Sample records for muscarinic receptor signal

  1. Molecular properties of muscarinic acetylcholine receptors

    PubMed Central

    HAGA, Tatsuya

    2013-01-01

    Muscarinic acetylcholine receptors, which comprise five subtypes (M1-M5 receptors), are expressed in both the CNS and PNS (particularly the target organs of parasympathetic neurons). M1-M5 receptors are integral membrane proteins with seven transmembrane segments, bind with acetylcholine (ACh) in the extracellular phase, and thereafter interact with and activate GTP-binding regulatory proteins (G proteins) in the intracellular phase: M1, M3, and M5 receptors interact with Gq-type G proteins, and M2 and M4 receptors with Gi/Go-type G proteins. Activated G proteins initiate a number of intracellular signal transduction systems. Agonist-bound muscarinic receptors are phosphorylated by G protein-coupled receptor kinases, which initiate their desensitization through uncoupling from G proteins, receptor internalization, and receptor breakdown (down regulation). Recently the crystal structures of M2 and M3 receptors were determined and are expected to contribute to the development of drugs targeted to muscarinic receptors. This paper summarizes the molecular properties of muscarinic receptors with reference to the historical background and bias to studies performed in our laboratories. PMID:23759942

  2. Muscarinic receptors acting at pre- and post-synaptic sites differentially regulate dopamine/DARPP-32 signaling in striatonigral and striatopallidal neurons.

    PubMed

    Kuroiwa, Mahomi; Hamada, Miho; Hieda, Eriko; Shuto, Takahide; Sotogaku, Naoki; Flajolet, Marc; Snyder, Gretchen L; Hendrick, Joseph P; Fienberg, Allen; Nishi, Akinori

    2012-12-01

    Muscarinic receptors, activated by acetylcholine, play critical roles in the functional regulation of medium spiny neurons in the striatum. However, the muscarinic receptor signaling pathways are not fully elucidated due to their complexity. In this study, we investigated the function of muscarinic receptors in the striatum by monitoring DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of M(r) 32 kDa) phosphorylation at Thr34 (the PKA-site) using mouse striatal slices. Treatment of slices with a non-selective muscarinic receptor agonist, oxotremorine (10 μM), rapidly and transiently increased DARPP-32 phosphorylation. The increase in DARPP-32 phosphorylation was completely abolished either by a dopamine D(1) receptor antagonist (SCH23390), tetrodotoxin, genetic deletion of M5 receptors, muscarinic toxins for M1 and M4 receptors, or 6-hydroxydopamine lesioning of dopaminergic neurons, whereas it was enhanced by nicotine. Analysis in D(1)-DARPP-32-Flag/D(2)-DARPP-32-Myc transgenic mice revealed that oxotremorine increases DARPP-32 phosphorylation selectively in D(1)-type/striatonigral, but not in D(2)-type/striatopallidal, neurons. When D(1) and D(2) receptors were blocked by selective antagonists to exclude the effects of released dopamine, oxotremorine increased DARPP-32 Thr34 phosphorylation only in D(2)-type/striatopallidal neurons. This increase required activation of M1 receptors and was dependent upon adenosine A(2A) receptor activity. The results demonstrate that muscarinic receptors, especially M5 receptors, act at presynaptic dopaminergic terminals, regulate the release of dopamine in cooperation with nicotinic receptors, and activate D(1) receptor/DARPP-32 signaling in the striatonigral neurons. Muscarinic M1 receptors expressed in striatopallidal neurons interact with adenosine A(2A) receptors and activate DARPP-32 signaling. Copyright © 2012 Elsevier Ltd. All rights reserved.

  3. Inhibition of m3 muscarinic acetylcholine receptors by local anaesthetics

    PubMed Central

    Hollmann, Markus W; Ritter, Carsten H; Henle, Philipp; de Klaver, Manuela; Kamatchi, Ganesan L; Durieux, Marcel E

    2001-01-01

    Muscarinic m1 receptors are inhibited by local anaesthetics (LA) at nM concentrations. To elucidate in more detail the site(s) of LA interaction, we compared these findings with LA effects on m3 muscarinic receptors. We expressed receptors in Xenopus oocytes. Using two-electrode voltage clamp, we measured the effects of lidocaine, QX314 (permanently charged) and benzocaine (permanently uncharged) on Ca2+-activated Cl−-currents (ICl(Ca)), elicited by acetyl-β-methylcholine bromide (MCh). We also characterized the interaction of lidocaine with [3H]-quinuclydinyl benzylate ([3H]-QNB) binding to m3 receptors. Antisense-injection was used to determine the role of specific G-protein α subunits in mediating the inhibitory effects of LA. Using chimeric receptor constructs we investigated which domains of the muscarinic receptors contribute to the binding site for LA. Lidocaine inhibited m3-signalling in a concentration-dependent, reversible, non-competitive manner with an IC50 of 370 nM, approximately 21 fold higher than the IC50 (18 nM) reported for m1 receptors. Intracellular inhibition of both signalling pathways by LA was similar, and dependent on the Gq- protein α subunit. In contrast to results reported for the m1 receptor, the m3 receptor lacks the major extracellular binding site for charged LA. The N-terminus and third extracellular loop of the m1 muscarinic receptor molecule were identified as requirements to obtain extracellular inhibition by charged LA. PMID:11325812

  4. Pharmacological approaches to targeting muscarinic acetylcholine receptors.

    PubMed

    Matera, Carlo; Tata, Ada M

    2014-01-01

    The presence of cholinergic system markers and muscarinic receptor subtypes in several tissues also of nonneuronal type has been largely demonstrated. Acetylcholine, synthesized in the nervous system, can locally contribute to modulate cell proliferation, survival and apoptosis. Considering that the cholinergic system functions are impaired in a number of disorders, the identification of new drugs regulating these functions appears of great clinical relevance. The possible involvement of muscarinic acetylcholine receptors in different pathologies has been proposed in recent years and is becoming an important area of study. However, the lack of selective muscarinic receptor ligands has for long time limited the therapeutic treatment based on muscarinic receptors as targets. To date, some muscarinic ligands such as xanomeline (patent, US5980933) or cevimeline (patents US4855290, US5571918) have been developed for the treatment of several pathologies (Alzheimer's and Sjogren's diseases). The present review will be focused on the potential effects produced by muscarinic receptor activation in different pathologies, including tumors. In fact, the potential use of muscarinic ligands in therapeutic protocols in cancer therapy will be discussed, considering that several muscarinic antagonists, already used in the treatment of genitourinary diseases (e.g. darifenacin, patent, US5096890, US6106864), have also been demonstrated to arrest the tumor growth in vivo. Moreover, the contribution of muscarinic receptors to analgesia is also reviewed. Finally, some of the most significant achievements in the field of bitopic/dualsteric ligands will be discussed and the molecules patented so far will be presented.

  5. Oxotremorine-M potentiates NMDA receptors by muscarinic receptor dependent and independent mechanisms.

    PubMed

    Zwart, Ruud; Reed, Hannah; Sher, Emanuele

    2018-01-01

    Muscarinic acetylcholine M1 receptors play an important role in synaptic plasticity in the hippocampus and cortex. Potentiation of NMDA receptors as a consequence of muscarinic acetylcholine M1 receptor activation is a crucial event mediating the cholinergic modulation of synaptic plasticity, which is a cellular mechanism for learning and memory. In Alzheimer's disease, the cholinergic input to the hippocampus and cortex is severely degenerated, and agonists or positive allosteric modulators of M1 receptors are therefore thought to be of potential use to treat the deficits in cognitive functions in Alzheimer's disease. In this study we developed a simple system in which muscarinic modulation of NMDA receptors can be studied in vitro. Human M1 receptors and NR1/2B NMDA receptors were co-expressed in Xenopus oocytes and various muscarinic agonists were assessed for their modulatory effects on NMDA receptor-mediated responses. As expected, NMDA receptor-mediated responses were potentiated by oxotremorine-M, oxotremorine or xanomeline when the drugs were applied between subsequent NMDA responses, an effect which was fully blocked by the muscarinic receptor antagonist atropine. However, in oocytes expressing NR1/2B NMDA receptors but not muscarinic M1 receptors, oxotremorine-M co-applied with NMDA also resulted in a potentiation of NMDA currents and this effect was not blocked by atropine, demonstrating that oxotremorine-M is able to directly potentiate NMDA receptors. Oxotremorine, which is a close analogue of oxotremorine-M, and xanomeline, a chemically distinct muscarinic agonist, did not potentiate NMDA receptors by this direct mechanism. Comparing the chemical structures of the three different muscarinic agonists used in this study suggests that the tri-methyl ammonium moiety present in oxotremorine-M is important for the compound's interaction with NMDA receptors. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Regulation of Neuronal Muscarinic Acetylcholine Receptors

    DTIC Science & Technology

    1989-01-01

    N1E - 115 cells with pertussis toxin blocks mAChR-mediated inhibition of adenylate cyclase but not mAChR-mediated stimulation of PI turnover...determine the effects of electrical depolarization on muscarinic acetylcholine receptors (mAChR) in the cultured neuroblastoma cell line, N E- 115 ...evidence that Gi and Go may differentially regulate cellular signaling mechanisms, these results suggest that depolarization may regulate specific

  7. Adhesion-Dependent Redistribution of MAP Kinase and MEK Promotes Muscarinic Receptor-Mediated Signaling to the Nucleus

    PubMed Central

    Slack, Barbara E.; Siniaia, Marina S.

    2008-01-01

    The mitogen-activated protein kinases (MAPKs) are activated by extracellular signals, and translocate to the nucleus where they modulate transcription. Integrin-mediated cell adhesion to extracellular matrix (ECM) proteins is required for efficient transmission of MAPK-based signals initiated by growth factors. However, the modulation of G protein-coupled receptor (GPCR) signaling by adhesion is less well understood. In the present study we assessed the impact of cell adhesion on MAPK activation by muscarinic M3 receptors. The muscarinic agonist carbachol more efficiently promoted stress fiber formation and tyrosine phosphorylation of focal adhesion-associated proteins in M3 receptor-expressing cells adherent to fibronectin or collagen type I, as compared to polylysine. Overall MAPK activation was robust in cells adherent to all three substrata. However, total levels of MAPK and mitogen-activated protein kinase kinase (MEK) in the nucleus were significantly greater in cells adherent to ECM proteins for 2.5 hours, and levels of activated MAPK and MEK in the nuclei of these cells were higher following carbachol stimulation, relative to levels in cells adherent to polylysine. MEK inhibitors did not prevent adhesion-dependent translocation of MAPK and MEK to the nucleus, and increased nuclear phospho-MEK levels in carbachol-stimulated cells. The results suggest that adhesion of cells to ECM triggers the redistribution of MAPK and MEK to the nucleus, possibly as a result of the cytoskeletal rearrangements that accompany cell spreading. This may represent a mechanism for priming the nucleus with MEK and MAPK, leading to more rapid and pronounced increases in intranuclear phospho-MAPK upon GPCR stimulation. PMID:15779001

  8. Muscarinic receptor regulates extracellular signal regulated kinase by two modes of arrestin binding.

    PubMed

    Jung, Seung-Ryoung; Kushmerick, Christopher; Seo, Jong Bae; Koh, Duk-Su; Hille, Bertil

    2017-07-11

    Binding of agonists to G-protein-coupled receptors (GPCRs) activates heterotrimeric G proteins and downstream signaling. Agonist-bound GPCRs are then phosphorylated by protein kinases and bound by arrestin to trigger desensitization and endocytosis. Arrestin plays another important signaling function. It recruits and regulates activity of an extracellular signal-regulated kinase (ERK) cascade. However, molecular details and timing of ERK activation remain fundamental unanswered questions that limit understanding of how arrestin-dependent GPCR signaling controls cell functions. Here we validate and model a system that tracks the dynamics of interactions of arrestin with receptors and of ERK activation using optical reporters. Our intermolecular FRET measurements in living cells are consistent with β-arrestin binding to M 1 muscarinic acetylcholine receptors (M 1 Rs) in two different binding modes, transient and stable. The stable mode persists for minutes after agonist removal. The choice of mode is governed by phosphorylation on key residues in the third intracellular loop of the receptor. We detect a similar intramolecular conformational change in arrestin in either binding mode. It develops within seconds of arrestin binding to the M 1 receptor, and it reverses within seconds of arrestin unbinding from the transient binding mode. Furthermore, we observed that, when stably bound to phosphorylated M 1 R, β-arrestin scaffolds and activates MEK-dependent ERK. In contrast, when transiently bound, β-arrestin reduces ERK activity via recruitment of a protein phosphatase. All this ERK signaling develops at the plasma membrane. In this scaffolding hypothesis, a shifting balance between the two arrestin binding modes determines the degree of ERK activation at the membrane.

  9. Muscarinic receptor regulates extracellular signal regulated kinase by two modes of arrestin binding

    PubMed Central

    Jung, Seung-Ryoung; Kushmerick, Christopher; Seo, Jong Bae; Koh, Duk-Su

    2017-01-01

    Binding of agonists to G-protein–coupled receptors (GPCRs) activates heterotrimeric G proteins and downstream signaling. Agonist-bound GPCRs are then phosphorylated by protein kinases and bound by arrestin to trigger desensitization and endocytosis. Arrestin plays another important signaling function. It recruits and regulates activity of an extracellular signal-regulated kinase (ERK) cascade. However, molecular details and timing of ERK activation remain fundamental unanswered questions that limit understanding of how arrestin-dependent GPCR signaling controls cell functions. Here we validate and model a system that tracks the dynamics of interactions of arrestin with receptors and of ERK activation using optical reporters. Our intermolecular FRET measurements in living cells are consistent with β-arrestin binding to M1 muscarinic acetylcholine receptors (M1Rs) in two different binding modes, transient and stable. The stable mode persists for minutes after agonist removal. The choice of mode is governed by phosphorylation on key residues in the third intracellular loop of the receptor. We detect a similar intramolecular conformational change in arrestin in either binding mode. It develops within seconds of arrestin binding to the M1 receptor, and it reverses within seconds of arrestin unbinding from the transient binding mode. Furthermore, we observed that, when stably bound to phosphorylated M1R, β-arrestin scaffolds and activates MEK-dependent ERK. In contrast, when transiently bound, β-arrestin reduces ERK activity via recruitment of a protein phosphatase. All this ERK signaling develops at the plasma membrane. In this scaffolding hypothesis, a shifting balance between the two arrestin binding modes determines the degree of ERK activation at the membrane. PMID:28652372

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Raufman, Jean-Pierre, E-mail: jraufman@medicine.umaryland.edu; Cheng, Kunrong; Saxena, Neeraj

    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 invasionmore » 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

  11. Muscarinic Receptor Activation Protects Cells from Apoptotic Effects of DNA Damage, Oxidative Stress, and Mitochondrial Inhibition*

    PubMed Central

    De Sarno, Patrizia; Shestopal, Svetlana A.; King, Taj D.; Zmijewska, Anna; Song, Ling; Jope, Richard S.

    2006-01-01

    The impact of muscarinic receptor stimulation was examined on apoptotic signaling induced by DNA damage, oxidative stress, and mitochondrial impairment. Exposure of human neuroblastoma SH-SY5Y cells to the DNA-damaging agent camptothecin increased p53 levels, activated caspase-3, and caused cell death. Pretreatment with oxotremorine-M, a selective agonist of muscarinic receptors that are expressed endogenously in these cells, did not affect the accumulation of p53 but greatly attenuated caspase-3 activation and protected from cell death to nearly the same extent as treatment with a general caspase inhibitor. Treatment with 50–200 μm H2O2 caused the activation of caspase-3 beginning after 2–3 h, followed by eventual cell death. Oxotremorine-M pretreatment protected cells from H2O2-inducedcaspase-3 activation and death, and this was equivalent to protection afforded by a caspase inhibitor. Muscarinic receptor stimulation also protected cells from caspase-3 activation induced by exposure to rotenone, a mitochondrial complex 1 inhibitor, but no protection was evident from staurosporine-induced caspase-3 activation. The mechanism of protection afforded by muscarinic receptor activation from camptothecin-induced apoptotic signaling involved blockade of mitochondrial cytochrome c release associated with a bolstering of mitochondrial bcl-2 levels and blockade of the translocation of Bax to mitochondria. Likely the most proximal of these events to muscarinic receptor activation, mitochondrial Bax accumulation, also was attenuated by oxotremorine-M treatment after treatment with H2O2 or rotenone. These results demonstrate that stimulation of muscarinic receptors provides substantial protection from DNA damage, oxidative stress, and mitochondrial impairment, insults that may be encountered by neurons in development, aging, or neurodegenerative diseases. These findings suggest that neurotransmitter-induced signaling bolsters survival mechanisms, and inadequate

  12. Muscarinic Control of MIN6 Pancreatic β Cells Is Enhanced by Impaired Amino Acid Signaling*

    PubMed Central

    Guerra, Marcy L.; Wauson, Eric M.; McGlynn, Kathleen; Cobb, Melanie H.

    2014-01-01

    We have shown recently that the class C G protein-coupled receptor T1R1/T1R3 taste receptor complex is an early amino acid sensor in MIN6 pancreatic β cells. Amino acids are unable to activate ERK1/2 in β cells in which T1R3 has been depleted. The muscarinic receptor agonist carbachol activated ERK1/2 better in T1R3-depleted cells than in control cells. Ligands that activate certain G protein-coupled receptors in pancreatic β cells potentiate glucose-stimulated insulin secretion. Among these is the M3 muscarinic acetylcholine receptor, the major muscarinic receptor in β cells. We found that expression of M3 receptors increased in T1R3-depleted MIN6 cells and that calcium responses were altered. To determine whether these changes were related to impaired amino acid signaling, we compared responses in cells exposed to reduced amino acid concentrations. M3 receptor expression was increased, and some, but not all, changes in calcium signaling were mimicked. These findings suggest that M3 acetylcholine receptors are increased in β cells as a mechanism to compensate for amino acid deficiency. PMID:24695728

  13. A specific multi-nutrient formulation enhances M1 muscarinic acetylcholine receptor responses in vitro.

    PubMed

    Savelkoul, Paul J M; Janickova, Helena; Kuipers, Almar A M; Hageman, Robert J J; Kamphuis, Patrick J; Dolezal, Vladimir; Broersen, Laus M

    2012-02-01

    Recent evidence indicates that supplementation with a specific combination of nutrients may affect cell membrane synthesis and composition. To investigate whether such nutrients may also modify the physical properties of membranes, and affect membrane-bound processes involved in signal transduction pathways, we studied the effects of nutrient supplementation on G protein-coupled receptor activation in vitro. In particular, we investigated muscarinic receptors, which are important for the progression of memory deterioration and pathology of Alzheimer's disease. Nerve growth factor differentiated pheochromocytoma cells that were supplemented with specific combinations of nutrients showed enhanced responses to muscarinic receptor agonists in a membrane potential assay. The largest effects were obtained with a combination of nutrients known as Fortasyn™ Connect, comprising docosahexaenoic acid, eicosapentaenoic acid, uridine monophosphate as a uridine source, choline, vitamin B6, vitamin B12, folic acid, phospholipids, vitamin C, vitamin E, and selenium. In subsequent experiments, it was shown that the effects of supplementation could not be attributed to single nutrients. In addition, it was shown that the agonist-induced response and the supplement-induced enhancement of the response were blocked with the muscarinic receptor antagonists atropine, telenzepine, and AF-DX 384. In order to determine whether the effects of Fortasyn™ Connect supplementation were receptor subtype specific, we investigated binding properties and activation of human muscarinic M1, M2 and M4 receptors in stably transfected Chinese hamster ovary cells after supplementation. Multi-nutrient supplementation did not change M1 receptor density in plasma membranes. However, M1 receptor-mediated G protein activation was significantly enhanced. In contrast, supplementation of M2- or M4-expressing cells did not affect receptor signaling. Taken together, these results indicate that a specific

  14. Acetylcholinesterase Inhibitors and Drugs Acting on Muscarinic Receptors- Potential Crosstalk of Cholinergic Mechanisms During Pharmacological Treatment

    PubMed Central

    Soukup, Ondrej; Winder, Michael; Killi, Uday Kumar; Wsol, Vladimir; Jun, Daniel; Kuca, Kamil; Tobin, Gunnar

    2017-01-01

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

  15. Muscarinic receptors in gastric mucosa are increased in peptic ulcer disease.

    PubMed Central

    Pfeiffer, A; Krömer, W; Friemann, J; Ruge, M; Herawi, M; Schätzl, M; Schwegler, U; May, B; Schatz, H

    1995-01-01

    Muscarinic receptors stimulate the secretion of acid pepsinogen and mucous in gastric mucosa. Whether muscarinic receptors are involved in the pathogenesis of benign gastric disease is unknown. Receptor changes in these conditions were therefore sought. An autoradiographic technique was developed to determine quantitatively muscarinic receptors in microtome sections of biopsy specimens obtained during gastroscopy. Muscarinic receptor density was mean (SEM) 18.4 (1.2) fmol/mg protein in the corpus and 8.9 (0.7) fmol/mg protein in the antrum (n = 53). Neither chronic nor active gastritis was associated with receptor changes in the antrum but chronic gastritis was associated with a receptor loss in the corpus. Patients with acute or recent duodenal or antral ulcers (n = 23) had significantly higher levels of muscarinic receptors in the corpus than controls (n = 25) (22.2 (1.5) v 16.9 (1.7) fmol/mg protein respectively (p < 0.025). These results suggest that muscarinic M3 receptor is overexpressed in duodenal ulcer disease and may play a part in its pathogenesis. Images Figure 2 PMID:7615265

  16. Role of the M3 muscarinic acetylcholine receptor in beta-cell function and glucose homeostasis.

    PubMed

    Gautam, D; Han, S-J; Duttaroy, A; Mears, D; Hamdan, F F; Li, J H; Cui, Y; Jeon, J; Wess, J

    2007-11-01

    The release of insufficient amounts of insulin in the presence of elevated blood glucose levels is one of the key features of type 2 diabetes. Various lines of evidence indicate that acetylcholine (ACh), the major neurotransmitter of the parasympathetic nervous system, can enhance glucose-stimulated insulin secretion from pancreatic beta-cells. Studies with isolated islets prepared from whole body M(3) muscarinic ACh receptor knockout mice showed that cholinergic amplification of glucose-dependent insulin secretion is exclusively mediated by the M(3) muscarinic receptor subtype. To investigate the physiological relevance of this muscarinic pathway, we used Cre/loxP technology to generate mutant mice that lack M(3) receptors only in pancreatic beta-cells. These mutant mice displayed impaired glucose tolerance and significantly reduced insulin secretion. In contrast, transgenic mice overexpressing M(3) receptors in pancreatic beta-cells showed a pronounced increase in glucose tolerance and insulin secretion and were resistant to diet-induced glucose intolerance and hyperglycaemia. These findings indicate that beta-cell M(3) muscarinic receptors are essential for maintaining proper insulin secretion and glucose homeostasis. Moreover, our data suggest that enhancing signalling through beta-cell M(3) muscarinic receptors may represent a new avenue in the treatment of glucose intolerance and type 2 diabetes.

  17. MOLECULAR PROBES FOR MUSCARINIC RECEPTORS: FUNCTIONALIZED CONGENERS OF SELECTIVE MUSCARINIC ANTAGONISTS

    PubMed Central

    Jacobson, Kenneth A.; Fischer, Bilha; van Rhee, A. Michiel

    2012-01-01

    Summary The muscarinic agonist oxotremorine and the tricyclic muscarinic antagonists pirenzepine and telenzepine have been derivatized using a functionalized congener approach for the purpose of synthesizing high affinity ligand probes that are suitable for conjugation with prosthetic groups, for receptor cross-linking, fluorescent and radioactive detection, etc. A novel fluorescent conjugate of TAC (telenzepine amine congener), an n-decylamino derivative of the ml-selective antagonist, with the fluorescent trisulfonated pyrene dye Cascade Blue may be useful for assaying the receptor as an alternative to radiotracers. In a rat m3 receptor mutant containing a single amino acid substitution in the sixth transmembrane domain (Asn507 to Ala) the parent telenzepine lost 636-fold in affinity, while TAC lost only 27-fold. Thus, the decylamino group of TAC stabilizes the bound state and thus enhances potency by acting as a distal anchor in the receptor binding site. We have built a computer-assisted molecular model of the transmembrane regions of muscarinic receptors based on homology with the G-protein coupled receptor rhodopsin, for which a low resolution structure is known. We have coordinated the antagonist pharmacophore (tricyclic and piperazine moieties) with residues of the third and seventh helices of the rat m3 receptor. Although the decylamino chain of TAC is likely to be highly flexible and may adopt many conformations, we located one possible site for a salt bridge formation with the positively charged −NH3+ group, i.e. Asp113 in helix II. PMID:10188781

  18. Muscarinic receptor agonists stimulate human colon cancer cell migration and invasion.

    PubMed

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

    2011-05-01

    Muscarinic receptors (CHRM) are overexpressed in colon cancer. To explore a role for muscarinic receptor signaling in colon cancer metastasis, we used human H508 and HT29 colon cancer cells that coexpress epidermal growth factor (ERBB) and CHRM3 receptors. In a wound closure model, following 8-h incubation of H508 cells with 100 μM ACh we observed a threefold increase in cell migration indistinguishable from the actions of epidermal growth factor (EGF). Atropine blocked the actions of ACh but not of EGF. In SNU-C4 colon cancer cells that express ERBB but not CHRM, EGF caused a threefold increase in migration; ACh had no effect. ACh-induced cell migration was attenuated by chemical inhibitors of ERBB1 activation, by anti-ERBB1 antibody, and by inhibitors of ERK and phosphatidylinositol 3-kinase (PI3K) signaling. Consistent with matrix metalloproteinase-7 (MMP7)-mediated release of an ERBB1 ligand, heparin binding epidermal growth factor-like growth factor (HBEGF), ACh-induced migration was inhibited by an MMP inhibitor and by anti-MMP7 and -HBEGF antibodies. ACh-induced cell migration was blocked by inhibiting RhoA and ROCK, key proteins that interact with the actin cytoskeleton. ACh-induced RhoA activation was attenuated by agents that inhibit ERBB1, ERK, and PI3K activation. Collectively, these findings indicate that ACh-induced cell migration is mediated by MMP7-mediated release of HBEGF, an ERBB ligand that activates ERBB1 and downstream ERK and PI3K signaling. In a cell invasion model, ACh-induced HT29 cell invasion was blocked by atropine. In concert with previous observations, these findings indicate that muscarinic receptor signaling plays a key role in colon cancer cell proliferation, survival, migration, and invasion.

  19. Muscarinic receptor agonists stimulate human colon cancer cell migration and invasion

    PubMed Central

    Belo, Angelica; Cheng, Kunrong; Chahdi, Ahmed; Shant, Jasleen; Xie, Guofeng; Khurana, Sandeep

    2011-01-01

    Muscarinic receptors (CHRM) are overexpressed in colon cancer. To explore a role for muscarinic receptor signaling in colon cancer metastasis, we used human H508 and HT29 colon cancer cells that coexpress epidermal growth factor (ERBB) and CHRM3 receptors. In a wound closure model, following 8-h incubation of H508 cells with 100 μM ACh we observed a threefold increase in cell migration indistinguishable from the actions of epidermal growth factor (EGF). Atropine blocked the actions of ACh but not of EGF. In SNU-C4 colon cancer cells that express ERBB but not CHRM, EGF caused a threefold increase in migration; ACh had no effect. ACh-induced cell migration was attenuated by chemical inhibitors of ERBB1 activation, by anti-ERBB1 antibody, and by inhibitors of ERK and phosphatidylinositol 3-kinase (PI3K) signaling. Consistent with matrix metalloproteinase-7 (MMP7)-mediated release of an ERBB1 ligand, heparin binding epidermal growth factor-like growth factor (HBEGF), ACh-induced migration was inhibited by an MMP inhibitor and by anti-MMP7 and -HBEGF antibodies. ACh-induced cell migration was blocked by inhibiting RhoA and ROCK, key proteins that interact with the actin cytoskeleton. ACh-induced RhoA activation was attenuated by agents that inhibit ERBB1, ERK, and PI3K activation. Collectively, these findings indicate that ACh-induced cell migration is mediated by MMP7-mediated release of HBEGF, an ERBB ligand that activates ERBB1 and downstream ERK and PI3K signaling. In a cell invasion model, ACh-induced HT29 cell invasion was blocked by atropine. In concert with previous observations, these findings indicate that muscarinic receptor signaling plays a key role in colon cancer cell proliferation, survival, migration, and invasion. PMID:21273532

  20. Effects of muscarinic receptor antagonists on cocaine discrimination in wild-type mice and in muscarinic receptor M1, M2, and M4 receptor knockout mice.

    PubMed

    Joseph, Lauren; Thomsen, Morgane

    2017-06-30

    Muscarinic M 1 /M 4 receptor stimulation can reduce abuse-related effects of cocaine and may represent avenues for treating cocaine addiction. Muscarinic antagonists can mimic and enhance effects of cocaine, including discriminative stimulus (S D ) effects, but the receptor subtypes mediating those effects are not known. A better understanding of the complex cocaine/muscarinic interactions is needed to evaluate and develop potential muscarinic-based medications. Here, knockout mice lacking M 1 , M 2 , or M 4 receptors (M 1 -/- , M 2 -/- , M 4 -/- ), as well as control wild-type mice and outbred Swiss-Webster mice, were trained to discriminate 10mg/kg cocaine from saline. Muscarinic receptor antagonists with no subtype selectivity (scopolamine), or preferential affinity at the M 1 , M 2 , or M 4 subtype (telenzepine, trihexyphenidyl; methoctramine, AQ-RA 741; tropicamide) were tested alone and in combination with cocaine. In intact animals, antagonists with high affinity at M 1 /M 4 receptors partially substituted for cocaine and increased the S D effect of cocaine, while M 2 -preferring antagonists did not substitute, and reduced the S D effect of cocaine. The cocaine-like effects of scopolamine were absent in M 1 -/- mice. The cocaine S D attenuating effects of methoctramine were absent in M 2 -/- mice and almost absent in M 1 -/- mice. The findings indicate that the cocaine-like S D effects of muscarinic antagonists are primarily mediated through M 1 receptors, with a minor contribution of M 4 receptors. The data also support our previous findings that stimulation of M 1 receptors and M 4 receptors can each attenuate the S D effect of cocaine, and show that this can also be achieved by blocking M 2 autoreceptors, likely via increased acetylcholine release. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. 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.

  2. Generalised smooth-muscle disease with defective muscarinic-receptor function.

    PubMed

    Bannister, R; Hoyes, A D

    1981-03-28

    A patient with widespread smooth-muscle disease presented with chronic intestinal pseudo-obstruction but had in addition defects of the bladder, pupils, sweating, and cardiovascular function. There was no evidence of a primary neural lesion, and minor changes in the muscle did not resemble those of a myopathy. In each organ affected muscarinic cholinergic function was at fault, but instead of supersensitivity to cholinergic drugs, which occurs in postganglionic autonomic neuropathies, there was a lack of response to cholinergic drugs and anticholinesterases. It was therefore concluded that the patient had a new type of defect of muscarinic-receptor function. The cause was unknown, but it may have been an autoimmune disease resembling myasthenia, in which there is a postjunctional defect of muscarinic receptors. In similar cases binding of muscarinic agonists and antagonists should be tested. When antibodies to purified human muscarinic receptors become available different patterns of smooth-muscle defect may be identifiable, enabling the lesion to be defined more precisely.

  3. Muscarinic and nicotinic receptors synergistically modulate working memory and attention in humans.

    PubMed

    Ellis, Julia R; Ellis, Kathryn A; Bartholomeusz, Cali F; Harrison, Ben J; Wesnes, Keith A; Erskine, Fiona F; Vitetta, Luis; Nathan, Pradeep J

    2006-04-01

    Functional abnormalities in muscarinic and nicotinic receptors are associated with a number of disorders including Alzheimer's disease and schizophrenia. While the contribution of muscarinic receptors in modulating cognition is well established in humans, the effects of nicotinic receptors and the interactions and possible synergistic effects between muscarinic and nicotinic receptors have not been well characterized in humans. The current study examined the effects of selective and simultaneous muscarinic and nicotinic receptor antagonism on a range of cognitive processes. The study was a double-blind, placebo-controlled, repeated measures design in which 12 healthy, young volunteers completed cognitive testing under four acute treatment conditions: placebo (P); mecamylamine (15 mg) (M); scopolamine (0.4 mg i.m.) (S); mecamylamine (15 mg)/scopolamine (0.4 mg i.m.) (MS). Muscarinic receptor antagonism with scopolamine resulted in deficits in working memory, declarative memory, sustained visual attention and psychomotor speed. Nicotinic antagonism with mecamylamine had no effect on any of the cognitive processes examined. Simultaneous antagonism of both muscarinic and nicotinic receptors with mecamylamine and scopolamine impaired all cognitive processes impaired by scopolamine and produced greater deficits than either muscarinic or nicotinic blockade alone, particularly on working memory, visual attention and psychomotor speed. These findings suggest that muscarinic and nicotinic receptors may interact functionally to have synergistic effects particularly on working memory and attention and suggests that therapeutic strategies targeting both receptor systems may be useful in improving selective cognitive processes in a number of disorders.

  4. Differential expression of muscarinic acetylcholine receptor subtypes in Jurkat cells and their signaling.

    PubMed

    Alea, Mileidys Perez; Borroto-Escuela, Dasiel O; Romero-Fernandez, Wilber; Fuxe, Kjell; Garriga, Pere

    2011-08-15

    Muscarinic acetylcholine receptors expression and signaling in the human Jurkat T cell line were investigated. Semiquantitative real-time PCR and radioligand binding studies, using a wide set of antagonist compounds, showed the co-existence of M(3), M(4), and M(5) subtypes. Stimulation of these subpopulations caused a concentration and time- dependent activation of second messengers and ERK signaling pathways, with a major contribution of the M(3) subtype in a G(q/11)-mediated response. In addition, we found that T-cell stimulation leads to increased expression of M(3) and M(5) both at transcriptional and protein levels in a PLC/PKCθ dependent manner. Our data clarifies the functional role of AChR subtypes in Jurkat cells and pave the way to future studies on the potential cross-talk among these subpopulations and their regulation of T lymphocytes immune function. Copyright © 2011 Elsevier B.V. All rights reserved.

  5. Role of muscarinic receptors in the regulation of immune and inflammatory responses

    PubMed Central

    Razani-Boroujerdi, Seddigheh; Behl, Muskaan; Hahn, Fletcher F.; Pena-Philippides, Juan Carlos; Hutt, Julie; Sopori, Mohan L.

    2008-01-01

    Leukocytes contain both nicotinic and muscarinic receptors, and while activation of nicotinic receptors suppresses immune/inflammatory responses, the role of muscarinic receptors in immunity is unclear. We examined the effects of a muscarinic receptor antagonist (atropine) and agonist (oxotremorine), administered chronically through miniosmotic pumps, on immune/inflammatory responses in the rat. Results show that while oxotremorine stimulated, atropine inhibited the antibody and T-cell proliferative responses. Moreover, atropine also suppressed the turpentine-induced leukocytic infiltration and tissue injury, and inhibited chemotaxis of leukocytes toward neutrophil and monocyte/lymphocyte chemoattractants. Thus, activation of nicotinic and muscarinic receptors has opposite effects on the immune/inflammatory responses. PMID:18190972

  6. 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.

  7. Central muscarinic cholinergic regulation of the systemic inflammatory response during endotoxemia

    PubMed Central

    Pavlov, Valentin A.; Ochani, Mahendar; Gallowitsch-Puerta, Margot; Ochani, Kanta; Huston, Jared M.; Czura, Christopher J.; Al-Abed, Yousef; Tracey, Kevin J.

    2006-01-01

    TNF has a critical mediator role in inflammation and is an important therapeutic target. We recently discovered that TNF production is regulated by neural signals through the vagus nerve. Activation of this “cholinergic antiinflammatory pathway” inhibits the production of TNF and other cytokines and protects animals from the inflammatory damage caused by endotoxemia and severe sepsis. Here, we describe a role for central muscarinic acetylcholine receptors in the activation of the cholinergic antiinflammatory pathway. Central muscarinic cholinergic activation by muscarine, the M1 receptor agonist McN-A-343, and the M2 receptor antagonist methoctramine inhibited serum TNF levels significantly during endotoxemia. Centrally administered methoctramine stimulated vagus-nerve activity measured by changes in instantaneous heart-rate variability. Blockade of peripheral muscarinic receptors did not abolish antiinflammatory signaling through the vagus nerve, indicating that peripheral muscarinic receptors on immune cells are not required for the cytokine-regulating activities of the cholinergic antiinflammatory pathway. The role of central muscarinic receptors in activating the cholinergic antiinflammatory pathway is of interest for the use of centrally acting muscarinic cholinergic enhancers as antiinflammatory agents. PMID:16549778

  8. Detection of muscarinic receptors in the human lung using PET.

    PubMed

    Visser, T J; van Waarde, A; van der Mark, T W; Kraan, J; Ensing, K; Willemsen, A T; Elsinga, P H; Vaalburg, W

    1999-08-01

    The characterization of pulmonary muscarinic receptors with PET is still in its infancy. Because approximately 70% of the lungs consists of air and pulmonary muscarinic receptor densities are low, ligands with high receptor affinity are required to obtain reasonable signal-to-noise ratios on PET images. Therefore, the potent 11C-labeled muscarinic antagonist N-methyl-piperidin-4-yl 2-cyclohexyl-2-hydroxy-2-phenylacetate methiodide ([R]-VC-002) was developed. We administered this radioligand to four healthy human volunteers to examine its suitability for studying pulmonary muscarinic receptors in vivo. [11C]VC-002 (185 MBq, specific activity > 7.4 TBq/mmol) was intravenously injected on 2 separate days, with an interval of at least 1 wk. On the first day the volunteers were not pretreated, but on the second day they received the anticholinergic glycopyrronium bromide (Robinul; 2 x 0.1 mg intravenous) 25 and 30 min before the injection of the radiopharmaceutical. C[15O]O scans (approximately 740 MBq [20 mCi] by inhalation) were acquired before the receptor scan to calculate pulmonary blood volume. On PET images of the thorax, the lungs were clearly visible. After the volunteer was pretreated with glycopyrronium bromide, pulmonary uptake of the radioligand was reduced to 32%+/-12% of the control value at 60 min postinjection and the lungs could no longer be seen. (R)-[11C]-VC-002 was rapidly cleared from plasma and was slowly metabolized during the time course (60 min) of the PET scan. The fraction of radioligand representing parent compound decreased from 99.9% at the time of injection to 82% at 40-60 min postinjection, both in the presence and absence of Robinul. Pulmonary tissue-to-plasma ratios, calculated on a count-per-minute-per-gram basis, reached a plateau value of 17.8+/-1.2 at 40-50 min postinjection. [11C]VC-002 appears to be suitable for in vivo studies of pulmonary cholinoceptors.

  9. Muscarinic Receptor Binding in Rat Bladder Urothelium and Detrusor Muscle by Intravesical Solifenacin.

    PubMed

    Ito, Yoshihiko; Kashiwabara, Michishi; Yoshida, Akira; Hikiyama, Eriko; Onoue, Satomi; Yamada, Shizuo

    2016-01-01

    Solifenacin is an antimuscarinic agent used to treat symptoms of overactive bladder. Pharmacologically significant amounts of solifenacin were excreted in the urine of humans taking a clinical dose of this drug. The aim of this study is to measure muscarinic receptor binding in the bladder urothelium and detrusor muscles of rats following the intravesical instillation of solifenacin. Muscarinic receptors were measured by radioreceptor assay using [N-methyl-(3)H]scopolamine methyl chloride ([(3)H]NMS), a selective radioligand of muscarinic receptors. Solifenacin showed concentration-dependent inhibition of specific [(3)H]NMS binding in the bladder urothelium and detrusor muscle of rats, with no significant difference in Ki values or Hill coefficients between these tissues. Following the intravesical instillation of solifenacin, there was significant muscarinic receptor binding (increase in Kd for specific [(3)H]NMS binding) in the bladder urothelium and detrusor muscle of rats. Similar bladder muscarinic receptor binding was observed by the intravesical instillation of oxybutynin, but not with trospium. In conclusion, the present study has demonstrated that solifenacin binds muscarinic receptors not only in the detrusor muscle but also in the bladder urothelium with high affinity. These bladder muscarinic receptors may be significantly affected by solifenacin excreted in the urine.

  10. Oxotremorine suppresses thalamocortical oscillations via thalamic muscarinic acetylcholine receptors.

    PubMed

    Puoliväli, J; Jäkälä, P; Koivisto, E; Riekkinen, P

    1998-12-01

    We investigated whether the local intrathalamic infusion of a muscarinic acetylcholine receptor agonist (oxotremorine) at either the reticular nucleus of thalamus (NRT) or the ventroposteromedial nucleus of thalamus (VPM) suppresses thalamocortically generated neocortical high-voltage spindles (HVSs). In addition, we studied whether the intracerebroventricular (ICV) infusion of a selective muscarinic M2 acetylcholine receptor antagonist (methoctramine) could block the suppression of HVSs induced by either systemic (IP) administration of an anticholinesterase drug [tetrahydroaminoacridine (THA)] or ICV infusion of oxotremorine in rats. Intrathalamic administration of oxotremorine at 3 and 15 microg in the NRT, and at 15 microg in the VPM suppressed HVSs. ICV oxotremorine at 30 and 100 microg and IP THA at 3 mg/kg decreased HVSs. ICV methoctramine at 100 microg increased HVSs and completely blocked the decrease in HVSs produced by oxotremorine 100 microg and THA 3 mg/kg. The results suggest that activation of muscarinic M2 acetylcholine receptors in thalamic nuclei (NRT and VPM) can suppress thalamocortical oscillations and that ICV or systemically administered drugs that activate either directly (oxotremorine and methoctramine) or indirectly (THA) the muscarinic M2 acetylcholine receptors may modulate neocortical HVSs via the thalamus.

  11. 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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. 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.

  13. Role of muscarinic receptor antagonists in urgency and nocturia.

    PubMed

    Michel, Martin C; de la Rosette, Jean J M C H

    2005-09-01

    The overactive bladder (OAB) syndrome is defined as urgency, with or without urgency incontinence, usually accompanied by frequency and nocturia. Muscarinic receptor antagonists are the most established form of treatment for OAB, but until recently their effectiveness was only confirmed for symptoms of incontinence and frequency. In recent studies, selected muscarinic antagonists, including darifenacin, solifenacin, tolterodine and trospium, significantly reduced the number of urgency episodes per day relative to placebo. While some data raise the possibility that certain of these agents may be more effective than others in this regard, this variability in their effect on urgency needs to be confirmed in future studies. Moreover, it remains to be determined whether counting the number of urgency episodes or assessing the subjective intensity of the sensation of urgency more adequately reflects patient needs and therapeutic efficacy. For nocturia, muscarinic receptor antagonists have only inconsistently shown statistically greater effects than placebo. This inconsistency may relate to the multifactorial nature of nocturia, which even in patients with OAB can have many causes, not all of which may respond/be sensitive to muscarinic receptor antagonism.

  14. 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).

  15. Regulation of Brain Muscarinic Receptors by Protein Kinase C

    DTIC Science & Technology

    1991-06-21

    esters or to high concentrations of muscarinic agonists. Neuronal mouse neuroblastoma cells maintained in culture (clone N1E - 115 ) were used as a...E.E. El-Fakahany: Inhibition of Cyclic AMP Formation in N1E - 115 Neuroblastoma Cells is Mediated by a Noncardiac M2 Muscarinic Receptor Subtype...Receptor-Mediated Second Messenger Responses in N1E - 115 Neuroblastoma Cells. Journal of Neurochemistry. 53, 1300-1308, 1989. 15. McKinney, M., D

  16. Immunolocalization of muscarinic M1 receptor in the rat medial prefrontal cortex

    PubMed Central

    Tsuneoka, Yousuke; Yoshida, Sachine; Adachi‐Akahane, Satomi; Ito, Masanori; Kuroda, Masaru; Funato, Hiromasa

    2018-01-01

    Abstract The medial prefrontal cortex (mPFC) has been considered to participate in many higher cognitive functions, such as memory formation and spatial navigation. These cognitive functions are modulated by cholinergic afferents via muscarinic acetylcholine receptors. Previous pharmacological studies have strongly suggested that the M1 receptor (M1R) is the most important subtype among muscarinic receptors to perform these cognitive functions. Actually, M1R is abundant in mPFC. However, the proportion of somata containing M1R among cortical cellular types, and the precise intracellular localization of M1R remain unclear. In this study, to clarify the precise immunolocalization of M1R in rat mPFC, we examined three major cellular types, pyramidal neurons, inhibitory neurons, and astrocytes. M1R immunopositivity signals were found in the majority of the somata of both pyramidal neurons and inhibitory neurons. In pyramidal neurons, strong M1R immunopositivity signals were usually found throughout their somata and dendrites including spines. On the other hand, the signal strength of M1R immunopositivity in the somata of inhibitory neurons significantly varied. Some neurons showed strong signals. Whereas about 40% of GAD67‐immunopositive neurons and 30% of parvalbumin‐immunopositive neurons (PV neurons) showed only weak signals. In PV neurons, M1R immunopositivity signals were preferentially distributed in somata. Furthermore, we found that many astrocytes showed substantial M1R immunopositivity signals. These signals were also mainly distributed in their somata. Thus, the distribution pattern of M1R markedly differs between cellular types. This difference might underlie the cholinergic modulation of higher cognitive functions subserved by mPFC. PMID:29424434

  17. 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

  18. 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

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Frey, K.A.; Ehrenkaufer, R.L.; Beaucage, S.

    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 withmore » 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.« less

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    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 (/supmore » 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.« less

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Scherer, R.W.

    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); eithermore » 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.« less

  2. Muscarinic receptor M4 positive allosteric modulators attenuate central effects of cocaine.

    PubMed

    Dall, Camilla; Weikop, Pia; Dencker, Ditte; Molander, Anna C; Wörtwein, Gitta; Conn, P Jeffrey; Fink-Jensen, Anders; Thomsen, Morgane

    2017-07-01

    Cocaine addiction is a chronic brain disease affecting neurotransmission. Muscarinic cholinergic receptors modulate dopaminergic signaling in the reward system, and muscarinic receptor stimulation can block direct reinforcing effects of cocaine. Here, we tested the hypothesis that specific muscarinic M 4 receptor stimulation can attenuate the discriminative stimulus effects and conditioned rewarding effects of cocaine, measures believed to predict the ability of cocaine and cocaine-associated cues to elicit relapse to drug taking. We tested the M 4 -selective positive allosteric modulators VU0152100 and VU0467154 in a drug discrimination assay and a conditioned place preference assay, including extinction and reinstatement of place preference. Specificity of the cocaine discrimination effect was verified using knockout mice lacking either M 1 or M 4 receptors (M 1 -/- , M 4 -/- ). We also replicated previous findings in cocaine-induced locomotor hyperactivity and striatal dopamine microdialysis assays. VU0152100 attenuated the discriminative stimulus effect of cocaine in wild-type mice and M 1 -/- mice, but not in M 4 -/- mice, without affecting rates of responding. As previously shown with VU0152100, VU0467154 almost eliminated cocaine-induced hyperactivity and striatal dopamine efflux. VU0467154 failed to attenuate acquisition of cocaine-conditioned place preference, but facilitated extinction and prevented reinstatement of the conditioned place preference. These findings further support the notion that M 4 receptors are promising targets for the treatment of cocaine addiction, by showing that results can be replicated using distinct ligands, and that in addition to blocking reinforcing effects of cocaine relevant to ongoing drug taking, M 4 positive allosteric modulators can also attenuate subjective and conditioned effects relevant to relapse. Copyright © 2017. Published by Elsevier B.V.

  3. Novel long‐acting antagonists of muscarinic ACh receptors

    PubMed Central

    Randáková, Alena; Rudajev, Vladimír; Doležal, Vladimír; Boulos, John

    2018-01-01

    Background and Purpose The aim of this study was to develop potent and long‐acting antagonists of muscarinic ACh receptors. The 4‐hexyloxy and 4‐butyloxy derivatives of 1‐[2‐(4‐oxidobenzoyloxy)ethyl]‐1,2,3,6‐tetrahydropyridin‐1‐ium were synthesized and tested for biological activity. Antagonists with long‐residence time at receptors are therapeutic targets for the treatment of several neurological and psychiatric human diseases. Their long‐acting effects allow for reduced daily doses and adverse effects. Experimental Approach The binding and antagonism of functional responses to the agonist carbachol mediated by 4‐hexyloxy compounds were investigated in CHO cells expressing individual subtypes of muscarinic receptors and compared with 4‐butyloxy analogues. Key Results The 4‐hexyloxy derivatives were found to bind muscarinic receptors with micromolar affinity and antagonized the functional response to carbachol with a potency ranging from 30 nM at M1 to 4 μM at M3 receptors. Under washing conditions to reverse antagonism, the half‐life of their antagonistic action ranged from 1.7 h at M2 to 5 h at M5 receptors. Conclusions and Implications The 4‐hexyloxy derivatives were found to be potent long‐acting M1‐preferring antagonists. In view of current literature, M1‐selective antagonists may have therapeutic potential for striatal cholinergic dystonia, delaying epileptic seizure after organophosphate intoxication or relieving depression. These compounds may also serve as a tool for research into cognitive deficits. PMID:29498041

  4. Demonstration of muscarinic and nicotinic receptor binding activities of distigmine to treat detrusor underactivity.

    PubMed

    Harada, Taketsugu; Fushimi, Kazumi; Kato, Aya; Ito, Yoshihiko; Nishijima, Saori; Sugaya, Kimio; Yamada, Shizuo

    2010-01-01

    The present study was undertaken to examine whether distigmine, a therapeutic agent used to treat detrusor underactivity, binds directly to muscarinic and nicotinic receptors. We used radioreceptor binding assays and compared the effects of distigmine with those of neostigmine and donepedil. The inhibitory effect of distigmine on the blood acetylcholinesterase (AChE) activity was significantly weaker than that of neostigmine. Distigmine, neostigmine, and donepezil competed for specific binding sites of [N-methyl-(3)H]scopolamine methyl chloride ([(3)H]NMS ) and [(3)H]oxotremorine-M in the bladder, submaxillary gland and cerebral cortex of rats in a concentration-dependent manner, indicating significant binding activity of muscarinic receptors. Distigmine displayed significantly higher affinity for binding sites of [(3)H]oxotremorine-M compared with those of [(3)H]NMS as revealed by large ratios of its K(i) value for [(3)H]NMS to that for [(3)H]oxotremorine-M, suggesting that it has preferential affinity for agonist sites of muscarinic receptors. Distigmine seemed to bind to the agonist sites of muscarinic receptors in a competitive manner. Repeated oral administration of distigmine caused a significant decrease in the maximal number of binding sites (B(max)) for [(3)H]NMS in the bladder and submaxillary gland but not cerebral cortex. Distigmine also bound to nicotinic receptors in the rat cerebral cortex. In conclusion, distigmine shows direct binding to muscarinic receptors in the rat bladder, and repeated oral administration of distigmine causes downregulation of muscarinic receptors in the rat bladder. The observed direct interaction of distigmine with the bladder muscarinic receptors may partly contribute to the therapeutic and/or side effects seen in the treatment of detrusor underactivity.

  5. Muscarinic supersensitivity and impaired receptor desensitization in G protein-coupled receptor kinase 5-deficient mice.

    PubMed

    Gainetdinov, R R; Bohn, L M; Walker, J K; Laporte, S A; Macrae, A D; Caron, M G; Lefkowitz, R J; Premont, R T

    1999-12-01

    G protein-coupled receptor kinase 5 (GRK5) is a member of a family of enzymes that phosphorylate activated G protein-coupled receptors (GPCR). To address the physiological importance of GRK5-mediated regulation of GPCRs, mice bearing targeted deletion of the GRK5 gene (GRK5-KO) were generated. GRK5-KO mice exhibited mild spontaneous hypothermia as well as pronounced behavioral supersensitivity upon challenge with the nonselective muscarinic agonist oxotremorine. Classical cholinergic responses such as hypothermia, hypoactivity, tremor, and salivation were enhanced in GRK5-KO animals. The antinociceptive effect of oxotremorine was also potentiated and prolonged. Muscarinic receptors in brains from GRK5-KO mice resisted oxotremorine-induced desensitization, as assessed by oxotremorine-stimulated [5S]GTPgammaS binding. These data demonstrate that elimination of GRK5 results in cholinergic supersensitivity and impaired muscarinic receptor desensitization and suggest that a deficit of GPCR desensitization may be an underlying cause of behavioral supersensitivity.

  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. Immunocytochemical localization of muscarinic, adrenergic and AT1 receptors.

    PubMed

    Schulze, W; Fu, M L

    1996-01-01

    By indirect immunofluorescence and post-embedding EM gold technique, the localization of alpha 1-adrenergic, M2-muscarinic and angiotensin II receptor-I (AT1) were determinated. With antipeptide antibodies directed against the second extracellular loops of all three receptors, these receptors were found to be localized at the sarcolemma of adult rat cardiomyocytes and at the surface membranes of cultivated neonatal heart cells. Additionally, M2 receptors were localized along T-tubule membranes of both rat and human adult cardiomyocytes. alpha 1-Adrenergic receptors were found intracellular near the surface of atrial granules (ANF-granules). By using M2 and alpha 1-adrenergic receptor antibodies the strongest fluorescence was found in the right atrium of the rat. Besides the localization in cardiomyocytes, AT1 receptors were also localized in outer plasma membranes and the endoplasmic reticulum of fibroblasts, and the surface of smooth muscle cells of the major arteries and veins. Likewise, the muscarinic M2 receptors were found along the outer membranes of endothelial cells from capillaries and endocardium.

  8. Nitric oxide/cGMP/PKG signaling pathway activated by M1-type muscarinic acetylcholine receptor cascade inhibits Na+-activated K+ currents in Kenyon cells

    PubMed Central

    Hasebe, Masaharu

    2016-01-01

    The interneurons of the mushroom body, known as Kenyon cells, are essential for the long-term memory of olfactory associative learning in some insects. Some studies have reported that nitric oxide (NO) is strongly related to this long-term memory in Kenyon cells. However, the target molecules and upstream and downstream NO signaling cascades are not completely understood. Here we analyzed the effect of the NO signaling cascade on Na+-activated K+ (KNa) channel activity in Kenyon cells of crickets (Gryllus bimaculatus). We found that two different NO donors, S-nitrosoglutathione (GSNO) and S-nitroso-N-acetyl-dl-penicillamine (SNAP), strongly suppressed KNa channel currents. Additionally, this inhibitory effect of GSNO on KNa channel activity was diminished by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase (sGC), and KT5823, an inhibitor of protein kinase G (PKG). Next, we analyzed the role of ACh in the NO signaling cascade. ACh strongly suppressed KNa channel currents, similar to NO donors. Furthermore, this inhibitory effect of ACh was blocked by pirenzepine, an M1 muscarinic ACh receptor antagonist, but not by 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP) and mecamylamine, an M3 muscarinic ACh receptor antagonist and a nicotinic ACh receptor antagonist, respectively. The ACh-induced inhibition of KNa channel currents was also diminished by the PLC inhibitor U73122 and the calmodulin antagonist W-7. Finally, we found that ACh inhibition was blocked by the nitric oxide synthase (NOS) inhibitor NG-nitro-l-arginine methyl ester (l-NAME). These results suggested that the ACh signaling cascade promotes NO production by activating NOS and NO inhibits KNa channel currents via the sGC/cGMP/PKG signaling cascade in Kenyon cells. PMID:26984419

  9. 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

  10. Identification of the Muscarinic Acetylcholine Receptor Subtype Mediating Cholinergic Vasodilation in Murine Retinal Arterioles

    PubMed Central

    Sniatecki, Jan J.; Goloborodko, Evgeny; Steege, Andreas; Zavaritskaya, Olga; Vetter, Jan M.; Grus, Franz H.; Patzak, Andreas; Wess, Jürgen; Pfeiffer, Norbert

    2011-01-01

    Purpose. To identify the muscarinic acetylcholine receptor subtype that mediates cholinergic vasodilation in murine retinal arterioles. Methods. Muscarinic receptor gene expression was determined in murine retinal arterioles using real-time PCR. To assess the functional relevance of muscarinic receptors for mediating vascular responses, retinal vascular preparations from muscarinic receptor–deficient mice were studied in vitro. Changes in luminal arteriole diameter in response to muscarinic and nonmuscarinic vasoactive substances were measured by video microscopy. Results. Only mRNA for the M3 receptor was detected in retinal arterioles. Thus, M3 receptor–deficient mice (M3R−/−) and respective wild-type controls were used for functional studies. Acetylcholine concentration-dependently dilated retinal arterioles from wild-type mice. In contrast, vasodilation to acetylcholine was almost completely abolished in retinal arterioles from M3R−/− mice, whereas responses to the nitric oxide (NO) donor nitroprusside were retained. Carbachol, an acetylcholinesterase-resistant analog of acetylcholine, also evoked dilation in retinal arterioles from wild-type, but not from M3R−/−, mice. Vasodilation responses from wild-type mice to acetylcholine were negligible after incubation with the non–subtype-selective muscarinic receptor blocker atropine or the NO synthase inhibitor Nω-nitro-l-arginine methyl ester, and were even reversed to contraction after endothelial damage with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Conclusions. These findings provide evidence that endothelial M3 receptors mediate cholinergic vasodilation in murine retinal arterioles via activation of NO synthase. PMID:21873683

  11. Muscarinic receptor immunoreactivity in the superior salivatory nucleus neurons innervating the salivary glands of the rat.

    PubMed

    Ueda, Hirotaka; Mitoh, Yoshihiro; Fujita, Masako; Kobashi, Motoi; Yamashiro, Takashi; Sugimoto, Tomosada; Ichikawa, Hiroyuki; Matsuo, Ryuji

    2011-07-15

    The superior salivatory nucleus (SSN) contains preganglionic parasympathetic neurons to the submandibular and sublingual salivary glands. Cevimeline, a muscarinic acetylcholine receptor agonist, stimulates the salivary glands and is presently used as sialogogue in the treatment of dry mouth. Since cevimeline passes through the blood-brain barrier, it is also able to act on muscarinic acetylcholine receptors in the central nervous system. Our preliminary experiment using the whole-cell patch-clamp technique has shown that cevimeline excites SSN neurons in rat brain slices, suggesting that SSN neurons have muscarinic acetylcholine receptors; however, it is unclear which subtypes of muscarinic acetylcholine receptors exist in SSN neurons. In the present study, we investigated immunohistochemically muscarinic acetylcholine receptor subtypes, M1 receptor (M1R), M2R, M3R, M4R, and M5R in SSN neurons. SSN neurons innervating the salivary glands, retrogradely labeled with a fluorescent tracer from the chorda-lingual nerve, mostly expressed M3R immunoreactivity (-ir) (92.3%) but not M1R-ir. About half of such SSN neurons also showed M2R- (40.1%), M4R- (54.0%) and M5R-ir (46.0%); therefore, it is probable that SSN neurons co-express M3R-ir with at least two of the other muscarinic receptor subtypes. This is the first report to show that SSN neurons contain muscarinic acetylcholine receptors. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

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

    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 capacitymore » 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.« less

  13. 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.

  14. Muscarinic receptor subtype selectivity of novel heterocyclic QNB analogues

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Baumgold, J.; Cohen, V.I.; Paek, R.

    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 potencymore » 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.« less

  15. 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.

  16. Muscarinic Receptors Modulate the Intrinsic Excitability of Infralimbic Neurons and Consolidation of Fear Extinction

    PubMed Central

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

    2012-01-01

    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. PMID:22510723

  17. 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. © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

  18. Action of AF64A on rat brain muscarinic receptors

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Eva, C.; Costa, E.

    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 butmore » 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.« less

  19. 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. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Transcriptional response to muscarinic acetylcholine receptor stimulation: regulation of Egr-1 biosynthesis by ERK, Elk-1, MKP-1, and calcineurin in carbachol-stimulated human neuroblastoma cells.

    PubMed

    Rössler, Oliver G; Henss, Isabell; Thiel, Gerald

    2008-02-01

    Carbachol-mediated activation of type M(3) muscarinic acetylcholine receptors induces the biosynthesis of the transcription factor Egr-1 in human SH-SY5Y neuroblastoma cells involving an activation of extracellular signal-regulated protein kinase. Carbachol triggered the phosphorylation of the ternary complex factor Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, and strikingly enhanced the transcriptional activation potential of Elk-1. Chromatin immunoprecipitation experiments revealed that Elk-1 binds in vivo to the 5'-upstream region of the Egr-1 gene in carbachol-stimulated neuroblastoma cells. Together, these data indicate that Elk-1 connects the intracellular signaling cascade elicited by activation of M(3) muscarinic acetylcholine receptors with the transcription of the Egr-1 gene. Lentiviral-mediated expression of either MAP kinase phosphatase-1 (MKP-1) or a constitutively active mutant of calcineurin A inhibited Egr-1 biosynthesis following carbachol stimulation, indicating that these phosphatases function as shut-off devices of muscarinic acetylcholine receptor signaling. Additionally, carbachol stimulation increased transcription of a chromatin-embedded collagenase promoter/reporter gene, showing that AP-1 activity is enhanced in carbachol-stimulated neuroblastoma. Expression experiments revealed that both MKP-1 and a constitutively active mutant of calcineurin A impaired carbachol-induced upregulation of AP-1 activity. The fact that carbachol stimulation of neuroblastoma cells activates the transcription factors Egr-1 and AP-1 suggests that changes in the gene expression pattern are an integral part of muscarinic acetylcholine receptor signaling.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Haga, Kazuko; Kruse, Andrew C.; Asada, Hidetsugu

    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 structuremore » 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.« less

  2. Heterologous desensitization of muscarinic receptors by P2Z purinoceptors in rat parotid acinar cells.

    PubMed

    Fukushi, Y

    1999-01-01

    We studied the heterologous desensitization of muscarinic receptors by ATP in fura-2-loaded rat parotid acinar cells. Exposure to ATP or 3'-o-(4-benzoyl) benzoyl-ATP shortened the duration and decreased the magnitude of acetylcholine-induced Ca2+ release from intracellular Ca2+ stores in a dose-dependent manner. The shortening was observed only in an early stage of desensitization (within 20 s), whereas the decrease in the magnitude of the response was dependent upon the time the cells were exposed to the nucleotides. Atropine induced a profound shortening during the progressive decrease in the magnitude of acetylcholine-induced Ca2+ release. 3'-o-(4-Benzoyl) benzoyl-ATP did not induce an increase in the cytosolic Ca2+ concentration when the cells were incubated in the Ca2+- and Na+-free medium, but it did induce a strong desensitization of muscarinic receptors. The specific protein kinase C inhibitor bisindoylmaleimide resensitized the 3'-o-(4-benzoyl) benzoyl-ATP-treated muscarinic receptors. Phorbol 12-myristate 13-acetate potentiated the desensitization of muscarinic receptors. Ceramides that prevent the activation of phospholipase D resensitized the 3'-o-(4-benzoyl) benzoyl-ATP-treated muscarinic receptors. These results suggest that ATP, acting through P2Z purinoceptor-mediated phospholipase D, may produce a Ca2+-independent protein kinase C. Heterologous desensitization of muscarinic receptors by protein kinase C may shorten the duration and decrease the magnitude of acetylcholine-induced Ca2+ release.

