receptor on bovine chromaffin cells that is coupled to secretion represents an unusual cholinergic receptor that has both nicotinic and muscarinic features.

  20. Quantitative autoradiography of muscarinic and benzodiazepine receptors in the forebrain of the turtle, Pseudemys scripta

    SciTech Connect

    Schlegel, J.R.; Kriegstein, A.R.

    1987-11-22

    The distribution of muscarinic and benzodiazepine receptors was investigated in the turtle forebrain by the technique of in vitro receptor autoradiography. Muscarinic binding sites were labeled with 1 nM /sup 3/H-quinuclidinyl benzilate (/sup 3/H-QNB), and benzodiazepine sites were demonstrated with the aid of 1 nM /sup 3/H-flunitrazepam (/sup 3/H-FLU). Autoradiograms generated on /sup 3/H-Ultrofilm apposed to tissue slices revealed regionally specific distributions of muscarinic and benzodiazepine binding sites that are comparable with those for mammalian brain. Dense benzodiazepine binding was found in the anterior olfactory nucleus, the lateral and dorsal cortices, and the dorsal ventricular ridge (DVR), a structure with no clear mammalian homologue. Muscarinic binding sites were most dense in the striatum, accumbens, DVR, lateral geniculate, and the anterior olfactory nucleus. Cortical binding sites were studied in greater detail by quantitative analysis of autoradiograms generated by using emulsion-coated coverslips. Laminar gradients of binding were observed that were specific for each radioligand; /sup 3/H-QNB sites were most dense in the inner molecular layer in all cortical regions, whereas /sup 3/H-FLU binding was generally most concentrated in the outer molecular layer and was least dense through all layers in the dorsomedial cortex. Because pyramidal cells are arranged in register in turtle cortex, the laminar patterns of receptor binding may reflect different receptor density gradients along pyramidal cell dendrites.

  1. Characterization of muscarinic receptors on isolated swine tracheal submucosal gland cells

    SciTech Connect

    Yang, C.M.; Dwyer, T.M.; Farley, J.M.

    1986-03-05

    Muscarinic receptors play an important role in the regulation of tracheobronchial secretion. Tracheal epithelium was cut into small pieces (approx.10 mm/sup 2/) and dissociated using collagenase in HEPES-Ringer solution at 37/sup 0/C. After dissociation the glands cells were isolated by discontinuous Percoll density gradient centrifugation. Submucosal gland cells concentrated above the layers with densities of 1.084 and 1.057 g/ml after centrifugation at x 500 g for 10 min at 15/sup 0/C. Cell viability was > 95% as determined by exclusion of trypan blue. Over 98% of the isolated cells were identified by periodic acid Schiff staining method to be gland cells. Muscarinic receptors on intact gland cells were characterized using the binding of specific muscarinic antagonist (/sup 3/H)quinuclidinyl benzilate ((/sup 3/H)QNB) binding. Scatchard plot analysis of saturation isotherms, showed that the maximal receptor density (B/sub max/) and dissociation constant (K/sub D/) were 7400 +/- 200 sites/cell and 100 +/- 20 pM, respectively (n = 3). These two parameters were less than those from cat tracheal gland cells, B/sub max/ = 42,000 sites/cell and K/sub D/ = 200 pM. In conclusion, this study provided a useful method to isolate tracheal gland cells and characterized the presence of muscarinic receptors on isolated intact cells.

  2. US -adrenergic and muscarinic receptor densities of rat submandibular main duct

    SciTech Connect

    Schneyer, C.A.; Humphreys-Beher, M.G.

    1987-05-01

    (TH)DHA binding studies show that main duct of rat submandibular has both US 1 and US 2 adrenoceptors, with the percentages of each being 69 and 31%, respectively, whereas whole submandibular gland has 90% sigma1 and 10% US 2 adrenoceptors. Muscarinic receptors of main duct are 25% less than that of whole submandibular gland.

  3. Beneficial effect of muscarinic-2 antagonist on dilated cardiomyopathy induced by autoimmune mechanism against muscarinic-2 receptor.

    PubMed

    Matsui, S; Fu, M L; Hayase, M; Katsuda, S; Yamaguchi, N; Teraoka, K; Kurihara, T; Takekoshi, N

    2001-10-01

    We have previously shown that a peptide corresponding to the sequence of the second extracellular loop of the human muscarinic-2 (M2) receptor (M2-peptide) was able to induce an autoimmune cardiomyopathy in rabbits. In this study, we investigated the effect of M2-antagonist (otenzepad) on M2-peptide-induced cardiomyopathy in rabbits. New Zealand White rabbits were divided into four groups: 1) control group, saline injection; 2) M2-peptide group, M2-peptide injection; 3) M2-antagonist group, otenzepad (30 mg/day) orally and saline injection; and (4) M2-antagonist + M2-peptide group, otenzepad (30 mg/day) orally and M2-peptide injection. The study duration was 1 year. Saline or peptide was injected once a month. All rabbits in both the M2-peptide group and the M2-antagonist + M2-peptide group had high titers of anti-M2-autoantibodies in their sera. Rabbits in the M2-peptide group showed an increase in heart weight, wall thinning and dilatation of the right ventricle. On the contrary, rabbits in the M2-antagonist + M2-peptide group had normal heart weight and shape. All rabbits in the M2-peptide group showed multifocal degeneration and necrosis of myocardial cells with moderate infiltration of inflammatory cells, while four rabbits in the M2-antagonist + M2-peptide group showed slight infiltration of inflammatory cells with normal myocardial cells and interstitium, and another three showed no histological changes in the hearts. In conclusion, M2-antagonist protects the myocardium from injury induced by autoimmune mechanism against M2-muscarinic receptor.

  4. Effect of paraoxon on muscarinic, dopamine and. gamma. -aminobutyric acid receptors of brain and sensitivity to muscarinic antagonists

    SciTech Connect

    Fernando, J.C.R.; Hoskins, B.; Ho, I.K.

    1986-03-05

    Several acetylcholinesterase (AChE) inhibitors decrease muscarinic cholinergic (mACh) receptors in the brain, alteration of dopamine (DA) and ..gamma..-aminobutyric acid (GABA) receptors after AChE inhibition was also reported. In view of the important interactions among DA, GABA and ACh systems, whether this is a common effect of AChE inhibitors should be established. They report the effect of the AChE inhibitor, paraoxon, on DA, GABA and mACh receptors in the rat. The binding of /sup 3/H-QNB (for mACh), /sup 3/H-spiperone (for DA) and /sup 3/H-muscimol (for GABA) to striatal and hippocampal membranes was analyzed. Also, behavioral sensitivity to atropine was studied. Twenty-four hr after a single dose (0.75 mg/kg, s.c.) of paraoxon, the density of mACh receptors in the striatum was decreased but, at 3 days, no change was seen. In the hippocampus, the mACh receptors were not affected. Repeated treatment with paraoxon (0.3 mg/kg, 48 hourly) for 2 weeks reduced the mACh receptor density in both regions. Neither single nor repeated paraoxon treatment had an effect on DA or GABA receptors. After single or repeated dosing with paraoxon, myoclonus induced by atropine (10 mg/kg, i.p.) was enhanced. The results show rapid downregulation of mACh receptors by paraoxon. DA or GABA, however, appear not to be affected under these treatment regimens.

  5. Muscarinic cholinergic receptors of B lymphocytes during the immune response in mice

    SciTech Connect

    Ado, A.D.; Gol'dshtein, M.M.; Kravchenko, S.A.; Fominova, T.I.

    1986-10-01

    The effect of a specific antigen on expression of muscarinic cholinergic (MC) receptors on the surface of splenic B lymphocytes of mice after their immunization was studied. To determine the number of MC receptors on the B lymphocytes, /sup 3/H-quinuclidinyl benzilate with specific radioactivity of 36 Ci/mmole, a specific blocker of these receptors, was used. The degree of specific binding was determined and the number of receptors on the surface of a B lymphocyte was calculated. The results are obtained are evidence of the possibility of stearic interaction between MC receptors and immunoglobulin receptors binding specific antigen on B lymphocytes during the immune response.

  6. Pharmacological characterization of muscarinic receptor subtypes mediating vasoconstriction of human umbilical vein

    PubMed Central

    Pujol Lereis, Virginia Andrea; Hita, Francisco Javier; Gobbi, Mauro Darío; Verdi, Marcela Gomez; Rodriguez, María Cecilia; Rothlin, Rodolfo Pedro

    2006-01-01

    The present study attempted to pharmacologically characterize the muscarinic receptor subtypes mediating contraction of human umbilical vein (HUV). HUV rings were mounted in organ baths and concentration–response curves were constructed for acetylcholine (ACh) (pEC50: 6.16±0.04; maximum response 80.00±1.98% of the responses induced by serotonin 10 μM). The absence of endothelium did not modify the contractile responses of ACh in this tissue. The role of cholinesterases was evaluated: neither neostigmine (acetylcholinesterase inhibitor) nor iso-OMPA (butyrylcholinesterase inhibitor) modified ACh responses. When both enzymes were simultaneously inhibited, a significantly but little potentiation was observed (control: pEC50 6.33±0.03; double inhibition: pEC50 6.57±0.05). Atropine, nonselective muscarinic receptors antagonist, inhibited ACh-induced contraction (pKB 9.67). The muscarinic receptors antagonists pirenzepine (M1), methoctramine (M2) and pFHHSiD (M3) also antagonized responses to ACh. The affinity values estimated for these antagonists against responses evoked by ACh were 7.58, 6.78 and 7.94, respectively. On the other hand, PD 102807 (M4 selective muscarinic receptors antagonist) was ineffective against ACh-induced contraction. In presence of a blocking concentration of pirenzepine, pFHHSiFD produced an additional antagonism activity on ACh-induced responses. The M1 muscarinic receptors agonist McN-A-343 produced similar maximum but less potent responses than ACh in HUV. The calculated pA2 for pirenzepine against McN-A-343 induced responses was 8.54. In conclusion, the data obtained in this study demonstrate the role of M1 muscarinic receptor subtypes and suggest the involvement of M3 muscarinic receptor subtypes in ACh-induced vasoconstriction in HUV rings. In addition, the vasomotor activity evoked by ACh does not seem to be modulated by endothelial factors, and their enzymatic degradation appears to have little functional relevance in this

  7. Binding and functional properties of hexocyclium and sila-hexocyclium derivatives to muscarinic receptor subtypes.

    PubMed Central

    Waelbroeck, M.; Camus, J.; Tastenoy, M.; Feifel, R.; Mutschler, E.; Tacke, R.; Strohmann, C.; Rafeiner, K.; Rodrigues de Miranda, J. F.; Lambrecht, G.

    1994-01-01

    1. We have compared the binding properties of several hexocyclium and sila-hexocyclium derivatives to muscarinic M1 receptors (in rat brain, human neuroblastoma (NB-OK 1) cells and calf superior cervical ganglia), rat heart M2 receptors, rat pancreas M3 receptors and M4 receptors in rat striatum, with their functional antimuscarinic properties in rabbit vas deferens (M1/M4-like), guinea-pig atria (M2), and guinea-pig ileum (M3) muscarinic receptors. 2. Sila-substitution (C/Si exchange) of hexocyclium (-->sila-hexocyclium) and demethyl-hexocyclium (-->demethyl-sila-hexocyclium) did not significantly affect their affinities for muscarinic receptors. By contrast, sila-substitution of o-methoxy-hexocyclium increased its affinity 2 to 3 fold for all the muscarinic receptor subtypes studied. 3. The p-fluoro- and p-chloro-derivatives of sila-hexocyclium had lower affinities than the parent compound at the four receptor subtypes, in binding and pharmacological studies. 4. In binding studies, o-methoxy-sila-hexocyclium (M1 = M4 > or = M3 > or = M2) had a much lower affinity than sila-hexocyclium for the four receptor subtypes, and discriminated the receptor subtypes more poorly than sila-hexocyclium (M1 = M3 > M4 > M2). This is in marked contrast with the very clear selectivity of o-methoxy-sila-hexocyclium for the prejunctional M1/M4-like heteroreceptors in rabbit vas deferens. 5. The tertiary amines demethyl-hexocyclium, demethyl-sila-hexocyclium and demethyl-o-methoxy-sila-hexocyclium had 10 to 30 fold lower affinities than the corresponding quaternary ammonium derivatives. PMID:8075869

  8. Cloned M1 muscarinic receptors mediate both adenylate cyclase inhibition and phosphoinositide turnover.

    PubMed Central

    Stein, R; Pinkas-Kramarski, R; Sokolovsky, M

    1988-01-01

    The rat M1 muscarinic receptor gene was cloned and expressed in a rat cell line lacking endogenous muscarinic receptors. Assignment of the cloned receptors to the M1 class was pharmacologically confirmed by their high affinity for the M1-selective muscarinic antagonist pirenzepine and low affinity for the M2-selective antagonist AF-DX-116. Guanylyl imidodiphosphate [Gpp(NH)p] converted agonist binding sites on the receptor, from high-affinity to the low-affinity state, thus indicating that the cloned receptors couple to endogenous G-proteins. The cloned receptors mediated both adenylate cyclase inhibition and phosphoinositide hydrolysis, but by different mechanisms. Pertussis toxin blocked the inhibition of adenylate cyclase (indicating coupling of the receptor to inhibitory G-protein), but did not affect phosphoinositide turnover. Furthermore, the stimulation of phosphoinositide hydrolysis was less efficient than the inhibition of adenylate cyclase. These findings demonstrate that cloned M1 receptors are capable of mediating multiple responses in the cell by coupling to different effectors, possibly to different G-proteins. Images PMID:2846274

  9. Graded activation and free energy landscapes of a muscarinic G-protein-coupled receptor.

    PubMed

    Miao, Yinglong; McCammon, J Andrew

    2016-10-25

    G-protein-coupled receptors (GPCRs) recognize ligands of widely different efficacies, from inverse to partial and full agonists, which transduce cellular signals at differentiated levels. However, the mechanism of such graded activation remains unclear. Using the Gaussian accelerated molecular dynamics (GaMD) method that enables both unconstrained enhanced sampling and free energy calculation, we have performed extensive GaMD simulations (∼19 μs in total) to investigate structural dynamics of the M2 muscarinic GPCR that is bound by the full agonist iperoxo (IXO), the partial agonist arecoline (ARC), and the inverse agonist 3-quinuclidinyl-benzilate (QNB), in the presence or absence of the G-protein mimetic nanobody. In the receptor-nanobody complex, IXO binding leads to higher fluctuations in the protein-coupling interface than ARC, especially in the receptor transmembrane helix 5 (TM5), TM6, and TM7 intracellular domains that are essential elements for GPCR activation, but less flexibility in the receptor extracellular region due to stronger binding compared with ARC. Two different binding poses are revealed for ARC in the orthosteric pocket. Removal of the nanobody leads to GPCR deactivation that is characterized by inward movement of the TM6 intracellular end. Distinct low-energy intermediate conformational states are identified for the IXO- and ARC-bound M2 receptor. Both dissociation and binding of an orthosteric ligand are observed in a single all-atom GPCR simulation in the case of partial agonist ARC binding to the M2 receptor. This study demonstrates the applicability of GaMD for exploring free energy landscapes of large biomolecules and the simulations provide important insights into the GPCR functional mechanism.

  10. Rate constants of agonist binding to muscarinic receptors in rat brain medulla. Evaluation by competition kinetics

    SciTech Connect

    Schreiber, G.; Henis, Y.I.; Sokolovsky, M.

    1985-07-25

    The method of competition kinetics, which measures the binding kinetics of an unlabeled ligand through its effect on the binding kinetics of a labeled ligand, was employed to investigate the kinetics of muscarinic agonist binding to rat brain medulla pons homogenates. The agonists studied were acetylcholine, carbamylcholine, and oxotremorine, with N-methyl-4-(TH)piperidyl benzilate employed as the radiolabeled ligand. Our results suggested that the binding of muscarinic agonists to the high affinity sites is characterized by dissociation rate constants higher by 2 orders of magnitude than those of antagonists, with rather similar association rate constants. Our findings also suggest that isomerization of the muscarinic receptors following ligand binding is significant in the case of antagonists, but not of agonists. Moreover, it is demonstrated that in the medulla pons preparation, agonist-induced interconversion between high and low affinity bindings sites does not occur to an appreciable extent.

  11. Muscarinic receptor subtypes mediating the mucosal response to neural stimulation of guinea pig ileum

    SciTech Connect

    Carey, H.V.; Tien, X.Y.; Wallace, L.J.; Cooke, H.J.

    1987-09-01

    Muscarinic receptors involved in the secretory response evoked by electrical stimulation of submucosal neutrons were investigated in muscle-stripped flat sheets of guinea pig ileum set up in flux chambers. Neural stimulation produced a biphasic increase in short-circuit current due to active chloride secretion. Atropine and 4-diphenylacetoxy-N-methylpiperadine methiodide (4-DAMP) (10/sup -7/ M) were more potent inhibitors of the cholinergic phase of the response than was pirenzepine. Dose-dependent increases in base-line short-circuit current were evoked by carbachol and bethanechol; 4-hydroxy-2-butynyl trimethylammonium chloride (McN A343) produced a much smaller effect. Tetrodotoxin abolished the effects of McN A343 but did not alter the responses of carbachol and bethanechol. McN A343 significantly reduced the cholinergic phase of the neurally evoked response and caused a rightward shift of the carbachol dose-response curve. All muscarinic compounds inhibited (/sup 3/H)quinuclidinyl benzilate binding to membranes from muscosal scrapings, with a rank order of potency of 4-DAMP > pirenzepine > McN A343 > carbachol > bethanechol. These results suggest that acetylcholine released from submucosal neurons mediates chloride secretion by interacting with muscarinic cholinergic receptors that display a high binding affinity for 4-DAMP. Activation of neural muscarinic receptors makes a relatively small contribution to the overall secretory response.

  12. Inactivation of muscarinic receptors impairs place and response learning: implications for multiple memory systems.

    PubMed

    Soares, Juliana Carlota Kramer; Oliveira, Maria Gabriela Menezes; Ferreira, Tatiana Lima

    2013-10-01

    Extensive research has shown that the hippocampus and striatum have dissociable roles in memory and are necessary for place and response learning, respectively. Additional evidence indicates that muscarinic cholinergic receptors in the hippocampus and striatum exert an important role in the modulation of these memory systems. In our experiments, we assessed whether intact hippocampal and striatal muscarinic cholinergic transmission may be essential and/or necessary for place and response learning. We addressed these questions using administration of the muscarinic receptor antagonist, scopolamine, on both place and response learning in a food-rewarded T-maze task. The administration of scopolamine (15 μg or 30 μg) directly into the dorsal hippocampus impaired the performance of rats subjected to both place and cue-rich response version of the task, but did not affect the response version, when the task was performed under cue-poor conditions. However, the administration of scopolamine in the dorsolateral striatum impaired the cue-poor response version of the T-maze task without interfering with the place version or cue-rich response version. Taken together, these results indicate that activation of muscarinic cholinergic receptors in the hippocampus and striatum facilitate the use of different strategies of learning, thus strengthening the hypothesis of multiple memory systems. Additionally, these results emphasize the importance of the environmental conditions under which tasks are performed.

  13. Role of Muscarinic Acetylcholine Receptor-2 in the Cerebellar Cortex in Cardiovascular Modulation in Anaesthetized Rats.

    PubMed

    Zhang, Changzheng; Sun, Tingzhe; Zhou, Peiling; Zhu, Qingfeng; Zhang, Liefeng

    2016-04-01

    Our previous investigations have demonstrated that microinjection of acetylcholine (ACh) or muscarinic ACh receptor activation in the cerebellar cortex induces a systemic blood pressure depressor response. This study aimed to determine the role of muscarinic ACh receptor-2 (M2 receptor) in the cerebellar cortex in cardiovascular function regulation in rats. A nonselective muscarinic receptor agonist (oxotremorine M, OXO; 30 mM), a selective M2 receptor agonist (arecaidine but-2-ynyl ester tosylate, ABET; 3, 10, and 30 mM), 30 mM OXO mixed with a selective M2 receptor antagonist (methoctramine hydrate, MCT; 0.3, 1, and 3 mM), and normal saline (0.9 % NaCl) were separately microinjected (0.5 µl/5 s) into the cerebellar cortex (lobule VI) of anaesthetized rats. We measured the mean arterial pressure (MAP), maximum change in MAP, and reactive time (RT; the duration required for the blood pressure to return to basal levels), heart rate (HR) and the maximum change in HR during the RT in response to drug activation. The results demonstrated that ABET dose-dependently decreased MAP and HR, increased the maximum change in MAP and the maximum change in HR, and prolonged the RT. Furthermore, MCT dose-dependently blocked the OXO-mediated cardiovascular depressor response. This study provides the first evidence that M2 receptors in the cerebellar cortex are involved in cardiovascular regulation, the activation of which evokes significant depressor and bradycardic responses.

  14. Muscarinic receptor/G-protein coupling is reduced in the dorsomedial striatum of cognitively impaired aged rats

    PubMed Central

    Nieves-Martinez, E.; Hayes, Katy; Childers, S.R.; Sonntag, W.E.; Nicolle, M. M.

    2011-01-01

    Behavioral flexibility, the ability to modify responses due to changing task demands, is detrimentally affected by aging with a shift towards increased cognitive rigidity. The neurobiological basis of this cognitive deficit is not clear although striatal cholinergic neurotransmission has been implicated. To investigate the possible association between striatal acetylcholine signaling with age-related changes in behavioral flexibility, young, middle-aged, and aged F344 X Brown Norway F1 rats were assessed using an attentional set-shifting task that includes two tests of behavioral flexibility: reversal learning and an extra-dimensional shift. Rats were also assessed in the Morris water maze to compare potential fronto-striatal-dependent deficits with hippocampal-dependent deficits. Behaviorally characterized rats were then assessed for acetylcholine muscarinic signaling within the striatum using oxotremorine-M-stimulated [35S]GTPγS binding and [3H]AFDX-384 receptor binding autoradiography. The results showed that by old age, cognitive deficits were pronounced across cognitive domains, suggesting deterioration of both hippocampal and fronto-striatal regions. A significant decline in oxotremorine-M-stimulated [35S]GTPγS binding was limited to the dorsomedial striatum of aged rats when compared to young and middle-aged rats. There was no effect of age on striatal [3H]AFDX-384 receptor binding. These results suggest that a decrease in M2/M4 muscarinic receptor coupling is involved in the age-associated decline in behavioral flexibility. PMID:22085876

  15. Expression of the human muscarinic receptor gene m2 in Dictyostelium discoideum

    SciTech Connect

    Voith, G.; Dingermann, T.

    1995-11-01

    We have expressed a functional human muscarinic M2 receptor, under the control of the homologous discoidin I{gamma} promoter, in the cellular slime mold Dictyostelium discoideum. The use of a contact site A leader peptide ensured insertion of the newly synthesized receptor protein into the plasma membrane. Due to the characteristics of the discoidin I{gamma} promoter, the M2 receptor is expressed during late growth and early development. The heterologously expressed M2 receptors show binding characteristics similar to authentic receptors. Membranes as well as whole cells can be used in ligand binding assays. 36 refs., 4 figs.

  16. Differential alterations in muscarinic receptor subtypes in Alzheimer's disease: implications for cholinergic-based therapies.

    PubMed

    Flynn, D D; Ferrari-DiLeo, G; Levey, A I; Mash, D C

    1995-01-01

    Molecular subtypes of muscarinic receptors (m1-m5) are novel targets for cholinergic replacement therapies in Alzheimer's disease (AD). However, knowledge concerning the relative distribution, abundance and functional status of these receptors in human brain and AD is incomplete. Recent data from our laboratory have demonstrated a defect in the ability of the M1 receptor subtype to form a high affinity agonist-receptor-G protein complex in AD frontal cortex. This defect is manifested by decreased M1 receptor-stimulated GTPgammaS binding and GTPase activity and by a loss in receptor-stimulated phospholipase C activity. Normal levels of G proteins suggest that the aberrant receptor-G protein interaction may result from an altered form of the m1 receptor in AD. The combined use of radioligand binding and receptor-domain specific antibodies has permitted the re-examination of the status of muscarinic receptor subtypes in the human brain. In AD, normal levels of m1 receptor [3H]-pirenzepine binding contrasted with diminished m1 immunoreactivity, further suggesting that there is an altered form of the m1 receptor in the disease. Reduced m2 immunoreactivity was consistent with decreased numbers of m2 binding sites. Increased levels of m4 receptors were observed in both binding and immunoreactivity measurements. These findings suggest one possible explanation for the relative ineffectiveness of cholinergic replacement therapies used to date and suggest potential new directions for development of effective therapeutic strategies for AD.

  17. Graded activation and free energy landscapes of a muscarinic G-protein–coupled receptor

    PubMed Central

    Miao, Yinglong; McCammon, J. Andrew

    2016-01-01

    G-protein–coupled receptors (GPCRs) recognize ligands of widely different efficacies, from inverse to partial and full agonists, which transduce cellular signals at differentiated levels. However, the mechanism of such graded activation remains unclear. Using the Gaussian accelerated molecular dynamics (GaMD) method that enables both unconstrained enhanced sampling and free energy calculation, we have performed extensive GaMD simulations (∼19 μs in total) to investigate structural dynamics of the M2 muscarinic GPCR that is bound by the full agonist iperoxo (IXO), the partial agonist arecoline (ARC), and the inverse agonist 3-quinuclidinyl-benzilate (QNB), in the presence or absence of the G-protein mimetic nanobody. In the receptor–nanobody complex, IXO binding leads to higher fluctuations in the protein-coupling interface than ARC, especially in the receptor transmembrane helix 5 (TM5), TM6, and TM7 intracellular domains that are essential elements for GPCR activation, but less flexibility in the receptor extracellular region due to stronger binding compared with ARC. Two different binding poses are revealed for ARC in the orthosteric pocket. Removal of the nanobody leads to GPCR deactivation that is characterized by inward movement of the TM6 intracellular end. Distinct low-energy intermediate conformational states are identified for the IXO- and ARC-bound M2 receptor. Both dissociation and binding of an orthosteric ligand are observed in a single all-atom GPCR simulation in the case of partial agonist ARC binding to the M2 receptor. This study demonstrates the applicability of GaMD for exploring free energy landscapes of large biomolecules and the simulations provide important insights into the GPCR functional mechanism. PMID:27791003

  18. Binding of N-methylscopolamine to the extracellular domain of muscarinic acetylcholine receptors

    PubMed Central

    Jakubík, Jan; Randáková, Alena; Zimčík, Pavel; El-Fakahany, Esam E.; Doležal, Vladimír

    2017-01-01

    Interaction of orthosteric ligands with extracellular domain was described at several aminergic G protein-coupled receptors, including muscarinic acetylcholine receptors. The orthosteric antagonists quinuclidinyl benzilate (QNB) and N-methylscopolamine (NMS) bind to the binding pocket of the muscarinic acetylcholine receptor formed by transmembrane α-helices. We show that high concentrations of either QNB or NMS slow down dissociation of their radiolabeled species from all five subtypes of muscarinic acetylcholine receptors, suggesting allosteric binding. The affinity of NMS at the allosteric site is in the micromolar range for all receptor subtypes. Using molecular modelling of the M2 receptor we found that E172 and E175 in the second extracellular loop and N419 in the third extracellular loop are involved in allosteric binding of NMS. Mutation of these amino acids to alanine decreased affinity of NMS for the allosteric binding site confirming results of molecular modelling. The allosteric binding site of NMS overlaps with the binding site of some allosteric, ectopic and bitopic ligands. Understanding of interactions of NMS at the allosteric binding site is essential for correct analysis of binding and action of these ligands. PMID:28091608

  19. Binding of N-methylscopolamine to the extracellular domain of muscarinic acetylcholine receptors

    NASA Astrophysics Data System (ADS)

    Jakubík, Jan; Randáková, Alena; Zimčík, Pavel; El-Fakahany, Esam E.; Doležal, Vladimír

    2017-01-01

    Interaction of orthosteric ligands with extracellular domain was described at several aminergic G protein-coupled receptors, including muscarinic acetylcholine receptors. The orthosteric antagonists quinuclidinyl benzilate (QNB) and N-methylscopolamine (NMS) bind to the binding pocket of the muscarinic acetylcholine receptor formed by transmembrane α-helices. We show that high concentrations of either QNB or NMS slow down dissociation of their radiolabeled species from all five subtypes of muscarinic acetylcholine receptors, suggesting allosteric binding. The affinity of NMS at the allosteric site is in the micromolar range for all receptor subtypes. Using molecular modelling of the M2 receptor we found that E172 and E175 in the second extracellular loop and N419 in the third extracellular loop are involved in allosteric binding of NMS. Mutation of these amino acids to alanine decreased affinity of NMS for the allosteric binding site confirming results of molecular modelling. The allosteric binding site of NMS overlaps with the binding site of some allosteric, ectopic and bitopic ligands. Understanding of interactions of NMS at the allosteric binding site is essential for correct analysis of binding and action of these ligands.

  20. Differential role of protein kinase C in desensitization of muscarinic receptor induced by phorbol esters and receptor agonists

    SciTech Connect

    Lai, Wi Sheung.

    1989-01-01

    PKC, a phorbol ester receptor, copurified with specific binding sites of ({sup 3}H)phorbol-12,13,-dibutyrate (({sup 3}H)PDBu). The specific binding of ({sup 3}H)PDBu to intact cells was saturable to a single class of binding sites. The PKC and phorbol ester receptors in N1E-115 cells can be down regulated by prolonged phorbol ester incubation. Phorbol 12-myristate 13-acetate (PMA) suppressed muscarinic receptor-mediated cyclic GMP response in a time-dependent and a concentration-dependent fashion and the suppressive effect of PMA could be attenuated by a protein kinase inhibitor, H-7, as well as by down-regulation of the PKC through long-term incubation with PDBu. Exposure of the cells to the muscarinic agonist carbamylcholine also desensitized subsequent CBC-mediated cyclic GMP response. However, pretreatment with carbamylcholine did not desensitize histamine-induced cyclic GMP formation while treatment with PMA suppressed this histamine-mediated response. Preincubation of the cells with CBC, but not with phorbol ester, resulted in down-regulation of muscarinic receptors. The loss of muscarinic receptors induced by agonist even occurred when the phosphoinositide hydrolysis response was suppressed.

  1. Muscarinic receptors are involved in LMM3 tumor cells proliferation and angiogenesis

    SciTech Connect

    Rimmaudo, Laura Elizabeth; Torre, Eulalia de la; Sacerdote de Lustig, Eugenia; Sales, Maria Elena . E-mail: mesales@2vias.com.ar

    2005-09-09

    Angiogenesis is a process of new blood vessel development from pre-existing vasculature and it plays an essential role in tumor growth and metastases. Here, we investigate the expression of muscarinic acetylcholine receptors (mAchR) and their participation in tumor cell proliferation and angiogenesis ability. Saturation binding assays with the tritiated muscarinic antagonist quinuclidinyl benzilate indicate that LMM3 cells derived from a murine mammary adenocarcinoma express a single class of functional mAchR. Competition binding assays with selective muscarinic antagonists indicate a predominance of M{sub 3} receptor subtype. The muscarinic agonist carbachol (CARB) stimulates LMM3 cell proliferation in a concentration dependent manner. The maximal effect induced by 10{sup -9} M CARB was totally blunted by atropine and by the selective M{sub 3} and M{sub 1} antagonists, para-fluoro hexahydro sila-difenidol (pf-HHSiD) and pirenzepine, respectively. In addition, pf-HHSiD completely blocked in vivo CARB-induced neovascular formation and vascular endothelial growth factor-A in LMM3 tumor cells. We can conclude that mAchR expressed in LMM3 mammary tumor cells positively regulate proliferation and angiogenesis required for tumor progression.

  2. Allosteric interaction of the neuromuscular blockers vecuronium and pancuronium with recombinant human muscarinic M2 receptors.

    PubMed

    Cembala, Thor M; Forde, Steven C O; Appadu, Balraj L; Lambert, David G

    2007-08-13

    Neuromuscular blocking drugs produce muscle weakness by interaction with nicotinic-acetylcholine receptors. Cardiovascular side effects have been reported. In this study the neuromuscular blocking drug vecuronium and the controls gallamine and pancuronium slowed the rate of atropine induced [(3)H]N-methylscopolamine dissociation from Chinese hamster ovary cells expressing recombinant human muscarinic M2 receptors K(off) values min(-1); vecuronium (125 nM), atropine 0.45+/-0.07+blocker 0.04+/-0.02; gallamine (21 nM), atropine 0.42+/-0.05+blocker 0.15+/-0.04; pancuronium(21 nM), atropine 0.36+/-0.03+blocker 0.03+/-0.01). These data indicate that vecuronium, gallamine and pancuronium interact with an allosteric site on the muscarinic M2 receptor (located on the heart) and this may explain some of their cardiac side effects.

  3. Autoantibodies against Muscarinic Receptors in Breast Cancer: Their Role in Tumor Angiogenesis

    PubMed Central

    Lombardi, María Gabriela; Negroni, María Pía; Pelegrina, Laura Tatiana; Castro, María Ester; Fiszman, Gabriel L.; Azar, María Eugenia; Morgado, Carlos Cresta; Sales, María Elena

    2013-01-01

    The presence of autoantibodies in cancer has become relevant in recent years. We demonstrated that autoantibodies purified from the sera of breast cancer patients activate muscarinic acetylcholine receptors in tumor cells. Immunoglobulin G (IgG) from breast cancer patients in T1N0Mx stage (tumor size≤2 cm, without lymph node metastasis) mimics the action of the muscarinic agonist carbachol stimulating MCF-7 cell proliferation, migration and invasion. Angiogenesis is a central step in tumor progression because it promotes tumor invasion and metastatic spread. Vascular endothelial growth factor-A (VEGF-A) is the main angiogenic mediator, and its levels have been correlated with poor prognosis in cancer. The aim of the present work was to investigate the effect of T1N0Mx-IgG on the expression of VEGF-A, and the in vivo neovascular response triggered by MCF-7 cells, via muscarinic receptor activation. We demonstrated that T1N0Mx-IgG (10−8 M) and carbachol (10−9 M) increased the constitutive expression of VEGF-A in tumor cells, effect that was reverted by the muscarinic antagonist atropine. We also observed that T1N0Mx-IgG and carbachol enhanced the neovascular response produced by MCF-7 cells in the skin of NUDE mice. The action of IgG or carbachol was reduced in the presence of atropine. In conclusion, T1N0Mx-IgG and carbachol may promote VEGF-A production and neovascularization induced by breast tumor cells via muscarinic receptors activation. These effects may be accelerating breast tumor progression. PMID:23460876

  4. Autoantibodies against muscarinic receptors in breast cancer: their role in tumor angiogenesis.

    PubMed

    Lombardi, María Gabriela; Negroni, María Pía; Pelegrina, Laura Tatiana; Castro, María Ester; Fiszman, Gabriel L; Azar, María Eugenia; Morgado, Carlos Cresta; Sales, María Elena

    2013-01-01

    The presence of autoantibodies in cancer has become relevant in recent years. We demonstrated that autoantibodies purified from the sera of breast cancer patients activate muscarinic acetylcholine receptors in tumor cells. Immunoglobulin G (IgG) from breast cancer patients in T1N0Mx stage (tumor size≤2 cm, without lymph node metastasis) mimics the action of the muscarinic agonist carbachol stimulating MCF-7 cell proliferation, migration and invasion. Angiogenesis is a central step in tumor progression because it promotes tumor invasion and metastatic spread. Vascular endothelial growth factor-A (VEGF-A) is the main angiogenic mediator, and its levels have been correlated with poor prognosis in cancer. The aim of the present work was to investigate the effect of T1N0Mx-IgG on the expression of VEGF-A, and the in vivo neovascular response triggered by MCF-7 cells, via muscarinic receptor activation. We demonstrated that T1N0Mx-IgG (10(-8) M) and carbachol (10(-9) M) increased the constitutive expression of VEGF-A in tumor cells, effect that was reverted by the muscarinic antagonist atropine. We also observed that T1N0Mx-IgG and carbachol enhanced the neovascular response produced by MCF-7 cells in the skin of NUDE mice. The action of IgG or carbachol was reduced in the presence of atropine. In conclusion, T1N0Mx-IgG and carbachol may promote VEGF-A production and neovascularization induced by breast tumor cells via muscarinic receptors activation. These effects may be accelerating breast tumor progression.

  5. Expression of muscarinic acetylcholine receptors M3 and M5 in osteoporosis.

    PubMed

    Kauschke, Vivien; Lips, Katrin Susanne; Heiss, Christian; Schnettler, Reinhard

    2014-05-27

    Cholinergic signaling via muscarinic acetylcholine receptors (mAChR) is known to influence various physiological functions. In bone, M3 mAChR and M5 mAChR were identified on the membrane of osteoblast-like cells. M3 mAChR seems to be particularly relevant for bone physiology, as signaling via this receptor was reported to increase bone formation and decrease bone resorption. Thus, in the present study we investigated the relative mRNA expression of M3 and M5 mAChR in bones of a rat osteoporosis model. Osteoporosis was induced in Sprague-Dawley rats by bilateral ovariectomy and additional feeding of a diet deficient in calcium, vitamins C, D2, D3, and phosphorus, and free of soy and phytoestrogen. After a period of 3, 12, and 14 months, relative mRNA expression of M3 mAChR and M5 mAChR was analyzed in the 11th thoracic vertebra by real-time RT-PCR. Relative mRNA expression of M3 mAChR was significantly reduced in bones of osteoporotic rats compared to sham operated animals that served as controls. Further, M3 mAChR mRNA expression was significantly down-regulated when comparing 14-month osteoporotic rats to 3-month osteoporotic rats. Relative M5 mAChR mRNA was expressed to a lesser extent than M3 mAChR and did not show significant differences in mRNA expression level between the experimental groups. M3 mAChR mRNA expression was reduced upon induction of osteoporosis and progression of disease was associated with further decrease of this receptor, indicating that M3 mAChR is involved in the development and regulation of osteoporosis.