  3. 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

  4. Muscarinic and nicotinic acetylcholine receptor agonists: current scenario in Alzheimer's disease therapy.

    PubMed

    Verma, Stuti; Kumar, Ashwini; Tripathi, Timir; Kumar, Awanish

    2018-04-16

    Alzheimer's disease (AD) has become the primary cause of dementia. It shows a progressive cognitive dysfunction with degenerating neurons. Acetylcholine receptors (AChRs) propagate the cognitive ability and it consists of two primary members namely muscarinic (mAChRs) and nicotinic receptors (nAChRs). Where mAChRs is G-protein coupled receptor, (nAChRs) are ligand-gated ion channels. The conventional therapeutic regimen for AD consists of three acetylcholinestearse inhibitors while a single NMDA receptor antagonist. Researchers around the globe are developing new and modifying the existing AChRs agonists to develop lead candidates with lower risk to benefit ratio where benefits clearly outweigh the adverse events. We have searched PubMed, MEDLINE, Google scholar, Science Direct and, Web of Science with keywords "Muscarinic/Nicotinic acetylcholine receptor, agonists and, AD". The literature search included articles written in English. Scientific relevance for clinical studies, basic science studies is eligibility criteria for articles referred in this paper. M1 is the primary muscarinic subtype while α7 is the primary nAChR subtype that is responsible for cognition and memory and these two have been the major recent experimental targets for mAChR agonist strategy. The last cholinergic receptor agonist to enter phase 3 trial was EVP-6124 (Enceniclin) but was withdrawn due to severe gastrointestinal adverse effects. We aim to present an overview of the efforts and achievements in targeting Muscarinic and Nicotinic acetylcholine receptor in the current review for development of better AD therapeutics. © 2018 Royal Pharmaceutical Society.

  5. Design, synthesis, and action of oxotremorine-related hybrid-type allosteric modulators of muscarinic acetylcholine receptors.

    PubMed

    Disingrini, Teresa; Muth, Mathias; Dallanoce, Clelia; Barocelli, Elisabetta; Bertoni, Simona; Kellershohn, Kerstin; Mohr, Klaus; De Amici, Marco; Holzgrabe, Ulrike

    2006-01-12

    A novel series of muscarinic receptor ligands of the hexamethonio-type was prepared which contained, on one side, the phthalimidopropane or 1,8-naphthalimido-2,2-dimethylpropane moiety typical for subtype selective allosteric antagonists and, on the other, the acetylenic fragment typical for the nonselective orthosteric muscarinic agonists oxotremorine, oxotremorine-M, and related muscarinic agonists. Binding experiments in M(2) receptors using [(3)H]N-methylscopolamine as an orthosteric probe proved an allosteric action of both groups of hybrids, 7a-10a and 8b-10b. The difference in activity between a-group and b-group hybrids corresponded with the activity difference between the allosteric parent compounds. In M(1)-M(3) muscarinic isolated organ preparations, most of the hybrids behaved as subtype selective antagonists. [(35)S]GTPgammaS binding assays using human M(2) receptors overexpressed in CHO cells revealed that a weak intrinsic efficacy was preserved in 8b-10b. Thus, attaching muscarinic allosteric antagonist moieties to orthosteric muscarinic agonists may lead to hybrid compounds in which functions of both components are mixed.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Pfeiffer, A.; Rochlitz, H.; Herz, A.

    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 withmore » 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.« less

  7. 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.

  8. Muscarinic receptor subtypes involved in carbachol-induced contraction of mouse uterine smooth muscle.

    PubMed

    Kitazawa, Takio; Hirama, Ryuichi; Masunaga, Kozue; Nakamura, Tatsuro; Asakawa, Koichi; Cao, Jinshan; Teraoka, Hiroki; Unno, Toshihiro; Komori, Sei-ichi; Yamada, Masahisa; Wess, Jürgen; Taneike, Tetsuro

    2008-06-01

    Functional muscarinic acetylcholine receptors present in the mouse uterus were characterized by pharmacological and molecular biological studies using control (DDY and wild-type) mice, muscarinic M2 or M3 single receptor knockout (M2KO, M3KO), and M2 and M3 receptor double knockout mice (M2/M3KO). Carbachol (10 nM-100 microM) increased muscle tonus and phasic contractile activity of uterine strips of control mice in a concentration-dependent manner. The maximum carbachol-induced contractions (Emax) differed between cervical and ovarian regions of the uterus. The stage of the estrous cycle had no significant effect on carbachol concentration-response relationships. Tetrodotoxin did not decrease carbachol-induced contractions, but the muscarinic receptor antagonists (11-[[2-[(diethylaminomethyl)-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3-b[2,3-b][1,4]benzodiazepin6-one (AF-DX116), N-[2-[2-[(dipropylamino)methyl]-1-piperidinyl]ethyl]-5,6-dihydro-6-oxo-11H-pyrido[2,3-b][1,4] benzodiazepine-11-carboxamide (AF-DX384), 4-diphenylacetoxy-N-methyl-piperidine(4-DAMP), para-fluoro-hexa hydro-sila-diphenidol (p-F-HHSiD), himbacine, methoctramine, pirenzepine, and tropicamide) inhibited carbachol-induced contractions in a competitive fashion. The pKb values for these muscarinic receptor antagonists correlated well with the known pKi values of these antagonists for the M3 muscarinic receptor. In uterine strips isolated from mice treated with pertussis toxin (100 microg/kg, i.p. for 96 h), Emax values for carbachol were significantly decreased, but effective concentration that caused 50% of Emax values (EC50) remained unchanged. In uterine strips treated with 4-DAMP mustard (30 nM) and AF-DX116 (1 microM), followed by subsequent washout of AF-DX116, neither carbachol nor N,N,N,-trimethyl-4-(2-oxo-1-pyrolidinyl)-2-butyn-1-ammonium iodide (oxotremorine-M) caused any contractile responses. Both M2 and M3 muscarinic receptor messenger RNAs were detected in the mouse uterus via

  9. Muscarinic Ca2+ responses resistant to muscarinic antagonists at perisynaptic Schwann cells of the frog neuromuscular junction.

    PubMed Central

    Robitaille, R; Jahromi, B S; Charlton, M P

    1997-01-01

    1. Acetylcholine causes a rise of intracellular Ca2+ in perisynaptic Schwann cells (PSCs) of the frog neuromuscular junction. The signalling pathway was characterized using the fluorescent Ca2+ indicator fluo-3 and fluorescence microscopy. 2. Nicotinic antagonists had no effect on Ca2+ responses evoked by ACh and no Ca2+ responses were evoked with the nicotinic agonist nicotine. The muscarinic agonists muscarine and oxotremorine-M induced Ca2+ signals in PSCs. 3. Ca2+ responses remained unchanged when extracellular Ca2+ was removed, indicating that they are due to the release of Ca2+ from internal stores. Incubation with pertussis toxin did not alter the Ca2+ signals induced by muscarine, but did block depression of transmitter release induced by adenosine and prevented Ca2+ responses in PSCs induced by adenosine. 4. The general muscarinic antagonists atropine, quinuclidinyl benzilate and N-methyl-scopolamine failed to block Ca2+ responses to muscarinic agonists. Atropine (at 20,000-fold excess concentration) also failed to reduce the proportion of cells responding to a threshold muscarine concentration sufficient to cause responses in less than 50% of cells. Only the allosteric, non-specific blocker, gallamine (1-10 microM) was effective in blocking muscarine-induced Ca2+ responses. 5. In preparations denervated 7 days prior to experiments, low concentrations of atropine reversibly and completely blocked Ca2+ responses to muscarine. 6. The lack of blockade by general muscarinic antagonists in innervated, in situ preparations suggests that muscarinic Ca2+ responses at PSCs are not mediated by any of the five known muscarinic receptors or that post-translational modification prevented antagonist binding. Images Figure 2 Figure 3 Figure 4 Figure 6 Figure 7 PMID:9365908

  10. Airborne fine particulate matter causes murine bronchial hyperreactivity via MAPK pathway-mediated M3 muscarinic receptor upregulation.

    PubMed

    Wang, Rong; Xiao, Xue; Shen, Zhenxing; Cao, Lei; Cao, Yongxiao

    2017-02-01

    Regarding the human health effects, airborne fine particulate matter 2.5 (PM 2.5 ) is an important environmental risk factor. However, the underlying molecular mechanisms are largely unknown. The present study examined the hypothesis that PM 2.5 causes bronchial hyperreactivity by upregulated muscarinic receptors via the mitogen-activated protein kinase (MAPK) pathway. The isolated rat bronchi segments were cultured with different concentration of PM 2.5 for different time. The contractile response of the bronchi segments were recorded by a sensitive myograph. The mRNA and protein expression levels of M 3 muscarinic receptors were studied by quantitative real-time PCR and immunohistochemistry, respectively. The muscarinic receptors agonist, carbachol induced a remarkable contractile response on fresh and DMSO cultured bronchial segments. Compared with the fresh or DMSO culture groups, 1.0 µg/mL of PM 2.5 cultured for 24 h significantly enhanced muscarinic receptor-mediated contractile responses in bronchi with a markedly increased maximal contraction. In addition, the expression levels of mRNA and protein for M 3 muscarinic receptors in bronchi of PM 2.5 group were higher than that of fresh or DMSO culture groups. SB203580 (p38 inhibitor) and U0126 (MEK1/2 inhibitor) significantly inhibited the PM 2.5 -induced enhanced contraction and increased mRNA and protein expression of muscarinic receptors. However, JNK inhibitor SP600125 had no effect on PM 2.5 -induced muscarinic receptor upregulation and bronchial hyperreactivity. In conclusion, airborne PM 2.5 upregulates muscarinic receptors, which causes subsequently bronchial hyperreactivity shown as enhanced contractility in bronchi. This process may be mediated by p38 and MEK1/2 MAPK pathways. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 371-381, 2017. © 2016 Wiley Periodicals, Inc.

  11. Fluoxetine induces vasodilatation of cerebral arterioles by co-modulating NO/muscarinic signalling

    PubMed Central

    Ofek, Keren; Schoknecht, Karl; Melamed-Book, Naomi; Heinemann, Uwe; Friedman, Alon; Soreq, Hermona

    2012-01-01

    Ischaemic stroke patients treated with Selective Serotonin Reuptake Inhibitors (SSRI) show improved motor, cognitive and executive functions, but the underlying mechanism(s) are incompletely understood. Here, we report that cerebral arterioles in the rat brain superfused with therapeutically effective doses of the SSRI fluoxetine showed consistent, dose-dependent vasodilatation (by 1.2 to 1.6-fold), suppressible by muscarinic and nitric oxide synthase (NOS) antagonists [atropine, NG-nitro-l-arginine methyl ester (l-NAME)] but resistant to nicotinic and serotoninergic antagonists (mecamylamine, methylsergide). Fluoxetine administered 10–30 min. following experimental vascular photo-thrombosis increased arterial diameter (1.3–1.6), inducing partial, but lasting reperfusion of the ischaemic brain. In brain endothelial b.End.3 cells, fluoxetine induced rapid muscarinic receptor-dependent increases in intracellular [Ca2+] and promoted albumin- and eNOS-dependent nitric oxide (NO) production and HSP90 interaction. In vitro, fluoxetine suppressed recombinant human acetylcholinesterase (rhAChE) activity only in the presence of albumin. That fluoxetine induces vasodilatation of cerebral arterioles suggests co-promotion of endothelial muscarinic and nitric oxide signalling, facilitated by albumin-dependent inhibition of serum AChE. PMID:22697296

  12. Attenuation in rats of impairments of memory by scopolamine, a muscarinic receptor antagonist, by mecamylamine, a nicotinic receptor antagonist.

    PubMed

    Newman, L A; Gold, P E

    2016-03-01

    Scopolamine, a muscarinic antagonist, impairs learning and memory for many tasks, supporting an important role for the cholinergic system in these cognitive functions. The findings are most often interpreted to indicate that a decrease in postsynaptic muscarinic receptor activation mediates the memory impairments. However, scopolamine also results in increased release of acetylcholine in the brain as a result of blocking presynaptic muscarinic receptors. The present experiments assess whether scopolamine-induced increases in acetylcholine release may impair memory by overstimulating postsynaptic cholinergic nicotinic receptors, i.e., by reaching the high end of a nicotinic receptor activation inverted-U dose-response function. Rats tested in a spontaneous alternation task showed dose-dependent working memory deficits with systemic injections of mecamylamine and scopolamine. When an amnestic dose of scopolamine (0.15 mg/kg) was co-administered with a subamnestic dose of mecamylamine (0.25 mg/kg), this dose of mecamylamine significantly attenuated the scopolamine-induced memory impairments. We next assessed the levels of acetylcholine release in the hippocampus in the presence of scopolamine and mecamylamine. Mecamylamine injections resulted in decreased release of acetylcholine, while scopolamine administration caused a large increase in acetylcholine release. These findings indicate that a nicotinic antagonist can attenuate impairments in memory produced by a muscarinic antagonist. The nicotinic antagonist may block excessive activation of nicotinic receptors postsynaptically or attenuate increases in acetylcholine release presynaptically. Either effect of a nicotinic antagonist-to decrease scopolamine-induced increases in acetylcholine output or to decrease postsynaptic acetylcholine receptor activation-may mediate the negative effects on memory of muscarinic antagonists.

  13. Attenuation in rats of impairments of memory by scopolamine, a muscarinic receptor antagonist, by mecamylamine, a nicotinic receptor antagonist

    PubMed Central

    Newman, L. A.

    2015-01-01

    Rationale Scopolamine, a muscarinic antagonist, impairs learning and memory for many tasks, supporting an important role for the cholinergic system in these cognitive functions. The findings are most often interpreted to indicate that a decrease in postsynaptic muscarinic receptor activation mediates the memory impairments. However, scopolamine also results in increased release of acetylcholine in the brain as a result of blocking presynaptic muscarinic receptors. Objectives The present experiments assess whether scopolamine-induced increases in acetylcholine release may impair memory by overstimulating postsynaptic cholinergic nicotinic receptors, i.e., by reaching the high end of a nicotinic receptor activation inverted-U dose-response function. Results Rats tested in a spontaneous alternation task showed dose-dependent working memory deficits with systemic injections of mecamylamine and scopolamine. When an amnestic dose of scopolamine (0.15 mg/kg) was co-administered with a subamnestic dose of mecamylamine (0.25 mg/kg), this dose of mecamylamine significantly attenuated the scopolamine-induced memory impairments. We next assessed the levels of acetylcholine release in the hippocampus in the presence of scopolamine and mecamylamine. Mecamylamine injections resulted in decreased release of acetylcholine, while scopolamine administration caused a large increase in acetylcholine release. Conclusions These findings indicate that a nicotinic antagonist can attenuate impairments in memory produced by a muscarinic antagonist. The nicotinic antagonist may block excessive activation of nicotinic receptors postsynaptically or attenuate increases in acetylcholine release presynaptically. Either effect of a nicotinic antagonist—to decrease scopolamine-induced increases in acetylcholine output or to decrease post-synaptic acetylcholine receptor activation—may mediate the negative effects on memory of muscarinic antagonists. PMID:26660295

  14. The allosteric site regulates the voltage sensitivity of muscarinic receptors.

    PubMed

    Hoppe, Anika; Marti-Solano, Maria; Drabek, Matthäus; Bünemann, Moritz; Kolb, Peter; Rinne, Andreas

    2018-01-01

    Muscarinic receptors (M-Rs) for acetylcholine (ACh) belong to the class A of G protein-coupled receptors. M-Rs are activated by orthosteric agonists that bind to a specific site buried in the M-R transmembrane helix bundle. In the active conformation, receptor function can be modulated either by allosteric modulators, which bind to the extracellular receptor surface or by the membrane potential via an unknown mechanism. Here, we compared the modulation of M 1 -Rs and M 3 -Rs induced by changes in voltage to their allosteric modulation by chemical compounds. We quantified changes in receptor signaling in single HEK 293 cells with a FRET biosensor for the G q protein cycle. In the presence of ACh, M 1 -R signaling was potentiated by voltage, similarly to positive allosteric modulation by benzyl quinolone carboxylic acid. Conversely, signaling of M 3 -R was attenuated by voltage or the negative allosteric modulator gallamine. Because the orthosteric site is highly conserved among M-Rs, but allosteric sites vary, we constructed "allosteric site" M 3 /M 1 -R chimeras and analyzed their voltage dependencies. Exchanging the entire allosteric sites eliminated the voltage sensitivity of ACh responses for both receptors, but did not affect their modulation by allosteric compounds. Furthermore, a point mutation in M 3 -Rs caused functional uncoupling of the allosteric and orthosteric sites and abolished voltage dependence. Molecular dynamics simulations of the receptor variants indicated a subtype-specific crosstalk between both sites, involving the conserved tyrosine lid structure of the orthosteric site. This molecular crosstalk leads to receptor subtype-specific voltage effects. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. 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. © 2013 Wiley Publishing Asia Pty Ltd.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    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) muscarinicallymore » 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.« less

  17. Activation of M1 muscarinic receptors triggers transmitter release from rat sympathetic neurons through an inhibition of M-type K+ channels.

    PubMed

    Lechner, Stefan G; Mayer, Martina; Boehm, Stefan

    2003-12-15

    Acetylcholine has long been known to excite sympathetic neurons via M1 muscarinic receptors through an inhibition of M-currents. Nevertheless, it remained controversial whether activation of muscarinic receptors is also sufficient to trigger noradrenaline release from sympathetic neurons. In primary cultures of rat superior cervical ganglia, the muscarinic agonist oxotremorine M inhibited M-currents with half-maximal effects at 1 microM and induced the release of previously incorporated [3H]noradrenaline with half-maximal effects at 10 microM. This latter action was not affected by the nicotinic antagonist mecamylamine which, however, abolished currents through nicotinic receptors elicited by high oxotremorine M concentrations. Ablation of the signalling cascades linked to inhibitory G proteins by pertussis toxin potentiated the release stimulating effect of oxotremorine M, and the half-maximal concentration required to stimulate noradrenaline release was decreased to 3 microM. Pirenzepine antagonized the inhibition of M-currents and the induction of release by oxotremorine M with identical apparent affinity, and both effects were abolished by the muscarinic toxin 7. These results indicate that one muscarinic receptor subtype, namely M1, mediates these two effects. Retigabine, which enhances M-currents, abolished the release induced by oxotremorine M, but left electrically induced release unaltered. Moreover, retigabine shifted the voltage-dependent activation of M-currents by about 20 mV to more negative potentials and caused 20 mV hyperpolarisations of the membrane potential. In the absence of retigabine, oxotremorine M depolarised the neurons and elicited action potential discharges in 8 of 23 neurons; in its presence, oxotremorine M still caused equal depolarisations, but always failed to trigger action potentials. Action potential waveforms caused by current injection were not affected by retigabine. These results indicate that the inhibition of M-currents is

  18. Corticotropin-releasing factor stimulates colonic motility via muscarinic receptors in the rat

    PubMed Central

    Kim, Kyung-Jo; Kim, Ki Bae; Yoon, Soon Man; Han, Joung-Ho; Chae, Hee Bok; Park, Seon Mee; Youn, Sei Jin

    2017-01-01

    AIM To measure exogenous corticotropin-releasing factor (CRF)-induced motility of the isolated rat colon and to demonstrate the effect of pharmacologic inhibition on CRF-induced motility. METHODS The isolated vascularly-perfused rat colon was used. Luminal pressure was monitored via microtip catheter pressure transducers in the proximal and distal colon. At first, exogenous CRF was administered in a stepwise manner and the concentration of CRF yielding maximal colonic motility was selected. After recording basal colonic motility, hexamethonium, phentolamine, propranolol, atropine and tetrodotoxin were infused into the isolated colon. Initially, only the test drug was infused; then, CRF was added. The motility index was expressed as percentage change over basal level. RESULTS Administration of 1.4, 14.4, 144 and 288 pmol/L CRF progressively increased colonic motility in the proximal and distal colon. Infusion of atropine or tetrodotoxin reduced CRF-induced motility of both the proximal and distal colon, whereas hexamethonium, phentolamine and propranolol had no effect. CONCLUSION CRF-induced colonic motility appears to be mediated by local cholinergic signaling via muscarinic receptors. Muscarinic receptors are potential targets for counteracting CRF-induced colonic hypermotility. PMID:28638222

  19. 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.

  20. 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.

  1. Antidepressant Effects of the Muscarinic Cholinergic Receptor Antagonist Scopolamine: A Review

    PubMed Central

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

    2014-01-01

    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. PMID:23200525

  2. Pituitary-adrenal responses to oxotremorine and acute stress in male and female M1 muscarinic receptor knockout mice: comparisons to M2 muscarinic receptor knockout mice.

    PubMed

    Rhodes, M E; Rubin, R T; McKlveen, J M; Karwoski, T E; Fulton, B A; Czambel, R K

    2008-05-01

    Both within the brain and in the periphery, M(1) muscarinic receptors function primarily as postsynaptic receptors and M(2) muscarinic receptors function primarily as presynaptic autoreceptors. In addition to classical parasympathetic effectors, cholinergic stimulation of central muscarinic receptors influences the release of adrenocorticotrophic hormone (ACTH) and corticosterone. We previously reported that oxotremorine administration to male and female M(2) receptor knockout and wild-type mice increased ACTH to a significantly greater degree in knockout males compared to all other groups, and that M(2) knockout mice of both sexes were significantly more responsive to the mild stress of saline injection than were wild-type mice. These results accord with the primary function of M(2) receptors as presynaptic autoreceptors. In the present study, we explored the role of the M(1) receptor in pituitary-adrenal responses to oxotremorine and saline in male and female M(1) knockout and wild-type mice. Because these mice responded differently to the mild stress of saline injection than did the M(2) knockout and wild-type mice, we also determined hormone responses to restraint stress in both M(1) and M(2) knockout and wild-type mice. Male and female M(1) knockout and wild-type mice were equally unresponsive to the stress of saline injection. Oxotremorine increased both ACTH and corticosterone in M(1) wild-type mice to a significantly greater degree than in knockout mice. In both M(1) knockout and wild-type animals, ACTH responses were greater in males compared to females, and corticosterone responses were greater in females compared to males. Hormone responses to restraint stress were increased in M(2) knockout mice and decreased in M(1) knockout mice compared to their wild-type counterparts. These findings suggest that M(1) and M(2) muscarinic receptor subtypes differentially influence male and female pituitary-adrenal responses to cholinergic stimulation and stress. The

  3. 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

  4. Bright light does not alter muscarinic receptor binding parameters.

    PubMed

    Giroux, M L; Malatynska, E; Dilsaver, S C

    1991-03-01

    Seasonal Affective Disorders (SADs) are disorders of mood characterized by recurrent episodes of illness with a fixed relationship to season. Winter depression is characterized by recurrent onset of depression in the fall or winter followed by spontaneous recovery in the spring. This syndrome is responsive to treatment with bright light. The pathophysiology of depressive disorders may involve central muscarinic mechanisms. This possibility led to a series of physiological studies. The authors now report that contrary to expectation, treatment with bright light did not decrease the density of muscarinic receptors in either the hypothalamus or striatum.

  5. Cloning and sequence analysis of the human brain beta-adrenergic receptor. Evolutionary relationship to rodent and avian beta-receptors and porcine muscarinic receptors.

    PubMed

    Chung, F Z; Lentes, K U; Gocayne, J; Fitzgerald, M; Robinson, D; Kerlavage, A R; Fraser, C M; Venter, J C

    1987-01-26

    Two cDNA clones, lambda-CLFV-108 and lambda-CLFV-119, encoding for the beta-adrenergic receptor, have been isolated from a human brain stem cDNA library. One human genomic clone, LCV-517 (20 kb), was characterized by restriction mapping and partial sequencing. The human brain beta-receptor consists of 413 amino acids with a calculated Mr of 46480. The gene contains three potential glucocorticoid receptor-binding sites. The beta-receptor expressed in human brain was homology with rodent (88%) and avian (52%) beta-receptors and with porcine muscarinic cholinergic receptors (31%), supporting our proposal [(1984) Proc. Natl. Acad. Sci. USA 81, 272 276] that adrenergic and muscarinic cholinergic receptors are structurally related. This represents the first cloning of a neurotransmitter receptor gene from human brain.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    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 memorymore » 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.« less

  7. Involvement of decreased muscarinic receptor function in prepulse inhibition deficits in mice reared in social isolation

    PubMed Central

    Koda, K; Ago, Y; Yano, K; Nishimura, M; Kobayashi, H; Fukada, A; Takuma, K; Matsuda, T

    2011-01-01

    BACKGROUND AND PURPOSE We have previously reported that galantamine, a weak acetylcholinesterase inhibitor, improves prepulse inhibition (PPI) deficits in mice reared in social isolation. ACh receptors are involved in the underlying mechanism of PPI, but whether rearing in social isolation causes dysfunction of the cholinergic system is unknown. In this study, we examined the involvement of muscarinic receptors in the improvement of PPI deficits induced by galantamine, and whether the cholinergic system is altered in mice reared in isolation. EXPERIMENTAL APPROACH Three-week-old male ddY mice were housed in isolated cages for 6 weeks before the initiation of experiments to create PPI deficits. Cholinergic functions were determined by measuring the behavioural and neurochemical responses to nicotinic and muscarinic receptor agonists. KEY RESULTS The improvement by galantamine of social isolation-induced PPI deficits was blocked by scopolamine, a non-selective muscarinic antagonist, and telenzepine, a preferential M1 receptor antagonist. Activation of M1 receptors improved social isolation-induced PPI deficits. Social isolation did not affect choline acetyltransferase and acetylcholinesterase activities in the prefrontal cortex and hippocampus, but it reduced the locomotor-suppressive response to muscarinic agonist oxotremorine, but not to nicotine. The isolation also attenuated the M1 receptor agonist N-desmethylclozapine-induced increase in prefrontal dopamine release. CONCLUSIONS AND IMPLICATIONS Galantamine improves PPI deficits of mice reared in social isolation via activation of M1 receptors. Social isolation reduces the muscarinic, especially M1, receptor function and this is involved in PPI deficits. PMID:20958289

  8. Crosstalk between beta-2-adrenoceptor and muscarinic acetylcholine receptors in the airway.

    PubMed

    Pera, Tonio; Penn, Raymond B

    2014-06-01

    The M3 and M2 muscarinic acetylcholine receptors (mAChRs) and beta-2-adrenoceptors (β2ARs) are important regulators of airway cell function, and drugs targeting these receptors are among the first line drugs in the treatment of the obstructive lung diseases asthma and chronic obstructive lung disease (COPD). Cross-regulation or crosstalk between mAChRs and β2ARs in airway smooth muscle (ASM) helps determine the contractile state of the muscle, thus airway diameter and resistance to airflow. In this review we will detail mAChR and β2AR-signaling and crosstalk, focusing on events in the ASM cell but also addressing the function of these receptors in other cell types that impact airway physiology. We conclude by discussing how recent advances in GPCR pharmacology offer a unique opportunity to fine tune mAChR and β2AR signaling and their crosstalk, and thereby produce superior therapeutics for obstructive lung and other diseases. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Activation of muscle nicotinic acetylcholine receptor channels by nicotinic and muscarinic agonists

    PubMed Central

    Akk, Gustav; Auerbach, Anthony

    1999-01-01

    The dose-response parameters of recombinant mouse adult neuromuscular acetylcholine receptor channels (nAChR) activated by carbamylcholine, nicotine, muscarine and oxotremorine were measured. Rate constants for agonist association and dissociation, and channel opening and closing, were estimated from single-channel kinetic analysis.The dissociation equilibrium constants were (mM): ACh (0.16)carbamylcholine (5.1)>oxotremorine M (0.6)>nicotine (0.5)>muscarine (0.15).Rat neuronal α4β2 nAChR can be activated by all of the agonists. However, detailed kinetic analysis was impossible because the recordings lacked clusters representing the activity of a single receptor complex. Thus, the number of channels in the patch was unknown and the activation rate constants could not be determined.Considering both receptor affinity and agonist efficacy, muscarine and oxotremorine are significant agonists of muscle-type nAChR. The results are discussed in terms of structure-function relationships at the nAChR transmitter binding site. PMID:10602325

  10. The pharmacological rationale for combining muscarinic receptor antagonists and β-adrenoceptor agonists in the treatment of airway and bladder disease☆

    PubMed Central

    Dale, Philippa R; Cernecka, Hana; Schmidt, Martina; Dowling, Mark R; Charlton, Steven J; Pieper, Michael P; Michel, Martin C

    2014-01-01

    Muscarinic receptor antagonists and β-adrenoceptor agonists are used in the treatment of obstructive airway disease and overactive bladder syndrome. Here we review the pharmacological rationale for their combination. Muscarinic receptors and β-adrenoceptors are physiological antagonists for smooth muscle tone in airways and bladder. Muscarinic agonism may attenuate β-adrenoceptor-mediated relaxation more than other contractile stimuli. Chronic treatment with one drug class may regulate expression of the target receptor but also that of the opposing receptor. Prejunctional β2-adrenoceptors can enhance neuronal acetylcholine release. Moreover, at least in the airways, muscarinic receptors and β-adrenoceptors are expressed in different locations, indicating that only a combined modulation of both systems may cause dilatation along the entire bronchial tree. While all of these factors contribute to a rationale for a combination of muscarinic receptor antagonists and β-adrenoceptor agonists, the full value of such combination as compared to monotherapy can only be determined in clinical studies. PMID:24682092

  11. 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

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

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

    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 bindingmore » 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.« less

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

    PubMed Central

    Greig, Nigel H.; Reale, Marcella; Tata, Ada Maria

    2016-01-01

    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

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Frey, K.A.; Hichwa, R.D.; Ehrenkaufer, R.L.

    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.more » 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.« less

  15. Muscarinic Receptors as Targets for Metronomic Therapy in Breast Cancer.

    PubMed

    Sales, María Elena

    2016-01-01

    It is actually known that acetylcholine works as a signaling molecule in non-neuronal cells and tissues, in addition to its neuronal function as neurotransmitter. It can act on two types of receptors nicotinic and muscarinic receptors (mAChRs). The latter belong to the G protein coupled receptor family and there are five subtypes genetically cloned. Their activation triggers classical and non-classical intracellular signals that could be linked to the proliferation of normal and/or transformed cells. The M3 subtype was identified in different types of tumors and its stimulation with agonists triggers cell proliferation, migration, invasion and metastasis. Our laboratory has extensively investigated the expression and function of mAChRs in breast tumors from animal and human origins. We found a profuse expression of mAChRs in breast tumors, but opposite to this, an absence of these receptors in normal breast cells and tissues. The stimulation of mAChRs with the cholinergic agonist carbachol for 20 h increased tumor cell death. Moreover, the combination of subthreshold concentrations of the agonist with paclitaxel potentiates cell death. The usage of low dose chemotherapy with short drug free intervals was named metronomic therapy and it has emerged as a novel regimen for cancer treatment with very low incidence of side effects. Our work and that of others indicate that mAChRs that are over-expressed in different types of tumor cells could be a useful target for metronomic therapy in cancer treatment.

  16. Pharmacological and molecular characterization of muscarinic receptor subtypes in human esophageal smooth muscle.

    PubMed

    Preiksaitis, H G; Krysiak, P S; Chrones, T; Rajgopal, V; Laurier, L G

    2000-12-01

    Esophageal peristalsis is dependent on activation of muscarinic receptors, but little is known about the roles of specific receptor subtypes in the human esophagus. We examined muscarinic receptor expression and function in human esophageal smooth muscle obtained from patients undergoing resection for cancer. [(3)H]Quinuclidinyl benzylate (QNB)-specific binding was similar in longitudinal muscle (B(max) = 106 +/- 22 fmol/mg of protein, K(d) = 68 +/- 9 pM) and circular muscle (B(max) = 81 +/- 16 fmol/mg of protein, K(d) = 79 +/- 15 pM). Subtype-selective antagonists inhibited [(3)H]QNB similarly in muscle from both layers. Further analysis of antagonist inhibition of [(3)H]QNB binding showed a major site (60-70%) with antagonist affinity profile consistent with the M2 subtype and a second site that could not be classified. Reverse transcription-polymerase chain reaction and immunoblotting demonstrated the presence of all five known muscarinic receptor subtypes, and immunocytochemistry on acutely isolated smooth muscle cells confirmed the expression of each subtype on the muscle cells. Subtype-selective antagonists had similar inhibitory effects on carbachol-evoked contractions in longitudinal muscle and circular muscle strips with pA(2) values of 9.5 +/- 0.1 and 9.6 +/- 0.2 for 4-diphenylacetoxy-N-methylpiperidine methiodide, 7.1 +/- 0.1 and 7.0 +/- 0.2 for pirenzepine, and 6.2 +/- 0.2 and 6.4 +/- 0.2 for methoctramine, respectively. We conclude that human esophageal smooth muscle expresses muscarinic receptor subtypes M1 through M5. The antagonist sensitivity profile for muscle contraction is consistent with activation of the M3 subtype.

  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. Characterization of muscarinic receptors mediating relaxation and contraction in the rat iris dilator muscle.

    PubMed Central

    Masuda, Y; Yamahara, N S; Tanaka, M; Ryang, S; Kawai, T; Imaizumi, Y; Watanabe, M

    1995-01-01

    1. The characteristics of muscarinic receptors mediating relaxation and/or contraction in the rat iris dilator muscle were examined. 2. Relaxation was induced in a dilator muscle by application of acetylcholine (ACh) at low doses (3 microM or less) and contraction was induced by high doses. Methacholine and carbachol also showed biphasic effects similar to those of ACh; in contrast, bethanechol, arecoline, pilocarpine and McN-A-343 induced mainly relaxation but no substantial contraction. 3. After parasympathetic denervation by ciliary ganglionectomy, the relaxant response to muscarinic agonists disappeared upon nerve stimulation. Application of McN-A-343 and pilocarpine induced only small contractions in denervated dilator muscles, indicating that these are partial agonists for contraction. 4. pA2 values of pirenzepine, methoctramine, AF-DX 116, himbacine, and 4-DAMP for antagonism to pilocarpine-induced relaxation in normal dilator muscles and those for antagonism to ACh-induced contraction in denervated dilator muscles were determined. The pA2 values for antagonism to relaxation of all these antagonists were most similar to those for M3-type muscarinic receptors. 5. Although pA2 values for contraction of these antagonists, except for methoctramine, were very close to those for relaxation, contraction was not significantly antagonized by methoctramine. Contraction might be mediated by M3-like receptors which have a very low affinity for methoctramine. 6. In conclusion, ACh-induced biphasic responses in rat iris dilator muscles were clearly distinguished from each other by specific muscarinic agonists and parasympathetic denervation, whereas muscarinic receptors could not be subclassified according to the pA2 values of 5 specific antagonists only. PMID:7539696

  19. M2 and M3 muscarinic receptors are involved in enteric nerve-mediated contraction of the mouse ileum: Findings obtained with muscarinic-receptor knockout mouse.

    PubMed

    Takeuchi, Tadayoshi; Tanaka, Keisuke; Nakajima, Hidemitsu; Matsui, Minoru; Azuma, Yasu-Taka

    2007-01-01

    The involvement of muscarinic receptors in neurogenic responses of the ileum was studied in wild-type and muscarinic-receptor (M-receptor) knockout (KO) mice. Electrical field stimulation to the wild-type mouse ileum induced a biphasic response, a phasic and sustained contraction that was abolished by tetrodotoxin. The sustained contraction was prolonged for an extended period after the termination of electrical field stimulation. The phasic contraction was completely inhibited by atropine. In contrast, the sustained contraction was enhanced by atropine. Ileal strips prepared from M2-receptor KO mice exhibited a phasic contraction similar to that seen in wild-type mice and a sustained contraction that was larger than that in wild-type mice. In M3-receptor KO mice, the phasic contraction was smaller than that observed in wild-type mice. Acetylcholine exogenously administrated induced concentration-dependent contractions in strips isolated from wild-type, M2- and M3-receptor KO mice. However, contractions in M3-receptor KO mice shifted to the right. The sustained contraction was inhibited by capsaicin and neurokinin NK2 receptor antagonist, suggesting that it is mediated by substance P (SP). SP-induced contraction of M2-receptor KO mice did not differ from that of wild-type mice. SP immunoreactivity was located in enteric neurons, colocalized with M2 receptor immunoreactivity. These results suggest that atropine-sensitive phasic contraction is mainly mediated via the M3 receptor, and SP-mediated sustained contraction is negatively regulated by the M2 receptor at a presynaptic level.

  20. The effects of abnormalities of glucose homeostasis on the expression and binding of muscarinic receptors in cerebral cortex of rats.

    PubMed

    Sherin, Antony; Peeyush, Kumar T; Naijil, George; Nandhu, Mohan Sobhana; Jayanarayanan, Sadanandan; Jes, Paul; Paulose, Cheramadathikudiyil Skaria

    2011-01-25

    Glucose homeostasis in humans is an important factor for the functioning of nervous system. Both hypo and hyperglycemia contributes to neuronal functional deficit. In the present study, effect of insulin induced hypoglycemia and streptozotocin induced diabetes on muscarinic receptor binding, cholinergic enzymes; AChE, ChAT expression and GLUT3 in the cerebral cortex of experimental rats were analysed. Total muscarinic, muscarinic M(1) receptor showed a significant decrease and muscarinic M(3) receptor subtype showed a significant increased binding in the cerebral cortex of hypoglycemic rats compared to diabetic and control. Real-Time PCR analysis of muscarinic M(1), M(3) receptor subtypes confirmed the receptor binding studies. Immunohistochemistry of muscarinic M(1), M(3) receptors using specific antibodies were also carried out. AChE and GLUT3 expression up regulated and ChAT expression down regulated in hypoglycemic rats compared to diabetic and control rats. Our results showed that hypo/hyperglycemia caused impaired glucose transport in neuronal cells as shown by altered expression of GLUT3. Increased AChE and decreased ChAT expression is suggested to alter cortical acetylcholine metabolism in experimental rats along with altered muscarinic receptor binding in hypo/hyperglycemic rats, impair cholinergic transmission, which subsequently lead to cholinergic dysfunction thereby causing learning and memory deficits. We observed a prominent cholinergic functional disturbance in hypoglycemic condition than in hyperglycemia. Hypoglycemia exacerbated the neurochemical changes in cerebral cortex induced by hyperglycemia. These findings have implications for both therapy and identification of causes contributing to neuronal dysfunction in diabetes. Copyright © 2010 Elsevier B.V. All rights reserved.

  1. Novel oxotremorine-related heterocyclic derivatives: Synthesis and in vitro pharmacology at the muscarinic receptor subtypes.

    PubMed

    Dallanoce, Clelia; De Amici, Marco; Barocelli, Elisabetta; Bertoni, Simona; Roth, Bryan L; Ernsberger, Paul; De Micheli, Carlo

    2007-12-15

    A set of novel heterocyclic ligands (6-27) structurally related to Oxotremorine 2 was designed, synthesized and tested at muscarinic receptor subtypes (mAChRs). In the binding experiments at cloned human receptors (hm1-5), compounds 7 and 15 evidenced a remarkable affinity and selectivity for the hm2 subtype. The in vitro functional assays, performed on a selected group of derivatives at M(1), M(2), and M(3) tissue preparations, singled out the 3-butynyloxy-5-methylisoxazole trimethylammonium salt 7 as a potent unselective muscarinic agonist [pEC(50): 7.40 (M(1)), 8.18 (M(2)), and 8.14 (M(3))], whereas its 5-phenyl analogue 12 behaved as a muscarinic antagonist, slightly selective for the M(1) subtype [pK(B): 6.88 (M(1)), 5.95 (M(2)), 5.53 (M(3))]. Moreover, the functional data put in evidence that the presence of the piperidine ring may generate a functional selectivity, e.g., an M(1) antagonist/M(2) partial agonist/M(3) full agonist profile (compound 21), at variance with the corresponding quaternary ammonium salt (compound 22) which behaved as a muscarinic agonist at all M(1-3) receptors, with an appreciable selectivity for the cardiac M(2) receptors.

  2. M1 muscarinic receptor facilitates cognitive function by interplay with AMPA receptor GluA1 subunit.

    PubMed

    Zhao, Lan-Xue; Ge, Yan-Hui; Xiong, Cai-Hong; Tang, Ling; Yan, Ying-Hui; Law, Ping-Yee; Qiu, Yu; Chen, Hong-Zhuan

    2018-03-06

    M1 muscarinic acetylcholine receptors (M1 mAChRs) are the most abundant muscarinic receptors in the hippocampus and have been shown to have procognitive effects. AMPA receptors (AMPARs), an important subtype of ionotropic glutamate receptors, are key components in neurocognitive networks. However, the role of AMPARs in procognitive effects of M1 mAChRs and how M1 mAChRs affect the function of AMPARs remain poorly understood. Here, we found that basal expression of GluA1, a subunit of AMPARs, and its phosphorylation at Ser845 were maintained by M1 mAChR activity. Activation of M1 mAChRs promoted membrane insertion of GluA1, especially to postsynaptic densities. Impairment of hippocampus-dependent learning and memory by antagonism of M1 mAChRs paralleled the reduction of GluA1 expression, and improvement of learning and memory by activation of M1 mAChRs was accompanied by the synaptic insertion of GluA1 and its increased phosphorylation at Ser845. Furthermore, abrogation of phosphorylation of Ser845 residue of GluA1 ablated M1 mAChR-mediated improvement of learning and memory. Taken together, these results show a functional correlation of M1 mAChRs and GluA1 and the essential role of GluA1 in M1 mAChR-mediated cognitive improvement.-Zhao, L.-X., Ge, Y.-H., Xiong, C.-H., Tang, L., Yan, Y.-H., Law, P.-Y., Qiu, Y., Chen, H.-Z. M1 muscarinic receptor facilitates cognitive function by interplay with AMPA receptor GluA1 subunit.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

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

    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.more » 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.« less

  4. Crystal structures of the M 1 and M 4 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

  5. Crystal structures of the M 1 and M 4 muscarinic acetylcholine receptors

    DOE Office of Scientific and Technical Information (OSTI.GOV)

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

    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

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

    PubMed

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

    2004-07-13

    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. 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. 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.

  7. Carbachol dimers as homobivalent modulators of muscarinic receptors.

    PubMed

    Matucci, Rosanna; Nesi, Marta; Martino, Maria Vittoria; Bellucci, Cristina; Manetti, Dina; Ciuti, Elisa; Mazzolari, Angelica; Dei, Silvia; Guandalini, Luca; Teodori, Elisabetta; Vistoli, Giulio; Romanelli, Maria Novella

    2016-05-15

    A series of homodimers of the well-known cholinergic agonist carbachol have been synthesized, showing the two agonist units symmetrically connected through a methylene chain of variable length. The new compounds have been tested on the five cloned muscarinic receptors (hM1-5) expressed in CHO cells by means of equilibrium binding studies, showing an increase in affinity by rising the number of methylene units up to 7 and 9. Functional experiments on guinea-pig ileum and assessment of ERK1/2 phosphorylation on hM1, hM2 and hM3 on CHO cells have shown that the new compounds are endowed with muscarinic antagonistic properties. Kinetic binding studies have revealed that some of the tested compounds are able to slow the rate of dissociation of NMS, suggesting a bitopic behavior. Docking simulations, performed on the hM1 and hM2 receptors, give a sound rationalization of the experimental data revealing how these compounds are able to interact with both orthosteric and allosteric binding sites depending on the length of their connecting chain. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Cooperativity and oligomeric status of cardiac muscarinic cholinergic receptors.

    PubMed

    Park, Paul S-H; Sum, Chi Shing; Pawagi, Asha B; Wells, James W

    2002-04-30

    Muscarinic cholinergic receptors can appear to be more numerous when labeled by [(3)H]quinuclidinylbenzilate (QNB) than by N-[(3)H]methylscopolamine (NMS). The nature of the implied heterogeneity has been studied with M(2) receptors in detergent-solubilized extracts of porcine atria. The relative capacity for [(3)H]NMS and [(3)H]QNB was about 1 in digitonin-cholate, 0.56 in cholate-NaCl, and 0.44 in Lubrol-PX. Adding digitonin to extracts in cholate-NaCl increased the absolute capacity for both radioligands, and the relative capacity increased to near 1. The latency cannot be attributed to a chemically impure radioligand, instability of the receptor, an irreversible effect of NMS, or a failure to reach equilibrium. Binding at near-saturating concentrations of [(3)H]QNB in cholate-NaCl or Lubrol-PX was blocked fully by unlabeled NMS, which therefore appeared to inhibit noncompetitively at sites inaccessible to radiolabeled NMS. Such an effect is inconsistent with the notion of functionally distinct, noninterconverting, and mutually independent sites. Both the noncompetitive effect of NMS on [(3)H]QNB and the shortfall in capacity for [(3)H]NMS can be described quantitatively in terms of cooperative interactions within a receptor that is at least tetravalent; no comparable agreement is possible with a receptor that is only di- or trivalent. The M(2) muscarinic receptor therefore appears to comprise at least four interacting sites, presumably within a tetramer or larger array, and ligands appear to bind in a cooperative manner under at least some conditions.

  9. 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

  10. Mutation analysis of the muscarinic cholinergic receptor genes in isolated growth hormone deficiency type IB.

    PubMed

    Mohamadi, Ali; Martari, Marco; Holladay, Cindy D; Phillips, John A; Mullis, Primus E; Salvatori, Roberto

    2009-07-01

    Isolated GH deficiency (IGHD) is familial in 5-30% of patients. The most frequent form (IGHD-IB) has autosomal recessive inheritance, and it is known that it can be caused by mutations in the GHRH receptor (GHRHR) gene or in the GH gene. However, most forms of IGHD-IB have an unknown genetic cause. In normal subjects, muscarinic cholinergic stimulation causes an increase in pituitary GH release, whereas its blockade has the opposite effect, suggesting that a muscarinic acetylcholine receptor (mAchR) is involved in stimulating GH secretion. Five types of mAchR (M(1)-M(5)) exist. A transgenic mouse in which the function of the M(3) receptor was selectively ablated in the central nervous system has isolated GH deficiency similar to animals with defective GHRH or GHRHR gene. We hypothesized that mAchR mutations may cause a subset of familial IGHD. After confirming the expression of M(1)-M(5) receptor mRNA in human hypothalamus, we analyzed the index cases of 39 families with IGHD-IB for mutations in the genes encoding for the five receptors. Coding sequences for each of the five mAchRs were subjected to direct sequencing. In one family, an affected member was homozygous for a M(3) change in codon 65 that replaces valine with isoleucine (V65I). The V65I receptor was expressed in CHO cells where it had normal ability to transmit methacholine signaling. mAchR mutations are absent or rare (less than 2.6%) in familial IGHD type IB.

  11. Desensitization and Down Regulation of Muscarinic Acetylcholine Receptors

    DTIC Science & Technology

    1988-06-22

    function, in vitro. This technique offers an easy method to obtain intact differentiated brain cells with minimal diffusion barriers. Preincubation of...neuroblastoma cells (clone NIE- 115 ). This treatment demonstrated that the muscarinic receptors in this neuronal clone can be divided into two types; one...mouse neuroblastoma NlE- 115 cells, and in other tissues, mediated an increase in phosphoinositide hydrolysis. Diacylglycerol is one of the important

  12. Adenosine receptors and muscarinic receptors cooperate in acetylcholine release modulation in the neuromuscular synapse.

    PubMed

    Santafe, M M; Priego, M; Obis, T; Garcia, N; Tomàs, M; Lanuza, M A; Tomàs, J

    2015-07-01

    Adenosine receptors (ARs) are present in the motor terminals at the mouse neuromuscular junction. ARs and the presynaptic muscarinic acetylcholine receptors (mAChRs) share the functional control of the neuromuscular junction. We analysed their mutual interaction in transmitter release modulation. In electrophysiological experiments with unaltered synaptic transmission (muscles paralysed by blocking the voltage-dependent sodium channel of the muscle cells with μ-conotoxin GIIIB), we found that: (i) a collaborative action between different AR subtypes reduced synaptic depression at a moderate activity level (40 Hz); (ii) at high activity levels (100 Hz), endogenous adenosine production in the synaptic cleft was sufficient to reduce depression through A1 -type receptors (A1 Rs) and A2 A-type receptors (A2 A Rs); (iii) when the non-metabolizable 2-chloroadenosine (CADO) agonist was used, both the quantal content and depression were reduced; (iv) the protective effect of CADO on depression was mediated by A1 Rs, whereas A2 A Rs seemed to modulate A1 Rs; (v) ARs and mAChRs absolutely depended upon each other for the modulation of evoked and spontaneous acetylcholine release in basal conditions and in experimental conditions with CADO stimulation; (vi) the purinergic and muscarinic mechanisms cooperated in the control of depression by sharing a common pathway although the purinergic control was more powerful than the muscarinic control; and (vii) the imbalance of the ARs created by using subtype-selective and non-selective inhibitory and stimulatory agents uncoupled protein kinase C from evoked transmitter release. In summary, ARs (A1 Rs, A2 A Rs) and mAChRs (M1 , M2 ) cooperated in the control of activity-dependent synaptic depression and may share a common protein kinase C pathway. © 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  13. Elimination of GRK2 from cholinergic neurons reduces behavioral sensitivity to muscarinic receptor activation.

    PubMed

    Daigle, Tanya L; Caron, Marc G

    2012-08-15

    Although G-protein-coupled receptor kinase 2 (GRK2) is the most widely studied member of a family of kinases that has been shown to exert powerful influences on a variety of G-protein-coupled receptors, its role in the brain remains largely unknown. Here we report the localization of GRK2 in the mouse brain and generate novel conditional knock-out (KO) mice to assess the physiological importance of this kinase in cholinergic neurons. Mice with the selective deletion of GRK2 in this cell population (ChAT(IRES-cre)Grk2(f/f) KO mice) exhibit reduced behavioral responsiveness to challenge with oxotremorine-M (Oxo-M), a nonselective muscarinic acetylcholine receptor agonist. Specifically, Oxo-M-induced hypothermia, hypolocomotion, and salivation were markedly reduced in these animals, while analgesic responses were unaltered. In contrast, we found that GRK2 deficiency in cholinergic neurons does not alter cocaine-induced psychomotor activation, behavioral sensitization, or conditioned place preference. These results demonstrate that the elimination of GRK2 in cholinergic neurons reduces sensitivity to select muscarinic-mediated behaviors, while dopaminergic effects remain intact and further suggests that GRK2 may selectively impair muscarinic acetylcholine receptor-mediated function in vivo.

  14. Colonic inflammation increases the contribution of muscarinic M2 receptors to carbachol-induced contraction of the rat colon.

    PubMed

    Jragh, Dina M; Khan, Islam; Oriowo, Mabayoje A

    2011-01-01

    Carbachol-induced contraction of the rat colon is impaired in rats with trinitrobenzene sulfonic acid (TNBS)-induced colitis. The main objective of this study was to examine the effect of colitis on the expression and function of muscarinic (M) receptor subtypes in the rat colon. Rats (n = 80) were treated with TNBS and used 5 days later for measurement of contractility, myeloperoxidase activity, histology and expression of muscarinic receptor isoforms using Western blot analysis. Carbachol produced concentration-dependent contractions of colonic segments from control (n = 40) and TNBS-treated (n = 40) rats with no significant difference in potency. However, the maximum response to carbachol was significantly reduced in colon segments of TNBS-treated rats. The selective muscarinic receptor antagonists 4-diphenylacetoxy-N-methyl piperidine (4-DAMP, M(3)), pirenzepine (M(1)) and methoctramine (M(2)) antagonized carbachol-induced contraction in control (9.1 ± 0.1, 6.7 ± 0.3 and 6.0 ± 0.1, respectively) and TNBS-treated rats (9.2 ± 0.2, 6.9 ± 0.2, 6.7 ± 0.2). The -logK(B) values in control rats are consistent with an action of carbachol on muscarinic M(3) receptors. There was no significant difference in -logK(B) values for 4-DAMP and pirenzepine in control and TNBS-treated rats, but methoctramine was fivefold more potent in TNBS-treated rats, possibly indicating an increased contribution of muscarinic M(2) receptors to carbachol-induced contraction in the inflamed colon. The expression of M(2) receptors was also significantly increased in colon segments from TNBS-treated rats, confirming the increased role of muscarinic M(2) receptors in the inflamed colon. The data show that while only M(3) receptors appeared to mediate carbachol-induced contraction in control segments, expression of both M(2) and M(3) receptors was increased in the inflamed rat colon. Copyright © 2011 S. Karger AG, Basel.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

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

    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, secretionmore » 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.« less

  16. Primary structure of rat cardiac beta-adrenergic and muscarinic cholinergic receptors obtained by automated DNA sequence analysis: further evidence for a multigene family.

    PubMed Central

    Gocayne, J; Robinson, D A; FitzGerald, M G; Chung, F Z; Kerlavage, A R; Lentes, K U; Lai, J; Wang, C D; Fraser, C M; Venter, J C

    1987-01-01

    Two cDNA clones, lambda RHM-MF and lambda RHB-DAR, encoding the muscarinic cholinergic receptor and the beta-adrenergic receptor, respectively, have been isolated from a rat heart cDNA library. The cDNA clones were characterized by restriction mapping and automated DNA sequence analysis utilizing fluorescent dye primers. The rat heart muscarinic receptor consists of 466 amino acids and has a calculated molecular weight of 51,543. The rat heart beta-adrenergic receptor consists of 418 amino acids and has a calculated molecular weight of 46,890. The two cardiac receptors have substantial amino acid homology (27.2% identity, 50.6% with favored substitutions). The rat cardiac beta receptor has 88.0% homology (92.5% with favored substitutions) with the human brain beta receptor and the rat cardiac muscarinic receptor has 94.6% homology (97.6% with favored substitutions) with the porcine cardiac muscarinic receptor. The muscarinic cholinergic and beta-adrenergic receptors appear to be as conserved as hemoglobin and cytochrome c but less conserved than histones and are clearly members of a multigene family. These data support our hypothesis, based upon biochemical and immunological evidence, that suggests considerable structural homology and evolutionary conservation between adrenergic and muscarinic cholinergic receptors. To our knowledge, this is the first report utilizing automated DNA sequence analysis to determine the structure of a gene. Images PMID:2825184

  17. Primary structure of rat cardiac beta-adrenergic and muscarinic cholinergic receptors obtained by automated DNA sequence analysis: further evidence for a multigene family.

    PubMed

    Gocayne, J; Robinson, D A; FitzGerald, M G; Chung, F Z; Kerlavage, A R; Lentes, K U; Lai, J; Wang, C D; Fraser, C M; Venter, J C

    1987-12-01

    Two cDNA clones, lambda RHM-MF and lambda RHB-DAR, encoding the muscarinic cholinergic receptor and the beta-adrenergic receptor, respectively, have been isolated from a rat heart cDNA library. The cDNA clones were characterized by restriction mapping and automated DNA sequence analysis utilizing fluorescent dye primers. The rat heart muscarinic receptor consists of 466 amino acids and has a calculated molecular weight of 51,543. The rat heart beta-adrenergic receptor consists of 418 amino acids and has a calculated molecular weight of 46,890. The two cardiac receptors have substantial amino acid homology (27.2% identity, 50.6% with favored substitutions). The rat cardiac beta receptor has 88.0% homology (92.5% with favored substitutions) with the human brain beta receptor and the rat cardiac muscarinic receptor has 94.6% homology (97.6% with favored substitutions) with the porcine cardiac muscarinic receptor. The muscarinic cholinergic and beta-adrenergic receptors appear to be as conserved as hemoglobin and cytochrome c but less conserved than histones and are clearly members of a multigene family. These data support our hypothesis, based upon biochemical and immunological evidence, that suggests considerable structural homology and evolutionary conservation between adrenergic and muscarinic cholinergic receptors. To our knowledge, this is the first report utilizing automated DNA sequence analysis to determine the structure of a gene.

  18. 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…

  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. Conformational changes in the M2 muscarinic receptor induced by membrane voltage and agonist binding

    PubMed Central

    Navarro-Polanco, Ricardo A; Galindo, Eloy G Moreno; Ferrer-Villada, Tania; Arias, Marcelo; Rigby, J Ryan; Sánchez-Chapula, José A; Tristani-Firouzi, Martin

    2011-01-01

    Abstract The ability to sense transmembrane voltage is a central feature of many membrane proteins, most notably voltage-gated ion channels. Gating current measurements provide valuable information on protein conformational changes induced by voltage. The recent observation that muscarinic G-protein-coupled receptors (GPCRs) generate gating currents confirms their intrinsic capacity to sense the membrane electrical field. Here, we studied the effect of voltage on agonist activation of M2 muscarinic receptors (M2R) in atrial myocytes and how agonist binding alters M2R gating currents. Membrane depolarization decreased the potency of acetylcholine (ACh), but increased the potency and efficacy of pilocarpine (Pilo), as measured by ACh-activated K+ current, IKACh. Voltage-induced conformational changes in M2R were modified in a ligand-selective manner: ACh reduced gating charge displacement while Pilo increased the amount of charge displaced. Thus, these ligands manifest opposite voltage-dependent IKACh modulation and exert opposite effects on M2R gating charge displacement. Finally, mutations in the putative ligand binding site perturbed the movement of the M2R voltage sensor. Our data suggest that changes in voltage induce conformational changes in the ligand binding site that alter the agonist–receptor interaction in a ligand-dependent manner. Voltage-dependent GPCR modulation has important implications for cellular signalling in excitable tissues. Gating current measurement allows for the tracking of subtle conformational changes in the receptor that accompany agonist binding and changes in membrane voltage. PMID:21282291

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

    PubMed

    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, M 1 -, M 2 - and M 4 -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 M 1 , M 2 and TrkB signaling individually increase axonal loss rate around P9. Here we show that although the M 1 and TrkB receptors cooperate and add their respective individual effects to increase axonal elimination rate even more, the effect of the M 2 receptor is largely independent of both M 1 and TrkB receptors. Thus both, cooperative and non-cooperative signaling mechanisms contribute to developmental synapse elimination.

  2. 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

  3. Ventricular, but not atrial, M2-muscarinic receptors increase in the canine pacing-overdrive model of heart failure.

    PubMed

    Wilkinson, M; Giles, A; Armour, J A; Cardinal, R

    1996-01-01

    To investigate the effects of heart failure induced by chronic rapid ventricular pacing (six weeks) on canine atrial and ventricular muscarinic receptors. Dogs (n = 4) were fitted with a bipolar pacing electrode connected to a Medtronic pacemaker set at 240 stimuli/min. Pacing was maintained for six weeks. Tissue samples obtained from the left atrium and ventral wall of the left ventricle were frozen at -70 degrees C. Control tissue was obtained from normal dogs (n = 6) following anesthesia and thoracotomy. M2-muscarinic receptors were characterized and quantified in tissue micropunches using the hydrophilic ligand [3H] N-methyl-scopolamine (NMS). Cardiac tissue bound [3H] NMS with the specificity of an M2 subtype. Tachycardia-induced heart failure did not affect atrial muscarinic receptors but signify left ventricular myocytes (control 160.0 +/- 10.0 fmol/mg protein versus heart failure 245.0 +/- 25.0 fmol/mg protein; P < 0.01). Canine ventricular muscarinic receptors display a specificity for the M2 subtype. In contrast to previous work, tachycardia-induced heart failure was accompanied by an increase (+ 53%) in ventricular, but not atrial, M2 receptors compared with normal dogs.

  4. Gender dependent contribution of muscarinic receptors in memory retrieval under sub-chronic stress.

    PubMed

    Rashid, Habiba; Ahmed, Touqeer

    2018-05-15

    Stress induces retrograde amnesia in humans and rodents. Muscarinic antagonism under normal physiological conditions causes gender dependent impairment in episodic memory retrieval. We aimed to explore the gender dependent role of muscarinic receptors in memory retrieval under sub-chronic stress condition. Male and female mice were trained for Morris water maze test and contextual fear conditioning, followed by 3 h restraint stress per day for five days. Stress was either given alone or in combination with a daily subcutaneous injection of scopolamine (1 mg/kg) or donepezil (1 mg/kg). Control mice were given saline without any stress. Sub-chronic stress (induced for five days) impaired spatial memory retrieval in males (P < 0.005) but not in females (P > 0.05). Stress induced spatial memory recall deficit in male mice was independent of muscarinic receptor activity (P > 0.05). However, stress induced contextual fear memory recall impairment was reversed by donepezil treatment in male (P < 0.005) and female (P < 0.0001) mice. These findings suggest that differential role of muscarinic activity in retrieving different types of memories under stress depends on gender of subjects. Copyright © 2018 Elsevier B.V. All rights reserved.

  5. Pirenzepine binding to membrane-bound, solubilized and purified muscarinic receptor subtypes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    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 themore » 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.« less

  6. Cholinergic nicotinic and muscarinic receptors in dementia of Alzheimer, Parkinson and Lewy body types.

    PubMed

    Perry, E K; Smith, C J; Court, J A; Perry, R H

    1990-01-01

    Cholinergic nicotinic and muscarinic receptor binding were measured in post mortem human brain tissue, using low (nM) concentrations of (3H)-nicotine to detect predominately the high affinity nicotinic site and (3H)-N-methylscopolamine in the presence and absence of 3 x 10(-4) M carbachol to measure both the low and high affinity agonist subtypes of the muscarinic receptor group. Consistent with most previous reports, the nicotinic but not muscarinic binding was reduced in the different forms of dementia associated with cortical cholinergic deficits, including Alzheimer's and Parkinson's disease, senile dementia of Lewy body type (SDLT) and Down's syndrome (over 50 years). Analysis of (3H)-nicotine binding displaced by a range of carbachol concentrations (10(-9)-10(-3) M) indicated 2 binding sites for nicotine and that the high affinity rather than low affinity site was reduced in Alzheimer's disease. In all 3 cortical areas investigated (temporal, parietal and occipital) there were increases in the low affinity muscarinic site in Parkinson's disease and SDLT but not Alzheimer's disease or middle-aged Down's syndrome. This observation raised the question of whether the presence of neurofibrillary tangles (evident in the latter but not former 2 disorders) is incompatible with denervation-induced muscarinic supersensitivity in cholinoceptive neurons which include cortical pyramids generally affeted by tangle formation.

  7. Comparative Effects of Oral Chlorpyrifos Exposure on Cholinesterase Activity and Muscarinic Receptor Binding in Neonatal and Adult Rat Heart

    PubMed Central

    Howard, Marcia D.; Mirajkar, Nikita; Karanth, Subramanya; Pope, Carey N.

    2010-01-01

    Organophosphorus (OP) pesticides elicit acute toxicity by inhibiting acetylcholinesterase (AChE), the enzyme responsible for inactivating acetylcholine (ACh) at cholinergic synapses. A number of OP toxicants have also been reported to interact directly with muscarinic receptors, in particular the M2 muscarinic subtype. Parasympathetic innervation to the heart primarily regulates cardiac function by activating M2 receptors in the sinus node, atrial-ventricular node and conducting tissues. Thus, OP insecticides can potentially influence cardiac function in a receptor–mediated manner indirectly by inhibiting acetylcholinesterase and directly by binding to muscarinic M2 receptors. Young animals are generally more sensitive than adults to the acute toxicity of OP insecticides and age related differences in potency of direct binding to muscarinic receptors by some OP toxicants have been reported. We thus compared the effects of the common OP insecticide chlorpyrifos (CPF) on functional signs of toxicity and cardiac ChE activity and muscarinic receptor binding in neonatal and adult rats. Dosages were based on acute lethality (i.e., 0.5 and 1 × LD10: neonates, 7.5 and 15 mg/kg; adults, 68 and 136 mg/kg). Dose- and time-related changes in body weight and cholinergic signs of toxicity (involuntary movements) were noted in both age groups. With 1 × LD10, relatively similar maximal reductions in ChE activity (95%) and muscarinic receptor binding (≈ 30%) were noted, but receptor binding reductions appeared earlier in adults and were more prolonged in neonates. In vitro inhibition studies indicated that ChE in neonatal tissues was markedly more sensitive to inhibition by the active metabolite of chlorpyrifos (i.e., chlorpyrifos oxon, CPO) than enzyme in adult tissues (IC50 values: neonates, 17 nM; adults, 200 nM). Chelation of free calcium with EDTA had relatively little effect on in vitro cholinesterase inhibition, suggesting that differential A-esterase activity was not

  8. Coupling of g proteins to reconstituted monomers and tetramers of the M2 muscarinic receptor.

    PubMed

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

    2014-08-29

    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-[(3)H]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. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Carbachol induces Ca(2+)-dependent contraction via muscarinic M2 and M3 receptors in rat intestinal subepithelial myofibroblasts.

    PubMed

    Iwanaga, Koichi; Murata, Takahisa; Okada, Muneyoshi; Hori, Masatoshi; Ozaki, Hiroshi

    2009-07-01

    Intestinal myofibroblasts (IMFs) that exist adjacent to the basement membrane of intestines have contractility and contribute to physical barriers of the intestine. Nerve endings distribute adjacent to IMFs, suggesting neurotransmitters may influence IMFs motility; however, there is no direct evidence showing the interaction. Here, we isolated IMFs from rat colon and investigated the effect of acetylcholine on IMFs contractility. In the collagen gel contraction assay, carbachol (1 - 10 microM) and the muscarinic receptor agonist bethanechol (30 - 300 microM) dose-dependently induced IMFs contraction. Pretreatment with the muscarinic receptor antagonist atropine (1 - 10 nM) inhibited carbachol-induced contraction. In RT-PCR, mRNA expression of all muscarinic receptor subtypes (M(1) - M(5)) was detected in IMFs. Subsequently we found pretreatment with the muscarinic M(2) receptor antagonist 11-([2-[(diethylamino)methyl]-1-piperdinyl]acetyl)-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one (AF-DX116) (10 and 30 nM) or the muscarinic M(3) receptor antagonist 4-diphenylacetoxy-N-methyl-piperidine (4-DAMP) (3 and 10 nM) dose-dependently inhibited carbachol-induced contraction. In Ca(2+) measurement, 1 - 10 microM carbachol and 30 - 300 microM bethanechol elevated the intracellular Ca(2+) concentration ([Ca(2+)](i)) in IMFs. Atropine (10 nM) eliminated carbachol-induced [Ca(2+)](i) elevation. The Ca(2+)-channel blocker LaCl(3) (3 microM) abolished carbachol-induced [Ca(2+)](i) elevation and contraction. Furthermore, AF-DX116 and 4-DAMP dose-dependently inhibited the carbachol-induced [Ca(2+)](i) elevation. These observations suggest that acetylcholine elicits Ca(2+)-dependent IMF contraction through muscarinic M(2) and M(3) receptors.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    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 withmore » 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.« less

  11. Muscarinic receptors mediate cold stress-induced detrusor overactivity in type 2 diabetes mellitus rats.

    PubMed

    Imamura, Tetsuya; Ishizuka, Osamu; Ogawa, Teruyuki; Yamagishi, Takahiro; Yokoyama, Hitoshi; Minagawa, Tomonori; Nakazawa, Masaki; Gautam, Sudha Silwal; Nishizawa, Osamu

    2014-10-01

    This study determined if muscarinic receptors could mediate the cold stress-induced detrusor overactivity induced in type 2 diabetes mellitus rats. Ten-week-old female Goto-Kakizaki diabetic rats (n = 12) and Wister Kyoto non-diabetic rats (n = 12) were maintained on a high-fat diet for 4 weeks. Cystometric investigations of the unanesthetized rats were carried out at room temperature (27 ± 2°C) for 20 min. They were intravenously administered imidafenacin (0.3 mg/kg, n = 6) or vehicle (n = 6). After 5 min, the rats were transferred to a low temperature (4 ± 2°C) for 40 min where the cystometry was continued. The rats were then returned to room temperature for the final cystometric measurements. Afterwards, expressions of bladder muscarinic receptor M3 and M2 messenger ribonucleic acids and proteins were assessed by reverse transcription polymerase chain reaction and immunohistochemistry. In non-diabetic Wister Kyoto rats, imidafenacin did not reduce cold stress-induced detrusor overactivity. In diabetic Goto-Kakizaki rats, just after transfer to a low temperature, the cold stress-induced detrusor overactivity in imidafenacin-treated rats was reduced compared with vehicle-treated rats. Within the urinary bladders, the ratio of M3 to M2 receptor messenger ribonucleic acid in the diabetic Goto-Kakizaki rats was significantly higher than that of the non-diabetic Wister Kyoto rats. The proportion of muscarinic M3 receptor-positive area within the detrusor in diabetic Goto-Kakizaki rats was also significantly higher than that in non-diabetic Wister Kyoto rats. Imidafenacin partially inhibits cold stress-induced detrusor overactivity in diabetic Goto-Kakizaki rats. In this animal model, muscarinic M3 receptors partially mediate cold stress-induced detrusor overactivity. © 2014 The Japanese Urological Association.

  12. 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.

  13. Functional acetylcholine muscarinic receptor subtypes in human brain microcirculation: identification and cellular localization.

    PubMed

    Elhusseiny, A; Cohen, Z; Olivier, A; Stanimirović, D B; Hamel, E

    1999-07-01

    Acetylcholine is an important regulator of local cerebral blood flow. There is, however, limited information available on the possible sites of action of this neurotransmitter on brain intraparenchymal microvessels. In this study, a combination of molecular and functional approaches was used to identify which of the five muscarinic acetylcholine receptors (mAChR) are present in human brain microvessels and their intimately associated astroglial cells. Microvessel and capillary fractions isolated from human cerebral cortex were found by reverse transcriptase-polymerase chain reaction to express m2, m3, and, occasionally, m1 and m5 receptor subtypes. To localize these receptors to a specific cellular compartment of the vessel wall, cultures of human brain microvascular endothelial and smooth muscle cells were used, together with cultured human brain astrocytes. Endothelial cells invariably expressed m2 and m5 receptors, and occasionally the m1 receptor; smooth muscle cells exhibited messages for all except the m4 mAChR subtypes, whereas messages for all five muscarinic receptors were identified in astrocytes. In all three cell types studied, acetylcholine induced a pirenzepine-sensitive increase (62% to 176%, P<0.05 to 0.01) in inositol trisphosphate, suggesting functional coupling of m1, m3, or m5 mAChR to a phospholipase C signaling cascade. Similarly, coupling of m2 or m4 mAChR to adenylate cyclase inhibition in endothelial cells and astrocytes, but not in smooth muscle cells, was demonstrated by the ability of carbachol to significantly reduce (44% to 50%, P<0.05 to 0.01) the forskolin-stimulated increase in cAMP levels. This effect was reversed by the mAChR antagonist AFDX 384. The results indicate that microvessels are able to respond to neurally released acetylcholine and that mAChR, distributed in different vascular and astroglial compartments, could regulate cortical perfusion and, possibly, blood-brain barrier permeability, functions that could become

  14. 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. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  15. Synergistic Action of Presynaptic Muscarinic Acetylcholine Receptors and Adenosine Receptors in Developmental Axonal Competition at the Neuromuscular Junction.

    PubMed

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

    2016-01-01

    The development of the nervous system involves the initial overproduction of synapses, which promotes connectivity. Hebbian competition between axons with different activities leads to the loss of roughly half of the overproduced elements and this refines connectivity. We used quantitative immunohistochemistry to investigate, in the postnatal day 7 (P7) to P9 neuromuscular junctions, the involvement of muscarinic receptors (muscarinic acetylcholine autoreceptors and the M1, M2, and M4 subtypes) and adenosine receptors (A1 and A2A subtypes) in the control of axonal elimination after the mouse levator auris longus muscle had been exposed to selective antagonists in vivo. In a previous study we analyzed the role of each of the individual receptors. Here we investigate the additive or occlusive effects of their inhibitors and thus the existence of synergistic activity between the receptors. The main results show that the A2A, M1, M4, and A1 receptors (in this order of ability) delayed axonal elimination at P7. M4 produces some occlusion of the M1 pathway and some addition to the A1 pathway, which suggests that they cooperate. M2 receptors may modulate (by allowing a permissive action) the other receptors, mainly M4 and A1. The continued action of these receptors (now including M2 but not M4) finally promotes axonal loss at P9. All 4 receptors (M2, M1, A1, and A2A, in this order of ability) are necessary. The M4 receptor (which in itself does not affect axon loss) seems to modulate the other receptors. We found a synergistic action between the M1, A1, and A2A receptors, which show an additive effect, whereas the potent M2 effect is largely independent of the other receptors (though can be modulated by M4). At P9, there is a full mutual dependence between the A1 and A2A receptors in regulating axon loss. In summary, postnatal axonal elimination is a regulated multireceptor mechanism that involves the cooperation of several muscarinic and adenosine receptor subtypes.

  16. Muscarinic acetylcholine M4 receptors play a critical role in oxotremorine-induced DARPP-32 phosphorylation at threonine 75 in isolated medium spiny neurons.

    PubMed

    Liu, Liqun; Huang, Yuqi; Huang, Qing; Zhao, Zhe; Yu, Jianqiang; Wang, Liyun

    2017-05-01

    Dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) play essential roles in dopamine (DA) transmission in the striatum. It is suggested that a link exists between muscarinic acetylcholine receptors (mAChRs) and DA/DARPP-32 signaling, but the molecular mechanisms mediating this relationship have not been elucidated. The predominant mAChRs subtypes in the striatum are M 1 and M 4 . In this study, we investigated the functions of these two receptors, particularly M 4 , in regulating cAMP production and DARPP-32 phosphorylation in rat striatal medium spiny neurons (MSNs). We used time-resolved fluorescence resonance energy transfer, immunofluorescence confocal microscopy, and western blot assays. In cultured intact MSNs, we confirmed that muscarinic M 1 and M 4 receptors were highly expressed. Notably, M 4 receptors were co-expressed with D 1 receptors in only a portion of the cultured MSNs. The nonselective muscarinic agonist oxotremorine M (OX) slightly enhanced cAMP production, but this effect was independent of M 1 or M 4 receptors. However, OX directly participated in DARPP-32 phosphorylation, phosphorylating DARPP-32 at Thr75 (the CDK5 site) and concomitantly de-phosphorylating DARPP-32 at Thr34 (the PKA site) in virtually cultured MSNs, whereas APO phosphorylated DARPP-32 at both Thr34 and Thr75. The OX-induced time-dependent increase in DARPP-32 phosphorylation at Thr75 was accompanied by increased p35 and CDK5 activity. Specifically, elevated immunoreactivity for phospho-DARPP-32-Thr75 and p35 was detected in M 4 receptor-expressing MSNs. Both genetic knockdown and pharmacologic inhibition of M 4 receptors with MT3, an M 4 receptor-selective antagonist, decreased the OX-induced DARPP-32-Thr75 phosphorylation in MSNs. These results indicate that the M 4 muscarinic receptor plays a critical role in modulating phosphorylation of DARPP-32-Thr75 in MSNs. The results suggest that M 4 receptor activation acts antagonistically with dopamine D 1 -like

  17. 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.

  18. Altered trafficking and unfolded protein response induction as a result of M3 muscarinic receptor impaired N-glycosylation.

    PubMed

    Romero-Fernandez, Wilber; Borroto-Escuela, Dasiel O; Alea, Mileidys Perez; Garcia-Mesa, Yoelvis; Garriga, Pere

    2011-12-01

    The human M(3) muscarinic acetylcholine receptor is present in both the central and peripheral nervous system, and it is involved in the pathophysiology of several neurodegenerative and autoimmune diseases. We suggested a possible N-glycosylation map for the M(3) muscarinic receptor expressed in COS-7 cells. Here, we examined the role that N-linked glycans play in the folding and in the cell surface trafficking of this receptor. The five potential asparagine-linked glycosylation sites in the muscarinic receptor were mutated and transiently expressed in COS-7 cells. The elimination of N-glycan attachment sites did not affect the cellular expression levels of the receptor. However, proper receptor localization to the plasma membrane was affected as suggested by reduced [(3)H]-N-methylscopolamine binding. Confocal microscopy confirmed this observation and showed that the nonglycosylated receptor was primarily localized in the intracellular compartments. The mutant variant showed an increase in phosphorylation of the α-subunit of eukaryote initiation factor 2, and other well-known endoplasmic reticulum stress markers of the unfolded protein response pathway, which further supports the proposal of the improper intracellular accumulation of the nonglycosylated receptor. The receptor devoid of glycans showed more susceptibility to events that culminate in apoptosis reducing cell viability. Our findings suggest up-regulation of pro-apoptotic Bax protein, down-regulation of anti-apoptotic Bcl-2, and cleavage of caspase-3 effectors. Collectively, our data provide experimental evidence of the critical role that N-glycan chains play in determining muscarinic receptor distribution, localization, as well as cell integrity. © The Author 2011. Published by Oxford University Press. All rights reserved.

  19. Functional significance of muscarinic receptor expression within the proximal and distal rat vagina.

    PubMed

    Basha, Maureen; Labelle, Edward F; Northington, Gina M; Wang, Tanchun; Wein, Alan J; Chacko, Samuel

    2009-11-01

    Information regarding the role of cholinergic nerves in mediating vaginal smooth muscle contraction is sparse, and in vitro studies of the effects of muscarinic agonists on vaginal smooth muscle are discrepant. The goal of this study was to determine the expression of muscarinic receptors in the vaginal wall of the rat. In addition, we sought to determine the effect of the muscarinic receptor agonist carbachol on contractility and inositol phosphate production of the proximal and distal rat vaginal muscularis. RT-PCR analysis indicated that both M(2) and M(3) receptor transcripts were expressed within the proximal and distal rat vagina. Carbachol dose-dependently (10(-7)-10(-4) M) contracted the rat vaginal muscularis with a greater maximal contractile response in the proximal vagina (P < 0.01) compared with the distal vagina. The contractile responses of the rat vaginal muscularis to carbachol were dose dependently inhibited by the M(3) antagonist para-fluoro-hexahydrosiladefenidol, and a pK(B) of 7.78 and 7.95 was calculated for the proximal and distal vagina, respectively. Inositol phosphate production was significantly increased in both regions of the vagina following 20-min exposure to 50 muM carbachol with higher levels detected in the proximal vagina compared with the distal (P < 0.05). Preliminary experiments indicated the presence of M(2) and M(3) receptors in the human vaginal muscularis as well as contraction of human vaginal muscularis to carbachol, indicating that our animal studies are relevant to human tissue. Our results provide strong evidence for the functional significance of M(3) receptor expression in the vaginal muscularis.

  20. Functional significance of muscarinic receptor expression within the proximal and distal rat vagina

    PubMed Central

    Basha, Maureen; LaBelle, Edward F.; Northington, Gina M.; Wang, Tanchun; Wein, Alan J.

    2009-01-01

    Information regarding the role of cholinergic nerves in mediating vaginal smooth muscle contraction is sparse, and in vitro studies of the effects of muscarinic agonists on vaginal smooth muscle are discrepant. The goal of this study was to determine the expression of muscarinic receptors in the vaginal wall of the rat. In addition, we sought to determine the effect of the muscarinic receptor agonist carbachol on contractility and inositol phosphate production of the proximal and distal rat vaginal muscularis. RT-PCR analysis indicated that both M2 and M3 receptor transcripts were expressed within the proximal and distal rat vagina. Carbachol dose-dependently (10−7–10−4 M) contracted the rat vaginal muscularis with a greater maximal contractile response in the proximal vagina (P < 0.01) compared with the distal vagina. The contractile responses of the rat vaginal muscularis to carbachol were dose dependently inhibited by the M3 antagonist para-fluoro-hexahydrosiladefenidol, and a pKB of 7.78 and 7.95 was calculated for the proximal and distal vagina, respectively. Inositol phosphate production was significantly increased in both regions of the vagina following 20-min exposure to 50 μM carbachol with higher levels detected in the proximal vagina compared with the distal (P < 0.05). Preliminary experiments indicated the presence of M2 and M3 receptors in the human vaginal muscularis as well as contraction of human vaginal muscularis to carbachol, indicating that our animal studies are relevant to human tissue. Our results provide strong evidence for the functional significance of M3 receptor expression in the vaginal muscularis. PMID:19741053

  1. Effects of fenoterol on beta-adrenoceptor and muscarinic M2 receptor function in bovine tracheal smooth muscle.

    PubMed

    De Vries, B; Roffel, A F; Kooistra, J M; Meurs, H; Zaagsma, J

    2001-05-11

    Prolonged (18 h) incubation of isolated bovine tracheal smooth muscle with the beta2-adrenoceptor agonist fenoterol (10 microM) induced desensitization of isoprenaline-induced adenylyl cyclase activity in bovine tracheal smooth muscle membranes, characterized by a 25% decrease in maximal effect (Emax) (P < 0.05), while the sensitivity to the agonist (pEC50) was unchanged. The Emax value of isoprenaline-induced smooth muscle relaxation of submaximal methacholine-induced contractile tones was similarly reduced by about 25% (P < 0.001), while the pEC50 value was diminished by 1.0 log unit (P < 0.001). As determined by 30 microM gallamine-induced muscarinic M2 receptor antagonism and pertussis toxin-induced inactivation of G(i alpha), muscarinic M2 receptor-mediated functional antagonism did not play a role in isoprenaline-induced relaxation of bovine tracheal smooth muscle contracted by methacholine, both in control and in 18-h fenoterol-treated tissue. In line with these observations, we found no enhanced muscarinic M2 receptor-mediated inhibition of 1 microM forskolin-stimulated adenylyl cyclase activity after 18-h fenoterol treatment. These data indicate that 18-h fenoterol treatment of bovine tracheal smooth muscle induces beta2-adrenoceptor desensitization and reduced functional antagonism of methacholine-induced contraction by beta-adrenoceptor agonists, without a change of muscarinic M2 receptor function.

  2. Volatile anesthetics interfere with muscarinic receptor-g protein interactions in rat heart

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Anthony, B.L.

    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 frommore » 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.« less

  3. The Cu-Zn superoxide dismutase (SOD1) inhibits ERK phosphorylation by muscarinic receptor modulation in rat pituitary GH3 cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Secondo, Agnese; De Mizio, Mariarosaria; Zirpoli, Laura

    The Cu-Zn superoxide dismutase (SOD1) belongs to a family of isoenzymes that are able to dismutate the oxygen superoxide in hydrogen peroxide and molecular oxygen. This enzyme is secreted by many cellular lines and it is also released trough a calcium-dependent depolarization mechanism involving SNARE protein SNAP 25. Using rat pituitary GH3 cells that express muscarinic receptors we found that SOD1 inhibits P-ERK1/2 pathway trough an interaction with muscarinic M1 receptor. This effect is strengthened by oxotremorine, a muscarinic M agonist and partially reverted by pyrenzepine, an antagonist of M1 receptor; moreover this effect is independent from increased intracellular calciummore » concentration induced by SOD1. Finally, P-ERK1/2 inhibition was accompanied by the reduction of GH3 cell proliferation. These data indicate that SOD1 beside the well studied antioxidant properties can be considered as a neuromodulator able to affect mitogen-activated protein kinase in rat pituitary cells trough a M1 muscarinic receptor.« less

  4. Muscarinic Acetylcholine Receptors Act in Synergy to Facilitate Learning and Memory

    ERIC Educational Resources Information Center

    Leaderbrand, Katherine; Chen, Helen J.; Corcoran, Kevin A.; Guedea, Anita L.; Jovasevic, Vladimir; Wess, Jurgen; Radulovic, Jelena

    2016-01-01

    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…

  5. M1-Muscarinic Receptors Promote Fear Memory Consolidation via Phospholipase C and the M-Current

    PubMed Central

    Young, Matthew B.

    2014-01-01

    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. PMID:24478341

  6. 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.

  7. Muscarinic and alpha 1-adrenergic receptor binding characteristics of saw palmetto extract in rat lower urinary tract.

    PubMed

    Suzuki, Mayumi; Oki, Tomomi; Sugiyama, Tomomi; Umegaki, Keizo; Uchida, Shinya; Yamada, Shizuo

    2007-06-01

    To elucidate the in vitro and ex vivo effects of saw palmetto extract (SPE) on autonomic receptors in the rat lower urinary tract. The in vitro binding affinities for alpha 1-adrenergic, muscarinic, and purinergic receptors in the rat prostate and bladder were measured by radioligand binding assays. Rats received vehicle or SPE (0.6 to 60 mg/kg/day) orally for 4 weeks, and alpha 1-adrenergic and muscarinic receptor binding in tissues of these rats were measured. Saw palmetto extract inhibited specific binding of [3H]prazosin and [N-methyl-3H]scopolamine methyl chloride (NMS) but not alpha, beta-methylene adenosine triphosphate [2,8-(3)H]tetrasodium salt in the rat prostate and bladder. The binding activity of SPE for muscarinic receptors was four times greater than that for alpha 1-adrenergic receptors. Scatchard analysis revealed that SPE significantly reduced the maximal number of binding sites (Bmax) for each radioligand in the prostate and bladder under in vitro condition. Repeated oral administration of SPE to rats brought about significant alteration in Bmax for prostatic [3H]prazosin binding and for bladder [3H]NMS binding. Such alteration by SPE was selective to the receptors in the lower urinary tract. Saw palmetto extract exerts significant binding activity on autonomic receptors in the lower urinary tract under in vitro and in vivo conditions.

  8. Signaling Pathways of Purinergic Receptors and Their Interactions with Cholinergic and Adrenergic Pathways in the Lacrimal Gland

    PubMed Central

    Hodges, Robin R.

    2016-01-01

    Abstract Purpose: Purinergic receptors play a key role in the function of the lacrimal gland (LG) as P1 purinergic receptors A1, A2A, and A2B, P2X1–7 receptors, and many of the P2Y receptors are expressed. Methods: This review examines the current knowledge of purinergic receptors in the LG as well as the signaling pathways activated by these receptors. Results: These receptors are expressed on the acinar, ductal, and myoepithelial cells. Considerable crosstalk exists between the pathways activated by P2X7 receptors with those activated by M3 muscarinic or α1D adrenergic receptors. The mechanism of the crosstalk between P2X7 and M3 muscarinic receptors differs from that of the crosstalk between P2X7 and α1D adrenergic receptors. Conclusions: Understanding purinergic receptors and how they modulate protein secretion could play a key role in normal and pathological responses of the LG. PMID:27463365

  9. 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.

  10. Evidence of paired M2 muscarinic receptors

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Potter, L.T.; Ballesteros, L.A.; Bichajian, L.H.

    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 withmore » 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.« less

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    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 manifestationmore » of mACh receptor supersensitivity and that sustained DA receptor subsensitivity may play some role in the alterations of mACh receptor sensitivity.« less

  12. Mechanism of the G-protein mimetic nanobody binding to a muscarinic G-protein-coupled receptor.

    PubMed

    Miao, Yinglong; McCammon, J Andrew

    2018-03-20

    Protein-protein binding is key in cellular signaling processes. Molecular dynamics (MD) simulations of protein-protein binding, however, are challenging due to limited timescales. In particular, binding of the medically important G-protein-coupled receptors (GPCRs) with intracellular signaling proteins has not been simulated with MD to date. Here, we report a successful simulation of the binding of a G-protein mimetic nanobody to the M 2 muscarinic GPCR using the robust Gaussian accelerated MD (GaMD) method. Through long-timescale GaMD simulations over 4,500 ns, the nanobody was observed to bind the receptor intracellular G-protein-coupling site, with a minimum rmsd of 2.48 Å in the nanobody core domain compared with the X-ray structure. Binding of the nanobody allosterically closed the orthosteric ligand-binding pocket, being consistent with the recent experimental finding. In the absence of nanobody binding, the receptor orthosteric pocket sampled open and fully open conformations. The GaMD simulations revealed two low-energy intermediate states during nanobody binding to the M 2 receptor. The flexible receptor intracellular loops contribute remarkable electrostatic, polar, and hydrophobic residue interactions in recognition and binding of the nanobody. These simulations provided important insights into the mechanism of GPCR-nanobody binding and demonstrated the applicability of GaMD in modeling dynamic protein-protein interactions.

  13. Myotropic Effects of Cholinergic Muscarinic Agonists and Antagonists in the Beetle Tenebrio molitor L.

    PubMed

    Chowanski, Szymon; Rosinski, Grzegorz

    2017-01-01

    In mammals, the cholinergic nervous system plays a crucial role in neuronal regulation of physiological processes. It acts on cells by two types of receptors - nicotinic and muscarinic receptors. Both signal transmission pathways also operate in the central and peripheral cholinergic nervous system of insects. In our pharmacological experiments, we studied the effects of two muscarinic agonists (carbachol, pilocarpine) and two muscarinic antagonists (atropine, scopolamine) on the muscle contractile activity of visceral organs in the beetle, Tenebrio molitor. Both antagonists, when injected to haemolymph at concentration 10-5 M, caused delayed and prolonged cardioinhibitory effects on heart contractility in ortho- and antidromic phases of heart activity in T. molitor pupa what was observed as negative chrono- and inotropic effects. Agonist of muscarinic receptors - carbachol evoked opposite effect and increased contraction rate but only in antidromic phase. Pilocarpine, the second agonist induced weak negative chronotropic effects in the antiand orthodromic phases of heart activity. However, neither agonists had an effect on semi-isolated beetle heart in vitro. Only atropine at the highest tested concentrations slightly decreased the frequency of myocardial contractions. These suggest the regulation of heart activity by muscarinic system indirectly. The tested compounds also affected the contractility of the oviduct and hindgut, but the responses of these organs were varied and depended on the concentration of the applied compounds. These pharmacological experiments suggest the possible modulation of insect visceral muscle contractility by the cholinergic nervous system and indirectly indicate the presence of muscarinic receptor(s) in the visceral organs of the beetle T. molitor. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  14. 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

  15. Muscarinic receptor M1 and M2 subtypes in the human eye: QNB, pirenzipine, oxotremorine, and AFDX-116 in vitro autoradiography.

    PubMed Central

    Gupta, N; McAllister, R; Drance, S M; Rootman, J; Cynader, M S

    1994-01-01

    Muscarinic cholinergic agents are used to lower intraocular pressure in the medical management of glaucoma and subtypes of muscarinic receptors have now been recognised in many tissues including the eye. To localise muscarinic receptors and their M1 and M2 subtypes in the human eye, in vitro ligand binding and autoradiographic techniques with densitometric quantitation on postmortem eye sections were used. As ligands, [3H] quinuclydinyl benzylate (QNB) (non-subtype specific muscarinic antagonist), [3H]pirenzipine (M1 antagonist), [3H]oxotremorine (M2 muscarinic agonist), [3H]AFDX-116(11[(2[diethylaminomethyl]1-piperidinyl)acetyl]5 , 11dihydro-6H-pyrido [2,3b][1,4]benzodiazepine-6-one) (M2 antagonist) were studied. Specific binding sites for QNB, pirenzipine, and AFDX-116 were localised in the entire ciliary muscle, the iris, and ciliary epithelium. [3H]oxotremorine localised only in the longitudinal portion of the ciliary muscle, and additionally, was not localised in the iris or ciliary epithelium. These results suggest that oxotremorine, by binding selectively to receptors on the longitudinal ciliary muscle and inducing its contraction, may modulate outflow facility independently from accommodation and miosis. Images PMID:7918268

  16. Anticholinesterase Effects on Number and Function of Brain Muscarinic Receptors and Central Cholinergic Activity: Drug Intervention.

    DTIC Science & Technology

    1986-04-11

    Leudee NWI 5th England 18. brain;striatum;hippocampus;cortex;brainstem;rat;hydrophilic drugs;hydrophobic drugs; oxotremorine ;physostigmine;choline...challenged with oxotremorine , marked cross-tolerance to the ACh-increasing action f the muscarinic receptor agonist was induced in both striatum and...responses except for slight tremor.A Fig. 2 shows the dose-response curves of the muscarinic agonists oxotremorine and the butynyl base, McN-A-343, a

  17. 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

  18. New analogues of oxotremorine and oxotremorine-M: estimation of their in vitro affinity and efficacy at muscarinic receptor subtypes.

    PubMed

    Barocelli, E; Ballabeni, V; Bertoni, S; Dallanoce, C; De Amici, M; De Micheli, C; Impicciatore, M

    2000-06-30

    Two subsets of tertiary amines (1a-6a) and methiodides (1b-6b) with a structural resemblance to oxotremorine and oxotremorine-M were tested at rabbit vas deferens (M1), guinea pig left atrium (M2), guinea pig ileum and urinary bladder (M3) muscarinic receptor subtypes. The pharmacological profile of the derivatives under study has been discussed by evaluating their potency, affinity and efficacy as well as the regional differences in muscarinic receptor occupancy.

  19. 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.

  20. LY2033298, a positive allosteric modulator at muscarinic M₄ receptors, enhances inhibition by oxotremorine of light-induced phase shifts in hamster circadian activity rhythms.

    PubMed

    Gannon, Robert L; Millan, Mark J

    2012-11-01

    Entrainment of circadian rhythms to the light-dark cycle is essential for restorative sleep, and abnormal sleep timing is implicated in central nervous system (CNS) disorders like depression, schizophrenia, and Alzheimer's disease. Many transmitters, including acetylcholine, that exerts its actions via muscarinic receptors modulate the suprachiasmatic nucleus, the master pacemaker. Since positive allosteric modulators of muscarinic M(4) receptors are candidates for treatment of mood and cognitive deficits of CNS disorders, it is important to evaluate their circadian actions. The effects of intraperitoneally applied muscarinic agents on circadian wheel-running rhythms were measured employing hamsters, a model organism for studying activity rhythms. Systemic administration of the muscarinic receptor agonist oxotremorine (0.01-0.04 mg/kg) inhibited light-induced phase delays and advances of hamster circadian wheel-running rhythms. The M₄ positive allosteric modulator, LY2033298 (10-40 mg/kg), had no effect on light-induced phase shifts when administered alone, yet significantly enhanced (at 20 mg/kg) the inhibitory influence of oxotremorine on light-induced phase delays. In addition, the muscarinic receptor antagonist, scopolamine, which was without effect on light-induced phase shifts when administered alone (0.001-0.1 mg/kg), antagonized (at 0.1 mg/kg) the inhibitory effect of oxotremorine and LY2033298 on light-induced phase delays. These results are the first to demonstrate that systemically applied muscarinic receptor agonists modulate circadian activity rhythms, and they also reveal a specific role for M₄ receptors. It will be of importance to evaluate circadian actions of psychotropic drugs acting via M₄ receptors, since they may display beneficial properties under pathological conditions.

  1. Effect of a muscarinic M3 receptor agonist on gastric motility.

    PubMed

    Chiba, Toshimi; Kudara, Norihiko; Sato, Masaki; Inomata, Masaaki; Orii, Seishi; Suzuki, Kazuyuki

    2007-11-01

    Muscarinic M3 receptors exist in the gastrointestinal wall in humans and the muscarinic M3 agonist cevimeline hydrochloride (Evoxac) is a candidate therapeutic agent for the treatment of xerostomia in Sjögren's syndrome. However, M3 receptor agonists are not known to show efficacy for diseases associated with abnormal gastrointestinal motility. Herein the effects are reported of cevimeline on gastric motility in two patients with non-ulcer dyspepsia. The patients both received long-term proton pump inhibitor therapy for 6 months, but their symptoms persisted. Then cevimeline was administered orally for 8 weeks at 30 mg three times daily (90 mg/day) and their dyspepsia symptoms improved. Electrogastrography was performed to examine gastric motility before and after administration of the M3 agonist. The fasting or nocturnal wave rate was significantly increased after administration compared with before administration, but no significant postprandial changes were seen. No adverse effects of cevimeline were observed. This drug might be a candidate therapeutic agent for non-ulcer dyspepsia. Because its postprandial effects on gastrointestinal motility are unclear, a dose-finding clinical study should be performed in the future.

  2. Hyperfunction of muscarinic receptor maintains long-term memory in 5-HT4 receptor knock-out mice.

    PubMed

    Segu, Luis; Lecomte, Marie-José; Wolff, Mathieu; Santamaria, Julie; Hen, René; Dumuis, Aline; Berrard, Sylvie; Bockaert, Joël; Buhot, Marie-Christine; Compan, Valérie

    2010-03-04

    Patients suffering from dementia of Alzheimer's type express less serotonin 4 receptors (5-HTR(4)), but whether an absence of these receptors modifies learning and memory is unexplored. In the spatial version of the Morris water maze, we show that 5-HTR(4) knock-out (KO) and wild-type (WT) mice performed similarly for spatial learning, short- and long-term retention. Since 5-HTR(4) control mnesic abilities, we tested whether cholinergic system had circumvented the absence of 5-HTR(4). Inactivating muscarinic receptor with scopolamine, at an ineffective dose (0.8 mg/kg) to alter memory in WT mice, decreased long-term but not short-term memory of 5-HTR(4) KO mice. Other changes included decreases in the activity of choline acetyltransferase (ChAT), the required enzyme for acetylcholine synthesis, in the septum and the dorsal hippocampus in 5-HTR(4) KO under baseline conditions. Training- and scopolamine-induced increase and decrease, respectively in ChAT activity in the septum in WT mice were not detected in the 5-HTR(4) KO animals. Findings suggest that adaptive changes in cholinergic systems may circumvent the absence of 5-HTR(4) to maintain long-term memory under baseline conditions. In contrast, despite adaptive mechanisms, the absence of 5-HTR(4) aggravates scopolamine-induced memory impairments. The mechanisms whereby 5-HTR(4) mediate a tonic influence on ChAT activity and muscarinic receptors remain to be determined.

  3. A critical role for beta cell M3 muscarinic acetylcholine receptors in regulating insulin release and blood glucose homeostasis in vivo.

    PubMed

    Gautam, Dinesh; Han, Sung-Jun; Hamdan, Fadi F; Jeon, Jongrye; Li, Bo; Li, Jian Hua; Cui, Yinghong; Mears, David; Lu, Huiyan; Deng, Chuxia; Heard, Thomas; Wess, Jürgen

    2006-06-01

    One of the hallmarks of type 2 diabetes is that pancreatic beta cells fail to release sufficient amounts of insulin in the presence of elevated blood glucose levels. Insulin secretion is modulated by many hormones and neurotransmitters including acetylcholine, the major neurotransmitter of the peripheral parasympathetic nervous system. The physiological role of muscarinic acetylcholine receptors expressed by pancreatic beta cells remains unclear at present. Here, we demonstrate that mutant mice selectively lacking the M3 muscarinic acetylcholine receptor subtype in pancreatic beta cells display impaired glucose tolerance and greatly reduced insulin release. In contrast, transgenic mice selectively overexpressing M3 receptors in pancreatic beta cells show a profound increase in glucose tolerance and insulin release. Moreover, these mutant mice are resistant to diet-induced glucose intolerance and hyperglycemia. These findings indicate that beta cell M3 muscarinic receptors play a key role in maintaining proper insulin release and glucose homeostasis.

  4. Altered coupling of muscarinic acetylcholine receptors in pancreatic acinar carcinoma of rat

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Chien, J.L.; Warren, J.R.

    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 highmore » 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.« less

  5. Biological sex influences learning strategy preference and muscarinic receptor binding in specific brain regions of prepubertal rats.

    PubMed

    Grissom, Elin M; Hawley, Wayne R; Hodges, Kelly S; Fawcett-Patel, Jessica M; Dohanich, Gary P

    2013-04-01

    According to the theory of multiple memory systems, specific brain regions interact to determine how the locations of goals are learned when rodents navigate a spatial environment. A number of factors influence the type of strategy used by rodents to remember the location of a given goal in space, including the biological sex of the learner. We recently found that prior to puberty male rats preferred a striatum-dependent stimulus-response strategy over a hippocampus-dependent place strategy when solving a dual-solution task, while age-matched females showed no strategy preference. Because the cholinergic system has been implicated in learning strategy and is known to be sexually dimorphic prior to puberty, we explored the relationship between learning strategy and muscarinic receptor binding in specific brain regions of prepubertal males and female rats. We confirmed our previous finding that at 28 days of age a significantly higher proportion of prepubertal males preferred a stimulus-response learning strategy than a place strategy to solve a dual-solution visible platform water maze task. Equal proportions of prepubertal females preferred stimulus-response or place strategies. Profiles of muscarinic receptor binding as assessed by autoradiography varied according to strategy preference. Regardless of biological sex, prepubertal rats that preferred stimulus-response strategy exhibited lower ratios of muscarinic receptor binding in the hippocampus relative to the dorsolateral striatum compared to rats that preferred place strategy. Importantly, much of the variance in this ratio was related to differences in the ventral hippocampus to a greater extent than the dorsal hippocampus. The ratios of muscarinic receptors in the hippocampus relative to the basolateral amygdala also were lower in rats that preferred stimulus-response strategy over place strategy. Results confirm that learning strategy preference varies with biological sex in prepubertal rats with males

  6. An Inotropic Action Caused by Muscarinic Receptor Subtype 3 in Canine Cardiac Purkinje Fibers

    PubMed Central

    Urushidani, Tetsuro; Tachibana, Shigehiro

    2013-01-01

    Objective. The objective of this study was to investigate the inotropic mechanisms and the related muscarinic receptor subtype of acetylcholine (ACh) in canine cardiac Purkinje fibers. Materials and Methods. Isolated Purkinje fiber bundles were used for the measurement of contraction. The receptor subtype was determined using PCR and real-time PCR methods. Results. ACh evoked a biphasic response with a transient negative inotropic effect followed by a positive inotropic effect in a concentration-dependent manner. The biphasic inotropic actions of ACh were inhibited by the pretreatment with atropine. Caffeine inhibited the positive inotropic effect of ACh. ACh increased inositol-1,4,5-trisphosphate content in the Purkinje fibers, which was abolished by atropine. Muscarinic subtypes 2 (M2) and 3 (M3) mRNAs were detected in the canine Purkinje fibers albeit the amount of M3 mRNA was smaller than M2 mRNA. M1 mRNA was not detected. Conclusion. These results suggest that the positive inotropic action of ACh may be mediated by the activation of IP3 receptors through the stimulation of M3 receptors in the canine cardiac Purkinje fibers. PMID:24260719

  7. Mechanisms and roles of muscarinic activation in guinea-pig adrenal medullary cells.

    PubMed

    Inoue, Masumi; Harada, Keita; Matsuoka, Hidetada; Nakamura, Jun; Warashina, Akira

    2012-09-15

    Muscarinic receptors are expressed in the adrenal medullary (AM) cells of various mammals, but their physiological roles are controversial. Therefore, the ionic mechanism for muscarinic receptor-mediated depolarization and the role of muscarinic receptors in neuronal transmission were investigated in dissociated guinea-pig AM cells and in the perfused guinea-pig adrenal gland. Bath application of muscarine induced an inward current at -60 mV. This inward current was partially suppressed by quinine with an IC(50) of 6.1 μM. The quinine-insensitive component of muscarine-induced currents changed the polarity at -78 mV and was inhibited by bupivacaine, a TWIK-related acid-sensitive K(+) (TASK) channel inhibitor. Conversely, the current-voltage relationship for the bupivacaine-insensitive component of muscarine currents showed a reversal potential of -5 mV and a negative slope below -40 mV. External application of La(3+) had a double action on muscarine currents of both enhancement and suppression. Immunoblotting and immunocytochemistry revealed expression of TASK1 channels and cononical transient receptor potential channels 1, 4, 5, and 7 in guinea-pig AM cells. Retrograde application of atropine reversibly suppressed transsynaptically evoked catecholamine secretion from the adrenal gland. The results indicate that muscarinic receptor stimulation in guinea-pig AM cells induces depolarization through inhibition of TASK channels and activation of nonselective cation channels and that muscarinic receptors are involved in neuronal transmission from the splanchnic nerve.

  8. Mechanisms and roles of muscarinic activation in guinea-pig adrenal medullary cells

    PubMed Central

    Harada, Keita; Matsuoka, Hidetada; Nakamura, Jun; Warashina, Akira

    2012-01-01

    Muscarinic receptors are expressed in the adrenal medullary (AM) cells of various mammals, but their physiological roles are controversial. Therefore, the ionic mechanism for muscarinic receptor-mediated depolarization and the role of muscarinic receptors in neuronal transmission were investigated in dissociated guinea-pig AM cells and in the perfused guinea-pig adrenal gland. Bath application of muscarine induced an inward current at −60 mV. This inward current was partially suppressed by quinine with an IC50 of 6.1 μM. The quinine-insensitive component of muscarine-induced currents changed the polarity at −78 mV and was inhibited by bupivacaine, a TWIK-related acid-sensitive K+ (TASK) channel inhibitor. Conversely, the current-voltage relationship for the bupivacaine-insensitive component of muscarine currents showed a reversal potential of −5 mV and a negative slope below −40 mV. External application of La3+ had a double action on muscarine currents of both enhancement and suppression. Immunoblotting and immunocytochemistry revealed expression of TASK1 channels and cononical transient receptor potential channels 1, 4, 5, and 7 in guinea-pig AM cells. Retrograde application of atropine reversibly suppressed transsynaptically evoked catecholamine secretion from the adrenal gland. The results indicate that muscarinic receptor stimulation in guinea-pig AM cells induces depolarization through inhibition of TASK channels and activation of nonselective cation channels and that muscarinic receptors are involved in neuronal transmission from the splanchnic nerve. PMID:22744007

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

    PubMed Central

    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.

    2017-01-01

    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. PMID:28094765

  10. 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

  11. Stereoselective L-(3H)quinuclidinyl benzilate-binding sites in nervous tissue of Aplysia californica: evidence for muscarinic receptors

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Murray, T.F.; Mpitsos, G.J.; Siebenaller, J.F.

    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 lessmore » 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.« less

  12. Role of ventrolateral orbital cortex muscarinic and nicotinic receptors in modulation of capsaicin-induced orofacial pain-related behaviors in rats.

    PubMed

    Tamaddonfard, Esmaeal; Erfanparast, Amir; Abbas Farshid, Amir; Delkhosh-Kasmaie, Fatmeh

    2017-11-15

    Acetylcholine, as a major neurotransmitter, mediates many brain functions such as pain. This study was aimed to investigate the effects of microinjection of muscarinic and nicotinic acetylcholine receptor antagonists and agonists into the ventrolateral orbital cortex (VLOC) on capsaicin-induced orofacial nociception and subsequent hyperalgesia. The right side of VLOC was surgically implanted with a guide cannula in anaesthetized rats. Orofacial pain-related behaviors were induced by subcutaneous injection of a capsaicin solution (1.5µg/20µl) into the left vibrissa pad. The time spent face rubbing with ipsilateral forepaw and general behavior were recorded for 10min, and then mechanical hyperalgesia was determined using von Frey filaments at 15, 30, 45 and 60min post-capsaicin injection. Alone intra-VLOC microinjection of atropine (a muscarinic acetylcholine receptor antagonist) and mecamylamine (a nicotinic acetylcholine receptor antagonist) at a similar dose of 200ng/site did not alter nocifensive behavior and hyperalgesia. Microinjection of oxotremorine (a muscarinic acetylcholine receptor agonist) at doses of 50 and 100ng/site and epibatidine (a nicotinic acetylcholine receptor agonist) at doses of 12.5, 25, 50 and 100ng/site into the VLOC suppressed pain-related behaviors. Prior microinjections of 200ng/site atropine and mecamylamine (200ng/site) prevented oxotremorine (100ng/site)-, and epibatidine (100ng/site)-induced antinociception, respectively. None of the above-mentioned chemicals changed general behavior. These results showed that the VLOC muscarinic and nicotinic acetylcholine receptors might be involved in modulation of orofacial nociception and hypersensitivity. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Muscarinic receptor subtypes involved in urothelium-derived relaxatory effects in the inflamed rat urinary bladder.

    PubMed

    Andersson, M; Aronsson, P; Doufish, D; Lampert, A; Tobin, G

    2012-09-25

    Functional studies have shown altered cholinergic mechanisms in the inflamed bladder, which partly depend on muscarinic receptor-induced release of nitric oxide (NO). The current study aimed to characterize which muscarinic receptor subtypes that are involved in the regulation of the nitrergic effects in the bladder cholinergic response during cystitis. For this purpose, in vitro examinations of carbachol-evoked contractions of inflamed and normal bladder preparations were performed. The effects of antagonists with different selectivity for the receptor subtypes were assessed on intact and urothelium-denuded bladder preparations. In preparations from cyclophosphamide (CYP; in order to induce cystitis) pre-treated rats, the response to carbachol was about 75% of that of normal preparations. Removal of the urothelium or administration of a nitric oxide synthase inhibitor re-established the responses in the inflamed preparations. Administration of 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) inhibited the carbachol-induced contractile responses of preparations from CYP pre-treated rats less potently than controls. Pirenzepine and p-fluoro-hexahydro-sila-diphenidol (pFHHSiD) affected the carbachol-induced contractile responses to similar extents in preparations of CYP pre-treated and control rats. However, the Schild slopes for the three antagonists were all significantly different from unity in the preparations from CYP pre-treated rats. Again, L-NNA or removal of the urothelium eliminated any difference compared to normal preparations. This study confirms that muscarinic receptor stimulation in the inflamed rat urinary bladder induces urothelial release of NO, which counteracts detrusor contraction. Copyright © 2012 Elsevier B.V. All rights reserved.

  14. Hydrostatic pressure and muscarinic receptors are involved in the release of inflammatory cytokines in human bladder smooth muscle cells.

    PubMed

    Liang, Zhou; Xin, Wei; Qiang, Liu; Xiang, Cai; Bang-Hua, Liao; Jin, Yang; De-Yi, Luo; Hong, Li; Kun-Jie, Wang

    2017-06-01

    Abnormal intravesical pressure results in a series of pathological changes. We investigated the effects of hydrostatic pressure and muscarinic receptors on the release of inflammatory cytokines in rat and human bladder smooth muscle cells (HBSMCs). Animal model of bladder outlet obstruction was induced by urethra ligation. HBSMCs were subjected to elevated hydrostatic pressure and/or acetylcholine (Ach). Macrophage infiltration in the bladder wall was determined by immunohistochemical staining. The expression of inflammatory genes was measured by RT-PCR, ELISA and immunofluorescence. In obstructed bladder, inflammatory genes and macrophage infiltration were remarkably induced. When HBSMCs were subjected to 200-300 cm H 2 O pressure for 2-24 h in vitro, the expressions of IL-6 and RANTES were significantly increased. Hydrostatic pressure promoted the protein levels of phospho-NFκB p65 and phospho-ERK1/2 as well as muscarinic receptors. Moreover, NFκB or ERK1/2 inhibitors suppressed pressure-induced inflammatory genes mRNA. When cells were treated with 1 μM acetylcholine for 6 h, a significant increase in IL-6 mRNA expression was detected. Acetylcholine also enhanced pressure-induced phospho-NFκB p65 and IL-6 protein expression. Additionally, pressure-induced IL-6 was partially suppressed by muscarinic receptors antagonists. Hydrostatic pressure and muscarinic receptors were involved in the secretion of inflammatory cytokines in HBSMCs, indicating a pro-inflammatory effect of the two factors in the pathological process of BOO. © 2016 Wiley Periodicals, Inc.

  15. Characterization of muscarinic and P2X receptors in the urothelium and detrusor muscle of the rat bladder.

    PubMed

    Ogoda, Masaki; Ito, Yoshihiko; Fuchihata, Yusuke; Onoue, Satomi; Yamada, Shizuo

    2016-05-01

    Muscarinic and purinergic (P2X) receptors play critical roles in bladder urothelium under physiological and pathological conditions. Aim of present study was to characterize these receptors in rat bladder urothelium and detrusor muscle using selective radioligands of [N-methyl-(3)H]scopolamine methyl chloride ([(3)H]NMS) and αβ-methylene ATP [2,8-(3)H]tetrasodium salt ([(3)H]αβ-MeATP). Similar binding parameters for each radioligand were observed in urothelium and detrusor muscle. Pretreatment with N-(2-chloroethyl)-4-piperidinyl diphenylacetate (4-DAMP mustard) mustard revealed co-existence of M2 and M3 receptors, with the number of M2 receptors being larger in the urothelium and detrusor muscle. Intravesical administration of imidafenacin and Dpr-P-4 (N → O) (active metabolite of propiverine) displayed significant binding of muscarinic receptors in the urothelium and detrusor muscle. The treatment with cyclophosphamide (CYP) or resiniferatoxin (RTX) resulted in a significant decrease in maximal number of binding sites (Bmax) for [(3)H]NMS and/or [(3)H]αβ-MeATP in the urothelium and detrusor muscle. These results demonstrated that 1) pharmacological characteristics of muscarinic and P2X receptors in rat bladder urothelium were similar to those in the detrusor muscle, 2) that densities of these receptors were significantly altered by pretreatments with CYP and RTX, and 3) that these receptors may be pharmacologically affected by imidafenacin and Dpr-P-4 (N → O) which are excreted in the urine. Copyright © 2016 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

  16. Acute Effects of Muscarinic M1 Receptor Modulation on AβPP Metabolism and Amyloid-β Levels in vivo: A Microdialysis Study.

    PubMed

    Welt, Tobias; Kulic, Luka; Hoey, Sarah E; McAfoose, Jordan; Späni, Claudia; Chadha, Antonella Santuccione; Fisher, Abraham; Nitsch, Roger M

    2015-01-01

    Indirect modulation of cholinergic activity by cholinesterase inhibition is currently a widely established symptomatic treatment for Alzheimer's disease (AD). Selective activation of certain muscarinic receptor subtypes has emerged as an alternative cholinergic-based amyloid-lowering strategy for AD, as selective muscarinic M1 receptor agonists can reduce amyloid-β (Aβ) production by shifting endoproteolytic amyloid-β protein precursor (AβPP) processing toward non-amyloidogenic pathways. In this study, we addressed the hypothesis that acute stimulation of muscarinic M1 receptors can inhibit Aβ production in awake and freely moving AβPP transgenic mice. By combining intracerebral microdialysis with retrodialysis, we determined hippocampal Aβ concentrations during simultaneous pharmacological modulation of brain M1 receptor function. Infusion with a M1 receptor agonist AF102B resulted in a rapid reduction of interstitial fluid (ISF) Aβ levels while treatment with the M1 antagonist dicyclomine increased ISF Aβ levels reaching significance within 120 minutes of treatment. The reduction in Aβ levels was associated with PKCα and ERK activation resulting in increased levels of the α-secretase ADAM17 and a shift in AβPP processing toward the non-amyloidogenic processing pathway. In contrast, treatment with the M1 receptor antagonist dicyclomine caused a decrease in levels of phosphorylated ERK that was independent of PKCα, and led to an elevation of β-secretase levels associated with increased amyloidogenic AβPP processing. The results of this study demonstrate rapid effects of in vivo M1 receptor modulation on the ISF pool of Aβ and suggest that intracerebral microdialysis with retrodialysis is a useful technical approach for monitoring acute treatment effects of muscarinic receptor modulators on AβPP/Aβ metabolism.

  17. Rapid Antidepressant Actions of Scopolamine: Role of Medial Prefrontal Cortex and M1-subtype Muscarinic Acetylcholine Receptors

    PubMed Central

    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.

    2015-01-01

    Clinical studies demonstrate that scopolamine, a nonselective muscarinic acetycholine 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) in the medial prefrontal cortex (mPFC). The present study extends these findings to determine the role of the mPFC and specific muscarinic acetylcholine receptor (M-AchR) subtypes in the actions of scopolamine. 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 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 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 scopolamine or VU0255035 administration blocked the anhedonic response caused by CUS, an effect that requires 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. PMID:26102021

  18. 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. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. 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

  20. Highly specific detection of muscarinic M3 receptor, G protein interaction and intracellular trafficking in human detrusor using Proximity Ligation Assay (PLA).

    PubMed

    Berndt-Paetz, Mandy; Herbst, Luise; Weimann, Annett; Gonsior, Andreas; Stolzenburg, Jens-Uwe; Neuhaus, Jochen

    2018-05-01

    Muscarinic acetylcholine receptors (mAChRs) regulate a number of important physiological functions. Alteration of mAChR expression or function has been associated in the etiology of several pathologies including functional bladder disorders (e.g bladder pain syndrome/interstitial cystitis - BPS/IC). In a previous study we found specific mAChR expression patterns associated with BPS/IC, while correlation between protein and gene expression was lacking. Posttranslational regulatory mechanisms, e.g. altered intracellular receptor trafficking, could explain those differences. In addition, alternative G protein (GP) coupling could add to the pathophysiology via modulation of muscarinic signaling. In our proof-of-principle study, we addressed these questions in situ. We established PLA in combination with confocal laserscanning microscopy (CLSM) and 3D object reconstruction for highly specific detection and analysis of muscarinic 3 receptors (M3), G protein (GP) coupling and intracellular trafficking in human detrusor samples. Paraffin sections of formalin-fixed bladder tissue (FFPE) of BPS/IC patients receiving transurethral biopsy were examined by Cy3-PLA for M3 expression, coupling of M3 to GPs (G αq/11 , G αs , G αi ) and interaction of M3 with endocytic regulator proteins. Membranes were labeled with wheat germ agglutinin-Alexa Fluor ® 488, nuclei were stained with DAPI. Object density and co-localization were analyzed in 3D-reconstruction of high resolution confocal z-stacks. Confocal image stack processing resulted in well demarcated objects. Calculated receptor densities correlated significantly with existing confocal expression data, while significantly improved specificity of M3 detection by PLA was verified using bladder tissue samples from transgenic mice. 50-60% of the M3 receptor complexes were plasma membrane associated in human bladder detrusor. Application of PLA for M3 and GPs allowed visualization of M3-GP interactions and revealed individual GP

  1. Muscarinic cholinergic receptor binding: in vivo depiction using single photon emission computed tomography and radioiodinated quinuclidinyl benzilate

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Drayer, B.; Jaszczak, R.; Coleman, E.

    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 striatummore » 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.« less

  2. Metabolic roles of the M3 muscarinic acetylcholine receptor studied with M3 receptor mutant mice: a review.

    PubMed

    Gautam, Dinesh; Jeon, Jongrye; Li, Jian Hua; Han, Sung-Jun; Hamdan, Fadi F; Cui, Yinghong; Lu, Huiyan; Deng, Chuxia; Gavrilova, Oksana; Wess, Jürgen

    2008-01-01

    The M(3) muscarinic acetylcholine (ACh) receptor (M(3) mAChR) is expressed in many central and peripheral tissues. It is a prototypic member of the superfamily of G protein-coupled receptors and preferentially activates G proteins of the G(q) family. Recent studies involving the use of newly generated mAChR mutant mice have revealed that the M(3) mAChR plays a key role in regulating many important metabolic functions. Phenotypic analyses of mutant mice that either selectively lacked or overexpressed M(3) receptors in pancreatic beta -cells indicated that beta -cell M(3) mAChRs are essential for maintaining proper insulin release and glucose homeostasis. The experimental data also suggested that strategies aimed at enhancing signaling through beta -cell M(3) mAChRs might be beneficial for the treatment of type 2 diabetes. Recent studies with whole body M(3) mAChR knockout mice showed that the absence of M(3) receptors protected mice against various forms of experimentally or genetically induced obesity and obesity-associated metabolic deficits. Under all experimental conditions tested, M(3) receptor-deficient mice showed greatly ameliorated impairments in glucose homeostasis and insulin sensitivity, reduced food intake, and a significant elevation in basal and total energy expenditure, most likely due to increased central sympathetic outflow and increased rate of fatty acid oxidation. These findings are of potential interest for the development of novel therapeutic approaches for the treatment of obesity and associated metabolic disorders.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Anthony, B.L.

    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 affinitymore » ({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.« less

  4. M1 muscarinic receptor activation mediates cell death in M1-HEK293 cells.

    PubMed

    Graham, E Scott; Woo, Kerhan K; Aalderink, Miranda; Fry, Sandie; Greenwood, Jeffrey M; Glass, Michelle; Dragunow, Mike

    2013-01-01

    HEK293 cells have been used extensively to generate stable cell lines to study G protein-coupled receptors, such as muscarinic acetylcholine receptors (mAChRs). The activation of M1 mAChRs in various cell types in vitro has been shown to be protective. To further investigate M1 mAChR-mediated cell survival, we generated stable HEK293 cell-lines expressing the human M1 mAChR. M1 mAChRs were efficiently expressed at the cell surface and efficiently internalised within 1 h by carbachol. Carbachol also induced early signalling cascades similar to previous reports. Thus, ectopically expressed M1 receptors behaved in a similar fashion to the native receptor over short time periods of analysis. However, substantial cell death was observed in HEK293-M1 cells within 24 h after carbachol application. Death was only observed in HEK cells expressing M1 receptors and fully blocked by M1 antagonists. M1 mAChR-stimulation mediated prolonged activation of the MEK-ERK pathway and resulted in prolonged induction of the transcription factor EGR-1 (>24 h). Blockade of ERK signalling with U0126 did not reduce M1 mAChR-mediated cell-death significantly but inhibited the acute induction of EGR-1. We investigated the time-course of cell death using time-lapse microscopy and xCELLigence technology. Both revealed the M1 mAChR cytotoxicity occurs within several hours of M1 activation. The xCELLigence assay also confirmed that the ERK pathway was not involved in cell-death. Interestingly, the MEK blocker did reduce carbachol-mediated cleaved caspase 3 expression in HEK293-M1 cells. The HEK293 cell line is a widely used pharmacological tool for studying G-protein coupled receptors, including mAChRs. Our results highlight the importance of investigating the longer term fate of these cells in short term signalling studies. Identifying how and why activation of the M1 mAChR signals apoptosis in these cells may lead to a better understanding of how mAChRs regulate cell-fate decisions.

  5. Regulation of muscarinic acetylcholine receptors in cultured guinea pig pancreatic acini

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hootman, S.R.; Brown, M.E.; Williams, J.A.

    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) wasmore » 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.« less

  6. Effects of muscarinic antagonists on ZENK expression in the chicken retina.

    PubMed

    Bitzer, Michaela; Kovacs, Beatrix; Feldkaemper, Marita; Schaeffel, Frank

    2006-03-01

    Muscarinic antagonists, particularly atropine, can inhibit myopia development in several animal models and also in children. However, the biochemical basis of the inhibition of axial eye growth remains obscure, and there are doubts whether muscarinic receptors are involved at all. Experiments in chickens and monkeys have shown that the synthesis of the transcription factor ZENK, also named Egr-1, in retinal glucagon amacrine cells is strongly associated with inhibition of axial eye growth (assumed to create a STOP signal). We have tested whether the muscarinic antagonists atropine, pirenzepine, oxyphenonium, gallamine, MT-3, himbacine, and 4-DAMP can stimulate ZENK expression so that the drugs' inhibitory effect on myopia development could be explained by an enhanced STOP signal. Because it is known that intravitreal quisqualic acid (QA) eliminates most cholinergic neurons in the retina within 6 or 7 days, in a second set of experiments, we tested whether these antagonists could still stimulate ZENK production, 6 days after QA was applied. Muscarinic antagonists, injected intravitreally at various concentrations, affected ZENK synthesis in various and unpredictable ways. Pirenzepine, oxyphenonium, and MT-3 increased the proportion of glucagon cells that were ZENK-immunoreactive, whereas himbacine decreased that proportion, and gallamine and 4-DAMP had no significant effect. Atropine caused an upregulation of ZENK only if all positive amacrine and bipolar cells were counted and therefore appeared to affect primarily cells other than glucagon amacrines. The pattern of results remained unchanged after ablation of most cholinergic neurons by QA. Our results suggest that at least some muscarinic antagonists do not activate cells that synthesize ZENK when they inhibit axial eye growth. Therefore, in line with other studies they also cast doubt on the assumption that muscarinic transmission is crucial, and they suggest that muscarinic antagonists may inhibit myopia

  7. Muscarinic M4 Receptors on Cholinergic and Dopamine D1 Receptor-Expressing Neurons Have Opposing Functionality for Positive Reinforcement and Influence Impulsivity.

    PubMed

    Klawonn, Anna M; Wilhelms, Daniel B; Lindström, Sarah H; Singh, Anand Kumar; Jaarola, Maarit; Wess, Jürgen; Fritz, Michael; Engblom, David

    2018-01-01

    The neurotransmitter acetylcholine has been implicated in reward learning and drug addiction. However, the roles of the various cholinergic receptor subtypes on different neuron populations remain elusive. Here we study the function of muscarinic M4 receptors (M4Rs) in dopamine D1 receptor (D1R) expressing neurons and cholinergic neurons (expressing choline acetyltransferase; ChAT), during various reward-enforced behaviors and in a "waiting"-impulsivity test. We applied cell-type-specific gene deletions targeting M4Rs in D1RCre or ChATCre mice. Mice lacking M4Rs in D1R-neurons displayed greater cocaine seeking and drug-primed reinstatement than their littermate controls in a Pavlovian conditioned place preference (CPP) paradigm. Furthermore, the M4R-D1RCre mice initiated significantly more premature responses (PRs) in the 5-choice-serial-reaction-time-task (5CSRTT) than their littermate controls, indicating impaired waiting impulse control. In contrast, mice lacking M4Rs in cholinergic neurons did not acquire cocaine Pavlovian conditioning. The M4R-ChATCre mice were also unable to learn positive reinforcement to either natural reward or cocaine in an operant runway paradigm. Immediate early gene (IEG) expression ( cFos and FosB ) induced by repeated cocaine injections was significantly increased in the forebrain of M4R-D1RCre mice, whereas it remained normal in the M4R-ChATCre mice. Our study illustrates that muscarinic M4Rs on specific neural populations, either cholinergic or D1R-expressing, are pivotal for learning processes related to both natural reward and drugs of abuse, with opposing functionality. Furthermore, we found that neurons expressing both M4Rs and D1Rs are important for signaling impulse control.