  6. Expression of muscarinic acetylcholine receptors M3 and M5 in osteoporosis

    PubMed Central

    Kauschke, Vivien; Lips, Katrin Susanne; Heiss, Christian; Schnettler, Reinhard

    2014-01-01

    Background Cholinergic signaling via muscarinic acetylcholine receptors (mAChR) is known to influence various physiological functions. In bone, M3 mAChR and M5 mAChR were identified on the membrane of osteoblast-like cells. M3 mAChR seems to be particularly relevant for bone physiology, as signaling via this receptor was reported to increase bone formation and decrease bone resorption. Thus, in the present study we investigated the relative mRNA expression of M3 and M5 mAChR in bones of a rat osteoporosis model. Material/Methods Osteoporosis was induced in Sprague-Dawley rats by bilateral ovariectomy and additional feeding of a diet deficient in calcium, vitamins C, D2, D3, and phosphorus, and free of soy and phytoestrogen. After a period of 3, 12, and 14 months, relative mRNA expression of M3 mAChR and M5 mAChR was analyzed in the 11th thoracic vertebra by real-time RT-PCR. Results Relative mRNA expression of M3 mAChR was significantly reduced in bones of osteoporotic rats compared to sham operated animals that served as controls. Further, M3 mAChR mRNA expression was significantly down-regulated when comparing 14-month osteoporotic rats to 3-month osteoporotic rats. Relative M5 mAChR mRNA was expressed to a lesser extent than M3 mAChR and did not show significant differences in mRNA expression level between the experimental groups. Conclusions M3 mAChR mRNA expression was reduced upon induction of osteoporosis and progression of disease was associated with further decrease of this receptor, indicating that M3 mAChR is involved in the development and regulation of osteoporosis. PMID:24866457

  7. Inhibition of muscarinic receptor-induced proliferation of astroglial cells by ethanol: mechanisms and implications for the fetal alcohol syndrome.

    PubMed

    Costa, Lucio G; Guizzetti, Marina

    2002-12-01

    In utero exposure to ethanol is deleterious to fetal brain development. Children born with the fetal alcohol syndrome (FAS) display a number of abnormalities, the most significant of which are central nervous system (CNS) dysfunctions, such as microencephaly and mental retardation. An interaction of ethanol with glial cells, particularly astrocytes, has been suggested to contribute to the developmental neurotoxicity of this alcohol. At low concentrations (10-100 mM) ethanol inhibits the proliferation of astroglial cells in vitro, particularly when stimulated by acetycholine through muscarinic M3 receptors. Of the several signal transduction pathways activated by these receptors in astrocytes or astrocytoma cells, which are involved in mitogenic signaling, only some (e.g. protein kinase C (PKC) zeta, p70S6 kinase) appear to be targeted by ethanol at the same low concentrations which effectively inhibit proliferation. Inhibition of astroglial proliferation by ethanol may contribute to the microencephaly seen in FAS.

  8. Ozone-induced hyperresponsiveness and blockade of M2 muscarinic receptors by eosinophil major basic protein.

    PubMed

    Yost, B L; Gleich, G J; Fryer, A D

    1999-10-01

    Control of airway smooth muscle is provided by parasympathetic nerves that release acetylcholine onto M(3) muscarinic receptors. Acetylcholine release is limited by inhibitory M(2) muscarinic receptors. In antigen-challenged guinea pigs, hyperresponsiveness is due to blockade of neuronal M(2) receptors by eosinophil major basic protein (MBP). Because exposure of guinea pigs to ozone also causes M(2) dysfunction and airway hyperresponsiveness, the role of eosinophils in ozone-induced hyperresponsiveness was tested. Animals were exposed to filtered air or to 2 parts/million ozone for 4 h. Twenty-four hours later, the muscarinic agonist pilocarpine no longer inhibited vagally induced bronchoconstriction in ozone-exposed animals, indicating M(2) dysfunction. M(2) receptor function in ozone-exposed animals was protected by depletion of eosinophils with antibody to interleukin-5 and by pretreatment with antibody to guinea pig MBP. M(2) function was acutely restored by removal of MBP with heparin. Ozone-induced hyperreactivity was also prevented by antibody to MBP and was reversed by heparin. These data show that loss of neuronal M(2) receptor function after ozone is due to release of eosinophil MBP.

  9. Muscarinic M2 receptors in acetylcholine-isoproterenol functional antagonism in human isolated bronchus.

    PubMed

    Sarria, Benjamin; Naline, Emmanuel; Zhang, Yong; Cortijo, Julio; Molimard, Mathieu; Moreau, Joelle; Therond, Patrice; Advenier, Charles; Morcillo, Esteban J

    2002-11-01

    The muscarinic functional antagonism of isoproterenol relaxation and the contribution of muscarinic M2 receptors were examined in human isolated bronchus. In intact tissues, acetylcholine (ACh) precontraction decreased isoproterenol potency and maximal relaxation (-log EC50 shift = -1.49 +/- 0.16 and E(max) inhibition for 100 microM ACh = 30%) more than the same levels of histamine contraction. The M2 receptor-selective antagonist methoctramine (1 microM) reduced this antagonism in ACh- but not histamine-contracted tissues. Similar results were obtained for forskolin-induced relaxation. After selective inactivation of M3 receptors with 4-diphenylacetoxy-N-(2-chloroethyl)piperadine hydrochloric acid (30 nM), demonstrated by abolition of contractile and inositol phosphate responses to ACh, muscarinic recontractile responses were obtained in U-46619-precontracted tissues fully relaxed with isoproterenol. Methoctramine antagonized recontraction, with pK(B) (6.9) higher than in intact tissues (5.4), suggesting participation of M2 receptors. In M3-inactivated tissues, methoctramine augmented the isoproterenol relaxant potency in U-46619-contracted bronchus and reversed the ACh-induced inhibition of isoproterenol cAMP accumulation. These results indicate that M2 receptors cause indirect contraction of human bronchus by reversing sympathetically mediated relaxation and contribute to cholinergic functional antagonism.

  10. Muscarinic Acetylcholine Receptor M3 Modulates Odorant Receptor Activity via Inhibition of β-Arrestin-2 Recruitment

    PubMed Central

    Jiang, Yue; Li, Yun Rose; Tian, Huikai; Ma, Minghong; Matsunami, Hiroaki

    2015-01-01

    The olfactory system in rodents serves a critical function in social, reproductive, and survival behaviors. Processing of chemosensory signals in the brain is dynamically regulated in part by an animal's physiological state. We previously reported that type 3 muscarinic acetylcholine receptors (M3-Rs) physically interact with odorant receptors (ORs) to promote odor-induced responses in a heterologous expression system. However, it is not known how M3-Rs affect the ability of olfactory sensory neurons (OSNs) to respond to odors. Here, we show that an M3-R antagonist attenuates odor-induced responses in OSNs from wild-type, but not M3-R-null mice. Using a novel molecular assay, we demonstrate that the activation of M3-Rs inhibits the recruitment of β-arrestin-2 to ORs, resulting in a potentiation of odor-induced response in OSNs. These results suggest a role for acetylcholine in modulating olfactory processing at the initial stages of signal transduction in the olfactory system. PMID:25800153

  11. Muscarinic cholinergic receptor binding sites differentiated by their affinity for pirenzepine do not interconvert

    SciTech Connect

    Gil, D.W.; Wolfe, B.B.

    1986-05-01

    Although it has been suggested by many investigators that subtypes of muscarinic cholinergic receptors exist, physical studies of solubilized receptors have indicated that only a single molecular species may exist. To test the hypothesis that the putative muscarinic receptor subtypes in rat forebrain are interconvertible states of the same receptor, the selective antagonist pirenzepine (PZ) was used to protect muscarinic receptors from blockade by the irreversible muscarinic receptor antagonist propylbenzilylcholine mustard (PBCM). If interconversion of high (M1) and low (M2) affinity binding sites for PZ occurs, incubation of cerebral cortical membranes with PBCM in the presence of PZ should not alter the proportions of M1 and M2 binding sites that are unalkylated (i.e., protected). If, on the other hand, the binding sites are not interconvertible, PZ should be able to selectively protect M1 sites and alter the proportions of unalkylated M1 and M2 binding sites. In the absence of PZ, treatment of cerebral cortical membranes with 20 nM PBCM at 4 degrees C for 50 min resulted in a 69% reduction in the density of M1 binding sites and a 55% reduction in the density of M2 binding sites with no change in the equilibrium dissociation constants of the radioligands (/sup 3/H)quinuclidinyl benzilate or (/sup 3/H)PZ. The reasons for this somewhat selective effect of PBCM are not apparent. In radioligand binding experiments using cerebral cortical membranes, PZ inhibited the binding of (/sup 3/H)quinuclidinyl benzilate in a biphasic manner.

  12. Differentiation of muscarinic cholinergic receptor subtypes in human cortex and pons - Implications for anti-motion sickness therapy

    NASA Technical Reports Server (NTRS)

    Mccarthy, Bruce G.; Peroutka, Stephen J.

    1988-01-01

    Radioligand binding studies were used to analyze muscarinic cholinergic receptor subtypes in human cortex and pons. Muscarinic cholinergic receptors were labeled by H-3-quinuclidinyl benzilate (H-3-QNB). Scopolamine was equipotent in both brain regions and did not discriminate subtypes of H-3-QNB binding. By contrast, the M1 selective antagonist pirenzepine was approximately 33-fold more potent in human cortex than pons. Carbachol, a putative M2 selective agonist, was more than 100-fold more potent in human pons than cortex. These results demonstrate that the human pons contains a relatively large proportion of carbachol-sensitive muscarinic cholinergic receptors. Drugs targeted to this subpopulation of muscarinic cholinergic receptors may prove to be effective anti-motion sickness agents with less side effects than scopolamine.

  13. Antagonism of Muscarinic Acetylcholine Receptors Alters Synaptic ERK Phosphorylation in the Rat Forebrain.

    PubMed

    Mao, Li-Min; Wang, Henry H; Wang, John Q

    2016-12-28

    Acetylcholine (ACh) is a key transmitter in the mesocorticolimbic circuit. By interacting with muscarinic ACh receptors (mAChR) enriched in the circuit, ACh actively regulates various neuronal and synaptic activities. The extracellular signal-regulated kinase (ERK) is one of members of the mitogen-activated protein kinase family and is subject to the regulation by dopamine receptors, although the regulation of ERKs by limbic mAChRs is poorly understood. In this study, we investigated the role of mAChRs in the regulation of ERK phosphorylation (activation) in the mesocorticolimbic system of adult rat brains in vivo. We targeted a sub-pool of ERKs at synaptic sites. We found that a systemic injection of the mAChR antagonist scopolamine increased phosphorylation of synaptic ERKs in the striatum (caudate putamen and nucleus accumbens) and medial prefrontal cortex (mPFC). Increases in ERK phosphorylation in both forebrain regions were rapid and transient. Notably, pretreatment with a dopamine D1 receptor (D1R) antagonist SCH23390 blocked the scopolamine-stimulated ERK phosphorylation in these brain regions, while a dopamine D2 receptor antagonist eticlopride did not. Scopolamine and SCH23390 did not change the amount of total ERK proteins. These results demonstrate that mAChRs inhibit synaptic ERK phosphorylation in striatal and mPFC neurons under normal conditions. Blockade of this inhibitory mAChR tone leads to the upregulation of ERK phosphorylation likely through a mechanism involving the level of D1R activity.

  14. Probing biochemical mechanisms of action of muscarinic M3 receptor antagonists with label-free whole cell assays.

    PubMed

    Deng, Huayun; Wang, Chaoming; Su, Ming; Fang, Ye

    2012-10-02

    Binding kinetics of drugs is increasingly recognized to be important for their in vivo efficacy and safety profiles. However, little is known about the effect of drug binding kinetics on receptor signaling in native cells. Here we used label-free whole cell dynamic mass redistribution (DMR) assays under persistent and duration-controlled stimulation conditions to investigate the influence of the binding kinetics of four antagonists on the signaling of endogenous muscarinic M3 receptor in native HT-29 cells. Results showed that DMR assays under different conditions differentiated the biochemical mechanisms of action of distinct M3 antagonists. When co-stimulated with acetylcholine, tiotropium, a relatively slow binding antagonist, was found to selectively block the late signaling of the receptor, suggesting that acetylcholine attains its binding equilibrium faster than tiotropium does, thereby still being able to initiate its rapid response until the antagonist draws up and fully blocks the signaling. Furthermore, DMR assays under microfluidics allowed estimation of the residence times of these antagonists acting at the receptor in native cells, which were found to be the determining factor for the blockage efficiency of M3 receptor signaling under duration-controlled conditions. This study demonstrates that DMR assays can be used to elucidate the functional consequence of kinetics-driven antagonist occupancy in native cells.

  15. Antipsychotic-like effect of the muscarinic acetylcholine receptor agonist BuTAC in non-human primates.

    PubMed

    Andersen, Maibritt B; Croy, Carrie Hughes; Dencker, Ditte; Werge, Thomas; Bymaster, Frank P; Felder, Christian C; Fink-Jensen, Anders

    2015-01-01

    Cholinergic, muscarinic receptor agonists exhibit functional dopamine antagonism and muscarinic receptors have been suggested as possible future targets for the treatment of schizophrenia and drug abuse. The muscarinic ligand (5R,6R)-6-(3-butylthio-1,2,5-thiadiazol-4-yl)-1-azabicyclo[3.2.1]octane (BuTAC) exhibits high affinity for muscarinic receptors with no or substantially less affinity for a large number of other receptors and binding sites, including the dopamine receptors and the dopamine transporter. In the present study, we wanted to examine the possible antipsychotic-like effects of BuTAC in primates. To this end, we investigated the effects of BuTAC on d-amphetamine-induced behaviour in antipsychotic-naive Cebus paella monkeys. Possible adverse events of BuTAC, were evaluated in the same monkeys as well as in monkeys sensitized to antipsychotic-induced extrapyramidal side effects. The present data suggests that, the muscarinic receptor ligand BuTAC exhibits antipsychotic-like behaviour in primates. The behavioural data of BuTAC as well as the new biochemical data further substantiate the rationale for the use of muscarinic M1/M2/M4-preferring receptor agonists as novel pharmacological tools in the treatment of schizophrenia.

  16. Investigation of the presence and antinociceptive function of muscarinic acetylcholine receptors in the African naked mole-rat (Heterocephalus glaber).

    PubMed

    Jørgensen, Kristine B; Krogh-Jensen, Karen; Pickering, Darryl S; Kanui, Titus I; Abelson, Klas S P

    2016-01-01

    The present study investigated the cholinergic system in the African naked mole-rat (Heterocephalus glaber) with focus on the muscarinic acetylcholine receptor subtypes M1 and M4. The protein sequences for the subtypes m 1-5 of the naked mole-rat were compared to that of the house mouse (Mus musculus) using basic local alignment search tool (BLAST). The presence and function of M1 and M4 was investigated in vivo, using the formalin test with the muscarinic receptor agonists xanomeline and VU0152100. Spinal cord tissue from the naked mole-rat was used for receptor saturation binding studies with [(3)H]-N-methylscopolamine. The BLAST test revealed 95 % protein sequence homology showing the naked mole-rat to have the genetic potential to express all five muscarinic acetylcholine receptor subtypes. A significant reduction in pain behavior was demonstrated after administration of 8.4 mg/kg in the formalin test. Administration of 50 mg/kg VU0152100 resulted in a non-significant tendency towards antinociception. The antinociceptive effects were reversed by the muscarinic acetylcholine receptor antagonist atropine. Binding studies indicated presence of muscarinic acetylcholine receptors with a radioligand affinity comparable to that reported in mice. In conclusion, muscarinic acetylcholine receptor subtypes are present in the naked mole-rat and contribute to antinociception in the naked mole-rat.

  17. A novel muscarinic receptor-independent mechanism of KCNQ2/3 potassium channel blockade by Oxotremorine-M.

    PubMed

    Zwart, Ruud; Reed, Hannah; Clarke, Sophie; Sher, Emanuele

    2016-11-15

    Inhibition of KCNQ (Kv7) potassium channels by activation of muscarinic acetylcholine receptors has been well established, and the ion currents through these channels have been long known as M-currents. We found that this cross-talk can be reconstituted in Xenopus oocytes by co-transfection of human recombinant muscarinic M1 receptors and KCNQ2/3 potassium channels. Application of the muscarinic acetylcholine receptor agonist Oxotremorine-methiodide (Oxo-M) between voltage pulses to activate KCNQ2/3 channels caused inhibition of the subsequent KCNQ2/3 responses. This effect of Oxo-M was blocked by the muscarinic acetylcholine receptor antagonist atropine. We also found that KCNQ2/3 currents were inhibited when Oxo-M was applied during an ongoing KCNQ2/3 response, an effect that was not blocked by atropine, suggesting that Oxo-M inhibits KCNQ2/3 channels directly. Indeed, also in oocytes that were transfected with only KCNQ2/3 channels, but not with muscarinic M1 receptors, Oxo-M inhibited the KCNQ2/3 response. These results show that besides the usual muscarinic acetylcholine receptor-mediated inhibition, Oxo-M also inhibits KCNQ2/3 channels by a direct mechanism. We subsequently tested xanomeline, which is a chemically distinct muscarinic acetylcholine receptor agonist, and oxotremorine, which is a close analogue of Oxo-M. Both compounds inhibited KCNQ2/3 currents via activation of M1 muscarinic acetylcholine receptors but, in contrast to Oxo-M, they did not directly inhibit KCNQ2/3 channels. Xanomeline and oxotremorine do not contain a positively charged trimethylammonium moiety that is present in Oxo-M, suggesting that such a charged moiety could be a crucial component mediating this newly described direct inhibition of KCNQ2/3 channels.

  18. Putative M2 muscarinic receptors of rat heart have high affinity for organophosphorus anticholinesterases

    SciTech Connect

    Silveira, C.L.; Eldefrawi, A.T.; Eldefrawi, M.E. )

    1990-05-01

    The M2 subtype of muscarinic receptor is predominant in heart, and such receptors were reported to be located in muscles as well as in presynaptic cholinergic and adrenergic nerve terminals. Muscarinic receptors of rat heart were identified by the high affinity binding of the agonist (+)-(3H)cis-methyldioxolane ((3H)CD), which has been used to label a high affinity population of M2 receptors. A single population of sites was detected and (3H)CD binding was sensitive to the M2 antagonist himbacine but much less so to pirenzepine, the M1 antagonist. These cardiac receptors had different sensitivities to NiCl2 and N-ethylmaleimide from brain muscarinic receptors, that were also labeled with (3H)CD and considered to be of the M2 subtype. Up to 70% of the (3H)CD-labeled cardiac receptors had high affinities for several organophosphate (OP) anticholinesterases. (3H)CD binding was inhibited by the nerve agents soman, VX, sarin, and tabun, with K0.5 values of 0.8, 2, 20, and 50 nM, respectively. It was also inhibited by echothiophate and paraoxon with K0.5 values of 100 and 300 nM, respectively. The apparent competitive nature of inhibition of (3H)CD binding by both sarin and paraoxon suggests that the OPs bind to the acetylcholine binding site of the muscarinic receptor. Other OP insecticides had lower potencies, inhibiting less than 50% of 5 nM (3H)CD binding by 1 microM of EPN, coumaphos, dioxathion, dichlorvos, or chlorpyriphos. There was poor correlation between the potencies of the OPs in reversibly inhibiting (3H)CD binding, and their anticholinesterase activities and toxicities. Acetylcholinesterases are the primary targets for these OP compounds because of the irreversible nature of their inhibition, which results in building of acetylcholine concentrations that activate muscarinic and nicotinic receptors and desensitize them, thereby inhibiting respiration.

  19. Characterization of (/sup 3/H)pirenzepine binding to muscarinic cholinergic receptors solubilized from rat brain

    SciTech Connect

    Luthin, G.R.; Wolfe, B.B.

    1985-07-01

    Membranes prepared from rat cerebral cortex were solubilized in buffer containing 1% digitonin. Material present in the supernatant after centrifugation at 147,000 X g was shown to contain binding sites for both (/sup 3/H)quinuclidinyl benzilate ((/sup 3/H)QNB) and (/sup 3/H)pirenzepine ((/sup 3/H)PZ). Recovery of binding sites was approximately 25% of the initial membrane-bound (/sup 3/H)QNB binding sites. The Kd values for (/sup 3/H)QNB and (/sup 3/H)PZ binding to solubilized receptors were 0.3 nM and 0.1 microM, respectively. As has been observed previously in membrane preparations, (/sup 3/H)PZ appeared to label fewer solubilized binding sites than did (/sup 3/H)QNB. Maximum binding values for (/sup 3/H)PZ and (/sup 3/H)QNB binding to solubilized receptors were approximately 400 and 950 fmol/mg of protein, respectively. Competition curves for PZ inhibiting the binding of (/sup 3/H)QNB, however, had Hill slopes of 1, with a Ki value of 0.24 microM. The k1 and k-1 for (/sup 3/H)PZ binding were 3.5 X 10(6) M-1 min-1 and 0.13 min-1, respectively. The muscarinic receptor antagonists atropine, scopolamine and PZ inhibited the binding of (/sup 3/H)QNB and (/sup 3/H)PZ to solubilized receptors with Hill slopes of 1, as did the muscarinic receptor agonist oxotremorine. The muscarinic receptor agonist carbachol competed for (/sup 3/H)QNB and (/sup 3/H)PZ binding with a Hill slope of less than 1 in cerebral cortex, but not in cerebellum. GTP did not alter the interactions of carbachol or oxotremorine with the solubilized receptor. Together, these data suggest that muscarinic receptor sites solubilized from rat brain retain their abilities to interact selectively with muscarinic receptor agonists and antagonists.

  20. Synaptic muscarinic response types in hippocampal CA1 interneurons depend on different levels of presynaptic activity and different muscarinic receptor subtypes.

    PubMed

    Bell, L Andrew; Bell, Karen A; McQuiston, A Rory

    2013-10-01

    Depolarizing, hyperpolarizing and biphasic muscarinic responses have been described in hippocampal inhibitory interneurons, but the receptor subtypes and activity patterns required to synaptically activate muscarinic responses in interneurons have not been completely characterized. Using optogenetics combined with whole cell patch clamp recordings in acute slices, we measured muscarinic responses produced by endogenously released acetylcholine (ACh) from cholinergic medial septum/diagonal bands of Broca inputs in hippocampal CA1. We found that depolarizing responses required more cholinergic terminal stimulation than hyperpolarizing ones. Furthermore, elevating extracellular ACh with the acetylcholinesterase inhibitor physostigmine had a larger effect on depolarizing versus hyperpolarizing responses. Another subpopulation of interneurons responded biphasically, and periodic release of ACh entrained some of these interneurons to rhythmically burst. M4 receptors mediated hyperpolarizing responses by activating inwardly rectifying K(+) channels, whereas the depolarizing responses were inhibited by the nonselective muscarinic antagonist atropine but were unaffected by M1, M4 or M5 receptor modulators. In addition, activation of M4 receptors significantly altered biphasic interneuron firing patterns. Anatomically, interneuron soma location appeared predictive of muscarinic response types but response types did not correlate with interneuron morphological subclasses. Together these observations suggest that the hippocampal CA1 interneuron network will be differentially affected by cholinergic input activity levels. Low levels of cholinergic activity will preferentially suppress some interneurons via hyperpolarization and increased activity will recruit other interneurons to depolarize, possibly because of elevated extracellular ACh concentrations. These data provide important information for understanding how cholinergic therapies will affect hippocampal network function

  1. Synaptic muscarinic response types in hippocampal CA1 interneurons depend on different levels of presynaptic activity and different muscarinic receptor subtypes

    PubMed Central

    Bell, L. Andrew; Bell, Karen A.; McQuiston, A. Rory

    2013-01-01

    Depolarizing, hyperpolarizing and biphasic muscarinic responses have been described in hippocampal inhibitory interneurons, but the receptor subtypes and activity patterns required to synaptically activate muscarinic responses in interneurons have not been completely characterized. Using optogenetics combined with whole cell patch clamp recordings in acute slices, we measured muscarinic responses produced by endogenously released acetylcholine (ACh) from cholinergic medial septum/diagonal bands of Broca inputs in hippocampal CA1. We found that depolarizing responses required more cholinergic terminal stimulation than hyperpolarizing ones. Furthermore, elevating extracellular ACh with the acetylcholinesterase inhibitor physostigmine had a larger effect on depolarizing versus hyperpolarizing responses. Another subpopulation of interneurons responded biphasically, and periodic release of ACh entrained some of these interneurons to rhythmically burst. M4 receptors mediated hyperpolarizing responses by activating inwardly rectifying K+ channels, whereas the depolarizing responses were inhibited by the nonselective muscarinic antagonist atropine but were unaffected by M1, M4 or M5 receptor modulators. In addition, activation of M4 receptors significantly altered biphasic interneuron firing patterns. Anatomically, interneuron soma location appeared predictive of muscarinic response types but response types did not correlate with interneuron morphological subclasses. Together these observations suggest that the hippocampal CA1 interneuron network will be differentially affected by cholinergic input activity levels. Low levels of cholinergic activity will preferentially suppress some interneurons via hyperpolarization and increased activity will recruit other interneurons to depolarize, possibly because of elevated extracellular ACh concentrations. These data provide important information for understanding how cholinergic therapies will affect hippocampal network function

  2. Muscarinic receptors as model targets and antitargets for structure-based ligand discovery.

    PubMed

    Kruse, Andrew C; Weiss, Dahlia R; Rossi, Mario; Hu, Jianxin; Hu, Kelly; Eitel, Katrin; Gmeiner, Peter; Wess, Jürgen; Kobilka, Brian K; Shoichet, Brian K

    2013-10-01

    G protein-coupled receptors (GPCRs) regulate virtually all aspects of human physiology and represent an important class of therapeutic drug targets. Many GPCR-targeted drugs resemble endogenous agonists, often resulting in poor selectivity among receptor subtypes and restricted pharmacologic profiles. The muscarinic acetylcholine receptor family exemplifies these problems; thousands of ligands are known, but few are receptor subtype-selective and nearly all are cationic in nature. Using structure-based docking against the M2 and M3 muscarinic receptors, we screened 3.1 million molecules for ligands with new physical properties, chemotypes, and receptor subtype selectivities. Of 19 docking-prioritized molecules tested against the M2 subtype, 11 had substantial activity and 8 represented new chemotypes. Intriguingly, two were uncharged ligands with low micromolar to high nanomolar Ki values, an observation with few precedents among aminergic GPCRs. To exploit a single amino-acid substitution among the binding pockets between the M2 and M3 receptors, we selected molecules predicted by docking to bind to the M3 and but not the M2 receptor. Of 16 molecules tested, 8 bound to the M3 receptor. Whereas selectivity remained modest for most of these, one was a partial agonist at the M3 receptor without measurable M2 agonism. Consistent with this activity, this compound stimulated insulin release from a mouse β-cell line. These results support the ability of structure-based discovery to identify new ligands with unexplored chemotypes and physical properties, leading to new biologic functions, even in an area as heavily explored as muscarinic pharmacology.

  3. SB 202026: a novel muscarinic partial agonist with functional selectivity for M1 receptors.

    PubMed

    Loudon, J M; Bromidge, S M; Brown, F; Clark, M S; Hatcher, J P; Hawkins, J; Riley, G J; Noy, G; Orlek, B S

    1997-12-01

    The finding that ascending cholinergic systems are severely degenerated in Alzheimer's disease has driven the search for a cholinomimetic therapy. Adverse effects observed with cholinesterase inhibitors and high-efficacy muscarinic agonists led us to design compounds with an improved profile. SB 202026 (R-(Z)-(+)-alpha-(methoxyimino)-1-azabicyclo[2.2.2] octane-3-acetonitrile) displaced [3H]-oxotremorine-M from muscarinic receptors in the rat brain with high affinity (IC50 = 14 nM), a potency similar to that of oxotremorine-M itself (IC50 = 13 nM), but exhibited low affinity for cholinergic nicotinic receptors and other neuroreceptors. In studies using cloned human muscarinic receptors, SB 202026 possessed approximately equal affinity in displacing [3H]-quinuclidinyl benzilate from all muscarinic receptor subtypes. In functional models in vitro, SB 202026 caused maximal depolarization of the rat superior cervical ganglion at low concentrations (300 nM) (M1-mediated effect), while producing a lower maximal effect than the high-efficacy agonists oxotremorine-M and carbachol on M2-mediated release of ACh and M3-mediated smooth muscle contraction (guinea pig ileum), respectively. The functional selectivity and partial agonist profile seen in vitro were reflected in vivo through potent cognition-related activity (M1-induced increase in hippocampal EEG power) combined with low efficacy, compared with arecoline or oxotremorine, on induction of bradycardia (M2-mediated response), hypotension (via M3-mediated vasorelaxation) and tremor (thought to be mediated by M3 receptors). The foregoing profile of SB 202026 predicted that cognition-enhancing activity would be achieved at doses below those that initiate undesirable side effects, and this has subsequently been demonstrated in rodents, marmosets and humans.

  4. Muscarinic receptors stimulate cell proliferation in the human urothelium-derived cell line UROtsa.

    PubMed

    Arrighi, Nicola; Bodei, Serena; Lucente, Alessandra; Michel, Martin C; Zani, Danilo; Simeone, Claudio; Cunico, Sergio Cosciani; Spano, PierFranco; Sigala, Sandra

    2011-10-01

    The widespread non-neuronal synthesis of acetylcholine (ACh) has changed the paradigm of ACh acting solely as a neurotransmitter. Indeed, the presence of ACh in many types of proliferating cells suggests a role for this neurotransmitter in the control of cell division. The parasympathetic system is a major pathway regulating micturition, but ACh-mediated control plays a more complex role than previously described, acting not only in the detrusor muscle, but also influencing detrusor function through the activity of urothelial muscarinic receptors. Here we investigated the role of muscarinic receptors in mediating cell proliferation in the human UROtsa cell line, which is a widely used experimental model to study urothelium physiology and pathophysiology. Our results demonstrate that UROtsa cells express the machinery for ACh synthesis and that muscarinic receptors, with the rank order of M3>M2>M5>M1=M4, are present and functionally linked to their known second messengers. Indeed, the cholinergic receptor agonist carbachol (CCh) (1-100 μM) concentration-dependently raised IP(3) levels, reaching 66±5% over basal. The forskolin-mediated adenylyl cyclase activation was reduced by CCh exposure (forskolin: 1.4±0.14 pmol/ml; forskolin+100 μM CCh: 0.84±0.12 pmol/ml). CCh (1-100 μM) concentration-dependently increased UROtsa cell proliferation and this effect was inhibited by the non-selective antagonist atropine and the M(3)-selective antagonists darifenacin and J104129. Finally, CCh-induced cell proliferation was blocked by selective PI-3 kinase and ERK activation inhibitors, strongly suggesting that these intracellular pathways mediate, at least in part, the muscarinic receptor-mediated cell proliferation.

  5. Effects of central galanin administration on muscarinic cholinergic and galanin receptor G protein coupling.

    PubMed

    Barreda-Gómez, G; Giralt, M T; Rodríguez-Puertas, R

    2005-06-01

    The neuropeptide galanin is expressed in the mammalian central nervous system and has been implicated in neurotrophic actions. Central galanin administration induces cognitive deficits in rodents and inhibits the release of acetylcholine in the hippocampus. In addition, a galanin hyperinnervation of the basal forebrain cholinergic cells in Alzheimer's disease patients has been reported. To evaluate the effect of galanin treatment on galanin and muscarinic cholinergic receptor G protein coupling, galanin was administered into the lateral ventricle of rats via an implanted cannula. Galanin or muscarinic receptor functional coupling to G proteins was quantified by galanin or carbachol stimulation of guanosine 5'-(gamma-[35S]thio)triphosphate binding in rat brain slices. Guanosine 5'-(gamma-[35S]thio)triphosphate basal binding in nucleus basalis of Meynert and thalamic nuclei was increased in the vehicle treated group. This effect was reverted by galanin treatment and indicates that the surgery increased receptor functional coupling to G proteins, which is restored by a possible neurotrophic action mediated by galanin. In addition, in galanin administered animals, galanin-stimulated binding was increased in the amygdala but decreased in the diagonal band, whilst binding stimulation mediated by carbachol was found to be increased in the amygdala, thalamic nuclei and diagonal band. These findings indicate that galanin treatment modulates the coupling of galanin and muscarinic cholinergic receptors to G proteins in specific regions of the rat central nervous system.

  6. Catestatin attenuates endoplasmic reticulum induced cell apoptosis by activation type 2 muscarinic acetylcholine receptor in cardiac ischemia/reperfusion

    PubMed Central

    Liao, Feng; Zheng, Yang; Cai, Junyan; Fan, Jinghui; Wang, Jing; Yang, Jichun; Cui, Qinghua; Xu, Guoheng; Tang, Chaoshu; Geng, Bin

    2015-01-01

    Catestatin (CST) is a catecholamine secretion inhibiting peptide as non-competitive inhibitor of nicotinic acetylcholine receptor. CST play a protective role in cardiac ischemia/reperfusion (I/R) but the molecular mechanism remains unclear. Cardiomyocytes endogenously produced CST and its expression was reduced after I/R. CST pretreatment decreased apoptosis especially endoplasmic reticulum (ER) stress response during I/R. The protection of CST was confirmed in H9c2 cardiomyoblasts under Anoxia/reoxygenation (A/R). In contrast, siRNA-mediated knockdown of CST exaggerated ER stress induced apoptosis. The protective effects of CST were blocked by extracellular signal-regulated kinases 1/2 (ERK1/2) inhibitor PD90895 and phosphoinositide 3-kinase (PI3 K) inhibitor wortmannin. CST also increased ERK1/2 and protein kinase B (Akt) phosphorylation and which was blocked by atropine and selective type 2 muscarinic acetylcholine (M2) receptor, but not type 1 muscarinic acetylcholine (M1) receptor antagonist. Receptor binding assay revealed that CST competitively bound to the M2 receptor with a 50% inhibitory concentration of 25.7 nM. Accordingly, CST inhibited cellular cAMP stimulated by isoproterenol or forskolin, and which was blocked by selective M2 receptor antagonist. Our findings revealed that CST binds to M2 receptor, then activates ERK1/2 and PI3 K/Akt pathway to inhibit ER stress-induced cell apoptosis resulting in attenuation cardiac I/R injury. PMID:26567709

  7. The M4 muscarinic receptor-selective effects on keratinocyte crawling locomotion.

    PubMed

    Chernyavsky, Alex I; Nguyen, Vu Thuong; Arredondo, Juan; Ndoye, Assane; Zia, Shaheen; Wess, Jürgen; Grando, Sergei A

    2003-03-28

    We have investigated how the cholinergic system of epidermal keratinocytes (KC) controls migratory function of these cells. Several molecular subtypes of muscarinic acetylcholine receptors (mAChRs) have been detected in KC. Early results suggested that M(4) is the predominant mAChR regulating cell motility. To determine muscarinic effects on lateral migration of KC, we used an agarose gel keratinocyte outgrowth system (AGKOS) which provides for measurements of the response of large cell populations (> 10(4) cells). Muscarine produced a dose-dependent stimulatory effect on cell migration (p < 0.05). This activity was abolished by atropine, which decreased migration distance when given alone. To identify the mAChR subtype(s) mediating these muscarinic effects, we substituted atropine with subtype-selective antagonists. Tropicamide (M(4)-selective) was more effective at decreasing the migration distance than pirenzepine and 4-DAMP at nanomolar concentrations. We then compared lateral migration of KC obtained from M(4) mAChR knockout mice with that of wild-type murine KC, using AGKOS. In the absence of M(4) mAChR, the migration distance of KC was significantly (p < 0.05) decreased. These results indicate that the M(4) mAChR plays a central role in mediating cholinergic control of keratinocyte migration by endogenous acetylcholine produced by these cells.

  8. Abundance, distribution, mobility and oligomeric state of M2 muscarinic acetylcholine receptors in live cardiac muscle

    PubMed Central

    Nenasheva, Tatiana A.; Neary, Marianne; Mashanov, Gregory I.; Birdsall, Nigel J.M.; Breckenridge, Ross A.; Molloy, Justin E.

    2013-01-01

    M2 muscarinic acetylcholine receptors modulate cardiac rhythm via regulation of the inward potassium current. To increase our understanding of M2 receptor physiology we used Total Internal Reflection Fluorescence Microscopy to visualize individual receptors at the plasma membrane of transformed CHOM2 cells, a cardiac cell line (HL-1), primary cardiomyocytes and tissue slices from pre- and post-natal mice. Receptor expression levels between individual cells in dissociated cardiomyocytes and heart slices were highly variable and only 10% of murine cardiomyocytes expressed muscarinic receptors. M2 receptors were evenly distributed across individual cells and their density in freshly isolated embryonic cardiomyocytes was ~ 1 μm− 2, increasing at birth (to ~ 3 μm− 2) and decreasing back to ~ 1 μm− 2 after birth. M2 receptors were primarily monomeric but formed reversible dimers. They diffused freely at the plasma membrane, moving approximately 4-times faster in heart slices than in cultured cardiomyocytes. Knowledge of receptor density and mobility has allowed receptor collision rate to be modeled by Monte Carlo simulations. Our estimated encounter rate of 5–10 collisions per second, may explain the latency between acetylcholine application and GIRK channel opening. PMID:23357106

  9. Influence of volatile anesthetics on muscarinic receptor adenylate cyclase coupling in brain and heart

    SciTech Connect

    Anthony, B.L.

    1988-01-01

    In the present study, the influence of four volatile anesthetics (enflurane, isoflurane, diethyl ether, and chloroform) on (1) muscarinic receptor binding parameters and (2) muscarnic regulation of adenylate cyclase activity was examined using membranes isolated from rat brain and heart. Membranes were equilibrated with each of the four anesthetics for 30 minutes and then during the binding assay. The data obtained can be summarized as follows: (1) volatile anesthetics increased receptor affinity for a radiolabeled antagonists, ({sup 3}H)N-methylscopolamine (({sup 3}H)MS), by decreasing its rate of dissociation in brain stem, but not in cardiac, membranes, (2) volatile anesthetics decreased high affinity ({sup 3}H)Oxotremorine-M binding, (3) volatile anesthetics depressed or eliminated the guanine nucleotide sensitivity of agonist binding. The influence of volatile anesthetics on muscarinic regulation of adenylate cyclase enzyme activity was studied using {alpha}({sup 32}P)ATP as the substrate.