  8. Ligands for SPECT and PET imaging of muscarinic-cholinergic receptors of the heart and brain

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    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 ofmore » 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.« less

  9. The interaction between tropomyosin-related kinase B receptors and presynaptic muscarinic receptors modulates transmitter release in adult rodent motor nerve terminals.

    PubMed

    Garcia, Neus; Tomàs, Marta; Santafé, Manel M; Besalduch, Nuria; Lanuza, Maria A; Tomàs, Josep

    2010-12-08

    The neurotrophin brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT-4) and the receptors tropomyosin-related kinase B (trkB) and p75(NTR) are present in the nerve terminals on the neuromuscular junctions (NMJs) of the levator auris longus muscle of the adult mouse. Exogenously added BDNF or NT-4 increased evoked ACh release after 3 h. This presynaptic effect (the size of the spontaneous potentials is not affected) is specific because it is not produced by neurotrophin-3 (NT-3) and is prevented by preincubation with trkB-IgG chimera or by pharmacological block of trkB [K-252a (C₂₇H₂₁N₃O₅)] or p75(NTR) [Pep5 (C₈₆H₁₁₁N₂₅O₁₉S₂] signaling. The effect of BDNF depends on the M₁ and M₂ muscarinic acetylcholine autoreceptors (mAChRs) because it is prevented by atropine, pirenzepine and methoctramine. We found that K-252a incubation reduces ACh release (~50%) in a short time (1 h), but the p75(NTR) signaling inhibitor Pep5 does not have this effect. The specificity of the K-252a blocking effect on trkB was confirmed with the anti-trkB antibody 47/trkB, which reduces evoked ACh release, like K-252a, whereas the nonpermeant tyrosine kinase blocker K-252b does not. Neither does incubation with the fusion protein trkB-IgG (to chelate endogenous BDNF/NT-4), anti-BDNF or anti-NT-4 change ACh release. Thus, the trkB receptor normally seems to be coupled to ACh release when there is no short-term local effect of neurotrophins at the NMJ. The normal function of the mAChR mechanism is a permissive prerequisite for the trkB pathway to couple to ACh release. Reciprocally, the normal function of trkB modulates M₁- and M₂-subtype muscarinic pathways.

  10. 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 M 2 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 M 2 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 M 2 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.

  11. Distinct pathways of ERK activation by the muscarinic agonists pilocarpine and carbachol in a human salivary cell line.

    PubMed

    Lin, Alan L; Zhu, Bing; Zhang, WanKe; Dang, Howard; Zhang, Bin-Xian; Katz, Michael S; Yeh, Chih-Ko

    2008-06-01

    Cholinergic-muscarinic receptor agonists are used to alleviate mouth dryness, although the cellular signals mediating the actions of these agents on salivary glands have not been identified. We examined the activation of ERK1/2 by two muscarinic agonists, pilocarpine and carbachol, in a human salivary cell line (HSY). Immunoblot analysis revealed that both agonists induced transient activation of ERK1/2. Whereas pilocarpine induced phosphorylation of the epidermal growth factor (EGF) receptor, carbachol did not. Moreover, ERK activation by pilocarpine, but not carbachol, was abolished by the EGF receptor inhibitor AG-1478. Downregulation of PKC by prolonged treatment of cells with the phorbol ester PMA diminished carbachol-induced ERK phosphorylation but had no effect on pilocarpine responsiveness. Depletion of intracellular Ca2+ ([Ca2+]i by EGTA did not affect ERK activation by either agent. In contrast to carbachol, pilocarpine did not elicit [Ca2+]i mobilization in HSY cells. Treatment of cells with the muscarinic receptor subtype 3 (M3) antagonist N-(3-chloropropyl)-4-piperidnyl diphenylacetate decreased ERK responsiveness to both agents, whereas the subtype 1 (M1) antagonist pirenzepine reduced only the carbachol response. Stimulation of ERKs by pilocarpine was also decreased by M3, but not M1, receptor small interfering RNA. The Src inhibitor PP2 blocked pilocarpine-induced ERK activation and EGF receptor phosphorylation, without affecting ERK activation by carbachol. Our results demonstrate that the actions of pilocarpine and carbachol in salivary cells are mediated through two distinct signaling mechanisms-pilocarpine acting via M3 receptors and Src-dependent transactivation of EGF receptors, and carbachol via M1/M3 receptors and PKC-converging on the ERK pathway.

  12. 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.

  13. 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...

  14. Impaired M3 and enhanced M2 muscarinic receptor contractile function in a streptozotocin model of mouse diabetic urinary bladder.

    PubMed

    Pak, K J; Ostrom, R S; Matsui, M; Ehlert, F J

    2010-05-01

    We investigated the contractile roles of M2 and M3 muscarinic receptors in urinary bladder from streptozotocin-treated mice. Wild-type and M2 muscarinic receptor knockout (M2 KO) mice were given a single injection of vehicle or streptozotocin (125 mg kg(-1)) 2-24 weeks prior to bladder assays. The effect of forskolin on contractions elicited to the muscarinic agonist, oxotremorine-M, was measured in isolated urinary bladder (intact or denuded of urothelium). Denuded urinary bladder from vehicle-treated wild-type and M2 KO mice exhibited similar contractile responses to oxotremorine-M, when contraction was normalized relative to that elicited by KCl (50 mM). Eight to 9 weeks after streptozotocin treatment, the EC(50) value of oxotremorine-M increased 3.1-fold in urinary bladder from the M2 KO mouse (N = 5) compared to wild type (N = 6; P < 0.001). Analogous changes were observed in intact bladder. In denuded urinary bladder from vehicle-treated mice, forskolin (5 microM) caused a much greater inhibition of contraction in M2 KO bladder compared to wild type. Following streptozotocin treatment, this forskolin effect increased 1.6-fold (P = 0.032). At the 20- to 24-week time point, the forskolin effect increased 1.7-fold for denuded as well as intact bladders (P = 0.036, 0.01, respectively). Although streptozotocin treatment inhibits M3 receptor-mediated contraction in denuded urinary bladder, muscarinic contractile function is maintained in wild-type bladder by enhanced M2 contractile function. M2 receptor activation opposes forskolin-induced relaxation of the urinary bladder, and this M(2) function is enhanced following streptozotocin treatment.

  15. Quantitative mRNA analysis of muscarinic acetylcholine receptors in the intestine of dairy cows with spontaneous caecal dilatation-dislocation.

    PubMed

    Ontsouka, E C; Steiner, A; Bruckmaier, R M; Blum, J W; Meylan, M

    2009-05-01

    Muscarinic receptors mediate acetylcholine-induced muscular contractions. In this study, mRNA levels of muscarinic receptor subtypes 2 and 3 (M(2) and M(3)) in the ileum, caecum, proximal loop of the ascending colon (PLAC) and external loop of the spiral colon (ELSC) were determined by quantitative polymerase chain reaction in seven cows with caecal dilatation-dislocation (CDD) and seven healthy control cows. Levels of M(2) were significantly lower in the caecum, PLAC and ELSC and levels of M(3) were significantly lower in the ileum, caecum, PLAC and ELSC of cows with CDD compared to healthy cows (P<0.05). Down-regulation of M(3) may play a role in the pathogenesis of CDD.

  16. Influence of differentiation on muscarinic receptors in N1E 115 neuroblastoma cells.

    PubMed

    Buyse, M A; Lefebvre, R A; Fraeyman, N H

    1989-01-01

    The effect of inducing morphological differentiation in N1E 115 mouse neuroblastoma cells on the number of muscarinic receptors and the ligand binding affinity was investigated using the lipophylic quinuclidinyl benzylate and the hydrophylic N-methylscopolamine as tritiated ligands. Induction of morphological differentiation was accompanied by a two- to three-fold increase of the number of receptors when assayed in a broken cell preparation; the ligand binding affinity was unaffected by differentiation. Using intact cells, this increase was not paralleled by a similar increase in binding sites accessible for N-methylscopolamine, which binds preferentially to extracellular sites.

  17. Targeting cholinesterase inhibitor poisoning with a novel blocker against both nicotinic and muscarinic receptors.

    PubMed

    Luo, Wangqian; Ge, Xulin; Cui, Wenyu; Wang, Hai

    2010-08-01

    Clinicians have been treating poisoning by acetylcholinesterase inhibitors (ChEI) for more than half a century. However, the current atropine-centered therapy still cannot protect completely against all ChEIs, and poisoning by ChEIs is fatal in more than 20% of cases. Various solutions that try to enhance atropine's antimuscarinic effects have been used, but these fail to increase the antidotal effect, and their too potent muscarinic antagonism may produce incapacitating side effects. We hypothesized that, in the treatment of ChEI poisoning, the high death rate may not be attributed to the insufficient muscarinic antagonism but to the lack of nicotinic antagonism. To test this hypothesis, we designed and synthesized benthiactzine, a drug that blocks both muscarinic acetylcholine receptors (mAChRs) and nicotinic acetylcholine receptors (nAChRs). A specific [(3)H]quinuclidinyl benzilate-binding assay showed that benthiactzine was much weaker than atropine in binding to five different mAChR subtypes or to mAChRs expressed in 14 different tissues. Electrophysiological measures were used to identify and characterize benthiactzine's antinicotinic effect on three typical neuronal nAChRs subtypes, alpha4beta2, alpha4beta4, and alpha7, which are expressed heterogenously in SH-EP1 cells. Finally, benthiactzine afforded better protection than atropine against the most lethal ChEI, VX or sarin, in a mouse model. These results indicate that the antidotal effect may not be directly related to the antidote's antimuscarinic effect and that the antinicotinic effect may provide additional protection against ChEI poisoning. This new drug may benefit future antidote discovery.

  18. Altered M(1) muscarinic acetylcholine receptor (CHRM1)-Galpha(q/11) coupling in a schizophrenia endophenotype.

    PubMed

    Salah-Uddin, Hasib; Scarr, Elizabeth; Pavey, Geoffrey; Harris, Kriss; Hagan, Jim J; Dean, Brian; Challiss, R A John; Watson, Jeannette M

    2009-08-01

    Alterations in muscarinic acetylcholine receptor (CHRM) populations have been implicated in the pathology of schizophrenia. Here we have assessed whether the receptor function of the M(1) subtype (CHRM1) is altered in a sub-population of patients with schizophrenia, defined by marked (60-80%) reductions in cortical [3H]-pirenzepine (PZP) binding, and termed 'muscarinic receptor-deficit schizophrenia' (MRDS). Using a [35S]-GTPgammaS-Galpha(q/11) immunocapture method we have assessed whether CHRM1 signalling in human cortex (Brodmann area 9 (BA9)) is altered in post mortem tissue from a MRDS group compared with a subgroup of patients with schizophrenia displaying normal PZP binding, and controls with no known history of psychiatric or neurological disorders. The CHRM agonist (oxotremorine-M) and a CHRM1-selective agonist (AC-42) increased Galpha(q/11)-[35S]-GTPgammaS binding, with AC-42 producing responses that were approximately 50% of those maximally evoked by the full agonist, oxotremorine-M, in control and subgroups of patients with schizophrenia. However, the potency of oxotremorine-M to stimulate Galpha(q/11)-[35S]-GTPgammaS binding was significantly decreased in the MRDS group (pEC(50) (M)=5.69+/-0.16) compared with the control group (6.17+/-0.10) and the non-MRDS group (6.05+/-0.07). The levels of Galpha(q/11) protein present in BA9 did not vary with diagnosis. Maximal oxotremorine-M-stimulated Galpha(q/11)-[35S]-GTPgammaS binding in BA9 membranes was significantly increased in the MRDS group compared with the control group. Similar, though non-statistically significant, trends were observed for AC-42. These data provide evidence that both orthosterically and allosterically acting CHRM agonists can stimulate a receptor-driven functional response ([35S]-GTPgammaS binding to Galpha(q/11)) in membranes prepared from post mortem human dorsolateral prefrontal cortex of patients with schizophrenia and controls . Furthermore, in a subgroup of patients with

  19. Acetylcholine modulates gamma frequency oscillations in the hippocampus by activation of muscarinic M1 receptors.

    PubMed

    Betterton, Ruth T; Broad, Lisa M; Tsaneva-Atanasova, Krasimira; Mellor, Jack R

    2017-06-01

    Modulation of gamma oscillations is important for the processing of information and the disruption of gamma oscillations is a prominent feature of schizophrenia and Alzheimer's disease. Gamma oscillations are generated by the interaction of excitatory and inhibitory neurons where their precise frequency and amplitude are controlled by the balance of excitation and inhibition. Acetylcholine enhances the intrinsic excitability of pyramidal neurons and suppresses both excitatory and inhibitory synaptic transmission, but the net modulatory effect on gamma oscillations is not known. Here, we find that the power, but not frequency, of optogenetically induced gamma oscillations in the CA3 region of mouse hippocampal slices is enhanced by low concentrations of the broad-spectrum cholinergic agonist carbachol but reduced at higher concentrations. This bidirectional modulation of gamma oscillations is replicated within a mathematical model by neuronal depolarisation, but not by reducing synaptic conductances, mimicking the effects of muscarinic M1 receptor activation. The predicted role for M1 receptors was supported experimentally; bidirectional modulation of gamma oscillations by acetylcholine was replicated by a selective M1 receptor agonist and prevented by genetic deletion of M1 receptors. These results reveal that acetylcholine release in CA3 of the hippocampus modulates gamma oscillation power but not frequency in a bidirectional and dose-dependent manner by acting primarily through muscarinic M1 receptors. © 2017 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  20. Neuronal M3 muscarinic acetylcholine receptors are essential for somatotroph proliferation and normal somatic growth.

    PubMed

    Gautam, Dinesh; Jeon, Jongrye; Starost, Matthew F; Han, Sung-Jun; Hamdan, Fadi F; Cui, Yinghong; Parlow, Albert F; Gavrilova, Oksana; Szalayova, Ildiko; Mezey, Eva; Wess, Jürgen

    2009-04-14

    The molecular pathways that promote the proliferation and maintenance of pituitary somatotrophs and other cell types of the anterior pituitary gland are not well understood at present. However, such knowledge is likely to lead to the development of novel drugs useful for the treatment of various human growth disorders. Although muscarinic cholinergic pathways have been implicated in regulating somatotroph function, the physiological relevance of this effect and the localization and nature of the receptor subtypes involved in this activity remain unclear. We report the surprising observation that mutant mice that selectively lack the M(3) muscarinic acetylcholine receptor subtype in the brain (neurons and glial cells; Br-M3-KO mice) showed a dwarf phenotype associated with a pronounced hypoplasia of the anterior pituitary gland and a marked decrease in pituitary and serum growth hormone (GH) and prolactin. Remarkably, treatment of Br-M3-KO mice with CJC-1295, a synthetic GH-releasing hormone (GHRH) analog, rescued the growth deficit displayed by Br-M3-KO mice by restoring normal pituitary size and normal serum GH and IGF-1 levels. These findings, together with results from M(3) receptor/GHRH colocalization studies and hypothalamic hormone measurements, support a model in which central (hypothalamic) M(3) receptors are required for the proper function of hypothalamic GHRH neurons. Our data reveal an unexpected and critical role for central M(3) receptors in regulating longitudinal growth by promoting the proliferation of pituitary somatotroph cells.

  1. Muscarinic Acetylcholine Receptors in Macaque V1 Are Most Frequently Expressed by Parvalbumin-Immunoreactive Neurons

    PubMed Central

    Disney, Anita A.; Aoki, Chiye

    2010-01-01

    Acetylcholine (ACh) is believed to underlie mechanisms of arousal and attention in mammals. ACh also has a demonstrated functional effect in visual cortex that is both diverse and profound. We have reported previously that cholinergic modulation in V1 of the macaque monkey is strongly targeted toward GABAergic interneurons. Here we examine the localization of m1 and m2 muscarinic receptor subtypes across subpopulations of GABAergic interneurons—identified by their expression of the calcium-binding proteins parvalbumin, calbindin, and calretinin—using dual-immunofluorescence confocal microscopy in V1 of the macaque monkey. In doing so, we find that the vast majority (87%) of parvalbumin-immunoreactive neurons express m1-type muscarinic ACh receptors. m1 receptors are also expressed by 60% of calbindin-immunoreactive neurons and 40% of calretinin-immunoreactive neurons. m2 AChRs, on the other hand, are expressed by only 31% of parvalbumin neurons, 23% of calbindin neurons, and 25% of calretinin neurons. Parvalbumin-immunoreactive cells comprise ≈75% of the inhibitory neuronal population in V1 and included in this large subpopulation are neurons known to veto and regulate the synchrony of principal cell spiking. Through the expression of m1 ACh receptors on nearly all of these PV cells, the cholinergic system avails itself of powerful control of information flow through and processing within the network of principal cells in the cortical circuit. PMID:18265004

  2. Muscarinic modulation of TREK currents in mouse sympathetic superior cervical ganglion neurons.

    PubMed

    Rivas-Ramírez, P; Cadaveira-Mosquera, A; Lamas, J A; Reboreda, A

    2015-07-01

    Muscarinic receptors play a key role in the control of neurotransmission in the autonomic ganglia, which has mainly been ascribed to the regulation of potassium M-currents and voltage-dependent calcium currents. Muscarinic agonists provoke depolarization of the membrane potential and a reduction in spike frequency adaptation in postganglionic neurons, effects that may be explained by M-current inhibition. Here, we report the presence of a riluzole-activated current (IRIL ) that flows through the TREK-2 channels, and that is also inhibited by muscarinic agonists in neurons of the mouse superior cervical ganglion (mSCG). The muscarinic agonist oxotremorine-M (Oxo-M) inhibited the IRIL by 50%, an effect that was abolished by pretreatment with atropine or pirenzepine, but was unaffected in the presence of himbacine. Moreover, these antagonists had similar effects on single-channel TREK-2 currents. IRIL inhibition was unaffected by pretreatment with pertussis toxin. The protein kinase C blocker bisindolylmaleimide did not have an effect, and neither did the inositol triphosphate antagonist 2-aminoethoxydiphenylborane. Nevertheless, the IRIL was markedly attenuated by the phospholipase C (PLC) inhibitor ET-18-OCH3. Finally, the phosphatidylinositol-3-kinase/phosphatidylinositol-4-kinase inhibitor wortmannin strongly attenuated the IRIL , whereas blocking phosphatidylinositol 4,5-bisphosphate (PIP2 ) depletion consistently prevented IRIL inhibition by Oxo-M. These results demonstrate that TREK-2 currents in mSCG neurons are inhibited by muscarinic agonists that activate M1 muscarinic receptors, reducing PIP2 levels via a PLC-dependent pathway. The similarities between the signaling pathways regulating the IRIL and the M-current in the same neurons reflect an important role of this new pathway in the control of autonomic ganglia excitability. © 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

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

    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 ofmore » 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.« less

  4. Urothelial/lamina propria spontaneous activity and the role of M3 muscarinic receptors in mediating rate responses to stretch and carbachol.

    PubMed

    Moro, Christian; Uchiyama, Jumpei; Chess-Williams, Russ

    2011-12-01

    To investigate the effects of tissue stretch and muscarinic receptor stimulation on the spontaneous activity of the urothelium/lamina propria and identify the specific receptor subtype mediating these responses. Isolated strips of porcine urothelium with lamina propria were set up for in vitro recording of contractile activity. Muscarinic receptor subtype-selective antagonists were used to identify the receptors influencing the contractile rate responses to stretch and stimulation with carbachol. Isolated strips of urothelium with lamina propria developed spontaneous contractions (3.7 cycles/min) that were unaffected by tetrodotoxin, Nω-nitro-L-arginine, or indomethacin. Carbachol (1 μM) increased the spontaneous contractile rate of these tissue strips by 122% ± 27% (P < .001). These responses were significantly depressed in the presence of the M3-selective muscarinic antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (10-30 nM) but were not affected by the M1-selective antagonist pirenzepine (30-100 nM) or the M2-selective antagonist methoctramine (0.1-1 μM). Stretching of the tissue also caused an increase in the spontaneous contractile rate, and these responses were abolished by atropine (1 μM) and low concentrations of 4-diphenylacetoxy-N-methylpiperidine methiodide (10 nM). Darifenacin, oxybutynin, tolterodine, and solifenacin (1 μM) all significantly depressed the frequency responses to carbachol (1 μM). The urothelium with the lamina propria exhibits a spontaneous contractile activity that is increased during stretch. The mechanism appears to involve endogenous acetylcholine release acting on M3 muscarinic receptors. Anticholinergic drugs used clinically depress the responses of these tissues, and this mechanism might represent an additional site of action for these drugs in the treatment of bladder overactivity. Copyright © 2011 Elsevier Inc. All rights reserved.

  5. The Effects of Nicotinic and Muscarinic Receptor Activation on Patch-Clamped Cells in the Optic Tectum of Rana Pipiens

    PubMed Central

    Yu, C.-J.; Debski, E. A.

    2008-01-01

    Both nicotinic and muscarinic cholinergic receptors are present in the optic tectum. To begin to understand how the activation of these receptors affects visual activity patterns, we have determined the types of physiological responses induced by their activation. Using tectal brain slices from the leopard frog, we found that application of nicotine (100 μM) evoked long-lasting responses in 60% of patch-clamped tectal cells. Thirty percent of these responses consisted of an increase in spontaneous postsynaptic currents (sPSCs) and had both a glutamatergic and GABAergic component as determined by the use of 6-cyano-7-nitroquinoxaline-2,3-dione (50 μM) and bicuculline (25 μM), respectively. Remaining response types consisted of an inward membrane current (16%) and an increase in sPSCs combined with an inward membrane current (14%). All responses could be elicited in the presence of tetrodotoxin (0.5 μM). Muscarinic receptor-mediated responses, induced by carbachol (100 μM) application after nicotinic receptor desensitization, produced responses in 70% of tectal cells. In contrast to responses elicited by nicotine, carbachol-induced responses could be evoked multiple times without significant decrement. Responses consisted of either an outward current (57%), a decrease in sPSCs (5%) or an increase in sPSCs, with (almost 6%) or without (almost 3%) an outward current. The response elicited by carbachol was not predicted by the response of the cell to nicotine. Our results suggest that nicotinic receptors are found predominantly at presynaptic locations in the optic tectum while muscarinic receptors are most often present at postsynaptic sites. We conclude that both of these receptor types could substantially modulate visual activity by changing either the input to tectal neurons or the level of their response to that input. PMID:12676145

  6. The effects of nicotinic and muscarinic receptor activation on patch-clamped cells in the optic tectum of Rana pipiens.

    PubMed

    Yu, C-J; Debski, E A

    2003-01-01

    Both nicotinic and muscarinic cholinergic receptors are present in the optic tectum. To begin to understand how the activation of these receptors affects visual activity patterns, we have determined the types of physiological responses induced by their activation. Using tectal brain slices from the leopard frog, we found that application of nicotine (100 microM) evoked long-lasting responses in 60% of patch-clamped tectal cells. Thirty percent of these responses consisted of an increase in spontaneous postsynaptic currents (sPSCs) and had both a glutamatergic and GABAergic component as determined by the use of 6-cyano-7-nitroquinoxaline-2,3-dione (50 microM) and bicuculline (25 microM), respectively. Remaining response types consisted of an inward membrane current (16%) and an increase in sPSCs combined with an inward membrane current (14%). All responses could be elicited in the presence of tetrodotoxin (0.5 microM). Muscarinic receptor-mediated responses, induced by carbachol (100 microM) application after nicotinic receptor desensitization, produced responses in 70% of tectal cells. In contrast to responses elicited by nicotine, carbachol-induced responses could be evoked multiple times without significant decrement. Responses consisted of either an outward current (57%), a decrease in sPSCs (5%) or an increase in sPSCs, with (almost 6%) or without (almost 3%) an outward current. The response elicited by carbachol was not predicted by the response of the cell to nicotine. Our results suggest that nicotinic receptors are found predominantly at presynaptic locations in the optic tectum while muscarinic receptors are most often present at postsynaptic sites. We conclude that both of these receptor types could substantially modulate visual activity by changing either the input to tectal neurons or the level of their response to that input.

  7. Tramadol state-dependent memory: involvement of dorsal hippocampal muscarinic acetylcholine receptors.

    PubMed

    Jafari-Sabet, Majid; Jafari-Sabet, Ali-Reza; Dizaji-Ghadim, Ali

    2016-08-01

    The effects on tramadol state-dependent memory of bilateral intradorsal hippocampal (intra-CA1) injections of physostigmine, an acetylcholinesterase inhibitor, and atropine, a muscarinic acetylcholine receptor antagonist, were examined in adult male NMRI mice. A single-trial step-down passive avoidance task was used for the assessment of memory retention. Post-training intra-CA1 administration of an atypical μ-opioid receptor agonist, tramadol (0.5 and 1 μg/mouse), dose dependently impaired memory retention. Pretest injection of tramadol (0.5 and 1 μg/mouse, intra-CA1) induced state-dependent retrieval of the memory acquired under the influence of post-training tramadol (1 μg/mouse, intra-CA1). A pretest intra-CA1 injection of physostigmine (1 μg/mouse) reversed the memory impairment induced by post-training administration of tramadol (1 μg/mouse, intra-CA1). Moreover, pretest administration of physostigmine (0.5 and 1 μg/mouse, intra-CA1) with an ineffective dose of tramadol (0.25 μg/mouse, intra-CA1) also significantly restored retrieval. Pretest administration of physostigmine (0.25, 0.5, and 1 μg/mouse, intra-CA1) by itself did not affect memory retention. A pretest intra-CA1 injection of the atropine (1 and 2 μg/mouse) 5 min before the administration of tramadol (1 μg/mouse, intra-CA1) dose dependently inhibited tramadol state-dependent memory. Pretest administration of atropine (0.5, 1, and 2 μg/mouse, intra-CA1) by itself did not affect memory retention. It can be concluded that dorsal hippocampal muscarinic acetylcholine receptor mechanisms play an important role in the modulation of tramadol state-dependent memory.

  8. Roles of threonine 192 and asparagine 382 in agonist and antagonist interactions with M1 muscarinic receptors

    PubMed Central

    Huang, Xi-Ping; Nagy, Peter I; Williams, Frederick E; Peseckis, Steven M; Messer, William S

    1999-01-01

    Conserved amino acids, such as Thr in transmembrane domains (TM) V and Asn in TM VI of muscarinic receptors, may be important in agonist binding and/or receptor activation. In order to determine the functional roles of Thr192 and Asn382 in human M1 receptors in ligand binding and receptor activation processes, we created and characterized mutant receptors with Thr192 or Asn382 substituted by Ala.HM1 wild-type (WT) and mutant receptors [HM1(Thr192Ala) and HM1(Asn382Ala)] were stably expressed in A9 L cells. The Kd values for 3H-(R)-QNB and Ki values for other classical muscarinic antagonists were similar at HM1(WT) and HM1(Thr192Ala) mutant receptors, yet higher at HM1(Asn382Ala) mutant receptors. Carbachol exhibited lower potency and efficacy in stimulating PI hydrolysis via HM1(Thr192Ala) mutant receptors, and intermediate agonist activity at the HM1(Asn382Ala) mutant receptors.The Asn382 residue in TM VI but not the Thr192 residue in TM V of the human M1 receptor appears to participate directly in antagonist binding. Both Thr192 and Asn382 residues are involved differentially in agonist binding and/or receptor activation processes, yet the Asn382 residue is less important than Thr192 in agonist activation of M1 receptors.Molecular modelling studies indicate that substitution of Thr192 or Asn382 results in the loss of hydrogen-bond interactions and changes in the agonist binding mode associated with an increase in hydrophobic interactions between ligand and receptor. PMID:10188986

  9. Impaired M3 and enhanced M2 muscarinic receptor contractile function in a streptozotocin model of mouse diabetic urinary bladder

    PubMed Central

    Pak, K. J.; Ostrom, R. S.; Matsui, M.

    2010-01-01

    We investigated the contractile roles of M2 and M3 muscarinic receptors in urinary bladder from streptozotocin-treated mice. Wild-type and M2 muscarinic receptor knockout (M2 KO) mice were given a single injection of vehicle or streptozotocin (125 mg kg−1) 2–24 weeks prior to bladder assays. The effect of forskolin on contractions elicited to the muscarinic agonist, oxotremorine-M, was measured in isolated urinary bladder (intact or denuded of urothelium). Denuded urinary bladder from vehicle-treated wild-type and M2 KO mice exhibited similar contractile responses to oxotremorine-M, when contraction was normalized relative to that elicited by KCl (50 mM). Eight to 9 weeks after streptozotocin treatment, the EC50 value of oxotremorine-M increased 3.1-fold in urinary bladder from the M2 KO mouse (N = 5) compared to wild type (N = 6; P < 0.001). Analogous changes were observed in intact bladder. In denuded urinary bladder from vehicle-treated mice, forskolin (5 µM) caused a much greater inhibition of contraction in M2 KO bladder compared to wild type. Following streptozotocin treatment, this forskolin effect increased 1.6-fold (P = 0.032). At the 20- to 24-week time point, the forskolin effect increased 1.7-fold for denuded as well as intact bladders (P = 0.036, 0.01, respectively). Although streptozotocin treatment inhibits M3 receptor-mediated contraction in denuded urinary bladder, muscarinic contractile function is maintained in wild-type bladder by enhanced M2 contractile function. M2 receptor activation opposes forskolin-induced relaxation of the urinary bladder, and this M2 function is enhanced following streptozotocin treatment. PMID:20349044

  10. Effects of methylmercury on muscarinic receptors in the mouse brain: A quantitative autoradiographic study

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lee, Haesung; Yee, S.; Geddes, J.

    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 ({supmore » 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.« less

  11. Muscarinic agonists for the treatment of cognition in schizophrenia.

    PubMed

    Sellin, Angela K; Shad, Mujeeb; Tamminga, Carol

    2008-11-01

    It is widely accepted that cholinergic activity at muscarinic receptors is required to maintain cognitive functions, including learning and memory. Memory domains are especially impaired in schizophrenia, which may explain difficulties in psychosocial rehabilitation of individuals with this illness. However, little is known about the mechanism of this impairment. To understand our current knowledge, we reviewed the literature since 1990 via a PubMed search for the terms "muscarinic", "schizophrenia", "cognition", "memory", "learning", and "agonist" in combination. We found 89 basic science/laboratory studies, case reports/series, case-control studies, cross-sectional studies, standardized controlled animal trials, standardized controlled human trials, and reviews. Although further research is required to fully understand the neuropharmacology of the cholinergic system in cognitive function in schizophrenia, we have examined the data currently available. In general, these data suggest that agonist activity at acetylcholine muscarinic type 1 (M1) receptors would enhance memory and learning in schizophrenia. We present an overview of likely side effects of muscarinic agonists. We outline the anticholinergic activity of several available antipsychotics and review the available M1 muscarinic agonists.

  12. Loss of M5 muscarinic acetylcholine receptors leads to cerebrovascular and neuronal abnormalities and cognitive deficits in mice.

    PubMed

    Araya, Runa; Noguchi, Takanori; Yuhki, Munehiro; Kitamura, Naohito; Higuchi, Makoto; Saido, Takaomi C; Seki, Kenjiro; Itohara, Shigeyoshi; Kawano, Masako; Tanemura, Kentaro; Takashima, Akihiko; Yamada, Kazuyuki; Kondoh, Yasushi; Kanno, Iwao; Wess, Jürgen; Yamada, Masahisa

    2006-11-01

    The M5 muscarinic acetylcholine receptor (M5R) has been shown to play a crucial role in mediating acetylcholine-dependent dilation of cerebral blood vessels. We show that male M5R-/- mice displayed constitutive constriction of cerebral arteries using magnetic resonance angiography in vivo. Male M5R-/- mice exhibited a significantly reduced cerebral blood flow (CBF) in the cerebral cortex, hippocampus, basal ganglia, and thalamus. Cortical and hippocampal pyramidal neurons from M5R-/- mice showed neuronal atrophy. Hippocampus-dependent spatial and nonspatial memory was also impaired in M5R-/- mice. In M5R-/- mice, CA3 pyramidal cells displayed a significantly attenuated frequency of the spontaneous postsynaptic current and long-term potentiation was significantly impaired at the mossy fiber-CA3 synapse. Our findings suggest that impaired M5R signaling may play a role in the pathophysiology of cerebrovascular deficits. The M5 receptor may represent an attractive novel therapeutic target to ameliorate memory deficits caused by impaired cerebrovascular function.

  13. Scopolamine Administration Modulates Muscarinic, Nicotinic and NMDA Receptor Systems

    PubMed Central

    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. PMID:22384146

  14. Effect of central muscarinic receptors on passive-avoidance learning deficits induced by prenatal pentylenetetrazol kindling in male offspring.

    PubMed

    Pourmotabbed, A; Mahmoodi, G; Mahmoodi, S; Mohammadi-Farani, A; Nedaei, S E; Pourmotabbed, T; Pourmotabbed, T

    2014-10-24

    Occurrence of the epileptic seizures during gestation might affect the neurodevelopment of the fetus resulting in cognitive problems for the child later in life. We have previously reported that prenatal pentylenetetrazol (PTZ)-kindling induces learning and memory deficits in the children born to kindled mothers, later in life but the mechanisms involved in this processes are unknown. The cholinergic system plays a major role in learning and memory. The present study was performed to investigate the possible involvement of central muscarinic cholinergic receptors on learning and memory deficits induced by prenatal PTZ-kindling in male offspring. Pregnant Wistar rats were kindled by repetitive i.p. injection of 25mg/kg of PTZ on day 13 of their pregnancy. The effect of intracerebroventricular (ICV) microinjection of scopolamine and pilocarpine, muscarinic cholinergic receptors antagonist and agonist, respectively on passive-avoidance learning of pups were tested at 12weeks of age using shuttle-box apparatus. Our data showed that the retention latencies of pups that received scopolamine (2 or 3μg) were significantly reduced compared to those received normal saline (p<0.05). Interestingly, post training ICV administration of pilocarpine (2μg) retrieved pups' memory deficits (p<0.001). These results demonstrate for the first time, the importance of the central muscarinic cholinergic receptors in learning and memory deficits in pups born to kindled dams and suggest a central mechanism for the cognitive and memory dysfunction, associated with seizures during pregnancy. Copyright © 2014. Published by Elsevier Ltd.

  15. Muscarinic receptor activation potentiates the effect of spinal cord stimulation on pain-related behavior in rats with mononeuropathy.

    PubMed

    Song, Zhiyang; Meyerson, Björn A; Linderoth, Bengt

    2008-05-02

    Spinal cord stimulation (SCS) has proven to be a valuable treatment in neuropathic pain. Our previous animal experiments performed on rat models of SCS and ensuing clinical trials have demonstrated that intrathecal (i.t.) administration of subeffective doses of certain drugs may enhance the pain relieving effect of SCS in cases with unsatisfactory SCS outcome. Recently, an augmented release of spinal acetylcholine acting on muscarinic receptors has been shown to be one of the mechanisms involved in SCS. The present study was performed to examine whether cold hypersensitivity and heat hyperalgesia in rats with partial sciatic nerve injuries can be attenuated by SCS in the same way as tactile hypersensitivity and to explore a possibly synergistic effect of SCS and a muscarinic receptor agonist, oxotremorine. Rats with signs of neuropathy were subjected to SCS applied in awake, freely moving condition. Oxotremorine was administered intrathecally. Tactile, cold and heat sensitivities were assessed by using von Frey filaments, cold spray and focused radiant heat, respectively. Oxotremorine i.t. dose-dependently suppressed the tactile hypersensitivity. SCS markedly increased withdrawal thresholds (WTs), withdrawal latencies and cold scores. When combining SCS with a subeffective dose of oxotremorine i.t., the suppressive effect of SCS on the pain-related symptoms was dramatically enhanced in rats failing to obtain a satisfactory effect with SCS alone. In conclusion, the combination of SCS and a drug with selective muscarinic receptor agonistic properties could be an optional therapy, when SCS per se has proven inefficient.

  16. Identification of four areas each enriched in a unique muscarinic receptor subtype

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hoss, W.; Ellerbrock, B.R.; Goldman, P.S.

    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 gyrusmore » 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.« less

  17. Prediction of consensus binding mode geometries for related chemical series of positive allosteric modulators of adenosine and muscarinic acetylcholine receptors.

    PubMed

    Sakkal, Leon A; Rajkowski, Kyle Z; Armen, Roger S

    2017-06-05

    Following insights from recent crystal structures of the muscarinic acetylcholine receptor, binding modes of Positive Allosteric Modulators (PAMs) were predicted under the assumption that PAMs should bind to the extracellular surface of the active state. A series of well-characterized PAMs for adenosine (A 1 R, A 2A R, A 3 R) and muscarinic acetylcholine (M 1 R, M 5 R) receptors were modeled using both rigid and flexible receptor CHARMM-based molecular docking. Studies of adenosine receptors investigated the molecular basis of the probe-dependence of PAM activity by modeling in complex with specific agonist radioligands. Consensus binding modes map common pharmacophore features of several chemical series to specific binding interactions. These models provide a rationalization of how PAM binding slows agonist radioligand dissociation kinetics. M 1 R PAMs were predicted to bind in the analogous M 2 R PAM LY2119620 binding site. The M 5 R NAM (ML-375) was predicted to bind in the PAM (ML-380) binding site with a unique induced-fit receptor conformation. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  18. Modulation of prepulse inhibition through both M1 and M4 muscarinic receptors in mice

    PubMed Central

    Thomsen, Morgane; Wess, Jürgen; Fulton, Brian S.; Fink-Jensen, Anders; Caine, S. Barak

    2014-01-01

    Rationale Muscarinic cholinergic M1 and M4 receptors may participate in schizophrenia's etiology, and have been proposed as targets for antipsychotic medications. Objective Here we investigated the involvement of these receptors in behavioral measures pertinent to schizophrenia using knockout mice lacking M1 receptors (M1−/−), M4 receptors (M4−/−) or both (M1−/−M4−/−). Methods We measured prepulse inhibition of startle (PPI) without drugs, and after treatment with scopolamine (0.32–1.8 mg/kg), xanomeline (3.2 mg/kg) oxotremorine (0.032–0.1 mg/kg), clozapine (1.0–5.6 mg/kg), or haloperidol (0.32–3.2 mg/kg). Results In female (but not male) mice, combined deletion of both M1 and M4 receptors decreased PPI relative to wild-type mice, while knockout of either receptor alone had no significant effect. Scopolamine disrupted PPI in wild-type and M4−/− mice, but not in female M1−/−M4−/− or female M1−/− mice. When administered before scopolamine, xanomeline restored PPI in wild-type mice and M1−/− mice, but not in M4−/− mice. In contrast, pretreatment with oxotremorine increased PPI regardless of genotype. Effects of clozapine and haloperidol on PPI were not hindered by either mutation. Conclusions Deletion of both M1 and M4 receptors can disrupt PPI, suggesting that (at least partially redundant) M1 and M4 receptor-dependent functions are involved in sensorimotor gating mechanisms. PPI-disrupting effects of muscarinic antagonists appeared dependent upon M1 receptor blockade. Our data also suggest that xanomeline exerts antipsychotic-like effects mainly through M4 receptor stimulation, while stimulation of non-M1/M4 subtypes may also have antipsychotic potential. Finally, our results do not support a role of M1/M4 receptors in mediating antipsychotic-like effects of clozapine. PMID:20013114

  19. Muscarinic acetylcholine receptor M1 and M3 subtypes mediate acetylcholine-induced endothelium-independent vasodilatation in rat mesenteric arteries.

    PubMed

    Tangsucharit, Panot; Takatori, Shingo; Zamami, Yoshito; Goda, Mitsuhiro; Pakdeechote, Poungrat; Kawasaki, Hiromu; Takayama, Fusako

    2016-01-01

    The present study investigated pharmacological characterizations of muscarinic acetylcholine receptor (AChR) subtypes involving ACh-induced endothelium-independent vasodilatation in rat mesenteric arteries. Changes in perfusion pressure to periarterial nerve stimulation and ACh were measured before and after the perfusion of Krebs solution containing muscarinic receptor antagonists. Distributions of muscarinic AChR subtypes in mesenteric arteries with an intact endothelium were studied using Western blotting. The expression level of M1 and M3 was significantly greater than that of M2. Endothelium removal significantly decreased expression levels of M2 and M3, but not M1. In perfused mesenteric vascular beds with intact endothelium and active tone, exogenous ACh (1, 10, and 100 nmol) produced concentration-dependent and long-lasting vasodilatations. In endothelium-denuded preparations, relaxation to ACh (1 nmol) disappeared, but ACh at 10 and 100 nmol caused long-lasting vasodilatations, which were markedly blocked by the treatment of pirenzepine (M1 antagonist) or 4-DAMP (M1 and M3 antagonist) plus hexamethonium (nicotinic AChR antagonist), but not methoctramine (M2 and M4 antagonist). These results suggest that muscarinic AChR subtypes, mainly M1, distribute throughout the rat mesenteric arteries, and that activation of M1 and/or M3 which may be located on CGRPergic nerves releases CGRP, causing an endothelium-independent vasodilatation. Copyright © 2015 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. All rights reserved.

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

    PubMed

    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.

  1. 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

  2. Muscarinic regulation of Kenyon cell dendritic arborizations in adult worker honey bees

    PubMed Central

    Dobrin, Scott E.; Herlihy, J. Daniel; Robinson, Gene E.; Fahrbach, Susan E.

    2011-01-01

    The experience of foraging under natural conditions increases the volume of mushroom body neuropil in worker honey bees. A comparable increase in neuropil volume results from treatment of worker honey bees with pilocarpine, an agonist for muscarinic-type cholinergic receptors. A component of the neuropil growth induced by foraging experience is growth of dendrites in the collar region of the calyces. We show here, via analysis of Golgi-impregnated collar Kenyon cells with wedge arborizations, that significant increases in standard measures of dendritic complexity were also found in worker honey bees treated with pilocarpine. This result suggests that signaling via muscarinic-type receptors promotes the increase in Kenyon cell dendritic complexity associated with foraging. Treatment of worker honey bees with scopolamine, a muscarinic inhibitor, inhibited some aspects of dendritic growth. Spine density on the Kenyon cell dendrites varied with sampling location, with the distal portion of the dendritic field having greater total spine density than either the proximal or medial section. This observation may be functionally significant because of the stratified organization of projections from visual centers to the dendritic arborizations of the collar Kenyon cells. Pilocarpine treatment had no effect on the distribution of spines on dendrites of the collar Kenyon cells. PMID:21262388

  3. Experiment K-6-18. Study of muscarinic and gaba (benzodiazepine) receptors in the sensory-motor cortex, hippcampus and spinal code

    NASA Technical Reports Server (NTRS)

    Daunton, N.; Damelio, F.; Krasnov, I.

    1990-01-01

    Frontal lobe samples of rat brains flown aboard Cosmos 1887 were processed for the study of muscarinic (cholinergic) and GABA (benzodiazepine) receptors and for immunocytochemical localization of the neurotransmitter gamma-aminobutyric acid (GABA) and glial fibrillary acidic protein (GFAP). Although radioactive labeling of both muscarinic cholinergic and GABA (benzodiazepine) receptors proved to be successful with the techniques employed, distinct receptor localization of individual laminae of the frontal neocortex was not possible since the sampling of the area was different in the various groups of animals. In spite of efforts made for proper orientation and regional identification of laminae, it was found that a densitometric (quantitation of autoradiograms) analysis of the tissue did not contribute to the final interpretation of the effects of weightlessness on these receptors. As to the immunocytochemical studies the use of both markers, GFAP and GABA antiserum, confirmed the suitability of the techniques for use in frozen material. However, similar problems to those encountered in the receptor studies prevented an adequate interpretation of the effects of micro-G exposure on the localization and distribution of GABA and GFAP. This study did, however, confirm the feasibility of investigating neurotransmitters and their receptors in future space flight experiments.

  4. Nucleus accumbens neurotransmission and effort-related choice behavior in food motivation: effects of drugs acting on dopamine, adenosine, and muscarinic acetylcholine receptors.

    PubMed

    Nunes, Eric J; Randall, Patrick A; Podurgiel, Samantha; Correa, Mercè; Salamone, John D

    2013-11-01

    Mesolimbic dopamine (DA) is a critical component of the brain circuitry regulating behavioral activation and effort-related processes. Although nucleus accumbens (NAc) DA depletions or antagonism leave aspects of appetite and primary food motivation intact, rats with impaired DA transmission reallocate their instrumental behavior away from food-reinforced tasks with high response requirements, and instead select less effortful food-seeking behaviors. Previous work showed that adenosine A2A antagonists can reverse the effects of DA D2 antagonists on effort-related choice, and that stimulation of adenosine A2A receptors produces behavioral effects that are similar to those induced by DA antagonism. The present review summarizes the literature on the role of NAc DA and adenosine in effort-related processes, and also presents original data on the effects of local stimulation of muscarinic acetylcholine receptors in NAc core. Local injections of the muscarinic agonist pilocarpine directly into NAc core produces shifts in effort-related choice behavior similar to those induced by DA antagonism or A2A receptor stimulation, decreasing lever pressing but increasing chow intake in rats responding on a concurrent fixed ratio/chow feeding choice task. In contrast, injections into a neostriatal control site dorsal to the NAc were ineffective. The actions of pilocarpine on this task were attenuated by co-administration of the muscarinic antagonist scopolamine. Thus, drugs that act on DA, adenosine A2A, and muscarinic receptors regulate effort-related choice behavior, which may have implications for the treatment of psychiatric symptoms such as psychomotor slowing, fatigue or anergia that can be observed in depression and other disorders. Copyright © 2013 Elsevier Ltd. All rights reserved.

  5. 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

  6. Muscarinic receptors as targets for anti-inflammatory therapy.

    PubMed

    Sales, María Elena

    2010-11-01

    ACh, the main neurotransmitter in the neuronal cholinergic system, is synthesized by pre-ganglionic fibers of the sympathetic and parasympathetic autonomic nervous system and by post-ganglionic parasympathetic fibers. There is increasing experimental evidence that ACh is widely expressed in prokaryotic and eukaryotic non-neuronal cells. The neuronal and non-neuronal cholinergic systems comprise ACh, choline acetyltransferase and cholinesterase, enzymes that synthesize and catabolize ACh, and the nicotinic and muscarinic ACh receptors (nAChRs and mAChRs, respectively), which are the targets for ACh action. This review analyzes the participation of the cholinergic system, particularly through mAChRs, in inflammation, and discusses the role of the different mAChR antagonists that have been used to treat skin inflammatory disorders, asthma and COPD, as well as intestinal inflammation and systemic inflammatory diseases, to assess the potential application of these compounds as therapeutic tools.

  7. Discrimination of putative M1 and M2 muscarinic receptor subtypes in rat brain by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ)

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Norman, A.B.; Creese, I.

    1986-03-01

    The EC/sub 50/ of EEDQ for the inhibition of (/sup 3/H)(-)QNB binding in vitro was approximately 3 fold lower for homogenates of hippocampus than brainstem (containing predominantly putative M/sub 1/ and M/sub 2/ muscarinic receptor subtypes respectively). Furthermore, the time-dependent loss of (/sup 3/H)(-)QNB binding produced by 100 ..mu..M EEDQ was faster in homogenates of hippocampus than brainstem. Administration of EEDQ (20 mg/kg i.p.) irreversibly reduced the Bmax of (/sup 3/H)(-)QNB binding by 56% and 34% in hippocampus and brainstem respectively. Pirenzepine competition for the remaining (/sup 3/H)(-)QNB binding sites following in vitro and in vivo treatment with EEDQ revealedmore » a significant increase in the proportion of (/sup 3/H)(-)QNB binding sites having low affinity for pirenzepine (M/sub 2/ receptors), indicating that the high affinity pirenzepine binding sites (M/sub 1/ receptors) were selectively and irreversibly lost. Thus, EEDQ discriminates the same putative M/sub 1/ and M/sub 2/ muscarinic receptor subtypes that are discriminated by pirenzepine. The reduction of (/sup 3/H)(-)QNB binding could be prevented both in vitro and in vivo by atropine or scopolamine. These data may indicate differences in the accessibility of these putative receptor subtypes to EEDQ or, alternatively, differences in the availability of carboxyl groups able to interact with EEDQ at the ligand recognition site of M/sub 1/ and M/sub 2/ muscarinic receptors.« less

  8. Muscarinic acetylcholine receptor expression in aganglionic bowel.

    PubMed

    Oue, T; Yoneda, A; Shima, H; Puri, P

    2000-01-01

    In Hirschsprung's disease (HD) there exists an overabundance of acetylcholine (ACh), which in turn stimulates excessive production of the enzyme acetylcholinesterase. Muscarinic ACh receptors (mAChRs) play an important role in smooth-muscle contraction. Recent studies have indicated five different subtypes of mAChRs encoded by five different genes, ml to m5. The purpose of this study was to investigate the expression of each mAChR subtype in aganglionic (AG) colon to further understand the pathophysiology of HD. Entire colon resected at the time of pull-through operation for HD was obtained from 14 patients. Specimens obtained at autopsy from 8 age-matched patients without gastrointestinal disease acted as controls. Frozen sections were used for indirect immunohistochemistry as well as in-situ hybridization. Immunohistochemistry was performed using specific antiserum against each mAChR subtype and in-situ hybridization was performed using specific oligonucleotide probes against ml to m5 subtypes. Messenger RNA (mRNA) was extracted from normoganglionic (NG) and AG bowel of HD patients and normal control bowel. Reverse transcription-polymerase chain reaction was performed to evaluate mRNA levels of each mAChR subtype. To adjust the levels of mRNA expression, a housekeeping gene G3PDH, known to be expressed normally, was used as an internal control. Strong m2 and m3 immunoreactivity was observed in the mucosal layer, smooth-muscle layers, and myenteric plexus of NG bowel, whereas ml immunoreactivity was only detected in the mucosal layer. The most striking finding was the abundance of m3-immunoreactive fibers in muscle layers of NG bowel while there was a total lack of m3 fibers in smooth-muscle of AG bowel. Intense mRNA signals encoding m2 and m3 and to a lesser degree ml were detected in NG bowel, and these signals were weak in AG bowel. Immunoreactivity and mRNA expression of m4 and m5 was not detected in NG or AG bowel. The lack of m3-immunoreactive fibers in the

  9. Participation of muscarinic receptors in memory consolidation in passive avoidance learning.

    PubMed

    Dobryakova, Yulia V; Gurskaya, Olga; Markevich, Vladimir A

    2014-01-01

    It is well-known that the cholinergic system and the muscarinic cholinergic receptors are associated with cognitive functions. Here we examined whether a non-selective muscarinic receptor antagonist scopolamine affects learning performance and/or synaptic plasticity during the memory consolidation period. Adult male Wistar rats (250-300 g) were injected with scopolamine (2 mg/kg) or saline immediately after training in a "passive avoidance" task. Memory retention test was conducted 24 h after training. The changes in the latency of the first entry into a dark compartment of a test chamber was chosen as a criterion of learning. The efficacy of synaptic transmission was estimated by the changes in the basal level of focal potentials (fEPSP amplitude and slope ratio) before training (baseline), 90 min after the training (consolidation period), and 24 hour after the training (retention period). We found that foot-shock presentation by itself had no effect on fEPSP within the first 90 min after training, but in 24 hour fEPSPs were decreased. In untrained rats administration of scopolamine had no effect on the fEPSP amplitude within the first 90 min after the injection, but in 24 h we observed an increase in the fEPSP amplitude. In trained animals, scopolamine decreased the fEPSP amplitude in the hippocampal CA1 area during first 1.5 h after the injection. However, the drug had no effect on the memory retention in the passive avoidance task. Taken together our data suggest that scopolamine modifies the synaptic placticity of the hippocampal network but does not induce significant changes in the retention of the passive avoidance skill.

  10. Electrophysiological evidence showing muscarinic agonist-antagonist activities of N-desmethylclozapine using hippocampal excitatory and inhibitory neurons.

    PubMed

    Sugawara, Yuto; Kikuchi, Yui; Yoneda, Mitsugu; Ohno-Shosaku, Takako

    2016-07-01

    The atypical antipsychotic clozapine is widely used for treatment-resistant schizophrenic patients. Clozapine and its major active metabolite, N-desmethylclozapine (NDMC), have complex pharmacological properties, and interact with various neurotransmitter receptors. There are several biochemical studies reporting that NDMC exhibits a partial agonist profile at the human recombinant M1 muscarinic receptors. However, direct electrophysiological evidence showing the ability of NDMC to activate native M1 receptors in intact neurons is poor. Using rat hippocampal neurons, we previously demonstrated that activation of muscarinic receptors by a muscarinic agonist, oxotremorine M (oxo-M), induces a decrease in outward K(+)current at -40mV. In the present study, using this muscarinic current response we assessed agonist and antagonist activities of clozapine and NDMC at native muscarinic receptors in intact hippocampal excitatory and inhibitory neurons. Suppression of the oxo-M-induced current response by the M1 antagonist pirenzepine was evident only in excitatory neurons, while the M3 antagonist darifenacin was effective in both types of neurons. Muscarinic agonist activity of NDMC was higher than that of clozapine, higher in excitatory neurons than in inhibitory neurons, sensitive to pirenzepine, and partially masked when co-applied with clozapine. Muscarinic antagonist activity of clozapine as well as NDMC was not different between excitatory and inhibitory neurons, but clozapine was more effective than NDMC. These results demonstrate that NDMC has the ability to activate native M1 receptors expressed in hippocampal excitatory neurons, but its agonist activity might be limited in clozapine-treated patients because of the presence of excessive clozapine with muscarinic antagonist activity. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. 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

  12. The contributions of muscarinic receptors and changes in plasma aldosterone levels to the anti-hypertensive effect of Tulbaghia violacea

    PubMed Central

    2013-01-01

    Background Tulbaghia violacea Harv. (Alliaceae) is used to treat various ailments, including hypertension (HTN) in South Africa. This study aims to evaluate the contributions of muscarinic receptors and changes in plasma aldosterone levels to its anti-hypertensive effect. Methods In the acute experiments, methanol leaf extracts (MLE) of T. violacea (30–120 mg/kg), muscarine (0.16 -10 μg/kg), and atropine (0.02 - 20.48 mg/kg), and/or the vehicle (dimethylsulfoxide (DMSO) and normal saline (NS)) were respectively and randomly administered intravenously in a group of spontaneously hypertensive (SHR) weighing 300 to 350 g and aged less than 5 months. Subsequently, T. violacea (60 mg/kg) or muscarine (2.5 μg/kg) was infused into eight SHRs, 20 min after atropine (5.12 mg/kg) pre-treatment. In the chronic (21 days) experiments, the SHRs were randomly divided into three groups, and given the vehicle (0.2 ml/day of DMSO and NS), T. violacea (60 mg/kg/day) and captopril (10 mg/kg/day) respectively into the peritoneum, to investigate their effects on blood pressure (BP), heart rate (HR), and plasma aldosterone levels. Systolic BP and HR were measured using tail-cuff plethysmography during the intervention. BP and HR were measured via a pressure transducer connecting the femoral artery and the Powerlab at the end of each intervention in the acute experiment; and on day 22 in the chronic experiment. Results In the acute experiments, T. violacea, muscarine, and atropine significantly (p < 0.05) reduced BP dose-dependently. T. violacea and muscarine produced dose-dependent decreases in HR, while the effect of atropine on HR varied. After atropine pre-treatment, dose-dependent increases in BP and HR were observed with T. violacea; while the BP and HR effects of muscarine were nullified. In the chronic experiments, the T. violacea-treated and captropril-treated groups had signicantly lower levels of aldosterone in plasma when compared to vehicle

  13. Activation of muscarinic M3 receptors inhibits large-conductance voltage- and Ca2+-activated K+ channels in rat urinary bladder smooth muscle cells

    PubMed Central

    Parajuli, Shankar P.

    2013-01-01

    Large conductance voltage- and Ca2+-activated K+ (BK) channels are key regulators of detrusor smooth muscle (DSM) contraction and relaxation during urine voiding and storage. Here, we explored whether BK channels are regulated by muscarinic receptors (M-Rs) in native freshly isolated rat DSM cells under physiological conditions using the perforated whole cell patch-clamp technique and pharmacological inhibitors. M-R activation with carbachol (1 μM) initially evoked large transient outward BK currents, followed by inhibition of the spontaneous transient outward BK currents (STBKCs) in DSM cells. Carbachol (1 μM) also inhibited the amplitude and frequency of spontaneous transient hyperpolarizations (STHs) and depolarized the DSM cell membrane potential. Selective inhibition of the muscarinic M3 receptors (M3-Rs) with 4-diphenylacetoxy-N-methylpiperidine (4-DAMP; 0.1 μM), but not muscarinic M2 receptors with methoctramine (1 μM), blocked the carbachol inhibitory effects on STBKCs. Furthermore, blocking the inositol 1,4,5-triphosphate (IP3) receptors with xestospongin-C (1 μM) inhibited the carbachol-induced large transient outward BK currents without affecting carbachol inhibitory effects on STBKCs. Upon pharmacological inhibition of all known cellular sources of Ca2+ for BK channel activation, carbachol (1 μM) did not affect the voltage-step-induced steady-state BK currents, suggesting that the muscarinic effects in DSM cells are mediated by mobilization of intracellular Ca2+. In conclusion, our findings provide strong evidence that activation of M3-Rs leads to inhibition of the STBKCs, STHs, and depolarization of DSM cells. Collectively, the data suggest the existence of functional interactions between BK channels and M3-Rs at a cellular level in DSM. PMID:23703523

  14. Variation of M3 muscarinic receptor expression in different prostate tissues and its significance.

    PubMed

    Song, Wei; Yuan, Mingzhen; Zhao, Shengtian

    2009-08-01

    To detect the expression of the muscarinic receptor (M receptor) in different prostate tissues and analyze the role of its subtype in prostatic oncogenesis. Thirty-six cases of normal prostate and benign prostatic hyperplasia, and 8 cases of prostatic tumor, were used in this study from the Shandong University, Shandong, China, between 2003-2006. The protein expressions of M1, M2, and M3 receptors in each group were determined by Western-blotting. The gene expressions of the M3 receptor and vascular endothelial growth factors (VEGF) in each group were determined by reverse transcriptase-polymerase chain reaction. The protein and gene expressions of the M3 receptor in the prostatic carcinoma group were higher than that of benign prostatic hyperplasia group (p=0.0001) and normal prostate group (p=0.0001). The M3 receptor and VEGF showed positive straight-line correlations of gene expressions with the 3 groups (r=0.4999, p=0.0001). The M3 receptor may have a close relationship with prostatic oncogenesis.

  15. Long-term depression of inhibitory synaptic transmission induced by spike-timing dependent plasticity requires coactivation of endocannabinoid and muscarinic receptors.

    PubMed

    Ahumada, Juan; Fernández de Sevilla, David; Couve, Alejandro; Buño, Washington; Fuenzalida, Marco

    2013-12-01

    The precise timing of pre-postsynaptic activity is vital for the induction of long-term potentiation (LTP) or depression (LTD) at many central synapses. We show in synapses of rat CA1 pyramidal neurons in vitro that spike timing dependent plasticity (STDP) protocols that induce LTP at glutamatergic synapses can evoke LTD of inhibitory postsynaptic currents or STDP-iLTD. The STDP-iLTD requires a postsynaptic Ca(2+) increase, a release of endocannabinoids (eCBs), the activation of type-1 endocananabinoid receptors and presynaptic muscarinic receptors that mediate a decreased probability of GABA release. In contrast, the STDP-iLTD is independent of the activation of nicotinic receptors, GABAB Rs and G protein-coupled postsynaptic receptors at pyramidal neurons. We determine that the downregulation of presynaptic Cyclic adenosine monophosphate/protein Kinase A pathways is essential for the induction of STDP-iLTD. These results suggest a novel mechanism by which the activation of cholinergic neurons and retrograde signaling by eCBs can modulate the efficacy of GABAergic synaptic transmission in ways that may contribute to information processing and storage in the hippocampus. Copyright © 2013 Wiley Periodicals, Inc.

  16. Topographical distribution of decrements and recovery in muscarinic receptors from rat brains repeatedly exposed to sublethal doses of soman

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Churchill, L.; Pazdernik, T.L.; Jackson, J.L.

    1984-08-01

    (3H)Quinuclidinyl benzilate binding to rat brain muscarinic receptors decreased after repeated exposure to soman, a potent organophosphorus cholinesterase inhibitor. The topographical distribution of this decrement was analyzed by quantitative receptor autoradiography. After 4 weeks of soman, three times a week, quinuclidinyl benzilate binding decreased to 67 to 80% of control in frontal and parietal cortex, caudate-putamen, lateral septum, hippocampal body, dentate gyrus, superior colliculus, nucleus of the fifth nerve, and central grey. Minor or no decreases were observed in thalamic or hypothalamic nuclei, reticular formation, pontine nuclei, inferior colliculus, nucleus of the seventh nerve, and cerebellum. Scatchard analyses of saturationmore » curves using frontal cortex sections from soman-treated rats revealed a decrease in maximal quinuclidinyl benzilate binding from that in control rats and a return toward control levels by 24 days without any significant change in affinity. These brain areas showing significant decrements in muscarinic receptors recovered with a similar time course. An estimate of the time for 50% recovery for some of the brain areas was 14 days for superior colliculus, 16 days for cortex, and 19 days for hippocampal body. The application of quantitative receptor autoradiography to analyze receptor alterations has been valuable in localizing the telencephalon as a region more susceptible to change in receptor concentration.« less

  17. Structure and dynamics of the M3 muscarinic acetylcholine receptor

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kruse, Andrew C.; Hu, Jianxin; Pan, Albert C.

    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 themore » 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.« less

  18. Stimulation of muscarinic receptors mimics experience-dependent plasticity in the honey bee brain

    PubMed Central

    Ismail, Nyla; Robinson, Gene E.; Fahrbach, Susan E.

    2006-01-01

    Honey bees begin life working in the hive. At ≈3 weeks of age, they shift to visiting flowers to forage for pollen and nectar. Foraging is a complex task associated with enlargement of the mushroom bodies, a brain region important in insects for certain forms of learning and memory. We report here that foraging bees had a larger volume of mushroom body neuropil than did age-matched bees confined to the hive. This result indicates that direct experience of the world outside the hive causes mushroom body neuropil growth in bees. We also show that oral treatment of caged bees with pilocarpine, a muscarinic agonist, induced an increase in the volume of the neuropil similar to that seen after a week of foraging experience. Effects of pilocarpine were blocked by scopolamine, a muscarinic antagonist. Our results suggest that signaling in cholinergic pathways couples experience to structural brain plasticity. PMID:16373504

  19. Lifetime of muscarinic receptor-G-protein complexes determines coupling efficiency and G-protein subtype selectivity.

    PubMed

    Ilyaskina, Olga S; Lemoine, Horst; Bünemann, Moritz

    2018-05-08

    G-protein-coupled receptors (GPCRs) are essential for the detection of extracellular stimuli by cells and transfer the encoded information via the activation of functionally distinct subsets of heterotrimeric G proteins into intracellular signals. Despite enormous achievements toward understanding GPCR structures, major aspects of the GPCR-G-protein selectivity mechanism remain unresolved. As this can be attributed to the lack of suitable and broadly applicable assays, we set out to develop a quantitative FRET-based assay to study kinetics and affinities of G protein binding to activated GPCRs in membranes of permeabilized cells in the absence of nucleotides. We measured the association and dissociation kinetics of agonist-induced binding of G i/o , G q/11 , G s , and G 12/13 proteins to muscarinic M 1 , M 2 , and M 3 receptors in the absence of nucleotides between fluorescently labeled G proteins and receptors expressed in mammalian cells. Our results show a strong quantitative correlation between not the on-rates of G-protein-M 3 -R interactions but rather the affinities of G q and G o proteins to M 3 -Rs, their GPCR-G-protein lifetime and their coupling efficiencies determined in intact cells, suggesting that the G-protein subtype-specific affinity to the activated receptor in the absence of nucleotides is, in fact, a major determinant of the coupling efficiency. Our broadly applicable FRET-based assay represents a fast and reliable method to quantify the intrinsic affinity and relative coupling selectivity of GPCRs toward all G-protein subtypes.

  20. Synthesis of dibenzodioxazocines and their effects on cholinesterases and muscarinic cholinergic receptors.

    PubMed

    Gaál, J; Batke, J; Borsodi, A; Rózsa, L; Somogyi, G

    1989-01-01

    A new family of tricyclic compounds, the dibenzodioxazocines were synthesized. These compounds were the following: 2-chloro-12-(2-piperidino-ethyl)-dibenzo d,g 1,3,6 dioxazocine hydrochloride: EGYT-2347, 2-chloro-12-(3-dimethylamino-2-methyl-propyl)-dibenzo [d,g] [1,3,6]-dibenzodioxazocine hydrochloride: EGYT-2509, 2-chloro-12-(3-dimethylamino-propyl)-dibenzo [d,g] [1,3,6] dioxazocine-maleate: EGYT-2474 and 2-chloro-12-2-(4-methyl-piperazino)-ethyl-dibenzo [d,g] [1,3,6]-dioxazocine-dihydrochloride: EGYT-2541. These compounds are inhibitors of both butyryl- and acetylcholinesterase to and they exhibited relatively good anticholinergic properties in receptor binding experiments. The most selective inhibitor of butyrylcholinesterase is the compound EGYT-2347 (Ki = 1.5 x 10(-7) M) which strongly binds to rat brain muscarinic cholinergic receptor (KD = 4.1 x 10(-8) M).

  1. Subcellular Distribution of M2-muscarinic Receptors in Relation to Dopaminergic Neurons of the Rat Ventral Tegmental Area

    PubMed Central

    Garzón, Miguel; Pickel, Virginia M.

    2008-01-01

    Acetylcholine can affect cognitive functions and reward, in part, through activation of muscarinic receptors in the ventral tegmental area (VTA) to evoke changes in mesocorticolimbic dopaminergic transmission. Of the known muscarinic receptor subtypes present in the VTA, the M2 receptor (M2R) is most implicated in autoregulation, and also may play a heteroreceptor role in regulation of the output of the dopaminergic neurons. We sought to determine the functionally relevant sites for M2R activation in relation to VTA dopaminergic neurons by examining the electron microscopic immunolabeling of M2R and the dopamine transporter (DAT) in the VTA of rat brain. The M2R was localized to endomembranes in DAT-containing somatodendritic profiles, but showed a more prominent, size-dependent plasmalemmal location in non-dopaminergic dendrites. M2R also was located on the plasma membrane of morphologically heterogenous axon terminals contacting unlabeled as well as M2R or DAT-labeled dendrites. Some of these terminals formed asymmetric synapses resembling those of cholinergic terminals in the VTA. The majority, however, formed symmetric, inhibitory-type synapses, or were apposed without recognized junctions. Our results provide the first ultrastructural evidence that the M2R is expressed, but largely not available for local activation, on the plasma membrane of VTA dopaminergic neurons. Instead, the M2R in this region has a distribution suggesting more indirect regulation of mesocorticolimbic transmission through autoregulation of acetylcholine release and changes in the physiological activity or release of other, largely inhibitory transmitters. These findings could have implications for understanding the muscarinic control of cognitive and goal-directed behaviors within the VTA. PMID:16927256

  2. Acetylcholine Attenuates Hydrogen Peroxide-Induced Intracellular Calcium Dyshomeostasis Through Both Muscarinic and Nicotinic Receptors in Cardiomyocytes.

    PubMed

    Palee, Siripong; Apaijai, Nattayaporn; Shinlapawittayatorn, Krekwit; Chattipakorn, Siriporn C; Chattipakorn, Nipon

    2016-01-01

    Oxidative stress induced intracellular Ca2+ overload plays an important role in the pathophysiology of several heart diseases. Acetylcholine (ACh) has been shown to suppress reactive oxygen species generation during oxidative stress. However, there is little information regarding the effects of ACh on the intracellular Ca2+ regulation in the presence of oxidative stress. Therefore, we investigated the effects of ACh applied before or after hydrogen peroxide (H2O2) treatment on the intracellular Ca2+ regulation in isolated cardiomyocytes. Single ventricular myocytes were isolated from the male Wistar rats for the intracellular Ca2+ transient study by a fluorimetric ratio technique. H2O2 significantly decreased both of intracellular Ca2+ transient amplitude and decay rate. ACh applied before, but not after, H2O2 treatment attenuated the reduction of intracellular Ca2+ transient amplitude and decay rate. Both atropine (a muscarinic acetylcholine receptor blocker) and mecamylamine (a nicotinic acetylcholine receptor blocker) significantly decreased the protective effects of acetylcholine on the intracellular Ca2+ regulation. Moreover, the combination of atropine and mecamylamine completely abolished the protective effects of acetylcholine on intracellular Ca2+ transient amplitude and decay rate. ACh pretreatment attenuates H2O2-induced intracellular Ca2+ dyshomeostasis through both muscarinic and nicotinic receptors. © 2016 The Author(s) Published by S. Karger AG, Basel.

  3. The interaction of trazodone with rat brain muscarinic cholinoceptors.

    PubMed

    Hyslop, D K; Taylor, D P

    1980-01-01

    The muscarinic receptor binding of trazodone, a new nontricyclic antidepressant, was compared with established tricyclic antidepressants. The ability to inhibit the binding of [3H]-quinuclidinyl benzilate in vitro was used for comparing atropine-like effects. Trazodone was found to have essentially no activity at the muscarinic acetylcholine binding site in comparison to the tricyclic antidepressants.

  4. The interaction of trazodone with rat brain muscarinic cholinoceptors.

    PubMed Central

    Hyslop, D. K.; Taylor, D. P.

    1980-01-01

    The muscarinic receptor binding of trazodone, a new nontricyclic antidepressant, was compared with established tricyclic antidepressants. The ability to inhibit the binding of [3H]-quinuclidinyl benzilate in vitro was used for comparing atropine-like effects. Trazodone was found to have essentially no activity at the muscarinic acetylcholine binding site in comparison to the tricyclic antidepressants. PMID:7470750

  5. Muscarinic receptor occupation and receptor activation in the guinea-pig ileum by some acetamides related to oxotremorine.

    PubMed Central

    Ringdahl, B.

    1984-01-01

    The dissociation constants (KD values) and relative efficacies of seven acetamide analogues of oxotremorine, including two enantiomeric pairs, at muscarinic receptors in the guinea-pig isolated ileum were determined. The method used involved analysis of dose-response data before and after fractional inactivation of receptors with propylbenzilylcholine mustard. All of the compounds studied had lower affinities than oxotremorine, but some had substantially higher relative efficacies. Replacement of the pyrrolidine ring of N-methyl-N-(4- pyrrolidino -2- butynyl )acetamide(I), the parent compound in the series, by a dimethylamino or a trimethylammonium group decreased the affinity 32 and 4.5 fold, respectively, whereas the relative efficacy increased 5.7-8.3 times. There was no correlation between relative efficacies and affinities of the compounds. The structural requirements for high affinity and high efficacy appeared to be quite different. PMID:6733356

  6. Quantitative properties and receptor reserve of the IP3 and calcium branch of Gq-coupled receptor signaling

    PubMed Central

    Dickson, Eamonn J.; Falkenburger, Björn H.

    2013-01-01

    Gq-coupled plasma membrane receptors activate phospholipase C (PLC), which hydrolyzes membrane phosphatidylinositol 4,5-bisphosphate (PIP2) into the second messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). This leads to calcium release, protein kinase C (PKC) activation, and sometimes PIP2 depletion. To understand mechanisms governing these diverging signals and to determine which of these signals is responsible for the inhibition of KCNQ2/3 (KV7.2/7.3) potassium channels, we monitored levels of PIP2, IP3, and calcium in single living cells. DAG and PKC are monitored in our companion paper (Falkenburger et al. 2013. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.201210887). The results extend our previous kinetic model of Gq-coupled receptor signaling to IP3 and calcium. We find that activation of low-abundance endogenous P2Y2 receptors by a saturating concentration of uridine 5′-triphosphate (UTP; 100 µM) leads to calcium release but not to PIP2 depletion. Activation of overexpressed M1 muscarinic receptors by 10 µM Oxo-M leads to a similar calcium release but also depletes PIP2. KCNQ2/3 channels are inhibited by Oxo-M (by 85%), but not by UTP (<1%). These differences can be attributed purely to differences in receptor abundance. Full amplitude calcium responses can be elicited even after PIP2 was partially depleted by overexpressed inducible phosphatidylinositol 5-phosphatases, suggesting that very low amounts of IP3 suffice to elicit a full calcium release. Hence, weak PLC activation can elicit robust calcium signals without net PIP2 depletion or KCNQ2/3 channel inhibition. PMID:23630337

  7. 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

  8. Changes in Muscarinic M2 Receptor Levels in the Cortex of Subjects with Bipolar Disorder and Major Depressive Disorder and in Rats after Treatment with Mood Stabilisers and Antidepressants

    PubMed Central

    Gibbons, Andrew Stuart; Jeon, Won Je; Scarr, Elizabeth; Dean, Brian

    2016-01-01

    Background: Increasingly, data are implicating muscarinic receptors in the aetiology and treatment of mood disorders. This led us to measure levels of different muscarinic receptor-related parameters in the cortex from people with mood disorders and the CNS of rats treated with mood stabilisers and antidepressant drugs. Methods: We measured [3H]AF-DX 384 binding in BA 46 and BA 24 from subjects with bipolar disorders (n = 14), major depressive disorders (n = 19), as well as age- and sex-matched controls (n = 19) and the CNS of rats treated with fluoxetine or imipramine. In addition, we used Western blots to measure levels of CHRM2 protein and oxotremorine-M stimulated [35S]GTPγS binding as a measure of CHRM 2 / 4 signaling. Results: Compared with controls, [3H]AF-DX 384 binding was lower in BA 24 and BA 46 in bipolar disorders and major depressive disorders, while CHRM2 protein and oxotremorine-M stimulated [35S]GTPγS binding was only lower in BA 24. Compared with vehicle, treatment with mood stabilisers, antidepressant drugs for 10 days, or imipramine for 28 days resulted in higher levels of in [3H]AF-DX 384 binding select regions of rat CNS. Conclusions: Our data suggest that levels of CHRM2 are lower in BA 24 from subjects with mood disorders, and it is possible that signalling by that receptor is also less in this cortical region. Our data also suggest increasing levels of CHRM2 may be involved in the mechanisms of action of mood stabilisers and tricyclic antidepressants. PMID:26475745

  9. An interspecies comparison of mercury inhibition on muscarinic acetylcholine receptor binding in the cerebral cortex and cerebellum

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Basu, Niladri; Department of Natural Resource Sciences, McGill University, Ste.-Anne-de-Bellevue, Quebec, H9X 3V9; Stamler, Christopher J.

    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,more » 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.« less

  10. Effect of progesterone-carbachol derivative on perfusion pressure and coronary resistance in isolated rat heart: via activation of the M2 muscarinic receptor.

    PubMed

    Figueroa-Valverde, Lauro; Diaz-Cedillo, Francisco; Garcia-Cervera, Elodia; Gomez, Eduardo Pool; Lopez-Ramos, Maria

    2014-01-01

    The present study was designed to investigate the effects of progesterone-carbachol derivative on perfusion pressure and coronary resistance in rats. An additional aim was to identify the molecular mechanisms involved. The Langendorff model was used to measure perfusion pressure and coronary resistance changes in isolated rat heart after progesterone-carbachol derivative alone and after the following compounds; mifepristone (progesterone receptor blocker), yohimbine (α2 adreno-receptor antagonist), ICI 118,551 (selective β2 receptor blocker), atropine (non-selective muscarinic receptor antagonist), methoctramine (antagonist of M2 receptor) and L-NAME (inhibitor of nitric oxide synthase). The results show that progesterone-carbachol derivative [10(-9) mM] significantly decreased perfusion pressure (P=0.005) and coronary resistance (P=0.006) in isolated rat heart. Additionally, the effect of progesterone-carbachol on perfusion pressure [10(-9) to 10(-4) mM] was only blocked in the presence of methoctramine and L-NAME. These data suggest that progesterone derivative exert its effect on perfusion pressure via activation of the M2 muscarinic. In addition, this phenomenon involves stimulation of nitric oxide synthase (NOS).

  11. Quantitative properties and receptor reserve of the IP(3) and calcium branch of G(q)-coupled receptor signaling.

    PubMed

    Dickson, Eamonn J; Falkenburger, Björn H; Hille, Bertil

    2013-05-01

    Gq-coupled plasma membrane receptors activate phospholipase C (PLC), which hydrolyzes membrane phosphatidylinositol 4,5-bisphosphate (PIP2) into the second messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). This leads to calcium release, protein kinase C (PKC) activation, and sometimes PIP2 depletion. To understand mechanisms governing these diverging signals and to determine which of these signals is responsible for the inhibition of KCNQ2/3 (KV7.2/7.3) potassium channels, we monitored levels of PIP2, IP3, and calcium in single living cells. DAG and PKC are monitored in our companion paper (Falkenburger et al. 2013. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.201210887). The results extend our previous kinetic model of Gq-coupled receptor signaling to IP3 and calcium. We find that activation of low-abundance endogenous P2Y2 receptors by a saturating concentration of uridine 5'-triphosphate (UTP; 100 µM) leads to calcium release but not to PIP2 depletion. Activation of overexpressed M1 muscarinic receptors by 10 µM Oxo-M leads to a similar calcium release but also depletes PIP2. KCNQ2/3 channels are inhibited by Oxo-M (by 85%), but not by UTP (<1%). These differences can be attributed purely to differences in receptor abundance. Full amplitude calcium responses can be elicited even after PIP2 was partially depleted by overexpressed inducible phosphatidylinositol 5-phosphatases, suggesting that very low amounts of IP3 suffice to elicit a full calcium release. Hence, weak PLC activation can elicit robust calcium signals without net PIP2 depletion or KCNQ2/3 channel inhibition.

  12. 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

  13. 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.

  14. 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.

  15. Reactive oxygen species potentiate the negative inotropic effect of cardiac M2-muscarinic receptor stimulation.

    PubMed

    Peters, S L; Sand, C; Batinik, H D; Pfaffendorf, M; van Zwieten, P A

    2001-08-01

    The aim of the present study was to investigate the influence of reactive oxygen species (ROS) on the contractile responses of rat isolated left atria to muscarinic receptor stimulation. ROS were generated by means of electrolysis (30 mA, 75 s) of the organ bath fluid. Twenty minutes after the electrolysis period, the electrically paced atria (3 Hz) were stimulated with the adenylyl cyclase activator forskolin (1 microM). Subsequently, cumulative acetylcholine concentration-response curves were constructed (0.01 nM-10 microM). In addition, phosphoinositide turnover and adenylyl cyclase activity under basal and stimulated conditions were measured. For these biochemical experiments we used the stable acetylcholine analogue carbachol. The atria exposed to reactive oxygen species were influenced more potently (pD2 control: 6.2 vs. 7.1 for electrolysis-treated atria, P<0.05) and more effectively (Emax control: 40% vs. 90% reduction of the initial amplitude, P<0.05) by acetylcholine. In contrast, ROS exposure did not alter the responses to adenosine, whose receptor is also coupled via a Gi-protein to adenylyl cyclase. The basal (40% vs. control, P<0.05) as well as the carbachol-stimulated (-85% vs. control, P<0.05) inositol-phosphate formation was reduced in atria exposed to ROS. The forskolin-stimulated adenylyl cyclase activity was identical in both groups but carbachol stimulation induced a more pronounced reduction in adenylyl cyclase activity in the electrolysis-treated atria. Accordingly we may conclude that ROS enhance the negative inotropic response of isolated rat atria to acetylcholine by both a reduction of the positive (inositide turnover) and increase of the negative (adenylyl cyclase inhibition) inotropic components of cardiac muscarinic receptor stimulation. This phenomenon is most likely M2-receptor specific, since the negative inotropic response to adenosine is unaltered by ROS exposure.

  16. Use of muscarinic agonists in the treatment of Sjögren's syndrome.

    PubMed

    Fox, R I; Konttinen, Y; Fisher, A

    2001-12-01

    Two muscarinic agonists (pilocarpine and cevimeline) have recently been approved for the treatment of symptoms of xerostomia in Sjögren's syndrome (SS). These agents stimulate the M1 and M3 receptors present on salivary glands, leading to increased secretory function. The use of these agents emphasizes the importance of neuroendocrine mechanisms in SS, which is considered an autoimmune disorder. We review recent studies on the release of cytokines and metalloproteinases in SS-affected glands and their influence on the release of and response to neurotransmitters. Also, we review the structure and function of muscarinic receptors as they may relate to SS and the potential use of novel muscarinic agonists in SS. (c)2001 Elsevier Science.

  17. Antipsychotic-like Effects of M4 Positive Allosteric Modulators Are Mediated by CB2 Receptor-Dependent Inhibition of Dopamine Release.

    PubMed

    Foster, Daniel J; Wilson, Jermaine M; Remke, Daniel H; Mahmood, M Suhaib; Uddin, M Jashim; Wess, Jürgen; Patel, Sachin; Marnett, Lawrence J; Niswender, Colleen M; Jones, Carrie K; Xiang, Zixiu; Lindsley, Craig W; Rook, Jerri M; Conn, P Jeffrey

    2016-09-21

    Muscarinic receptors represent a promising therapeutic target for schizophrenia, but the mechanisms underlying the antipsychotic efficacy of muscarinic modulators are not well understood. Here, we report that activation of M4 receptors on striatal spiny projection neurons results in a novel form of dopaminergic regulation resulting in a sustained depression of striatal dopamine release that is observed more than 30 min after removal of the muscarinic receptor agonist. Furthermore, both the M4-mediated sustained inhibition of dopamine release and the antipsychotic-like efficacy of M4 activators were found to require intact signaling through CB2 cannabinoid receptors. These findings highlight a novel mechanism by which striatal cholinergic and cannabinoid signaling leads to sustained reductions in dopaminergic transmission and concurrent behavioral effects predictive of antipsychotic efficacy. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Behavioral impact of neurotransmitter-activated GPCRs: Muscarinic and GABAB receptors regulate C. elegans locomotion

    PubMed Central

    Dittman, Jeremy S; Kaplan, Joshua M

    2008-01-01

    Neurotransmitter released from presynaptic terminals activates both ligand-gated ion channels (ionotropic receptors) and a variety of G protein-coupled receptors (GPCRs). These neurotransmitter receptors are expressed on both pre- and postsynaptic cells. Thus, each neurotransmitter acts on multiple receptor classes, generating a large repertoire of physiological responses. The impact of many ionotropic receptors on neuronal activity and behavior has been clearly elucidated; however, much less is known about how neurotransmitter-gated GPCRs regulate neurons and circuits. In C. elegans, both Acetylcholine (ACh) and GABA are released in the nerve cord and mediate fast neuromuscular excitation and inhibition during locomotion. Here we identify a muscarinic receptor (GAR-2) and the GABAB receptor dimer (GBB-1/2) that detect synaptically released ACh and GABA, respectively. Both GAR-2 and GBB-1/2 inhibited cholinergic motor neurons when ACh and GABA levels were enhanced. Loss of either GPCR resulted in movement defects, suggesting that these receptors are activated during locomotion. When the negative feedback provided by GAR-2 was replaced with positive feedback, animals became highly sensitive to ACh levels and locomotion was severely impaired. Thus, conserved GPCRs act in the nematode motor circuit to provide negative feedback and to regulate locomotory behaviors that underlie navigation. PMID:18614679

  19. Role of Nicotinic and Muscarinic Receptors on Synaptic Plasticity and Neurological Diseases.

    PubMed

    Fuenzalida, Marco; Pérez, Miguel Ángel; Arias, Hugo R

    2016-01-01

    The cholinergic activity in the brain is fundamental for cognitive functions. The modulatory activity of the neurotransmitter acetylcholine (ACh) is mediated by activating a variety of nicotinic acetylcholine receptors (nAChR) and muscarinic acetylcholine receptors (mAChR). Accumulating evidence indicates that both nAChR and mAChRs can modulate the release of several other neurotransmitters, modify the threshold of long-term plasticity, finally improving learning and memory processes. Importantly, the expression, distribution, and/or function of these systems are altered in several neurological diseases. The aim of this review is to discuss our current knowledge on cholinergic receptors and their regulating synaptic functions and neuronal network activities as well as their use as targets for the development of new and clinically useful cholinergic ligands. These new therapies involve the development of novel and more selective cholinergic agonists and allosteric modulators as well as selective cholinesterase inhibitors, which may improve cognitive and behavioral symptoms, and also provide neuroprotection in several brain diseases. The review will focus on two nAChR receptor subtypes found in the mammalian brain and the most commonly targeted in drug discovery programs for neuropsychiatric disorder, the ligands of α4β2 nAChR and α7 nAChRs.

  20. The selective positive allosteric M1 muscarinic receptor modulator PQCA attenuates learning and memory deficits in the Tg2576 Alzheimer's disease mouse model.

    PubMed

    Puri, Vanita; Wang, Xiaohai; Vardigan, Joshua D; Kuduk, Scott D; Uslaner, Jason M

    2015-01-01

    We have recently shown that the M1 muscarinic receptor positive allosteric modulator, PQCA, improves cognitive performance in rodents and non-human primates administered the muscarinic receptor antagonist scopolamine. The purpose of the present experiments was to characterize the effects of PQCA in a model more relevant to the disease pathology of Alzheimer's disease. Tg2576 transgenic mice that have elevated Aβ were tested in the novel object recognition task to characterize recognition memory as a function of age and treatment with the PQCA. The effects of PQCA were compared to the acetylcholinesterase inhibitor donepezil, the standard of care for Alzheimer's disease. In addition, the effect of co-administering PQCA and donepezil was evaluated. Aged Tg2576 mice demonstrated a deficit in recognition memory that was significantly attenuated by PQCA. The positive control donepezil also reversed the deficit. Furthermore, doses of PQCA and donepezil that were inactive on their own were found to improve recognition memory when given together. These studies suggest that M1 muscarinic receptor positive allosteric modulation can ameliorate memory deficits in disease relevant models of Alzheimer's disease. These data, combined with our previous findings demonstrating PQCA improves scopolamine-induced cognitive deficits in both rodents and non-human primates, suggest that M1 positive allosteric modulators have therapeutic potential for the treatment of Alzheimer's disease. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Design and functional characterization of a novel, arrestin-biased designer G protein-coupled receptor.

    PubMed

    Nakajima, Ken-ichiro; Wess, Jürgen

    2012-10-01

    Mutational modification of distinct muscarinic receptor subtypes has yielded novel designer G protein-coupled receptors (GPCRs) that are unable to bind acetylcholine (ACh), the endogenous muscarinic receptor ligand, but can be efficiently activated by clozapine-N-oxide (CNO), an otherwise pharmacologically inert compound. These CNO-sensitive designer GPCRs [alternative name: designer receptors exclusively activated by designer drug (DREADDs)] have emerged as powerful new tools to dissect the in vivo roles of distinct G protein signaling pathways in specific cell types or tissues. As is the case with other GPCRs, CNO-activated DREADDs not only couple to heterotrimeric G proteins but can also recruit proteins of the arrestin family (arrestin-2 and -3). Accumulating evidence suggests that arrestins can act as scaffolding proteins to promote signaling through G protein-independent signaling pathways. To explore the physiological relevance of these arrestin-dependent signaling pathways, the availability of an arrestin-biased DREADD would be highly desirable. In this study, we describe the development of an M₃ muscarinic receptor-based DREADD [Rq(R165L)] that is no longer able to couple to G proteins but can recruit arrestins and promote extracellular signal-regulated kinase-1/2 phosphorylation in an arrestin- and CNO-dependent fashion. Moreover, CNO treatment of mouse insulinoma (MIN6) cells expressing the Rq(R165L) construct resulted in a robust, arrestin-dependent stimulation of insulin release, directly implicating arrestin signaling in the regulation of insulin secretion. This newly developed arrestin-biased DREADD represents an excellent novel tool to explore the physiological relevance of arrestin signaling pathways in distinct tissues and cell types.

  2. Changes in Muscarinic M2 Receptor Levels in the Cortex of Subjects with Bipolar Disorder and Major Depressive Disorder and in Rats after Treatment with Mood Stabilisers and Antidepressants.

    PubMed

    Gibbons, Andrew Stuart; Jeon, Won Je; Scarr, Elizabeth; Dean, Brian

    2016-04-01

    Increasingly, data are implicating muscarinic receptors in the aetiology and treatment of mood disorders. This led us to measure levels of different muscarinic receptor-related parameters in the cortex from people with mood disorders and the CNS of rats treated with mood stabilisers and antidepressant drugs. We measured [(3)H]AF-DX 384 binding in BA 46 and BA 24 from subjects with bipolar disorders (n = 14), major depressive disorders (n = 19), as well as age- and sex-matched controls (n = 19) and the CNS of rats treated with fluoxetine or imipramine. In addition, we used Western blots to measure levels of CHRM2 protein and oxotremorine-M stimulated [(35)S]GTPγS binding as a measure of CHRM 2 / 4 signaling. Compared with controls, [(3)H]AF-DX 384 binding was lower in BA 24 and BA 46 in bipolar disorders and major depressive disorders, while CHRM2 protein and oxotremorine-M stimulated [(35)S]GTPγS binding was only lower in BA 24. Compared with vehicle, treatment with mood stabilisers, antidepressant drugs for 10 days, or imipramine for 28 days resulted in higher levels of in [(3)H]AF-DX 384 binding select regions of rat CNS. Our data suggest that levels of CHRM2 are lower in BA 24 from subjects with mood disorders, and it is possible that signalling by that receptor is also less in this cortical region. Our data also suggest increasing levels of CHRM2 may be involved in the mechanisms of action of mood stabilisers and tricyclic antidepressants. © The Author 2015. Published by Oxford University Press on behalf of CINP.

  3. Learning and Memory Impairments in a Congenic C57BL/6 Strain of Mice That Lacks the M2 Muscarinic Acetylcholine Receptor Subtype

    PubMed Central

    Bainbridge, Natalie K.; Koselke, Lisa R.; Jeon, Jongrye; Bailey, Kathleen R.; Wess, Jürgen; Crawley, Jacqueline N.; Wrenn, Craige C.

    2009-01-01

    The neurotransmitter acetylcholine is an important modulator of cognitive functions including attention, learning, and memory. The actions of acetylcholine are mediated by five distinct muscarinic acetylcholine receptor subtypes (M1-M5). The lack of drugs with a high degree of selectivity for these subtypes has impeded the determination of which subtypes mediate which components of cholinergic neurotransmission relevant to cognitive abilities. The present study examined the behavioral functions of the M2 muscarinic receptor subtype by utilizing congenic C57BL/6 mice possessing a null-mutation in the M2 muscarinic receptor gene (M2−/− mice). Comprehensive assessment of general health and neurological function found no major differences between M2−/− and wild-type (M2+/+) mice. In tests of learning and memory, M2−/− mice were impaired in the acquisition (trials to criterion), but not the retention (72 hr) of a passive avoidance task. In a novel open field, M2−/− mice were impaired in between-sessions, but not within-session habituation. In a holeboard test of spatial memory, M2−/− mice committed more errors in working memory than M2+/+ mice. Reference memory did not differ between the genotypes. M2−/− mice showed no impairments in either cued or contextual fear conditioning. These findings replicate and extend earlier findings in a hybrid strain and solidify the interpretation that the M2 receptor plays a critical role in specific components of cognitive abilities. PMID:18346798

  4. Accelerated molecular dynamics simulations of ligand binding to a muscarinic G-protein-coupled receptor.

    PubMed

    Kappel, Kalli; Miao, Yinglong; McCammon, J Andrew

    2015-11-01

    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.

  5. Differential inhibition of [3H]-oxotremorine-M and [3H]-quinuclinidyl benzilate binding to muscarinic receptors in rat brain membranes with acetylcholinesterase inhibitors.

    PubMed

    Lockhart, B; Closier, M; Howard, K; Steward, C; Lestage, P

    2001-04-01

    The potential interaction of acetylcholinesterase inhibitors with cholinergic receptors may play a significant role in the therapeutic and/or side-effects associated with this class of compound. In the present study, the capacity of acetylcholinesterase inhibitors to interact with muscarinic receptors was assessed by their ability to displace both [3H]-oxotremorine-M and [3H]-quinuclinidyl benzilate binding in rat brain membranes. The [3H]-quinuclinidyl benzilate/[3H]-oxotremorine-M affinity ratios permitted predictions to be made of either the antagonist or agonist properties of the different compounds. A series of compounds, representative of the principal classes of acetylcholinesterase inhibitors, displaced [3H]-oxotremorine-M binding with high-to-moderate potency (ambenonium>neostigmine=pyridostigmine=tacrine>physostigmine> edrophonium=galanthamine>desoxypeganine) whereas only ambenonium and tacrine displaced [3H]-quinuclinidyl benzilate binding. Inhibitors such as desoxypeganine, parathion and gramine demonstrated negligible inhibition of the binding of both radioligands. Scatchard plots constructed from the inhibition of [3H]-oxotremorine-M binding in the absence and presence of different inhibitors showed an unaltered Bmax and a reduced affinity constant, indicative of potential competitive or allosteric mechanisms. The capacity of acetylcholinesterase inhibitors, with the exception of tacrine and ambenonium, to displace bound [3H]-oxotremorine-M in preference to [3H]quinuclinidyl benzilate predicts that the former compounds could act as potential agonists at muscarinic receptors. Moreover, the rank order for potency in inhibiting acetylcholinesterase (ambenonium>neostigmine=physostigmine =tacrine>pyridostigmine=edrophonium=galanthamine >desoxypeganine>parathion>gramine) indicated that the most effective inhibitors of acetylcholinesterase also displaced [3H]-oxotremorine-M to the greatest extent. The capacity of these inhibitors to displace [3H

  6. Role of M2 Muscarinic Receptor in the Airway Response to Methacholine of Mice Selected for Minimal or Maximal Acute Inflammatory Response

    PubMed Central

    Castro, Juciane Maria de Andrade; Resende, Rodrigo R.; Florsheim, Esther; Albuquerque, Layra Lucy; Lino-dos-Santos-Franco, Adriana; Gomes, Eliane; Tavares de Lima, Wothan; de Franco, Marcelo; Ribeiro, Orlando Garcia

    2013-01-01

    Airway smooth muscle constriction induced by cholinergic agonists such as methacholine (MCh), which is typically increased in asthmatic patients, is regulated mainly by muscle muscarinic M3 receptors and negatively by vagal muscarinic M2 receptors. Here we evaluated basal (intrinsic) and allergen-induced (extrinsic) airway responses to MCh. We used two mouse lines selected to respond maximally (AIRmax) or minimally (AIRmin) to innate inflammatory stimuli. We found that in basal condition AIRmin mice responded more vigorously to MCh than AIRmax. Treatment with a specific M2 antagonist increased airway response of AIRmax but not of AIRmin mice. The expression of M2 receptors in the lung was significantly lower in AIRmin compared to AIRmax animals. AIRmax mice developed a more intense allergic inflammation than AIRmin, and both allergic mouse lines increased airway responses to MCh. However, gallamine treatment of allergic groups did not affect the responses to MCh. Our results confirm that low or dysfunctional M2 receptor activity is associated with increased airway responsiveness to MCh and that this trait was inherited during the selective breeding of AIRmin mice and was acquired by AIRmax mice during allergic lung inflammation. PMID:23691511

  7. BK channel β1 subunits regulate airway contraction secondary to M2 muscarinic acetylcholine receptor mediated depolarization.

    PubMed

    Semenov, Iurii; Wang, Bin; Herlihy, Jeremiah T; Brenner, Robert

    2011-04-01

    The large conductance calcium- and voltage-activated potassium channel (BK channel) and its smooth muscle-specific β1 subunit regulate excitation–contraction coupling in many types of smooth muscle cells. However, the relative contribution of BK channels to control of M2- or M3-muscarinic acetylcholine receptor mediated airway smooth muscle contraction is poorly understood. Previously, we showed that knockout of the BK channel β1 subunit enhances cholinergic-evoked trachea contractions. Here, we demonstrate that the enhanced contraction of the BK β1 knockout can be ascribed to a defect in BK channel opposition of M2 receptor-mediated contractions. Indeed, the enhanced contraction of β1 knockout is eliminated by specific M2 receptor antagonism. The role of BK β1 to oppose M2 signalling is evidenced by a greater than fourfold increase in the contribution of L-type voltage-dependent calcium channels to contraction that otherwise does not occur with M2 antagonist or with β1 containing BK channels. The mechanism through which BK channels oppose M2-mediated recruitment of calcium channels is through a negative shift in resting voltage that offsets, rather than directly opposes, M2-mediated depolarization. The negative shift in resting voltage is reduced to similar extents by BK β1 knockout or by paxilline block of BK channels. Normalization of β1 knockout baseline voltage with low external potassium eliminated the enhanced M2-receptor mediated contraction. In summary, these findings indicate that an important function of BK/β1 channels is to oppose cholinergic M2 receptor-mediated depolarization and activation of calcium channels by restricting excitation–contraction coupling to more negative voltage ranges.

  8. Alpha-lipoic acid-mediated activation of muscarinic receptors improves hippocampus- and amygdala-dependent memory.

    PubMed

    Mahboob, Aamra; Farhat, Syeda Mehpara; Iqbal, Ghazala; Babar, Mustafeez Mujtaba; Zaidi, Najam-us-Sahar Sadaf; Nabavi, Seyed Mohammad; Ahmed, Touqeer

    2016-04-01

    Aluminum (Al) is a neurotoxic agent which readily crosses the blood-brain-barrier (BBB) and accumulates in the brain leading to neurodegenerative disorders, characterised by cognitive impairment. Alpha-lipoic acid (ALA) is an antioxidant and has a potential to improve cognitive functions. This study aimed to evaluate the neuroprotective effect of ALA in AlCl3-induced neurotoxicity mouse model. Effect of ALA (25mg/kg/day) was evaluated in the AlCl3-induced neurotoxicity (AlCl3 150 mg/kg/day) mouse model on learning and memory using behaviour tests and on the expression of muscarinic receptor genes (using RT-PCR), in hippocampus and amygdala. Following ALA treatment, the expression of muscarinic receptor genes M1, M2 and choline acetyltransferase (ChaT) were significantly improved (p<0.05) relative to AlCl3-treated group. ALA enhanced fear memory (p<0.01) and social novelty preference (p<0.001) comparative to the AlCl3-treated group. Fear extinction memory was remarkably restored (p<0.001) in ALA-treated group demonstrated by reduced freezing response as compared to the AlCl3-treated group which showed higher freezing. In-silico analysis showed that racemic mixture of ALA has higher binding affinity for M1 and M2 compared to acetylcholine. These novel findings highlight the potential role of ALA in cognitive functions and cholinergic system enhancement thus presenting it an enviable therapeutic candidate for the treatment of neurodegenerative disorders. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. IN VITRO EFFECTS OF CHLORPYRIFOS, PARATHION, METHYL PARATHION AND THEIR OXONS ON CARDIAC MUSCARINIC RECEPTOR BINDING IN NEONATAL AND ADULT RATS. (R825811)

    EPA Science Inventory

    Organophosphorus insecticides elicit toxicity by inhibiting acetylcholinesterase. Young animals are generally more sensitive than adults to these toxicants. A number of studies reported that some organophosphorus agents also bind directly to muscarinic receptors, in particular...

  10. Human urothelial cell lines as potential models for studying cannabinoid and excitatory receptor interactions in the urinary bladder.

    PubMed

    Bakali, Evangelia; Elliott, Ruth A; Taylor, Anthony H; Lambert, David G; Willets, Jonathon M; Tincello, Douglas G

    2014-06-01

    To characterize human urothelial cell lines' cannabinoid receptor expression and evaluate their possible use for studying signalling interactions with purinergic and muscarinic receptor activation. PCR was used to detect cannabinoid (CB), muscarinic and purinergic receptor transcripts in HCV29 and UROtsa cells, whilst immunofluorescence evaluated protein expression and localization of cannabinoid receptors. The effect of CB1 agonist (ACEA) on carbachol- and ATP-induced changes in intracellular calcium ([Ca(2+)]i) levels was measured using fluorimetry. The ability of ACEA to reduce intracellular cAMP was investigated in HCV29 cells. CB1 and GPR55 receptor transcripts were detected in HCV29 and UROtsa cells, respectively. Immunofluorescence showed positive staining for CB1 in the HCV29 cells. Both cell lines expressed transcript levels for muscarinic receptors, but carbachol did not raise [Ca(2+)]i levels indicating a lack or low expression of G(q)-coupled muscarinic receptors. Transcripts for purinergic receptors were detected; ATP significantly increased [Ca(2+)]i in HCV29 and UROtsa cells by 395 ± 61 and 705 ± 100 nM (mean ± SEM, n = 6), respectively. ACEA did not alter ATP-induced [Ca(2+)]i or cAMP levels in HCV29 cells. Whilst HCV29 cells expressed CB1 and UROtsa cells expressed GPR55 receptors, these were not functionally coupled to the existing purinergic-driven increase in Ca2+ as such they do not represent a good model to study signalling interactions.

  11. Muscarinic Cholinergic Modulation of Long-Lasting Synaptic Plasticity in the Rat Dentate Gyrus

    DTIC Science & Technology

    1990-12-14

    ability to block GABAB-mediated responses, which are PTx-sensitive. The effects of the GABAg receptor agonist baclofen on evoked responses were analyzed...both in slices previously exposed to 10/iM muscarine (n=4), and nonexposed slices (n=2). The disinhibitory effects of baclofen usually seen in...20 min washout of muscarine always preceeded the baclofen exposure, to allow for washout of muscarine. There were no differences in the responses to

  12. The guinea pig ileum lacks the direct, high-potency, M(2)-muscarinic, contractile mechanism characteristic of the mouse ileum.

    PubMed

    Griffin, Michael T; Matsui, Minoru; Ostrom, Rennolds S; Ehlert, Frederick J

    2009-10-01

    We explored whether the M(2) muscarinic receptor in the guinea pig ileum elicits a highly potent, direct-contractile response, like that from the M(3) muscarinic receptor knockout mouse. First, we characterized the irreversible receptor-blocking activity of 4-DAMP mustard in ileum from muscarinic receptor knockout mice to verify its M(3) selectivity. Then, we used 4-DAMP mustard to inactivate M(3) responses in the guinea pig ileum to attempt to reveal direct, M(2) receptor-mediated contractions. The muscarinic agonist, oxotremorine-M, elicited potent contractions in ileum from wild-type, M(2) receptor knockout, and M(3) receptor knockout mice characterized by negative log EC(50) (pEC (50)) values +/- SEM of 6.75 +/- 0.03, 6.26 +/- 0.05, and 6.99 +/- 0.08, respectively. The corresponding E (max) values in wild-type and M(2) receptor knockout mice were approximately the same, but that in the M(3) receptor knockout mouse was only 36% of wild type. Following 4-DAMP mustard treatment, the concentration-response curve of oxotremorine-M in wild-type ileum resembled that of the M(3) knockout mouse in terms of its pEC (50), E (max), and inhibition by selective muscarinic antagonists. Thus, 4-DAMP mustard treatment appears to inactivate M(3) responses selectively and renders the muscarinic contractile behavior of the wild-type ileum similar to that of the M(3) knockout mouse. Following 4-DAMP mustard treatment, the contractile response of the guinea pig ileum to oxotremorine-M exhibited low potency and a competitive-antagonism profile consistent with an M(3) response. The guinea pig ileum, therefore, lacks a direct, highly potent, M(2)-contractile component but may have a direct, lower potency M(2) component.

  13. Muscarinic Stimulation Facilitates Sarcoplasmic Reticulum Ca Release by Modulating Ryanodine Receptor 2 Phosphorylation Through Protein Kinase G and Ca/Calmodulin-Dependent Protein Kinase II.

    PubMed

    Ho, Hsiang-Ting; Belevych, Andriy E; Liu, Bin; Bonilla, Ingrid M; Radwański, Przemysław B; Kubasov, Igor V; Valdivia, Héctor H; Schober, Karsten; Carnes, Cynthia A; Györke, Sándor

    2016-11-01

    Although the effects and the underlying mechanism of sympathetic stimulation on cardiac Ca handling are relatively well established both in health and disease, the modes of action and mechanisms of parasympathetic modulation are poorly defined. Here, we demonstrate that parasympathetic stimulation initiates a novel mode of excitation-contraction coupling that enhances the efficiency of cardiac sarcoplasmic reticulum Ca store utilization. This efficient mode of excitation-contraction coupling involves reciprocal changes in the phosphorylation of ryanodine receptor 2 at Ser-2808 and Ser-2814. Specifically, Ser-2808 phosphorylation was mediated by muscarinic receptor subtype 2 and activation of PKG (protein kinase G), whereas dephosphorylation of Ser-2814 involved activation of muscarinic receptor subtype 3 and decreased reactive oxygen species-dependent activation of CaMKII (Ca/calmodulin-dependent protein kinase II). The overall effect of these changes in phosphorylation of ryanodine receptor 2 is an increase in systolic Ca release at the low sarcoplasmic reticulum Ca content and a paradoxical reduction in aberrant Ca leak. Accordingly, cholinergic stimulation of cardiomyocytes isolated from failing hearts improved Ca cycling efficiency by restoring altered ryanodine receptor 2 phosphorylation balance. © 2016 American Heart Association, Inc.

  14. Inositol trisphosphate mediates cloned muscarinic receptor-activated conductances in transfected mouse fibroblast A9 L cells.

    PubMed Central

    Jones, S V; Barker, J L; Goodman, M B; Brann, M R

    1990-01-01

    1. The mechanism by which cloned m1 and m3 muscarinic receptor subtypes activate Ca2+-dependent channels was investigated with whole-cell and cell-attached patch-clamp recording techniques and with Fura-2 Ca2+ indicator dye measurements in cultured A9 L cells transfected with rat m1 and m3 cDNAs. 2. The Ca2+-dependent K+ and Cl- currents induced by muscarinic receptor stimulation were dependent on GTP. Responses were reduced when GTP was excluded from the intracellular recording solution or when GDP-beta-S was added. Intracellular GTP-gamma-S activated spontaneous fluctuations and permitted only one acetylcholine-(ACh) induced current response. These results implicate GTP-binding proteins (G protein) in the signal transduction pathway. This G protein is probably not pertussis toxin-sensitive as the ACh-induced electrical response was not abolished by pertussis toxin treatment. 3. Cell-attached single-channel recordings revealed activation of ion channels within the patch during application of ACh outside the patch, implying that second messengers might be involved in the ACh-induced response. Two types of K+ channel were activated, a discrete channel of 36 pS and channel activity calculated to be about 5 pS. 4. Application of 8-bromo cyclic AMP or 1-oleoyl-1,2-acetylglycerol (OAG) produced no electrical response and did not affect the ACh-induced responses. Phorbol myristic acetate (PMA) evoked no electrical response, but reduced the ACh-induced responses. 5. Inclusion of inositol 1,4,5-trisphosphate (IP3) in the intracellular pipette solution activated outward currents at -50 mV associated with an increase in conductance. The IP3-induced current response reversed polarity at -65 mV and showed a dependence on K+. Increasing the intracellular free Ca2+ concentration ([Ca2+]i) from 20 nM to 1 microM also induced an outward current response associated with an increase in conductance. Inclusion of inositol 1,3,4,5-tetrakisphosphate (IP4) in the intracellular solution

  15. Design and Functional Characterization of a Novel, Arrestin-Biased Designer G Protein-Coupled Receptor

    PubMed Central

    Nakajima, Ken-ichiro

    2012-01-01

    Mutational modification of distinct muscarinic receptor subtypes has yielded novel designer G protein-coupled receptors (GPCRs) that are unable to bind acetylcholine (ACh), the endogenous muscarinic receptor ligand, but can be efficiently activated by clozapine-N-oxide (CNO), an otherwise pharmacologically inert compound. These CNO-sensitive designer GPCRs [alternative name: designer receptors exclusively activated by designer drug (DREADDs)] have emerged as powerful new tools to dissect the in vivo roles of distinct G protein signaling pathways in specific cell types or tissues. As is the case with other GPCRs, CNO-activated DREADDs not only couple to heterotrimeric G proteins but can also recruit proteins of the arrestin family (arrestin-2 and -3). Accumulating evidence suggests that arrestins can act as scaffolding proteins to promote signaling through G protein-independent signaling pathways. To explore the physiological relevance of these arrestin-dependent signaling pathways, the availability of an arrestin-biased DREADD would be highly desirable. In this study, we describe the development of an M3 muscarinic receptor-based DREADD [Rq(R165L)] that is no longer able to couple to G proteins but can recruit arrestins and promote extracellular signal-regulated kinase-1/2 phosphorylation in an arrestin- and CNO-dependent fashion. Moreover, CNO treatment of mouse insulinoma (MIN6) cells expressing the Rq(R165L) construct resulted in a robust, arrestin-dependent stimulation of insulin release, directly implicating arrestin signaling in the regulation of insulin secretion. This newly developed arrestin-biased DREADD represents an excellent novel tool to explore the physiological relevance of arrestin signaling pathways in distinct tissues and cell types. PMID:22821234

  16. Urtica dioica leaves modulates muscarinic cholinergic system in the hippocampus of streptozotocin-induced diabetic mice.

    PubMed

    Patel, Sita Sharan; Parashar, Arun; Udayabanu, Malairaman

    2015-06-01

    Diabetes mellitus is a chronic metabolic disorder and has been associated with cognitive dysfunction. In our earlier study, chronic Urtica dioica (UD) treatment significantly ameliorated diabetes induced associative and spatial memory deficit in mice. The present study was designed to explore the effect of UD leaves extract on muscarinic cholinergic system, which has long been known to be involved in cognition. Streptozotocin (STZ) (50 mg/kg, i.p., consecutively for 5 days) was used to induce diabetes followed by treatment with UD extract (50 mg/kg, oral) or rosiglitazone (5 mg/kg, oral) for 8 weeks. STZ-induced diabetic mice showed significant reduction in hippocampal muscarinic acetylcholine receptor-1 and choline acetyltransferase expressions. Chronic diabetes significantly up-regulated the protein expression of acetylcholinesterase associated with oxidative stress in hippocampus. Besides, STZ-induced diabetic mice showed hypolocomotion with up-regulation of muscarinic acetylcholine receptor-4 expression in striatum. Chronic UD treatment significantly attenuated the cholinergic dysfunction and oxidative stress in the hippocampus of diabetic mice. UD had no effect on locomotor activity and muscarinic acetylcholine receptor-4 expression in striatum. In conclusion, UD leaves extract has potential to reverse diabetes mediated alteration in muscarinic cholinergic system in hippocampus and thereby improve memory functions.

  17. Reductions of {sup 56}Fe heavy-particle irradiation-induced deficits in striatal muscarinic receptor sensitivity by selective cross-activation/inhibition of second-messenger systems

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Joseph, J.A.; Villalobos-Molina, R.; Rabin, B.M.

    Recent experiments have revealed radiation-induced losses of sensitivity of rodent neostriatal muscarinic receptors to stimulation by cholinergic agonists that appears as reduction in oxotremorine enhancement of K{sup +}-evoked dopamine release. These losses were postulated to be the result of radiation-induced alterations early in phosphoinositide-mediated signal transduction. Additional findings indicated that if the ligand-receptor-G protein interface was bypassed no radiation deficits were seen. In the present study, radiation-induced deficits in K{sup +}-evoked dopamine release were examined in perifused striatal tissue obtained from rats exposed to 0,0.1 or 1.0 Gy of {sup 56}Fe particles. Results showed that these deficits could be reducedmore » by co-applying combinations of various pharmacological agents that were known to have differential effects on various second messengers such as 1,4,5-inositoltrisphosphate (IP{sub 3}). Combinations included oxotremorine-carbachol, and either oxotremorine or carbachol with arginine vasopressin or arachidonic acid. These results are discussed in terms of putative radiation-induced changes in receptor-containing membranes which alter receptor-G protein coupling/uncoupling. 49 refs., 4 figs.« less

  18. Differing time dependencies of object recognition memory impairments produced by nicotinic and muscarinic cholinergic antagonism in perirhinal cortex

    PubMed Central

    Tinsley, Chris J.; Fontaine-Palmer, Nadine S.; Vincent, Maria; Endean, Emma P.E.; Aggleton, John P.; Brown, Malcolm W.; Warburton, E. Clea

    2011-01-01

    The roles of muscarinic and nicotinic cholinergic receptors in perirhinal cortex in object recognition memory were compared. Rats' discrimination of a novel object preference test (NOP) test was measured after either systemic or local infusion into the perirhinal cortex of the nicotinic receptor antagonist methyllycaconitine (MLA), which targets alpha-7 (α7) amongst other nicotinic receptors or the muscarinic receptor antagonists scopolamine, AFDX-384, and pirenzepine. Methyllycaconitine administered systemically or intraperirhinally before acquisition impaired recognition memory tested after a 24-h, but not a 20-min delay. In contrast, all three muscarinic antagonists produced a similar, unusual pattern of impairment with amnesia after a 20-min delay, but remembrance after a 24-h delay. Thus, the amnesic effects of nicotinic and muscarinic antagonism were doubly dissociated across the 20-min and 24-h delays. The same pattern of shorter-term but not longer-term memory impairment was found for scopolamine whether the object preference test was carried out in a square arena or a Y-maze and whether rats of the Dark Agouti or Lister-hooded strains were used. Coinfusion of MLA and either scopolamine or AFDX-384 produced an impairment profile matching that for MLA. Hence, the antagonists did not act additively when coadministered. These findings establish an important role in recognition memory for both nicotinic and muscarinic cholinergic receptors in perirhinal cortex, and provide a challenge to simple ideas about the role of cholinergic processes in recognition memory: The effects of muscarinic and nicotinic antagonism are neither independent nor additive. PMID:21693636

  19. Differing time dependencies of object recognition memory impairments produced by nicotinic and muscarinic cholinergic antagonism in perirhinal cortex.

    PubMed

    Tinsley, Chris J; Fontaine-Palmer, Nadine S; Vincent, Maria; Endean, Emma P E; Aggleton, John P; Brown, Malcolm W; Warburton, E Clea

    2011-01-01

    The roles of muscarinic and nicotinic cholinergic receptors in perirhinal cortex in object recognition memory were compared. Rats' discrimination of a novel object preference test (NOP) test was measured after either systemic or local infusion into the perirhinal cortex of the nicotinic receptor antagonist methyllycaconitine (MLA), which targets alpha-7 (α7) amongst other nicotinic receptors or the muscarinic receptor antagonists scopolamine, AFDX-384, and pirenzepine. Methyllycaconitine administered systemically or intraperirhinally before acquisition impaired recognition memory tested after a 24-h, but not a 20-min delay. In contrast, all three muscarinic antagonists produced a similar, unusual pattern of impairment with amnesia after a 20-min delay, but remembrance after a 24-h delay. Thus, the amnesic effects of nicotinic and muscarinic antagonism were doubly dissociated across the 20-min and 24-h delays. The same pattern of shorter-term but not longer-term memory impairment was found for scopolamine whether the object preference test was carried out in a square arena or a Y-maze and whether rats of the Dark Agouti or Lister-hooded strains were used. Coinfusion of MLA and either scopolamine or AFDX-384 produced an impairment profile matching that for MLA. Hence, the antagonists did not act additively when coadministered. These findings establish an important role in recognition memory for both nicotinic and muscarinic cholinergic receptors in perirhinal cortex, and provide a challenge to simple ideas about the role of cholinergic processes in recognition memory: The effects of muscarinic and nicotinic antagonism are neither independent nor additive.

  20. Treatment with LPS plus INF-γ induces the expression and function of muscarinic acetylcholine receptors, modulating NIH3T3 cell proliferation: participation of NOS and COX.

    PubMed

    Español, A J; Maddaleno, M O; Lombardi, M G; Cella, M; Martínez Pulido, P; Sales, M E

    2014-11-01

    LPS and IFN-γ are potent stimuli of inflammation, a process in which fibroblasts are frequently involved. We analysed the effect of treatment with LPS plus IFN-γ on the expression and function of muscarinic acetylcholine receptors in NIH3T3 fibroblasts with regards to proliferation of these cells. We also investigated the participation of NOS and COX, and the role of NF-κB in this process. NIH3T3 cells were treated with LPS (10 ng·mL(-1)) plus IFN-γ (0.5 ng·mL(-1)) for 72 h (iNIH3T3 cells). Cell proliferation was evaluated with MTT and protein expression by Western blot analysis. NOS and COX activities were measured by the Griess method and radioimmunoassay respectively. The cholinoceptor agonist carbachol was more effective at stimulating proliferation in iNIH3T3 than in NIH3T3 cells, probably due to the de novo induction of M3 and M5 muscarinic receptors independently of NF-κB activation. iNIH3T3 cells produced higher amounts of NO and PGE2 than NIH3T3 cells, concomitantly with an up-regulation of NOS1 and COX-2, and with the de novo induction of NOS2/3 in inflamed cells. We also found a positive feedback between NOS and COX that could potentiate inflammation. Inflammation induced the expression of muscarinic receptors and, therefore,stimulated carbachol-induced proliferation of fibroblasts. Inflammation also up-regulated the expression of NOS and COX-2, thus potentiating the effect of carbachol on NO and PGE2 production. A positive crosstalk between NOS and COX triggered by carbachol in inflamed cells points to muscarinic receptors as potential therapeutic targets in inflammation. © 2014 The British Pharmacological Society.

  1. Treatment with LPS plus INF-γ induces the expression and function of muscarinic acetylcholine receptors, modulating NIH3T3 cell proliferation: participation of NOS and COX

    PubMed Central

    Español, A J; Maddaleno, M O; Lombardi, M G; Cella, M; Martínez Pulido, P; Sales, M E

    2014-01-01

    Background and Purpose LPS and IFN-γ are potent stimuli of inflammation, a process in which fibroblasts are frequently involved. We analysed the effect of treatment with LPS plus IFN-γ on the expression and function of muscarinic acetylcholine receptors in NIH3T3 fibroblasts with regards to proliferation of these cells. We also investigated the participation of NOS and COX, and the role of NF-κB in this process. Experimental Approach NIH3T3 cells were treated with LPS (10 ng·mL−1) plus IFN-γ (0.5 ng·mL−1) for 72 h (iNIH3T3 cells). Cell proliferation was evaluated with MTT and protein expression by Western blot analysis. NOS and COX activities were measured by the Griess method and radioimmunoassay respectively. Key Results The cholinoceptor agonist carbachol was more effective at stimulating proliferation in iNIH3T3 than in NIH3T3 cells, probably due to the de novo induction of M3 and M5 muscarinic receptors independently of NF-κB activation. iNIH3T3 cells produced higher amounts of NO and PGE2 than NIH3T3 cells, concomitantly with an up-regulation of NOS1 and COX-2, and with the de novo induction of NOS2/3 in inflamed cells. We also found a positive feedback between NOS and COX that could potentiate inflammation. Conclusions and Implications Inflammation induced the expression of muscarinic receptors and, therefore,stimulated carbachol-induced proliferation of fibroblasts. Inflammation also up-regulated the expression of NOS and COX-2, thus potentiating the effect of carbachol on NO and PGE2 production. A positive crosstalk between NOS and COX triggered by carbachol in inflamed cells points to muscarinic receptors as potential therapeutic targets in inflammation. PMID:24990429

  2. Bitopic Binding Mode of an M1 Muscarinic Acetylcholine Receptor Agonist Associated with Adverse Clinical Trial Outcomes

    PubMed Central

    Bradley, Sophie J.; Molloy, Colin; Bundgaard, Christoffer; Mogg, Adrian J.; Thompson, Karen J.; Dwomoh, Louis; Sanger, Helen E.; Crabtree, Michael D.; Brooke, Simon M.; Sexton, Patrick M.; Felder, Christian C.; Christopoulos, Arthur; Broad, Lisa M.

    2018-01-01

    The realization of the therapeutic potential of targeting the M1 muscarinic acetylcholine receptor (mAChR) for the treatment of cognitive decline in Alzheimer’s disease has prompted the discovery of M1 mAChR ligands showing efficacy in alleviating cognitive dysfunction in both rodents and humans. Among these is GSK1034702 (7-fluoro-5-methyl-3-[1-(oxan-4-yl)piperidin-4-yl]-1H-benzimidazol-2-one), described previously as a potent M1 receptor allosteric agonist, which showed procognitive effects in rodents and improved immediate memory in a clinical nicotine withdrawal test but induced significant side effects. Here we provide evidence using ligand binding, chemical biology and functional assays to establish that rather than the allosteric mechanism claimed, GSK1034702 interacts in a bitopic manner at the M1 mAChR such that it can concomitantly span both the orthosteric and an allosteric binding site. The bitopic nature of GSK1034702, together with the intrinsic agonist activity and a lack of muscarinic receptor subtype selectivity reported here, all likely contribute to the adverse effects of this molecule in clinical trials. Although they impart beneficial effects on learning and memory, we conclude that these properties are undesirable in a clinical candidate due to the likelihood of adverse side effects. Rather, our data support the notion that “pure” positive allosteric modulators showing selectivity for the M1 mAChR with low levels of intrinsic activity would be preferable to provide clinical efficacy with low adverse responses. PMID:29695609

  3. Bitopic Binding Mode of an M1 Muscarinic Acetylcholine Receptor Agonist Associated with Adverse Clinical Trial Outcomes.

    PubMed

    Bradley, Sophie J; Molloy, Colin; Bundgaard, Christoffer; Mogg, Adrian J; Thompson, Karen J; Dwomoh, Louis; Sanger, Helen E; Crabtree, Michael D; Brooke, Simon M; Sexton, Patrick M; Felder, Christian C; Christopoulos, Arthur; Broad, Lisa M; Tobin, Andrew B; Langmead, Christopher J

    2018-06-01

    The realization of the therapeutic potential of targeting the M 1 muscarinic acetylcholine receptor (mAChR) for the treatment of cognitive decline in Alzheimer's disease has prompted the discovery of M 1 mAChR ligands showing efficacy in alleviating cognitive dysfunction in both rodents and humans. Among these is GSK1034702 (7-fluoro-5-methyl-3-[1-(oxan-4-yl)piperidin-4-yl]-1 H -benzimidazol-2-one), described previously as a potent M 1 receptor allosteric agonist, which showed procognitive effects in rodents and improved immediate memory in a clinical nicotine withdrawal test but induced significant side effects. Here we provide evidence using ligand binding, chemical biology and functional assays to establish that rather than the allosteric mechanism claimed, GSK1034702 interacts in a bitopic manner at the M 1 mAChR such that it can concomitantly span both the orthosteric and an allosteric binding site. The bitopic nature of GSK1034702, together with the intrinsic agonist activity and a lack of muscarinic receptor subtype selectivity reported here, all likely contribute to the adverse effects of this molecule in clinical trials. Although they impart beneficial effects on learning and memory, we conclude that these properties are undesirable in a clinical candidate due to the likelihood of adverse side effects. Rather, our data support the notion that "pure" positive allosteric modulators showing selectivity for the M 1 mAChR with low levels of intrinsic activity would be preferable to provide clinical efficacy with low adverse responses. Copyright © 2018 by The Author(s).

  4. Spinal α2-adrenergic and muscarinic receptors and the NO release cascade mediate supraspinally produced effectiveness of gabapentin at decreasing mechanical hypersensitivity in mice after partial nerve injury

    PubMed Central

    Takasu, Keiko; Honda, Motoko; Ono, Hideki; Tanabe, Mitsuo

    2006-01-01

    After partial nerve injury, the central analgesic effect of systemically administered gabapentin is mediated by both supraspinal and spinal actions. We further evaluate the mechanisms related to the supraspinally mediated analgesic actions of gabapentin involving the descending noradrenergic system. Intracerebroventricularly (i.c.v.) administered gabapentin (100 μg) decreased thermal and mechanical hypersensitivity in a murine chronic pain model that was prepared by partial ligation of the sciatic nerve. These effects were abolished by intrathecal (i.t.) injection of either yohimbine (3 μg) or idazoxan (3 μg), α2-adrenergic receptor antagonists. Pretreatment with atropine (0.3 mg kg−1, i.p. or 0.1 μg, i.t.), a muscarinic receptor antagonist, completely suppressed the effect of i.c.v.-injected gabapentin on mechanical hypersensitivity, whereas its effect on thermal hypersensitivity remained unchanged. Similar effects were obtained with pirenzepine (0.1 μg, i.t.), a selective M1-muscarinic receptor antagonist, but not with methoctramine (0.1 and 0.3 μg, i.t.), a selective M2-muscarinic receptor antagonist. The cholinesterase inhibitor neostigmine (0.3 ng, i.t.) potentiated only the analgesic effect of i.c.v. gabapentin on mechanical hypersensitivity, confirming spinal acetylcholine release downstream of the supraspinal action of gabapentin. Moreover, the effect of i.c.v. gabapentin on mechanical but not thermal hypersensitivity was reduced by i.t. injection of L-NAME (3 μg) or L-NMMA (10 μg), both of which are nitric oxide (NO) synthase inhibitors. Systemically administered naloxone (10 mg kg−1, i.p.), an opioid receptor antagonist, failed to suppress the analgesic actions of i.c.v. gabapentin, indicating that opioid receptors are not involved in activation of the descending noradrenergic system by gabapentin. Thus, the supraspinally mediated effect of gabapentin on mechanical hypersensitivity involves activation of spinal α2

  5. BK channel β1 subunits regulate airway contraction secondary to M2 muscarinic acetylcholine receptor mediated depolarization

    PubMed Central

    Semenov, Iurii; Wang, Bin; Herlihy, Jeremiah T; Brenner, Robert

    2011-01-01

    Abstract The large conductance calcium- and voltage-activated potassium channel (BK channel) and its smooth muscle-specific β1 subunit regulate excitation–contraction coupling in many types of smooth muscle cells. However, the relative contribution of BK channels to control of M2- or M3-muscarinic acetylcholine receptor mediated airway smooth muscle contraction is poorly understood. Previously, we showed that knockout of the BK channel β1 subunit enhances cholinergic-evoked trachea contractions. Here, we demonstrate that the enhanced contraction of the BK β1 knockout can be ascribed to a defect in BK channel opposition of M2 receptor-mediated contractions. Indeed, the enhanced contraction of β1 knockout is eliminated by specific M2 receptor antagonism. The role of BK β1 to oppose M2 signalling is evidenced by a greater than fourfold increase in the contribution of L-type voltage-dependent calcium channels to contraction that otherwise does not occur with M2 antagonist or with β1 containing BK channels. The mechanism through which BK channels oppose M2-mediated recruitment of calcium channels is through a negative shift in resting voltage that offsets, rather than directly opposes, M2-mediated depolarization. The negative shift in resting voltage is reduced to similar extents by BK β1 knockout or by paxilline block of BK channels. Normalization of β1 knockout baseline voltage with low external potassium eliminated the enhanced M2-receptor mediated contraction. In summary, these findings indicate that an important function of BK/β1 channels is to oppose cholinergic M2 receptor-mediated depolarization and activation of calcium channels by restricting excitation–contraction coupling to more negative voltage ranges. PMID:21300746

  6. Muscarinic Receptors Modulate Dendrodendritic Inhibitory Synapses to Sculpt Glomerular Output

    PubMed Central

    Shao, Zuoyi; Puche, Adam; Wachowiak, Matt; Rothermel, Markus

    2015-01-01

    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. PMID:25855181

  7. 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. Copyright © 2015 the authors 0270-6474/15/355680-13$15.00/0.