  10. Identification of a ligand-dependent switch within a muscarinic receptor.

    PubMed

    Spalding, T A; Burstein, E S; Henderson, S C; Ducote, K R; Brann, M R

    1998-08-21

    G-protein-coupled receptors spontaneously switch between active and inactive conformations. Agonists stabilize the active conformation, whereas antagonists stabilize the inactive conformation. In a systematic search for residues that participate in receptor function, several regions of the m5 muscarinic receptor were randomly mutated and tested for their functional properties. Mutations spanning one face of transmembrane 6 (TM6) were found to induce high levels of receptor activity in the absence of agonists (constitutive activity). The same face of TM6 contained several residues crucial for receptor activation by agonists and one residue identified as a contact site for both agonists and antagonists. In addition, one mutation induced agonist-like responses from the receptor when exposed to classical antagonists. These results suggest that TM6 is a switch that defines the activation state of the receptor, and that ligand interactions with TM6 stabilize the receptor in either an active or an inactive conformation.

  11. Effect of aging on airway remodeling and muscarinic receptors in a murine acute asthma model

    PubMed Central

    Kang, Ji Young; Lee, Sook Young; Rhee, Chin Kook; Kim, Seung Joon; Kwon, Soon Seog; Kim, Young Kyoon

    2013-01-01

    Background and objectives The influence of aging on the development of asthma has not been studied thoroughly. The aim of this study was to investigate age-related airway responses involving lung histology and expression of muscarinic receptors in a murine model of acute asthma. Methods Female BALB/c mice at the ages of 6 weeks and 6, 9, and 12 months were sensitized and challenged with ovalbumin (OVA) for 1 month (n = 8–12 per group). We analyzed inflammatory cells and T-helper (Th)2 cytokines in bronchoalveolar lavage (BAL) fluid and parameters of airway remodeling and expression of muscarinic receptors in lung tissue. Results Among the OVA groups, total cell and eosinophil numbers in BAL fluid were significantly higher in the older (6-, 9-, and 12-month-old) mice than in the young (6-week-old) mice. Interleukin (IL) 4 (IL-4) concentration increased, but IL-5 and IL-13 concentrations showed a decreased tendency, with age. IL-17 concentration tended to increase with age, which did not reach statistical significance. Periodic acid-Schiff (PAS) staining area, peribronchial collagen deposition, and area of α-smooth muscle staining were significantly higher in the 6-month older OVA group than in the young OVA group. The expression of the M3 and M2 muscarinic receptors tended to increase and decrease, respectively, with age. Conclusion The aged mice showed an active and unique pattern not only on airway inflammation, but also on airway remodeling and expression of the muscarinic receptors during the development of acute asthma compared with the young mice. These findings suggest that the aging process affects the pathogenesis of acute asthma and age-specific approach might be more appropriate for better asthma control in a clinical practice. PMID:24204129

  12. Ventral tegmental area muscarinic receptors modulate depression and anxiety-related behaviors in rats

    PubMed Central

    Small, Keri M.; Nunes, Eric; Hughley, Shannon; Addy, Nii A.

    2016-01-01

    Cholinergic and dopaminergic mechanisms within the mesolimbic dopamine system are suggested to play a role in the manifestation of depression and anxiety-related disorders. However, despite the fact that cholinergic mechanisms in the ventral tegmental area (VTA) highly regulate dopamine activity, the role of VTA cholinergic mechanisms in depression-related behaviors is relatively unknown. Here we sought to determine whether enhancing cholinergic tone in the VTA would alter depression and anxiety-related behavior in the forced swim test (FST), elevated plus maze (EPM) and sucrose preference test (SPT). Adult Sprague Dawley male rats received VTA infusion of the acetylcholinesterase inhibitor, physostigmine (0, 1, 2 μg/side), immediately prior to the FST, EPM, or SPT. Physostigmine administration increased immobility time in the FST, decreased time spent on open arms in the EPM, and decreased sucrose preference. In a separate cohort of rats, we also examined whether activation of VTA muscarinic receptors was sufficient to alter behavior in the FST and EPM. Similar to physostigmine, VTA infusion of the muscarinic receptor agonist, pilocarpine (0, 3, 30 μg/side), increased immobility time in the FST and decreased time spent on open arms in the EPM. These data suggest that enhanced VTA cholinergic tone promotes pro-depressive and anxiogenic-like effects and demonstrate that specific activation of VTA muscarinic receptors is also sufficient to induce pro-depressive and anxiogenic responses. Together, these findings reveal a novel role of VTA cholinergic, and specifically muscarinic receptor, mechanisms in mediating responses to stress and anxiety. PMID:26828299

  13. Muscarinic and PACAP receptor interactions at pontine level in the rat: significance for REM sleep regulation.

    PubMed

    Ahnaou, A; Laporte, A M; Ballet, S; Escourrou, P; Hamon, M; Adrien, J; Bourgin, P

    2000-12-01

    Cholinergic and PACAPergic systems within the oral pontine reticular nucleus (PnO) play a critical role in REM sleep generation in rats. In this present work, we have investigated whether REM sleep enhancement induced by carbachol (a cholinergic agonist) or PACAP, depends on an interaction between muscarinic and PACAP receptors. This hypothesis was tested by recording sleep-wake cycles in freely moving rats injected into the PnO with PACAP in combination with the muscarinic receptor antagonist atropine, or with carbachol in combination with the PACAP receptor antagonist PACAP6-27. When administered alone, PACAP (3 pmol) or carbachol (110 pmol) induced an enhancement of REM sleep during 8 h (+61%, n = 8; +70%, n = 5), which was totally prevented by infusion of atropine (290 pmol) for PACAP, or of PACAP6-27 (3 pmol) for carbachol. Quantitative autoradiographic studies indicated that (i) PACAP (10-9-10-7 M) induced in the PnO an increase (+35%) of the specific binding of the muscarinic antagonist [3H]quinuclidinyl benzylate, which could be completely prevented by PACAP6-27 (IC50 = 8 x 10-8 M) and (ii) both carbachol and PACAP enhanced [35S]GTP-gamma-S binding in a concentration-dependent manner in the PnO. The maximal increase due to carbachol was significantly higher in the presence (+126%) than in the absence (+102%) of PACAP (0.1 microM). These data showed that interactions between muscarinic and PACAP receptors do exist within the PnO and play a role in the local mechanisms of REM sleep control in the rat.

  14. Ventral tegmental area muscarinic receptors modulate depression and anxiety-related behaviors in rats.

    PubMed

    Small, Keri M; Nunes, Eric; Hughley, Shannon; Addy, Nii A

    2016-03-11

    Cholinergic and dopaminergic mechanisms within the mesolimbic dopamine system are suggested to play a role in the manifestation of depression and anxiety-related disorders. However, despite the fact that cholinergic mechanisms in the ventral tegmental area (VTA) highly regulate dopamine activity, the role of VTA cholinergic mechanisms in depression-related behaviors is relatively unknown. Here we sought to determine whether enhancing cholinergic tone in the VTA would alter depression and anxiety-related behavior in the forced swim test (FST), elevated plus maze (EPM) and sucrose preference test (SPT). Adult Sprague Dawley male rats received VTA infusion of the acetylcholinesterase inhibitor, physostigmine (0, 1, 2μg/side), immediately prior to the FST, EPM, or SPT. Physostigmine administration increased immobility time in the FST, decreased time spent on open arms in the EPM, and decreased sucrose preference. We also examined whether activation of VTA muscarinic receptors was sufficient to alter behavior in the FST and EPM. Similar to physostigmine, VTA infusion of the muscarinic receptor agonist, pilocarpine (0, 3, 30μg/side), increased immobility time in the FST and decreased time spent on open arms in the EPM. These data suggest that enhanced VTA cholinergic tone promotes pro-depressive and anxiogenic-like effects and demonstrate that specific activation of VTA muscarinic receptors is also sufficient to induce pro-depressive and anxiogenic responses. Together, these findings reveal a novel role of VTA cholinergic, and specifically muscarinic receptor, mechanisms in mediating responses to stress and anxiety. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  15. Antidepressant effects of the muscarinic cholinergic receptor antagonist scopolamine: a review.

    PubMed

    Drevets, Wayne C; Zarate, Carlos A; Furey, Maura L

    2013-06-15

    The muscarinic cholinergic receptor system has been implicated in the pathophysiology of depression, with physiological evidence indicating this system is overactive or hyperresponsive in depression and with genetic evidence showing that variation in genes coding for receptors within this system are associated with higher risk for depression. In studies aimed at assessing whether a reduction in muscarinic cholinergic receptor function would improve depressive symptoms, the muscarinic receptor antagonist scopolamine manifested antidepressant effects that were robust and rapid relative to conventional pharmacotherapies. Here, we review the data from a series of randomized, double-blind, placebo-controlled studies involving subjects with unipolar or bipolar depression treated with parenteral doses of scopolamine. The onset and duration of the antidepressant response are considered in light of scopolamine's pharmacokinetic properties and an emerging literature that characterizes scopolamine's effects on neurobiological systems beyond the cholinergic system that appear relevant to the neurobiology of mood disorders. Scopolamine infused at 4.0 μg/kg intravenously produced robust antidepressant effects versus placebo, which were evident within 3 days after the initial infusion. Placebo-adjusted remission rates were 56% and 45% for the initial and subsequent replication studies, respectively. While effective in male and female subjects, the change in depression ratings was greater in female subjects. Clinical improvement persisted more than 2 weeks following the final infusion. The timing and persistence of the antidepressant response to scopolamine suggest a mechanism beyond that of direct muscarinic cholinergic antagonism. These temporal relationships suggest that scopolamine-induced changes in gene expression and synaptic plasticity may confer the therapeutic mechanism.

  16. Chronic treatment with simvastatin upregulates muscarinic M1/4 receptor binding in the rat brain.

    PubMed

    Wang, Q; Zengin, A; Ying, W; Newell, K A; Wang, P; Yeo, W; Wong, P T-H; Yenari, M A; Huang, X-F

    2008-06-26

    Statins are increasingly being used for the treatment of a variety of conditions beyond their original indication for cholesterol lowering. We previously reported that simvastatin affected the dopaminergic system in the rat brain. This study aims to investigate regional changes of muscarinic M1/4 receptors in the rat brain after 4-week administration of simvastatin (1 or 10 mg/kg/day). M1/4 receptor distribution and alterations in the post-mortem rat brain were detected by [(3)H]pirenzepine binding autoradiography. Simvastatin (1 mg/kg/day) increased [(3)H]pirenzepine binding, predominantly in the prefrontal cortex (171%, P<0.001), primary motor cortex (153%, P=0.001), cingulate cortex (109%, P<0.001), hippocampus (138%, P<0.001), caudate putamen (122%, P=0.002) and nucleus accumbens (170%, P<0.001) compared with controls; while lower but still significant increases of [(3)H]pirenzepine binding were observed in the examined regions following simvastatin (10 mg/kg/day) treatment. Our results also provide strong evidence that chronic simvastatin administration, especially at a low dosage, up-regulates M1/4 receptor binding, which is likely to be independent of its muscarinic agonist-like effect. Alterations in [(3)H]pirenzepine binding in the examined brain areas may represent the specific regions that mediate the clinical effects of simvastatin treatment on cognition and memory via the muscarinic cholinergic system. These findings contribute to a better understanding of the critical roles of simvastatin in treating neurodegenerative disorders, via muscarinic receptors.

  17. The structure of the third intracellular loop of the muscarinic acetylcholine receptor M2 subtype.

    PubMed

    Ichiyama, Susumu; Oka, Yoshiaki; Haga, Kazuko; Kojima, Shuichi; Tateishi, Yukihiro; Shirakawa, Masahiro; Haga, Tatsuya

    2006-01-09

    We have examined whether the long third intracellular loop (i3) of the muscarinic acetylcholine receptor M2 subtype has a rigid structure. Circular dichroism (CD) and nuclear magnetic resonance spectra of M2i3 expressed in and purified from Escherichia coli indicated that M2i3 consists mostly of random coil. In addition, the differential CD spectrum between the M2 and M2deltai3 receptors, the latter of which lacks most of i3 except N- and C-terminal ends, gave no indication of secondary structure. These results suggest that the central part of i3 of the M2 receptor has a flexible structure.

  18. Suitability of Nicotinic Acetylcholine Receptor α7 and Muscarinic Acetylcholine Receptor 3 Antibodies for Immune Detection

    PubMed Central

    Rommel, Frank R.; Raghavan, Badrinarayanan; Paddenberg, Renate; Kummer, Wolfgang; Tumala, Susanne; Lochnit, Günter; Gieler, Uwe

    2015-01-01

    Recent evidence reveals a crucial role for acetylcholine and its receptors in the regulation of inflammation, particularly of nicotinic acetylcholine receptor α7 (Chrna7) and muscarinic acetylcholine receptor 3 (Chrm3). Immunohistochemistry is a key tool for their cellular localization in functional tissues. We evaluated nine different commercially available antibodies on back skin tissue from wild-type (Wt) and gene-deficient (KO) mice. In the immunohistochemical analysis, we focused on key AChR-ligand sensitive skin cells (mast cells, nerve fibers and keratinocytes). All five antibodies tested for Chrm3 and the first three Chrna7 antibodies stained positive in both Wt and respective KO skin. With the 4th antibody (ab23832) nerve fibers were unlabeled in the KO mice. By western blot analysis, this antibody detected bands in both Wt and Chrna7 KO skin and brain. qRT-PCR revealed mRNA amplification with a primer set for the undeleted region in both Wt and KO mice, but none with a primer set for the deleted region in KO mice. By 2D electrophoresis, we found β-actin and β-enolase cross reactivity, which was confirmed by double immunolabeling. In view of the present results, the tested antibodies are not suitable for immunolocalization in skin and suggest thorough control of antibody specificity is required if histomorphometry is intended. PMID:25673288

  19. FRET-Based Detection of M1 Muscarinic Acetylcholine Receptor Activation by Orthosteric and Allosteric Agonists

    PubMed Central

    Markovic, Danijela; Holdich, Jonathan; Al-Sabah, Suleiman; Mistry, Rajendra; Krasel, Cornelius; Mahaut-Smith, Martyn P.; Challiss, R. A. John

    2012-01-01

    Background and Objective Muscarinic acetylcholine receptors (mAChRs) are 7-transmembrane, G protein-coupled receptors that regulate a variety of physiological processes and represent potentially important targets for therapeutic intervention. mAChRs can be stimulated by full and partial orthosteric and allosteric agonists, however the relative abilities of such ligands to induce conformational changes in the receptor remain unclear. To gain further insight into the actions of mAChR agonists, we have developed a fluorescently tagged M1 mAChR that reports ligand-induced conformational changes in real-time by changes in Förster resonance energy transfer (FRET). Methods Variants of CFP and YFP were inserted into the third intracellular loop and at the end of the C-terminus of the mouse M1 mAChR, respectively. The optimized FRET receptor construct (M1-cam5) was expressed stably in HEK293 cells. Results The variant CFP/YFP-receptor chimera expressed predominantly at the plasma membrane of HEK293 cells and displayed ligand-binding affinities comparable with those of the wild-type receptor. It also retained an ability to interact with Gαq/11 proteins and to stimulate phosphoinositide turnover, ERK1/2 phosphorylation and undergo agonist-dependent internalization. Addition of the full agonist methacholine caused a reversible decrease in M1 FRET (FEYFP/FECFP) that was prevented by atropine pre-addition and showed concentration-dependent amplitude and kinetics. Partial orthosteric agonists, arecoline and pilocarpine, as well as allosteric agonists, AC-42 and 77-LH-28-1, also caused atropine-sensitive decreases in the FRET signal, which were smaller in amplitude and significantly slower in onset compared to those evoked by methacholine. Conclusion The M1 FRET-based receptor chimera reports that allosteric and orthosteric agonists induce similar conformational changes in the third intracellular loop and/or C-terminus, and should prove to be a valuable molecular reagent for

  20. Crystal structures of the M1 and M4 muscarinic acetylcholine receptors

    DOE PAGES

    Thal, David M.; Sun, Bingfa; Feng, Dan; ...

    2016-03-09

    Muscarinic M1–M5 acetylcholine receptors are G-protein-coupled receptors that regulate many vital functions of the central and peripheral nervous systems. In particular, the M1 and M4 receptor subtypes have emerged as attractive drug targets for treatments of neurological disorders, such as Alzheimer’s disease and schizophrenia, but the high conservation of the acetylcholine-binding pocket has spurred current research into targeting allosteric sites on these receptors. In this paper, we report the crystal structures of the M1 and M4 muscarinic receptors bound to the inverse agonist, tiotropium. Comparison of these structures with each other, as well as with the previously reported M2 andmore » M3 receptor structures, reveals differences in the orthosteric and allosteric binding sites that contribute to a role in drug selectivity at this important receptor family. Finally, we also report identification of a cluster of residues that form a network linking the orthosteric and allosteric sites of the M4 receptor, which provides new insight into how allosteric modulation may be transmitted between the two spatially distinct domains.« less

  1. The binding of pirenzepine to digitonin-solubilized muscarinic acetylcholine receptors from the rat myocardium.

    PubMed Central

    Birdsall, N. J.; Hulme, E. C.; Keen, M.

    1986-01-01

    The binding of pirenzepine to digitonin-solubilized rat myocardial muscarinic acetylcholine receptors has been examined at 4 degrees C. Solubilization produced only small changes in the binding of N-methylscopolamine and atropine. In contrast to the low affinity binding of pirenzepine found to be present in in the membranes, high affinity binding was detected in the soluble preparation. In both preparations, pirenzepine binding was complex. High affinity pirenzepine binding (KD approximately 3 X 10(-8)M) to the soluble myocardial receptors could be monitored directly using [3H]-pirenzepine. [3H]-pirenzepine-labelled soluble myocardial receptors have a sedimentation coefficient of 11.1 s. This indicates that [3H]-pirenzepine binds predominantly to the uncoupled form of the receptor. However, [3H]-pirenzepine-agonist competition experiments indicated that the high affinity pirenzepine binding sites are capable of coupling with a guanosine 5'-triphosphate (GTP)-binding protein. Pirenzepine affinities for the soluble myocardial receptors were unaffected by their state of association with the GTP-binding proteins found in the heart. The equilibrium binding properties of the soluble cortical and myocardial receptors were very similar. However, the binding kinetics of the myocardial receptor were much slower. It appears that the membrane environment can affect the affinity of pirenzepine for the rat myocardial muscarinic receptor. Removal of the constraint by solubilization allows the expression of high affinity pirenzepine binding. PMID:3754173

  2. Crystal Structures of the M1 and M4 Muscarinic Acetylcholine Receptors

    PubMed Central

    Thal, David M.; Sun, Bingfa; Feng, Dan; Nawaratne, Vindhya; Leach, Katie; Felder, Christian C.; Bures, Mark G.; Evans, David A.; Weis, William I.; Bachhawat, Priti; Kobilka, Tong Sun; Sexton, Patrick M.; Kobilka, Brian K.; Christopoulos, Arthur

    2016-01-01

    Summary Muscarinic M1–M5 acetylcholine receptors are G protein-coupled receptors (GPCRs) that regulate many vital functions of the central and peripheral nervous systems. In particular, the M1 and M4 receptor subtypes have emerged as attractive drug targets for treatments of neurological disorders, such as Alzheimer's disease and schizophrenia, but the high conservation of the acetylcholine-binding pocket has spurred current research into targeting allosteric sites on these receptors. Here, we report the first crystal structures of the M1 and M4 muscarinic receptors bound to the inverse agonist, tiotropium. Comparison of these structures to each other, as well as the previously reported M2 and M3 receptor structures, reveals differences in the orthosteric and allosteric binding sites that contribute to a role in drug selectivity at this important receptor family. We also report identification of a cluster of residues that form a network linking the orthosteric and allosteric sites of the M4 receptor, which provides new insight into how allosteric modulation may be transmitted between the two spatially distinct domains. PMID:26958838

  3. The muscarinic antagonists scopolamine and atropine are competitive antagonists at 5-HT3 receptors.

    PubMed

    Lochner, Martin; Thompson, Andrew J

    2016-09-01

    Scopolamine is a high affinity muscarinic antagonist that is used for the prevention of post-operative nausea and vomiting. 5-HT3 receptor antagonists are used for the same purpose and are structurally related to scopolamine. To examine whether 5-HT3 receptors are affected by scopolamine we examined the effects of this drug on the electrophysiological and ligand binding properties of 5-HT3A receptors expressed in Xenopus oocytes and HEK293 cells, respectively. 5-HT3 receptor-responses were reversibly inhibited by scopolamine with an IC50 of 2.09 μM. Competitive antagonism was shown by Schild plot (pA2 = 5.02) and by competition with the 5-HT3 receptor antagonists [(3)H]granisetron (Ki = 6.76 μM) and G-FL (Ki = 4.90 μM). The related molecule, atropine, similarly inhibited 5-HT evoked responses in oocytes with an IC50 of 1.74 μM, and competed with G-FL with a Ki of 7.94 μM. The reverse experiment revealed that granisetron also competitively bound to muscarinic receptors (Ki = 6.5 μM). In behavioural studies scopolamine is used to block muscarinic receptors and induce a cognitive deficit, and centrally administered concentrations can exceed the IC50 values found here. It is therefore possible that 5-HT3 receptors are also inhibited. Studies that utilise higher concentrations of scopolamine should be mindful of these potential off-target effects.

  4. Comparative evaluation of exocrine muscarinic receptor binding characteristics and inhibition of salivation of solifenacin in mice.

    PubMed

    Oki, Tomomi; Takeuchi, Chihiro; Yamada, Shizuo

    2006-07-01

    Anticholinergic agents such as oxybutynin are clinically useful in the treatment of overactive bladder. However, oral administration of oxybutynin is frequently accompanied by side effects such as dry mouth, and novel bladder-selective anticholinergic agents such as solifenacin and tolterodine are now under development. The aim of the present study was to characterize the suppression of cholinergic salivation and exocrine muscarinic receptor binding of solifenacin on oral administration to mice in comparison with those of oxybutynin. Results showed that both drugs produced a significant increase in K(d) values for specific [N-Methyl-(3)H]scopolamine methyl chloride ([(3)H]NMS) binding in the mouse submaxillary gland, compared with control values. However, this enhancement in K(d) values was significantly smaller with solifenacin than with oxybutynin. Moreover, the inhibitory effect of solifenacin on pilocarpine-induced salivary secretion was significantly weaker than that of oxybutynin. Solifenacin dissociated more readily from muscarinic receptors in the mouse submaxillary gland than oxybutynin. In conclusion, the present study indicates that the weak suppression of cholinergic salivation by solifenacin compared with oxybutynin may be partially attributed to its relatively fast dissociation kinetics from exocrine muscarinic receptors.

  5. Muscarinic cholinergic receptor (M2) plays a crucial role in the development of myopia in mice.

    PubMed

    Barathi, Veluchamy A; Kwan, Jia Lin; Tan, Queenie S W; Weon, Sung Rhan; Seet, Li Fong; Goh, Liang Kee; Vithana, Eranga N; Beuerman, Roger W

    2013-09-01

    Myopia is a huge public health problem worldwide, reaching the highest incidence in Asia. Identification of susceptible genes is crucial for understanding the biological basis of myopia. In this paper, we have identified and characterized a functional myopia-associated gene using a specific mouse-knockout model. Mice lacking the muscarinic cholinergic receptor gene (M2; also known as Chrm2) were less susceptible to lens-induced myopia compared with wild-type mice, which showed significantly increased axial length and vitreous chamber depth when undergoing experimental induction of myopia. The key findings of this present study are that the sclera of M2 mutant mice has higher expression of collagen type I and lower expression of collagen type V than do wild-type mice and mice that are mutant for other muscarinic subtypes, and, therefore, M2 mutant mice were resistant to the development of experimental myopia. Pharmacological blockade of M2 muscarinic receptor proteins retarded myopia progression in the mouse. These results suggest for the first time a role of M2 in growth-related changes in extracellular matrix genes during myopia development in a mammalian model. M2 receptor antagonists might thus provide a targeted therapeutic approach to the management of this refractive error.

  6. Muscarinic cholinergic receptor (M2) plays a crucial role in the development of myopia in mice

    PubMed Central

    Barathi, Veluchamy A.; Kwan, Jia Lin; Tan, Queenie S. W.; Weon, Sung Rhan; Seet, Li Fong; Goh, Liang Kee; Vithana, Eranga N.; Beuerman, Roger W.

    2013-01-01

    SUMMARY Myopia is a huge public health problem worldwide, reaching the highest incidence in Asia. Identification of susceptible genes is crucial for understanding the biological basis of myopia. In this paper, we have identified and characterized a functional myopia-associated gene using a specific mouse-knockout model. Mice lacking the muscarinic cholinergic receptor gene (M2; also known as Chrm2) were less susceptible to lens-induced myopia compared with wild-type mice, which showed significantly increased axial length and vitreous chamber depth when undergoing experimental induction of myopia. The key findings of this present study are that the sclera of M2 mutant mice has higher expression of collagen type I and lower expression of collagen type V than do wild-type mice and mice that are mutant for other muscarinic subtypes, and, therefore, M2 mutant mice were resistant to the development of experimental myopia. Pharmacological blockade of M2 muscarinic receptor proteins retarded myopia progression in the mouse. These results suggest for the first time a role of M2 in growth-related changes in extracellular matrix genes during myopia development in a mammalian model. M2 receptor antagonists might thus provide a targeted therapeutic approach to the management of this refractive error. PMID:23649821

  7. Regulation of molecular clock oscillations and phagocytic activity via muscarinic Ca2+ signaling in human retinal pigment epithelial cells

    PubMed Central

    Ikarashi, Rina; Akechi, Honami; Kanda, Yuzuki; Ahmad, Alsawaf; Takeuchi, Kouhei; Morioka, Eri; Sugiyama, Takashi; Ebisawa, Takashi; Ikeda, Masaaki; Ikeda, Masayuki

    2017-01-01

    Vertebrate eyes are known to contain circadian clocks, however, the intracellular mechanisms regulating the retinal clockwork remain largely unknown. To address this, we generated a cell line (hRPE-YC) from human retinal pigmental epithelium, which stably co-expressed reporters for molecular clock oscillations (Bmal1-luciferase) and intracellular Ca2+ concentrations (YC3.6). The hRPE-YC cells demonstrated circadian rhythms in Bmal1 transcription. Also, these cells represented circadian rhythms in Ca2+-spiking frequencies, which were canceled by dominant-negative Bmal1 transfections. The muscarinic agonist carbachol, but not photic stimulation, phase-shifted Bmal1 transcriptional rhythms with a type-1 phase response curve. This is consistent with significant M3 muscarinic receptor expression and little photo-sensor (Cry2 and Opn4) expression in these cells. Moreover, forskolin phase-shifted Bmal1 transcriptional rhythm with a type-0 phase response curve, in accordance with long-lasting CREB phosphorylation levels after forskolin exposure. Interestingly, the hRPE-YC cells demonstrated apparent circadian rhythms in phagocytic activities, which were abolished by carbachol or dominant-negative Bmal1 transfection. Because phagocytosis in RPE cells determines photoreceptor disc shedding, molecular clock oscillations and cytosolic Ca2+ signaling may be the driving forces for disc-shedding rhythms known in various vertebrates. In conclusion, the present study provides a cellular model to understand molecular and intracellular signaling mechanisms underlying human retinal circadian clocks. PMID:28276525

  8. Muscarinic acetylcholine receptor activation increases transcellular transport of macromolecules across mouse and human intestinal epithelium in vitro.

    PubMed

    Cameron, H L; Perdue, M H

    2007-01-01

    The intestinal epithelium acts as a barrier restricting uptake of luminal macromolecules such as dietary antigens and microbes. Here, we examined the role of cholinergic signalling in the regulation of permeability to macromolecules. Mouse jejunum was mounted in Ussing chambers and permeability was determined by measuring the flux of the antigen-sized protein, horseradish peroxidase (HRP), across the tissue. Baseline HRP permeability was significantly reduced by neural blockade with tetrodotoxin or cholinergic muscarinic antagonism with atropine, suggesting that ongoing release of endogenous acetylcholine from enteric nerves regulates barrier function. Exogenous addition of the muscarinic agonist bethanechol caused significant increases in both HRP flux and the area of HRP-containing endosomes in enterocytes. Bethanechol-enhanced HRP flux was abrogated by the M3 receptor antagonist, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), the phospholipase A(2) inhibitor quinacrine, and the cyclooxygenase inhibitor indomethacin. Complementary in vitro studies showed direct effects of bethanechol on T84 epithelial cells, where increased HRP uptake was associated with increased F-actin, and increased cytosolic phospholipase A(2) (cPLA(2)) phosphorylation. Taken together, these results provide evidence for cholinergic regulation of transepithelial transport of macromolecules, mainly mediated by activation of M3 receptors with subsequent involvement of phospholipase A(2) and cyclooxygenase products.

  9. Characterization of muscarinic receptor binding and inhibition of salivation after oral administration of tolterodine in mice.

    PubMed

    Oki, Tomomi; Maruyama, Shuji; Takagi, Yukiko; Yamamura, Henry I; Yamada, Shizuo

    2006-01-04

    The current study was undertaken to characterize the effects of oral administration of tolterodine on muscarinic receptor binding in the bladder and submaxillary gland and on salivation in mice. In the in vitro experiment, tolterodine and its metabolite (5-hydroxymethyl metabolite: 5-HM) competed concentration-dependently with [N-methyl-(3)H]-scopolamine ([(3)H]NMS) in the mouse bladder, submaxillary gland and heart, and the potencies of both agents were greater than that of oxybutynin. After oral administration of tolterodine (6.31, 21.0 micromol/kg) and oxybutynin (76.1 micromol/kg), there was a dose and time-dependent increase in K(d) values for specific [(3)H]NMS binding in the bladder, prostate, submaxillary gland, heart, colon and lung, compared with control values, suggesting significant muscarinic receptor binding in each tissue. The K(d) increase in each tissue by oral oxybutynin reached a maximum value of 0.5 h after oral administration and then rapidly declined, while that by tolterodine was greatest 2 h after the administration and it was maintained for at least 6 or 12 h, depending on the dose and on the tissue. Thus, muscarinic receptor binding of oral tolterodine was slower in onset and of a longer duration than that of oxybutynin. Also, oral oxybutynin showed relatively greater receptor binding in the submaxillary gland as compared with other tissues, but such high selectivity to the exocrine gland muscarinic receptors was not observed by oral tolterodine. Oral administration of tolterodine and oxybutynin reduced significantly the pilocarpine-induced salivary secretion in mice, and the attenuation of oral tolterodine appeared more slowly and it was more persistent than that of oral oxybutynin. The antagonistic effect of oral tolterodine on the dose-response curves to pilocarpine was significantly weaker than that of oxybutynin. These data suggest that oral tolterodine, compared with the case of oral oxybutynin, binds more selectively to muscarinic

  10. Evidence for a M1 muscarinic receptor on the endothelium of human pulmonary veins

    PubMed Central

    Walch, Laurence; Gascard, Jean-Pierre; Dulmet, Elisabeth; Brink, Charles; Norel, Xavier

    2000-01-01

    To characterize the muscarinic receptors on human pulmonary veins associated with the acetylcholine (ACh)-induced relaxation, isolated venous and arterial preparations were pre-contracted with noradrenaline (10 μM) and were subsequently challenged with ACh in the absence or presence of selective muscarinic antagonists.ACh relaxed venous preparations derived from human lung with a pD2 value of 5.82±0.09 (n=16). In venous preparations where the endothelium had been removed, the ACh relaxations were abolished (n=4). ACh relaxed arterial preparations with a pD2 value of 7.06±0.14 (n=5).Atropine (1 μM), the non selective antagonist for muscarinic receptors, inhibited ACh-induced relaxations in human pulmonary veins. The affinity value (pKB value) for atropine was: 8.64±0.10 (n=5). The selective muscarinic antagonists (darifenacin (M3), himbacine (M2,M4), methoctramine (M2) and pFHHSiD (M1,M3)) also inhibited ACh-induced relaxations in venous preparations. The pKB values obtained for these antagonists were not those predicted for the involvement of M2–5 receptors in the ACh-induced relaxation in human pulmonary veins.The pKB value for darifenacin (1 μM) was significantly greater in human pulmonary arterial (8.63±0.14) than in venous (7.41±0.20) preparations derived from three lung samples.In human pulmonary veins, the pKB values for pirenzepine (0.5 and 1 μM), a selective antagonist for M1 receptors, were: 7.89±0.24 (n=7) and 8.18±0.22 (n=5), respectively. In the venous preparations, the pKB values derived from the functional studies with all the different muscarinic antagonists used were correlated (r=0.89; P=0.04; slope=0.78) with the affinity values (pKi values) previously published for human cloned m1 receptors in CHO cells.These results suggest that the relaxations induced by ACh are due to the activation of M1 receptors on endothelial cells in isolated human pulmonary veins. PMID:10781000

  11. Muscarinic receptor subtypes controlling the cationic current in guinea-pig ileal smooth muscle

    PubMed Central

    Zholos, Alexander V; Bolton, Thomas B

    1997-01-01

    The effects of muscarinic antagonists on cationic current evoked by activating muscarinic receptors with the stable agonist carbachol were studied by use of patch-clamp recording techniques in guinea-pig single ileal smooth muscle cells. Ascending concentrations of carbachol (3–300 μM) activated the cationic conductance in a concentration-dependent manner with conductance at a maximally effective carbachol concentration (Gmax) of 27.4±1.4 nS and a mean −log EC50 of 5.12±0.03 (mean±s.e.mean) (n=114). Muscarinic antagonists with higher affinity for the M2 receptor, methoctramine, himbacine and tripitramine, produced a parallel shift of the carbachol concentration-effect curve to the right in a concentration-dependent manner with pA2 values of 8.1, 8.0 and 9.1, respectively. All M3 selective muscarinic antagonists tested, 4-DAMP, p-F-HHSiD and zamifenacin, reduced the maximal response in a concentration-dependent and non-competitive manner. This effect could be observed even at concentrations which did not produce any increase in the EC50 for carbachol. At higher concentrations M3 antagonists shifted the agonist curve to the right, increasing the EC50, and depressed the maximum conductance response. Atropine, a non-selective antagonist, produced both reduction in Gmax (M3 effect) and significant increase in the EC50 (M2 effect) in the same concentration range. The depression of the conductance by 4-DAMP, zamifenacin and atropine could not be explained by channel block as cationic current evoked by adding GTPγS to the pipette (without application of carbachol) was unaffected. The results support the hypothesis that carbachol activates M2 muscarinic receptors so initiating the opening of cationic channels which cause depolarization; this effect is potentiated by an unknown mechanism when carbachol activates M3 receptors. As an increasing fraction of M3 receptors are blocked by an antagonist, the effects on cationic current of an increasing proportion of

  12. Agonist and antagonist binding to rat brain muscarinic receptors: influence of aging

    SciTech Connect

    Gurwitz, D.; Egozi, Y.; Henis, Y.I.; Kloog, Y.; Sokolovsky, M.

    1987-03-01

    The objective of the present study was to determine the binding properties of muscarinic receptors in six brain regions in mature and old rats of both sexes by employing direct binding of (/sup 3/H)-antagonist as well as of the labeled natural neurotransmitter, (/sup 3/H)-acetylcholine (( /sup 3/H)-AcCh). In addition, age-related factors were evaluated in the modulation processes involved in agonist binding. The results indicate that as the rat ages the density of the muscarinic receptors is altered differently in the various brain regions: it is decreased in the cerebral cortex, hippocampus, striatum and olfactory bulb of both male and female rats, but is increased (58%) in the brain stem of senescent males while no significant change is observed for females. The use of the highly sensitive technique measuring direct binding of (/sup 3/H)-AcCh facilitated the separate detection of age-related changes in the two classes (high- and low-affinity) of muscarinic agonist binding sites. In old female rats the density of high-affinity (/sup 3/H)-AcCh binding sites was preserved in all tissues studied, indicating that the decreases in muscarinic receptor density observed with (/sup 3/H)-antagonist represent a loss of low-affinity agonist binding sites. In contrast, (/sup 3/H)-AcCh binding is decreased in the hypothalamus and increased in the brain stem of old male rats. These data imply sexual dimorphism of the aging process in central cholinergic mechanisms.

  13. Muscarinic cholinergic and histamine H1 receptor binding of phenothiazine drug metabolites

    SciTech Connect

    Hals, P.A.; Hall, H.; Dahl, S.G.

    1988-01-01

    In vitro binding affinities of chlorpromazine, fluphenazine, levomepromazine, perphenazine and some of their metabolites for dopamine D2 receptors, ..cap alpha../sub 1/- and ..cap alpha../sup 2/ adrenoceptors in rat brain were previously reported from out laboratories. The present study reports the in vitro binding affinities of the same compounds for muscarinic cholinergic receptors and for histamine H1 receptors in rat brain, using /sup 3/H-quinuclidinyl benzilate and /sup 3/H-mepyramine as radioligands. Chlorpromazine, levomepromazine, and their metabolites had 5-30 times higher binding affinities for muscarinic cholinergic receptors than fluphenazine, perphenazine and their metabolites. Levomepromazine was the most potent and fluphenazine the least potent of the four drugs in histamine H1 receptor binding. 7-Hydroxy levomepromazine, 3-hydroxy levomepromazine and 7-hydroxy fluphenazine had only 10% of the potency of the parent drug in histamine H1 receptor binding, while the 7-hydroxy-metabolites of chlorpromazine and perphenazine had about 75% of the potency of the parent drug in this binding system. This histamine H1 receptor binding affinities indicate that metabolites may contribute to the sedative effects of chlorpromazine and levomepromazine.

  14. Relationship between muscarinic receptor occupancy and adenylate cyclase inhibition in the rabbit myocardium

    SciTech Connect

    Ehlert, F.J.