  8. Determinants of positive cooperativity between strychnine-like allosteric modulators and N-methylscopolamine at muscarinic receptors.

    PubMed

    Jakubík, Jan; Dolezal, Vladimír

    2006-01-01

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

  9. 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.

  10. Bisquaternary dimers of strychnine and brucine. A new class of potent enhancers of antagonist binding to muscarinic M2 receptors.

    PubMed

    Zlotos, D P; Buller, S; Holzgrabe, U; Mohr, K

    2003-06-12

    Bisquaternary dimers of strychnine and brucine were synthesized and their allosteric effect on muscarinic acetylcholine M(2) receptors was examined. The compounds retarded the dissociation of the antagonist [(3)H]N-methylscopolamine ([(3)H]NMS) from porcine cardiac cholinoceptors. This action indicated ternary complex formation. All compounds exhibited higher affinity to the allosteric site of [(3)H]NMS-occupied M(2) receptors than the monomeric strychnine and brucine, while the positive cooperativity with NMS was fully maintained. SAR studies revealed the unchanged strychnine ring as an important structural feature for high allosteric potency.

  11. 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.

  12. An Antibody Biosensor Establishes the Activation of the M1 Muscarinic Acetylcholine Receptor during Learning and Memory.

    PubMed

    Butcher, Adrian J; Bradley, Sophie J; Prihandoko, Rudi; Brooke, Simon M; Mogg, Adrian; Bourgognon, Julie-Myrtille; Macedo-Hatch, Timothy; Edwards, Jennifer M; Bottrill, Andrew R; Challiss, R A John; Broad, Lisa M; Felder, Christian C; Tobin, Andrew B

    2016-04-22

    Establishing the in vivo activation status of G protein-coupled receptors would not only indicate physiological roles of G protein-coupled receptors but would also aid drug discovery by establishing drug/receptor engagement. Here, we develop a phospho-specific antibody-based biosensor to detect activation of the M1 muscarinic acetylcholine receptor (M1 mAChR) in vitro and in vivo Mass spectrometry phosphoproteomics identified 14 sites of phosphorylation on the M1 mAChR. Phospho-specific antibodies to four of these sites established that serine at position 228 (Ser(228)) on the M1 mAChR showed extremely low levels of basal phosphorylation that were significantly up-regulated by orthosteric agonist stimulation. In addition, the M1 mAChR-positive allosteric modulator, 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, enhanced acetylcholine-mediated phosphorylation at Ser(228) These data supported the hypothesis that phosphorylation at Ser(228) was an indicator of M1 mAChR activation. This was further supported in vivo by the identification of phosphorylated Ser(228) on the M1 mAChR in the hippocampus of mice following administration of the muscarinic ligands xanomeline and 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid. Finally, Ser(228) phosphorylation was seen to increase in the CA1 region of the hippocampus following memory acquisition, a response that correlated closely with up-regulation of CA1 neuronal activity. Thus, determining the phosphorylation status of the M1 mAChR at Ser(228) not only provides a means of establishing receptor activation following drug treatment both in vitro and in vivo but also allows for the mapping of the activation status of the M1 mAChR in the hippocampus following memory acquisition thereby establishing a link between M1 mAChR activation and hippocampus-based memory and learning. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Dynamic regulation of glycinergic input to spinal dorsal horn neurones by muscarinic receptor subtypes in rats.

    PubMed

    Wang, Xiu-Li; Zhang, Hong-Mei; Li, De-Pei; Chen, Shao-Rui; Pan, Hui-Lin

    2006-03-01

    Activation of spinal muscarinic acetylcholine receptors (mAChRs) inhibits nociception. However, the cellular mechanisms of this action are not fully known. In this study, we determined the role of mAChR subtypes in regulation of synaptic glycine release in the spinal cord. Whole-cell voltage-clamp recordings were performed on lamina II neurones in the rat spinal cord slices. The mAChR agonist oxotremorine-M significantly increased the frequency of glycinergic sIPSCs but not mIPSCs. Surprisingly, the effect of oxotremorine-M on sIPSCs was largely attenuated at a higher concentration. On the other hand, 1-10 microm oxotremorine-M dose-dependently increased the frequency of sIPSCs in rats pretreated with intrathecal pertussis toxin. Furthermore, oxotremorine-M also dose-dependently increased the frequency of sIPSCs in the presence of himbacine (an M2/M4 mAChR antagonist) or AF-DX116 (an M2 mAChR antagonist). The M3 mAChR antagonist 4-DAMP abolished the stimulatory effect of oxotremorine-M on sIPSCs. Interestingly, the GABA(B) receptor antagonist CGP55845 potentiated the stimulatory effect of oxotremorine-M on sIPSCs. In the presence of CGP55845, both himbacine and AF-DX116 similarly reduced the potentiating effect of oxotremorine-M on sIPSCs. Collectively, these data suggest that the M3 subtype is present on the somatodendritic site of glycinergic neurones and is mainly responsible for muscarinic potentiation of glycinergic input to spinal dorsal horn neurones. Concurrent stimulation of mAChRs on adjacent GABAergic interneurones attenuates synaptic glycine release through presynaptic GABA(B) receptors on glycinergic interneurones. This study illustrates a complex dynamic interaction between GABAergic and glycinergic synapses in the spinal cord dorsal horn.

  14. Stereochemical requirements for central and peripheral muscarinic and antimuscarinic activity of some acetylenic compounds related to oxotremorine.

    PubMed Central

    Dahlbom, R.; Jenden, D. J.; Resul, B.; Ringdahl, B.

    1982-01-01

    1 The enantiomers of some analogues of the central muscarinic agent, oxotremorine, were prepared and investigated for tremorogenic and tremorolytic activity in intact mice and for muscarinic and antimuscarinic activity on the isolated ileum of the guinea-pig. 2 The R-isomers were more potent than the S-isomers both in vivo and in vitro regardless of whether the compounds are agonists, partial agonists or competitive antagonists. 3 It is suggested that in the oxotremorine series, agonists and antagonists interact with a common receptor site, in contrast to classical muscarinic antagonists which are believed to bind also to accessory receptor areas, located close to the agonist binding site. PMID:7093587

  15. Specificity of arrestin subtypes in regulating airway smooth muscle G protein-coupled receptor signaling and function.

    PubMed

    Pera, Tonio; Hegde, Akhil; Deshpande, Deepak A; Morgan, Sarah J; Tiegs, Brian C; Theriot, Barbara S; Choi, Yeon H; Walker, Julia K L; Penn, Raymond B

    2015-10-01

    Arrestins have been shown to regulate numerous G protein-coupled receptors (GPCRs) in studies employing receptor/arrestin overexpression in artificial cell systems. Which arrestin isoforms regulate which GPCRs in primary cell types is poorly understood. We sought to determine the effect of β-arrestin-1 or β-arrestin-2 inhibition or gene ablation on signaling and function of multiple GPCRs endogenously expressed in airway smooth muscle (ASM). In vitro [second messenger (calcium, cAMP generation)], ex vivo (ASM tension generation in suspended airway), and in vivo (invasive airway resistance) analyses were performed on human ASM cells and murine airways/whole animal subject to β-arrestin-1 or -2 knockdown or knockout (KO). In both human and murine model systems, knockdown or KO of β-arrestin-2 relative to control missense small interfering RNA or wild-type mice selectively increased (40-60%) β2-adrenoceptor signaling and function. β-arrestin-1 knockdown or KO had no effect on signaling and function of β2-adrenoceptor or numerous procontractile GPCRs, but selectively inhibited M3 muscarinic acetylcholine receptor signaling (∼50%) and function (∼25% ex vivo, >50% in vivo) without affecting EC50 values. Arrestin subtypes differentially regulate ASM GPCRs and β-arrestin-1 inhibition represents a novel approach to managing bronchospasm in obstructive lung diseases. © FASEB.

  16. Critical role of CA1 muscarinic receptors on memory acquisition deficit induced by total (TSD) and REM sleep deprivation (RSD).

    PubMed

    Javad-Moosavi, Bibi-Zahra; Vaezi, Gholamhassan; Nasehi, Mohammad; Haeri-Rouhani, Seyed-Ali; Zarrindast, Mohammad-Reza

    2017-10-03

    Despite different theories regarding sleep physiological function, an overall census indicates that sleep is useful for neural plasticity which eventually strengthens cognition and brain performance. Different studies show that sleep deprivation (SD) leads to impaired learning and hippocampus dependent memory. According to some studies, cholinergic system plays an important role in sleep (particularly REM sleep), learning, memory, and its retrieval. So this study has been designed to investigate the effect of CA1 Cholinergic Muscarinic Receptors on memory acquisition deficit induced by total sleep deprivation (TSD) and REM sleep deprivation (RSD). A modified water box (locomotor activity may be provide a limiting factor in this method of SD) or multiple platforms were used for induction of TSD or RSD, respectively. Inhibitory passive avoidance apparatus has been used to determine the effects of SD and its changes by physostigmine (as cholinesterase inhibitor) or scopolamine (muscarinic receptor antagonist) on memory formation. Because locomotor activity and pain perception induce critical roles in passive avoidance memory formation, we also measured these factors by open field and hot-plate instruments, respectively. The results showed that TSD and RSD for 24 hours impaired memory formation but they did not alter locomotor activity. TSD also induced analgesia effect, but RSD did not alter it. Intra-CA1 injection of physostigmine (0.0001μg/rat) and scopolamine (0.01μg/rat) did not alter memory acquisition in the sham-TSD or sham-RSD, by themselves. Moreover, intra-CA1 injection of sub-threshold dose of physostigmine (0.0001μg/rat) and scopolamine (0.01μg/rat) could restore the memory acquisition deficit induced by RSD, while scopolamine could restore TSD-induced amnesia. Both drugs reversed analgesia induced by TSD. None of previous interventions altered locomotor activity. According to this study, CA1 cholinergic muscarinic receptors play an important role in

  17. Pharmacological and ionic characterizations of the muscarinic receptors modulating (/sup 3/H)acetylcholine release from rat cortical synaptosomes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Meyer, E.M.; Otero, D.H.

    The muscarinic receptors that modulate acetylcholine release from rat cortical synaptosomes were characterized with respect to sensitivity to drugs that act selectively at M1 or M2 receptor subtypes, as well as to changes in ionic strength and membrane potential. The modulatory receptors appear to be of the M2 type, since they are activated by carbachol, acetylcholine, methacholine, oxotremorine, and bethanechol, but not by pilocarpine, and are blocked by atropine, scopolamine, and gallamine (at high concentrations), but not by pirenzepine or dicyclomine. The ED50S for carbachol, acetylcholine, and oxotremorine are less than 10 microM, suggesting that the high affinity state ofmore » the receptor is functional. High ionic strength induced by raising the NaCl concentration has no effect on agonist (oxotremorine) potency, but increases the efficacy of this compound, which disagrees with receptor-binding studies. On the other hand, depolarization with either KCl or with veratridine (20 microM) reduces agonist potencies by approximately an order of magnitude, suggesting a potential mechanism for receptor regulation.« less

  18. Cooperative ethylene receptor signaling

    PubMed Central

    Liu, Qian; Wen, Chi-Kuang

    2012-01-01

    The gaseous plant hormone ethylene is perceived by a family of five ethylene receptor members in the dicotyledonous model plant Arabidopsis. Genetic and biochemical studies suggest that the ethylene response is suppressed by ethylene receptor complexes, but the biochemical nature of the receptor signal is unknown. Without appropriate biochemical measures to trace the ethylene receptor signal and quantify the signal strength, the biological significance of the modulation of ethylene responses by multiple ethylene receptors has yet to be fully addressed. Nevertheless, the ethylene receptor signal strength can be reflected by degrees in alteration of various ethylene response phenotypes and in expression levels of ethylene-inducible genes. This mini-review highlights studies that have advanced our understanding of cooperative ethylene receptor signaling. PMID:22827938

  19. 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.

  20. Signaling mechanisms mediating muscarinic enhancement of GABAergic synaptic transmission in the spinal cord.

    PubMed

    Zhang, H-M; Chen, S-R; Cai, Y-Q; Richardson, T E; Driver, L C; Lopez-Berestein, G; Pan, H-L

    2009-02-18

    Activation of muscarinic acetylcholine receptors (mAChRs) inhibits spinal nociceptive transmission by potentiation of GABAergic tone through M(2), M(3), and M(4) subtypes. To study the signaling mechanisms involved in this unique mAChR action, GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) of lamina II neurons were recorded using whole-cell patch clamp techniques in rat spinal cord slices. The mAChR agonist oxotremorine-M caused a profound increase in the frequency of GABAergic sIPSCs, which was abolished in the Ca(2+)-free solution. Inhibition of voltage-gated Ca(2+) channels with Cd(2+) and Ni(2+) largely reduced the effect of oxotremorine-M on sIPSCs. Blocking nonselective cation channels (NSCCs) with SKF96365 or 2-APB also largely attenuated the effect of oxotremorine-M. However, the KCNQ channel blocker XE991 and the adenylyl cyclase inhibitor MDL12330A had no significant effect on oxotremorine-M-induced increases in sIPSCs. Furthermore, the phosphoinositide-3-kinase (PI3K) inhibitor wortmannin or LY294002 significantly reduced the potentiating effect of oxotremorine-M on sIPSCs. In the spinal cord in which the M(3) subtype was specifically knocked down by intrathecal small interfering RNA (siRNA) treatment, SKF96365 and wortmannin still significantly attenuated the effect of oxotremorine-M. In contrast, SKF96365 and wortmannin both failed to alter the effect of oxotremorine-M on sIPSCs when the M(2)/M(4) mAChRs were blocked. Therefore, our study provides new evidence that activation of mAChRs increases synaptic GABA release through Ca(2+) influx and voltage-gated Ca(2+) channels. The PI3K-NSCC signaling cascade is primarily involved in the excitation of GABAergic interneurons by the M(2)/M(4) mAChRs in the spinal dorsal horn.

  1. An Antibody Biosensor Establishes the Activation of the M1 Muscarinic Acetylcholine Receptor during Learning and Memory*♦

    PubMed Central

    Butcher, Adrian J.; Bradley, Sophie J.; Prihandoko, Rudi; Brooke, Simon M.; Mogg, Adrian; Bourgognon, Julie-Myrtille; Macedo-Hatch, Timothy; Edwards, Jennifer M.; Bottrill, Andrew R.; Challiss, R. A. John; Broad, Lisa M.; Felder, Christian C.; Tobin, Andrew B.

    2016-01-01

    Establishing the in vivo activation status of G protein-coupled receptors would not only indicate physiological roles of G protein-coupled receptors but would also aid drug discovery by establishing drug/receptor engagement. Here, we develop a phospho-specific antibody-based biosensor to detect activation of the M1 muscarinic acetylcholine receptor (M1 mAChR) in vitro and in vivo. Mass spectrometry phosphoproteomics identified 14 sites of phosphorylation on the M1 mAChR. Phospho-specific antibodies to four of these sites established that serine at position 228 (Ser228) on the M1 mAChR showed extremely low levels of basal phosphorylation that were significantly up-regulated by orthosteric agonist stimulation. In addition, the M1 mAChR-positive allosteric modulator, 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, enhanced acetylcholine-mediated phosphorylation at Ser228. These data supported the hypothesis that phosphorylation at Ser228 was an indicator of M1 mAChR activation. This was further supported in vivo by the identification of phosphorylated Ser228 on the M1 mAChR in the hippocampus of mice following administration of the muscarinic ligands xanomeline and 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid. Finally, Ser228 phosphorylation was seen to increase in the CA1 region of the hippocampus following memory acquisition, a response that correlated closely with up-regulation of CA1 neuronal activity. Thus, determining the phosphorylation status of the M1 mAChR at Ser228 not only provides a means of establishing receptor activation following drug treatment both in vitro and in vivo but also allows for the mapping of the activation status of the M1 mAChR in the hippocampus following memory acquisition thereby establishing a link between M1 mAChR activation and hippocampus-based memory and learning. PMID:26826123

  2. 3-(2-Benzofuranyl)quinuclidin-2-ene derivatives: novel muscarinic antagonists.

    PubMed

    Nordvall, G; Sundquist, S; Johansson, G; Glas, G; Nilvebrant, L; Hacksell, U

    1996-08-16

    A series of 26 derivatives of the novel muscarinic antagonist 3-(2-benzofuranyl)quinuclidin-2-ene (1) has been synthesized and evaluated for muscarinic and antimuscarinic properties. The affinity of the compounds was determined by competition experiments in homogenates of cerebral cortex, heart, parotid gland, and urinary bladder from guinea pigs using (-)-[3H]-3-quinuclidinyl benzilate as the radioligand, and the antimuscarinic-potency was determined in a functional assay on isolated guinea pig urinary bladder using carbachol as the agonist. The 5-fluorobenzofuranyl derivative was slightly more potent than 1. The 7-bromo-substituted 8 displayed a 14-fold tissue selectivity ratio for muscarinic receptors in the cortex versus the parotid gland. Comparative molecular field analysis and quantitative structure-activity relationship models were developed for this series of substituted benzofuranyl derivatives.

  3. Concomitant alteration in number and affinity of P2X and muscarinic receptors are associated with bladder dysfunction in early stage of diabetic rats.

    PubMed

    Yoshizawa, Tsuyoshi; Hayashi, Yukio; Yoshida, Akira; Yoshida, Shohei; Ito, Yoshihiko; Yamaguchi, Kenya; Yamada, Shizuo; Takahashi, Satoru

    2018-03-01

    To investigate time course of bladder dysfunction and concurrent changes in number and affinity of the muscarinic and P 2 X receptor in the early stage of streptozotocin (STZ)-induced diabetic rats. Diabetic rats were prepared by the intraperitoneal injection of 50 mg/kg of STZ to 7-week-old female Wistar rats. We performed recording of 24-h voiding behavior and cystometry at 1, 4, 8, and 12 weeks after the induction of diabetes. A muscle strip experiments with electrical field stimulation (EFS), carbachol, and α,β-methylene adenosine 5'-triphosphate (α,β-MeATP) were also performed at the same time-points. Additionally, concurrent changes in number and affinity of bladder muscarinic and P 2 X receptor were measured by a radioreceptor assay using [N-methyl- 3 H] scopolamine methyl chloride ([ 3 H]NMS) and α,β-methylene-ATP (2,8- 3 H) tetrasodium salt ([ 3 H]α,β-MeATP). In STZ-induced diabetic rats, polydipsic polyuric pollakiuria were noted on recording of 24-h voiding behavior from early stage. Also, the residual urine volume markedly increased in diabetic rats on cystometry. In the muscle strip experiment, the detrusor contractions induced by EFS, carbachol, and α,β-MeATP were enhanced in STZ-induced diabetic rats. Based on the radioreceptor assay, the maximum number of sites (Bmax) for the specific binding of [ 3 H]NMS and [ 3 H]α,β-MeATP was concurrently increased in the bladder from diabetic rats. Increased bladder contractility is found in early stage of diabetic rats. Then, bladder dysfunction is associated with increased number of muscarinic and P 2 X receptors in STZ-induced diabetic rats.

  4. Selective Activation of M4 Muscarinic Acetylcholine Receptors Reverses MK-801-Induced Behavioral Impairments and Enhances Associative Learning in Rodents

    PubMed Central

    2015-01-01

    Positive allosteric modulators (PAMs) of the M4 muscarinic acetylcholine receptor (mAChR) represent a novel approach for the treatment of psychotic symptoms associated with schizophrenia and other neuropsychiatric disorders. We recently reported that the selective M4 PAM VU0152100 produced an antipsychotic drug-like profile in rodents after amphetamine challenge. Previous studies suggest that enhanced cholinergic activity may also improve cognitive function and reverse deficits observed with reduced signaling through the N-methyl-d-aspartate subtype of the glutamate receptor (NMDAR) in the central nervous system. Prior to this study, the M1 mAChR subtype was viewed as the primary candidate for these actions relative to the other mAChR subtypes. Here we describe the discovery of a novel M4 PAM, VU0467154, with enhanced in vitro potency and improved pharmacokinetic properties relative to other M4 PAMs, enabling a more extensive characterization of M4 actions in rodent models. We used VU0467154 to test the hypothesis that selective potentiation of M4 receptor signaling could ameliorate the behavioral, cognitive, and neurochemical impairments induced by the noncompetitive NMDAR antagonist MK-801. VU0467154 produced a robust dose-dependent reversal of MK-801-induced hyperlocomotion and deficits in preclinical models of associative learning and memory functions, including the touchscreen pairwise visual discrimination task in wild-type mice, but failed to reverse these stimulant-induced deficits in M4 KO mice. VU0467154 also enhanced the acquisition of both contextual and cue-mediated fear conditioning when administered alone in wild-type mice. These novel findings suggest that M4 PAMs may provide a strategy for addressing the more complex affective and cognitive disruptions associated with schizophrenia and other neuropsychiatric disorders. PMID:25137629

  5. Muscarinic acetylcholine receptor subtype expression in avian vestibular hair cells, nerve terminals and ganglion cells.

    PubMed

    Li, G Q; Kevetter, G A; Leonard, R B; Prusak, D J; Wood, T G; Correia, M J

    2007-04-25

    Muscarinic acetylcholine receptors (mAChRs) are widely expressed in the CNS and peripheral nervous system and play an important role in modulating the cell activity and function. We have shown that the cholinergic agonist carbachol reduces the pigeon's inwardly rectifying potassium channel (pKir2.1) ionic currents in native vestibular hair cells. We have cloned and sequenced pigeon mAChR subtypes M2-M5 and we have studied the expression of all five mAChR subtypes (M1-M5) in the pigeon vestibular end organs (semicircular canal ampullary cristae and utricular maculae), vestibular nerve fibers and the vestibular (Scarpa's) ganglion using tissue immunohistochemistry (IH), dissociated single cell immunocytochemistry (IC) and Western blotting (WB). We found that vestibular hair cells, nerve fibers and ganglion cells each expressed all five (M1-M5) mAChR subtypes. Two of the three odd-numbered mAChRs (M1, M5) were present on the hair cell cilia, supporting cells and nerve terminals. And all three odd numbered mAChRs (M1, M3 and M5) were expressed on cuticular plates, myelin sheaths and Schwann cells. Even-numbered mAChRs were seen on the nerve terminals. M2 was also shown on the cuticular plates and supporting cells. Vestibular efferent fibers and terminals were not identified in our studies. Results from WB of the dissociated vestibular epithelia, nerve fibers and vestibular ganglia were consistent with the results from IH and IC. Our findings suggest that there is considerable co-expression of the subtypes on the neural elements of the labyrinth. Further electrophysiological and pharmacological studies should delineate the mechanisms of action of muscarinic acetylcholine receptors on structures in the labyrinth.

  6. Effect of hyperglycaemia on muscarinic M3 receptor expression and secretory sensitivity to cholinergic receptor activation in islets.

    PubMed

    Hauge-Evans, A C; Reers, C; Kerby, A; Franklin, Z; Amisten, S; King, A J; Hassan, Z; Vilches-Flores, A; Tippu, Z; Persaud, S J; Jones, P M

    2014-10-01

    Islets are innervated by parasympathetic nerves which release acetylcholine (ACh) to amplify glucose-induced insulin secretion, primarily via muscarinic M3 receptors (M3R). Here we investigate the consequence of chronic hyperglycaemia on islet M3R expression and secretory sensitivity of mouse islets to cholinergic receptor activation. The impact of hyperglycaemia was studied in (i) islets isolated from ob/ob mice, (ii) alginate-encapsulated mouse islets transplanted intraperitoneally into streptozotocin-induced diabetic mice and (iii) mouse and human islets maintained in vitro at 5.5 or 16 mmol/l glucose. Blood glucose levels were assessed by a commercial glucose meter, insulin content by RIA and M3R expression by qPCR and immunohistochemistry. M3R mRNA expression was reduced in both ob/ob islets and islets maintained at 16 mmol/l glucose for 3 days (68 and 50% control, respectively). In all three models of hyperglycaemia the secretory sensitivity to the cholinergic receptor agonist, carbachol, was reduced by 60-70% compared to control islets. Treatment for 72 h with the irreversible PKC activator, PMA, or the PKC inhibitor, Gö6983, did not alter islet M3R mRNA expression nor did incubation with the PI3K-inhibitor, LY294002, although enhancement of glucose-induced insulin secretion by LY294002 was reduced in islets maintained at 16 mmol/l glucose, as was mRNA expression of the PI3K regulatory subunit, p85α. Cholinergic regulation of insulin release is impaired in three experimental islet models of hyperglycaemia consistent with reduced expression of M3 receptors. Our data suggest that the receptor downregulation is a PKC- and PI3K-independent consequence of the hyperglycaemic environment, and they imply that M3 receptors could be potential targets in the treatment of type 2 diabetes. © 2014 John Wiley & Sons Ltd.

  7. The size of the hydroxyl group and its contribution to the affinity of atropine for muscarine-sensitive acetylcholine receptors.

    PubMed Central

    Barlow, R. B.; Ramtoola, S.

    1980-01-01

    1 From measurements of the affinity constants of hydratropyltropine and its methiodide for muscarine-sensitive acetylcholine receptors in the guinea-pig ileum, the increment in log K for the hydroxyl group in atropine is 2.06 and in the methiodide it is 2.16. These effects are slightly bigger than any so far recorded with these receptors. 2 The estimate of the increment in apparent molal volume for the hydroxyl group is 1.1 cm3/mol in atropine and 1.0 cm3/mol in the methobromide. 3 The large effect of the group on affinity may be linked to its small apparent size in water as suggested in the previous paper. PMID:7470742

  8. Muscarinic binding sites in cultured bovine pulmonary arterial endothelial cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Aronstam, R.S.; Catravas, J.D.; Ryan, U.S.

    The authors have previously reported a) the presence of muscarinic binding sites on cultured bovine pulmonary arterial endothelial cells (BPAE; 2,000 sites/cell) and b) that acetylcholine inhibits the release of thromboxane B/sub 2/ fro BPAE. Since the authors findings could reflect muscarinic receptors (mAChR) on BPAE, they have further investigated the nature of BPAE muscarinic binding sites and contrast them to those of known functional mAChR. Muscarinic binding sites on BPAE resembled mAChR in that a) the binding of 3 nM /sup 3/H QNB was inhibited by muscarinic agonists and antagonists; b) /sup 3/H QNB binding was 30 times moremore » sensitive to R(-)- than to S(+)-QNB; c) carbamylcholine binding was resolved into high and low affinity components (IC50's = 0.04 and 2 ..mu..M; d) 5'-guanylylimidodiphosphate (100 ..mu..M) shifted agonist binding curves to the right by a factor of 3; 4) the atropine-sensitive binding of /sup 3/H oxotremorine-M (/sup 3/H-OXO-M) was depressed by the guanine nucleotide (IC50 + 60 ..mu..M). However, although gallamine allosterically regulates mAChR binding in other tissues, it did not affect the rates of dissociation of /sup 3/H QNB, /sup 3/H methylscopolamine or /sup 3/H OXO-M from BPAE binding sites. Thus, BPAE muscarinic binding sites posses many but not all of the properties associated with functional mAChR.« less

  9. Nicotinic and muscarinic cholinergic receptors are recruited by acetylcholine-mediated neurotransmission within the locus coeruleus during the organisation of post-ictal antinociception.

    PubMed

    de Oliveira, Rithiele Cristina; de Oliveira, Ricardo; Biagioni, Audrey Franceschi; Falconi-Sobrinho, Luiz Luciano; Dos Anjos-Garcia, Tayllon; Coimbra, Norberto Cysne

    2016-10-01

    Post-ictal antinociception is characterised by an increase in the nociceptive threshold that accompanies tonic and tonic-clonic seizures (TCS). The locus coeruleus (LC) receives profuse cholinergic inputs from the pedunculopontine tegmental nucleus. Different concentrations (1μg, 3μg and 5μg/0.2μL) of the muscarinic cholinergic receptor antagonist atropine and the nicotinic cholinergic receptor antagonist mecamylamine were microinjected into the LC of Wistar rats to investigate the role of cholinergic mechanisms in the severity of TCS and the post-ictal antinociceptive response. Five minutes later, TCS were induced by systemic administration of pentylenetetrazole (PTZ) (64mg/kg). Seizures were recorded inside the open field apparatus for an average of 10min. Immediately after seizures, the nociceptive threshold was recorded for 130min using the tail-flick test. Pre-treatment of the LC with 1μg, 3μg and 5μg/0.2μL concentrations of both atropine and mecamylamine did not cause a significant effect on seizure severity. However, the same treatments decreased the post-ictal antinociceptive phenomenon. In addition, mecamylamine caused an earlier decrease in the post-ictal antinociception compared to atropine. These results suggest that muscarinic and mainly nicotinic cholinergic receptors of the LC are recruited to organise tonic-clonic seizure-induced antinociception. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Muscarinic contribution to the acute cortical effects of vagus nerve stimulation

    NASA Astrophysics Data System (ADS)

    Nichols, Justin A.

    2011-12-01

    Electrical stimulation of the vagus nerve (VNS) has been used to treat more than 60,000 patients with drug-resistant epilepsy and is under investigation as a treatment for several other neurological disorders and conditions. Among these, VNS increases memory performance and enhances recovery of motor and cognitive function in animal models of traumatic brain injury. Recent research indicates that pairing brief VNS with tones multiple-times a day for several weeks induces long-term, input specific cortical plasticity, which can be used to re-normalize the pathological cortical reorganization and eliminate a behavioral correlate of chronic tinnitus in noise exposed rats. Despite the therapeutic potential, the mechanisms of action of VNS remain speculative. In chapter 2 of this dissertation, the acute effects of VNS on cortical synchrony, excitability, and temporal processing are examined. In anesthetized rats implanted with multi-electrode arrays, VNS increased and decorrelated spontaneous multi-unit activity, and suppressed entrainment to repetitive noise burst stimulation at 6 to 8 Hz, but not after systemic administration of the muscarinic antagonist scopolamine. Chapter 3 focuses on VNS-tone pairing induced cortical plasticity. Pairing VNS with a tone one hundred times in anesthetized rats resulted in frequency specific plasticity in 31% of the auditory cortex sites. Half of these sites exhibited a frequency specific increase in firing rate and half exhibited a frequency specific decrease. Muscarinic receptor blockade with scopolamine almost entirely prevented the frequency specific increases, but not decreases. Collectively, these experiments demonstrate the capacity for VNS to not only acutely influence cortical synchrony, and excitability, but to also influence temporal and spectral tuning via muscarinic receptor activation. These results strengthen the hypothesis that acetylcholine and muscarinic receptors are involved in the mechanisms of action of VNS and

  11. Urinary Retention, Incontinence, and Dysregulation of Muscarinic Receptors in Male Mice Lacking Mras.

    PubMed

    Ehrhardt, Annette; Wang, Bin; Yung, Andrew C; Wang, Yanni; Kozlowski, Piotr; van Breemen, Cornelis; Schrader, John W

    2015-01-01

    Here we show that male, but not female mice lacking expression of the GTPase M-Ras developed urinary retention with distention of the bladder that exacerbated with age but occurred in the absence of obvious anatomical outlet obstruction. There were changes in detrusor morphology in Mras-/- males: Smooth muscle tissue, which exhibited a compact organization in WT mice, appeared disorganized and became increasingly 'layered' with age in Mras-/- males, but was not fibrotic. Bladder tissue near the apex of bladders of Mras-/- males exhibited hypercontractility in response to the cholinergic agonist carbachol in in vitro, while responses in Mras-/- females were normal. In addition, spontaneous phasic contractions of detrusors from Mras-/- males were increased, and Mras-/- males exhibited urinary incontinence. We found that expression of the muscarinic M2 and M3 receptors that mediate the cholinergic contractile stimuli of the detrusor muscle was dysregulated in both Mras-/- males and females, although only males exhibited a urinary phenotype. Elevated expression of M2R in young males lacking M-Ras and failure to upregulate M3R with age resulted in significantly lower ratios of M3R/M2R expression that correlated with the bladder abnormalities. Our data suggests that M-Ras and M3R are functionally linked and that M-Ras is an important regulator of male bladder control in mice. Our observations also support the notion that bladder control is sexually dimorphic and is regulated through mechanisms that are largely independent of acetylcholine signaling in female mice.

  12. Urinary Retention, Incontinence, and Dysregulation of Muscarinic Receptors in Male Mice Lacking Mras

    PubMed Central

    Ehrhardt, Annette; Wang, Bin; Yung, Andrew C.; Wang, Yanni; Kozlowski, Piotr; van Breemen, Cornelis; Schrader, John W.

    2015-01-01

    Here we show that male, but not female mice lacking expression of the GTPase M-Ras developed urinary retention with distention of the bladder that exacerbated with age but occurred in the absence of obvious anatomical outlet obstruction. There were changes in detrusor morphology in Mras -/- males: Smooth muscle tissue, which exhibited a compact organization in WT mice, appeared disorganized and became increasingly ‘layered’ with age in Mras -/- males, but was not fibrotic. Bladder tissue near the apex of bladders of Mras -/- males exhibited hypercontractility in response to the cholinergic agonist carbachol in in vitro, while responses in Mras -/- females were normal. In addition, spontaneous phasic contractions of detrusors from Mras -/- males were increased, and Mras -/- males exhibited urinary incontinence. We found that expression of the muscarinic M2 and M3 receptors that mediate the cholinergic contractile stimuli of the detrusor muscle was dysregulated in both Mras -/- males and females, although only males exhibited a urinary phenotype. Elevated expression of M2R in young males lacking M-Ras and failure to upregulate M3R with age resulted in significantly lower ratios of M3R/M2R expression that correlated with the bladder abnormalities. Our data suggests that M-Ras and M3R are functionally linked and that M-Ras is an important regulator of male bladder control in mice. Our observations also support the notion that bladder control is sexually dimorphic and is regulated through mechanisms that are largely independent of acetylcholine signaling in female mice. PMID:26516777

  13. FRET-based sensors for the human M1-, M3-, and M5-acetylcholine receptors.

    PubMed

    Ziegler, Nicole; Bätz, Julia; Zabel, Ulrike; Lohse, Martin J; Hoffmann, Carsten

    2011-02-01

    Based on the recently developed approach to generate fluorescence resonance energy transfer (FRET)-based sensors to measure GPCR activation, we generated sensor constructs for the human M(1)-, M(3)-, and M(5)-acetylcholine receptor. The receptors were labeled with cyan fluorescent protein (CFP) at their C-terminus, and with fluorescein arsenical hairpin binder (FlAsH) via tetra-cysteine tags inserted in the third intracellular loop. We then measured FRET between the donor CFP and the acceptor FlAsH in living cells and real time. Agonists like acetylcholine, carbachol, or muscarine activate each receptor construct with half-maximal activation times between 60 and 70ms. Removal of the agonist caused the reversal of the signal. Compared with all other agonists, oxotremorine M differed in two major aspects: it caused significantly slower signals at M(1)- and M(5)-acetylcholine receptors and the amplitude of these signals was larger at the M(1)-acetylcholine receptor. Concentration-response curves for the agonists reveal that all agonists tested, with the mentioned exception of oxotremorine M, caused similar maximal FRET-changes as acetylcholine for the M(1)-, M(3)- and M(5)-acetylcholine receptor constructs. Taken together our data support the notion that orthosteric agonists behave similar at different muscarinic receptor subtypes but that kinetic differences can be observed for receptor activation. Copyright © 2010 Elsevier Ltd. All rights reserved.

  14. Structural Modifications to Tetrahydropyridine-3-Carboxylate Esters en route to the Discovery of M5-Preferring Muscarinic Receptor Orthosteric Antagonists

    PubMed Central

    Zheng, Guangrong; Smith, Andrew M.; Huang, Xiaoqin; Subramanian, Karunai L.; Siripurapu, Kiran B.; Deaciuc, Agripina; Zhan, Chang-Guo; Dwoskin, Linda P.

    2013-01-01

    The M5 muscarinic acetylcholine receptor is suggested to be a potential pharmacotherapeutic target for the treatment of drug abuse. We describe herein the discovery of a series of M5-preferring orthosteric antagonists based on the scaffold of 1,2,5,6-tetrahydropyridine-3-carboxylic acid. Compound 56, the most selective compound in this series, possesses an 11-fold selectivity for the M5 over M1 receptor, and shows little activity at M2–M4. This compound, although exhibiting modest affinity (Ki = 2.24 μM) for the [3H]N-methylscopolamine binding site on the M5 receptor, is potent (IC50 = 0.45 nM) in inhibiting oxotremorine-evoked [3H]DA release from rat striatal slices. Further, a homology model of human M5 receptor based on the crystal structure of the rat M3 receptor was constructed, and docking studies of compounds 28 and 56 were performed in an attempt to understand the possible binding mode of these novel analogues to the receptor. PMID:23379472

  15. The Vagal Nerve Stimulates Activation of the Hepatic Progenitor Cell Compartment via Muscarinic Acetylcholine Receptor Type 3

    PubMed Central

    Cassiman, David; Libbrecht, Louis; Sinelli, Nicoletta; Desmet, Valeer; Denef, Carl; Roskams, Tania

    2002-01-01

    In the rat the hepatic branch of the nervus vagus stimulates proliferation of hepatocytes after partial hepatectomy and growth of bile duct epithelial cells after bile duct ligation. We studied the effect of hepatic vagotomy on the activation of the hepatic progenitor cell compartment in human and rat liver. The number of hepatic progenitor cells and atypical reactive ductular cells in transplanted (denervated) human livers with hepatitis was significantly lower than in innervated matched control livers and the number of oval cells in vagotomized rat livers with galactosamine hepatitis was significantly lower than in livers of sham-operated rats with galactosamine hepatitis. The expression of muscarinic acetylcholine receptors (M1-M5 receptor) was studied by immunohistochemistry and reverse transcriptase-polymerase chain reaction. In human liver, immunoreactivity for M3 receptor was observed in hepatic progenitor cells, atypical reactive ductules, intermediate hepatocyte-like cells, and bile duct epithelial cells. mRNA for the M1-M3 and the M5 receptor, but not the M4 receptor, was detected in human liver homogenates. In conclusion, the hepatic vagus branch stimulates activation of the hepatic progenitor cell compartment in diseased liver, most likely through binding of acetylcholine to the M3 receptor expressed on these cells. These findings may be of clinical importance for patients with a transplant liver. PMID:12163377

  16. Effects of muscarinic blockade in perirhinal cortex during visual recognition

    PubMed Central

    Tang, Yi; Mishkin, Mortimer; Aigner, Thomas G.

    1997-01-01

    Stimulus recognition in monkeys is severely impaired by destruction or dysfunction of the perirhinal cortex and also by systemic administration of the cholinergic-muscarinic receptor blocker, scopolamine. These two effects are shown here to be linked: Stimulus recognition was found to be significantly impaired after bilateral microinjection of scopolamine directly into the perirhinal cortex, but not after equivalent injections into the laterally adjacent visual area TE or into the dentate gyrus of the overlying hippocampal formation. The results suggest that the formation of stimulus memories depends critically on cholinergic-muscarinic activation of the perirhinal area, providing a new clue to how stimulus representations are stored. PMID:9356507

  17. Induction of Anti-Hebbian LTP in CA1 Stratum Oriens Interneurons: Interactions between Group I Metabotropic Glutamate Receptors and M1 Muscarinic Receptors

    PubMed Central

    Savary, Etienne; Kullmann, Dimitri M.; Miles, Richard

    2015-01-01

    An anti-Hebbian form of LTP is observed at excitatory synapses made with some hippocampal interneurons. LTP induction is facilitated when postsynaptic interneurons are hyperpolarized, presumably because Ca2+ entry through Ca2+-permeable glutamate receptors is enhanced. The contribution of modulatory transmitters to anti-Hebbian LTP induction remains to be established. Activation of group I metabotropic receptors (mGluRs) is required for anti-Hebbian LTP induction in interneurons with cell bodies in the CA1 stratum oriens. This region receives a strong cholinergic innervation from the septum, and muscarinic acetylcholine receptors (mAChRs) share some signaling pathways and cooperate with mGluRs in the control of neuronal excitability. We therefore examined possible interactions between group I mGluRs and mAChRs in anti-Hebbian LTP at synapses which excite oriens interneurons in rat brain slices. We found that blockade of either group I mGluRs or M1 mAChRs prevented the induction of anti-Hebbian LTP by pairing presynaptic activity with postsynaptic hyperpolarization. Blocking either receptor also suppressed long-term effects of activation of the other G-protein coupled receptor on interneuron membrane potential. However, no crossed blockade was detected for mGluR or mAchR effects on interneuron after-burst potentials or on the frequency of miniature EPSPs. Paired recordings between pyramidal neurons and oriens interneurons were obtained to determine whether LTP could be induced without concurrent stimulation of cholinergic axons. Exogenous activation of mAChRs led to LTP, with changes in EPSP amplitude distributions consistent with a presynaptic locus of expression. LTP, however, required noninvasive presynaptic and postsynaptic recordings. SIGNIFICANCE STATEMENT In the hippocampus, a form of NMDA receptor-independent long-term potentiation (LTP) occurs at excitatory synapses made on some inhibitory neurons. This is preferentially induced when postsynaptic

  18. Control of Insulin Secretion by Cholinergic Signaling in the Human Pancreatic Islet

    PubMed Central

    Molina, Judith; Rodriguez-Diaz, Rayner; Fachado, Alberto; Jacques-Silva, M. Caroline

    2014-01-01

    Acetylcholine regulates hormone secretion from the pancreatic islet and is thus crucial for glucose homeostasis. Little is known, however, about acetylcholine (cholinergic) signaling in the human islet. We recently reported that in the human islet, acetylcholine is primarily a paracrine signal released from α-cells rather than primarily a neural signal as in rodent islets. In this study, we demonstrate that the effects acetylcholine produces in the human islet are different and more complex than expected from studies conducted on cell lines and rodent islets. We found that endogenous acetylcholine not only stimulates the insulin-secreting β-cell via the muscarinic acetylcholine receptors M3 and M5, but also the somatostatin-secreting δ-cell via M1 receptors. Because somatostatin is a strong inhibitor of insulin secretion, we hypothesized that cholinergic input to the δ-cell indirectly regulates β-cell function. Indeed, when all muscarinic signaling was blocked, somatostatin secretion decreased and insulin secretion unexpectedly increased, suggesting a reduced inhibitory input to β-cells. Endogenous cholinergic signaling therefore provides direct stimulatory and indirect inhibitory input to β-cells to regulate insulin secretion from the human islet. PMID:24658304

  19. Antihypertensive effect of the methanolic extract from Eruca sativa Mill., (Brassicaceae) in rats: muscarinic receptor-linked vasorelaxant and cardiotonic effects.

    PubMed

    Salma, Umme; Khan, Taous; Shah, Abdul Jabbar

    2018-06-15

    Eruca sativa Mill., (Brassicaceae) is a popular remedy for the treatment of hypertension in Pakistan. However, direct effect of the extract and its fractions on blood pressure and vascular tone are unknown. This investigation was aimed to explore the pharmacological base for the traditional use of E. sativa in hypertension. In-vivo blood pressure study was carried out using normotensive and high salt-induced hypertensive rats under anaesthesia. The cardiovascular mechanisms were explored using rat aorta and atria in-vitro. Preliminary phytochemical analysis, spectrophotometric detection of total phenols, flavonoids and HPLC analysis of crude extract were performed using quercetin and erucin as marker compounds. Intravenous injection of crude extract induced a fall in mean arterial pressure (MAP) in both normotensive (max fall: 41.79±1.55% mmHg) and hypertensive (max fall: 58.25±0.91% mmHg) rats. Atropine (1mg/kg) pretreatment attenuated this effect significantly (p < 0.001), suggesting the involvement of muscarinic receptor in its antihypertensive effect. Fractions also induced atropine-sensitive antihypertensive effect. Similarly, oral administration of crude and aqueous extracts resulted a fall in MAP in the hypertensive rats. In isolated rat aortic rings from normotensive rats, crude extract and fractions induced an endothelium-dependent relaxation. This relaxation was partially inhibited with L- NAME and atropine pretreatment and with denudation of aortic rings, indicating involvement of muscarinic receptor-linked nitric oxide (NO). In aorta from the hypertensive rats, crude extract and fractions induced endothelium-independent relaxation. This relaxation was not affected by pretreatment with L- NAME or atropine. Crude extract and fractions also suppressed phenylephrine contractions in Ca +2 free/EGTA medium. In isolated rat atrial preparations, crude extract and fractions induced negative inotropic and chronotropic effects with a positive inotropic effect by

  20. Acetylcholine receptors in the human retina

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hutchins, J.B.; Hollyfield, J.G.

    1985-11-01

    Evidence for a population of acetylcholine (ACh) receptors in the human retina is presented. The authors have used the irreversible ligand TH-propylbenzilylcholine mustard (TH-PrBCM) to label muscarinic receptors. TH- or SVI-alpha-bungarotoxin (alpha-BTx) was used to label putative nicotinic receptors. Muscarinic receptors are apparently present in the inner plexiform layer of the retina. Autoradiographic grain densities are reduced in the presence of saturating concentrations of atropine, quinuclidinyl benzilate or scopolamine; this indicates that TH-PrBCM binding is specific for a population of muscarinic receptors in the human retina. Binding sites for radiolabeled alpha-BTx are found predominantly in the inner plexiform layer ofmore » the retina. Grain densities are reduced in the presence of d-tubocurarine, indicating that alpha-BTx may bind to a pharmacologically relevant nicotinic ACh receptor. This study provides evidence for cholinergic neurotransmission in the human retina.« less

  1. Degradation of submandibular gland AQP5 by parasympathetic denervation of chorda tympani and its recovery by cevimeline, an M3 muscarinic receptor agonist.

    PubMed

    Li, Xuefei; Azlina, Ahmad; Karabasil, Mileva Ratko; Purwanti, Nunuk; Hasegawa, Takahiro; Yao, Chenjuan; Akamatsu, Tetsuya; Hosoi, Kazuo

    2008-07-01

    By chorda tympani denervation (CTD, parasympathectomy), the aquaporin 5 (AQP5), but not AQP1, protein level in the rat submandibular gland (SMG) was significantly decreased, dropping to 37% of that of the contralateral gland at 4 wk. The protein levels of AQP5 and AQP1 were not significantly affected by denervation of the cervical sympathetic trunk (sympathectomy). Administration of cevimeline hydrochloride, an M3 muscarinic receptor agonist (10 mg/kg for 7 days po), but not pilocarpine (0.3 mg/kg for 7 days po), recovered the AQP5 protein level reduced by CTD and increased the AQP1 protein level above the control one. The mRNA level of AQP5 was scarcely affected by CTD and cevimeline hydrochloride administration. Administration of chloroquine (50 mg/kg for 7 days po), a denaturant of lysosomes, increased the AQP5 protein level reduced by CTD. An extract obtained from the submandibular lysosomal fraction degraded the AQP5 protein in the total membrane fraction in vitro. These results suggest the possible regulation of the AQP5 protein level in the SMG by the parasympathetic nerves/M3 muscarinic receptor agonist and imply the involvement of lysosomal enzymes, but not a transcriptional mechanism, in this regulation.

  2. Pharmacological profiles of the subtypes of muscarinic cholinoceptors that mediate aggregation of pigment in the melanophores of two species of catfish.

    PubMed

    Hayashi, H; Fujii, R

    1994-06-01

    Using selective antagonists, including pirenzepine, adiphenine, AF-DX 116, gallamine, and 4-DAMP, we attempted to characterize the muscarinic cholinoceptors on the melanophores of the translucent glass catfish Kryptopterus bicirrhis and the mailed catfish Corydoras paleatus. The M3 receptor-selective antagonist, 4-DAMP, potently inhibited the acetylcholine-induced aggregation of pigment in both species. It appeared, therefore, that the receptors that mediated the cholinergically evoked aggregation of melanosomes in these species were of the M3 muscarinic subtype.

  3. Role of Orai1 and store-operated calcium entry in mouse lacrimal gland signalling and function.

    PubMed

    Xing, Juan; Petranka, John G; Davis, Felicity M; Desai, Pooja N; Putney, James W; Bird, Gary S

    2014-03-01

    Lacrimal glands function to produce an aqueous layer, or tear film, that helps to nourish and protect the ocular surface. Lacrimal glands secrete proteins, electrolytes and water, and loss of gland function can result in tear film disorders such as dry eye syndrome, a widely encountered and debilitating disease in ageing populations. To combat these disorders, understanding the underlying molecular signalling processes that control lacrimal gland function will give insight into corrective therapeutic approaches. Previously, in single lacrimal cells isolated from lacrimal glands, we demonstrated that muscarinic receptor activation stimulates a phospholipase C-coupled signalling cascade involving the inositol trisphosphate-dependent mobilization of intracellular calcium and the subsequent activation of store-operated calcium entry (SOCE). Since intracellular calcium stores are finite and readily exhausted, the SOCE pathway is a critical process for sustaining and maintaining receptor-activated signalling. Recent studies have identified the Orai family proteins as critical components of the SOCE channel activity in a wide variety of cell types. In this study we characterize the role of Orai1 in the function of lacrimal glands using a mouse model in which the gene for the calcium entry channel protein, Orai1, has been deleted. Our data demonstrate that lacrimal acinar cells lacking Orai1 do not exhibit SOCE following activation of the muscarinic receptor. In comparison with wild-type and heterozygous littermates, Orai1 knockout mice showed a significant reduction in the stimulated tear production following injection of pilocarpine, a muscarinic receptor agonist. In addition, calcium-dependent, but not calcium-independent exocytotic secretion of peroxidase was eliminated in glands from knockout mice. These studies indicate a critical role for Orai1-mediated SOCE in lacrimal gland signalling and function.

  4. Effects of Saw Palmetto Extract on Urodynamic Parameters, Bladder Muscarinic and Purinergic Receptors and Urinary Cytokines in Rats with Cyclophosphamide-Induced Cystitis.

    PubMed

    Nasrin, Sweety; Masuda, Eiji; Kugaya, Haruna; Osano, Ayaka; Ito, Yoshihiko; Yamada, Shizuo

    2014-01-01

    To clarify the effect of saw palmetto extract (SPE), a phytotherapeutic agent, on urodynamic parameters, bladder muscarinic and purinergic receptors, and urinary cytokines in rats with cystitis induced by cyclophosphamide (CYP). Saw palmetto extract (60 mg/kg per day) was administered orally twice a day for 7 days to rats. The urodynamic parameters in CYP (150 mg/kg i.p.)-treated rats were monitored by a cystometric method under anesthesia. The muscarinic and purinergic receptors in the bladder and submaxillary gland were measured by radioreceptor assays using [N-methyl-(3) H] scopolamine chloride([(3) H]NMS) and αβ-methylene-ATP [2,8-(3) H] tetrasodium salt ([(3) H]αβ-MeATP), respectively. Urinary cytokines (interleukin-1β [IL-1β], IL-6 and L-17) were measured with enzyme linked immunosorbent assay kits. Micturition interval and micturition volume were significantly decreased and the frequency of micturition and basal pressure were significantly increased in the CYP-treated rats compared with sham-operated rats. Orally administered SPE significantly increased the micturition interval and micturition volume and decreased the frequency of micturition and basal pressure. The maximal number of sites (Bmax ) for the specific binding of [(3) H]NMS and [(3) H]αβ-MeATP was significantly decreased in the bladder. The decrease in receptors was attenuated by repeated treatment with SPE. An elevation in urinary cytokine (IL-1β and IL-17) levels were seen, and this increase was effectively suppressed by SPE treatment. Saw palmetto extract attenuates the alteration of urodynamic parameters, pharmacologically relevant receptors, and urinary cytokines in CYP-treated rats. Therefore, SPE may be a potential therapeutic agent for improving the clinical symptoms of cystitis. © 2013 Wiley Publishing Asia Pty Ltd.

  5. Nucleus Accumbens Acetylcholine Receptors Modulate Dopamine and Motivation.

    PubMed

    Collins, Anne L; Aitken, Tara J; Greenfield, Venuz Y; Ostlund, Sean B; Wassum, Kate M

    2016-11-01

    Environmental reward-predictive cues can motivate reward-seeking behaviors. Although this influence is normally adaptive, it can become maladaptive in disordered states, such as addiction. Dopamine release in the nucleus accumbens core (NAc) is known to mediate the motivational impact of reward-predictive cues, but little is known about how other neuromodulatory systems contribute to cue-motivated behavior. Here, we examined the role of the NAc cholinergic receptor system in cue-motivated behavior using a Pavlovian-to-instrumental transfer task designed to assess the motivating influence of a reward-predictive cue over an independently-trained instrumental action. Disruption of NAc muscarinic acetylcholine receptor activity attenuated, whereas blockade of nicotinic receptors augmented cue-induced invigoration of reward seeking. We next examined a potential dopaminergic mechanism for this behavioral effect by combining fast-scan cyclic voltammetry with local pharmacological acetylcholine receptor manipulation. The data show evidence of opposing modulation of cue-evoked dopamine release, with muscarinic and nicotinic receptor antagonists causing suppression and augmentation, respectively, consistent with the behavioral effects of these manipulations. In addition to demonstrating cholinergic modulation of naturally-evoked and behaviorally-relevant dopamine signaling, these data suggest that NAc cholinergic receptors may gate the expression of cue-motivated behavior through modulation of phasic dopamine release.

  6. Retinal co-mediator acetylcholine evokes muscarinic inhibition of recurrent excitation in frog tectum column.

    PubMed

    Baginskas, Armantas; Kuras, Antanas

    2016-08-26

    Acetylcholine receptors contribute to the control of neuronal and neuronal network activity from insects to humans. We have investigated the action of acetylcholine receptors in the optic tectum of Rana temporaria (common frog). Our previous studies have demonstrated that acetylcholine activates presynaptic nicotinic receptors, when released into the frog optic tectum as a co-mediator during firing of a single retinal ganglion cell, and causes: a) potentiation of retinotectal synaptic transmission, and b) facilitation of transition of the tectum column to a higher level of activity. In the present study we have shown that endogenous acetylcholine also activates muscarinic receptors, leading to a delayed inhibition of recurrent excitatory synaptic transmission in the tectum column. The delay of muscarinic inhibition was evaluated to be of ∼80ms, with an extent of inhibition of ∼2 times. The inhibition of the recurrent excitation determines transition of the tectum column back to its resting state, giving a functional sense for the inhibition. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  7. The change in muscarinic receptor subtypes in different brain regions of rats treated with fluoxetine or propranolol in a model of post-traumatic stress disorder.

    PubMed

    Aykaç, Aslı; Aydın, Banu; Cabadak, Hülya; Gören, M Zafer

    2012-06-15

    This study shows the possible contribution of muscarinic receptors in the pathophysiology of post-traumatic stress disorder. Sprague-Dawley rats of both sexes were exposed to dirty cat litter (trauma) for 10 min and the protocol was repeated 1 week later with a trauma reminder (clean litter). The rats also received intraperitoneal fluoxetine (2.5, 5 or 10 mg/kg/day), propranolol (10 mg/kg/day) or saline for 7 days between two exposure sessions. Functional behavioral experiments were performed using elevated plus maze, following exposure to trauma reminder. Western blot analyses for M(1), M(2), M(3), M(4) and M(5) receptor proteins were employed in the homogenates of the hippocampus, the frontal cortex and the amygdaloid complex. The anxiety indices increased from 0.63±0.02 to 0.89±0.04 in rats exposed to the trauma reminder. The freezing times were also recorded as 47±6 and 133±12 s, in control and test animals respectively. Fluoxetine or propranolol treatments restored the increases in the anxiety indices and the freezing times. Female rats had higher anxiety indices compared to males. Western blot data showed increases in M(2) and M(5) expression in the frontal cortex. Expression of M(1) receptors increased and M(4) subtype decreased in the hippocampus. In the amygdaloid complex of rats, we also detected a down-regulation of M(4) receptors. Fluoxetine and propranolol only corrected the changes occurred in the frontal cortex. These results may imply that muscarinic receptors are involved in this experimental model of post-traumatic stress disorder. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. Claudin-4 is required for modulation of paracellular permeability by muscarinic acetylcholine receptor in epithelial cells.

    PubMed

    Cong, Xin; Zhang, Yan; Li, Jing; Mei, Mei; Ding, Chong; Xiang, Ruo-Lan; Zhang, Li-Wei; Wang, Yun; Wu, Li-Ling; Yu, Guang-Yan

    2015-06-15

    The epithelial cholinergic system plays an important role in water, ion and solute transport. Previous studies have shown that activation of muscarinic acetylcholine receptors (mAChRs) regulates paracellular transport of epithelial cells; however, the underlying mechanism is still largely unknown. Here, we found that mAChR activation by carbachol and cevimeline reduced the transepithelial electrical resistance (TER) and increased the permeability of paracellular tracers in rat salivary epithelial SMG-C6 cells. Carbachol induced downregulation and redistribution of claudin-4, but not occludin or ZO-1 (also known as TJP1). Small hairpin RNA (shRNA)-mediated claudin-4 knockdown suppressed, whereas claudin-4 overexpression retained, the TER response to carbachol. Mechanistically, the mAChR-modulated claudin-4 properties and paracellular permeability were triggered by claudin-4 phosphorylation through ERK1/2 (also known as MAPK3 and MAPK1, respectively). Mutagenesis assay demonstrated that S195, but not S199, S203 or S207, of claudin-4, was the target for carbachol. Subsequently, the phosphorylated claudin-4 interacted with β-arrestin2 and triggered claudin-4 internalization through the clathrin-dependent pathway. The internalized claudin-4 was further degraded by ubiquitylation. Taken together, these findings suggested that claudin-4 is required for mAChR-modulated paracellular permeability of epithelial cells through an ERK1/2, β-arrestin2, clathrin and ubiquitin-dependent signaling pathway. © 2015. Published by The Company of Biologists Ltd.

  9. Characterization of cholinergic muscarinic receptor-stimulated phosphoinositide metabolism in brain from immature rats

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Balduini, W.; Murphy, S.D.; Costa, L.G.

    Hydrolysis of phosphoinositides elicited by stimulation of cholinergic muscarinic receptors has been studied in brain from neonatal (7-day-old) rats in order to determine: (1) whether the neonatal rat could provide a good model system to study this signal-transduction pathway; and (2) whether potential differences with adult nerve tissue would explain the differential, age-related effects of cholinergic agonists. Accumulation of (3H) inositol phosphates in (3H)inositol prelabeled slices from neonatal and adult rats was measured as an index of phosphoinositide metabolism. Full (acetylcholine, methacholine, carbachol) and partial (oxotremorine, bethanechol) agonists had qualitatively similar, albeit quantitatively different, effects in neonatal and adult rats.more » Atropine and pirenzepine effectively blocked the carbachol-induced response with inhibition constants of 1.2 and 20.7 nM, respectively. In all brain areas, response to all agonists was higher in neonatal than adult rats, and in hippocampus and cerebral cortex the response was higher than in cerebellum or brainstem. The relative intrinsic activity of partial agonists was higher in the latter two areas (0.6-0.7) than in the former two (0.3-0.4). Carbachol-stimulated phosphoinositide metabolism in brain areas correlated well with the binding of (3H)QNB (r2 = 0.627) and, particularly, with (3H)pirenzepine (r2 = 0.911). In cerebral cortex the effect of carbachol was additive to that of norepinephrine and glutamate. The presence of calcium (250-500 microM) was necessary for maximal response to carbachol to be elicited; the EC50 value for Ca2+ was 65.4 microM. Addition of EDTA completely abolished the response. Removal of sodium ions from the incubation medium reduced the response to carbachol by 50%.« less

  10. Activation of central muscarinic receptors causes respiratory stimulation in conscious animals.

    PubMed Central

    Weinstock, M.

    1981-01-01

    1 Oxotremorine (10 microgram/kg) injected intravenously into conscious rabbits pretreated with atropine-methyl-nitrate (ATMN, 0.5 mg/kg) caused significant increases in respiration rate from 94 to 131 per min, and in PaO2 from 13.8 to 15.4 kPa, and a decrease in PaCO2 from 3.30 to 2.09 pKa within 15 min. Blood pH fell from 7.44 to 7.16. 2 Blood pressure increased by 11.6%, 5 min after oxotremorine injection. 3 The acidosis was shown to be due to an increase in blood lactic acid from 41 to 132 mg/100 ml. 4 Pretreatment with propranolol (5 mg/kg s.c.) prevented the lactic acidosis and fall in pH but did not alter the respiratory stimulation induced by oxotremorine. 5 It is suggested that the lactic acidosis induced by oxotremorine results from stimulation of beta-adrenoceptors in skeletal muscle by catecholamines released from the adrenal medulla and sympathetic nerves. 6 Since all the above effects of oxotremorine are antagonized by hyoscine (5 mg/kg) but not by ATMN (0.5 mg/kg), it is concluded that oxotremorine can stimulate respiration by a direct action on muscarinic receptors in the central nervous system. PMID:7296163

  11. A 100K well screen for a muscarinic receptor using the Epic label-free system--a reflection on the benefits of the label-free approach to screening seven-transmembrane receptors.

    PubMed

    Dodgson, K; Gedge, L; Murray, D C; Coldwell, M

    2009-01-01

    Seven-transmembrane receptors (7TMRs) are a family of proteins of great interest as therapeutic targets because of their abundance on the cell surface, diverse effects in modulating cell behavior and success as a key class of drugs. We have evaluated the Epic label-free system for the purpose of identifying antagonists of the muscarinic M3 receptor. We compared the data generated from the label-free technology with data for the same compounds in a calcium flux assay. We have shown that this technology can be used for high throughput screening (HTS) of 7TMRs and as an orthogonal approach to enable rapid evaluation of HTS outputs. A number of compounds have been identified which were not found in a functional HTS measuring the output from a single pathway, which may offer new approaches to inhibiting responses through this receptor.

  12. Effects of acute administration of nicotinic and muscarinic cholinergic agonists and antagonists on performance in different cost-benefit decision making tasks in rats.

    PubMed

    Mendez, Ian A; Gilbert, Ryan J; Bizon, Jennifer L; Setlow, Barry

    2012-12-01

    Alterations in cost-benefit decision making accompany numerous neuropsychiatric conditions, including schizophrenia, attention deficit hyperactivity disorder, and addiction. Central cholinergic systems have been linked to the etiology and/or treatment of many of these conditions, but little is known about the role of cholinergic signaling in cost-benefit decision making. The goal of these experiments was to determine how cholinergic signaling is involved in cost-benefit decision making, using a behavioral pharmacological approach. Male Long-Evans rats were trained in either "probability discounting" or "delay discounting" tasks, in which rats made discrete-trial choices between a small food reward and a large food reward associated with either varying probabilities of omission or varying delays to delivery, respectively. The effects of acute administration of different doses of nicotinic and muscarinic acetylcholine receptor agonists and antagonists were assessed in each task. In the probability discounting task, acute nicotine administration (1.0 mg/kg) significantly increased choice of the large risky reward, and control experiments suggested that this was due to robust nicotine-induced impairments in behavioral flexibility. In the delay discounting task, the muscarinic antagonists scopolamine (0.03, 0.1, and 0.3 mg/kg) and atropine (0.3 mg/kg) both significantly increased choice of the small immediate reward. Neither mecamylamine nor oxotremorine produced reliable effects on either of the decision making tasks. These data suggest that cholinergic receptors play multiple roles in decision making contexts which include consideration of reward delay or probability. These roles should be considered when targeting these receptors for therapeutic purposes.

  13. Muscarinic acetylcholine receptors control baseline activity and Hebbian stimulus timing-dependent plasticity in fusiform cells of the dorsal cochlear nucleus.

    PubMed

    Stefanescu, Roxana A; Shore, Susan E

    2017-03-01

    Cholinergic modulation contributes to adaptive sensory processing by controlling spontaneous and stimulus-evoked neural activity and long-term synaptic plasticity. In the dorsal cochlear nucleus (DCN), in vitro activation of muscarinic acetylcholine receptors (mAChRs) alters the spontaneous activity of DCN neurons and interacts with N -methyl-d-aspartate (NMDA) and endocannabinoid receptors to modulate the plasticity of parallel fiber synapses onto fusiform cells by converting Hebbian long-term potentiation to anti-Hebbian long-term depression. Because noise exposure and tinnitus are known to increase spontaneous activity in fusiform cells as well as alter stimulus timing-dependent plasticity (StTDP), it is important to understand the contribution of mAChRs to in vivo spontaneous activity and plasticity in fusiform cells. In the present study, we blocked mAChRs actions by infusing atropine, a mAChR antagonist, into the DCN fusiform cell layer in normal hearing guinea pigs. Atropine delivery leads to decreased spontaneous firing rates and increased synchronization of fusiform cell spiking activity. Consistent with StTDP alterations observed in tinnitus animals, atropine infusion induced a dominant pattern of inversion of StTDP mean population learning rule from a Hebbian to an anti-Hebbian profile. Units preserving their initial Hebbian learning rules shifted toward more excitatory changes in StTDP, whereas units with initial suppressive learning rules transitioned toward a Hebbian profile. Together, these results implicate muscarinic cholinergic modulation as a factor in controlling in vivo fusiform cell baseline activity and plasticity, suggesting a central role in the maladaptive plasticity associated with tinnitus pathology. NEW & NOTEWORTHY This study is the first to use a novel method of atropine infusion directly into the fusiform cell layer of the dorsal cochlear nucleus coupled with simultaneous recordings of neural activity to clarify the contribution of

  14. General pharmacological profile of the novel muscarinic receptor agonist SNI-2011, a drug for xerostomia in Sjögren's syndrome. 1st communication: effects on general behavior and central nervous system.

    PubMed

    Arisawa, Hirohiko; Imai, Eiichi; Fujise, Nobuaki; Fukui, Kenji; Masunaga, Hiroaki

    2002-01-01

    A novel muscarinic receptor agonist, SNI-2011 ((+/-)-cis-2-methylspiro[1,3-oxathiolane-5,3'-quinuclidine] monohydrochloride hemihydrate, cevimeline, CAS 153504-70-2), is a candidate therapeutic drug for xerostomia in Sjögren's syndrome. The general pharmacological properties of this drug on general behavior and the central nervous system were investigated in mice, rats and cats. 1. General behavior: When SNI-2011 was administered orally to mice at 100 mg/kg, mydriasis, a decrease of spontaneous motor activity, tremor, convulsions, salivation, abnormal posture, abnormal gait, reduced grip strength and reduced response against external stimulating were observed, and 2 out of 6 animals died. At 10 mg/kg or lower, no particular sign was observed except mydriasis, which appeared to be caused via the peripheral muscarinic acetylcholine receptors. 2. Central nervous system: SNI-2011 had no effect on the motor coordination in mice. Hypothermia was observed in rats and reduced spontaneous motor activity, analgesia and enhanced maximum electroshock-induced convulsions were observed in mice after oral administration of 30 mg/kg SNI-2011. Slight increase in the rate of theta-wave band in the hippocampal EEG of rats and spinal multisynaptic reflexes in cats were observed after intravenous injection of 10 mg/kg SNI-2011. At an oral dose of 10 mg/kg, prolongation of thiopental-induced sleeping time in mice was observed. The prolongation of sleeping time was inhibited by a peripheral muscarinic antagonist. These results suggest that SNI-2011 has muscarinic effects on general behavior and the central nervous system at the doses approximately 10-fold higher than the effective doses needed for saliva secretion.

  15. Acetylcholine is released from taste cells, enhancing taste signalling

    PubMed Central

    Dando, Robin; Roper, Stephen D

    2012-01-01

    Acetylcholine (ACh), a candidate neurotransmitter that has been implicated in taste buds, elicits calcium mobilization in Receptor (Type II) taste cells. Using RT-PCR analysis and pharmacological interventions, we demonstrate that the muscarinic acetylcholine receptor M3 mediates these actions. Applying ACh enhanced both taste-evoked Ca2+ responses and taste-evoked afferent neurotransmitter (ATP) secretion from taste Receptor cells. Blocking muscarinic receptors depressed taste-evoked responses in Receptor cells, suggesting that ACh is normally released from taste cells during taste stimulation. ACh biosensors confirmed that, indeed, taste Receptor cells secrete acetylcholine during gustatory stimulation. Genetic deletion of muscarinic receptors resulted in significantly diminished ATP secretion from taste buds. The data demonstrate a new role for acetylcholine as a taste bud transmitter. Our results imply specifically that ACh is an autocrine transmitter secreted by taste Receptor cells during gustatory stimulation, enhancing taste-evoked responses and afferent transmitter secretion. PMID:22570381

  16. Evidence for inhibitory nicotinic and facilitatory muscarinic receptors in cholinergic nerve terminals of the rat urinary bladder.

    PubMed

    Somogyi, G T; de Groat, W C

    1992-02-01

    Cholinergic prejunctional modulatory receptors on parasympathetic nerves in the rat urinary bladder were studied by measuring 3H-acetylcholine (ACh) release in muscle strips from the bladder body. Electrical field stimulation markedly increased 3H-ACh overflow in strips preloaded with 3H-choline. Oxotremorine (1 microM), an M2 receptor agonist and DMPP (10 microM) a nicotinic (N) receptor agonist decreased the release of ACh (50% and 55% respectively); whereas McN-A 343 (50 microM) an M1 receptor agonist increased the release (33%), indicating the presence of three types of modulatory receptors. The anticholinesterase agent, physostigmine in concentrations of 1, 5 and 25 microM and neostigmine (5 microM) increased ACh release (44-710%). However a low concentration of physostigmine (0.05 microM) decreased release. Pirenzepine, an M1 muscarinic antagonist or atropine blocked the increased ACh release in physostigmine-treated strips, but in normal strips pirenzepine did not change release and atropine increased release. McN-A 343 or prolonged application (15 min) of DMPP increased ACh release (376% and 391% respectively) in physostigmine-treated strips. The response to McN-A 343 was blocked by pirenzepine. d-Tubocurarine (DTC), a nicotinic receptor blocker, enhanced ACh release in the presence of physostigmine but proved to be ineffective in normal preparations. These findings suggest that all three cholinergic receptors (M1 facilitatory, N inhibitory and M2 inhibitory) are activated by endogenous ACh in physostigmine treated preparations whereas only M2-inhibitory receptors are activated in normal preparations. It will be important in future studies to determine whether M1 and M2 mechanisms can also be activated under more physiological conditions in the bladder and whether they are present at other cholinergic synapses.

  17. COMBINED USE OF α-ADRENERGIC AND MUSCARINIC ANTAGONISTS FOR THE TREATMENT OF VOIDING DYSFUNCTION

    PubMed Central

    RUGGIERI, MICHAEL R.; BRAVERMAN, ALAN S.; PONTARI, MICHEL A.

    2012-01-01

    Purpose We provide an overview of the medical literature supporting the combined use of muscarinic and α-adrenergic antagonist therapy for the treatment of voiding dysfunction. Materials and Methods The MEDLINE database (1966 to 2004) of the United States National Library of Medicine was searched for pertinent studies. Results Although the mechanism of action of α-adrenergic antagonist therapy for voiding dysfunction has traditionally been assumed to be relaxation of the periurethral, prostatic and bladder neck smooth muscle, substantial evidence supports action at extraprostatic sites involved in micturition, including the bladder dome smooth muscle, peripheral ganglia, spinal cord and brain. Likewise the mechanism of action of anticholinergic therapy has been traditionally assumed to be inhibition of the M3 muscarinic receptor subtypes that mediate normal bladder contractions. However, M2 receptor mediates hypertrophied bladder contractions and there is evidence for an M2 component to the suprasacral control of voiding. Conclusions Based on the physiology of α-adrenergic and muscarinic receptors the inhibition of each one would be expected to be more beneficial than that of either alone because they would work on 2 components of detrusor function. Patients who would likely benefit from this combination therapy are men with lower urinary tract symptoms, women with urgency/frequency syndrome (overactive bladder), patients with uninhibited bladder contractions due to neurogenic bladder, and patients with pelvic pain and voiding symptoms, ie interstitial cystitis and chronic prostatitis/chronic pelvic pain syndrome. PMID:16217275

  18. Activation of Muscarinic M1 Acetylcholine Receptors Induces Long-Term Potentiation in the Hippocampus

    PubMed Central

    Dennis, Siobhan H.; Pasqui, Francesca; Colvin, Ellen M.; Sanger, Helen; Mogg, Adrian J.; Felder, Christian C.; Broad, Lisa M.; Fitzjohn, Steve M.; Isaac, John T.R.; Mellor, Jack R.

    2016-01-01

    Muscarinic M1 acetylcholine receptors (M1Rs) are highly expressed in the hippocampus, and their inhibition or ablation disrupts the encoding of spatial memory. It has been hypothesized that the principal mechanism by which M1Rs influence spatial memory is by the regulation of hippocampal synaptic plasticity. Here, we use a combination of recently developed, well characterized, selective M1R agonists and M1R knock-out mice to define the roles of M1Rs in the regulation of hippocampal neuronal and synaptic function. We confirm that M1R activation increases input resistance and depolarizes hippocampal CA1 pyramidal neurons and show that this profoundly increases excitatory postsynaptic potential-spike coupling. Consistent with a critical role for M1Rs in synaptic plasticity, we now show that M1R activation produces a robust potentiation of glutamatergic synaptic transmission onto CA1 pyramidal neurons that has all the hallmarks of long-term potentiation (LTP): The potentiation requires NMDA receptor activity and bi-directionally occludes with synaptically induced LTP. Thus, we describe synergistic mechanisms by which acetylcholine acting through M1Rs excites CA1 pyramidal neurons and induces LTP, to profoundly increase activation of CA1 pyramidal neurons. These features are predicted to make a major contribution to the pro-cognitive effects of cholinergic transmission in rodents and humans. PMID:26472558

  19. 3D Reconstructed Cyto-, Muscarinic M2 Receptor, and Fiber Architecture of the Rat Brain Registered to the Waxholm Space Atlas

    PubMed Central

    Schubert, Nicole; Axer, Markus; Schober, Martin; Huynh, Anh-Minh; Huysegoms, Marcel; Palomero-Gallagher, Nicola; Bjaalie, Jan G.; Leergaard, Trygve B.; Kirlangic, Mehmet E.; Amunts, Katrin; Zilles, Karl

    2016-01-01

    High-resolution multiscale and multimodal 3D models of the brain are essential tools to understand its complex structural and functional organization. Neuroimaging techniques addressing different aspects of brain organization should be integrated in a reference space to enable topographically correct alignment and subsequent analysis of the various datasets and their modalities. The Waxholm Space (http://software.incf.org/software/waxholm-space) is a publicly available 3D coordinate-based standard reference space for the mapping and registration of neuroanatomical data in rodent brains. This paper provides a newly developed pipeline combining imaging and reconstruction steps with a novel registration strategy to integrate new neuroimaging modalities into the Waxholm Space atlas. As a proof of principle, we incorporated large scale high-resolution cyto-, muscarinic M2 receptor, and fiber architectonic images of rat brains into the 3D digital MRI based atlas of the Sprague Dawley rat in Waxholm Space. We describe the whole workflow, from image acquisition to reconstruction and registration of these three modalities into the Waxholm Space rat atlas. The registration of the brain sections into the atlas is performed by using both linear and non-linear transformations. The validity of the procedure is qualitatively demonstrated by visual inspection, and a quantitative evaluation is performed by measurement of the concordance between representative atlas-delineated regions and the same regions based on receptor or fiber architectonic data. This novel approach enables for the first time the generation of 3D reconstructed volumes of nerve fibers and fiber tracts, or of muscarinic M2 receptor density distributions, in an entire rat brain. Additionally, our pipeline facilitates the inclusion of further neuroimaging datasets, e.g., 3D reconstructed volumes of histochemical stainings or of the regional distributions of multiple other receptor types, into the Waxholm Space

  20. 3D Reconstructed Cyto-, Muscarinic M2 Receptor, and Fiber Architecture of the Rat Brain Registered to the Waxholm Space Atlas.

    PubMed

    Schubert, Nicole; Axer, Markus; Schober, Martin; Huynh, Anh-Minh; Huysegoms, Marcel; Palomero-Gallagher, Nicola; Bjaalie, Jan G; Leergaard, Trygve B; Kirlangic, Mehmet E; Amunts, Katrin; Zilles, Karl

    2016-01-01

    High-resolution multiscale and multimodal 3D models of the brain are essential tools to understand its complex structural and functional organization. Neuroimaging techniques addressing different aspects of brain organization should be integrated in a reference space to enable topographically correct alignment and subsequent analysis of the various datasets and their modalities. The Waxholm Space (http://software.incf.org/software/waxholm-space) is a publicly available 3D coordinate-based standard reference space for the mapping and registration of neuroanatomical data in rodent brains. This paper provides a newly developed pipeline combining imaging and reconstruction steps with a novel registration strategy to integrate new neuroimaging modalities into the Waxholm Space atlas. As a proof of principle, we incorporated large scale high-resolution cyto-, muscarinic M2 receptor, and fiber architectonic images of rat brains into the 3D digital MRI based atlas of the Sprague Dawley rat in Waxholm Space. We describe the whole workflow, from image acquisition to reconstruction and registration of these three modalities into the Waxholm Space rat atlas. The registration of the brain sections into the atlas is performed by using both linear and non-linear transformations. The validity of the procedure is qualitatively demonstrated by visual inspection, and a quantitative evaluation is performed by measurement of the concordance between representative atlas-delineated regions and the same regions based on receptor or fiber architectonic data. This novel approach enables for the first time the generation of 3D reconstructed volumes of nerve fibers and fiber tracts, or of muscarinic M2 receptor density distributions, in an entire rat brain. Additionally, our pipeline facilitates the inclusion of further neuroimaging datasets, e.g., 3D reconstructed volumes of histochemical stainings or of the regional distributions of multiple other receptor types, into the Waxholm Space

  1. Bidirectional modulation of visual plasticity by cholinergic receptor subtypes in the frog optic tectum

    PubMed Central

    Yu, Chuan-Jiang; Butt, Christopher M.; Debski, Elizabeth A.

    2008-01-01

    Cholinergic input to the optic tectum is necessary for visual map maintenance. To understand why, we examined the effects of activation of the different cholinergic receptor subtypes in tectal brain slices and determined whether the retinotectal map was affected by manipulations of their activity in vivo. Both α-bungarotoxin sensitive and insensitive nicotinic receptor agonists increased spontaneous postsynaptic currents (sPSCs) in a subpopulation of patch-clamped tectal cells; application of subtype selective receptor antagonists reduced nicotine-induced increases in sPSCs. Activation of α-bungarotoxin insensitive nicotinic receptors also induced substantial inward current in some cells. Muscarinic receptor mediated outward current responses were blocked by the M2-like muscarinic receptor antagonists himbacine or AF-DX 384 and mimicked by application of the M2-like agonist oxotremorine. A less frequently observed muscarinic response involving a change in sPSC frequency appeared to be mediated by M1-like muscarinic receptors. In separate experiments, pharmacological manipulation of cholinergic receptor subtype activation led to changes in the activity-dependent visual map created in the tectum by retinal ganglion cell terminals. Chronic exposure of the tectum to either α-bungarotoxin insensitive, α-bungarotoxin sensitive or M1-like receptor antagonists resulted in map disruption. However, treatment with the M2-like receptor antagonist, AF-DX 384, compressed the map. We conclude that nicotinic or M1-like muscarinic receptors control input to tectal cells while α-bungarotoxin insensitive nicotinic receptors and M2-like muscarinic receptors change tectal cell responses to that input. Blockade of the different cholinergic receptor subtypes can have opposing effects on map topography that are consistent with expected effects on tectal cell activity levels. PMID:12670313

  2. The interleukin-4 receptor: signal transduction by a hematopoietin receptor.

    PubMed

    Keegan, A D; Pierce, J H

    1994-02-01

    Over the last several years, the receptors for numerous cytokines have been molecularly characterized. Analysis of their amino acid sequences shows that some of these receptors bear certain motifs in their extracellular domains that define a family of receptors called the Hematopoietin receptor superfamily. Significant advances in characterizing the structure, function, and mechanisms of signal transduction have been made for several members of this family. The purpose of this review is to discuss the recent advances made for one of the family members, the interleukin (IL) 4 receptor. Other receptor systems have recently been reviewed elsewhere. The IL-4 receptor consists of, at the minimum, the cloned 140 kDa IL-4-binding chain with the potential for associating with other chains. The IL-4 receptor transduces its signal by activating a tyrosine kinase that phosphorylates cellular substrates, including the receptor itself, and the 170 kDa substrate called 4PS. Phosphorylated 4PS interacts with the SH2 domain of the enzyme PI-3'-kinase and increases its enzymatic activity. These early events in the IL-4 receptor initiated signaling pathway may trigger a series of signals that will ultimately lead to an IL-4 specific biologic outcome.

  3. Acetylcholine-induced activation of M3 muscarinic receptors stimulates robust matrix metalloproteinase gene expression in human colon cancer cells.

    PubMed

    Xie, Guofeng; Cheng, Kunrong; Shant, Jasleen; Raufman, Jean-Pierre

    2009-04-01

    Previously, we showed that ACh-induced proliferation of human colon cancer cells is mediated by transactivation of epidermal growth factor (EGF) receptors (EGFRs). In the present study, we elucidate the molecular mechanism underlying this action. ACh-induced proliferation of H508 colon cancer cells, which express exclusively M3 muscarinic receptors (M3Rs), was attenuated by anti-EGFR ligand binding domain antibody, a broad-spectrum matrix metalloproteinase (MMP) inhibitor, anti-MMP7 antibody, a diphtheria toxin analog that blocks release of an EGFR ligand [heparin-binding EGF-like growth factor (HBEGF)], and anti-HBEGF antibody. Conditioned media from ACh-treated H508 cells induced proliferation of SNU-C4 colon cancer cells that express EGFR but not M3R. These actions were attenuated by an EGFR inhibitor and by anti-EGFR and anti-HBEGF antibodies. In H508, but not SNU-C4, colon cancer cells, ACh caused a striking dose- and time-dependent increase in levels of MMP7 mRNA and MMP7 protein. Similarly, ACh induced robust MMP1 and MMP10 gene transcription. ACh-induced MMP1, MMP7, and MMP10 gene transcription was attenuated by atropine, anti-EGFR antibody, and chemical inhibitors of EGFR and ERK activation. In contrast, inhibitors of phosphatidylinositol 3-kinase and NF-kappaB activation did not alter MMP gene transcription. Collectively, these findings indicate that MMP7-catalyzed release of HBEGF mediates ACh-induced transactivation of EGFR and consequent proliferation of colon cancer cells. ACh-induced activation of EGFR and downstream ERK signaling also regulates transcriptional activation of MMP7, thereby identifying a novel feed-forward mechanism for neoplastic cell proliferation.

  4. Acetylcholine-induced activation of M3 muscarinic receptors stimulates robust matrix metalloproteinase gene expression in human colon cancer cells

    PubMed Central

    Xie, Guofeng; Cheng, Kunrong; Shant, Jasleen; Raufman, Jean-Pierre

    2009-01-01

    Previously, we showed that ACh-induced proliferation of human colon cancer cells is mediated by transactivation of epidermal growth factor (EGF) receptors (EGFRs). In the present study, we elucidate the molecular mechanism underlying this action. ACh-induced proliferation of H508 colon cancer cells, which express exclusively M3 muscarinic receptors (M3Rs), was attenuated by anti-EGFR ligand binding domain antibody, a broad-spectrum matrix metalloproteinase (MMP) inhibitor, anti-MMP7 antibody, a diphtheria toxin analog that blocks release of an EGFR ligand [heparin-binding EGF-like growth factor (HBEGF)], and anti-HBEGF antibody. Conditioned media from ACh-treated H508 cells induced proliferation of SNU-C4 colon cancer cells that express EGFR but not M3R. These actions were attenuated by an EGFR inhibitor and by anti-EGFR and anti-HBEGF antibodies. In H508, but not SNU-C4, colon cancer cells, ACh caused a striking dose- and time-dependent increase in levels of MMP7 mRNA and MMP7 protein. Similarly, ACh induced robust MMP1 and MMP10 gene transcription. ACh-induced MMP1, MMP7, and MMP10 gene transcription was attenuated by atropine, anti-EGFR antibody, and chemical inhibitors of EGFR and ERK activation. In contrast, inhibitors of phosphatidylinositol 3-kinase and NF-κB activation did not alter MMP gene transcription. Collectively, these findings indicate that MMP7-catalyzed release of HBEGF mediates ACh-induced transactivation of EGFR and consequent proliferation of colon cancer cells. ACh-induced activation of EGFR and downstream ERK signaling also regulates transcriptional activation of MMP7, thereby identifying a novel feed-forward mechanism for neoplastic cell proliferation. PMID:19221016

  5. Bombesin and muscarinic receptor activation in rat pancreas generate cyclic inositol monophosphate: possible involvement of different phospholipase C isoenzymes.

    PubMed

    Sekar, M C; Sambandam, V; McDonald, J M

    1993-05-14

    Phospholipase C isoenzymes can generate different proportions of cyclic and non-cyclic inositol phosphates. Stimulation of [3H]-inositol labeled pancreatic minilobules by buffer, bombesin, neuromedin B or carbachol in presence of 10 mM lithium, followed by separation of inositol phosphates, yielded the following results for cyclic inositol monophosphate (cIP) [DPM/mg protein; Mean +/- SEM (n)]: control [21 +/- 6, (9)]; bombesin [145 +/- 24, (12)]; neuromedin B (99 +/- 22 (9)] and carbachol [512 +/- 60, (12)]. The generation of cIP and IP were significantly correlated [r2 = 0.72 (p < 0.05)] following carbachol activation, while no significant correlation was obtained following bombesin receptor activation by either bombesin or neuromedin B. Presence of zinc (100 microM) in the final incubation medium failed to amplify the bombesin-stimulated cIP accumulation. Based on our studies we postulate that different phospholipase C isoenzymes may be activated following muscarinic and bombesin receptor stimulation in pancrea.

  6. A G Protein-biased Designer G Protein-coupled Receptor Useful for Studying the Physiological Relevance of Gq/11-dependent Signaling Pathways.

    PubMed

    Hu, Jianxin; Stern, Matthew; Gimenez, Luis E; Wanka, Lizzy; Zhu, Lu; Rossi, Mario; Meister, Jaroslawna; Inoue, Asuka; Beck-Sickinger, Annette G; Gurevich, Vsevolod V; Wess, Jürgen

    2016-04-08

    Designerreceptorsexclusivelyactivated by adesignerdrug (DREADDs) are clozapine-N-oxide-sensitive designer G protein-coupled receptors (GPCRs) that have emerged as powerful novel chemogenetic tools to study the physiological relevance of GPCR signaling pathways in specific cell types or tissues. Like endogenous GPCRs, clozapine-N-oxide-activated DREADDs do not only activate heterotrimeric G proteins but can also trigger β-arrestin-dependent (G protein-independent) signaling. To dissect the relative physiological relevance of G protein-mediatedversusβ-arrestin-mediated signaling in different cell types or physiological processes, the availability of G protein- and β-arrestin-biased DREADDs would be highly desirable. In this study, we report the development of a mutationally modified version of a non-biased DREADD derived from the M3muscarinic receptor that can activate Gq/11with high efficacy but lacks the ability to interact with β-arrestins. We also demonstrate that this novel DREADD is activein vivoand that cell type-selective expression of this new designer receptor can provide novel insights into the physiological roles of G protein (Gq/11)-dependentversusβ-arrestin-dependent signaling in hepatocytes. Thus, this novel Gq/11-biased DREADD represents a powerful new tool to study the physiological relevance of Gq/11-dependent signaling in distinct tissues and cell types, in the absence of β-arrestin-mediated cellular effects. Such studies should guide the development of novel classes of functionally biased ligands that show high efficacy in various pathophysiological conditions but display a reduced incidence of side effects. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Muscarinic type 2 receptors in the lateral dorsal tegmental area modulate cocaine and food seeking behavior in rats.

    PubMed

    Shabani, S; Foster, R; Gubner, N; Phillips, T J; Mark, G P

    2010-10-13

    The cholinergic input from the lateral dorsal tegmental area (LDTg) modulates the dopamine cells of the ventral tegmental area (VTA) and plays an important role in cocaine taking. Specific pharmacological agents that block or stimulate muscarinic receptors in the LDTg change acetylcholine (ACh) levels in the VTA. Furthermore, manipulations of cholinergic input in the VTA can change cocaine taking. In the current study, the ACh output from the LDTg was attenuated by treatment with the selective muscarinic type 2 (M2) autoreceptor agonist oxotremorine.sesquifumarate (OxoSQ). We hypothesized that OxoSQ would reduce the motivation of rats to self-administer both natural and drug rewards. Animals were tested on progressive ratio (PR) schedules of reinforcement for food pellets and cocaine. On test days, animals on food and on cocaine schedules were bilaterally microinjected prior to the test. Rats received either LDTg OxoSQ infusions or LDTg artificial cerebrospinal fluid (aCSF) infusions in a within-subjects design. In addition, infusions were delivered into a dorsal brain area above the LDTg as an anatomical control region. OxoSQ microinjection in the LDTg, compared to aCSF, significantly reduced both the number of self-administered pellets and cocaine infusions during the initial half of the session; this reduction was dose-dependent. OxoSQ microinjections into the area just dorsal to the LDTg had no significant effect on self-administration of food pellets or cocaine. Animals were also tested in locomotor activity chambers for motor effects following the above microinjections. Locomotor activity was mildly increased by OxoSQ microinjection into the LDTg during the initial half of the session. Overall, these data suggest that LDTg cholinergic neurons play an important role in modifying the reinforcing value of natural and drug rewards. These effects cannot be attributed to significant alterations of locomotor behavior and are likely accomplished through LDTg

  8. Effects of stimulation of muscarinic receptors on bladder afferent nerves in the in vitro bladder-pelvic afferent nerve preparation of the rat.

    PubMed

    Yu, Yongbei; de Groat, William C

    2010-11-18

    Effects of a muscarinic receptor agonist oxotremorine-M (oxo-M) on bladder afferent nerve (BAN) activity were studied in an in vitro bladder-pelvic nerve preparation. Distension of the bladder induced rhythmic bladder contractions that were accompanied by multiunit afferent firing. Intravesical administration of 25 and 50 μM oxo-M significantly increased afferent firing from 41 ± 2 spikes/s to 51 ± 4 spikes/s and 60.5 ± 5 spikes/s, respectively, but did not change the maximum amplitude of spontaneous bladder contractions. The afferent nerve firing induced by isotonic distension of the bladder (10-40 cmH(2)O) was increased 22-100% by intravesical administration of 50 μM oxo-M. Electrical stimulation on the surface of the bladder elicited action potentials (AP) in BAN. Oxo-M significantly decreased the voltage threshold by 40% (p<0.05) and increased by 157% (p<0.05) the area of the AP evoked at a submaximal stimulus intensity. These effects were blocked by intravesical injection of 5 μM atropine methyl nitrate (AMN). Intravesical administration of 5 μM AMN alone did not alter BAN firing or the amplitude of bladder contractions. The facilitatory effects induced by oxo-M on BAN activity were also suppressed (p<0.05) by intravesical administration of 2',3'-0-trinitrophenyl-ATP (TNP-ATP) (30 μM). In preparations pretreated with capsaicin (125 mg/kg, s.c.) the facilitatory effects of 50 μM oxo-M on BAN activity were absent. These results suggest that activation of muscarinic receptors facilitates mechano-sensitive, capsaicin-sensitive BAN activity in part by mechanisms involving purinergic receptors located near the luminal surface of the bladder and ATP release which presumably occurs in the urothelium. Copyright © 2010 Elsevier B.V. All rights reserved.

  9. A comparison of β-adrenoceptors and muscarinic cholinergic receptors in tissues of brown bullhead catfish (Ameiurus nebulosus) from the black river and old woman creek, Ohio

    USGS Publications Warehouse

    Steevens, Jeffery A.; Baumann, Paul C.; Jones, Susan B.

    1996-01-01

    β-Adrenoceptors (βARs) and muscarinic cholinergic receptors were measured in brain, gill, and heart tissues of brown bullhead catfish exposed to polycyclic aromatic hydrocarbons in the Black River, Ohio, USA, and were compared to values from Old Woman Creek, Ohio, a reference site. A decreased number of βARs were found in the gill from Black River fish, possibly indicating a compensatory response subsequent to chemical stress.

  10. Thermodynamics of antagonist binding to rat muscarinic M2 receptors: antimuscarinics of the pridinol, sila-pridinol, diphenidol and sila-diphenidol type.

    PubMed Central

    Waelbroeck, M.; Camus, J.; Tastenoy, M.; Lambrecht, G.; Mutschler, E.; Kropfgans, M.; Sperlich, J.; Wiesenberger, F.; Tacke, R.; Christophe, J.

    1993-01-01

    1. We studied the effect of temperature on the binding to rat heart M2 muscarinic receptors of antagonists related to the carbon/silicon pairs pridinol/sila-pridinol and diphenidol/sila-diphenidol (including three germanium compounds) and six structurally related pairs of enantiomers [(R)- and (S)-procyclidine, (R)- and (S)-trihexyphenidyl, (R)- and (S)-tricyclamol, (R)- and (S)-trihexyphenidyl methiodide, (R)- and (S)-hexahydro-diphenidol and (R)- and (S)-hexbutinol]. Binding affinities were determined in competition experiments using [3H]-N-methyl-scopolamine chloride as radioligand. The reference drugs were scopolamine and N-methyl-scopolamine bromide. 2. The affinity of the antagonists either increased or decreased with temperature. van't Hoff plots were linear in the 278-310 degrees K temperature range. Binding of all antagonists was entropy driven. Enthalpy changes varied from large negative values (down to -29 kJ mol-1) to large positive values (up to +30 kJ mol-1). 3. (R)-configurated drugs had a 10 to 100 fold greater affinity for M2 receptors than the corresponding (S)-enantiomers. Enthalpy and entropy changes of the respective enantiomers were different but no consistent pattern was observed. 4. When silanols (R3SiOH) were compared to carbinols (R3COH), the affinity increase caused by C/Si exchange varied between 3 and 10 fold for achiral drugs but was negligible in the case of chiral drugs. Silanols induced more favourable enthalpy and less favourable entropy changes than the corresponding carbinols when binding. Organogermanium compounds (R4Ge) when compared to their silicon counterparts (R4Si) showed no significant difference in affinity as well as in enthalpy and entropy changes. 5. Exchange of a cyclohexyl by a phenyl moiety was associated with an increase or a decrease in drug affinity (depending on the absolute configuration in the case of chiral drugs) and generally also with a more favourable enthalpy change and a less favourable entropy change

  11. Effects of acute administration of nicotinic and muscarinic cholinergic agonists and antagonists on performance in different cost–benefit decision making tasks in rats

    PubMed Central

    Mendez, Ian A.; Gilbert, Ryan J.; Bizon, Jennifer L.

    2012-01-01

    Rationale Alterations in cost–benefit decision making accompany numerous neuropsychiatric conditions, including schizophrenia, attention deficit hyperactivity disorder, and addiction. Central cholinergic systems have been linked to the etiology and/or treatment of many of these conditions, but little is known about the role of cholinergic signaling in cost–benefit decision making. Objectives The goal of these experiments was to determine how cholinergic signaling is involved in cost–benefit decision making, using a behavioral pharmacological approach. Methods Male Long-Evans rats were trained in either “probability discounting” or “delay discounting” tasks, in which rats made discrete-trial choices between a small food reward and a large food reward associated with either varying probabilities of omission or varying delays to delivery, respectively. The effects of acute administration of different doses of nicotinic and muscarinic acetylcholine receptor agonists and antagonists were assessed in each task. Results In the probability discounting task, acute nicotine administration (1.0 mg/kg) significantly increased choice of the large risky reward, and control experiments suggested that this was due to robust nicotine-induced impairments in behavioral flexibility. In the delay discounting task, the muscarinic antagonists scopolamine (0.03, 0.1, and 0.3 mg/kg) and atropine (0.3 mg/kg) both significantly increased choice of the small immediate reward. Neither mecamylamine nor oxotremorine produced reliable effects on either of the decision making tasks. Conclusions These data suggest that cholinergic receptors play multiple roles in decision making contexts which include consideration of reward delay or probability. These roles should be considered when targeting these receptors for therapeutic purposes. PMID:22760484

  12. 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

  13. The influences of metabotropic receptor activation on cellular signaling and synaptic function in amacrine cells.

    PubMed

    Gleason, Evanna

    2012-01-01

    Amacrine cells receive glutamatergic input from bipolar cells and GABAergic, glycinergic, cholinergic, and dopaminergic input from other amacrine cells. Glutamate, GABA, glycine, and acetylcholine (ACh) interact with ionotropic receptors and it is these interactions that form much of the functional circuitry in the inner retina. However, glutamate, GABA, ACh, and dopamine also activate metabotropic receptors linked to second messenger pathways that have the potential to modify the function of individual cells as well as retinal circuitry. Here, the physiological effects of activating dopamine receptors, metabotropic glutamate receptors, GABAB receptors, and muscarinic ACh receptors on amacrine cells will be discussed. The retina also expresses metabotropic receptors and the biochemical machinery associated with the synthesis and degradation of endocannabinoids and sphingosine-1-phosphate (S1P). The effects of activating cannabinoid receptors and S1P receptors on amacrine cell function will also be addressed. Copyright © Cambridge University Press, 2012

  14. Guanosine 5'-triphosphate binding protein (G/sub i/) and two additional pertussis toxin substrates associated with muscarinic receptors in rat heart myocytes: characterization and age dependency

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Moscona-Amir, E.; Henis, Y.I.; Sokolovsky, M.

    1988-07-12

    The coupling of muscarinic receptors with G-proteins was investigated in cultured myocytes prepared from the hearts of newborn rats. The coupling was investigated in both young (5 days after plating) and aged (14 days after plating) cultures, in view of the completely different effects of 5'-guanylyl imidodiphosphate (Gpp(NH)p) on muscarinic agonist binding to homogenates from young vs aged cultures. Pretreatment of cultures from both ages by Bordetella pertussis toxin (IAP) was found to eliminate any Gpp(NH)p effect on carbamylcholine binding. IAP by itself induced a rightward shift in the carbamylcholine competition curve in homogenates from aged cultures, but no suchmore » effect was observed in homogenates from young cultures. IAP-catalyzed (/sup 32/P)ADP-ribosylation of membrane preparations from young and aged cultures revealed major differences between them. Young cultures exhibited a major IAP substrate at 40 kDa, which was also recognized by anti-..cap alpha../sub i/ antibodies, and two novel IAP substrates at 28 and 42 kDa, which were weakly ADP-ribosylated by the toxin and were not recognized with either anti-..cap alpha../sub i/ or anti-..cap alpha../sub 0/ antibodies. In aged cultures, only the 40-kDa band (ribosylated to a lower degree) was detected. The parallel age-dependent changes in the three IAP substrates (28, 40, and 42 kDa) and in the interactions of the G-protein(s) with the muscarinic receptors strongly suggest close association between the two phenomena. All of these age-dependent changes in the G-protein related parameters were prevented by phosphatidylcholine-liposome treatment of the aged cultures. The role of the membrane lipid composition in these phenomena is discussed.« less

  15. Insulin oversecretion in MSG-obese rats is related to alterations in cholinergic muscarinic receptor subtypes in pancreatic islets.

    PubMed

    Miranda, Rosiane A; Agostinho, Aryane R; Trevenzoli, Isis H; Barella, Luiz F; Franco, Claudinéia C S; Trombini, Amanda B; Malta, Ananda; Gravena, Clarice; Torrezan, Rosana; Mathias, Paulo C F; de Oliveira, Júlio C

    2014-01-01

    Impaired pancreatic beta cell function and insulin secretion/action are a link between obesity and type 2 diabetes, which are worldwide public health burdens. We aimed to characterize the muscarinic acetylcholine receptor (mAChR) M1-M4 subtypes in isolated pancreatic islets from pre-diabetic obese rats that had been treated neonatally with monosodium L-glutamate (MSG). At 90 days of age, both the MSG and the control groups underwent biometric and biochemical evaluation. Anti-muscarinic drugs were used to study mAChR function either in vivo or in vitro. The results demonstrated that atropine treatment reduced insulin secretion in the MSG-treated and control groups, whereas treatment with an M2mAChR-selective antagonist increased secretion. Moreover, the insulinostatic effect of an M3mAChR-selective antagonist was significantly higher in the MSG-treated group. M1mAChR and M3mAChR expression was increased in the MSG-obese group by 55% and 73%, respectively. In contrast, M2mAChR expression decreased by 25% in the MSG group, whereas M4mAChR expression was unchanged. Functional changes in and altered content of the mAChR (M1-M4) subtypes are pivotal to the demand for high pancreatic beta cell insulin secretion in MSG-obese rats, which is directly associated with vagal hyperactivity and peripheral insulin resistance. © 2014 S. Karger AG, Basel.

  16. Tachykinin receptor expression and function in human esophageal smooth muscle.

    PubMed

    Kovac, Jason R; Chrones, Tom; Preiksaitis, Harold G; Sims, Stephen M

    2006-08-01

    Tachykinins are present in enteric nerves of the gastrointestinal tract and cause contraction of esophageal smooth muscle; however, the mechanisms involved are not understood. Our aim was to characterize tachykinin signaling in human esophageal smooth muscle. We investigated functional effects of tachykinins on human esophageal smooth muscle using tension recordings and isolated cells, receptor expression with reverse transcription (RT)-polymerase chain reaction (PCR) and immunoblotting, intracellular Ca2+ responses using fluorescent indicator dyes, and membrane currents with patch-clamp electrophysiology. The mammalian tachykinins [substance P and neurokinin (NK) A and NKB] elicited concentration-dependent contractions of human esophageal smooth muscle. These responses were not affected by muscarinic receptor or neuronal blockade indicating a direct effect on smooth muscle cells (SMCs). Immunofluorescence and RT-PCR identified tachykinin receptors (NK1, NK2, and NK3) on SMCs. Contraction was mediated through a combination of Ca2+ release from intracellular stores and influx through L-type Ca2+ channels. NK2 receptor blockade inhibited the largest proportion of tachykinin-evoked responses. NKA evoked a nonselective cation current (I(NSC)) with properties similar to that elicited by muscarinic stimulation. The following paradigm is suggested: tachykinin receptor binding to SMCs releases Ca2+ from stores along with activation of I(NSC), which in turn results in membrane depolarization, L-type Ca2+ channel opening, rise of Ca2+ concentration, and contraction. These studies reveal new aspects of tachykinin signaling in human esophageal SMCs. Excitatory tachykinin pathways may represent targets for pharmacological intervention in disorders of esophageal dysmotility.

  17. The muscarinic inhibition of the potassium M-current modulates the action-potential discharge in the vestibular primary-afferent neurons of the rat.

    PubMed

    Pérez, C; Limón, A; Vega, R; Soto, E

    2009-02-18

    There is consensus that muscarinic and nicotinic receptors expressed in vestibular hair cells and afferent neurons are involved in the efferent modulation of the electrical activity of the afferent neurons. However the underlying mechanisms of postsynaptic control in neurons are not well understood. In our work we show that the activation of muscarinic receptors in the vestibular neurons modulates the potassium M-current modifying the activity of afferent neurons. Whole-cell patch-clamp recordings were made on vestibular-afferent neurons isolated from Wistar rats (postnatal days 7-10) and held in primary culture (18-24 h). The M-current was studied during its deactivation after depolarizing voltage-clamp pulses. In 68% of the cells studied, those of larger capacitance, the M-current antagonists linopirdine and XE-991 reduced the amplitude of the M-current by 54%+/-7% and 50%+/-3%. The muscarinic-receptor agonist oxotremorine-M also significantly reduced the M-current by 58%+/-12% in the cells. The action of oxotremorine-M was blocked by atropine, thus indicating its cholinergic nature. The erg-channel blocker E-4031 did not significantly modify the M-current amplitude. In current-clamp experiments, linopirdine, XE-991, and oxotremorine-M modified the discharge response to current pulses from single spike to multiple spiking, reducing the adaptation of the electrical discharge. Our results indicate that large soma-size cultured vestibular-afferent neurons (most probably calyx-bearing neurons) express the M-current and that the modulation of this current by activation of muscarinic-receptor reduces its spike-frequency adaptation.

  18. Comparison of (/sup 3/H)pirenzepine and (/sup 3/H)quinuclidinylbenzilate binding to muscarinic cholinergic receptors in rat brain

    DOE Office of Scientific and Technical Information (OSTI.GOV)

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

    The properties of (/sup 3/H)quinuclidinylbenzilate ( (/sup 3/H)QNB) binding and (/sup 3/H)pirenzepine ( (/sup 3/H)PZ) binding to various regions of rat brain were compared. (/sup 3/H)PZ appeared to bind with high affinity to a single site, with a Kd value of approximately 15 nM in the cerebral cortex. The rank order of potencies of muscarinic drugs to inhibit binding of either (/sup 3/H)QNB or (/sup 3/H)PZ was QNB greater than atropine . scopolamine greater than pirenzepine greater than oxotremorine greater than bethanechol. Muscarinic antagonists (except PZ) inhibited both (/sup 3/H)PZ and (/sup 3/H)QNB binding with Hill coefficients of approximately 1.more » PZ inhibited (/sup 3/H)QNB binding in cortex with a Hill coefficient of 0.7, but inhibited (/sup 3/H)PZ binding with a Hill coefficient of 1.0. Hill coefficients for agonists were less than 1. The density of (/sup 3/H)PZ binding sites was approximately half the density of (/sup 3/H)QNB binding sites in cortex, striatum and hippocampus. In pons-medulla and cerebellum, the densities of (/sup 3/H)PZ binding sites were 20 and 0%, respectively, relative to the densities of (/sup 3/H)QNB binding sites. When unlabeled PZ was used to compete for (/sup 3/H)QNB binding, the relative number of high-affinity PZ binding sites in cortex, pons and cerebellum agreed with the relative number of (/sup 3/H)PZ binding sites in those regions. The binding of (/sup 3/H)PZ and (/sup 3/H)QNB was nonadditive in cortex. GTP inhibited high-affinity oxotremorine binding, but not PZ binding. Together, these data suggest that (/sup 3/H)PZ binds to a subset of (/sup 3/H)QNB binding sites. Whether this subset reflects the existence of subtypes of muscarinic receptors or is a consequence of coupling to another membrane protein remains to be seen.« less

  19. Cigarette Smoke Disturbs the Survival of CD8+ Tc/Tregs Partially through Muscarinic Receptors-Dependent Mechanisms in Chronic Obstructive Pulmonary Disease.

    PubMed

    Chen, Gang; Zhou, Mei; Chen, Long; Meng, Zhao-Ji; Xiong, Xian-Zhi; Liu, Hong-Ju; Xin, Jian-Bao; Zhang, Jian-Chu

    2016-01-01

    CD8+ T cells (Cytotoxic T cells, Tc) are known to play a critical role in the pathogenesis of smoking related airway inflammation including chronic obstructive pulmonary disease (COPD). However, how cigarette smoke directly impacts systematic CD8+ T cell and regulatory T cell (Treg) subsets, especially by modulating muscarinic acetylcholine receptors (MRs), has yet to be well elucidated. Circulating CD8+ Tc/Tregs in healthy nonsmokers (n = 15), healthy smokers (n = 15) and COPD patients (n = 18) were evaluated by flow cytometry after incubating with anti-CD3, anti-CD8, anti-CD25, anti-Foxp3 antibodies. Peripheral blood T cells (PBT cells) from healthy nonsmokers were cultured in the presence of cigarette smoke extract (CSE) alone or combined with MRs agonist/antagonist for 5 days. Proliferation and apoptosis were evaluated by flow cytometry using Ki-67/Annexin-V antibodies to measure the effects of CSE on the survival of CD8+ Tc/Tregs. While COPD patients have elevated circulating percentage of CD8+ T cells, healthy smokers have higher frequency of CD8+ Tregs. Elevated percentages of CD8+ T cells correlated inversely with declined FEV1 in COPD. CSE promoted the proliferation and inhibited the apoptosis of CD8+ T cells, while facilitated both the proliferation and apoptosis of CD8+ Tregs. Notably, the effects of CSE on CD8+ Tc/Tregs can be mostly simulated or attenuated by muscarine and atropine, the MR agonist and antagonist, respectively. However, neither muscarine nor atropine influenced the apoptosis of CD8+ Tregs. The results imply that cigarette smoking likely facilitates a proinflammatory state in smokers, which is partially mediated by MR dysfunction. The MR antagonist may be a beneficial drug candidate for cigarette smoke-induced chronic airway inflammation.

  20. AT1 receptor signaling pathways in the cardiovascular system.

    PubMed

    Kawai, Tatsuo; Forrester, Steven J; O'Brien, Shannon; Baggett, Ariele; Rizzo, Victor; Eguchi, Satoru

    2017-11-01

    The importance of the renin angiotensin aldosterone system in cardiovascular physiology and pathophysiology has been well described whereas the detailed molecular mechanisms remain elusive. The angiotensin II type 1 receptor (AT1 receptor) is one of the key players in the renin angiotensin aldosterone system. The AT1 receptor promotes various intracellular signaling pathways resulting in hypertension, endothelial dysfunction, vascular remodeling and end organ damage. Accumulating evidence shows the complex picture of AT1 receptor-mediated signaling; AT1 receptor-mediated heterotrimeric G protein-dependent signaling, transactivation of growth factor receptors, NADPH oxidase and ROS signaling, G protein-independent signaling, including the β-arrestin signals and interaction with several AT1 receptor interacting proteins. In addition, there is functional cross-talk between the AT1 receptor signaling pathway and other signaling pathways. In this review, we will summarize an up to date overview of essential AT1 receptor signaling events and their functional significances in the cardiovascular system. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Membrane receptor location defines receptor interaction with signaling proteins in a polarized epithelium.

    PubMed

    Amsler, K; Kuwada, S K

    1999-01-01

    Signal transduction from receptors is mediated by the interaction of activated receptors with proximate downstream signaling proteins. In polarized epithelial cells, the membrane is divided into subdomains: the apical and basolateral membranes. Membrane receptors may be present in one or both subdomains. Using a combination of immunoprecipitation and Western blot analyses, we tested the hypothesis that a tyrosine kinase growth factor receptor, epidermal growth factor receptor (EGFR), interacts with distinct signaling proteins when present at the apical vs. basolateral membrane of a polarized renal epithelial cell. We report here that tyrosine phosphorylation of phospholipase C-gamma (PLC-gamma) was induced only when basolateral EGFR was activated. In contrast, tyrosine phosphorylation of several other signaling proteins was increased by activation of receptor at either surface. All signaling proteins were distributed diffusely throughout the cytoplasm; however, PLC-gamma protein also displayed a concentration at lateral cell borders. These results demonstrate that in polarized epithelial cells the array of signaling pathways initiated by activation of a membrane receptor is defined, at least in part, by the membrane location of the receptor.

  2. Distribution of the muscarinic K+ channel proteins Kir3.1 and Kir3.4 in the ventricle, atrium, and sinoatrial node of heart.

    PubMed

    Dobrzynski, H; Marples, D D; Musa, H; Yamanushi, T T; Henderson, Z; Takagishi, Y; Honjo, H; Kodama, I; Boyett, M R

    2001-10-01

    The functionally important effects on the heart of ACh released from vagal nerves are principally mediated by the muscarinic K+ channel. The aim of this study was to determine the abundance and cellular location of the muscarinic K+ channel subunits Kir3.1 and Kir3.4 in different regions of heart. Western blotting showed a very low abundance of Kir3.1 in rat ventricle, although Kir3.1 was undetectable in guinea pig and ferret ventricle. Although immunofluorescence on tissue sections showed no labeling of Kir3.1 in rat, guinea pig, and ferret ventricle and Kir3.4 in rat ventricle, immunofluorescence on single ventricular cells from rat showed labeling in t-tubules of both Kir3.1 and Kir3.4. Kir3.1 was abundant in the atrium of the three species, as shown by Western blotting and immunofluorescence, and Kir3.4 was abundant in the atrium of rat, as shown by immunofluorescence. Immunofluorescence showed Kir3.1 expression in SA node from the three species and Kir3.4 expression in the SA node from rat. The muscarinic K+ channel is activated by ACh via the m2 muscarinic receptor and, in atrium and SA node from ferret, Kir3.1 labeling was co-localized with m2 muscarinic receptor labeling throughout the outer cell membrane.

  3. Purinergic and muscarinic modulation of ATP release from the urothelium and its paracrine actions

    PubMed Central

    Sui, Guiping; Fry, Chris H.; Montgomery, Bruce; Roberts, Max; Wu, Rui

    2013-01-01

    The urothelium is a newly recognized sensory structure that detects bladder fullness. Pivotal to this sensory role is the release of ATP from the urothelium. However, the routes for urothelial ATP release, its modulation by receptor-mediated pathways, and the autocrine/paracrine role of ATP are poorly understood, especially in native tissue. We examined the action of key neurotransmitters: purinergic and muscarinic agonists on ATP release and its paracrine effect. Guinea pig and human urothelial mucosa were mounted in a perfusion trough; superfusate ATP was measured using a luciferin-luciferase assay, and tissue contractions were recorded with a tension transducer. Intracellular Ca2+ was measured in isolated urothelial cells with fura-2. The P2Y agonist UTP but not the P2X agonist α,β-methylene-ATP generated ATP release. The muscarinic agonist carbachol and the M2-preferential agonist oxotremorine also generated ATP release, which was antagonized by the M2-specific agent methoctramine. Agonist-evoked ATP release was accompanied by mucosal contractions. Urothelial ATP release was differentially mediated by intracellular Ca2+ release, cAMP, exocytosis, or connexins. Urothelium-attached smooth muscle exhibited spontaneous contractions that were augmented by subthreshold concentrations of carbachol, which had little direct effect on smooth muscle. This activity was attenuated by desensitizing P2X receptors on smooth muscle. Urothelial ATP release was increased in aging bladders. Purinergic and muscarinic agents produced similar effects in human urothelial tissue. This is the first demonstration of specific modulation of urothelial ATP release in native tissue by purinergic and muscarinic neurotransmitters via distinct mechanisms. Released ATP produces paracrine effects on underlying tissues. This process is altered during aging and has relevance to human bladder pathologies. PMID:24285497

  4. Purinergic and muscarinic modulation of ATP release from the urothelium and its paracrine actions.

    PubMed

    Sui, Guiping; Fry, Chris H; Montgomery, Bruce; Roberts, Max; Wu, Rui; Wu, Changhao

    2014-02-01

    The urothelium is a newly recognized sensory structure that detects bladder fullness. Pivotal to this sensory role is the release of ATP from the urothelium. However, the routes for urothelial ATP release, its modulation by receptor-mediated pathways, and the autocrine/paracrine role of ATP are poorly understood, especially in native tissue. We examined the action of key neurotransmitters: purinergic and muscarinic agonists on ATP release and its paracrine effect. Guinea pig and human urothelial mucosa were mounted in a perfusion trough; superfusate ATP was measured using a luciferin-luciferase assay, and tissue contractions were recorded with a tension transducer. Intracellular Ca²⁺ was measured in isolated urothelial cells with fura-2. The P2Y agonist UTP but not the P2X agonist α,β-methylene-ATP generated ATP release. The muscarinic agonist carbachol and the M₂-preferential agonist oxotremorine also generated ATP release, which was antagonized by the M₂-specific agent methoctramine. Agonist-evoked ATP release was accompanied by mucosal contractions. Urothelial ATP release was differentially mediated by intracellular Ca²⁺ release, cAMP, exocytosis, or connexins. Urothelium-attached smooth muscle exhibited spontaneous contractions that were augmented by subthreshold concentrations of carbachol, which had little direct effect on smooth muscle. This activity was attenuated by desensitizing P2X receptors on smooth muscle. Urothelial ATP release was increased in aging bladders. Purinergic and muscarinic agents produced similar effects in human urothelial tissue. This is the first demonstration of specific modulation of urothelial ATP release in native tissue by purinergic and muscarinic neurotransmitters via distinct mechanisms. Released ATP produces paracrine effects on underlying tissues. This process is altered during aging and has relevance to human bladder pathologies.

  5. Interactions of agonists with an allosteric antagonist at muscarinic acetylcholine M2 receptors.

    PubMed

    Lanzafame, A; Christopoulos, A; Mitchelson, F

    1996-11-28

    The interaction of heptane-1,7-bis(dimethyl-3'-phthalimidopropylammonium bromide) (C7/3'-phth), with several agonists, was investigated at the muscarinic M2 receptor in guinea-pig left atria. C7/3'-phth shifted concentration-response curves for the agonists, carbachol, oxotremorine-M and (+)-cis-dioxolane, to the right in a parallel fashion. Arunlakshana-Schild regressions of the data yielded slopes significantly different to unity, suggesting non-competitive antagonism. Non-linear regression analysis, using an equation based on allosteric modulation, gave quantitative estimates of co-operativity (alpha values) and the dissociation constant of C7/3'-phth (KB). In all cases, the KB estimates for C7/3'-phth were not significantly different. Increasing the carbachol contact time 10-fold did not significantly influence the KB or the alpha value obtained with C7/3'-phth. Changing from Krebs to Tyrode solution did not significantly alter the KB for C7/3'-phth, although alpha values obtained were consistently lower in Tyrode solution, suggesting that the allosteric action may be sensitive to buffer composition. A 4-fold higher degree of negative, heterotropic co-operativity between C7/3'-phth and agonists than between C7/3'-phth and competitive antagonists was also found.

  6. Activation of muscarinic receptors by a hydroalcoholic extract of Dicksonia sellowiana Presl. HooK (Dicksoniaceae) induces vascular relaxation and hypotension in rats.

    PubMed

    Rattmann, Yanna D; Crestani, Sandra; Lapa, Fernanda R; Miguel, Obdúlio G; Marques, Maria C A; da Silva-Santos, J Eduardo; Santos, Adair R S

    2009-01-01

    Dicksonia sellowiana (Presl.) Hook is a native plant from the Central and South Americas that contain high levels of polyphenols, antioxidant compounds involved in protection against inflammation, cancer and cardiovascular risk. A phytomedicinal preparation obtained from aerial parts of D. sellowiana is currently under clinical evaluation in Brazil against asthma, and has been associated with several other beneficial effects. This study demonstrates that a hydroalcoholic extract obtained from D. sellowiana leaves (HEDS) fully relax, in a concentration-dependent manner, rat aortic rings precontracted with phenylephrine. Moreover, administration of HEDS (10, 20 and 40 mg/kg, i.v.) in anaesthetized rats resulted in a strong but reversible hypotension. Aortic relaxation induced by HEDS was abolished by endothelium removal, by incubation of the nitric oxide synthase inhibitor L-NAME, or the soluble guanylate cyclase inhibitor ODQ. In addition, this effect was partially inhibited by indomethacin (a cyclooxygenase inhibitor) and KT 5730 (a PKA inhibitor). The potassium channels blockade by either tetraethylammonium or charybdotoxin also resulted in a potent inhibition of HEDS-induced aortic relaxation, whereas apamine only slightly reduced it. In addition HEDS-induced relaxation was unchanged by 4-amynopiridine and glibenclamide. The selective muscarinic receptor antagonist atropine counteracted both aortic relaxation and blood pressure reduction generated by HEDS. Experiments using HPLC revealed the presence of high amounts of phenolic compounds in this extract. Taken together, our results reveal that the D. sellowiana possess substances with both in vivo and in vitro activities and that the vascular effect of HEDS involves activation of muscarinic receptors, stimulation of the nitric oxide pathway and opening of calcium-activated potassium channels.

  7. Deletion of muscarinic type 1 acetylcholine receptors alters splenic lymphocyte functions and splenic noradrenaline concentration.

    PubMed

    Hainke, Susanne; Wildmann, Johannes; Del Rey, Adriana

    2015-11-01

    The existence of interactions between the immune and the sympathetic nervous systems is well established. Noradrenaline can promote or inhibit the immune response, and conversely, the immune response itself can affect noradrenaline concentration in lymphoid organs, such as the spleen. It is also well known that acetylcholine released by pre-ganglionic neurons can modulate noradrenaline release by the postsynaptic neuron. The spleen does not receive cholinergic innervation, but it has been reported that lymphocytes themselves can produce acetylcholine, and express acetylcholine receptors and acetylcholinesterase. We found that the spleen of not overtly immunized mice in which muscarinic type 1 acetylcholine receptors have been knocked out (M1KO) has higher noradrenaline concentrations than that of the wildtype mice, without comparable alterations in the heart, in parallel to a decreased number of IgG-producing B cells. Splenic lymphocytes from M1KO mice displayed increased in vitro-induced cytotoxicity, and this was observed only when CD4(+) T cells were present. In contrast, heterozygous acetylcholinesterase (AChE+/-) mice, had no alterations in splenic noradrenaline concentration, but the in vitro proliferation of AChE+/- CD4(+) T cells was increased. It is theoretically conceivable that reciprocal effects between neuronally and non-neuronally derived acetylcholine and noradrenaline might contribute to the results reported. Our results emphasize the need to consider the balance between the effects of these mediators for the final immunoregulatory outcome. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. A novel cell-based assay for inhibitory anti-muscarinic type 3 receptor antibodies in primary Sjögren's syndrome.

    PubMed

    Bastian, Isabell; Gordon, Tom P; Jackson, Michael W

    2015-12-01

    Inhibitory autoantibodies acting at the muscarinic acetylcholine receptor type 3 (M3R) are postulated to mediate autonomic dysfunction, including decreased salivary and lacrimal gland output and extra-glandular manifestations, in patients with primary Sjögren's syndrome. However, the contention that anti-M3R antibodies are pathogenic in patients remains untested, due to a lack of assays both sophisticated enough to detect inhibitory anti-M3R antibodies yet suitable for screening large patient cohorts. In the current study, we have established a cell-based bioassay of M3R activity, based on dual transfection of the M3R and a luciferase reporter gene. The bioassay is capable of capturing real-time agonist-mediated signalling of the M3R, which is inhibited specifically by patient IgG that have previously been demonstrated to have anti-M3R activity. The assay can be run in multi-well culture plates, and analysed using simple luminescence readers. As such, the new bioassay incorporating M3R-mediated luciferase transduction is the first assay adaptable to common diagnostic platforms that is capable of determining the presence in patient serum of functionally active anti-M3R autoantibodies. The new bioassay should prove useful for large cohort screening studies aiming to correlate the presence in patients of inhibitory anti-M3R antibodies with symptoms of both glandular and extra-glandular autonomic dysfunction. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Toll-like receptor ligands sensitize B-cell receptor signalling by reducing actin-dependent spatial confinement of the receptor.

    PubMed

    Freeman, Spencer A; Jaumouillé, Valentin; Choi, Kate; Hsu, Brian E; Wong, Harikesh S; Abraham, Libin; Graves, Marcia L; Coombs, Daniel; Roskelley, Calvin D; Das, Raibatak; Grinstein, Sergio; Gold, Michael R

    2015-02-03

    Integrating signals from multiple receptors allows cells to interpret the physiological context in which a signal is received. Here we describe a mechanism for receptor crosstalk in which receptor-induced increases in actin dynamics lower the threshold for signalling by another receptor. We show that the Toll-like receptor ligands lipopolysaccharide and CpG DNA, which are conserved microbial molecules, enhance signalling by the B-cell antigen receptor (BCR) by activating the actin-severing protein cofilin. Single-particle tracking reveals that increased severing of actin filaments reduces the spatial confinement of the BCR within the plasma membrane and increases BCR mobility. This allows more frequent collisions between BCRs and greater signalling in response to low densities of membrane-bound antigen. These findings implicate actin dynamics as a means of tuning receptor signalling and as a mechanism by which B cells distinguish inert antigens from those that are accompanied by indicators of microbial infection.

  10. Toll-like receptor ligands sensitize B-cell receptor signalling by reducing actin-dependent spatial confinement of the receptor

    PubMed Central

    Freeman, Spencer A.; Jaumouillé, Valentin; Choi, Kate; Hsu, Brian E.; Wong, Harikesh S.; Abraham, Libin; Graves, Marcia L.; Coombs, Daniel; Roskelley, Calvin D.; Das, Raibatak; Grinstein, Sergio; Gold, Michael R.