    1985-11-01

    The muscarinic receptor-binding properties of a series of muscarinic drugs were compared with their effects on adenylate cyclase in membranes of the rabbit myocardium. When measured by competitive inhibition of (TH)-N-methylscopolamine binding, the competition curves of the various agonists were adequately described by the ternary complex model. This model assumes that the receptor can bind reversibly with a guanine nucleotide binding protein in the membrane and that the affinity of the agonist for the receptor-guanine nucleotide-binding protein complex is higher than that for the free receptor. A satisfactory fit of the ternary complex model to the data could only be achieved assuming that very little receptor is precoupled with the guanine nucleotide-binding protein in the absence of agonist. There was good agreement between the efficacy of each agonist as measured by inhibition of adenylate cyclase and the estimate of the positive cooperativity between the binding of the agonist receptor complex and the guanine nucleotide-binding protein. Guanosine 5'-triphosphate (0.1 mM) had no significant effect on the binding of (TH)N-methylscopolamine but caused an increase in the concentration of the various agonists required for half-maximal receptor occupancy. There was good correlation between efficacy as measured by inhibition of adenylate cyclase and the influence of guanosine 5'-triphosphate on binding properties.

  15. Identification of three muscarinic receptor subtypes in rat lung using binding studies with selective antagonists

    SciTech Connect

    Fryer, A.D.; El-Fakahany, E.E. )

    1990-01-01

    Heterogeneity of the muscarinic receptor population in the rat central and peripheral lung was found in competition binding experiments against ({sup 3}H)quinuclidinyl benzilate (({sup 3}H)QNB) using the selective antagonists pirenzepine, AF-DX 116 and hexahydrosiladifenidol (HHSiD). Pirenzepine displaced ({sup 3}H)QNB with low affinity from preparations of central airways indicating the absence of M{sub 1} receptors in the trachea and bronchi. Muscarinic receptors in the central airways are comprised of both M{sub 2} and M{sub 3} receptors since AF-DX 116, an M{sub 2}-selective antagonist, bound with high affinity to 70% of the available sites while HHSiD, an M{sub 3}-selective antagonist bound with high affinity to the remaining binding sites. In the peripheral lung, pirenzepine bound with high affinity to 14% of the receptor population, AF-DX 116 bound with high affinity 79% of the binding sites while HHSiD bound with high affinity to 18% of the binding sites. The presence of M{sub 1} receptors in the peripheral airways but not in the central airways was confirmed using ({sup 3}H)telenzepine, an M{sub 1} receptor ligand. ({sup 3}H)Telenzepine showed specific saturable binding to 8% of ({sup 3}H)QNB labeled binding sites in homogenates of rat peripheral lung, while there was no detectable specific binding in homogenates of rat trachea or heart.

  16. In vivo characterization of muscarinic receptors in peripheral tissues: evaluation of bladder selectivity of anticholinergic agents to treat overactive bladder.

    PubMed

    Maruyama, Shuji; Hasuike, Naoki; Suzuki, Kazuhiro; Yamada, Shizuo

    2008-06-01

    The present study was undertaken to characterize in vivo muscarinic receptors in peripheral tissues (urinary bladder, submaxillary gland, colon, stomach, heart) of mice, and further to evaluate bladder-selectivity of anticholinergic agents to treat overactive bladder. Following i.v. injection of [3H]QNB in mice, the radioactivity in peripheral tissues was exclusively detected as the unchanged form. The in vivo specific [3H]QNB binding in particulate fraction of tissue homogenates of mice showed a pharmacological specificity which characterized muscarinic receptors. Binding parameters (Kd and Bmax) for in vivo specific [3H]QNB binding differed between mouse tissues. Oral administration of oxybutynin attenuated significantly in vivo specific [3H]QNB binding in all tissues of mice. From ratios of AUCurinary bladder/AUCother tissues of time-dependent muscarinic receptor occupancy, oral oxybutynin has been shown to exert little urinary bladder selectivity. Following oral administration of propiverine, there was a significant reduction of in vivo specific [3H]QNB binding in the urinary bladder, colon and submaxillary gland, but not in the stomach and heart. From the ratios of AUCurinary bladder to AUCsubmaxillary gland or AUCheart, it has been shown that oral propiverine exerts higher selectivity to muscarinic receptors in the urinary bladder than in the submaxillary gland and heart. Similarly, tolterodine displayed high selectivity to muscarinic receptors in the urinary bladder than in the submaxillary gland. Thus, the present study has demonstrated that [3H]QNB may be a useful ligand for in vivo characterization of muscarinic receptor binding of anticholinergic agents to treat overactive bladder. Propiverine and tolterodine have exhibited in vivo selectivity of muscarinic receptor in the mouse urinary bladder rather than in the submaxillary gland, and such receptor binding specificity may be the reason of lower incidence of dry mouth.

  17. Comparative evaluation of central muscarinic receptor binding activity by oxybutynin, tolterodine and darifenacin used to treat overactive bladder.

    PubMed

    Oki, Tomomi; Kageyama, Aiko; Takagi, Yukiko; Uchida, Shinya; Yamada, Shizuo

    2007-02-01

    We characterized muscarinic receptor binding in the mouse cerebral cortex after oral administration of anticholinergic agents used to treat overactive bladder. Muscarinic receptors in the mouse cerebral cortex and bladder after oral administration of anticholinergic agents were measured using [(3)H]N-methylscopolamine. In vitro binding affinities of tolterodine and its metabolite 5-hydroxymethyl metabolite in the mouse cerebral cortex and bladder were considerably greater than those of oxybutynin and darifenacin. Also, muscarinic receptor binding affinity of oxybutynin and its metabolite N-desethyl-oxybutynin in the cerebral cortex compared with that in the bladder was 2 to 3 times higher, whereas that of tolterodine and 5-hydroxymethyl metabolite was approximately 2 times lower. Oral administration of oxybutynin (76.1 micromol/kg), tolterodine (6.31 micromol/kg) and darifenacin (59.1 micromol/kg) showed binding activity that was approximately equal to that of bladder muscarinic receptors. Oral administration of oxybutynin (76.1 micromol/kg) showed significant binding of cerebral cortical muscarinic receptors in mice, as indicated by about a 2-fold increase in K(d) values for specific [(3)H]N-methylscopolamine binding 0.5 and 2 hours later. On the other hand, tolterodine and darifenacin given at oral doses that would exert a similar extent of bladder receptor binding activity as oxybutynin showed only a low level of binding activity of central muscarinic receptors in mice. Significant binding of brain muscarinic receptors in mice was observed by the oral administration of oxybutynin but not tolterodine and darifenacin.

  18. Urinary Excretion Contributes to Long-Lasting Blockade of Bladder Muscarinic Receptors by Imidafenacin: Effect of Bilateral Ureteral Ligation.

    PubMed

    Ito, Yoshihiko; Kuraoka, Shiori; Endo, Soma; Takahashi, Ayaka; Onoue, Satomi; Yamada, Shizuo

    2017-01-01

    Imidafenacin is a potent and selective antagonist of M1 and M3 muscarinic receptors that is safe, efficacious, and well tolerated for controlling the symptoms of overactive bladder (OAB). However, the precise mechanisms responsible for the bladder-selective pharmacological effects of this agent remain unclear. The in vivo pharmacologic effects of imidafenacin result from receptor occupancy. Therefore, the present study was performed to characterize in vivo muscarinic receptor binding by tritium-labeled imidafenacin with high specific activity ([(3)H]imidafenacin) in the bladder and other tissues of mice, and to clarify the mechanisms underlying selective binding of imidafenacin to bladder muscarinic receptors. After intravenous injection of [(3)H]imidafenacin, its binding to muscarinic receptors in the bladder and other tissues of mice was assessed by a radioligand binding assay. [(3)H]Imidafenacin showed a significantly longer duration of binding to muscarinic receptors in the bladder than in other tissues, and muscarinic receptor binding of [(3)H]imidafenacin was markedly suppressed in the bladder alone after bilateral ligation of the ureters. After intravenous injection, the [(3)H]imidafenacin concentration was markedly higher in the urine than in the plasma, suggesting that urinary excretion may contribute significantly to the selective and long-lasting binding of imidafenacin to bladder muscarinic receptors. These findings suggest that the intravesicular concentration of an antimuscarinic agent and its active metabolites may have a substantial influence on its pharmacological effect and duration of action in patients with OAB. In addition, factors that modulate urine production may influence the efficacy and safety of antimuscarinic agents. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  19. Identification of M1 muscarinic receptors in pulmonary sympathetic nerves in the guinea-pig by use of pirenzepine.

    PubMed Central

    Maclagan, J.; Fryer, A. D.; Faulkner, D.

    1989-01-01

    1. The effect of pirenzepine, a muscarinic antagonist considered to be selective for M1 receptors, was studied on bronchoconstriction and bradycardia elicited by preganglionic stimulation of the parasympathetic vagal nerves and by i.v. injections of acetylcholine (ACh) in anaesthetized guinea-pigs. 2. Pirenzepine was equipotent in the heart and lung as an antagonist of the effects of i.v. ACh at postjunctional muscarinic receptors. Doses of pirenzepine in excess of 1 mumol kg-1 abolished all muscarinic responses consistent with non-selective blockade of M3 receptors on airway smooth muscle and M2 receptors on atrial cells. 3. In the lung, low doses of pirenzepine (1-100 nmol kg-1) increased vagally-induced bronchoconstriction despite concurrent partial blockade of the postjunctional receptors. This suggests blockade of neuronal muscarinic receptors. 4. Propranolol (1 mg kg-1) increased control bronchoconstrictor responses elicited by ACh and vagal stimulation but did not alter the potency of pirenzepine for postjunctional receptors in heart or lung. However, pirenzepine-induced enhancement of vagally-induced bronchoconstriction was abolished by propranolol, suggesting that pirenzepine may be an antagonist for muscarinic receptors located in the sympathetic nerves innervating airway smooth muscle. 5. These results confirm that bronchoconstrictor stimuli indirectly initiate activation of an opposing sympathetic reflex in the guinea-pig lung. This response is facilitated by muscarinic receptors located in the sympathetic nervous pathway. 6. The high potency of pirenzepine for the neuronal receptors in the sympathetic nerves suggests that these are M1 receptors. In contrast, the parasympathetic nerves innervating airway smooth muscle in this species contain M2 receptors which inhibit neurotransmission. PMID:2758228

  20. Two types of muscarinic acetylcholine receptors in Drosophila and other arthropods.

    PubMed

    Collin, Caitlin; Hauser, Frank; Gonzalez de Valdivia, Ernesto; de Valdivia, Ernesto Gonzalez; Li, Shizhong; Reisenberger, Julia; Carlsen, Eva M M; Khan, Zaid; Hansen, Niels O; Puhm, Florian; Søndergaard, Leif; Niemiec, Justyna; Heninger, Magdalena; Ren, Guilin R; Grimmelikhuijzen, Cornelis J P

    2013-09-01

    Muscarinic acetylcholine receptors (mAChRs) play a central role in the mammalian nervous system. These receptors are G protein-coupled receptors (GPCRs), which are activated by the agonists acetylcholine and muscarine, and blocked by a variety of antagonists. Mammals have five mAChRs (m1-m5). In this study, we cloned two structurally related GPCRs from the fruit fly Drosophila melanogaster, which, after expression in Chinese hamster ovary cells, proved to be muscarinic acetylcholine receptors. One mAChR (the A-type; encoded by gene CG4356) is activated by acetylcholine (EC50, 5 × 10(-8) M) and muscarine (EC50, 6 × 10(-8) M) and blocked by the classical mAChR antagonists atropine, scopolamine, and 3-quinuclidinyl-benzilate (QNB), while the other (the B-type; encoded by gene CG7918) is also activated by acetylcholine, but has a 1,000-fold lower sensitivity to muscarine, and is not blocked by the antagonists. A- and B-type mAChRs were also cloned and functionally characterized from the red flour beetle Tribolium castaneum. Recently, Haga et al. (Nature 2012, 482: 547-551) published the crystal structure of the human m2 mAChR, revealing 14 amino acid residues forming the binding pocket for QNB. These residues are identical between the human m2 and the D. melanogaster and T. castaneum A-type mAChRs, while many of them are different between the human m2 and the B-type receptors. Using bioinformatics, one orthologue of the A-type and one of the B-type mAChRs could also be found in all other arthropods with a sequenced genome. Protostomes, such as arthropods, and deuterostomes, such as mammals and other vertebrates, belong to two evolutionarily distinct lineages of animal evolution that split about 700 million years ago. We found that animals that originated before this split, such as cnidarians (Hydra), had two A-type mAChRs. From these data we propose a model for the evolution of mAChRs.

  1. Desensitization and Down Regulation of Muscarinic Acetylcholine Receptors

    DTIC Science & Technology

    1988-06-22

    decrease in the relative densities ofNMlImuscarinic receptors which have a high affinity for the selective antagonist pirenzepine . Activation of...which is readily susceptible to regu- lation by receptor agonists, while the other is resistant in this regard. In control cells, both pirenzepine and... pirenzepine and carbamylcholine with [3H]N-methylsco- polamine showed that the receptor sites remaining upon desensitization are composed mainly of pirenzepine

  2. Role of internalization of M2 muscarinic receptor via clathrin-coated vesicles in desensitization of the muscarinic K+ current in heart.

    PubMed

    Yamanushi, T T; Shui, Z; Leach, R N; Dobrzynski, H; Claydon, T W; Boyett, M R

    2007-04-01

    In the heart, ACh activates the ACh-activated K(+) current (I(K,ACh)) via the M(2) muscarinic receptor. The relationship between desensitization of I(K,ACh) and internalization of the M(2) receptor has been studied in rat atrial cells. On application of the stable muscarinic agonist carbachol for 2 h, I(K,ACh) declined by approximately 62% with time constants of 1.5 and 26.9 min, whereas approximately 83% of the M(2) receptor was internalized from the cell membrane with time constants of 2.9 and 51.6 min. Transfection of the cells with beta-adrenergic receptor kinase 1 (G protein-receptor kinase 2) and beta-arrestin 2 significantly increased I(K,ACh) desensitization and M(2) receptor internalization during a 3-min application of agonist. Internalized M(2) receptor in cells exposed to carbachol for 2 h was colocalized with clathrin and not caveolin. It is concluded that a G protein-receptor kinase 2- and beta-arrestin 2-dependent internalization of the M(2) receptor into clathrin-coated vesicles could play a major role in I(K,ACh) desensitization.

  3. A fluorescence anisotropy assay for the muscarinic M1 G-protein-coupled receptor.

    PubMed

    Huwiler, Kristin G; De Rosier, Therese; Hanson, Bonnie; Vogel, Kurt W

    2010-06-01

    In the search for new chemical entities that interact with G-proteincoupled receptors (GPCRs), assays that quantify efficacy and affinity are employed. Traditional methods for measuring affinity involve radiolabeled ligands. To address the need for homogeneous biochemical fluorescent assays to characterize orthosteric ligand affinity and dissociation rates, we have developed a fluorescence anisotropy (FA) assay for the muscarinic M1 receptor that can be conducted in a 384-well plate. We used membranes from a muscarinic M1 cell line optimized for high-throughput functional assays and the previously characterized fluorescent antagonist BODIPY FL pirenzepine. The affinities of reference compounds were determined in the competitive FA assay and compared with those obtained with a competitive filter-based radioligand-binding assay using [(3)H] N-methylscopolamine. The IC(50) values produced from the FA assay were well-correlated with the radioligand-binding K(i) values (R(2) = 0.98). The dissociation of the BODIPY FL pirenzepine was readily monitored in real time using the FA assay and was sensitive to the presence of the allosteric modulator gallamine. This M1 FA assay offers advantages over traditional radioligandbinding assays as it eliminates radioactivity while allowing investigation of orthosteric or allosteric muscarinic M1 ligands in a homogeneous format.

  4. Myocardial muscarinic receptor upregulation and normal response to isoproterenol in denervated hearts by familial amyloid polyneuropathy.

    PubMed

    Delahaye, N; Le Guludec, D; Dinanian, S; Delforge, J; Slama, M S; Sarda, L; Dollé, F; Mzabi, H; Samuel, D; Adams, D; Syrota, A; Merlet, P

    2001-12-11

    Patients with familial amyloid polyneuropathy, a rare hereditary form of amyloidosis, have progressive autonomic neuropathy. The disease usually does not induce heart failure but is associated with sudden death, conduction disturbances, and an increased risk of complications during anesthesia. Although cardiac sympathetic denervation has been clearly demonstrated, the postsynaptic status of the cardiac autonomic nervous system remains unelucidated. Twenty-one patients were studied (age, 39+/-11 years; normal coronary arteries; left ventricular ejection fraction 68+/-9%). To evaluate the density and affinity constants of myocardial muscarinic receptors, PET with (11)C-MQNB (methylquinuclidinyl benzilate), a specific hydrophilic antagonist, was used. Cardiac beta-receptor functional efficiency was studied by the heart rate (HR) response to intravenous infusion of isoproterenol (5 minutes after 2 mg of atropine, 5, 10, and 15 ng/kg per minute during 5 minutes per step). The mean muscarinic receptor density was higher in patients than in control subjects (B'(max), 35.5+/-8.9 versus 26.1+/-6.7 pmol/mL, P=0.003), without change in receptor affinity. The increase in HR after injection of atropine as well as of MQNB was lower in patients compared with control subjects despite a similar basal HR (DeltaHR after atropine, 11+/-21% versus 62+/-17%; P<0.001), consistent with parasympathetic denervation. Incremental infusion of isoproterenol induced a similar increase in HR in patients and control subjects. Cardiac autonomic denervation in familial amyloid polyneuropathy results in an upregulation of myocardial muscarinic receptors but without change in cardiac beta-receptor responsiveness to catecholamines.

  5. Muscarinic receptors mediate the endocrine-disrupting effects of an organophosphorus insecticide in zebrafish.

    PubMed

    Santos da Rosa, João Gabriel; Alcântara Barcellos, Heloísa Helena de; Fagundes, Michele; Variani, Cristiane; Rossini, Mainara; Kalichak, Fabiana; Koakoski, Gessi; Acosta Oliveira, Thiago; Idalencio, Renan; Frandoloso, Rafael; Piato, Angelo L; José Gil Barcellos, Leonardo

    2017-07-01

    The glucocorticoid cortisol, the end product of hypothalamus-pituitary-interrenal axis in zebrafish (Danio rerio), is synthesized via steroidogenesis and promotes important physiological regulations in response to a stressor. The failure of this axis leads to inability to cope with environmental challenges preventing adaptive processes in order to restore homeostasis. Pesticides and agrichemicals are widely used, and may constitute an important class of environmental pollutants when reach aquatic ecosystems and nontarget species. These chemical compounds may disrupt hypothalamus-pituitary-interrenal axis by altering synthesis, structure or function of its constituents. We present evidence that organophosphorus exposure disrupts stress response by altering the expression of key genes of the neural steroidogenesis, causing downregulation of star, hsp70, and pomc genes. This appears to be mediated via muscarinic receptors, since the muscarinic antagonist scopolamine blocked these effects. © 2017 Wiley Periodicals, Inc.

  6. Muscarinic receptor activation tunes mouse stratum oriens interneurones to amplify spike reliability

    PubMed Central

    Lawrence, J Josh; Grinspan, Zachary M; Statland, Jeffrey M; McBain, Chris J

    2006-01-01

    Cholinergic activation of hippocampal targets can initiate and sustain network oscillations in vivo and in vitro, yet the impact of cholinergic modulation on the oscillatory properties of interneurones remains virtually unexplored. Using whole cell current clamp recordings in acute hippocampal slices, we investigated the influence of muscarinic receptor (mAChR) activation on the oscillatory properties of CA1 stratum oriens (SO) interneurones in vitro. In response to suprathreshold oscillatory input, mAChR activation increased spike reliability and precision, and extended the bandwidth that interneurone firing phase-locked. These suprathreshold effects were largest at theta frequencies, indicating that mAChR activation tunes active conductances to enhance firing reliability and precision to theta frequency input. Muscarinic tuning of the intrinsic oscillatory properties of interneurones is a novel mechanism that may be crucial for the genesis of the theta rhythm. PMID:16439425

  7. [Effect of microinjections of a selective blocker of M1-muscarinic receptors pirenzepine into the neostriatum on the rat motor activity].

    PubMed

    Shapovalova, K B; Kamkina, Iu V; Mysovskiĭ, D A

    2004-02-01

    In simulated discrimination conditioned reflex of active avoidance (CRAA) in T-maze, the effect of bilateral microinjections of the muscarinic receptor M1 selective blocker pirenzepine on the CRAA formation and behaviour in the "open filed" test, was studied in rats. A sharp worsening of the CRAA learning and a significant increase in the motor activity were shown to occur in rats following the microinjections as compared with control rats. The change in the motor responses seems to account for the worsening of the CRAA learning. Another reason of the phenomenon could involve a disorder in perception of conditioned signals and their poor differentiation. The data obtained and the literature data suggest a complex character of changes induced by the blockade of the M1 muscarinic receptors of the neostriatum.

  8. Volatile anesthetics interfere with muscarinic receptor-g protein interactions in rat heart

    SciTech Connect

    Anthony, B.L.

    1987-01-01

    The influence of halothane and enflurane (0.5-8%) on muscarinic receptor binding in rat atrium was studied using (/sup 3/H) methylscopolamine ((/sup 3/H)MS). Anesthetic-gas mixtures were blown over membrane suspensions for 20 min before and during the binding assays. Halothane and enflurane increased the affinity of cardiac muscarinic receptors for (/sup 3/H)MS by slowing the rate of dissociation. These anesthetics did not affect the affinity of the receptor for carbamylcholine, but significantly reduced the sensitivity of agonist binding to regulation by guanine nucleotides. For example, the fraction of receptors displaying high affinity agonist binding was decreased by a GTP analog from 0.64 to 0.43 in the absence, but only to 0.52 in the presence of 2% halothane. The binding of a radiolabeled agonist, (/sup 3/H)oxotremorine-M, was reduced by 50% by halothane, while its sensitivity to guanine nucleotides was reduced by at least 100 fold. The diminution of the guanine nucleotide effect may reflect a stabilization of the receptor-G proteincomplex due to either a direct action on the receptor complex or to an alteration of the physical state of the membrane. It is also possible that the ability of the G protein to bind guanine nucleotides is adversely affected by anesthetic agents.

  9. Modelling of the binding site of the human m1 muscarinic receptor: Experimental validation and refinement

    NASA Astrophysics Data System (ADS)

    Bourdon, Hélène; Trumpp-Kallmeyer, Susanne; Schreuder, Herman; Hoflack, , Jan; Hibert, Marcel; Wermuth, Camille-Georges

    1997-07-01

    Our model of the human m1 muscarinic receptor has been refined on the basis of the recently published projection map of bovine rhodopsin. The refined model has a slightly different helix arrangement, which reveals the presence of an extra hydrophobic pocket located between helices 3, 4 and 5. The interaction of series of agonists and antagonists with the m1 muscarinic receptor has been studied experimentally by site-directed mutagenesis. In order to account for the observed results, three-dimensional models of m1 ligands docked in the target receptor are proposed. Qualitatively, the obtained models are in good agreement with the experimental observations. Agonists and partial agonists have a relatively small size. They can bind to the same region of the receptor using, however, different anchoring receptor residues. Antagonists are usually larger molecules, filling almost completely the same pocket as agonists. They can usually produce much stronger interactions with aromatic residues. Experimental data combined with molecular modelling studies highlight how subtle and diverse receptor-ligand interactions could be.

  10. Muscarinic receptors: their roles in disorders of the central nervous system and potential as therapeutic targets.

    PubMed

    Scarr, Elizabeth

    2012-05-01

    Phylogenetically, acetylcholine is an ancient neurochemical. Therefore, it is not surprising that cholinergic neurons project extensively throughout the central nervous system, innervating a wide range of structures within the brain. In fact, acetylcholine is involved in processes that underpin some of our most basic central functions. Both muscarinic and nicotinic receptor families, which mediate cholinergic transmission, have been implicated in the pathophysiology of psychiatric and neurological disorders. The question that remains to be definitively answered is whether or not these receptors are viable targets for the development of future therapeutic agents.

  11. Pharmacologic study of muscarinic receptor subtypes and arteriolar dilations: a comparison of conducted and local responses.

    PubMed

    Rivers, R J

    1999-03-01

    Arteriolar relaxation caused by the application of muscarinic agonists is mediated by multiple factors. One factor causes dilation only at the point of drug microapplication (local response), and a second factor causes responses remote (500 microm away) from the site of application (conducted response). This study was performed to determine if different muscarinic subtypes mediate the two responses. Arterioles of anesthetized hamster cheek pouch were studied with videomicroscopy. Muscarinic antagonists methscopolamine, scopolamine, pirenzepine, 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide), and AFDX-116 [(11-2[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5, 11-dihydro-6H-pyrido [2,3-b][1,4]benzodiazepin-6-one)] were cumulatively applied, and the K(B) for each antagonist was determined for the local and conducted responses caused by methacholine microapplication (10(-4) M, 5 s). The pK(B) (local, conducted) were not significantly different for the two responses when using scopolamine (10.5, 10.4). When the antagonist AFDX-116 (5.6, 6.3), selective for muscarinic receptor (m2) subtype was applied, the K(B) was greater for the conducted response. The pK(B) was greater, however, for the local response when the m1 subtype-selective pirenzepine (7.7, 6.9) or m3 subtype-selective 4-DAMP (10.1, 9.8) was applied. Thus the antagonist pK(B) ratio for on the local and conducted responses depends on the subtype selectivity of the antagonist. These data strongly suggest that different receptors are involved in the two responses.

  12. Differential effects of M1 muscarinic receptor blockade and nicotinic receptor blockade in the dorsomedial striatum on response reversal learning

    PubMed Central

    Tzavos, Arianna; Jih, Jane; Ragozzino, Michael E.

    2011-01-01

    The present studies determined whether blockade of M1-like muscarinic or nicotinic cholinergic receptors in the dorsomedial striatum affects acquisition or reversal learning of a response discrimination. Testing occurred in a modified cross-maze across two consecutive sessions. In the acquisition phase, a rat learned to turn to the left or to the right. In the reversal learning phase, a rat learned to turn in the opposite direction as required during acquisition. Experiment 1 investigated the effects of the M1-like muscarinic receptor antagonist, pirenzepine infused into the dorsomedial striatum on acquisition and reversal learning. Experiment 2 examined the effects of the nicotinic cholinergic antagonist, mecamylamine injected into the dorsomedial striatum on acquisition and reversal learning. Bilateral injections of pirenzepine at 10 µg, but not 1 µg, selectively impaired reversal learning. Analysis of the errors indicated that pirenzepine treatment did not impair the initial shift, but increased reversions back to the original response choice following the initial shift. Bilateral injections of mecamylamine, 6 or 18 µg, did not affect acquisition or reversal learning. The results suggest that activation of M1 muscarinic cholinergic receptors, but not nicotinic cholinergic receptors, in the dorsomedial striatum is important for facilitating the flexible shifting of response patterns. PMID:15302131

  13. Further evidence for the heterogeneity of functional muscarinic receptors in guinea pig gallbladder.

    PubMed

    Akici, A; Karaalp, A; Iskender, E; Christopoulos, A; El-Fakahany, E E; Oktay, S

    2000-01-24

    Previous studies have suggested the presence of multiple muscarinic receptor subtypes in guinea pig gallbladder smooth muscle, although the relative abundance and functional role of these subtypes remains an area of significant research efforts. The present study utilized both radioligand kinetic and functional experiments to further probe the nature of the muscarinic receptors in gallbladder smooth muscle and their mode of coupling to intra- and extra-cellular Ca(2+) sources. Dissociation kinetic studies using [3H]N-methylscopolamine ([3H]NMS) indicated that the binding profile in guinea pig gallbladder smooth muscle could not be reconciled with that expected for a single muscarinic receptor subtype, the latter determined in parallel experiments conducted on the cloned muscarinic M(1)-M(5) subtypes in Chinese hamster ovary (CHO) cells. Furthermore, comparison of the gallbladder data with the dissociation characteristics of [3H]NMS in guinea pig urinary bladder revealed a significantly different kinetic profile, with the urinary bladder, but not the gallbladder, demonstrating biphasic radioligand dissociation kinetics. In functional experiments, carbachol caused a concentration-dependent contraction of guinea pig gallbladder smooth muscle strips in Ca(2+)-free or 5 mM Sr(2+)-substituted physiological salt solutions (PSS) with amplitudes of the maximal contractions corresponding to 45.8+/-8.0% and 33.2+/-6.6% of control responses in normal PSS, respectively. Furthermore, the stimulus-response characteristics of carbachol-mediated contraction appeared significantly altered in Ca(2+)-free PSS relative to normal or Sr(2+)-substituted PSS. The antagonist, methoctramine (1x10(-7)-3x10(-5) M), exerted only a slight inhibition of carbachol (10(-5) M)-induced contractions in 5 mM Sr(2+)-substituted medium, whereas it was significantly more potent in antagonizing gallbladder contractions in response to 10(-5) M carbachol in the absence of extracellular Ca(2+). Both atropine

  14. Pirenzepine binding to membrane-bound, solubilized and purified muscarinic receptor subtypes

    SciTech Connect

    Baumgold, J.

    1986-05-01

    Muscarinic receptors were purified to near-homogeneity from bovine cortex, an area rich in the putative M1 subtype, and from bovine pons/medulla, an area rich in the putative M2 subtype. In both cases, the receptors were solubilized in digitonin and purified over an affinity column. Both the cortical and pons/medulla preparations yielded receptor proteins of 70,000 daltons. Pirenzepine binding was deduced from its competition with /sup 3/H-N-methyl scopolamine. The binding of pirenzepine to membrane-bound receptors from cortex was best described by a two site model, with approximately half the sites having a Ki of 6.4 x 10/sup -9/ M and the remaining sites having a Ki of 3.5 x 10/sup -7/ M. Membrane-bound receptors from pons/medulla bound pirenzepine according to a one-site model with a Ki of 1.1 x 10/sup -7/ M. After solubilization the two-site binding of cortical receptors became a one-site binding, Ki = 1.1 x 10/sup -7/M. This value was still five-fold lower than that of soluble receptors from pons/medulla. After purification however the affinity of pirenzepine for the pons/medulla receptor increased so that the two putative subtypes bound pirenzepine with approximately the same affinity. These findings suggest that the different pirenzepine binding characteristics used to define muscarinic receptor subtypes are not inherent in the receptor protein itself but may be due to coupling factors associated with the receptor.

  15. M1 and m2 muscarinic receptor subtypes regulate antidepressant-like effects of the rapidly acting antidepressant scopolamine.

    PubMed

    Witkin, J M; Overshiner, C; Li, X; Catlow, J T; Wishart, G N; Schober, D A; Heinz, B A; Nikolayev, A; Tolstikov, V V; Anderson, W H; Higgs, R E; Kuo, M-S; Felder, C C

    2014-11-01

    Scopolamine produces rapid and significant symptom improvement in patients with depression, and most notably in patients who do not respond to current antidepressant treatments. Scopolamine is a nonselective muscarinic acetylcholine receptor antagonist, and it is not known which one or more of the five receptor subtypes in the muscarinic family are mediating these therapeutic effects. We used the mouse forced-swim test, an antidepressant detecting assay, in wild-type and transgenic mice in which each muscarinic receptor subtype had been genetically deleted to define the relevant receptor subtypes. Only the M1 and M2 knockout (KO) mice had a blunted response to scopolamine in the forced-swim assay. In contrast, the effects of the tricyclic antidepressant imipramine were not significantly altered by gene deletion of any of the five muscarinic receptors. The muscarinic antagonists biperiden, pirenzepine, and VU0255035 (N-[3-oxo-3-[4-(4-pyridinyl)-1-piper azinyl]propyl]-2,1,3-benzothiadiazole-4-sulfonamide) with selectivity for M1 over M2 receptors also demonstrated activity in the forced-swim test, which was attenuated in M1 but not M2 receptor KO mice. An antagonist with selectivity of M2 over M1 receptors (SCH226206 [(2-amino-3-methyl-phenyl)-[4-[4-[[4-(3 chlorophenyl)sulfonylphenyl]methyl]-1-piperidyl]-1-piperidyl]methanone]) was also active in the forced-swim assay, and the effects were deleted in M2 (-/-) mice. Brain exposure and locomotor activity in the KO mice demonstrated that these behavioral effects of scopolamine are pharmacodynamic in nature. These data establish muscarinic M1 and M2 receptors as sufficient to generate behavioral effects consistent with an antidepressant phenotype and therefore as potential targets in the antidepressant effects of scopolamine.

  16. M1 and M2 muscarinic receptors mediate excitation and inhibition of guinea-pig intracardiac neurones in culture.

    PubMed Central

    Allen, T G; Burnstock, G

    1990-01-01

    -pig intracardiac neurones results from reduction of a potassium conductance similar to the M-conductance, through activation of M1 muscarinic receptors. The hyperpolarization results from an increase in potassium conductance, through activation of M2 muscarinic receptors. PMID:2352188

  17. Comparative characterization of lung muscarinic receptor binding after intratracheal administration of tiotropium, ipratropium, and glycopyrrolate.

    PubMed

    Ogoda, Masaki; Niiya, Ryo; Koshika, Tadatsura; Yamada, Shizuo

    2011-01-01

    The aim of the current study was to characterize comparatively the binding of muscarinic receptor in the lung of rats intratracheally administered anticholinergic agents (tiotropium, ipratropium, glycopyrrolate) used clinically to treat chronic obstructive pulmonary disease (COPD) and asthma. Binding parameters of [N-methyl-(3)H]scopolamine methyl chloride ([(3)H]NMS) were determined in tissues (lung, bladder, submaxillary gland) of rats intratracheally administered tiotropium, ipratropium, and glycopyrrolate. The in vitro binding affinity of tiotropium for the receptors was 10-11-fold higher than those of ipratropium and glycopyrrolate. Intratracheal administration of tiotropium (0.6-6.4 nmol/kg) caused sustained (lasting at least 24 h) increase in the apparent dissociation constant (K(d)) for [(3)H]NMS binding in rat lung compared with the control value. Concomitantly, there was a long-lasting decrease in the maximal number of binding sites (B(max)) for [(3)H]NMS. Similary, ipratropium and glycopyrrolate at 7.3 and 7.5 nmol/kg, respectively, brought about a significant increase in K(d) for [(3)H]NMS binding. The effect by ipratropium was observed at 2 h but not 12 h, and that by glycopyrrolate lasted for 24 h. Both agents had little influence on the muscarinic receptors in the bladder and submaxillary gland. The present study provides the first evidence that tiotropium, ipratropium, and glycopyrrolate administered intratracheally in rats selectively bound muscarinic receptors of the lung, and tiotropium and glycopyrrolate had a much longer-lasting effect than ipratropium.

  18. Distribution of muscarinic acetylcholine receptors on processes of isolated retinal cells.

    PubMed

    Townes-Anderson, E; Vogt, B A

    1989-12-15

    Binding of propylbenzilylcholine mustard, a muscarinic acetylcholine receptor antagonist, to isolated retinal cells was examined with light microscopic autoradiography. Dissociation of the adult tiger salamander retina yielded identifiable rod, cone, horizontal, bipolar, amacrine/ganglion, and Müller cells. Preservation of fine structure was assessed with conventional electron microscopy. For all cell types, the plasmalemma was intact and free of adhering debris; in addition, presynaptic ribbon complexes were present in photoreceptor and bipolar axon terminals indicating that synaptic structures were retained. Specific binding to cell bodies and processes was analyzed separately by using morphometric and statistical techniques. The highest grain densities occurred on processes of amacrine/ganglion cells and axons and 2 degrees and 3 degrees dendrites of bipolar neurons. Bipolar cells, however, seemed to be a heterogeneous population because there was great variation in the density of binding sites on both their axons and distal dendrites. Intermediate levels of binding were found on bipolar 1 degree dendrites and horizontal cells. No specific binding was detected on Müller cells and most parts of photoreceptors. Comparisons between cells showed that grain densities were similar for bipolar axons and amacrine/ganglion cell processes but bipolar dendrites were richer in binding sites than horizontal cell dendrites. Thus, muscarinic receptors in the salamander retina are located on amacrine/ganglion, bipolar, and horizontal cells and primarily confined to the processes which compose the two synaptic layers. In the inner plexiform layer, muscarinic receptors reside on processes from all three inner retinal neurons: in the outer synaptic layer, receptors are only on second-order cells and are more numerous along bipolar than horizontal cell dendrites.

  19. The prelimbic cortex muscarinic M₃ receptor-nitric oxide-guanylyl cyclase pathway modulates cardiovascular responses in rats.

    PubMed

    Fassini, Aline; Antero, Leandro S; Corrêa, Fernando M A; Joca, Sâmia R; Resstel, Leonardo B M

    2015-05-01

    The prelimbic cortex (PL), a limbic structure, sends projections to areas involved in the control of cardiovascular responses. Stimulation of the PL with acetylcholine (ACh) evokes depressor and tachycardiac responses mediated by local PL muscarinic receptors. Early studies demonstrated that stimulation of muscarinic receptors induced nitric oxide (NO) synthesis and cyclic guanosine cyclic monophosphate (cGMP) formation. Hence, this study investigates which PL muscarinic receptor subtype is involved in the cardiovascular response induced by ACh and tests the hypothesis that cardiovascular responses caused by muscarinic receptor stimulation in the PL are mediated by local NO and cGMP formation. PL pretreatment with J104129 (an M3 receptor antagonist) blocked the depressor and tachycardiac response evoked by injection of ACh into the PL. Pretreatment with either pirenzepine (an M1 receptor antagonist) or AF-DX 116 (an M2 and M4 receptor antagonist) did not affect cardiovascular responses evoked by ACh. Moreover, similarly to the antagonism of PL M3 receptors, pretreatment with N(ω)-propyl-L-arginine (an inhibitor of neuronal NO synthase), carboxy-PTIO(S)-3-carboxy-4-hydroxyphenylglicine (an NO scavenger), or 1H-[1,2,4]oxadiazolol-[4,3-a]quinoxalin-1-one (a guanylate cyclase inhibitor) blocked both the depressor and the tachycardiac response evoked by ACh. The current results demonstrate that cardiovascular responses evoked by microinjection of ACh into the PL are mediated by local activation of the M3 receptor-NO-guanylate cyclase pathway.

  20. Ligands for SPECT and PET imaging of muscarinic-cholinergic receptors of the heart and brain

    SciTech Connect

    Knapp, F.F. Jr.; McPherson, D.W.; Luo, H.

    1995-06-01

    Interest in the potential use of cerebral SPECT and PET imaging for determination of the density and activity of muscarinic-cholinergic receptors (mAChR) has been stimulated by the changes in these receptors which occur in many neurological diseases. In addition, the important involvement of mAChR in modulating negative inotropic cardiac activity suggests that such receptor ligands may have important applications in evaluation of changes which may occur in cardiac disease. In this paper, the properties of several key muscarinic receptor ligands being developed or which have been used for clinical SPECT and PET are discussed. In addition, the ORNL development of the new iodinated IQNP ligand based on QNB and the results of in vivo biodistribution studies in rats, in vitro competitive binding studies and ex vivo autoradiographic experiments are described. The use of radioiodinated IQNP may offer several advantages in comparison to IQNB because of its easy and high yield preparation and high brain uptake and the potential usefulness of the {open_quotes}partial{close_quotes} subtype selective IONP isomers. We also describe the development of new IQNP-type analogues which offer the opportunity for radiolabeling with positron-emitting radioisotopes (carbon-11, fluorine-18 and bromine-76) for potential use with PET.

  1. Alterations in alpha-adrenergic and muscarinic cholinergic receptor binding in rat brain following nonionizing radiation

    SciTech Connect

    Gandhi, V.C.; Ross, D.H.

    1987-01-01

    Microwave radiation produces hyperthermia. The mammalian thermoregulatory system defends against changes in temperature by mobilizing diverse control mechanisms. Neurotransmitters play a major role in eliciting thermoregulatory responses. The involvement of adrenergic and muscarinic cholinergic receptors was investigated in radiation-induced hyperthermia. Rats were subjected to radiation at 700 MHz frequency and 15 mW/cm/sup 2/ power density and the body temperature was raised by 2.5 degrees C. Of six brain regions investigated only the hypothalamus showed significant changes in receptor states, confirming its pivotal role in thermoregulation. Adrenergic receptors, studied by (/sup 3/H)clonidine binding, showed a 36% decrease in binding following radiation after a 2.5 degrees C increase in body temperature, suggesting a mechanism to facilitate norepinephrine release. Norepinephrine may be speculated to maintain thermal homeostasis by activating heat dissipation. Muscarinic cholinergic receptors, studied by (3H)quinuclidinyl benzilate binding, showed a 65% increase in binding at the onset of radiation. This may be attributed to the release of acetylcholine in the hypothalamus in response to heat cumulation. The continued elevated binding during the period of cooling after radiation was shut off may suggest the existence of an extra-hypothalamic heat-loss pathway.

  2. Proliferative actions of muscarinic receptors expressed in macrophages derived from normal and tumor bearing mice.

    PubMed

    de la Torre, Eulalia; Genaro, Ana M; Ribeiro, María L; Pagotto, Romina; Pignataro, Omar P; Sales, María E

    2008-02-01

    Macrophages (Mps) are essential cellular components of the innate immune system. They are released from the bone marrow as immature monocytes and after circulating in the blood stream, migrate into tissues to undergo final differentiation into resident Mps. In general terms Mps behavior in breast tumors, was described as being either for or against tumor growth. Under certain well defined circumstances Mps are able to kill cells in two ways: direct tumor cytotoxicity or antibody dependent cytotoxicity. We had previously demonstrated that peritoneal Mps from LMM3 mammary tumor bearing mice (TMps) enhanced in vivo the LMM3 induced angiogenesis, promoting tumor growth while Mps from normal BALB/c mice (NMps) did not. In this work, we demonstrate that Mps, expressing functional muscarinic acetylcholine receptors, are able to proliferate in vitro in response to the muscarinic agonist carbachol. These peritoneal cells use two distinct metabolic pathways: TMps are primed by tumor presence and they proliferate mainly by activating arginase pathway and by producing high levels of prostaglandin E(2) via M(1)-M(3) receptors activation. In NMps, carbachol stimulates M(2) receptors function, triggering protein kinase C activity and induces moderate prostaglandin E(2) liberation via M(1) receptor.

  3. 5-HT1A and muscarinic acetylcholine receptors jointly regulate passive avoidance behavior.

    PubMed

    Riekkinen, P

    1994-09-01

    The present study was designed to investigate the effects of combined stimulation of 5-HT1A or 5-HT2 receptors and blockade of muscarinic acetylcholine receptors on passive avoidance behavior. Administration of 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), a 5-HT1A receptor agonist, and 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), a 5-HT2 receptor agonist, impaired passive avoidance acquisition (pre-training injections) and consolidation (post-training injections) performance. Ketanserin, a 5-HT2 receptor antagonist, blocked the performance-impairing effect of DOI on passive avoidance consolidation. Interestingly, 5-HT receptor agonists may affect passive avoidance consolidation only during the immediate post-training period, as passive avoidance testing performance was not modulated by 8-OH-DPAT or DOI injected 30 min after the training trial. Furthermore, passive avoidance retention (pre-testing injections) performance was impaired only by the highest dose of 8-OH-DPAT, and DOI had no effect on passive avoidance retention. Next, the effects of combined 5-HT and acetylcholine receptor manipulations on passive avoidance behavior were studied. The effects on passive avoidance behavior of a combination of subthreshold doses of scopolamine, a muscarinic acetylcholine receptor antagonist, and 8-OH-DPAT were compared to those of a single high dose of scopolamine. A combination of small doses of scopolamine and 8-OH-DPAT impaired acquisition and consolidation of passive avoidance performance, but a single high dose of scopolamine impaired only acquisition performance. The small dose of 8-OH-DPAT also aggravated medial septal lesion-induced passive avoidance acquisition and consolidation failure. The combination of small doses of scopolamine and DOI had no effect on passive avoidance behavior. Peripherally acting scopolamine methylbromide alone or in combination with 8-OH-DPAT had no effect on passive avoidance performance. Motor activity in a swimming pool

  4. Ovulation requires the activation on proestrus of M₁ muscarinic receptors in the left ovary.

    PubMed

    Cruz, M E; Flores, A; Alvarado, B E; Hernández, C G; Zárate, A; Chavira, R; Cárdenas, M; Arrieta-Cruz, I; Gutiérrez-Juárez, R

    2015-08-01

    We analyzed the effects of chemically blocking type 1 muscarinic receptors (M1R) on either the left or right ovary on ovulation rate, number of ova shed and steroid hormones levels. M1R were unilaterally blocked in ovary with the M1R selective antagonist pirenzepine (PZP). PZP was delivered into the bursa ovarica of the left or right ovary of adult rats at 13:00 h on proestrus day. PZP treatment in the left but not in the right ovary blocked ovulation. PZP did not modify the number of ova shed, nor progesterone or 17β-estradiol serum levels. The surge of luteinizing hormone levels was diminished while that of follicle-stimulating hormone did not change in animals treated with PZP in the left ovary. Interestingly, treatment with either synthetic luteinizing hormone-releasing hormone or human chorionic gonadotropin 1 h after PZP administration in the left ovary restored ovulation in both ovaries. The presence of M1R protein in the theca cells of the ovarian follicles as well as in cells of the corpus luteum was detected on proestrus day. These results suggest that M1R activation in the left ovary is required for pre-ovulatory gonadotropin-releasing hormone (GnRH) secretion and ovulation. Furthermore, these results also suggest that M1R in the left ovary might be regulating ovulation asymmetrically through a stimulatory neural signal relayed to the hypothalamus via the vagus nerve to induce the GnRH secretion which then triggers ovulation.

  5. Muscarinic acetylcholine receptor-mediated effects in slices from human epileptogenic cortex.

    PubMed

    Gigout, S; Wierschke, S; Lehmann, T-N; Horn, P; Dehnicke, C; Deisz, R A

    2012-10-25

    Acetylcholine has been implicated in higher cortical functions such as learning, memory and cognition, yet the cellular effects of muscarinic acetylcholine receptor (mAChR) activation are poorly understood in the human cortex. Here we investigated the effect of the mAChR agonist carbachol (CCh) and various mAChR antagonists in human cortical slices (from tissue removed during neurosurgical treatment of epilepsy) by intracellular and extracellular recordings. CCh increased neuronal firing, which was antagonised by atropine (non-selective mAChR antagonist) and pirenzepine (M(1)/M(4) mAChRs antagonist) when applied before or after CCh application. AF-DX 116 (M(2)/M(4) mAChRs antagonist) had no effect on CCh-induced increase of firing. CCh also reduced evoked excitatory postsynaptic potentials (EPSP), and the CCh-induced depression of EPSP was fully reversed by atropine. Pirenzepine reversed the depression of CCh on EPSP, but failed to prevent the depression when applied before CCh. AF-DX 116 prevented the CCh-induced depression of evoked EPSP when applied before CCh. CCh also depressed GABAergic transmission and this effect was antagonised by AF-DX 116. Xanomeline (M(1)/M(4) mAChR agonist) increased neuronal firing and decreased EPSP, but had no effect on GABAergic transmission. Reduction (with linopirdine) and enhancement (with retigabine) of the M-current (mediated by K(V)7 channels), increased and decreased neuronal firing, respectively, but had marginal effects on the evoked EPSP. Our results indicate that three pharmacologically distinct mAChRs modulate neuronal firing, glutamatergic and GABAergic transmissions in the human epileptogenic neocortex. The data are discussed towards possible implications of altered mAChR signalling in hyperexcitability and cognitive functions in the human neocortex. Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

  6. Tuning the allosteric regulation of artificial muscarinic and dopaminergic ligand-gated potassium channels by protein engineering of G protein-coupled receptors

    PubMed Central

    Moreau, Christophe J.; Revilloud, Jean; Caro, Lydia N.; Dupuis, Julien P.; Trouchet, Amandine; Estrada-Mondragón, Argel; Nieścierowicz, Katarzyna; Sapay, Nicolas; Crouzy, Serge; Vivaudou, Michel

    2017-01-01

    Ligand-gated ion channels enable intercellular transmission of action potential through synapses by transducing biochemical messengers into electrical signal. We designed artificial ligand-gated ion channels by coupling G protein-coupled receptors to the Kir6.2 potassium channel. These artificial channels called ion channel-coupled receptors offer complementary properties to natural channels by extending the repertoire of ligands to those recognized by the fused receptors, by generating more sustained signals and by conferring potassium selectivity. The first artificial channels based on the muscarinic M2 and the dopaminergic D2L receptors were opened and closed by acetylcholine and dopamine, respectively. We find here that this opposite regulation of the gating is linked to the length of the receptor C-termini, and that C-terminus engineering can precisely control the extent and direction of ligand gating. These findings establish the design rules to produce customized ligand-gated channels for synthetic biology applications. PMID:28145461

  7. The Role of Hippocampal NMDA Receptors in Long-Term Emotional Responses following Muscarinic Receptor Activation.

    PubMed

    Hoeller, Alexandre A; Costa, Ana Paula R; Bicca, Maíra A; Matheus, Filipe C; Lach, Gilliard; Spiga, Francesca; Lightman, Stafford L; Walz, Roger; Collingridge, Graham L; Bortolotto, Zuner A; de Lima, Thereza C M

    2016-01-01

    Extensive evidence indicates the influence of the cholinergic system on emotional processing. Previous findings provided new insights into the underlying mechanisms of long-term anxiety, showing that rats injected with a single systemic dose of pilocarpine--a muscarinic receptor (mAChR) agonist--displayed persistent anxiogenic-like responses when evaluated in different behavioral tests and time-points (24 h up to 3 months later). Herein, we investigated whether the pilocarpine-induced long-term anxiogenesis modulates the HPA axis function and the putative involvement of NMDA receptors (NMDARs) following mAChRs activation. Accordingly, adult male Wistar rats presented anxiogenic-like behavior in the elevated plus-maze (EPM) after 24 h or 1 month of pilocarpine injection (150 mg/kg, i.p.). In these animals, mAChR activation disrupted HPA axis function inducing a long-term increase of corticosterone release associated with a reduced expression of hippocampal GRs, as well as consistently decreased NMDAR subunits expression. Furthermore, in another group of rats injected with memantine--an NMDARs antagonist (4 mg/kg, i.p.)--prior to pilocarpine, we found inhibition of anxiogenic-like behaviors in the EPM but no further alterations in the pilocarpine-induced NMDARs downregulation. Our data provide evidence that behavioral anxiogenesis induced by mAChR activation effectively yields short- and long-term alterations in hippocampal NMDARs expression associated with impairment of hippocampal inhibitory regulation of HPA axis activity. This is a novel mechanism associated with anxiety-like responses in rats, which comprise a putative target to future translational studies.

  8. Characterization of the novel positive allosteric modulator, LY2119620, at the muscarinic M(2) and M(4) receptors.

    PubMed

    Croy, Carrie H; Schober, Douglas A; Xiao, Hongling; Quets, Anne; Christopoulos, Arthur; Felder, Christian C

    2014-07-01

    The M(4) receptor is a compelling therapeutic target, as this receptor modulates neural circuits dysregulated in schizophrenia, and there is clinical evidence that muscarinic agonists possess both antipsychotic and procognitive efficacy. Recent efforts have shifted toward allosteric ligands to maximize receptor selectivity and manipulate endogenous cholinergic and dopaminergic signaling. In this study, we present the pharmacological characterization of LY2119620 (3-amino-5-chloro-N-cyclopropyl-4-methyl-6-[2-(4-methylpiperazin-1-yl)-2-oxoethoxy] thieno[2,3-b]pyridine-2-carboxamide), a M(2)/M(4) receptor-selective positive allosteric modulator (PAM), chemically evolved from hits identified through a M4 allosteric functional screen. Although unsuitable as a therapeutic due to M(2) receptor cross-reactivity and, thus, potential cardiovascular liability, LY2119620 surpassed previous congeners in potency and PAM activity and broadens research capabilities through its development into a radiotracer. Characterization of LY2119620 revealed evidence of probe dependence in both binding and functional assays. Guanosine 5'-[γ-(35)S]-triphosphate assays displayed differential potentiation depending on the orthosteric-allosteric pairing, with the largest cooperativity observed for oxotremorine M (Oxo-M) LY2119620. Further [(3)H]Oxo-M saturation binding, including studies with guanosine-5'-[(β,γ)-imido]triphosphate, suggests that both the orthosteric and allosteric ligands can alter the population of receptors in the active G protein-coupled state. Additionally, this work expands the characterization of the orthosteric agonist, iperoxo, at the M(4) receptor, and demonstrates that an allosteric ligand can positively modulate the binding and functional efficacy of this high efficacy ligand. Ultimately, it was the M(2) receptor pharmacology and PAM activity with iperoxo that made LY2119620 the most suitable allosteric partner for the M(2) active-state structure recently solved

  9. Type 3 muscarinic receptors contribute to intestinal mucosal homeostasis and clearance of nippostrongylus brasiliensis through induction of Th2 cytokines

    USDA-ARS?s Scientific Manuscript database

    Despite increased appreciation for the role of nicotinic receptors in the modulation of and response to inflammation, the contribution of muscarinic receptors to mucosal homeostasis, clearance of enteric pathogens, and modulation of immune cell function remains relatively undefined. Uninfected and N...

  10. Effects of dopamine D1-like and D2-like antagonists on cocaine discrimination in muscarinic receptor knockout mice.

    PubMed

    Thomsen, Morgane; Caine, Simon Barak

    2016-04-05

    Muscarinic and dopamine brain systems interact intimately, and muscarinic receptor ligands, like dopamine ligands, can modulate the reinforcing and discriminative stimulus (S(D)) effects of cocaine. To enlighten the dopamine/muscarinic interactions as they pertain to the S(D) effects of cocaine, we evaluated whether muscarinic M1, M2 or M4 receptors are necessary for dopamine D1 and/or D2 antagonist mediated modulation of the S(D) effects of cocaine. Knockout mice lacking M1, M2, or M4 receptors, as well as control wild-type mice and outbred Swiss-Webster mice, were trained to discriminate 10mg/kg cocaine from saline in a food-reinforced drug discrimination procedure. Effects of pretreatments with the dopamine D1 antagonist SCH 23390 and the dopamine D2 antagonist eticlopride were evaluated. In intact mice, both SCH 23390 and eticlopride attenuated the cocaine discriminative stimulus effect, as expected. SCH 23390 similarly attenuated the cocaine discriminative stimulus effect in M1 knockout mice, but not in mice lacking M2 or M4 receptors. The effects of eticlopride were comparable in each knockout strain. These findings demonstrate differences in the way that D1 and D2 antagonists modulate the S(D) effects of cocaine, D1 modulation being at least partially dependent upon activity at the inhibitory M2/M4 muscarinic subtypes, while D2 modulation appeared independent of these systems. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Short-term regulation of muscarinic acetylcholine receptors: An assessment utilizing mouse brain and mouse neuroblastoma cells

    SciTech Connect

    Cioffi, C.L.

    1988-01-01

    The effects of muscarinic agonists and diisopropylfluorophosphate (DFP) on muscarinic receptor density and muscarinic receptor-mediated responses was assessed in mouse brain and mouse neuroblastoma cells (clone N1E-115). Utilizing the antagonist ({sup 3}H)quinuclidinyl benzilate (({sup 3}H)QNB), there was no difference in the maximal binding capacity (B{sub max}) or equilibrium dissociation constant (K{sub d}) between untreated and 24 hour DFP-treated mice. However, one administration of DFP produced a 24% and 33% decrease in B{sub max} measured by ({sup 3}H)N-methylscopolamine (({sup 3}H)NMS) after 18 and 24 hours which was rapidly reversible within 36 hours after DFP treatment. The loss of ({sup 3}H)NMS binding sites following acute DFP treatment was not accompanied by a change in a particular muscarinic receptor binding conformation. Furthermore, the magnitude of muscarinic receptor-mediated phosphoinositide hydrolysis was unchanged following short-term DFP treatment.

  12. Comparison of two radiolabeled quinuclidinyl benzilate ligands for the characterization of the human peripheral lung muscarinic receptor

    SciTech Connect

    Joad, J.P.; Casale, T.B.

    1987-09-28

    Quinuclidinyl benzilate, a muscarinic antagonist, has previously been used in its tritiated form ((/sup 3/H)-QNB) to study the lung muscarinic receptor. The authors investigated whether a newer iodinated form of QNB ((/sup 125/I)-QNB) of higher specific activity would be an appropriate ligand to study the human peripheral lung muscarinic receptor. Both the tritiated and iodinated ligands bound specifically to human lung at 23/sup 0/C. At 37/sup 0/C the specific binding of (/sup 3/H)-QNB increased slightly, but no specific binding of (/sup 125/I)-QNB was found. The data from multiple equilibrium binding experiments covering a wide range of radiolabeled QNB concentrations were combined and analyzed using the computer modeling program, LIGAND. The tritiated QNB identified a single affinity human lung binding site with a Kd of 46 +/- 9 pM. The iodinated QNB identified a single higher affinity human lung binding site of much smaller quantity. Competition studies comparing the binding of unlabeled QNB relative to labeled QNB indicated that unlabeled QNB had the same Kd as that measured for (/sup 3/H)-QNB, but a 5 log greater Kd than that measured for (/sup 125/I)-QNB. Other muscarinic receptor agonists and antagonists competed with (/sup 3/H)QNB, but not (/sup 125/I)-QNB for binding to muscarinic receptors with the expected magnitude and rank order of potency. 17 references, 2 figures, 2 tables.

  13. Organophosphorus Pesticides Decrease M2 Muscarinic Receptor Function in Guinea Pig Airway Nerves via Indirect Mechanisms

    PubMed Central

    Proskocil, Becky J.; Bruun, Donald A.; Thompson, Charles M.; Fryer, Allison D.; Lein, Pamela J.

    2010-01-01

    Background Epidemiological studies link organophosphorus pesticide (OP) exposures to asthma, and we have shown that the OPs chlorpyrifos, diazinon and parathion cause airway hyperreactivity in guinea pigs 24 hr after a single subcutaneous injection. OP-induced airway hyperreactivity involves M2 muscarinic receptor dysfunction on airway nerves independent of acetylcholinesterase (AChE) inhibition, but how OPs inhibit neuronal M2 receptors in airways is not known. In the central nervous system, OPs interact directly with neurons to alter muscarinic receptor function or expression; therefore, in this study we tested whether the OP parathion or its oxon metabolite, paraoxon, might decrease M2 receptor function on peripheral neurons via similar direct mechanisms. Methodology/Principal Findings Intravenous administration of paraoxon, but not parathion, caused acute frequency-dependent potentiation of vagally-induced bronchoconstriction and increased electrical field stimulation (EFS)-induced contractions in isolated trachea independent of AChE inhibition. However, paraoxon had no effect on vagally-induced bradycardia in intact guinea pigs or EFS-induced contractions in isolated ileum, suggesting mechanisms other than pharmacologic antagonism of M2 receptors. Paraoxon did not alter M2 receptor expression in cultured cells at the mRNA or protein level as determined by quantitative RT-PCR and radio-ligand binding assays, respectively. Additionally, a biotin-labeled fluorophosphonate, which was used as a probe to identify molecular targets phosphorylated by OPs, did not phosphorylate proteins in guinea pig cardiac membranes that were recognized by M2 receptor antibodies. Conclusions/Significance These data indicate that neither direct pharmacologic antagonism nor downregulated expression of M2 receptors contributes to OP inhibition of M2 function in airway nerves, adding to the growing evidence of non-cholinergic mechanisms of OP neurotoxicity. PMID:20479945

  14. Modulation of direct pathway striatal projection neurons by muscarinic M₄-type receptors.

    PubMed

    Hernández-Flores, Teresa; Hernández-González, Omar; Pérez-Ramírez, María B; Lara-González, Esther; Arias-García, Mario A; Duhne, Mariana; Pérez-Burgos, Azucena; Prieto, G Aleph; Figueroa, Alejandra; Galarraga, Elvira; Bargas, José

    2015-02-01

    Models of basal ganglia (BG) function posit a dynamic balance between two classes of striatal projection neurons (SPNs): direct pathway neurons (dSPNs) that facilitate movements, and indirect pathway neurons (iSPNs) that repress movement execution. Two main modulatory transmitters regulate the output of these neurons: dopamine (DA) and acetylcholine (ACh). dSPNs express D1-type DA, M1-and M4-type ACh receptors, while iSPNs express D2-type DA and M1-type ACh receptors. Actions of M1-, D1-, and D2-receptors have been extensively reported, but we still ignore most actions of muscarinic M4-type receptors. Here, we used whole-cell recordings in acutely dissociated neurons, pharmacological tools such as mamba-toxins, and BAC D(1 or 2)-eGFP transgenic mice to show that activation of M4-type receptors with bath applied muscarine enhances Ca(2+)-currents through CaV1-channels in dSPNs and not in iSPNs. This action increases excitability of dSPNs after both direct current injection and synaptically driven stimulation. The increases in Ca(2+)-current and excitability were blocked specifically by mamba toxin-3, suggesting mediation via M4-type receptors. M4-receptor activation also increased network activity of dSPNs but not of iSPNs as seen with calcium-imaging techniques. Moreover, actions of D1-type and M4-type receptors may add to produce a larger enhancement of excitability of dSPNs or, paradoxically, oppose each other depending on the order of their activation. Possible implications of these findings are discussed. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. The muscarinic receptor agonist xanomeline has an antipsychotic-like profile in the rat.

    PubMed

    Stanhope, K J; Mirza, N R; Bickerdike, M J; Bright, J L; Harrington, N R; Hesselink, M B; Kennett, G A; Lightowler, S; Sheardown, M J; Syed, R; Upton, R L; Wadsworth, G; Weiss, S M; Wyatt, A

    2001-11-01

    The muscarinic receptor agonist xanomeline was examined and compared with the antipsychotics clozapine and/or haloperidol in the following in vivo rat models: apomorphine-induced disruption of prepulse inhibition (PPI), amphetamine-induced hyperlocomotion, and the conditioned emotional response (CER) test. The effects of xanomeline were also assessed ex vivo on dopamine turnover in the rat medial prefrontal cortex. Under conditions of varying dose and prepulse intensity, xanomeline, like haloperidol, had no effect on PPI. In contrast, the muscarinic receptor antagonist scopolamine and the muscarinic receptor agonist pilocarpine both induced significant dose-dependent deficits in PPI. Haloperidol and xanomeline, but not pilocarpine, dose dependently reversed apomorphine-induced disruption of PPI. Thus, xanomeline induced a clear antipsychotic-like effect in PPI, whereas pilocarpine appeared to induce a psychotomimetic-like effect. Xanomeline attenuated amphetamine-induced hyperactivity at doses that had no effect on spontaneous activity, possibly indicating a separation between attenuation of limbic hyperdopaminergic function and the induction of hypolocomotion. Haloperidol and clozapine also reversed amphetamine-induced hyperlocomotion, but at similar doses to those that reduced spontaneous locomotion. Clozapine, but not haloperidol had an anxiolytic-like effect in the CER test. The effects of xanomeline in the CER test were similar to those of clozapine, although at the anxiolytic dose it tended to disrupt baseline levels of lever pressing. Finally, haloperidol, clozapine, pilocarpine, and xanomeline, all induced an increase in dopamine turnover in medial prefrontal cortex. The antipsychotic-like effects of xanomeline in the animal models used here suggest that it may be a useful treatment for psychosis.

  16. Muscarinic acetylcholine receptors regulating cell cycle progression are expressed in human gingival keratinocytes.

    PubMed

    Arredondo, J; Hall, L L; Ndoye, A; Chernyavsky, A I; Jolkovsky, D L; Grando, S A

    2003-02-01

    We have previously reported the presence in human gingival keratinocytes (GKC) of choline acetyltransferase, the acetylcholine (ACh) synthesizing enzyme, acetylcholinesterase, the ACh degrading enzyme, and alpha 3, alpha 5, alpha 7, beta 2 as well as alpha 9 nicotinic ACh receptor subunits. To expand the knowledge about the role of ACh in oral biology, we investigated the presence of the muscarinic ACh receptor (mAChR) subtypes in GKC. RT-PCR demonstrated the presence of m2, m3, m4, and m5 mRNA transcripts. Synthesis of the respective proteins was verified by immunoblotting with the subtype-specific antibodies that revealed receptor bands at the expected molecular weights. The antibodies mapped mAChR subtypes in the epithelium of human attached gingiva and also visualized them on the cell membrane of cultured GKC. The whole cell radioligand binding assay revealed that GKC have specific binding sites for the muscarinic ligand [3H]quinuclidinyl benzilate, Bmax = 222.9 fmol/106 cells with a Kd of 62.95 pM. The downstream coupling of the mAChRs to regulation of cell cycle progression in GKC was studied using quantitative RT-PCR and immunoblotting assays. Incubation of GKC for 24 h with 10 micro m muscarine increased relative amounts of Ki-67, PCNA and p53 mRNAs and PCNA, cyclin D1, p21 and p53 proteins. These effects were abolished in the presence of 50 micro m atropine. The finding in GKC of mAChRs coupled to regulation of the cell cycle progression demonstrate further the structure/function of the non-neuronal cholinergic system operating in human oral epithelium. The results obtained in this study help clarify the role for keratinocyte ACh axis in the physiologic control of oral gingival homeostasis.

  17. Altered coupling of muscarinic acetylcholine receptors in pancreatic acinar carcinoma of rat

    SciTech Connect

    Chien, J.L.; Warren, J.R.

    1986-03-05

    The structure and function of muscarinic acetylcholine receptors (mAChR) in acinar carcinoma cells have been compared to mAChR in normal pancreatic acinar cells. Similar 80 kD proteins identified by SDS-PAGE of tumor and normal mAChR affinity-labeled with the muscarinic antagonist /sup 3/H-propylbenzilyl-choline mustards, and identical binding of the antagonist N-methylscopolamine to tumor and normal cells (K/sub D/approx.4x10/sup -10/ M), indicate conservation of mAChR proteins in carcinoma cells. Carcinoma mAChR display homogeneous binding of the agonists carbamylcholine (CCh), K/sub D/approx.3x10/sup -5/ M, and oxotremorine (Oxo), K/sub D/approx.x10/sup -6/ M, whereas normal cells display heterogeneous binding, with a minor component of high affinity interactions for CCh, K/sub D/approx.3x10/sup -6/ M, and Oxo, K/sub D/approx.2x/sup -17/ M, and a major component of low affinity interactions for CCh, K/sub D/approx.1x10/sup -4/ M, and Oxo, K/sub D/approx.2x10/sup -5/ M. Both carcinoma and normal cells exhibit concentration-dependent CCh-stimulated increase in cytosolic free Ca/sup 2 +/, as measured by intracellular Quin 2 fluorescence and /sup 45/Ca/sup 2 +/ efflux. However, carcinoma cells demonstrate 50% maximal stimulation of intracellular Ca/sup 2 +/ release at a CCh concentration (EC/sub 50/approx.6x10/sup -7/ M) one log below that observed for normal cells. The authors propose an altered coupling of mAChR to intracellular Ca/sup 2 +/ homeostasis in carcinoma cells, which is manifest as a single activated receptor state for agonist binding, and increased sensitivity to muscarinic receptor stimulation of Ca/sup 2 +/ release.

  18. Long-acting muscarinic receptor antagonists for the treatment of respiratory disease.

    PubMed

    Cazzola, Mario; Page, Clive; Matera, Maria Gabriella

    2013-06-01

    The use of muscarinic receptor antagonists in the treatment of chronic obstructive pulmonary disease (COPD) is well established. More recently, the potential for long-acting muscarinic receptor antagonists (LAMAs) in the treatment of asthma has also been investigated. While LAMAs offer advantages over short-acting muscarinic receptor antagonists, in terms of a reduced dosing frequency, there remains a need for therapies that improve symptom control throughout both the day and night, provide better management of exacerbations and deliver improved health-related quality of life. Furthermore, the potential for unwanted anticholinergic side effects, particularly cardiovascular effects, remains a concern for this class of compounds. Novel LAMAs in clinical development for the treatment of respiratory disease include: aclidinium bromide, NVA237 (glycopyrronium bromide), GP-MDI, EP-101, CHF-5259, umeclidinium bromide, CHF-5407, TD-4208, AZD8683 and V-0162. These compounds offer potential advantages in terms of onset of action, symptom control and safety. In addition, a number of LAMAs are also being developed as combination treatments with long-acting β2-agonists (LABAs) or inhaled glucocorticosteroids, potentially important treatment options for patients who require combination therapy to achieve an optimal therapeutic response as their disease progresses. More recently, compounds such as GSK961081 and THRX-198321 have been identified that combine LAMA and LABA activity in the same molecule, and have the potential to offer the benefits of combination therapy in a single compound. Here, we review novel LAMAs and dual action compounds in clinical development, with a particular focus on how they may address the current unmet clinical needs in the treatment of respiratory disease, particularly COPD. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. Effect of pirenzepine, a muscarinic M1 receptor antagonist, on amygdala kindling in rat.

    PubMed

    Eşkazan, E; Aker, R; Onat, F; Köseoğlu, S; Gören, M Z; Hasanoğlu, A

    1999-11-01

    Kindling, an animal model of complex partial seizures with secondary generalization, is performed by daily application of low-intensity electrical brain stimulation. The purpose of this study was to investigate the role of muscarinic M1 receptors on amygdala kindling in the rat. Bipolar nichrome stimulation and recording electrodes were stereotaxically implanted into the right and left basolateral amygdala. Extradural recording electrodes were also placed bilaterally in the skull over the cortex. Amygdala stimulation was applied twice daily at the current intensity of afterdischarge threshold. Seizure intensity was graded by using Racine's standard five-stage scale. In the first group of experiments, saline or pirenzepine (10, 25, 50 and 100 nmol), a muscarinic M1 receptor antagonist, was injected intracerebroventricularly 1 h before the electrical stimulation. In the second group of experiments, rats were kindled to full stage 5 seizures. After a recovery period, 50 nmol of pirenzepine was administered intracerebroventricularly to kindled animals. In the first group of experiments, none of the animals pretreated with the doses of 50 and 100 nmol of pirenzepine reached a stage 5 seizure. Pirenzepine significantly retarded kindling seizure development and increased the total number of stimulations required to reach the first stage 5 seizure. Afterdischarge duration was also reduced in the pirenzepine 10 nmol group as compared with that in the saline-pretreated group. In the second group, seizure stage and afterdischarge duration were not affected by pirenzepine in fully-kindled animals. The findings of this study suggest that muscarinic M1 receptors may have a critical role in the development of kindling epileptic activity, but not in already kindled seizures.

  20. Rapid antidepressant actions of scopolamine: Role of medial prefrontal cortex and M1-subtype muscarinic acetylcholine receptors.

    PubMed

    Navarria, Andrea; Wohleb, Eric S; Voleti, Bhavya; Ota, Kristie T; Dutheil, Sophie; Lepack, Ashley E; Dwyer, Jason M; Fuchikami, Manabu; Becker, Astrid; Drago, Filippo; Duman, Ronald S

    2015-10-01

    Clinical studies demonstrate that scopolamine, a non-selective muscarinic acetylcholine receptor (mAchR) antagonist, produces rapid therapeutic effects in depressed patients, and preclinical studies report that the actions of scopolamine require glutamate receptor activation and the mechanistic target of rapamycin complex 1 (mTORC1). The present study extends these findings to determine the role of the medial prefrontal cortex (mPFC) and specific muscarinic acetylcholine receptor (M-AchR) subtypes in the actions of scopolamine. The administration of scopolamine increases the activity marker Fos in the mPFC, including the infralimbic (IL) and prelimbic (PrL) subregions. Microinfusions of scopolamine into either the IL or the PrL produced significant antidepressant responses in the forced swim test, and neuronal silencing of IL or PrL blocked the antidepressant effects of systemic scopolamine. The results also demonstrate that the systemic administration of a selective M1-AChR antagonist, VU0255035, produced an antidepressant response and stimulated mTORC1 signaling in the PFC, similar to the actions of scopolamine. Finally, we used a chronic unpredictable stress model as a more rigorous test of rapid antidepressant actions and found that a single dose of scopolamine or VU0255035 blocked the anhedonic response caused by CUS, an effect that requires the chronic administration of typical antidepressants. Taken together, these findings indicate that mPFC is a critical mediator of the behavioral actions of scopolamine and identify the M1-AChR as a therapeutic target for the development of novel and selective rapid-acting antidepressants.

  1. Pharmacology, Distribution and Development of Muscarinic Acetylcholine Receptor Subtypes in the Optic Tectum of Rana Pipiens

    PubMed Central

    Butt, C. M.; Pauly, J. R.; Wilkins, L. H.; Dwoskin, L. P.; Debski, E. A.

    2008-01-01

    Visually evoked behaviors mediated by the frog optic tectum require cholinergic activity, but the receptor subtypes through which acetylcholine acts are not yet identified. Using quantitative autoradiography and scintillation spectrometry, we examined the binding of [3H]pirenzepine and [3H]AF-DX 384 in the laminated optic tectum of the frog. In mammalian systems, these substances bind excitatory (m1 and m3 subtypes) and inhibitory (m2 and m4 subtypes) muscarinic acetylcholine receptors, respectively. Pharmacological analyses, including the use of specific muscarinic toxins, confirmed the subtype selectivity of the radioligands in the frog brain. Binding sites for [3H]pirenzepine were distinct from those for [3H]AF-DX 384. In the adult tectum, [3H]pirenzepine demonstrated specific binding in tectal layers 5–9. [3H]Pirenzepine binding was also present in tadpoles as young as stage V, but all sampled stages of tadpole tectum had significantly less binding when compared to adults. Lesioning of the optic nerve had no effect on [3H]pirenzepine binding. Specific [3H]AF-DX 384 binding was found in all layers of the adult tectum. All sampled tadpole stages exhibited binding sites for [3H]AF-DX 384, but the densities of these sites were also significantly higher in adults than they were in developing stages. Short-term lesions of the optic nerve reduced [3H]AF-DX 384 binding in all tectal layers of the deafferented lobe when compared to the afferented one. Long-term lesions decreased [3H]AF-DX 384 sites in both lobes. These results indicate that multiple muscarinic acetylcholine receptor binding sites reside in the frog optic tectum at all stages of development, and their pharmacology resembles that of mammalian m1/m3, m2 and m4 subtypes. Our data indicate that few, if any, of these receptors are likely to be located on retinal ganglion cell terminals. Furthermore, the expression of inhibitory muscarinic subtypes seems to be regulated by different mechanisms than that for

  2. Synergistic regulation of m2 muscarinic acetylcholine receptor desensitization and sequestration by G protein-coupled receptor kinase-2 and beta-arrestin-1.

    PubMed

    Schlador, M L; Nathanson, N M

    1997-07-25

    The m2 muscarinic acetylcholine receptor (m2 mAChR) belongs to the superfamily of G protein-coupled receptors and is regulated by many processes that attenuate signaling following prolonged stimulation by agonist. We used a heterologous expression system to examine the ability of G protein-coupled receptor kinase-2 (GRK2) and beta-arrestin-1 to regulate the phosphorylation state and to promote desensitization and sequestration of the m2 mAChR. Treatment of JEG-3 cells transiently expressing the m2 mAChR with a muscarinic agonist induced an approximately 4- or 8-fold increase in receptor phosphorylation in the absence or presence of cotransfected GRK2, respectively, compared with untreated cells transfected with receptor alone. Using the expression of a cAMP-regulated reporter gene to measure receptor function, we found that transiently transfected m2 mAChRs underwent functional desensitization following exposure to agonist. Transfected GRK2 enhanced agonist-induced functional desensitization in a manner that was synergistically enhanced by cotransfection of beta-arrestin-1, which had no effect on m2 mAChR function when coexpressed in the absence of GRK2. Finally, GRK2 and beta-arrestin-1 synergistically enhanced both the rate and extent of agonist-induced m2 mAChR sequestration. These results are the first to demonstrate that agonist-induced desensitization and sequestration of the m2 mAChR in the intact cell can be enhanced by the presence of GRK2 and beta-arrestin-1 and show that these molecules have multiple actions on the m2 mAChR.

  3. Human eosinophil major basic protein is an endogenous allosteric antagonist at the inhibitory muscarinic M2 receptor.

    PubMed Central

    Jacoby, D B; Gleich, G J; Fryer, A D

    1993-01-01

    The effect of human eosinophil major basic protein (MBP) as well as other eosinophil proteins, on binding of [3H]N-methyl-scopolamine ([3H]NMS: 1 x 10(-10) M) to muscarinic M2 receptors in heart membranes and M3 receptors in submandibular gland membranes was studied. MBP inhibited specific binding of [3H]NMS to M2 receptors but not to M3 receptors. MBP also inhibited atropine-induced dissociation of [3H]NMS-receptor complexes in a dose-dependent fashion, demonstrating that the interaction of MBP with the M2 muscarinic receptor is allosteric. This effect of MBP suggests that it may function as an endogenous allosteric inhibitor of agonist binding to the M2 muscarinic receptor. Inhibition of [3H]NMS binding by MBP was reversible by treatment with heparin, which binds and neutralizes MBP. Eosinophil peroxidase (EPO) also inhibited specific binding of [3H]NMS to M2 receptors but not to M3 receptors and inhibited atropine-induced dissociation of [3H]NMS-receptor complexes. On a molar basis, EPO is less potent than MBP. Neither eosinophil cationic protein nor eosinophil-derived neurotoxin affected binding of [3H]NMS to M2 receptors. Thus both MBP and EPO are selective allosteric antagonists at M2 receptors. The effects of these proteins may be important causes of M2 receptor dysfunction and enhanced vagally mediated bronchoconstriction in asthma. Images PMID:8473484

  4. Mice Lacking M1 and M3 Muscarinic Acetylcholine Receptors Have Impaired Odor Discrimination and Learning

    PubMed Central

    Chan, Wilson; Singh, Sanmeet; Keshav, Taj; Dewan, Ramita; Eberly, Christian; Maurer, Robert; Nunez-Parra, Alexia; Araneda, Ricardo C.

    2017-01-01

    The cholinergic system has extensive projections to the olfactory bulb (OB) where it produces a state-dependent regulation of sensory gating. Previous work has shown a prominent role of muscarinic acetylcholine (ACh) receptors (mAChRs) in regulating the excitability of OB neurons, in particular the M1 receptor. Here, we examined the contribution of M1 and M3 mAChR subtypes to olfactory processing using mice with a genetic deletion of these receptors, the M1−/− and the M1/M3−/− knockout (KO) mice. Genetic ablation of the M1 and M3 mAChRs resulted in a significant deficit in odor discrimination of closely related molecules, including stereoisomers. However, the discrimination of dissimilar molecules, social odors (e.g., urine) and novel object recognition was not affected. In addition the KO mice showed impaired learning in an associative odor-learning task, learning to discriminate odors at a slower rate, indicating that both short and long-term memory is disrupted by mAChR dysfunction. Interestingly, the KO mice exhibited decreased olfactory neurogenesis at younger ages, a deficit that was not maintained in older animals. In older animals, the olfactory deficit could be restored by increasing the number of new born neurons integrated into the OB after exposing them to an olfactory enriched environment, suggesting that muscarinic modulation and adult neurogenesis could be two different mechanism used by the olfactory system to improve olfactory processing. PMID:28210219

  5. Effects of absolute configuration of IQNP on muscarinic receptor subtype selectivity in vitro and in vivo

    SciTech Connect

    McPherson, D.W.; Lambert, C.R.; Knapp, F.F.

    1994-05-01

    IQNP, a high affinity muscarinic ligand with high cerebral uptake and long retention, contains two chiral centers in addition to vinyl iodide sterochemistry. The various diastereomers, in which the 3-quinuclidinyl moiety has the R configuration, have been prepared and evaluated in vitro and in vivo. These data show that muscarinic receptor subtype selectivity is dramatically affected by the configuration of the acetate center and vinyl iodide. In vitro studies show that E-(R,R)-IQNP is 100 times more selective for ml than m2 subtype as compared to E-(R,S), which was confirmed by in vivo results. In contrast, in vivo, Z-(R,R) has high uptake in m2 rich tissues (heart and cerebellum). In vitro studies are being performed on the Z isomers. Blocking studies with subtype-specific ligands confirm these data which illustrate the importance of molecular configuration on receptor subtype selectivity. These combined studies demonstrate that these isomers of IQNP are good candidates for future studies of receptor subtypes.

  6. Interleukin-1β Mediates Virus-Induced M2 Muscarinic Receptor Dysfunction and Airway Hyperreactivity

    PubMed Central

    Rynko, Abby E.; Fryer, Allison D.

    2014-01-01

    Respiratory viral infections are associated with the majority of asthma attacks. Inhibitory M2 receptors on parasympathetic nerves, which normally limit acetylcholine (ACh) release, are dysfunctional after respiratory viral infection. Because IL-1β is up-regulated during respiratory viral infections, we investigated whether IL-1β mediates M2 receptor dysfunction during parainfluenza virus infection. Virus-infected guinea pigs were pretreated with the IL-1β antagonist anakinra. In the absence of anakinra, viral infection increased bronchoconstriction in response to vagal stimulation but not to intravenous ACh, and neuronal M2 muscarinic receptors were dysfunctional. Pretreatment with anakinra prevented virus-induced increased bronchoconstriction and M2 receptor dysfunction. Anakinra did not change smooth muscle M3 muscarinic receptor response to ACh, lung viral loads, or blood and bronchoalveolar lavage leukocyte populations. Respiratory virus infection decreased M2 receptor mRNA expression in parasympathetic ganglia extracted from infected animals, and this was prevented by blocking IL-1β or TNF-α. Treatment of SK-N-SH neuroblastoma cells or primary cultures of guinea pig parasympathetic neurons with IL-1β directly decreased M2 receptor mRNA, and this was not synergistic with TNF-α treatment. Treating guinea pig trachea segment with TNF-α or IL-1β in vitro increased tracheal contractions in response to activation of airway nerves by electrical field stimulation. Blocking IL-1β during TNF-α treatment prevented this hyperresponsiveness. These data show that virus-induced hyperreactivity and M2 dysfunction involves IL-1β and TNF-α, likely in sequence with TNF-α causing production of IL-1β. PMID:24735073

  7. The molecular basis of oligomeric organization of the human M3 muscarinic acetylcholine receptor.

    PubMed

    Liste, María José Varela; Caltabiano, Gianluigi; Ward, Richard J; Alvarez-Curto, Elisa; Marsango, Sara; Milligan, Graeme

    2015-06-01

    G protein-coupled receptors, including the M3 muscarinic acetylcholine receptor, can form homo-oligomers. However, the basis of these interactions and the overall organizational structure of such oligomers are poorly understood. Combinations of site-directed mutagenesis and homogenous time-resolved fluorescence resonance energy transfer studies that assessed interactions between receptor protomers at the surface of transfected cells indicated important contributions of regions of transmembrane domains I, IV, V, VI, and VII as well as intracellular helix VIII to the overall organization. Molecular modeling studies based on both these results and an X-ray structure of the inactive state of the M3 receptor bound by the antagonist/inverse agonist tiotropium were then employed. The results could be accommodated fully by models in which a proportion of the cell surface M3 receptor population is a tetramer with rhombic, but not linear, orientation. This is consistent with previous studies based on spectrally resolved, multiphoton fluorescence resonance energy transfer. Modeling studies furthermore suggest an important role for molecules of cholesterol at the dimer + dimer interface of the tetramer, which is consistent with the presence of cholesterol at key locations in many G protein-coupled receptor crystal structures. Mutants that displayed disrupted quaternary organization were often poorly expressed and showed immature N-glycosylation. Sustained treatment of cells expressing such mutants with the muscarinic receptor inverse agonist atropine increased cellular levels and restored both cell surface delivery and quaternary organization to many of the mutants. These observations suggest that organization as a tetramer may occur before plasma membrane delivery and may be a key step in cellular quality control assessment.

  8. [Interaction of chagasic autoantibodies with the third extracellular domain of the human heart muscarinic receptor. Functional and pathological implications].

    PubMed

    Goin, J C; Pérez Leirós, C; Borda, E; Sterin-Borda, L

    1996-01-01

    Herein we demonstrate by ELISA and immunoblotting the presence in the sera of chagasic patients of circulating autoantibodies against the third extracellular domain of human muscarinic acetylcholine receptors by using a synthetic peptide corresponding to the sequence 169-192 of the receptor. Immunoaffinity purified antipeptide antibodies displayed cardiac muscarinic activity as decreased contractility and cAMP production and increased cGMP levels. These effects were specifically blocked by the synthetic peptide and by atropine. A strong association between the existence of circulating autoantibodies and the presence of dysautonomia was shown, making these autoantibodies an appropriate marker of heart autonomic dysfunction.

  9. Knife cuts of entorhinal cortex: effects on development of amygdaloid kindling and seizure-induced decrease of muscarinic cholinergic receptors

    SciTech Connect

    Savage, D.D.; Rigsbee, L.C.; McNamara, J.O.

    1985-02-01

    This report examines the effect of transection of the entorhinal hippocampal projection on amygdaloid kindling. We found that: bilateral knife cuts of entorhinal cortex but not of dorsal neocortex antagonize the development of amygdaloid kindling; and bilateral knife cuts of entorhinal cortex eliminate the seizure-induced decrease in number of muscarinic receptors of dentate granule cells. We suggest the following interpretations of these data: the hippocampal formation circuitry facilitates the development of amygdaloid kindling; and the decline of muscarinic receptors after kindled seizures is due to excessive activation of granule cells by axons from entorhinal cortex, a noncholinergic afferent.

  10. Cholinergic stimulation of pancreatic amylase release and muscarinic receptors: effect of ionophore A23187

    SciTech Connect

    Larose, L.; Morisset, J.

    1985-07-22

    Dispersed rat pancreatic acini were incubated in 0.5 mM calcium medium with increasing concentrations of carbamylcholine, with or without the ionophore A23187 (10/sup -6/M). Addition of the ionophore reduced maximal amylase release, increased the maximal effective concentration of carbamylcholine and dramatically impaired the agonist's capacity to induce enzyme secretion at low concentration. The ionophore also abolished the inhibition of secretion observed at high carbamylcholine concentrations. These effects of the ionophore on the cholinergic secretory response cannot be explained by interaction at the muscarinic receptor since neither the Bmax, the affinity of the receptor for the (/sup 3/H)QNB nor the binding of carbamylcholine were affected by the ionophore. It is suggested that for the conditions studied, the ionophore can interact with the secretory process at one or several points ulterior to the initial recognition site of carbamylcholine on its receptor. 30 references, 3 figures.

  11. Arrestin interactions with G protein-coupled receptors. Direct binding studies of wild type and mutant arrestins with rhodopsin, beta 2-adrenergic, and m2 muscarinic cholinergic receptors.

    PubMed

    Gurevich, V V; Dion, S B; Onorato, J J; Ptasienski, J; Kim, C M; Sterne-Marr, R; Hosey, M M; Benovic, J L

    1995-01-13

    Arrestins play an important role in quenching signal transduction initiated by G protein-coupled receptors. To explore the specificity of arrestin-receptor interaction, we have characterized the ability of various wild-type arrestins to bind to rhodopsin, the beta 2-adrenergic receptor (beta 2AR), and the m2 muscarinic cholinergic receptor (m2 mAChR). Visual arrestin was found to be the most selective arrestin since it discriminated best between the three different receptors tested (highest binding to rhodopsin) as well as between the phosphorylation and activation state of the receptor (> 10-fold higher binding to the phosphorylated light-activated form of rhodopsin compared to any other form of rhodopsin). While beta-arrestin and arrestin 3 were also found to preferentially bind to the phosphorylated activated form of a given receptor, they only modestly discriminated among the three receptors tested. To explore the structural characteristics important in arrestin function, we constructed a series of truncated and chimeric arrestins. Analysis of the binding characteristics of the various mutant arrestins suggests a common molecular mechanism involved in determining receptor binding selectivity. Structural elements that contribute to arrestin binding include: 1) a C-terminal acidic region that serves a regulatory role in controlling arrestin binding selectivity toward the phosphorylated and activated form of a receptor, without directly participating in receptor interaction; 2) a basic N-terminal domain that directly participates in receptor interaction and appears to serve a regulatory role via intramolecular interaction with the C-terminal acidic region; and 3) two centrally localized domains that are directly involved in determining receptor binding specificity and selectivity. A comparative structure-function model of all arrestins and a kinetic model of beta-arrestin and arrestin 3 interaction with receptors are proposed.

  12. Regulation of muscarinic acetylcholine receptor-mediated synaptic responses by adenosine receptors in the rat hippocampus.

    PubMed Central

    Morton, R A; Davies, C H

    1997-01-01

    1. Intracellular current clamp recordings were made from CA1 pyramidal neurones in rat hippocampal slices. Experiments were performed in the presence of ionotropic glutamate receptor antagonists and gamma-aminobutyric acid (GABA) receptor antagonists to block all fast excitatory and inhibitory synaptic transmission. A single stimulus, delivered extracellularly in the stratum oriens, caused a reduction in spike frequency adaptation in response to a depolarizing current step delivered 2 s after the stimulus. A 2- to 10-fold increase in stimulus intensity evoked a slow excitatory postsynaptic potential (EPSP) which was associated with a small increase in input resistance. The peak amplitude of the EPSP occurred approximately 2.5 s after the stimulus and its magnitude (up to 30 mV) and duration (10-50 s) increased with increasing stimulus intensity. 2. The slow EPSP was unaffected by the metabotropic glutamate receptor antagonist (+)-alpha-methyl-4-carboxyphenylglycine ((+)-MCPG; 1000 microM) but was greatly enhanced by the acetylcholinesterase inhibitor physostigmine (1-5 microM). Both the slow EPSP and the stimulus-evoked reduction in spike frequency adaptation were inhibited by the muscarinic acetylcholine receptor (mAChR) antagonist atropine (1-5 microM). These results are consistent with these effects being mediated by mAChRs. 3. Both the mAChR-mediated EPSP (EPSPm) and the associated reduction in spike frequency adaptation were reversibly depressed (up to 97%) by either adenosine (100 microM) or its non-hydrolysable analogue 2-chloroadenosine (CADO; 0.1-5.0 microM). These effects were often accompanied by postsynaptic hyperpolarization (up to 8 mV) and a reduction in input resistance (up to 11%). The selective adenosine A1 receptor agonists 2-chloro-N6-cyclopentyladenosine (CCPA; 0.1-0.4 microM) and R(-)N6-(2-phenylisopropyl)-adenosine (R-PIA; 1 microM) both depressed the EPSPm. In contrast, the adenosine A2A receptor agonist 2-p-(2-carboxyethyl)-phenethylamino-5

  13. Ionic mechanism of the slow afterdepolarization induced by muscarinic receptor activation in rat prefrontal cortex.

    PubMed

    Haj-Dahmane, S; Andrade, R

    1998-09-01

    The mammalian prefrontal cortex receives a dense cholinergic innervation from subcortical regions. We previously have shown that cholinergic stimulation of layer V pyramidal neurons of the rat prefrontal cortex results in a depolarization and the appearance of a slow afterdepolarization (sADP). In the current report we examine the mechanism underlying the sADP with the use of sharp microelectrode and whole cell recording techniques in in vitro brain slices. The ability of acetylcholine (ACh) and carbachol to induce the appearance of an sADP in pyramidal cells of layer V of prefrontal cortex is antagonized in a surmountable manner by atropine and is mimicked by application of muscarine or oxotremorine. These results indicate that ACh acts on muscarinic receptors to induce the sADP. In many cell types afterpotentials are triggered by calcium influx into the cell. Therefore we examined the possibility that calcium influx might be the trigger for the generation of the sADP. Consistent with this possibility, buffering intracellular calcium reduced or abolished the sADP but had little effect on the direct muscarinic receptor-induced depolarization also seen in these cells. These results, coupled to the previous observation that calcium channel blockers inhibit the sADP, indicated that the sADP results from a rise in intracellular calcium secondary to calcium influx into the cell. The ionic basis for the current underlying the sADP (IsADP) was examined with the use of ion substitution experiments. The amplitude of IsADP was found to be reduced in a graded fashion by replacement of extracellular sodium with N-methyl-D-glucamine (NMDG). In contrast no clear evidence for the involvement of potassium or chloride channels in the generation of the sADP or IsADP could be found. This result indicated that IsADP is carried by sodium ions flowing into the cell. However, the dependence of IsADP on extracellular sodium was less pronounced than expected for a pure sodium current. We

  14. The modulatory role of M2 muscarinic receptor on apomorphine-induced yawning and genital grooming.

    PubMed

    Gamberini, Maria Thereza; Bolognesi, Maria Laura; Nasello, Antonia Gladys

    2012-12-07

    The interaction between dopaminergic and cholinergic pathways in the induction of behavioral responses has been previously established. In the brain, M2 receptors are found predominantly in presynaptic cholinergic neurons as autoreceptors, and in dopaminergic neurons as heteroceptors, suggesting a control role of acetylcholine and dopamine release, respectively. Our aim was to investigate the role of M2 receptors on the yawning and genital grooming of rats induced by apomorphine, a dopaminergic receptor agonist, focusing on the interaction between cholinergic and dopaminergic pathways. Initially, the effect of atropine, a non-selective muscarinic antagonist, on yawning and genital grooming induced by apomorphine (100 μg/kg s.c.) was analyzed. Atropine doses of 0.5, 1 and 2 mg/kg i.p. were administered to Wistar rats 30 min before induction of the behavioral responses by apomorphine. Number of yawns and time spent genital grooming were quantified over a 60 min period. Apomorphine-induced yawning was increased by low dose (0.5 mg/kg i.p.) but not by high doses (1 and 2 mg/kg, i.p.) of atropine. Genital grooming was antagonized by 2 mg/kg i.p. of atropine and showed no changes at the other doses tested. Tripitramine, a selective M2 cholinergic antagonist, was used as a tool for distinguishing between M2 and all other muscarinic receptor subtypes in yawning and genital grooming. Tripitramine doses of 0.01, 0.02 and 0.04 μmol/kg i.p. were administered to Wistar rats 30 min before apomorphine (100 μg/kg s.c.). Number of yawns and time spent genital grooming were also quantified over a 60 min period. Tripitramine 0.01 μmol/kg increased all parameters. Higher doses, which possibly block all subtypes of muscarinic receptor, did not modify the response of apomorphine, suggesting a non-selective effect of tripitramine at these doses. Given that low doses of tripitramine increased the behavioral responses induced by apomorphine and that the main distribution of the M2

  15. Postsynaptic muscarinic m2 receptors at cholinergic and glutamatergic synapses of mouse brainstem motoneurons.

    PubMed

    Csaba, Zsolt; Krejci, Eric; Bernard, Véronique

    2013-06-15

    In many brain areas, few cholinergic synapses are identified. Acetylcholine is released into the extracellular space and acts through diffuse transmission. Motoneurons, however, are contacted by numerous cholinergic terminals, indicating synaptic cholinergic transmission on them. The muscarinic m2 receptor is the major acetylcholine receptor subtype of motoneurons; therefore, we analyzed the localization of the m2 receptor in correlation with synapses by electron microscopic immunohistochemistry in the mouse trigeminal, facial, and hypoglossal motor nuclei. In all nuclei, m2 receptors were localized at the membrane of motoneuronal perikarya and dendrites. The m2 receptors were concentrated at cholinergic synapses located on the perikarya and most proximal dendrites. However, m2 receptors at cholinergic synapses represented only a minority (<10%) of surface m2 receptors. The m2 receptors were also enriched at glutamatergic synapses in both motoneuronal perikarya and dendrites. A relatively large proportion (20-30%) of plasma membrane-associated m2 receptors were located at glutamatergic synapses. In conclusion, the effect of acetylcholine on motoneuron populations might be mediated through a synaptic as well as diffuse type of transmission.

  16. α1-Adrenoceptors and muscarinic receptors in voiding function – binding characteristics of therapeutic agents in relation to the pharmacokinetics

    PubMed Central

    Yamada, Shizuo; Ito, Yoshihiko; Tsukada, Hideo

    2011-01-01

    In vivo and ex vivo binding of α1-adrenoceptor and muscarinic receptors involved in voiding function is reviewed with therapeutic agents (α1-adrenoceptor antagonists: prazosin, tamsulosin and silodosin; and muscarinic receptor antagonists: oxybutynin, tolterodine, solifenacin, propiverine, imiafenacin and darifenacin) in lower urinary tract symptoms. This approach allows estimation of the inhibition of a well-characterized selective (standard) radioligand by unlabelled potential drugs or direct measurement of the distribution and receptor binding of a standard radioligand or radiolabelled form of a novel drug. In fact, these studies could be conducted in various tissues from animals pretreated with radioligands and/or unlabelled novel drugs, by conventional radioligand binding assay, radioactivity measurement, autoradiography and positron emission tomography. In vivo and ex vivo receptor binding with α1-adrenoceptor antagonists and muscarinic receptor antagonists have been proved to be useful in predicting the potency, organ selectivity and duration of action of drugs in relation to their pharmacokinetics. Such evaluations of drug–receptor binding reveal that adverse effects could be avoided by the use of new α1-adrenoceptor antagonists and muscarinic receptor antagonists for the treatment of lower urinary tract symptoms. Thus, the comparative analysis of α1-adrenoceptor and muscarinic receptor binding characteristics in the lower urinary tract and other tissues after systemic administration of therapeutic agents allows the rationale for their pharmacological characteristics from the integrated viewpoint of pharmacokinetics and pharmacodynamics. The current review emphasizes the usefulness of in vivo and ex vivo receptor binding in the discovery and development of novel drugs for the treatment of not only urinary dysfunction but also other disorders. PMID:21265873

  17. α1-Adrenoceptors and muscarinic receptors in voiding function - binding characteristics of therapeutic agents in relation to the pharmacokinetics.

    PubMed

    Yamada, Shizuo; Ito, Yoshihiko; Tsukada, Hideo

    2011-08-01

    In vivo and ex vivo binding of α(1)-adrenoceptor and muscarinic receptors involved in voiding function is reviewed with therapeutic agents (α(1)-adrenoceptor antagonists: prazosin, tamsulosin and silodosin; and muscarinic receptor antagonists: oxybutynin, tolterodine, solifenacin, propiverine, imiafenacin and darifenacin) in lower urinary tract symptoms. This approach allows estimation of the inhibition of a well-characterized selective (standard) radioligand by unlabelled potential drugs or direct measurement of the distribution and receptor binding of a standard radioligand or radiolabelled form of a novel drug. In fact, these studies could be conducted in various tissues from animals pretreated with radioligands and/or unlabelled novel drugs, by conventional radioligand binding assay, radioactivity measurement, autoradiography and positron emission tomography. In vivo and ex vivo receptor binding with α(1)-adrenoceptor antagonists and muscarinic receptor antagonists have been proved to be useful in predicting the potency, organ selectivity and duration of action of drugs in relation to their pharmacokinetics. Such evaluations of drug-receptor binding reveal that adverse effects could be avoided by the use of new α(1)-adrenoceptor antagonists and muscarinic receptor antagonists for the treatment of lower urinary tract symptoms. Thus, the comparative analysis of α(1)-adrenoceptor and muscarinic receptor binding characteristics in the lower urinary tract and other tissues after systemic administration of therapeutic agents allows the rationale for their pharmacological characteristics from the integrated viewpoint of pharmacokinetics and pharmacodynamics. The current review emphasizes the usefulness of in vivo and ex vivo receptor binding in the discovery and development of novel drugs for the treatment of not only urinary dysfunction but also other disorders. © 2011 The Authors. British Journal of Clinical Pharmacology © 2011 The British Pharmacological

  18. Effects of methylmercury on muscarinic receptors in the mouse brain: A quantitative autoradiographic study

    SciTech Connect

    Lee, Haesung; Yee, S.; Geddes, J.; Choi, Byung, H. Univ. of California, Irvine )

    1991-03-11

    Methylmercury (MeHg) is reported to inhibit several stages of cholinergic neurotransmission in brain tissue in-vitro and in-vivo. To examine whether or not behavioral disturbances and/or selective vulnerability of specific neuronal groups in MeHg poisoning may be related to MeHg effects on cholinergic receptors in specific regions of the brain, the density and distribution of muscarinic receptors in the brains of C57BL/6J mice were determined following repeated injections of 5 mg/kg of methylmercuric chloride (MMC). The receptor densities in six cortical laminae of seven cerebral cortical regions, hippocampus and striatum were quantitated by computer-assisted imaging system following in-vitro labeling with ({sup 3}H)-pirenzepine (M1) and ({sup 3}H)N-methyl scopolamine (M2). The results showed heterogeneous distribution of M1 and M2 sites in different regions of the brain, and significant reduction in the density of both receptor subtypes following MeHg poisoning in many cortical and subcortical regions. However, the changes in the density were variable in different laminae even in the same cortical regions. Prominent reductions in M1 densities were noted in the temporal and entorhinal cortices, CA3 and hilar regions of the hippocampus as compared to control, whereas the reduction in M2 receptor density was most prominently noted in the frontal, perirhinal and entorhinal cortices, and CA1 and hilar regions of the hippocampus. Thus, it is apparent that MeHg significantly affects muscarinic receptors in the mouse brain, and that these data when used in conjunction with immunocytochemical and other morphological studies would provide further insights into the mechanisms of neurotoxic effects of MeHg.

  19. L-689,660, a novel cholinomimetic with functional selectivity for M1 and M3 muscarinic receptors.

    PubMed Central

    Hargreaves, R. J.; McKnight, A. T.; Scholey, K.; Newberry, N. R.; Street, L. J.; Hutson, P. H.; Semark, J. E.; Harley, E. A.; Patel, S.; Freedman, S. B.

    1992-01-01

    1. L-689,660, 1-azabicyclo[2.2.2]octane, 3-(6-chloropyrazinyl)maleate, a novel cholinomimetic, demonstrated high affinity binding (pKD (apparent) 7.42) at rat cerebral cortex muscarinic receptors. L-689,660 had a low ratio (34) of pKD (apparent) values for the displacement of binding of the antagonist ([3H]-N-methylscopolamine ([3H]-NMS) compared with the displacement of the agonist [3H]-oxotremorine-M ([3H]-Oxo-M), in rat cerebral cortex. Low NMS/Oxo-M ratios have been shown previously to be a characteristic of compounds that are low efficacy partial agonists with respect to stimulation of phosphatidyl inositol turnover in the cerebral cortex. 2. L-689,660 showed no muscarinic receptor subtype selectivity in radioligand binding assays but showed functional selectivity in pharmacological assays. At M1 muscarinic receptors in the rat superior cervical ganglion, L-689,660 was a potent (pEC50 7.3 +/- 0.2) full agonist in comparison with (+/-)-muscarine. At M3 receptors in the guinea-pig ileum myenteric plexus-longitudinal muscle or in trachea, L-689,660 was again a potent agonist (pEC50 7.5 +/- 0.2 and 7.7 +/- 0.3 respectively) but had a lower maximum response than carbachol. In contrast L-689,660 was an antagonist at M2 receptors in guinea-pig atria (pA2 7.2 (95% confidence limits 7, 7.4)) and at muscarinic autoreceptors in rat hippocampal slices. 3. The putative M1-selective muscarinic agonist, AF102B (cis-2-methylspiro-(1,3-oxathiolane 5,3')-quinuclidine hydrochloride) was found to have a profile similar to L-689,660 but had up to 100 times less affinity in binding and functional assays.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1422595

  20. Antagonism of nucleus accumbens M(2) muscarinic receptors disrupts operant responding for sucrose under a progressive ratio reinforcement schedule.

    PubMed

    Cousens, Graham A; Beckley, Jacob T

    2007-07-19

    Diverse cholinergic signaling mechanisms regulate the excitability of striatal principal neurons and modulate striatal-dependent behavior. These effects are mediated, in part, by action at muscarinic receptors (mAChR), subtypes of which exhibit distinct patterns of expression across striatal neuronal populations. Non-selective mAChR blockade within the nucleus accumbens (NAc) has been shown to disrupt operant responding for food and to inhibit food consumption. However, the specific receptor subtypes mediating these effects are not known. Thus, we evaluated effects of intra-NAc infusions of pirenzepine and methoctramine, mAChR antagonisits with distinct binding affinity profiles, on operant responding for sucrose reward under a progressive ratio (PR) reinforcement schedule. Moderate to high doses of methoctramine disrupted operant responding and reduced behavioral breakpoint. In contrast, pirenzepine failed to impact operant performance at any dose tested. Methoctramine failed to affect latencies to complete appetitive-consummatory response sequences or to impact measures of acoustic startle, suggesting that its' disruptive effects on operant behavior were not consequent to gross motor impairment. Since methoctramine has a greater affinity for M(2) receptors compared to pirenzepine, which has a greater relative affinity for M(1) and M(3) receptors, these findings suggest that M(2) mAChRs within the NAc regulate behavioral processes underling the acquisition of reward.

  1. Inverse agonist activity of pirenzepine at M2 muscarinic acetylcholine receptors.

    PubMed

    Daeffler, L; Schmidlin, F; Gies, J P; Landry, Y

    1999-03-01

    1. The intrinsic properties of muscarinic ligands were studied through their binding properties and their abilities to modulate the GTPase activity of G proteins coupled to muscarinic M2 receptors in pig atrial sarcolemma. 2. Competition binding experiments were performed with [3H]-oxotremorine-M to assess the affinity of receptors coupled to G proteins (R*), with [3H]-N-methylscopolamine ([3H]-NMS) to estimate the affinities of coupled and uncoupled receptors (R*+R) and with [3H]-NMS in the presence of GppNHp to assess the affinity of uncoupled receptors (R). 3. The ranking of Ki values for the agonist carbachol was R*R*+R>R (174, 155, 115 nM), suggesting inverse agonism. 4. The Vmax of the basal high affinity GTPase activity of pig atrial sarcolemma was increased by mastoparan and decreased by GPAnt-2 indicating the relevance of this activity to G proteins coupled to receptors (R*). The K(M) value (0.26-0.33 microM) was not modified by mastoparan or GPAnt-2. 5. Carbachol increased the Vmax of GTP hydrolysis (EC50 8.1+/-0.3 microM), whereas atropine and AF-DX 116, up to 1 mM, did not modify it. Pirenzepine decreased the Vmax of GTP hydrolysis (EC50 77.5+/-10.3 microM). This effect was enhanced when KCI was substituted for NaCl (EC50 11.0+/-0.8 microM) and was antagonized by atropine and AF-DX 116 (IC50 0.91+/-0.71 and 197+/-85 nM). 6. Pirenzepine is proposed as an inverse agonist and atropine and AF-DX 116 as neutral antagonists at the muscarinic M2 receptor.

  2. Inverse agonist activity of pirenzepine at M2 muscarinic acetylcholine receptors

    PubMed Central

    Daeffler, Laurent; Schmidlin, Fabien; Gies, Jean-Pierre; Landry, Yves

    1999-01-01

    The intrinsic properties of muscarinic ligands were studied through their binding properties and their abilities to modulate the GTPase activity of G proteins coupled to muscarinic M2 receptors in pig atrial sarcolemma. Competition binding experiments were performed with [3H]-oxotremorine-M to assess the affinity of receptors coupled to G proteins (R*), with [3H]-N-methylscopolamine ([3H]-NMS) to estimate the affinities of coupled and uncoupled receptors (R*+R) and with [3H]-NMS in the presence of GppNHp to assess the affinity of uncoupled receptors (R). The ranking of Ki values for the agonist carbachol was R*<R*+R>R (174, 155, 115 nM), suggesting inverse agonism. The Vmax of the basal high affinity GTPase activity of pig atrial sarcolemma was increased by mastoparan and decreased by GPAnt-2 indicating the relevance of this activity to G proteins coupled to receptors (R*). The KM value (0.26–0.33 μM) was not modified by mastoparan or GPAnt-2. Carbachol increased the Vmax of GTP hydrolysis (EC50 8.1±0.3 μM), whereas atropine and AF-DX 116, up to 1 mM, did not modify it. Pirenzepine decreased the Vmax of GTP hydrolysis (EC50 77.5±10.3 μM). This effect was enhanced when KCl was substituted for NaCl (EC50 11.0±0.8 μM) and was antagonized by atropine and AF-DX 116 (IC50 0.91±0.71 and 197±85 nM). Pirenzepine is proposed as an inverse agonist and atropine and AF-DX 116 as neutral antagonists at the muscarinic M2 receptor. PMID:10205015

  3. A role for muscarinic receptors in neutrophil extracellular trap formation and levamisole-induced autoimmunity

    PubMed Central

    Carmona-Rivera, Carmelo; Purmalek, Monica M.; Moore, Erica; Waldman, Meryl; Walter, Peter J.; Garraffo, H. Martin; Phillips, Karran A.; Preston, Kenzie L.; Graf, Jonathan; Grayson, Peter C.

    2017-01-01

    Levamisole, an anthelmintic drug with cholinergic properties, has been implicated in cases of drug-induced vasculitis when added to cocaine for profit purposes. Neutrophil extracellular trap (NET) formation is a cell death mechanism characterized by extrusion of chromatin decorated with granule proteins. Aberrant NET formation and degradation have been implicated in idiopathic autoimmune diseases that share features with levamisole-induced autoimmunity as well as in drug-induced autoimmunity. This study’s objective was to determine how levamisole modulates neutrophil biology and its putative effects on the vasculature. Murine and human neutrophils exposed to levamisole demonstrated enhanced NET formation through engagement of muscarinic subtype 3 receptor. Levamisole-induced NETosis required activation of Akt and the RAF/MEK/ERK pathway, ROS induction through the nicotinamide adenine dinucleotide phosphate oxidase, and peptidylarginine deiminase activation. Sera from two cohorts of patients actively using levamisole-adulterated cocaine displayed autoantibodies against NET components. Cutaneous biopsy material obtained from individuals exposed to levamisole suggests that neutrophils produce NETs in areas of vasculitic inflammation and thrombosis. NETs generated by levamisole were toxic to endothelial cells and impaired endothelium-dependent vasorelaxation. Stimulation of muscarinic receptors on neutrophils by cholinergic agonists may contribute to the pathophysiology observed in drug-induced autoimmunity through the induction of inflammatory responses and neutrophil-induced vascular damage. PMID:28194438

  4. Effect of muscarinic and nicotinic receptor antagonism on rat gastric motor activity.

    PubMed

    Janssen, Pieter; Karlsson, Lisa K C; Nielsen, Maria Astin; Gillberg, Per-Göran; Hultin, Leif

    2010-01-01

    Our aim was to investigate whether muscarinic and nicotinic receptors mediate nitric oxide release during motor events in the rat stomach. Isolated rat stomach volume changes were monitored in an organ bath setup with an intragastric balloon coupled to a barostat and studied in basal conditions and during electrical vagal stimulation (EVS). In conscious rats, the intragastric pressure (IGP) was measured during test meal infusion. In the presence of N(G)-nitro-L-arginine methyl ester (L-NAME; 0.1 mmol/l), EVS induced significant gastric contractions (mean +/- SEM = 0.27 +/- 0.04 ml; n = 6) that could be blocked by atropine (3 micromol/l) and hexamethonium (0.1 mmol/l). In the presence of atropine and/or hexamethonium, EVS-induced relaxations could not be blocked by L-NAME, while exogenous nitric oxide could still relax the stomach. In conscious rats, atropine (1 mg kg(-1)) initially decreased IGP, while during further distension it increased IGP. In the presence of L-NAME (30 mg kg(-1)) atropine consistently decreased IGP. L-NAME alone significantly increased IGP during the test meal infusion, but this effect was reduced in the presence of atropine. These findings indicate a role for nicotinic and muscarinic receptors in the vagal-stimulation-induced activation of nitrergic nerves in the rat stomach. Copyright 2010 S. Karger AG, Basel.

  5. Local heating of human skin causes hyperemia without mediation by muscarinic cholinergic receptors or prostanoids.

    PubMed

    Golay, Sandrine; Haeberli, Christian; Delachaux, Anne; Liaudet, Lucas; Kucera, Paul; Waeber, Bernard; Feihl, François

    2004-11-01

    Local changes in surface temperature have a powerful influence on the perfusion of human skin. Heating increases local skin blood flow, but the mechanisms and mediators of this response (thermal hyperemia response) are incompletely elucidated. In the present study, we examined the possible dependence of the thermal hyperemia response on stimulation of muscarinic cholinergic receptors and on production of vasodilator prostanoids. In 13 male healthy subjects aged 20-30 yr, a temperature-controlled chamber was positioned on the volar face of one forearm and used to raise surface temperature from 34 to 41 degrees C. The time course of the resulting thermal hyperemia response was recorded with a laser-Doppler imager. In one experiment, each of eight subjects received an intravenous bolus of the antimuscarinic agent glycopyrrolate (4 microg/kg) on one visit and saline on the other. The thermal hyperemia response was determined within the hour after the injections. Glycopyrrolate effectively inhibited the skin vasodilation induced by iontophoresis of acetylcholine but did not influence the thermal hyperemia response. In a second experiment, conducted in five other subjects, 1 g of the cyclooxygenase inhibitor aspirin administered orally totally abolished the vasodilation induced in the skin by anodal current but also failed to modify the thermal hyperemia response. The present study excludes the stimulation of muscarinic receptors and the production of vasodilator prostaglandins as essential and nonredundant mechanisms for the vasodilation induced by local heating in human forearm skin.

  6. On homology modeling of the M2 muscarinic acetylcholine receptor subtype

    NASA Astrophysics Data System (ADS)

    Jakubík, Jan; Randáková, Alena; Doležal, Vladimír

    2013-06-01

    Twelve homology models of the human M2 muscarinic receptor using different sets of templates have been designed using the Prime program or the modeller program and compared to crystallographic structure (PDB:3UON). The best models were obtained using single template of the closest published structure, the M3 muscarinic receptor (PDB:4DAJ). Adding more (structurally distant) templates led to worse models. Data document a key role of the template in homology modeling. The models differ substantially. The quality checks built into the programs do not correlate with the RMSDs to the crystallographic structure and cannot be used to select the best model. Re-docking of the antagonists present in crystallographic structure and relative binding energy estimation by calculating MM/GBSA in Prime and the binding energy function in YASARA suggested it could be possible to evaluate the quality of the orthosteric binding site based on the prediction of relative binding energies. Although estimation of relative binding energies distinguishes between relatively good and bad models it does not indicate the best one. On the other hand, visual inspection of the models for known features and knowledge-based analysis of the intramolecular interactions allows an experimenter to select overall best models manually.

  7. Decreased levels of muscarinic receptors in bladders from the alcohol preferring rat line

    SciTech Connect

    Smyth, R.J.; Ruggieri, M.R. ); Kiianmaa, K. )

    1992-01-01

    The B{sub max} for ({sup 3}H)QNB binding in the bladders of alcohol preferring (AA) rats was only approximately 60% of that in the alcohol non-preferring (ANA) rats. No significant change in B{sub max} for ({sup 3}H)QNB binding in bladder was observed between alcohol insensitive (AT) and alcohol sensitive (ANT) rats. No significant change in K{sub d} for ({sup 3}H)QNB binding in bladder was observed between the four different rat lines studied. Therefore, alcohol preference but not sensitivity is associated with a decrease in muscarinic receptor density in the rat bladder. Because all of the rats used in this study were ethanol-naive, the decrease in muscarinic receptor density in the bladders of alcohol preferring rats is associated genetic factors inherent to this rat line. Further studies are needed to determine if these observations are tissue specific or specific to the m2 subtype, which predominates in the rat bladder.

  8. M1 muscarinic receptors are necessary for retrieval of remote context fear memory.

    PubMed

    Patricio, Rafael Rodisanski; Soares, Juliana Carlota Kramer; Oliveira, Maria Gabriela Menezes

    2017-02-01

    Several studies have investigated the transition of consolidation of recent memory to remote memory in aversively motivated tasks, such as contextual fear conditioning (CFC) and inhibitory avoidance (IA). However, the mechanisms that serve the retrieval of remote memories, has not yet been fully understood. Some evidences suggest that the central cholinergic system appears be involved in the modulation of these processes. Therefore, the present study aimed to investigate the effects of a pre-test administration of dicyclomine, a high-affinity M1 muscarinic receptor antagonist, on the retrieval of remote memories in fear conditioning and IA tasks. Male Wistar rats were trained, and after 1 or 28days, the rats received dicyclomine (16 or 32mg/kg, intraperitoneally, i.p.) and were tested in CFC, tone fear conditioning (TFC) and IA tasks. At both time intervals, 32mg/kg dicyclomine induced impairment of CFC. In TFC task only the performance of the rats 28days after training was impaired. The IA task was not affected in any of the studied intervals. These findings suggest a differential contribution of muscarinic receptors on recent and remote memories retrieval revealing a more generalized role in remote memory.

  9. Permanent alterations in muscarinic receptors and pupil size produced by chronic atropinization in kittens

    SciTech Connect

    Smith, E.L.; Redburn, D.A.; Harwerth, R.S.; Maguire, G.W.

    1984-02-01

    Chronic mydriasis was induced in six kittens (four monocular, two binocular) and two adult cats (both monocular) by the daily topical application of atropine. Both the kittens and the adult cats were atropinized for a 13-week period with the treatment regimen beginning at the time of eye opening for the kittens. Pupil size measurements, obtained 1 year after the atropinization were discontinued, revealed that, although the pupils of the adult cats were normal, the pupils of the kittens' treated eyes were consistently smaller than pupils in control eyes. The status of the muscarinic receptors in the kittens' irides was investigated using /sup 3/H-QNB binding assays. In comparison with iris muscle homogenates from the control eyes, those from the treated eyes demonstrated an eightfold increase in the number of receptor binding sites. The results indicate that pupil size can be altered permanently by chronic mydriasis initiated early in the life of a kitten and that the permanent change in pupil size may result, in part, from a type of permanent supersensitivity response in the muscle following chronic blockade of muscarinic transmission by atropine.

  10. Desensitization of human muscarinic acetylcholine receptor m2 subtypes is caused by their sequestration/internalization.

    PubMed

    Tsuga, H; Kameyama, K; Haga, T

    1998-10-01

    Desensitization of human muscarinic acetylcholine receptor m2 subtypes (hm2 receptors) stably expressed in chinese hamster ovary cells was measured as decreases in the carbamylcholine-stimulated [35S]GTPgammaS binding activity in membrane preparations after pre-treatment of cells with carbamylcholine. The extent of carbamylcholine-stimulated [35S]GTPgammaS binding activity was found to decrease to 64% following pretreatment of cells with 10 microM carbamylcholine for 30 min, and under the same conditions 51-59% of hm2 receptors were sequestered/internalized as assessed by decreases in the [3H]N-methylscopolamine binding activity on the cell surface. A similar reduction in the carbamylcholine-stimulated [35S]GTPgammaS binding activity was observed by pretreatment of cells with 5 nM propylbenzylylcholine mustard, which irreversibly bound to and inactivated 58% of the hm2 receptors. When the cells were pretreated with 10 microM carbamylcholine in the presence of 0.32 M sucrose, which is known to inhibit clathrin-mediated endocytosis, no sequestration/internalization of hm2 receptors was observed, and the extent of carbamylcholine-stimulated [35S]GTPgammaS binding activity did not change. These results indicate that desensitization of hm2 receptors may be caused by reduction of receptor number on the cell surface through sequestration/internalization rather than by loss of the function of receptors.

  11. Muscarinic receptors: evidence for a nonuniform distribution in tracheal smooth muscle and exocrine glands

    SciTech Connect

    Basbaum, C.B.; Grillo, M.A.; Widdicombe, J.H.

    1984-02-01

    Muscarinic receptor distribution in smooth muscle, exocrine glands, and epithelium of the ferret trachea was determined using (3H)propylbenzilylcholine mustard ((3H)PrBCM) binding and autoradiography. Specific, atropine-sensitive (3H)PrBCM binding was quantified autoradiographically in the trachealis muscle (approximately 21 binding sites/microns2), surface epithelium (approximately 6 binding sites/microns2), and submucosal glands (approximately 5 binding sites/microns2). Serous and mucous cells in the glands did not differ in receptor density. Binding sites on gland and epithelial cells were associated with basolateral membranes. In the trachealis muscle, a gradient in receptor density was observed, with outer layers of muscle containing 3 to 10 times more receptors per unit area than inner layers. Receptor distribution in both glands and muscle paralleled the distribution of cholinergic axons. However, at the light microscope level, there was no evidence for the presence of receptor ''hot spots'' related to the position of individual axons. The parallelism in the distribution of axons and receptors suggests the possibility of neural control of the genesis and/or maintenance of receptor distribution in these tissues.

  12. Subcellular redistribution of m2 muscarinic acetylcholine receptors in striatal interneurons in vivo after acute cholinergic stimulation.

    PubMed

    Bernard, V; Laribi, O; Levey, A I; Bloch, B

    1998-12-01

    The purpose of our work was to investigate how the cholinergic environment influences the targeting and the intracellular trafficking of the muscarinic receptor m2 (m2R) in vivo. To address this question, we have used immunohistochemical approaches at light and electron microscopic levels to detect the m2R in control rats and rats treated with muscarinic receptor agonists. In control animals, m2Rs were located mostly at postsynaptic sites at the plasma membrane of perikarya and dendrites of cholinergic and NPY-somatostatin interneurons as autoreceptors and heteroreceptors, respectively. Presynaptic receptors were also detected in boutons. The m2Rs were usually detected at extrasynaptic sites, but they could be found rarely in association with symmetrical synapses, suggesting that the cholinergic transmission mediated by m2R occurs via synaptic and nonsynaptic mechanisms. The stimulation of muscarinic receptors with oxotremorine provoked a dramatic alteration of m2R compartmentalization, including endocytosis with a decrease of the density of m2R at the membrane (-63%) and an increase of those associated with endosomes (+86%) in perikarya. The very strong increase of m2R associated with multivesicular bodies (+732%) suggests that oxotremorine activated degradation. The slight increase in the Golgi apparatus (+26%) suggests that the m2R stimulation had an effect on the maturation of m2R. The substance P receptor located at the membrane of the same neurons was unaffected by oxotremorine. Our data demonstrate that cholinergic stimulation dramatically influences the subcellular distribution of m2R in striatal interneurons in vivo. These events may have key roles in controlling abundance and availability of muscarinic receptors via regulation of receptor endocytosis, degradation, and/or neosynthesis. Further, the control of muscarinic receptor trafficking may influence the activity of striatal interneurons, including neurotransmitter release and/or electric activity.

  13. Label-free cell phenotypic assessment of the biased agonism and efficacy of agonists at the endogenous muscarinic M3 receptors

    PubMed Central

    Deng, Huayun; Sun, Haiyan; Fang, Ye

    2013-01-01

    Introduction Efficacy describes the property of a ligand that enables the receptor to change its behavior towards the host cell, while biased agonism defines the ability of a ligand to differentially activate some of the vectorial pathways over others mediated through the receptor. However, little is known about the molecular basis defining the efficacy of ligands at G protein-coupled receptors. Here we characterize the biased agonism and cell phenotypic efficacy of seven agonists at the endogenous muscarinic M3 receptors in six different cell lines including HT-29, PC-3, HeLa, SF268, CCRF-CEM and HCT-15 cells. Methods Quantitative real-time PCR and multiple label-free whole cell dynamic mass redistribution (DMR) assays were used to determine the functional muscarinic receptors in each cell line. DMR pathway deconvolution assay was used to determine the pathway biased activity of the muscarinic agonists. Operational agonism model was used to quantify the pathway bias, while macro-kinetic data reported in literature was used to analyze the biochemical mechanism of action of these agonists. Results Quantitative real-time PCR and ligand pharmacology studies showed that all the native cell lines endogenously express functional M3 receptors. Furthermore, different agonists triggered distinct DMR signals in a specific cell line as well as in different cell lines. DMR pathway deconvolution using known G protein modulators revealed that the M3 receptor in all the six cell lines signals through multiple G protein-mediated pathways, and certain agonists display biased agonism in a cell line-dependent manner. The whole cell efficacy and potency of these agonists were found to be sensitive to the assay time as well as the cell background. Correlation analysis suggested that the whole cell efficacy of agonists is correlated well with their macro-dissociation rate constants. Discussion This study implicates that the endogenous M3 receptors are coupled to multiple pathways, and

  14. Negative cooperativity in binding of muscarinic receptor agonists and GDP as a measure of agonist efficacy

    PubMed Central

    Jakubík, J; Janíčková, H; El-Fakahany, EE; Doležal, V

    2011-01-01

    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 Gi/o G-proteins but only its dissociation from Gs/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 Gi/o versus Gs/olf G-proteins that are not identified by conventional GTPγS binding. PMID:20958290

  15. Negative cooperativity in binding of muscarinic receptor agonists and GDP as a measure of agonist efficacy.

    PubMed

    Jakubík, J; Janíčková, H; El-Fakahany, E E; Doležal, V

    2011-03-01

    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. 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. 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. 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. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

  16. Activation of m1 muscarinic acetylcholine receptor induces surface transport of KCNQ channels through a CRMP-2-mediated pathway

    PubMed Central

    Jiang, Ling; Kosenko, Anastasia; Yu, Clinton; Huang, Lan; Li, Xuejun; Hoshi, Naoto

    2015-01-01

    ABSTRACT Neuronal excitability is strictly regulated by various mechanisms, including modulation of ion channel activity and trafficking. Stimulation of m1 muscarinic acetylcholine receptor (also known as CHRM1) increases neuronal excitability by suppressing the M-current generated by the Kv7/KCNQ channel family. We found that m1 muscarinic acetylcholine receptor stimulation also triggers surface transport of KCNQ subunits. This receptor-induced surface transport was observed with KCNQ2 as well as KCNQ3 homomeric channels, but not with Kv3.1 channels. Deletion analyses identified that a conserved domain in a proximal region of the N-terminal tail of KCNQ protein is crucial for this surface transport – the translocation domain. Proteins that bind to this domain were identified as α- and β-tubulin and collapsin response mediator protein 2 (CRMP-2; also known as DPYSL2). An inhibitor of casein kinase 2 (CK2) reduced tubulin binding to the translocation domain, whereas an inhibitor of glycogen synthase kinase 3 (GSK3) facilitated CRMP-2 binding to the translocation domain. Consistently, treatment with the GSK3 inhibitor enhanced receptor-induced KCNQ2 surface transport. M-current recordings from neurons showed that treatment with a GSK3 inhibitor shortened the duration of muscarinic suppression and led to over-recovery of the M-current. These results suggest that m1 muscarinic acetylcholine receptor stimulates surface transport of KCNQ channels through a CRMP-2-mediated pathway. PMID:26446259

  17. Activation of m1 muscarinic acetylcholine receptor induces surface transport of KCNQ channels through a CRMP-2-mediated pathway.

    PubMed

    Jiang, Ling; Kosenko, Anastasia; Yu, Clinton; Huang, Lan; Li, Xuejun; Hoshi, Naoto

    2015-11-15

    Neuronal excitability is strictly regulated by various mechanisms, including modulation of ion channel activity and trafficking. Stimulation of m1 muscarinic acetylcholine receptor (also known as CHRM1) increases neuronal excitability by suppressing the M-current generated by the Kv7/KCNQ channel family. We found that m1 muscarinic acetylcholine receptor stimulation also triggers surface transport of KCNQ subunits. This receptor-induced surface transport was observed with KCNQ2 as well as KCNQ3 homomeric channels, but not with Kv3.1 channels. Deletion analyses identified that a conserved domain in a proximal region of the N-terminal tail of KCNQ protein is crucial for this surface transport--the translocation domain. Proteins that bind to this domain were identified as α- and β-tubulin and collapsin response mediator protein 2 (CRMP-2; also known as DPYSL2). An inhibitor of casein kinase 2 (CK2) reduced tubulin binding to the translocation domain, whereas an inhibitor of glycogen synthase kinase 3 (GSK3) facilitated CRMP-2 binding to the translocation domain. Consistently, treatment with the GSK3 inhibitor enhanced receptor-induced KCNQ2 surface transport. M-current recordings from neurons showed that treatment with a GSK3 inhibitor shortened the duration of muscarinic suppression and led to over-recovery of the M-current. These results suggest that m1 muscarinic acetylcholine receptor stimulates surface transport of KCNQ channels through a CRMP-2-mediated pathway. © 2015. Published by The Company of Biologists Ltd.

  18. Amnesic effects of the anticholinergic drugs, trihexyphenidyl and biperiden: differences in binding properties to the brain muscarinic receptor.

    PubMed

    Kimura, Y; Ohue, M; Kitaura, T; Kihira, K

    1999-07-10

    An amnesic effect of anticholinergic drugs was previously described from several behavioral studies. We examined this effect induced by trihexyphenidyl and biperiden, clinically used in the parkinsonism and schizophrenic patients, by using passive avoidance tasks. Both of these drugs (0.1-10 mg/kg, s.c.) showed dose-dependent amnesic effects in the acquisition and retrieval phases. However, the effect induced by trihexyphenidyl was transient, whereas that of biperiden was long-lasting. To clarify the reason for the different duration of the amnesic activity, binding to the muscarinic receptor was examined. In the Scatchard analysis, trihexyphenidyl competed with [(3)H]quinuclidinyl benzilate ([(3)H]QNB) on the muscarinic receptor (showed increased K(d) and unchanged B(max) value), while biperiden decreased [(3)H]QNB binding (B(max) value) significantly. Furthermore, in an exchange assay for receptor inactivation, trihexyphenidyl binding to muscarinic receptors was exchanged by [(3)H]QNB completely, but biperiden decreased the exchangeable binding of [(3)H]QNB in a dose dependent manner (0.1-100 nM). These results suggested that the binding of trihexyphenidyl and biperiden to muscarinic receptor might be completely reversible and partially irreversible, respectively, whereas the K(i) values of these two drugs were similar. In conclusion, this difference in binding property may explain the difference in the time-course of the amnesic effect induced by trihexyphenidyl and biperiden.

  19. Muscarinic receptors participation in angiogenic response induced by macrophages from mammary adenocarcinoma-bearing mice

    PubMed Central

    de la Torre, Eulalia; Davel, Lilia; Jasnis, María A; Gotoh, Tomomi; de Lustig, Eugenia Sacerdote; Sales, María E

    2005-01-01

    Introduction The role of macrophages in tumor progression has generated contradictory evidence. We had previously demonstrated the ability of peritoneal macrophages from LMM3 murine mammary adenocarcinoma-bearing mice (TMps) to increase the angiogenicity of LMM3 tumor cells, mainly through polyamine synthesis. Here we investigate the ability of the parasympathetic nervous system to modulate angiogenesis induced by TMps through the activation of the muscarinic acetylcholine receptor (mAchR). Methods Peritoneal macrophages from female BALB/c mice bearing a 7-day LMM3 tumor were inoculated intradermally (3 × 105 cells per site) into syngeneic mice. Before inoculation, TMps were stimulated with the muscarinic agonist carbachol in the absence or presence of different muscarinic antagonists or enzyme inhibitors. Angiogenesis was evaluated by counting vessels per square millimeter of skin. The expression of mAchR, arginase and cyclo-oxygenase (COX) isoforms was analyzed by Western blotting. Arginase and COX activities were evaluated by urea and prostaglandin E2 (PGE2) production, respectively. Results TMps, which stimulate neovascularization, express functional mAchR, because carbachol-treated TMps potently increased new blood vessels formation. This response was completely blocked by preincubating TMps with pirenzepine and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP), M1 and M3 receptor antagonists, and partly by the M2 receptor antagonist methoctramine. M1 receptor activation by carbachol in TMps triggers neovascularization through arginase products because Nω-hydroxy-L-arginine reversed the agonist action. Preincubation of TMps with methoctramine partly prevented carbachol-stimulated urea formation. In addition, COX-derived liberation of PGE2 is responsible for the promotion of TMps angiogenic activity by M3 receptor. We also detected a higher expression of vascular endothelial growth factor (VEGF) in TMps than in macrophages from normal mice. Carbachol

  20. Muscarinic receptors participation in angiogenic response induced by macrophages from mammary adenocarcinoma-bearing mice.

    PubMed

    de la Torre, Eulalia; Davel, Lilia; Jasnis, María A; Gotoh, Tomomi; de Lustig, Eugenia Sacerdote; Sales, María E

    2005-01-01

    The role of macrophages in tumor progression has generated contradictory evidence. We had previously demonstrated the ability of peritoneal macrophages from LMM3 murine mammary adenocarcinoma-bearing mice (TMps) to increase the angiogenicity of LMM3 tumor cells, mainly through polyamine synthesis. Here we investigate the ability of the parasympathetic nervous system to modulate angiogenesis induced by TMps through the activation of the muscarinic acetylcholine receptor (mAchR). Peritoneal macrophages from female BALB/c mice bearing a 7-day LMM3 tumor were inoculated intradermally (3 x 10(5) cells per site) into syngeneic mice. Before inoculation, TMps were stimulated with the muscarinic agonist carbachol in the absence or presence of different muscarinic antagonists or enzyme inhibitors. Angiogenesis was evaluated by counting vessels per square millimeter of skin. The expression of mAchR, arginase and cyclo-oxygenase (COX) isoforms was analyzed by Western blotting. Arginase and COX activities were evaluated by urea and prostaglandin E2 (PGE2) production, respectively. TMps, which stimulate neovascularization, express functional mAchR, because carbachol-treated TMps potently increased new blood vessels formation. This response was completely blocked by preincubating TMps with pirenzepine and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP), M1 and M3 receptor antagonists, and partly by the M2 receptor antagonist methoctramine. M1 receptor activation by carbachol in TMps triggers neovascularization through arginase products because Nomega-hydroxy-L-arginine reversed the agonist action. Preincubation of TMps with methoctramine partly prevented carbachol-stimulated urea formation. In addition, COX-derived liberation of PGE2 is responsible for the promotion of TMps angiogenic activity by M3 receptor. We also detected a higher expression of vascular endothelial growth factor (VEGF) in TMps than in macrophages from normal mice. Carbachol significantly increased VEGF

  1. The Effects of Repeated Low-Level Sarin Exposure on Muscarinic M1 Receptor Binding, Amyloid Precursor Protein Levels and Neuropathology

    DTIC Science & Technology

    2005-08-01

    Muscarinic; Nerve agents; Organophosphorus; Pirenzepine ; Receptor Binding; Sarin 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT...either Bmax (receptor density) or Kd (receptor affinity) following cortical M1 muscarinic receptor binding using [3H]- Pirenzepine , across all five...binding assays using [3H]- Pirenzepine (m1AChR ligand; Hammer et al., 1980), Western blotting using an antibody to APP in cortex, and neuropathological

  2. ADP-ribosylation factor-dependent phospholipase D activation by the M3 muscarinic receptor.

    PubMed

    Mitchell, Rory; Robertson, Derek N; Holland, Pamela J; Collins, Daniel; Lutz, Eve M; Johnson, Melanie S

    2003-09-05

    G protein-coupled receptors can potentially activate phospholipase D (PLD) by a number of routes. We show here that the native M3 muscarinic receptor in 1321N1 cells and an epitope-tagged M3 receptor expressed in COS7 cells substantially utilize an ADP-ribosylation factor (ARF)-dependent route of PLD activation. This pathway is activated at the plasma membrane but appears to be largely independent of G, phospholipase C, Ca2+ q/11, protein kinase C, tyrosine kinases, and phosphatidyl inositol 3-kinase. We report instead that it involves physical association of ARF with the M3 receptor as demonstrated by co-immunoprecipitation and by in vitro interaction with a glutathione S-transferase fusion protein of the receptor's third intracellular loop domain. Experiments with mutant constructs of ARF1/6 and PLD1/2 indicate that the M3 receptor displays a major ARF1-dependent route of PLD1 activation with an additional ARF6-dependent pathway to PLD1 or PLD2. Examples of other G protein-coupled receptors assessed in comparison display alternative pathways of protein kinase C- or ARF6-dependent activation of PLD2.

  3. Muscarinic Acetylcholine Receptor Localization and Activation Effects on Ganglion Response Properties

    PubMed Central

    Renna, Jordan M.; Amthor, Franklin R.; Keyser, Kent T.

    2010-01-01

    Purpose. The activation and blockade of muscarinic acetylcholine receptors (mAChRs) affects retinal ganglion cell light responses and firing rates. This study was undertaken to identify the full complement of mAChRs expressed in the rabbit retina and to assess mAChR distribution and the functional effects of mAChR activation and blockade on retinal response properties. Methods. RT-PCR, Western blot analysis, and immunohistochemistry were used to identify the complement and distribution of mAChRs in the rabbit retina. Extracellular electrophysiology was used to determine the effects of the activation or blockade of mAChRs on ganglion cell response properties. Results. RT-PCR of whole neural retina resulted in the amplification of mRNA transcripts for the m1 to m5 mAChR subtypes. Western blot and immunohistochemical analyses confirmed that all five mAChR subtypes were expressed by subpopulations of bipolar, amacrine, and ganglion cells in the rabbit retina, including subsets of cells in cholinergic and glycinergic circuits. Nonspecific muscarinic activation and blockade resulted in the class-specific modulation of maintained ganglion cell firing rates and light responses. Conclusions. The expression of mAChR subtypes on subsets of bipolar, amacrine, and ganglion cells provides a substrate for both enhancement and suppression of retinal responses via activation by cholinergic agents. Thus, the muscarinic cholinergic system in the retina may contribute to the modulation of complex stimuli. Understanding the distribution and function of mAChRs in the retina has the potential to provide important insights into the visual changes that are caused by decreased ACh in the retinas of Alzheimer's patients and the potential visual effects of anticholinergic treatments for ocular diseases. PMID:20042645

  4. An interspecies comparison of mercury inhibition on muscarinic acetylcholine receptor binding in the cerebral cortex and cerebellum

    SciTech Connect

    Basu, Niladri; Stamler, Christopher J.; Loua, Kovana Marcel; Chan, H.M. . E-mail: laurie.chan@mcgill.ca

    2005-05-15

    Mercury (Hg) is a ubiquitous pollutant that can disrupt neurochemical signaling pathways in mammals. It is well documented that inorganic Hg (HgCl{sub 2}) and methyl Hg (MeHg) can inhibit the binding of radioligands to the muscarinic acetylcholine (mACh) receptor in rat brains. However, little is known concerning this relationship in specific anatomical regions of the brain or in other species, including humans. The purpose of this study was to explore the inhibitory effects of HgCl{sub 2} and MeHg on [{sup 3}H]-quinuclidinyl benzilate ([{sup 3}H]-QNB) binding to the mACh receptor in the cerebellum and cerebral cortex regions from human, rat, mouse, mink, and river otter brain tissues. Saturation binding curves were obtained from each sample to calculate receptor density (B {sub max}) and ligand affinity (K {sub d}). Subsequently, samples were exposed to HgCl{sub 2} or MeHg to derive IC50 values and inhibition constants (K {sub i}). Results demonstrate that HgCl{sub 2} is a more potent inhibitor of mACh receptor binding than MeHg, and the receptors in the cerebellum are more sensitive to Hg-mediated mACh receptor inhibition than those in the cerebral cortex. Species sensitivities, irrespective of Hg type and brain region, can be ranked from most to least sensitive: river otter > rat > mink > mouse > humans. In summary, our data demonstrate that Hg can inhibit the binding [{sup 3}H]-QNB to the mACh receptor in a range of mammalian species. This comparative study provides data on interspecies differences and a framework for interpreting results from human, murine, and wildlife studies.

  5. Vitamin D3 restores altered cholinergic and insulin receptor expression in the cerebral cortex and muscarinic M3 receptor expression in pancreatic islets of streptozotocin induced diabetic rats.

    PubMed

    Kumar, Peeyush T; Antony, Sherin; Nandhu, Mohan S; Sadanandan, Jayanarayanan; Naijil, George; Paulose, Chiramadathikudiyil S

    2011-05-01

    Nutritional therapy is a challenging but necessary dimension in the management of diabetes and neurodegenerative changes associated with it. The study evaluates the effect of vitamin D(3) in preventing the altered function of cholinergic, insulin receptors and GLUT3 in the cerebral cortex of diabetic rats. Muscarinic M3 acetylcholine receptors in pancreas control insulin secretion. Vitamin D(3) treatment in M3 receptor regulation in the pancreatic islets was also studied. Radioreceptor binding assays and gene expression was done in the cerebral cortex of male Wistar rats. Immunocytochemistry of muscarinic M3 receptor was studied in the pancreatic islets using specific antibodies. Y-maze was used to evaluate the exploratory and spatial memory. Diabetes induced a decrease in muscarinic M1, insulin and vitamin D receptor expression and an increase in muscarinic M3, α7 nicotinic acetylcholine receptor, acetylcholine esterase and GLUT3 expression. Vitamin D(3) and insulin treatment reversed diabetes-induced alterations to near control. Diabetic rats showed a decreased Y-maze performance while vitamin D(3) supplementation improved the behavioural deficit. In conclusion, vitamin D(3) shows a potential therapeutic effect in normalizing diabetes-induced alterations in cholinergic, insulin and vitamin D receptor and maintains a normal glucose transport and utilisation in the cortex. In addition vitamin D(3) modulated muscarinic M3 receptors activity in pancreas and plays a pivotal role in controlling insulin secretion. Hence our findings proved, vitamin D(3) supplementation as a potential nutritional therapy in ameliorating diabetes mediated cortical dysfunctions and suggest an interaction between vitamin D(3) and muscarinic M3 receptors in regulating insulin secretion from pancreas.

  6. Role of muscarinic and nicotinic cholinergic receptors in an experimental model of epilepsy-induced analgesia.

    PubMed

    de Freitas, Renato Leonardo; de Oliveira, Rithiele Cristina; de Carvalho, Andressa Daiane; Felippotti, Tatiana Tocchini; Bassi, Gabriel Shimizu; Elias-Filho, Daoud Hibrahim; Coimbra, Norberto Cysne

    2004-10-01

    The blockade of GABA-mediated Cl(-) influx with pentylenetetrazol (PTZ) was used in the present work to induce seizures in animals. The neurotransmission in the postictal period has been the focus of many studies, and there is evidence suggesting antinociceptive mechanisms following tonic-clonic seizures in both animals and men. The aim of this work was to study the involvement of acetylcholine in the antinociception induced by convulsions elicited by peripheral administration of PTZ (64 mg/kg). Analgesia was measured by the tail-flick test in eight albino Wistar rats per group. Convulsions were followed by significant increases in tail-flick latencies (TFLs) at least for 120 min of the postictal period. Peripheral administration of atropine (0.25, 1 and 4 mg/kg) caused a significant dose-dependent decrease in the TFL in seizing animals, as compared to controls. These data were corroborated by peripheral administration of mecamylamine, a nicotinic cholinergic receptor blocker, at the same doses (0.25, 1 and 4 mg/kg) used for the muscarinic cholinergic receptor antagonist. The recruitment of the muscarinic receptor was made 10 min postconvulsions and in subsequent periods of postictal analgesia, whereas the involvement of the nicotinic cholinergic receptor was implicated only after 30 min postseizures. The cholinergic antagonists caused a minimal reduction in body temperature, but did not impair baseline TFL, spontaneous exploration or motor coordination in the rotarod test at the maximal dose of 4 mg/kg. These results indicate that acetylcholine may be involved as a neurotransmitter in postictal analgesia.

  7. Muscarinic receptor modulation of GABA-mediated giant depolarizing potentials in the neonatal rat hippocampus

    PubMed Central

    Avignone, Elena; Cherubini, Enrico

    1999-01-01

    The whole-cell patch clamp technique was used to study the role of muscarinic receptors in regulating the frequency of giant depolarizing potentials (GDPs) in CA3 hippocampal neurones in slices from postnatal (P) P1-P8 rats. Atropine (1 μM) reduced the frequency of GDPs by 64·2 ± 2·9%. The acetylcholinesterase inhibitor edrophonium (20 μM) increased the frequency of GDPs in a developmentally regulated way. This effect was antagonized by the M1 muscarinic receptor antagonist pirenzepine. In the presence of edrophonium, tetanic stimulation of cholinergic fibres induced either an enhancement of GDP frequency (179 ± 79%) or a membrane depolarization (27 ± 16 mV) associated with an increase in synaptic noise. These effects were prevented by atropine. Application of carbachol (3 μM) produced an increase in GDP frequency that at P5-P6 was associated with a membrane depolarization and an increase in synaptic noise. These effects were prevented by atropine, pirenzepine (3 μM) and bicuculline (10 μM). In the presence of pirenzepine, carbachol reduced GDP frequency by 50 ± 4%. Conversely, in the presence of methoctramine (3 μM), carbachol enhanced GDP frequency by 117 ± 4%. It is concluded that endogenous acetylcholine, through the activation of M1 receptors, enhances the release of γ-aminobutyric acid (GABA), in a developmentally regulated way. On the other hand, carbachol exerts both an up- and downregulation of GABA release through the activation of M1 and M2 receptors, respectively. PMID:10373692

  8. Regulation of muscarinic acetylcholine receptors in cultured guinea pig pancreatic acini

    SciTech Connect

    Hootman, S.R.; Brown, M.E.; Williams, J.A.; Logsdon, C.D.

    1986-07-01

    Regulation of muscarinic receptors in cultured guinea pig pancreatic acini was investigated by assessing the effects of cholinergic agonists on binding of (N-methyl-TH)scopolamine ((TH)NMS) and on amylase release. Freshly dispersed acini bound (TH)NMS with a K/sub d/ of 74 pM and a maximal binding level (B/sub max/) of 908 fmol/mg DNA. Carbachol (CCh) stimulated amylase secretion and inhibited (TH)NMS binding. Incubation of acini for 30 min with 0.1 mM CCh decreased the subsequent efficacy of CCh in stimulating amylase release by threefold but had no effect on its potency. In contrast, amylase release in response to cholecystokinin octapeptide (CCK-8) was not altered by CCh preincubation. (TH)NMS binding to acini was decreased only 15-20% after 30-min incubation with CCh. However, culture of acini with 0.1 mM CCh decreased (TH)NMS binding by 50% at 3-4 h and by 85-90% at 24 h. This decrease was attributable primarily to a reduction in B/sub max/ (TH)NMS binding also was decreased to a similar extent by the cholinergic agonists bethanechol and methacholine but not by other secretagogues. The decrease in antagonist binding induced by CCh was dose dependent, with the IC50, 5.8 M, approximating the EC50 for amylase release, 4.3 M. Cultured of acini for 24 h with CCh abolished subsequent amylase release in response to CCh but not to CCK-8. The results indicate that muscarinic receptor turnover in the pancreatic acinus is regulated by receptor activation and that both a decease in receptor numbers and sensitivity to agonists follows prolonged cholinergic agonist exposure.

  9. Fesoterodine, its active metabolite, and tolterodine bind selectively to muscarinic receptors in human bladder mucosa and detrusor muscle.

    PubMed

    Yoshida, Akira; Fuchihata, Yusuke; Kuraoka, Shiori; Osano, Ayaka; Otsuka, Atsushi; Ozono, Seiichiro; Takeda, Masayuki; Masuyama, Keisuke; Araki, Isao; Yamada, Shizuo

    2013-04-01

    To comparatively characterize the binding activity of fesoterodine, its active metabolite (5-hydroxymethyl tolterodine [5-HMT]), and tolterodine in the human bladder mucosa, detrusor muscle, and parotid gland. Muscarinic receptors in the homogenates of human bladder mucosa, detrusor muscle, and parotid gland were measured by a radioligand binding assay using [N-methyl-(3)H] scopolamine methyl chloride. Fesoterodine, 5-HMT, and tolterodine competed with [N-methyl-(3)H] scopolamine methyl chloride for binding sites in the bladder mucosa, detrusor muscle, and parotid gland in a concentration-dependent manner. The affinity for muscarinic receptors of these agents was significantly greater in the bladder than in the parotid gland, suggesting pharmacologic selectivity for the bladder over the parotid gland. The bladder selectivity was larger for fesoterodine and 5-HMT than for tolterodine. Fesoterodine, 5-HMT, and tolterodine resulted in significantly increased (two- to five-fold) values of the apparent dissociation constant for specific [N-methyl-(3)H] scopolamine methyl chloride binding in the detrusor muscle and parotid gland, with little effect on the corresponding values of the maximal number of binding sites. This finding indicates that these agents bind to the human muscarinic receptors in a competitive and reversible manner. Fesoterodine and 5-HMT bind to the muscarinic receptors with greater affinity in the human bladder mucosa and detrusor muscle than in the parotid gland in a competitive and reversible manner. Copyright © 2013 Elsevier Inc. All rights reserved.

  10. Characteristics of muscarinic receptors that selectively couple to inhibition of adenylate cyclase or stimulation of phospholipase C on NG108-15 and 1321N1 cells

    SciTech Connect

    Liang, M.

    1988-01-01

    The purpose of this dissertation was to establish whether different muscarinic receptor proteins selectively couple to different second messenger response system. Although both second messenger response systems are fully functional in both cell lines, activation of muscarinic cholinergic receptors only results in inhibition of adenylate cyclase in NG108-15 neuroblastoma {times} glioma cells and stimulation of phosphoinositide hydrolysis in 1321N1 human astrocytoma cells. Muscarinic receptors on both cell types were covalently labeled with ({sup 3}H)Propylbenzilylcholine mustard (({sup 3}H)PBCM) and the mobilities of the ({sup 3}H)PBCM-labelled species of both cells were compared by SDS-PAGE. 1321N1 and NG108-15 cells each primarily expressed a single ({sup 3}H)PBCM-labelled species with an apparent size of approximately 92,000 and 66,000 Da, respectively. ({sup 3}H)PBCM labelling was completely inhibited by 1 {mu}M atropine or by down-regulation of muscarinic receptors by an overnight incubation with carbachol. The apparent size of the ({sup 3}H)PBCM-labelled species of both cell lines was not altered by treatment with a series of protease inhibitors or by treatment with dithiothreitol and iodoacetamide. Another approach for determining differences in the muscarinic receptors of 2 cells lines was to study agonist-induced alteration of muscarinic receptor number. Exposure of both cell types to agonists resulted in rapid loss of muscarinic receptors from cell surface without change of total cellular muscarinic receptors followed by subsequently loss of receptors from cells. Muscarinic receptors on both cell lines were regulated by agonist with similar properties.

  11. Guanylpirenzepine distinguishes between neuronal ml and m4 muscarinic receptor subtypes

    SciTech Connect

    Monferini, E.; Cereda, E.; Ladinsky, H.; Donetti, A.; Giraldo, E. )

    1990-01-01

    Guanylpirenzepine, a polar, non-quaternary analog of pirenzepine, exhibited a novel binding behavior in rat brain regions: in competition binding experiments against (3H)pirenzepine labeling the M1 receptor in membranes from cerebral cortex, hippocampus and striatum, the compound, differently from pirenzepine, displayed heterogeneous binding curves. Computer assisted analysis of these curves, evidenced the existence of two populations of binding sites: a large proportion (84-89%) of high affinity receptors (KH = 64-92 nM) and a remainder with very low affinity (KL = 19-28 microM). Like pirenzepine, guanylpirenzepine showed low affinity for the glandular M3 and the cardiac M2 receptors when (3H)N-methylscopolamine was used to label the receptors in membranes from these two tissues; affinity values for guanylpirenzepine were 1336 and 5790 nM respectively, vs 323 and 683 nM for pirenzepine. We conclude that guanylpirenzepine is able to discriminate between m1 and m4 receptor subtypes and may represent a new tool for deeper studies on muscarinic receptors classification.

  12. Muscarinic receptor-mediated inositol tetrakisphosphate response in bovine adrenal chromaffin cells

    SciTech Connect

    Sanborn, B.B.; Schneider, A.S. )

    1990-01-01

    Inositol trisphosphate (IP{sub 3}), a product of the phosphoinositide cycle, mobilizes intracellular Ca{sup 2+} in many cell types. New evidence suggests that inositol tetrakisphosphate (IP{sub 4}), an IP{sub 3} derivative, may act as another second messenger to further alter calcium homeostasis. However, the function and mechanism of action of IP{sub 4} are presently unresolved. We now report evidence of muscarinic receptor-mediated accumulation of IP{sub 4} in bovine adrenal chromaffin cells, a classic neurosecretory system in which calcium movements have been well studied. Muscarine stimulated an increase in ({sup 3}H)IP{sub 4} and ({sup 3}H)IP{sub 3} accumulation in chromaffin cells and this effect was completely blocked by atropine. ({sup 3}H)IP{sub 4} accumulation was detectable within 15 sec, increased to a maximum by 30 sec and thereafter declined. 2,3-diphosphoglycerate, an inhibitor of IP{sub 3} and IP{sub 4} hydrolysis, enhanced accumulation of these inositol polyphosphates. The results provide the first evidence of a rapid inositol tetrakisphosphate response in adrenal chromaffin cells, which should facilitate the future resolution of the relationship between IP{sub 4} and calcium homeostasis.

  13. Endocannabinoids Mediate Muscarinic Acetylcholine Receptor-Dependent Long-Term Depression in the Adult Medial Prefrontal Cortex.

    PubMed

    Martin, Henry G S; Bernabeu, Axel; Lassalle, Olivier; Bouille, Clément; Beurrier, Corinne; Pelissier-Alicot, Anne-Laure; Manzoni, Olivier J

    2015-01-01

    Cholinergic inputs into the prefrontal cortex (PFC) are associated with attention and cognition; however there is evidence that acetylcholine also has a role in PFC dependent learning and memory. Muscarinic acetylcholine receptors (mAChR) in the PFC can induce synaptic plasticity, but the underlying mechanisms remain either opaque or unresolved. We have characterized a form of mAChR mediated long-term depression (LTD) at glutamatergic synapses of layer 5 principal neurons in the adult medial PFC. This mAChR LTD is induced with the mAChR agonist carbachol and inhibited by selective M1 mAChR antagonists. In contrast to other cortical regions, we find that this M1 mAChR mediated LTD is coupled to endogenous cannabinoid (eCB) signaling. Inhibition of the principal eCB CB1 receptor blocked carbachol induced LTD in both rats and mice. Furthermore, when challenged with a sub-threshold carbachol application, LTD was induced in slices pretreated with the monoacylglycerol lipase (MAGL) inhibitor JZL184, suggesting that the eCB 2-arachidonylglyerol (2-AG) mediates M1 mAChR LTD. Yet, when endogenous acetylcholine was released from local cholinergic afferents in the PFC using optogenetics, it failed to trigger eCB-LTD. However coupling patterned optical and electrical stimulation to generate local synaptic signaling allowed the reliable induction of LTD. The light-electrical pairing induced LTD was M1 mAChR and CB1 receptor mediated. This shows for the first time that connecting excitatory synaptic activity with coincident endogenously released acetylcholine controls synaptic gain via eCB signaling. Together these results shed new light on the mechanisms of synaptic plasticity in the adult PFC and expand on the actions of endogenous cholinergic signaling.

  14. Endocannabinoids Mediate Muscarinic Acetylcholine Receptor-Dependent Long-Term Depression in the Adult Medial Prefrontal Cortex

    PubMed Central

    Martin, Henry G. S.; Bernabeu, Axel; Lassalle, Olivier; Bouille, Clément; Beurrier, Corinne; Pelissier-Alicot, Anne-Laure; Manzoni, Olivier J.

    2015-01-01

    Cholinergic inputs into the prefrontal cortex (PFC) are associated with attention and cognition; however there is evidence that acetylcholine also has a role in PFC dependent learning and memory. Muscarinic acetylcholine receptors (mAChR) in the PFC can induce synaptic plasticity, but the underlying mechanisms remain either opaque or unresolved. We have characterized a form of mAChR mediated long-term depression (LTD) at glutamatergic synapses of layer 5 principal neurons in the adult medial PFC. This mAChR LTD is induced with the mAChR agonist carbachol and inhibited by selective M1 mAChR antagonists. In contrast to other cortical regions, we find that this M1 mAChR mediated LTD is coupled to endogenous cannabinoid (eCB) signaling. Inhibition of the principal eCB CB1 receptor blocked carbachol induced LTD in both rats and mice. Furthermore, when challenged with a sub-threshold carbachol application, LTD was induced in slices pretreated with the monoacylglycerol lipase (MAGL) inhibitor JZL184, suggesting that the eCB 2-arachidonylglyerol (2-AG) mediates M1 mAChR LTD. Yet, when endogenous acetylcholine was released from local cholinergic afferents in the PFC using optogenetics, it failed to trigger eCB-LTD. However coupling patterned optical and electrical stimulation to generate local synaptic signaling allowed the reliable induction of LTD. The light—electrical pairing induced LTD was M1 mAChR and CB1 receptor mediated. This shows for the first time that connecting excitatory synaptic activity with coincident endogenously released acetylcholine controls synaptic gain via eCB signaling. Together these results shed new light on the mechanisms of synaptic plasticity in the adult PFC and expand on the actions of endogenous cholinergic signaling. PMID:26648844

  15. Activation of midbrain presumed dopaminergic neurones by muscarinic cholinergic receptors: an in vivo electrophysiological study in the rat

    PubMed Central

    Gronier, B; Rasmussen, K

    1998-01-01

    Extracellular single-unit recording and iontophoresis were used to examine the effects of different cholinoceptor agonists and antagonists on the firing rate and firing pattern of A9 and A10 presumed dopaminergic neurones in the anaesthetized rat.Administration of low currents (1–5 nA) of the selective muscarinic agonists oxotremorine M (Oxo M) and muscarine and of the non-selective muscarinic/nicotinic agonist carbamylcholine (CCh) produced a dose-dependent increase in firing rate in most of the A9 and A10 presumed dopaminergic neurones tested. Oxo M-induced activation could be completely blocked by iontophoretic application of the muscarinic antagonist butyl-scopolamine or systemic administration of the muscarinic antagonist scopolamine (300 μg kg−1, i.v.).Iontophoretic application of the selective nicotinic agonist methylcarbamylcholine (MCCh), but not nicotine, induced a consistent increase in firing rate. Surprisingly, the excitatory effect of MCCh was significantly reduced by the selective muscarinic antagonist scopolamine (300 μg kg−1, i.v.), but not by the selective nicotinic antagonist mecamylamine (2.2 mg kg−1, i.v.). Mecamylamine (3 mg kg−1, i.v.) was also ineffective in reducing the CCh-induced activation of presumed dopamine neurones, suggesting that both CCh and MCCh increased the activity of dopamine neurones via an interaction with muscarinic receptors.Iontophoretic application of the endogenous agonist acetylcholine (ACh) had no or little effect on the firing activity of A10 presumed dopaminergic neurones. However, concomitant application of neostigmine, a potent cholinesterase inhibitor, with acetylcholine induced a substantial activation of these neurones. This activation consisted of two components; one, which was prevalent, was scopolamine (300 μg kg−1, i.v.)-sensitive, and the other was mecamylamine (2 mg kg−1, i.v.)-sensitive.In addition to their effect on firing activity, Oxo M, muscarine and

  16. Functional and biochemical characteristics of urinary bladder muscarinic receptors in long-term alloxan diabetic rats

    PubMed Central

    Rocha, Jeová Nina

    2015-01-01

    Objective To re-examine the function of the urinary bladder in vivo as well as to determine the functional and biochemical characteristics of bladder muscarinic receptors in long-term alloxan-induced diabetes rats. Methods Two-month-old male Wistar rats were injected with alloxan and the animals showing blood glucose levels >300mg/dL together with age-paired untreated animals were kept for 11 months. Body weight, bladder weight, blood glucose, and urinary volume over a period of 24 hours were determined in both groups of animals. A voiding cystometry in conscious control and diabetic rats was performed to determine maximal micturition pressure, micturition contraction interval and duration as well as voided and post-voiding residual volume. In addition, concentration-response curves for bethanechol in isolated bladder strips, as well as [3H]-N methyl-scopolamine binding site characteristics in bladder homogenates were determined. Results Mean bladder weight was 162.5±21.2mg versus 290±37.9mg in control and treated animals, respectively (p<0.05). Micturition contraction amplitude (34.6±4.7mmHg versus 49.6±2.5mmHg), duration (14.5±1.7 seconds versus 23.33±4.6 seconds) and interval (87.5±17.02 seconds versus 281.11±20.24 seconds) were significantly greater in alloxan diabetic rats. Voided urine volume per micturition contraction was also significantly higher in diabetic animals. However the post-voiding residual volume was not statistically different. Bethanechol potency (EC50 3µM versus 5µM) and maximal effect (31.2±5.9g/g versus 36.1±6.8g/g) in isolated bladder strips as well as number (169±4fmol/mg versus 176±3fmol/mg protein) and affinity (0.69±0.1nM versus 0.57±0.1nM) of bladder muscarinic receptors were also not statistically different. Conclusion Bladder function in vivo is altered in chronic alloxan-induced diabetes rats without changes in functional and biochemical characteristics of bladder muscarinic receptors. PMID:26466064

  17. Flurbiprofen, a cyclooxygenase inhibitor, reduces the brain arachidonic acid signal in response to the cholinergic muscarinic agonist, arecoline, in awake rats.

    PubMed

    Basselin, Mireille; Villacreses, Nelly E; Lee, Ho-Joo; Bell, Jane M; Rapoport, Stanley I

    2007-11-01

    Cholinergic muscarinic receptors, when stimulated by arecoline, can activate cytosolic phospholipase A(2) (cPLA(2)) to release arachidonic acid (AA) from membrane phospholipid. This signal can be imaged in the brain in vivo using quantitative autoradiography following the intravenous injection of radiolabeled AA, as an increment in a regional brain AA incorporation coefficient k*. Arecoline increases k* significantly in brain regions having muscarinic M(1,3,5) receptors in wild-type but not in cyclooxygenase (COX)-2 knockout mice. To further clarify the roles of COX enzymes in the AA signal, in this paper we imaged k* following arecoline (5 mg/kg i.p.) or saline in each of 81 brain regions of unanesthetized rats pretreated 6 h earlier with the non-selective COX inhibitor flurbiprofen (FB, 60 mg/kg s.c.) or with vehicle. Baseline values of k* were unaffected by FB treatment, which however reduced by 80% baseline brain concentrations of prostaglandin E(2) (PGE(2)) and thromboxane B(2) (TXB(2)), eicosanoids preferentially derived from AA via COX-2 and COX-1, respectively. In vehicle-pretreated rats, arecoline increased the brain PGE(2) but not TXB(2) concentration, as well as values for k* in 77 of the 81 brain regions. FB-pretreatment prevented these arecoline-provoked changes. These results and those reported in COX-2 knockout mice suggest that the AA released in brain following muscarinic receptor-mediated activation is lost via COX-2 to PGE(2) but not via COX-1 to TXB(2), and that increments in k* following arecoline largely represent replacement by unesterified plasma AA of this loss.

  18. Distinct muscarinic acetylcholine receptor subtypes contribute to stability and growth, but not compensatory plasticity, of neuromuscular synapses.

    PubMed

    Wright, Megan C; Potluri, Srilatha; Wang, Xueyong; Dentcheva, Eva; Gautam, Dinesh; Tessler, Alan; Wess, Jürgen; Rich, Mark M; Son, Young-Jin

    2009-11-25

    Muscarinic acetylcholine receptors (mAChRs) modulate synaptic function, but whether they influence synaptic structure remains unknown. At neuromuscular junctions (NMJs), mAChRs have been implicated in compensatory sprouting of axon terminals in paralyzed or denervated muscles. Here we used pharmacological and genetic inhibition and localization studies of mAChR subtypes at mouse NMJs to demonstrate their roles in synaptic stability and growth but not in compensatory sprouting. M(2) mAChRs were present solely in motor neurons, whereas M(1), M(3), and M(5) mAChRs were associated with Schwann cells and/or muscle fibers. Blockade of all five mAChR subtypes with atropine evoked pronounced effects, including terminal sprouting, terminal withdrawal, and muscle fiber atrophy. In contrast, methoctramine, an M(2/4)-preferring antagonist, induced terminal sprouting and terminal withdrawal, but no muscle fiber atrophy. Consistent with this observation, M(2)(-/-) but no other mAChR mutant mice exhibited spontaneous sprouting accompanied by extensive loss of parental terminal arbors. Terminal sprouting, however, seemed not to be the causative defect because partial loss of terminal branches was common even in the M(2)(-/-) NMJs without sprouting. Moreover, compensatory sprouting after paralysis or partial denervation was normal in mice deficient in M(2) or other mAChR subtypes. We also found that many NMJs of M(5)(-/-) mice were exceptionally small and reduced in proportion to the size of parental muscle fibers. These findings show that axon terminals are unstable without M(2) and that muscle fiber growth is defective without M(5). Subtype-specific muscarinic signaling provides a novel means for coordinating activity-dependent development and maintenance of the tripartite synapse.

  19. Identification of four areas each enriched in a unique muscarinic receptor subtype

    SciTech Connect

    Hoss, W.; Ellerbrock, B.R.; Goldman, P.S.; Collins, D.A.; Messer, W.S. Jr. )

    1990-01-01

    The affinities of muscarinic agonists and antagonists were determined by autoradiography and image analysis in selected areas of the rat brain. IC{sub 50} values and Hill coefficients for the inhibition of the binding of 0.2 nM ({sup 3}H)-QNB to dentate gyrus, superior colliculus, rhomboid thalamus and substantia nigra were measured in coronal sections. Pirenzepine displayed a high affinity for receptors in the dentate gyrus and AF-DX 116, the superior colliculus. Both pirenzepine and AF-DX 116 had high affinities for the substantia nigra and low affinities for the rhomboid thalamus. Gallamine displayed a 50-fold preference for superior colliculus over dentate gyrus receptors. Amitriptyline was less selective, showing a modest preference for substantia nigra receptors and 4-DAMP was essentially nonselective. Carbachol was the most selective agonist with a 4000-fold preference for superior colliculus over dentate gyrus receptors. Other agonists except RS 86 were also selective for superior colliculus receptors in the order carbachol >> arecoline > bethanechol > McN A343 = oxotremorine = pilocarpine.

  20. Classical and atypical agonists activate M1 muscarinic acetylcholine receptors through common mechanisms.

    PubMed

    Randáková, Alena; Dolejší, Eva; Rudajev, Vladimír; Zimčík, Pavel; Doležal, Vladimír; El-Fakahany, Esam E; Jakubík, Jan

    2015-07-01

    We mutated key amino acids of the human variant of the M1 muscarinic receptor that target ligand binding, receptor activation, and receptor-G protein interaction. We compared the effects of these mutations on the action of two atypical M1 functionally preferring agonists (N-desmethylclozapine and xanomeline) and two classical non-selective orthosteric agonists (carbachol and oxotremorine). Mutations of D105 in the orthosteric binding site and mutation of D99 located out of the orthosteric binding site decreased affinity of all tested agonists that was translated as a decrease in potency in accumulation of inositol phosphates and intracellular calcium mobilization. Mutation of D105 decreased the potency of the atypical agonist xanomeline more than that of the classical agonists carbachol and oxotremorine. Mutation of the residues involved in receptor activation (D71) and coupling to G-proteins (R123) completely abolished the functional responses to both classical and atypical agonists. Our data show that both classical and atypical agonists activate hM1 receptors by the same molecular switch that involves D71 in the second transmembrane helix. The principal difference among the studied agonists is rather in the way they interact with D105 in the orthosteric binding site. Furthermore, our data demonstrate a key role of D105 in xanomeline wash-resistant binding and persistent activation of hM1 by wash-resistant xanomeline. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  1. Muscarinic acetylcholine receptors act in synergy to facilitate learning and memory.

    PubMed

    Leaderbrand, Katherine; Chen, Helen J; Corcoran, Kevin A; Guedea, Anita L; Jovasevic, Vladimir; Wess, Jurgen; Radulovic, Jelena

    2016-11-01

    Understanding how episodic memories are formed and retrieved is necessary if we are to treat disorders in which they malfunction. Muscarinic acetylcholine receptors (mAChR) in the hippocampus and cortex underlie memory formation, but there is conflicting evidence regarding their role in memory retrieval. Additionally, there is no consensus on which mAChR subtypes are critical for memory processing. Using pharmacological and genetic approaches, we found that (1) encoding and retrieval of contextual memory requires mAChR in the dorsal hippocampus (DH) and retrosplenial cortex (RSC), (2) memory formation requires hippocampal M3 and cooperative activity of RSC M1 and M3, and (3) memory retrieval is more impaired by inactivation of multiple M1-M4 mAChR in DH or RSC than inactivation of individual receptor subtypes. Contrary to the view that acetylcholine supports learning but is detrimental to memory retrieval, we found that coactivation of multiple mAChR is required for retrieval of both recently and remotely acquired context memories. Manipulations with higher receptor specificity were generally less potent than manipulations targeting multiple receptor subtypes, suggesting that mAChR act in synergy to regulate memory processes. These findings provide unique insight into the development of therapies for amnestic symptoms, suggesting that broadly acting, rather than receptor-specific, mAchR agonists and positive allosteric modulators may be the most effective therapeutic approach. © 2016 Leaderbrand et al.; Published by Cold Spring Harbor Laboratory Press.

  2. Accelerated Molecular Dynamics Simulations of Ligand Binding to a Muscarinic G-protein Coupled Receptor

    PubMed Central

    Kappel, Kalli; Miao, Yinglong; McCammon, J. Andrew

    2017-01-01

    Elucidating the detailed process of ligand binding to a receptor is pharmaceutically important for identifying druggable binding sites. With the ability to provide atomistic detail, computational methods are well poised to study these processes. Here, accelerated molecular dynamics (aMD) is proposed to simulate processes of ligand binding to a G-protein coupled receptor (GPCR), in this case the M3 muscarinic receptor, which is a target for treating many human diseases, including cancer, diabetes and obesity. Long-timescale aMD simulations were performed to observe the binding of three chemically diverse ligand molecules: antagonist tiotropium (TTP), partial agonist arecoline (ARc), and full agonist acetylcholine (ACh). In comparison with earlier microsecond-timescale conventional MD simulations, aMD greatly accelerated the binding of ACh to the receptor orthosteric ligand-binding site and the binding of TTP to an extracellular vestibule. Further aMD simulations also captured binding of ARc to the receptor orthosteric site. Additionally, all three ligands were observed to bind in the extracellular vestibule during their binding pathways, suggesting that it is a metastable binding site. This study demonstrates the applicability of aMD to protein-ligand binding, especially the drug recognition of GPCRs. PMID:26537408

  3. Stereoselective L-(3H)quinuclidinyl benzilate-binding sites in nervous tissue of Aplysia californica: evidence for muscarinic receptors

    SciTech Connect

    Murray, T.F.; Mpitsos, G.J.; Siebenaller, J.F.; Barker, D.L.

    1985-12-01

    The muscarinic antagonist L-(/sup 3/H)quinuclidinyl benzilate (L-(/sup 3/H)QNB) binds with a high affinity (Kd = 0.77 nM) to a single population of specific sites (Bmax = 47 fmol/mg of protein) in nervous tissue of the gastropod mollusc, Aplysia. The specific L-(/sup 3/H)QNB binding is displaced stereoselectively by the enantiomers of benzetimide, dexetimide, and levetimide. The pharmacologically active enantiomer, dexetimide, is more potent than levetimide as an inhibitor of L-(/sup 3/H)QNB binding. Moreover, the muscarinic cholinergic ligands, scopolamine, atropine, oxotremorine, and pilocarpine are effective inhibitors of the specific L-(/sup 3/H)QNB binding, whereas nicotinic receptor antagonists, decamethonium and d-tubocurarine, are considerably less effective. These pharmacological characteristics of the L-(/sup 3/H)QNB-binding site provide evidence for classical muscarinic receptors in Aplysia nervous tissue. The physiological relevance of the dexetimide-displaceable L-(/sup 3/H)QNB-binding site was supported by the demonstration of the sensitivity of the specific binding to thermal denaturation. Specific binding of L-(/sup 3/H)QNB was also detected in nervous tissue of another marine gastropod, Pleurobranchaea californica. The characteristics of the Aplysia L-(/sup 3/H)QNB-binding site are in accordance with studies of numerous vertebrate and invertebrate tissues indicating that the muscarinic cholinergic receptor site has been highly conserved through evolution.

  4. Dynamic Regulation of Quaternary Organization of the M1 Muscarinic Receptor by Subtype-selective Antagonist Drugs*

    PubMed Central

    Pediani, John D.; Ward, Richard J.; Godin, Antoine G.; Marsango, Sara

    2016-01-01

    Although rhodopsin-like G protein-coupled receptors can exist as both monomers and non-covalently associated dimers/oligomers, the steady-state proportion of each form and whether this is regulated by receptor ligands are unknown. Herein we address these topics for the M1 muscarinic acetylcholine receptor, a key molecular target for novel cognition enhancers, by using spatial intensity distribution analysis. This method can measure fluorescent particle concentration and assess oligomerization states of proteins within defined regions of living cells. Imaging and analysis of the basolateral surface of cells expressing some 50 molecules·μm−2 human muscarinic M1 receptor identified a ∼75:25 mixture of receptor monomers and dimers/oligomers. Both sustained and shorter term treatment with the selective M1 antagonist pirenzepine resulted in a large shift in the distribution of receptor species to favor the dimeric/oligomeric state. Although sustained treatment with pirenzepine also resulted in marked up-regulation of the receptor, simple mass action effects were not the basis for ligand-induced stabilization of receptor dimers/oligomers. The related antagonist telenzepine also produced stabilization and enrichment of the M1 receptor dimer population, but the receptor subtype non-selective antagonists atropine and N-methylscopolamine did not. In contrast, neither pirenzepine nor telenzepine altered the quaternary organization of the related M3 muscarinic receptor. These data provide unique insights into the selective capacity of receptor ligands to promote and/or stabilize receptor dimers/oligomers and demonstrate that the dynamics of ligand regulation of the quaternary organization of G protein-coupled receptors is markedly more complex than previously appreciated. This may have major implications for receptor function and behavior. PMID:27080256

  5. Dynamic Regulation of Quaternary Organization of the M1 Muscarinic Receptor by Subtype-selective Antagonist Drugs.

    PubMed

    Pediani, John D; Ward, Richard J; Godin, Antoine G; Marsango, Sara; Milligan, Graeme

    2016-06-17

    Although rhodopsin-like G protein-coupled receptors can exist as both monomers and non-covalently associated dimers/oligomers, the steady-state proportion of each form and whether this is regulated by receptor ligands are unknown. Herein we address these topics for the M1 muscarinic acetylcholine receptor, a key molecular target for novel cognition enhancers, by using spatial intensity distribution analysis. This method can measure fluorescent particle concentration and assess oligomerization states of proteins within defined regions of living cells. Imaging and analysis of the basolateral surface of cells expressing some 50 molecules·μm(-2) human muscarinic M1 receptor identified a ∼75:25 mixture of receptor monomers and dimers/oligomers. Both sustained and shorter term treatment with the selective M1 antagonist pirenzepine resulted in a large shift in the distribution of receptor species to favor the dimeric/oligomeric state. Although sustained treatment with pirenzepine also resulted in marked up-regulation of the receptor, simple mass action effects were not the basis for ligand-induced stabilization of receptor dimers/oligomers. The related antagonist telenzepine also produced stabilization and enrichment of the M1 receptor dimer population, but the receptor subtype non-selective antagonists atropine and N-methylscopolamine did not. In contrast, neither pirenzepine nor telenzepine altered the quaternary organization of the related M3 muscarinic receptor. These data provide unique insights into the selective capacity of receptor ligands to promote and/or stabilize receptor dimers/oligomers and demonstrate that the dynamics of ligand regulation of the quaternary organization of G protein-coupled receptors is markedly more complex than previously appreciated. This may have major implications for receptor function and behavior.

  6. Starvation activates MAP kinase through the muscarinic acetylcholine pathway in Caenorhabditis elegans pharynx

    PubMed Central

    You, Young-jai; Kim, Jeongho; Cobb, Melanie; Avery, Leon

    2012-01-01

    Summary Starvation activates MAPK in the pharyngeal muscles of C. elegans through a muscarinic acetylcholine receptor, Gqα, and nPKC as shown by the following results: (1) Starvation causes phosphorylation of MAPK in pharyngeal muscle. (2) In a sensitized genetic background in which Gqα signaling cannot be downregulated, activation of the pathway by a muscarinic agonist causes lethal changes in pharyngeal muscle function. Starvation has identical effects. (3) A muscarinic antagonist blocks the effects of starvation on sensitized muscle. (4) Mutations and drugs that block any step of signaling from the muscarinic receptor to MAPK also block the effects of starvation on sensitized muscle. (5) Overexpression of MAPK in wild-type pharyngeal muscle mimics the effects of muscarinic agonist and of starvation on sensitized muscle. We suggest that, during starvation, the muscarinic pathway to MAPK is activated to change the pharyngeal muscle physiology to enhance ingestion of food when food becomes available. PMID:16581001

  7. Muscarinic cholinergic receptor binding: in vivo depiction using single photon emission computed tomography and radioiodinated quinuclidinyl benzilate

    SciTech Connect

    Drayer, B.; Jaszczak, R.; Coleman, E.; Storni, A.; Greer, K.; Petry, N.; Lischko, M.; Flanagan, S.

    1982-06-01

    An attempt was made to characterize, in vivo, specific binding to the muscarinic cholinergic receptor in the calf using the radioiodinated ligand quinuclidinyl benzilate (/sup 123/I-OH-QNB) and single photon detection emission computed tomography (SPECT). The supratentorial brain activity was significantly increased after the intravenous infusion of /sup 123/I-OH-QNB as compared to free /sup 123/I. Scopolamine, a muscarinic cholinergic receptor antagonist, decreased the measured brain activity when infused prior to /sup 123/I-OH-QNB consistent with pharmacologic blockade of specific receptor binding. Quantitative in vitro tissue distribution studies obtained following SPECT imaging were consistent with regionally distinct specific receptor binding in the striatum and cortical gray matter, nonspecific binding in the cerebellum, and pharmacologic blockade of specific binding sites with scopolamine. Although /sup 123/I-OH-QNB is not the ideal radioligand, our limited success will hopefully encourage the development of improved binding probes for SPECT imaging and quantitation.

  8. Rivastigmine improves isolation rearing-induced prepulse inhibition deficits via muscarinic acetylcholine receptors in mice.

    PubMed

    Higashino, Kosuke; Ago, Yukio; Umeki, Takahiro; Hasebe, Shigeru; Onaka, Yusuke; Hashimoto, Hitoshi; Takuma, Kazuhiro; Matsuda, Toshio

    2016-02-01

    The acetylcholinesterase inhibitors donepezil, galantamine, and rivastigmine are used for the treatment of Alzheimer's disease. We previously demonstrated that donepezil and galantamine differentially affect isolation rearing-induced prepulse inhibition (PPI) deficits and that this might be due to differential effects on brain muscarinic acetylcholine (mACh) receptor function in mice. We examined the effects of rivastigmine on isolation rearing-induced PPI deficits, brain ACh levels, and mACh receptor function in mice. Acoustic startle responses were measured in a startle chamber. Microdialysis was performed, and the levels of dopamine and ACh in the prefrontal cortex were measured. Rivastigmine (0.3 mg/kg) improved PPI deficits, and this improvement was antagonized by the mACh receptor antagonist telenzepine but not by the nicotinic ACh receptor antagonist mecamylamine. Rivastigmine increased extracellular ACh levels by approximately 2-3-fold, less than the increase produced by galantamine. Rivastigmine enhanced the effect of the mACh receptor agonist N-desmethylclozapine on prefrontal dopamine release, a marker of mACh receptor function, and this increase was blocked by telenzepine. In contrast, galantamine did not affect N-desmethylclozapine-induced dopamine release. Furthermore, rivastigmine did not affect cortical dopamine release induced by the serotonin1A receptor agonist osemozotan, suggesting that the effect of rivastigmine has specificity for mACh receptors. Taken together with our previous finding that marked increases in ACh levels are required for the PPI deficit improvement induced by galantamine, our present results suggest that rivastigmine improves isolation rearing-induced PPI deficits by increasing ACh levels and by concomitantly enhancing mACh receptor function.

  9. Effect of propylbenzilylcholine mustard on contraction and radioligand binding parameters of muscarinic receptors in guinea pig ileum

    SciTech Connect

    Rodrigues de Miranda, J.; Salden, H.J.M.; van Ginneken C.A.M.

    1987-10-26

    The receptor occupancy-biological effect relationship for muscarinic receptors in guinea pig ileal smooth muscle has been studied by comparison of radioligand binding and contractile response. Muscarinic receptors in homogenates of ileal smooth muscle were labeled with (/sub 3/H)-1-Quinuclidinyl benzilate. Treatment with propylbenzilylcholine mustard (PrBCM), to inactivate irreversibly muscarinic receptors, caused a large dose dependent rightward shift of the dose-response curve to three agonistic furtrethonium derivatives with a concomitant decrease in maximal response. Using those data, the fraction of receptors remaining unoccupied (q-values) and true affinity constants (-log K/sub A/-values) were calculated. Exposure to 20 or 60 nM PrBCM for 15 minutes resulted in a 39% and a 61% reduction in specific (/sup 3/H)-1-Quinuclidinyl benzilate binding sites respectively to be compared with a 62% and a 85% decrease expected from calculated q-values. K/sub A/-values for the methyl and ethyl derivative agreed well with the dissociation constants for the high affinity agonist sites determined from displacement of (/sup 3/H-)-1-Quinuclidinyl benzilate. The K/sub A/-value for the propylfurtrethonium corresponds to the low affinity agonist dissociation constant. The fraction of receptors in the high affinity agonist state differs considerably for the three furtrethonium derivatives investigated. Neither the fraction of receptors in the high affinity agonist state, nor the ratio of dissociation constants for these states is affected by the alkylation of 85% of the functional muscarinic receptors. The inactivation of components of the effector system by PrBCM seems unlikely. 22 references, 3 figures, 3 tables.

  10. Signaling by Sensory Receptors

    PubMed Central

    Julius, David; Nathans, Jeremy

    2012-01-01

    Sensory systems detect small molecules, mechanical perturbations, or radiation via the activation of receptor proteins and downstream signaling cascades in specialized sensory cells. In vertebrates, the two principal categories of sensory receptors are ion channels, which mediate mechanosensation, thermosensation, and acid and salt taste; and G-protein-coupled receptors (GPCRs), which mediate vision, olfaction, and sweet, bitter, and umami tastes. GPCR-based signaling in rods and cones illustrates the fundamental principles of rapid activation and inactivation, signal amplification, and gain control. Channel-based sensory systems illustrate the integration of diverse modulatory signals at the receptor, as seen in the thermosensory/pain system, and the rapid response kinetics that are possible with direct mechanical gating of a channel. Comparisons of sensory receptor gene sequences reveal numerous examples in which gene duplication and sequence divergence have created novel sensory specificities. This is the evolutionary basis for the observed diversity in temperature- and ligand-dependent gating among thermosensory channels, spectral tuning among visual pigments, and odorant binding among olfactory receptors. The coding of complex external stimuli by a limited number of sensory receptor types has led to the evolution of modality-specific and species-specific patterns of retention or loss of sensory information, a filtering operation that selectively emphasizes features in the stimulus that enhance survival in a particular ecological niche. The many specialized anatomic structures, such as the eye and ear, that house primary sensory neurons further enhance the detection of relevant stimuli. PMID:22110046

  11. Changes in acetylcholine content, release and muscarinic receptors in rat hippocampus under cold stress

    SciTech Connect

    Fatranska, M.; Budai, D.; Gulya, K; Kvetnansky, R.

    1989-01-01

    The aim was to study the mechanism of the previously established decrease in acetylcholine (ACh) concentration in the rat hippocampus under cold stress. Male rats were exposed for 14 days to cold (5/degree/C) or kept (controls) at room temperature (24/degree/C). Acetylcholine content, release and muscarinic receptor binding were investigated in the hippocampus. Cold exposure resulted in a decrease of ACh concentration in the dorsal hippocampus. Moreover, the potassium-evoked release of ACh from hippocampal slices was increased and an increase of maximal binding capacity of (/sup 3/H)(-) quinuclidinyl benzilate in the dorsal hippocampus of cold exposed animals was also observed. Thus the decrease of hippocampal ACh concentration under cold exposure is probably due to its increased release. On balance then, our results demonstrate that cold stress in the rat induces significant activation of the hippocampal cholinergic system.

  12. The Role of Muscarinic Receptors in the Pathophysiology of Mood Disorders: A Potential Novel Treatment?

    PubMed Central

    Jeon, Won Je; Dean, Brian; Scarr, Elizabeth; Gibbons, Andrew

    2015-01-01

    The central cholinergic system has been implicated in the pathophysiology of mood disorders. An imbalance in central cholinergic neurotransmitter activity has been proposed to contribute to the manic and depressive episodes typical of these disorders. Neuropharmacological studies into the effects of cholinergic agonists and antagonists on mood state have provided considerable support for this hypothesis. Furthermore, recent clinical studies have shown that the pan-CHRM antagonist, scopolamine, produces rapid-acting antidepressant effects in individuals with either major depressive disorder (MDD) or bipolar disorder (BPD), such as bipolar depression, contrasting the delayed therapeutic response of conventional mood stabilisers and antidepressants. This review presents recent data from neuroimaging, post-mortem and genetic studies supporting the involvement of muscarinic cholinergic receptors (CHRMs), particularly CHRM2, in the pathophysiology of MDD and BPD. Thus, novel drugs that selectively target CHRMs with negligible effects in the peripheral nervous system might produce more rapid and robust clinical improvement in patients with BPD and MDD. PMID:26630954

  13. Fesoterodine: a novel muscarinic receptor antagonist for the treatment of overactive bladder syndrome.

    PubMed

    Michel, Martin C

    2008-07-01

    Fesoterodine is a newly approved drug for the treatment of overactive bladder syndrome. The aim of this study was to review the preclinical and clinical data on fesoterodine. The study involved a search of the Medline database and the proceedings volumes of urological congresses. Fesoterodine functions as an orally active prodrug that is converted to the active metabolite 5-hydroxymethyltolterodine by non-specific esterases. 5-Hydroxymethyltolterodine is a muscarinic receptor antagonist. Fesoterodine is primarily eliminated as inactive metabolites along with significant renal excretion as the unchanged active metabolite 5-hydroxymethyltolterodine. Fesoterodine is indicated for use at doses of 4 and 8 mg once daily. In clinical studies both doses of fesoterodine were consistently superior to placebo in improving the symptoms of overactive bladder syndrome, with 8 mg/day having significantly greater effects than 4 mg/day.

  14. Muscarinic M1 receptors modulate endotoxemia-induced loss of synaptic plasticity.

    PubMed

    Zivkovic, Aleksandar R; Sedlaczek, Oliver; von Haken, Rebecca; Schmidt, Karsten; Brenner, Thorsten; Weigand, Markus A; Bading, Hilmar; Bengtson, C Peter; Hofer, Stefan

    2015-11-04

    Septic encephalopathy is associated with rapid deterioration of cortical functions. Using magnetic resonance imaging (MRI) we detected functional abnormalities in the hippocampal formation of patients with septic delirium. Hippocampal dysfunction was further investigated in an animal model for sepsis using lipopolysaccharide (LPS) injections to induce endotoxemia in rats, followed by electrophysiological recordings in brain slices. Endotoxemia induced a deficit in long term potentiation which was completely reversed by apamin, a blocker of small conductance calcium-activated potassium (SK) channels, and partly restored by treatment with physostigmine (eserine), an acetylcholinesterase inhibitor, or TBPB, a selective M1 muscarinic acetylcholine receptor agonist. These results suggest a novel role for SK channels in the etiology of endotoxemia and explain why boosting cholinergic function restores deficits in synaptic plasticity. Drugs which enhance cholinergic or M1 activity in the brain may prove beneficial in treatment of septic delirium in the intensive care unit.

  15. Differentiation-associated decrease in muscarinic receptor sensitivity in human neuroblastoma cells

    SciTech Connect

    Heikkilae, J.E.; Scott, J.G.; Suominen, L.A.; Akerman, K.E.O.

    1987-01-01

    Muscarinic receptor-linked increases in intracellular free Ca/sup 2 +/ as measured with quin-2 and Ca/sup 2 +/ release from monolayers of cells have been measured in the human neuroblastoma cell line SH-SY5Y. Induction of differentiation with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) leads to a decrease in the sensitivity of the cells to low concentrations of agonists with respect to the induced increase in cytosolic free Ca/sup 2 +/ and stimulation of Ca/sup 2 +/ efflux. No decrease in agonist binding affinity was observed when the displacement of a labelled antagonist, /sup 3/H-NMS, by a non-labelled agonist was studied.

  16. [Propofol-induced myocardial depression: possible role of atrial muscarinic cholinergic receptors].

    PubMed

    Aguero Peña, R E; Pascuzzo-Lima, C; Granado Duque, A E; Bonfante-Cabarcas, R A

    2008-02-01

    To investigate the possible role of muscarinic cholinergic receptors (MCRs) in the depression of myocardial function induced by propofol, an intravenous anesthetic chemically unrelated to other drugs. Although adverse effects are rare, bradycardia has been reported and this can lead to cardiac arrest in some patients. The mechanism behind this effect is still unknown but a possible role for MCRs has been suggested. The interaction of propofol with human atrial MCRs was determined by means of inhibition tests using [3H] quinuclidinyl benzilate ([3H] QNB). The displacement of [3H] QNB binding to human atrial MCRs by propofol was concentration dependent but the observed effect was not consistent with a model of simple competition between propofol and [3H] QNB. Propofol appears to have the ability to modify the