    2015-01-01

    Integrating signals from multiple receptors allows cells to interpret the physiological context in which a signal is received. Here we describe a mechanism for receptor crosstalk in which receptor-induced increases in actin dynamics lower the threshold for signalling by another receptor. We show that the Toll-like receptor ligands lipopolysaccharide and CpG DNA, which are conserved microbial molecules, enhance signalling by the B-cell antigen receptor (BCR) by activating the actin-severing protein cofilin. Single-particle tracking reveals that increased severing of actin filaments reduces the spatial confinement of the BCR within the plasma membrane and increases BCR mobility. This allows more frequent collisions between BCRs and greater signalling in response to low densities of membrane-bound antigen. These findings implicate actin dynamics as a means of tuning receptor signalling and as a mechanism by which B cells distinguish inert antigens from those that are accompanied by indicators of microbial infection. PMID:25644899

  11. A Fluorescent Live Imaging Screening Assay Based on Translocation Criteria Identifies Novel Cytoplasmic Proteins Implicated in G Protein-coupled Receptor Signaling Pathways*

    PubMed Central

    Lecat, Sandra; Matthes, Hans W.D.; Pepperkok, Rainer; Simpson, Jeremy C.; Galzi, Jean-Luc

    2015-01-01

    Several cytoplasmic proteins that are involved in G protein-coupled receptor signaling cascades are known to translocate to the plasma membrane upon receptor activation, such as beta-arrestin2. Based on this example and in order to identify new cytoplasmic proteins implicated in the ON-and-OFF cycle of G protein-coupled receptor, a live-imaging screen of fluorescently labeled cytoplasmic proteins was performed using translocation criteria. The screening of 193 fluorescently tagged human proteins identified eight proteins that responded to activation of the tachykinin NK2 receptor by a change in their intracellular localization. Previously we have presented the functional characterization of one of these proteins, REDD1, that translocates to the plasma membrane. Here we report the results of the entire screening. The process of cell activation was recorded on videos at different time points and all the videos can be visualized on a dedicated website. The proteins BAIAP3 and BIN1, partially translocated to the plasma membrane upon activation of NK2 receptors. Proteins ARHGAP12 and PKM2 translocated toward membrane blebs. Three proteins that associate with the cytoskeleton were of particular interest : PLEKHH2 rearranged from individual dots located near the cell-substrate adhesion surface into lines of dots. The speriolin-like protein, SPATC1L, redistributed to cell-cell junctions. The Chloride intracellular Channel protein, CLIC2, translocated from actin-enriched plasma membrane bundles to cell-cell junctions upon activation of NK2 receptors. CLIC2, and one of its close paralogs, CLIC4, were further shown to respond with the same translocation pattern to muscarinic M3 and lysophosphatidic LPA receptors. This screen allowed us to identify potential actors in signaling pathways downstream of G protein-coupled receptors and could be scaled-up for high-content screening. PMID:25759509

  12. A Fluorescent Live Imaging Screening Assay Based on Translocation Criteria Identifies Novel Cytoplasmic Proteins Implicated in G Protein-coupled Receptor Signaling Pathways.

    PubMed

    Lecat, Sandra; Matthes, Hans W D; Pepperkok, Rainer; Simpson, Jeremy C; Galzi, Jean-Luc

    2015-05-01

    Several cytoplasmic proteins that are involved in G protein-coupled receptor signaling cascades are known to translocate to the plasma membrane upon receptor activation, such as beta-arrestin2. Based on this example and in order to identify new cytoplasmic proteins implicated in the ON-and-OFF cycle of G protein-coupled receptor, a live-imaging screen of fluorescently labeled cytoplasmic proteins was performed using translocation criteria. The screening of 193 fluorescently tagged human proteins identified eight proteins that responded to activation of the tachykinin NK2 receptor by a change in their intracellular localization. Previously we have presented the functional characterization of one of these proteins, REDD1, that translocates to the plasma membrane. Here we report the results of the entire screening. The process of cell activation was recorded on videos at different time points and all the videos can be visualized on a dedicated website. The proteins BAIAP3 and BIN1, partially translocated to the plasma membrane upon activation of NK2 receptors. Proteins ARHGAP12 and PKM2 translocated toward membrane blebs. Three proteins that associate with the cytoskeleton were of particular interest : PLEKHH2 rearranged from individual dots located near the cell-substrate adhesion surface into lines of dots. The speriolin-like protein, SPATC1L, redistributed to cell-cell junctions. The Chloride intracellular Channel protein, CLIC2, translocated from actin-enriched plasma membrane bundles to cell-cell junctions upon activation of NK2 receptors. CLIC2, and one of its close paralogs, CLIC4, were further shown to respond with the same translocation pattern to muscarinic M3 and lysophosphatidic LPA receptors. This screen allowed us to identify potential actors in signaling pathways downstream of G protein-coupled receptors and could be scaled-up for high-content screening. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. The C. elegans VIG-1 and FRM-1 modulate carbachol-stimulated ERK1/2 activation in chinese hamster ovary cells expressing the muscarinic acetylcholine receptor GAR-3.

    PubMed

    Shin, Youngmi; Cho, Nam Jeong

    2014-04-01

    Many neurotransmitter receptors are known to interact with a variety of intracellular proteins that modulate signaling processes. In an effort to understand the molecular mechanism by which acetylcholine (ACh) signaling is modulated, we searched for proteins that interact with GAR-3, the Caenorhabditis elegans homolog of muscarinic ACh receptors. We isolated two proteins, VIG-1 and FRM-1, in a yeast two-hybrid screen of a C. elegans cDNA library using the third intracellular (i3) loop of GAR-3 as bait. To test whether these proteins regulate ACh signaling, we utilized Chinese hamster ovary (CHO) cells stably expressing GAR-3 (GAR-3/CHO cells). Previously we have shown that the cholinergic agonist carbachol stimulates extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation in an atropine-sensitive manner in this cell line. When VIG-1 was transiently expressed in GAR-3/CHO cells, carbachol-stimulated ERK1/2 activation was substantially reduced. In contrast, transient expression of FRM-1 significantly enhanced carbachol-stimulated ERK1/2 activation. Neither VIG-1 nor FRM-1 expression appeared to alter the affinity between GAR-3 and carbachol. In support of this notion, expression of these proteins did not affect GAR-3-mediated phospholipase C activation. To verify the modulation of ERK1/2 activity by VIG-1 and FRM-1, we used an i3 loop deletion mutant of GAR-3 (termed GAR-3Δi3). Carbachol treatment evoked robust ERK1/2 activation in CHO cells stably expressing the deletion mutant (GAR-3Δi3/CHO cells). However, transient expression of either VIG-1 or FRM-1 had little effect on carbachol-stimulated ERK1/2 activation in GAR-3Δi3/CHO cells. Taken together, these results indicate that VIG-1 and FRM-1 regulate GAR-3-mediated ERK1/2 activation by interacting with the i3 loop of GAR-3.

  14. Nucleus accumbens acetylcholine and food intake: decreased muscarinic tone reduces feeding but not food-seeking.

    PubMed

    Pratt, Wayne E; Blackstone, Kaitlin

    2009-03-02

    Separate groups of food-deprived rats were given 2h access to food after receiving bilateral nucleus accumbens infusions of the muscarinic antagonist scopolamine methyl bromide (at 0, 1.0, and 10.0 microg/side), the M2-preferring agonist oxotremorine sesquifumarate (Oxo-S; at 0, 1.0, or 10.0 microg/side) or the M2 antagonist AFDX-116 (at 0, 0.2, or 1.0 microg/side). Injections of scopolamine or Oxo-S, but not AFDX-116, reduced food consumption across the 2h. These experiments confirm a critical role for Acb acetylcholine in promoting food ingestion, and suggest that decreased acetylcholine tone at post-synaptic muscarinic receptors disrupts normal consummatory behavior.

  15. Inhibition of muscarinic receptor-induced inositol phospholipid hydrolysis by caffeine, beta-adrenoceptors and protein kinase C in intestinal smooth muscle.

    PubMed Central

    Prestwich, S A; Bolton, T B

    1995-01-01

    1. The effects of caffeine, isoprenaline, dibutyryl cyclic AMP, isobutylmethylxanthine (IBMX), 12-O-tetradecanoylphorbol-13-acetate (TPA) or 1-oleoyl-2-acetylglycerol (OAG), (protein kinase C (PKC) activators), 2-methoxy verapamil (D600), thapsigargin and ryanodine on muscarinic acetylcholine receptor (AChR)-stimulated inositol phospholipid hydrolysis were studied in smooth muscle fragments from the longitudinal layer of the small intestine of the guinea-pig. 2. Incubation of the fragments with the muscarinic agonist, carbachol (CCh) (100 microM) resulted in rapid increases in the levels of all the inositol phosphate isomers with maximal increases in the [3H]-inositol (1,4,5) trisphosphate ([3H]-Ins(1,4,5)P3) isomer occurring 10 s following incubation. 3. The beta-adrenoceptor agonist, isoprenaline (10 microM) and dibutyryl cyclic AMP (10 microM), a membrane permeant analogue of cyclic AMP both reduced the CCh stimulation, but not the basal levels of [3H]-inositol phosphates. This inhibition by dibutyryl cyclic AMP was enhanced in the presence of the phosphodiesterase inhibitor, IBMX. CCh inhibited the isoprenaline-induced increases in the levels of cyclic AMP and this was via a pertussi toxin (PTX)-sensitive G-protein mechanism. 4. TPA (1 microM) and OAG (100 microM) a 1,2-diacylglycerol (DAG) analogue both reduced the CCh-induced increases in [3H]-inositol phosphates levels but neither affected basal values nor the basal levels of cyclic AMP. 5. D600 (10 microM), which blocks voltage-dependent Ca2+ channels, also reduced the CCh-stimulated levels of [3H]-inositol phosphates suggesting that some of the agonist-induced increases are due to a potentiating effect of Ca2+ entering the cell. 6. Caffeine (0.5-30 mM) significantly inhibited both the basal and CCh-induced increases in all the [3H]-inositol phosphate isomers. Its inhibitory action was not due to increases in cyclic AMP since caffeine had no effect on the levels of cyclic AMP at concentrations up to 30 m

  16. Lithium modulates the muscarinic facilitation of synaptic plasticity and theta-gamma coupling in the hippocampal-prefrontal pathway.

    PubMed

    Ruggiero, Rafael N; Rossignoli, Matheus T; Lopes-Aguiar, Cleiton; Leite, João P; Bueno-Junior, Lezio S; Romcy-Pereira, Rodrigo N

    2018-06-01

    Mood disorders are associated to functional unbalance in mesolimbic and frontal cortical circuits. As a commonly used mood stabilizer, lithium acts through multiple biochemical pathways, including those activated by muscarinic cholinergic receptors crucial for hippocampal-prefrontal communication. Therefore, here we investigated the effects of lithium on prefrontal cortex responses under cholinergic drive. Lithium-treated rats were anesthetized with urethane and implanted with a ventricular cannula for muscarinic activation, a recording electrode in the medial prefrontal cortex (mPFC), and a stimulating electrode in the intermediate hippocampal CA1. Either of two forms of synaptic plasticity, long-term potentiation (LTP) or depression (LTD), were induced during pilocarpine effects, which were monitored in real time through local field potentials. We found that lithium attenuates the muscarinic potentiation of cortical LTP (<20 min) but enhances the muscarinic potentiation of LTD maintenance (>80 min). Moreover, lithium treatment promoted significant cross-frequency coupling between CA1 theta (3-5 Hz) and mPFC low-gamma (30-55 Hz) oscillations. Interestingly, lithium by itself did not affect any of these measures. Thus, lithium pretreatment and muscarinic activation synergistically modulate the hippocampal-prefrontal connectivity. Because these alterations varied with time, oscillatory parameters, and type of synaptic plasticity, our study suggests that lithium influences prefrontal-related circuits through intricate dynamics, informing future experiments on mood disorders. Copyright © 2018. Published by Elsevier Inc.

  17. Phosphorylation and regulation of a Gq/11-coupled receptor by casein kinase 1alpha.

    PubMed

    Budd, D C; McDonald, J E; Tobin, A B

    2000-06-30

    Agonist-mediated receptor phosphorylation by one or more of the members of the G-protein receptor kinase (GRK) family is an established model for G-protein-coupled receptor (GPCR) phosphorylation resulting in receptor desensitization. Our recent studies have, however, suggested that an alternative route to GPCR phosphorylation may be an operation involving casein kinase 1alpha (CK1alpha). In the current study we investigate the involvement of CK1alpha in the phosphorylation of the human m3-muscarinic receptor in intact cells. We show that expression of a catalytically inactive mutant of CK1alpha, designed to act in a dominant negative manner, inhibits agonist-mediated receptor phosphorylation by approximately 40% in COS-7 and HEK-293 cells. Furthermore, we present evidence that a peptide corresponding to the third intracellular loop of the m3-muscarinic receptor (Ser(345)-Leu(463)) is an inhibitor of CK1alpha due to its ability to both act as a pseudo-substrate for CK1alpha and form a high affinity complex with CK1alpha. Expression of this peptide was able to reduce both basal and agonist-mediated m3-muscarinic receptor phosphorylation in intact cells. These results support the notion that CK1alpha is able to mediate GPCR phosphorylation in an agonist-dependent manner and that this may provide a novel mechanism for GPCR phosphorylation. The functional role of phosphorylation was investigated using a mutant of the m3-muscarinic receptor that showed an approximately 80% reduction in agonist-mediated phosphorylation. Surprisingly, this mutant underwent agonist-mediated desensitization suggesting that, unlike many GPCRs, desensitization of the m3-muscarinic receptor is not mediated by receptor phosphorylation. The inositol (1,4, 5)-trisphosphate response did, however, appear to be dramatically potentiated in the phosphorylation-deficient mutant indicating that phosphorylation may instead control the magnitude of the initial inositol phosphate response.

  18. Wild-type NM23-H1, but not its S120 mutants, suppresses desensitization of muscarinic potassium current.

    PubMed

    Otero, A S; Doyle, M B; Hartsough, M T; Steeg, P S

    1999-03-08

    NM23 (NDP kinase) modulates the gating of muscarinic K+ channels by agonists through a mechanism distinct from GTP regeneration. To better define the function of NM23 in this pathway and to identify sites in NM23 that are important for its role in muscarinic K+ channel function, we utilized MDA-MB-435 human breast carcinoma cells that express low levels of NM23-H1. M2 muscarinic receptors and GIRK1/GIRK4 channel subunits were co-expressed in cells stably transfected with vector only (control), wild-type NM23-H1, or several NM23-H1 mutants. Lysates from all cell lines tested exhibit comparable nucleoside diphosphate (NDP) kinase activity. Whole cell patch clamp recordings revealed a substantial reduction of the acute desensitization of muscarinic K+ currents in cells overexpressing NM23-H1. The mutants NM23-H1P96S and NM23-H1S44A resembled wild-type NM23-H1 in their ability to reduce desensitization. In contrast, mutants NM23-H1S120G and NM23-H1S120A completely abolished the effect of NM23-H1 on desensitization of muscarinic K+ currents. Furthermore, NM23-H1S120G potentiated acute desensitization, indicating that this mutant retains the ability to interact with the muscarinic pathway, but has properties antithetical to those of the wild-type protein. We conclude that NM23 acts as a suppressor of the processes leading to the desensitization of muscarinic K+ currents, and that Ser-120 is essential for its actions.

  19. Impact of Muscarinic M3 Receptor Antagonism on the Risk of Type 2 Diabetes in Antidepressant-Treated Patients: A Case-Controlled Study.

    PubMed

    Tran, Yen-Hao; Schuiling-Veninga, Catharina C M; Bergman, Jorieke E H; Groen, Henk; Wilffert, Bob

    2017-06-01

    M 3 muscarinic receptor antagonism has been associated with glucose intolerance and disturbance of insulin secretion. Our objective was to examine the risk of type 2 diabetes mellitus (T2DM) in patients using antidepressants with and without M 3 muscarinic receptor antagonism (AD_antaM 3 and AD_nonantaM 3 , respectively). We designed a case-control study using a pharmacy prescription database. We selected a cohort of patients who initiated antidepressant use between the ages of 20 and 40 years and who did not receive any anti-diabetic prescriptions at baseline. Cases were defined as those who developed T2DM [i.e., receiving oral anti-diabetic medication, Anatomical Therapeutic Chemical (ATC) code A10B] during the follow-up period (1994-2014), and ten random controls were picked for each case from the cohort of patients who did not develop T2DM. A total of 530 cases with incident T2DM and 5300 controls were included. Compared with no use of antidepressants during the previous 2 years, recent (within the last 6 months) exposure to AD_antaM 3 was associated with a moderately increased risk of T2DM: adjusted odds ratio 1.55 (95% confidence interval 1.18-2.02). In the stratified analyses, this association was dose dependent (>365 defined daily doses) and significant for patients who were in the younger age group (<45 years at the end of follow-up), were female and had no co-morbidity. On the other hand, recent exposure to AD_nonantaM 3 was not associated with a risk for T2DM in any of our analyses. Our results suggest that exposure to AD_antaM 3 was associated with the development of T2DM among antidepressant users.

  20. Tertiapin-Q removes a mechanosensitive component of muscarinic control of the sinoatrial pacemaker in the rat.

    PubMed

    Han, SuYoung; Wilson, Suzanne J; Bolter, Chris P

    2010-09-01

    1. In an isolated right atrial preparation, an increase in right atrial pressure (RAP) produces an increase in atrial rate. This rate response is larger and occurs faster when there is background vagal or muscarinic stimulation. 2. We hypothesized that in the latter situation, an increase in RAP antagonizes the effect of muscarinic stimulation through stretch inactivation of the mechanosensitive muscarinic potassium current I(K,ACh). 3. In two groups of bath-mounted right atria isolated from male Wistar rats (control n = 12; 300 nmol/L tertiapin-Q treated (to block I(K,ACh)) n = 10), we examined the change in atrial rate when RAP was raised from 2 to 8 mmHg; oxotremorine-M (oxo-M; from 10 to 500 nmol/L) was added to incrementally activate muscarinic receptors. 4. In both control and tertiapin-Q-treated groups, oxo-M reduced atrial rate, but its effect was less ( approximately 40-50%) in the latter group (P < 0.001). In control preparations, responses to an increase in RAP became progressively larger and quicker as the concentration of oxo-M was increased, whereas in tertiapin-Q treated preparations oxo-M did not affect either the amplitude or the speed of the response (P < 0.0001 for both). 5. The results support the hypothesis that atrial stretch antagonizes muscarinic slowing by its effect on I(K,ACh). We suggest that through this mechanism, parasympathetic control of heart rate may be modulated continuously by RAP.

  1. Effects of muscarinic receptor agonists and antagonists on alpha 2-adrenoceptors in rat brain.

    PubMed

    Hollingsworth, P J; Smith, C B

    1989-09-13

    The specific binding of [3H]clonidine to alpha 2-adrenoceptors on neural membranes isolated from six brain areas was determined with rats treated for various periods of time with the muscarinic agonists, oxotremorine or pilocarpine, or with the muscarinic antagonists atropine, atropine methyl nitrate, scopolamine and scopolamine methyl bromide. Administration of pilocarpine, 10 mg/kg, twice daily i.p. for 1 and 14 days increased markedly the number of alpha 2-adrenoceptors on neural membranes from all six brain areas. In contrast, oxotremorine, 0.3 mg/kg, twice daily i.p., for 7 days decreased the number of alpha 2-adrenoceptors on membranes from all brain areas except the brainstem and caudate nucleus. Both atropine and scopolamine increased the density of alpha 2-adrenoceptors in specific brain areas. Neither atropine methyl nitrate nor scopolamine methyl bromide had an appreciable effect upon the specific binding of [3H]clonidine to neural membranes from most brain areas.

  2. Inflammation triggers constitutive activity and agonist-induced negative responses at M(3) muscarinic receptor in dental pulp.

    PubMed

    Sterin-Borda, Leonor; Orman, Betina; De Couto Pita, Alejandra; Borda, Enri

    2011-02-01

    The purpose of this study was to investigate whether the inflammation of rat dental pulp induces the muscarinic acetylcholine receptor (mAChR) constitutive receptor activity. Pulpitis was induced with bacterial lipolysaccharide in rat incisors dental pulp. Saturation assay with [(3)H]-quinuclidinyl benzilate ([(3)H] QNB), competitive binding with different mAChR antagonist subtypes, and nitric oxide synthase (NOS) activity were performed. A drastic change in expression and response to mAChR subtypes was observed in pulpitis. Inflamed pulp expressed high number of M(3) mAChR of high affinity, whereas the M(1) mAChR is the main subtype displayed in normal pulp. Consistent with the identification of the affinity constant (Ki) of M(3) and Ki of M(1) in both pulpitis and in normal pulps are the differences in the subtype functionality of these cells. In pulpitis, pilocarpine (1 × 10(-11) mol/L to 5 × 10(-9) mol/L) exerted an inhibitory action on NOS activity that was blocked by J 104129 fumarate (highest selective affinity to M(3) mAChR). In normal pulps, pilocarpine (1 × 10(-11) mol/L to 5 × 10(-9) mol/L) has no effect. NOS basal activity was 5.9 times as high in pulpitis as in the normal pulp as a result of the activation of inducible NOS. The irreversible pulpitis could induce a mAChR alteration, increasing the high-affinity receptor density and transduction-coupling efficiency of inducible NOS activity, leading to a spontaneously active conformation of the receptor. Pilocarpine acting as an inverse agonist might be useful therapeutically to prevent necrosis and subsequent loss of dental pulp. Copyright © 2011 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  3. Muscarinic acetylcholine receptor in cerebellar cortex participates in acetylcholine-mediated blood depressor response in rats.

    PubMed

    Zhou, Peiling; Zhu, Qingfeng; Liu, Ming; Li, Jing; Wang, Yong; Zhang, Changzheng; Hua, Tianmiao

    2015-04-23

    Our previous investigations have revealed that cerebellar cholinergic innervation is involved in cardiovascular regulation. This study was performed to examine the effects of the muscarinic cholinergic receptor (mAChR) in the cerebellar cortex on blood pressure (BP) modulation in rats. Acetylcholine (ACh, 100mM), nonselective mAChR agonist (oxotremorine M; Oxo-M, 10, 30 and 100mM) and 100mM ACh mixed with nonselective mAChR antagonist atropine (1, 3 and 10mM) were microinjected into the cerebellar cortex of anesthetized rats. Mean arterial pressure (MAP), maximal decreased MAP (MDMAP), and reaction time (duration required for BP to return to basal values) were measured and analyzed. The results showed that Oxo-M dose-dependently decreased MAP, increased MDMAP, and prolonged reaction time, which displayed a homodromous effect of ACh-mediated blood depressor response; meanwhile, atropine concentration-dependently blocked the effect of ACh on the BP regulation. In conclusion, the present study showed for the first time that mAChRs in cerebellar cortex could modulate somatic BP by participation in ACh-mediated depressor response. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  4. Caveolin-1: Functional Insights into Its Role in Muscarine- and Serotonin-Induced Smooth Muscle Constriction in Murine Airways

    PubMed Central

    Keshavarz, Maryam; Schwarz, Heike; Hartmann, Petra; Wiegand, Silke; Skill, Melanie; Althaus, Mike; Kummer, Wolfgang; Krasteva-Christ, Gabriela

    2017-01-01

    An increased bronchoconstrictor response is a hallmark in the progression of obstructive airway diseases. Acetylcholine and 5-hydroxytryptamine (5-HT, serotonin) are the major bronchoconstrictors. There is evidence that both cholinergic and serotonergic signaling in airway smooth muscle (ASM) involve caveolae. We hypothesized that caveolin-1 (cav-1), a structural protein of caveolae, plays an important regulatory role in ASM contraction. We analyzed airway contraction in different tracheal segments and extra- and intrapulmonary bronchi in cav-1 deficient (cav-1−/−) and wild-type mice using organ bath recordings and videomorphometry of methyl-beta-cyclodextrin (MCD) treated and non-treated precision-cut lung slices (PCLS). The presence of caveolae was investigated by electron microscopy. Receptor subtypes driving 5-HT-responses were studied by RT-PCR and videomorphometry after pharmacological inhibition with ketanserin. Cav-1 was present in tracheal epithelium and ASM. Muscarine induced a dose dependent contraction in all airway segments. A significantly higher Emax was observed in the caudal trachea. Although, caveolae abundancy was largely reduced in cav-1−/− mice, muscarine-induced airway contraction was maintained, albeit at diminished potency in the middle trachea, in the caudal trachea and in the bronchus without changes in the maximum efficacy. MCD-treatment of PLCS from cav-1−/− mice reduced cholinergic constriction by about 50%, indicating that cholesterol-rich plasma domains account for a substantial portion of the muscarine-induced bronchoconstriction. Notably, cav-1-deficiency fully abrogated 5-HT-induced contraction of extrapulmonary airways. In contrast, 5-HT-induced bronchoconstriction was fully maintained in cav-1-deficient intrapulmonary bronchi, but desensitization upon repetitive stimulation was enhanced. RT-PCR analysis revealed 5-HT1B, 5-HT2A, 5-HT6, and 5-HT7 receptors as the most prevalent subtypes in the airways. The 5-HT

  5. Plant cell surface receptor-mediated signaling - a common theme amid diversity.

    PubMed

    He, Yunxia; Zhou, Jinggeng; Shan, Libo; Meng, Xiangzong

    2018-01-29

    Sessile plants employ a diverse array of plasma membrane-bound receptors to perceive endogenous and exogenous signals for regulation of plant growth, development and immunity. These cell surface receptors include receptor-like kinases (RLKs) and receptor-like proteins (RLPs) that harbor different extracellular domains for perception of distinct ligands. Several RLK and RLP signaling pathways converge at the somatic embryogenesis receptor kinases (SERKs), which function as shared co-receptors. A repertoire of receptor-like cytoplasmic kinases (RLCKs) associate with the receptor complexes to relay intracellular signaling. Downstream of the receptor complexes, mitogen-activated protein kinase (MAPK) cascades are among the key signaling modules at which the signals converge, and these cascades regulate diverse cellular and physiological responses through phosphorylation of different downstream substrates. In this Review, we summarize the emerging common theme that underlies cell surface receptor-mediated signaling pathways in Arabidopsis thaliana : the dynamic association of RLKs and RLPs with specific co-receptors and RLCKs for signal transduction. We further discuss how signaling specificities are maintained through modules at which signals converge, with a focus on SERK-mediated receptor signaling. © 2018. Published by The Company of Biologists Ltd.

  6. Androgen Receptor Signaling in Bladder Cancer

    PubMed Central

    Li, Peng; Chen, Jinbo; Miyamoto, Hiroshi

    2017-01-01

    Emerging preclinical findings have indicated that steroid hormone receptor signaling plays an important role in bladder cancer outgrowth. In particular, androgen-mediated androgen receptor signals have been shown to correlate with the promotion of tumor development and progression, which may clearly explain some sex-specific differences in bladder cancer. This review summarizes and discusses the available data, suggesting the involvement of androgens and/or the androgen receptor pathways in urothelial carcinogenesis as well as tumor growth. While the precise mechanisms of the functions of the androgen receptor in urothelial cells remain far from being fully understood, current evidence may offer chemopreventive or therapeutic options, using androgen deprivation therapy, in patients with bladder cancer. PMID:28241422

  7. Development of radiohalogenated muscarinic ligands for the in vivo imaging of m-AChR by nuclear medicine techniques

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    McPherson, D.W.; Luo, H.; Knapp, F.F. Jr.

    1994-06-01

    Alterations in the density of acetylcholinergic muscarinic receptors (m-AChR) have been observed in various dementias. This has spurred interest in the development of radiohalogenated ligands which can be used for the non-invasive in vivo detection of m-AChR by nuclear medicine techniques. We have developed a new ligand 1-azabicyclo[2.2.2]oct-3-yl ({alpha}-hydroxy-{alpha}-(1-iodo-1-propen-3-yl)-{alpha}-phenylacetate (IQNP,12) which demonstrates high affinity for the muscarinic receptor. When labeled with radioiodine it has been shown to be selective and specific for m-ACHR. Initial studies on the separation and in vivo evaluation of the various isomers of IQNP have shown that the stereochemistry of the chiral centers and the configurationmore » around the double bond play an important role in m-AChR subtype specificity. In vivo evaluation of these stereoisomers demonstrate that E-(R,R)-IQNP has a high affinity for the M{sub 1} muscarinic subtype while Z-(R,R)-IQNP demonstrate a high affinity for M{sub 1} and M{sub 2} receptor subtypes. These data demonstrate IQNP (12) has potential for use in the non-evasive in vivo detection of m-AChR by single photon emission computed tomography (SPECT). A brominated analogue, ``BrQNP,`` in which the iodine has been replaced by a bromine atom, has also been prepared and was shown to block the in vivo uptake of IQNP in the brain and heart and therefore has potential for positron emission tomographic (PET) studies of m-AChR.« less

  8. Brain regional acetylcholinesterase activity and muscarinic acetylcholine receptors in rats after repeated administration of cholinesterase inhibitors and its withdrawal

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kobayashi, Haruo; Suzuki, Tadahiko; Sakamoto, Maki

    Activity of acetylcholinesterase (AChE) and specific binding of [{sup 3}H]quinuclidinyl benzilate (QNB), [{sup 3}H]pirenzepine (PZP) and [{sup 3}H]AF-DX 384 to muscarinic acetylcholine receptor (mAChR) preparations in the striatum, hippocampus and cortex of rats were determined 1, 6 and 11 days after the last treatment with an organophosphate DDVP, a carbamate propoxur or a muscarinic agonist oxotremorine as a reference for 7 and 14 days. AChE activity was markedly decreased in the three regions 1 day after the treatment with DDVP for 7 and 14 days with a gradual recovery 6 to 11 days, and much less decreased 1, 6 andmore » 11 days after the treatment with propoxur for 7 days but not for 14 days in the hippocampus and cortex. The binding of [{sup 3}H]-QNB, PZP and AF-DX 384 in the three regions was generally decreased by the treatment with DDVP for 7 and 14 days. Such down-regulations were generally restored 6 or 11 days after the treatment for 7 but not for 14 days. The down-regulation or up-regulation as measured by [{sup 3}H]-QNB, PZP and AF-DX 384 was observed 1, 6 or 11 days after treatment with propoxur for 7 days and/or 14 days. Repeated treatment with oxotremorine produced similar effects except AChE activity to DDVP. These results suggest that repeated inhibition of AChE activity may usually cause down-regulation of mAChRs with some exception in the hippocampus when a reversible antiChE propoxur is injected.« less

  9. Exposure to Gulf War Illness chemicals induces functional muscarinic receptor maladaptations in muscle nociceptors.

    PubMed

    Cooper, B Y; Johnson, R D; Nutter, T J

    2016-05-01

    Chronic pain is a component of the multisymptom disease known as Gulf War Illness (GWI). There is evidence that pain symptoms could have been a consequence of prolonged and/or excessive exposure to anticholinesterases and other GW chemicals. We previously reported that rats exposed, for 8 weeks, to a mixture of anticholinesterases (pyridostigmine bromide, chlorpyrifos) and a Nav (voltage activated Na(+) channel) deactivation-inhibiting pyrethroid, permethrin, exhibited a behavior pattern that was consistent with a delayed myalgia. This myalgia-like behavior was accompanied by persistent changes to Kv (voltage activated K(+)) channel physiology in muscle nociceptors (Kv7, KDR). In the present study, we examined how exposure to the above agents altered the reactivity of Kv channels to a muscarinic receptor (mAChR) agonist (oxotremorine-M). Comparisons between muscle nociceptors harvested from vehicle and GW chemical-exposed rats revealed that mAChR suppression of Kv7 activity was enhanced in exposed rats. Yet in these same muscle nociceptors, a Stromatoxin-insensitive component of the KDR (voltage activated delayed rectifier K(+) channel) exhibited decreased sensitivity to activation of mAChR. We have previously shown that a unique mAChR-induced depolarization and burst discharge (MDBD) was exaggerated in muscle nociceptors of rats exposed to GW chemicals. We now provide evidence that both muscle and vascular nociceptors of naïve rats exhibit MDBD. Examination of the molecular basis of the MDBD in naïve animals revealed that while the mAChR depolarization was independent of Kv7, the action potential burst was modulated by Kv7 status. mAChR depolarizations were shown to be dependent, in part, on TRPA1. We argue that dysfunction of the MDBD could be a functional convergence point for maladapted ion channels and receptors consequent to exposure to GW chemicals. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Non-ionotropic signaling by the NMDA receptor: controversy and opportunity.

    PubMed

    Gray, John A; Zito, Karen; Hell, Johannes W

    2016-01-01

    Provocative emerging evidence suggests that the N-methyl-d-aspartate (NMDA) receptor can signal in the absence of ion flux through the receptor. This non-ionotropic signaling is thought to be due to agonist-induced conformational changes in the receptor, independently of channel opening. Non-ionotropic NMDA receptor signaling has been proposed to be sufficient to induce synaptic long-term depression (LTD), directly challenging the decades-old model that prolonged low-level calcium influx is required to induce LTD. Here, we briefly review these recent findings, focusing primarily on the potential role of non-ionotropic signaling in NMDA receptor-mediated LTD. Further reports concerning additional roles of non-ionotropic NMDA receptor signaling are also discussed. If validated, this new view of NMDA receptor-mediated signaling will usher in an exciting new era of exploring synapse function and dysfunction.

  11. Non-ionotropic signaling by the NMDA receptor: controversy and opportunity

    PubMed Central

    Gray, John A.; Zito, Karen; Hell, Johannes W.

    2016-01-01

    Provocative emerging evidence suggests that the N-methyl-d-aspartate (NMDA) receptor can signal in the absence of ion flux through the receptor. This non-ionotropic signaling is thought to be due to agonist-induced conformational changes in the receptor, independently of channel opening. Non-ionotropic NMDA receptor signaling has been proposed to be sufficient to induce synaptic long-term depression (LTD), directly challenging the decades-old model that prolonged low-level calcium influx is required to induce LTD. Here, we briefly review these recent findings, focusing primarily on the potential role of non-ionotropic signaling in NMDA receptor-mediated LTD. Further reports concerning additional roles of non-ionotropic NMDA receptor signaling are also discussed. If validated, this new view of NMDA receptor-mediated signaling will usher in an exciting new era of exploring synapse function and dysfunction. PMID:27303637

  12. Erythropoietin Receptor Signaling Is Membrane Raft Dependent

    PubMed Central

    McGraw, Kathy L.; Fuhler, Gwenny M.; Johnson, Joseph O.; Clark, Justine A.; Caceres, Gisela C.; Sokol, Lubomir; List, Alan F.

    2012-01-01

    Upon erythropoietin (Epo) engagement, Epo-receptor (R) homodimerizes to activate JAK2 and Lyn, which phosphorylate STAT5. Although recent investigations have identified key negative regulators of Epo-R signaling, little is known about the role of membrane localization in controlling receptor signal fidelity. Here we show a critical role for membrane raft (MR) microdomains in creation of discrete signaling platforms essential for Epo-R signaling. Treatment of UT7 cells with Epo induced MR assembly and coalescence. Confocal microscopy showed that raft aggregates significantly increased after Epo stimulation (mean, 4.3±1.4(SE) vs. 25.6±3.2 aggregates/cell; p≤0.001), accompanied by a >3-fold increase in cluster size (p≤0.001). Raft fraction immunoblotting showed Epo-R translocation to MR after Epo stimulation and was confirmed by fluorescence microscopy in Epo stimulated UT7 cells and primary erythroid bursts. Receptor recruitment into MR was accompanied by incorporation of JAK2, Lyn, and STAT5 and their activated forms. Raft disruption by cholesterol depletion extinguished Epo induced Jak2, STAT5, Akt and MAPK phosphorylation in UT7 cells and erythroid progenitors. Furthermore, inhibition of the Rho GTPases Rac1 or RhoA blocked receptor recruitment into raft fractions, indicating a role for these GTPases in receptor trafficking. These data establish a critical role for MR in recruitment and assembly of Epo-R and signal intermediates into discrete membrane signaling units. PMID:22509308

  13. Dynamic control of glutamatergic synaptic input in the spinal cord by muscarinic receptor subtypes defined using knockout mice.

    PubMed

    Chen, Shao-Rui; Chen, Hong; Yuan, Wei-Xiu; Wess, Jürgen; Pan, Hui-Lin

    2010-12-24

    Activation of muscarinic acetylcholine receptors (mAChRs) in the spinal cord inhibits pain transmission. At least three mAChR subtypes (M(2), M(3), and M(4)) are present in the spinal dorsal horn. However, it is not clear how each mAChR subtype contributes to the regulation of glutamatergic input to dorsal horn neurons. We recorded spontaneous excitatory postsynaptic currents (sEPSCs) from lamina II neurons in spinal cord slices from wild-type (WT) and mAChR subtype knock-out (KO) mice. The mAChR agonist oxotremorine-M increased the frequency of glutamatergic sEPSCs in 68.2% neurons from WT mice and decreased the sEPSC frequency in 21.2% neurons. Oxotremorine-M also increased the sEPSC frequency in ∼50% neurons from M(3)-single KO and M(1)/M(3) double-KO mice. In addition, the M(3) antagonist J104129 did not block the stimulatory effect of oxotremorine-M in the majority of neurons from WT mice. Strikingly, in M(5)-single KO mice, oxotremorine-M increased sEPSCs in only 26.3% neurons, and J104129 abolished this effect. In M(2)/M(4) double-KO mice, but not M(2)- or M(4)-single KO mice, oxotremorine-M inhibited sEPSCs in significantly fewer neurons compared with WT mice, and blocking group II/III metabotropic glutamate receptors abolished this effect. The M(2)/M(4) antagonist himbacine either attenuated the inhibitory effect of oxotremorine-M or potentiated the stimulatory effect of oxotremorine-M in WT mice. Our study demonstrates that activation of the M(2) and M(4) receptor subtypes inhibits synaptic glutamate release to dorsal horn neurons. M(5) is the predominant receptor subtype that potentiates glutamatergic synaptic transmission in the spinal cord.

  14. Dynamic Control of Glutamatergic Synaptic Input in the Spinal Cord by Muscarinic Receptor Subtypes Defined Using Knockout Mice*

    PubMed Central

    Chen, Shao-Rui; Chen, Hong; Yuan, Wei-Xiu; Wess, Jürgen; Pan, Hui-Lin

    2010-01-01

    Activation of muscarinic acetylcholine receptors (mAChRs) in the spinal cord inhibits pain transmission. At least three mAChR subtypes (M2, M3, and M4) are present in the spinal dorsal horn. However, it is not clear how each mAChR subtype contributes to the regulation of glutamatergic input to dorsal horn neurons. We recorded spontaneous excitatory postsynaptic currents (sEPSCs) from lamina II neurons in spinal cord slices from wild-type (WT) and mAChR subtype knock-out (KO) mice. The mAChR agonist oxotremorine-M increased the frequency of glutamatergic sEPSCs in 68.2% neurons from WT mice and decreased the sEPSC frequency in 21.2% neurons. Oxotremorine-M also increased the sEPSC frequency in ∼50% neurons from M3-single KO and M1/M3 double-KO mice. In addition, the M3 antagonist J104129 did not block the stimulatory effect of oxotremorine-M in the majority of neurons from WT mice. Strikingly, in M5-single KO mice, oxotremorine-M increased sEPSCs in only 26.3% neurons, and J104129 abolished this effect. In M2/M4 double-KO mice, but not M2- or M4-single KO mice, oxotremorine-M inhibited sEPSCs in significantly fewer neurons compared with WT mice, and blocking group II/III metabotropic glutamate receptors abolished this effect. The M2/M4 antagonist himbacine either attenuated the inhibitory effect of oxotremorine-M or potentiated the stimulatory effect of oxotremorine-M in WT mice. Our study demonstrates that activation of the M2 and M4 receptor subtypes inhibits synaptic glutamate release to dorsal horn neurons. M5 is the predominant receptor subtype that potentiates glutamatergic synaptic transmission in the spinal cord. PMID:20940295

  15. Analysis of erectile responses to BAY 41-8543 and muscarinic receptor stimulation in the rat.

    PubMed

    Lasker, George F; Pankey, Edward A; Allain, Alexander V; Dhaliwal, Jasdeep S; Stasch, Johannes-Peter; Murthy, Subramanyam N; Kadowitz, Philip J

    2013-03-01

    Soluble guanylate cyclase (sGC) is the receptor for nitric oxide (NO) and in pathophysiologic conditions where NO formation or bioavailability is impaired, erectile dysfunction (ED) occurs. The aim of this study was to investigate erectile responses to the sGC stimulator BAY 41-8543 in physiologic and pathophysiologic conditions. Increases in intracavernosal pressure (ICP) in response to intracavernosal (ic) injections of BAY 41-8543 were investigated in the anesthetized rat. Increases in ICP/MAP in response to ic injections of BAY 41-8543 and the interaction of BAY 41-8543 with exogenous and endogenously released NO were investigated and the effect of the sGC stimulator on cavernosal nerve injury was assessed. The mechanism of the increase in ICP/MAP in response to ic injection of acetylcholine was investigated. The ic injections of BAY 41-8543 increased ICP/MAP and the duration of the response. BAY 41-8543 was less potent than sodium nitroprusside (SNP) and ic injections of BAY 41-8543 and SNP produced a larger response than the algebraic sum of responses to either agent alone. Simultaneous ic injection of BAY 41-8543 and cavernosal nerve stimulation produced a greater response than either intervention alone. Atropine and cavernosal nerve crush injury decreased the response to nerve stimulation and ic injection of BAY 41-8543 restored the response. These data show that BAY 41-8543 has significant erectile activity and can synergize with exogenous and endogenously released NO. This study shows that atropine and nerve crush attenuate the response to cavernosal nerve stimulation and that BAY 41-8543 can restore the response. The results with atropine, L-NAME and hexamethonium indicate that the response to ic injection of acetylcholine is mediated by muscarinic receptors and the release of NO with no significant role for nicotinic receptors. These results suggest that BAY 41-8543 would be useful in the treatment of ED. © 2012 International Society for Sexual

  16. Muscarinic acetylcholine receptor subtype 4 is essential for cholinergic stimulation of duodenal bicarbonate secretion in mice - relationship to D cell/somatostatin.

    PubMed

    Takeuchi, K; Kita, K; Takahashi, K; Aihara, E; Hayashi, S

    2015-06-01

    We investigated the roles of muscarinic (M) acetylcholine receptor subtype in the cholinergic stimulation of duodenal HCO3(-) secretion using knockout (KO) mice. Wild-type and M1-M5 KO C57BL/6J mice were used. The duodenal mucosa was mounted on an Ussing chamber, and HCO3(-) secretion was measured at pH 7.0 using a pH-stat method in vitro. Carbachol (CCh) or other agents were added to the serosal side. CCh dose-dependently stimulated HCO3(-) secretion in wild-type mice, and this effect was completely inhibited in the presence of atropine. The HCO3(-) response to CCh in wild-type mice was also inhibited by pirenzepine (M1 antagonist), 4DAMP (M3 antagonist), and tropicamide (M4 antagonist), but not by methoctramine (M2 antagonist). CCh stimulated HCO3(-) secretion in M2 and M5 KO animals as effectively as in WT mice; however, this stimulatory effect was significantly attenuated in M1, M3, and M4 KO mice. The decrease observed in the CCh-stimulated HCO3(-) response in M4 KO mice was reversed by the co-application of CYN154806, a somatostatin receptor type 2 (SST2) antagonist. Octreotide (a somatostatin analogue) decreased the basal and CCh-stimulated secretion of HCO3(-) in wild-type mice. The co-localized expression of somatostatin and M4 receptors was confirmed immunohistologically in the duodenum. We concluded that the duodenal HCO3(-) response to CCh was directly mediated by M1/M3 receptors and indirectly modified by M4 receptors. The activation of M4 receptors was assumed to inhibit the release of somatostatin from D cells and potentiate the HCO3(-) response by removing the negative influence of somatostatin via the activation of SST2 receptors.

  17. Autoantibodies against Muscarinic Type 3 Receptor in Sjögren's Syndrome Inhibit Aquaporin 5 Trafficking

    PubMed Central

    Lee, Byung Ha; Gauna, Adrienne E.; Perez, Geidys; Park, Yun-jong; Pauley, Kaleb M.; Kawai, Toshihisa; Cha, Seunghee

    2013-01-01

    Sjögren's syndrome (SjS) is a chronic autoimmune disease that mainly targets the salivary and lacrimal glands. It has been controversial whether anti-muscarinic type 3 receptor (α-M3R) autoantibodies in patients with SjS inhibit intracellular trafficking of aquaporin-5 (AQP5), water transport protein, leading to secretory dysfunction. To address this issue, GFP-tagged human AQP5 was overexpressed in human salivary gland cells (HSG-hAQP5) and monitored AQP5 trafficking to the plasma membrane following carbachol (CCh, M3R agonist) stimulation. AQP5 trafficking was indeed mediated by M3R stimulation, shown in partial blockage of trafficking by M3R-antagonist 4-DAMP. HSG-hAQP5 pre-incubated with SjS plasma for 24 hours significantly reduced AQP5 trafficking with CCh, compared with HSG-hAQP5 pre-incubated with healthy control (HC) plasma. This inhibition was confirmed by monoclonal α-M3R antibody and pre-absorbed plasma. Interestingly, HSG-hAQP5 pre-incubated with SjS plasma showed no change in cell volume, compared to the cells incubated with HC plasma showing shrinkage by twenty percent after CCh-stimulation. Our findings clearly indicate that binding of anti-M3R autoantibodies to the receptor, which was verified by immunoprecipitation, suppresses AQP5 trafficking to the membrane and contribute to impaired fluid secretion in SjS. Our current study urges further investigations of clinical associations between SjS symptoms, such as degree of secretory dysfunction, cognitive impairment, and/or bladder irritation, and different profiles (titers, isotypes, and/or specificity) of anti-M3R autoantibodies in individuals with SjS. PMID:23382834

  18. Glucocorticoid receptor signaling in health and disease

    PubMed Central

    Kadmiel, Mahita; Cidlowski, John A.

    2013-01-01

    Glucocorticoids are steroid hormones regulated in a circadian and stres-associated manner to maintain various metabolic and homeostatic functions that are necessary for life. Synthetic glucocorticoids are widely prescribed drugs for many conditions including asthma, chronic obstructive pulmonary disease (COPD), and inflammatory disorders of the eye. Research in the last few years has begun to unravel the profound complexity of glucocorticoid signaling and has contributed remarkably to improved therapeutic strategies. Glucocorticoids signal through the glucocorticoid receptor, a member of the superfamily of nuclear receptors, in both genomic and non-genomic ways in almost every tissue in the human body. In this review, we will provide an update on glucocorticoid receptor signaling and highlight the role of GR signaling in physiological and pathophysiological conditions in the major organ systems in the human body. PMID:23953592

  19. MRAP2 regulates ghrelin receptor signaling and hunger sensing.

    PubMed

    Srisai, Dollada; Yin, Terry C; Lee, Abigail A; Rouault, Alix A J; Pearson, Nicole A; Grobe, Justin L; Sebag, Julien A

    2017-09-28

    Ghrelin is the only known circulating orexigenic hormone. It is primarily secreted by the stomach and acts at its receptor, the growth hormone secretagogue receptor 1a (GHSR1a), in the hypothalamus to signal hunger and promote food intake. The melanocortin receptor accessory protein 2 (MRAP2) was previously shown to regulate energy homeostasis through the modulation of the activity of the melanocortin-4 receptor and prokineticin receptors. In this study we identify MRAP2 as a partner of ghrelin-GHSR1a signaling. We show that MRAP2 interacts with GHSR1a and potentiates ghrelin-stimulated signaling both in vitro and in vivo. We demonstrate that in the absence of MRAP2, fasting fails to activate agouti-related protein neurons. In addition, we show that the orexigenic effect of ghrelin is lost in mice lacking MRAP2. Our results suggest that MRAP2 is an important modulator of the energy homeostasis machinery that operates through the regulation of multiple GPCRs throughout the hypothalamus.Melanocortin receptor accessory protein 2 (MRAP2) is an adaptor protein that contributes to melanocortin-4 receptor and prokineticin receptor 1 signalling. Here the authors show that MRAP2 also regulates ghrelin receptor signalling in the hypothalamus and starvation sensing in mice.

  20. The effect of a muscarinic receptor 1 gene variant on grey matter volume in schizophrenia.

    PubMed

    Cropley, Vanessa L; Scarr, Elizabeth; Fornito, Alex; Klauser, Paul; Bousman, Chad A; Scott, Rodney; Cairns, Murray J; Tooney, Paul A; Pantelis, Christos; Dean, Brian

    2015-11-30

    Previous research has demonstrated that individuals with schizophrenia who are homozygous at the c.267C>A single nucleotide polymorphism (rs2067477) within the cholinergic muscarinic M1 receptor (CHRM1) perform less well on the Wisconsin Card Sorting Test (WCST) than those who are heterozygous. This study sought to determine whether variation in the rs2067477 genotype was associated with differential changes in brain structure. Data from 227 patients with established schizophrenia or schizoaffective disorder were obtained from the Australian Schizophrenia Research Bank. Whole-brain voxel-based morphometry was performed to compare regional grey matter volume (GMV) between the 267C/C (N=191) and 267C/A (N=36) groups. Secondary analyses tested for an effect of genotype on cognition (the WCST was not available). Individuals who were homozygous (267C/C) demonstrated significantly reduced GMV in the right precentral gyrus compared to those who were heterozygous (267C/A). These preliminary results suggest that the rs2067477 genotype is associated with brain structure in the right precentral gyrus in individuals with schizophrenia/schizoaffective disorder. Future studies are required to replicate these results and directly link the volumetric reductions with specific cognitive processes. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  1. Fibroblast growth factor receptor signaling crosstalk in skeletogenesis.

    PubMed

    Miraoui, Hichem; Marie, Pierre J

    2010-11-02

    Fibroblast growth factors (FGFs) play important roles in the control of embryonic and postnatal skeletal development by activating signaling through FGF receptors (FGFRs). Germline gain-of-function mutations in FGFR constitutively activate FGFR signaling, causing chondrocyte and osteoblast dysfunctions that result in skeletal dysplasias. Crosstalk between the FGFR pathway and other signaling cascades controls skeletal precursor cell differentiation. Genetic analyses revealed that the interplay of WNT and FGFR1 determines the fate and differentiation of mesenchymal stem cells during mouse craniofacial skeletogenesis. Additionally, interactions between FGFR signaling and other receptor tyrosine kinase networks, such as those mediated by the epidermal growth factor receptor and platelet-derived growth factor receptor α, were associated with excessive osteoblast differentiation and bone formation in the human skeletal dysplasia called craniosynostosis, which is a disorder of skull development. We review the roles of FGFR signaling and its crosstalk with other pathways in controlling skeletal cell fate and discuss how this crosstalk could be pharmacologically targeted to correct the abnormal cell phenotype in skeletal dysplasias caused by aberrant FGFR signaling.

  2. The roles of bile acids and sphingosine-1-phosphate signaling in the hepatobiliary diseases

    PubMed Central

    Nagahashi, Masayuki; Yuza, Kizuki; Hirose, Yuki; Nakajima, Masato; Ramanathan, Rajesh; Hait, Nitai C.; Hylemon, Phillip B.; Zhou, Huiping; Takabe, Kazuaki; Wakai, Toshifumi

    2016-01-01

    Based on research carried out over the last decade, it has become increasingly evident that bile acids act not only as detergents, but also as important signaling molecules that exert various biological effects via activation of specific nuclear receptors and cell signaling pathways. Bile acids also regulate the expression of numerous genes encoding enzymes and proteins involved in the synthesis and metabolism of bile acids, glucose, fatty acids, and lipoproteins, as well as energy metabolism. Receptors activated by bile acids include, farnesoid X receptor α, pregnane X receptor, vitamin D receptor, and G protein-coupled receptors, TGR5, muscarinic receptor 2, and sphingosine-1-phosphate receptor (S1PR)2. The ligand of S1PR2, sphingosine-1-phosphate (S1P), is a bioactive lipid mediator that regulates various physiological and pathophysiological cellular processes. We have recently reported that conjugated bile acids, via S1PR2, activate and upregulate nuclear sphingosine kinase 2, increase nuclear S1P, and induce genes encoding enzymes and transporters involved in lipid and sterol metabolism in the liver. Here, we discuss the role of bile acids and S1P signaling in the regulation of hepatic lipid metabolism and in hepatobiliary diseases. PMID:27459945

  3. Effects of sex steroids on muscarinic sties in the rat brain

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Al-Dahan, M.I.

    1986-03-01

    The level of binding sites for (/sup 3/H)scopolamine in the rat hypothalamus and amygdala (but not elsewhere in the brain) is modified by hormonal status. In females, there is an inverse relation between the level of sites and estrogen (E/sub 2/) and progesterone (P) concentration. Binding is high in metoestrous (Met) and in ovariectomized (Ovx) animals but low in proestrous (Pro). Hormone replacement in ovariectomized animals lowers the level of the sites. Castration (Cast) of males reduces the level of sites but subsequent testosterone (T) treatment restores normal levels. The results support a role of hormones in sexual behavior viamore » alteration in levels of muscarinic receptors: male hormone increases and female hormones decrease receptor levels.« less

  4. Engineered Context-Sensitive Agonism: Tissue-Selective Drug Signaling through a G Protein-Coupled Receptor.

    PubMed

    Seemann, Wiebke K; Wenzel, Daniela; Schrage, Ramona; Etscheid, Justine; Bödefeld, Theresa; Bartol, Anna; Warnken, Mareille; Sasse, Philipp; Klöckner, Jessica; Holzgrabe, Ulrike; DeAmici, Marco; Schlicker, Eberhard; Racké, Kurt; Kostenis, Evi; Meyer, Rainer; Fleischmann, Bernd K; Mohr, Klaus

    2017-02-01

    Drug discovery strives for selective ligands to achieve targeted modulation of tissue function. Here we introduce engineered context-sensitive agonism as a postreceptor mechanism for tissue-selective drug action through a G protein-coupled receptor. Acetylcholine M 2 -receptor activation is known to mediate, among other actions, potentially dangerous slowing of the heart rate. This unwanted side effect is one of the main reasons that limit clinical application of muscarinic agonists. Herein we show that dualsteric (orthosteric/allosteric) agonists induce less cardiac depression ex vivo and in vivo than conventional full agonists. Exploration of the underlying mechanism in living cells employing cellular dynamic mass redistribution identified context-sensitive agonism of these dualsteric agonists. They translate elevation of intracellular cAMP into a switch from full to partial agonism. Designed context-sensitive agonism opens an avenue toward postreceptor pharmacologic selectivity, which even works in target tissues operated by the same subtype of pharmacologic receptor. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

  5. Molecular Mechanisms of Opioid Receptor-Dependent Signaling and Behavior

    PubMed Central

    Al-Hasani, Ream; Bruchas, Michael R.

    2013-01-01

    Opioid receptors have been targeted for the treatment of pain and related disorders for thousands of years, and remain the most widely used analgesics in the clinic. Mu (μ), kappa (κ), and delta (δ) opioid receptors represent the originally classified receptor subtypes, with opioid receptor like-1 (ORL1) being the least characterized. All four receptors are G-protein coupled, and activate inhibitory G-proteins. These receptors form homo- and hetereodimeric complexes, signal to kinase cascades, and scaffold a variety of proteins. In this review, we discuss classical mechanisms and developments in understanding opioid tolerance, opioid receptor signaling, and highlight advances in opioid molecular pharmacology, behavioral pharmacology, and human genetics. We put into context how opioid receptor signaling leads to the modulation of behavior with the potential for therapeutic intervention. Finally, we conclude that there is a continued need for more translational work on opioid receptors in vivo. PMID:22020140

  6. [Signal transduction mechanisms of hormones through membrane receptors].

    PubMed

    Yasufuku-Takano, Junko; Takano, Koji

    2002-02-01

    Hormones exert their effect on cells either via membrane receptors or intracellular receptors. This paper aims to review membrane receptors and the intracellular signal transduction mechanisms. Membrane receptors could be classified according to their structural characteristics and the way they initiate the intracellular signal transduction. These include 1) Seven transmembrane(or G-protein coupled) receptors--heterotrimeric G-proteins--effector, system, 2) Receptor tyrosine kinases--protein-protein interaction through SH2, SH3, and PTB domain--MAP kinase cascades and PI3-kinase pathways, 3) Cytokine receptors--JAK--STAT pathways, 4) Receptors of the TGF- beta superfamily--SMAD pathways, 5) Apoptosis-related receptors--caspase pathways, and 6) ligand-gated ion channels. There are growing knowledge of cross-talks between these pathways. It is being recognized that steroid hormones have distinct membrane receptors, which mediate rapid, nongenomic effect.

  7. General pharmacological profile of the novel muscarinic receptor agonist SNI-2011, a drug for xerostomia in Sjögren's syndrome. 2nd communication: effects on somatic nervous system and on autonomic nervous system and smooth muscle.

    PubMed

    Arisawa, Hirohiko; Fukui, Kenji; Fujise, Nobuaki; Masunaga, Hiroaki

    2002-01-01

    A novel muscarinic receptor agonist SNI-2011 ((+/-)-cis-2-methylspirol[1,3-oxathiolane-5,3'-quinuclidine] monohydrochloride hemihydrate, cevimeline, CAS 153504-70-2), is a candidate therapeutic drug for xerostomia in Sjögren's syndrome. The general pharmacological properties of this drug on the somatic nervous system and on the autonomic nervous system and smooth muscle were investigated in mice, rats, guinea pigs, rabbits and cats. 1. Somatic nervous system: SNI-2011 had no effect on the neuromuscular junction in rats and no muscle relaxant effect in mice. No surface anesthetic effect was observed in guinea pigs, but infiltration anesthetic effect was found after intracutaneous injection of solution (1% or higher). 2. Autonomic nervous system and smooth muscle: SNI-2011 tended to cause mydriasis at 3 mg/kg i.v. or higher in rabbits and dose-dependently caused mydriasis at 10 mg/kg p.o. or higher in rats. Mydriasis in rats was also observed by ophthalmic instillation, caused via the peripheral muscarinic acetylcholine receptors. SNI-2011 elevated the base line tension of nictitating membrane in cats when it was injected intravenously at 3 mg/kg or higher. In the smooth muscle, SNI-2011 increased the spontaneous movement of isolated rabbit ileum (1 x 10(-6) mol/l or higher), contractions of isolated guinea pig ileum (1 x 10(-6) mol/l or higher) and isolated guinea pig trachea (3 x 10(-6) mol/l or higher). SNI-2011 relaxed the histamine- and noradrenaline-induced contractions of isolated guinea pig aorta and augmented noradrenaline- and phenylephrine-induced contractions of isolated rat vas deferens. These effects were induced by relatively higher concentrations only i.e. 1 x 10(-5) mol/l or higher. From these results, SNI-2011 has muscarinic side effects on the somatic nervous system and on the autonomic nervous system and smooth muscle, however, in the case of oral administration, that is clinical administration route, SNI-2011 caused no muscarinic side effect at

  8. Deficits in acetylcholine homeostasis, receptors and behaviors in choline transporter heterozygous mice.

    PubMed

    Bazalakova, M H; Wright, J; Schneble, E J; McDonald, M P; Heilman, C J; Levey, A I; Blakely, R D

    2007-07-01

    Cholinergic neurons elaborate a hemicholinium-3 (HC-3) sensitive choline transporter (CHT) that mediates presynaptic, high-affinity choline uptake (HACU) in support of acetylcholine (ACh) synthesis and release. Homozygous deletion of CHT (-/-) is lethal shortly after birth (Ferguson et al. 2004), consistent with CHT as an essential component of cholinergic signaling, but precluding functional analyses of CHT contributions in adult animals. In contrast, CHT+/- mice are viable, fertile and display normal levels of synaptosomal HACU, yet demonstrate reduced CHT protein and increased sensitivity to HC-3, suggestive of underlying cholinergic hypofunction. We find that CHT+/- mice are equivalent to CHT+/+ siblings on measures of motor co-ordination (rotarod), general activity (open field), anxiety (elevated plus maze, light/dark paradigms) and spatial learning and memory (Morris water maze). However, CHT+/- mice display impaired performance as a result of physical challenge in the treadmill paradigm, as well as reduced sensitivity to challenge with the muscarinic receptor antagonist scopolamine in the open field paradigm. These behavioral alterations are accompanied by significantly reduced brain ACh levels, elevated choline levels and brain region-specific decreased expression of M1 and M2 muscarinic acetylcholine receptors. Our studies suggest that CHT hemizygosity results in adequate baseline ACh stores, sufficient to sustain many phenotypes, but normal sensitivities to physical and/or pharmacological challenge require full cholinergic signaling capacity.

  9. Dynamics of the actin cytoskeleton mediates receptor cross talk: An emerging concept in tuning receptor signaling

    PubMed Central

    Mattila, Pieta K.; Batista, Facundo D.

    2016-01-01

    Recent evidence implicates the actin cytoskeleton in the control of receptor signaling. This may be of particular importance in the context of immune receptors, such as the B cell receptor, where dysregulated signaling can result in autoimmunity and malignancy. Here, we discuss the role of the actin cytoskeleton in controlling receptor compartmentalization, dynamics, and clustering as a means to regulate receptor signaling through controlling the interactions with protein partners. We propose that the actin cytoskeleton is a point of integration for receptor cross talk through modulation of protein dynamics and clustering. We discuss the implication of this cross talk via the cytoskeleton for both ligand-induced and low-level constitutive (tonic) signaling necessary for immune cell survival. PMID:26833785

  10. Design principles of nuclear receptor signaling: how complex networking improves signal transduction

    PubMed Central

    Kolodkin, Alexey N; Bruggeman, Frank J; Plant, Nick; Moné, Martijn J; Bakker, Barbara M; Campbell, Moray J; van Leeuwen, Johannes P T M; Carlberg, Carsten; Snoep, Jacky L; Westerhoff, Hans V

    2010-01-01

    The topology of nuclear receptor (NR) signaling is captured in a systems biological graphical notation. This enables us to identify a number of ‘design' aspects of the topology of these networks that might appear unnecessarily complex or even functionally paradoxical. In realistic kinetic models of increasing complexity, calculations show how these features correspond to potentially important design principles, e.g.: (i) cytosolic ‘nuclear' receptor may shuttle signal molecules to the nucleus, (ii) the active export of NRs may ensure that there is sufficient receptor protein to capture ligand at the cytoplasmic membrane, (iii) a three conveyor belts design dissipating GTP-free energy, greatly aids response, (iv) the active export of importins may prevent sequestration of NRs by importins in the nucleus and (v) the unspecific nature of the nuclear pore may ensure signal-flux robustness. In addition, the models developed are suitable for implementation in specific cases of NR-mediated signaling, to predict individual receptor functions and differential sensitivity toward physiological and pharmacological ligands. PMID:21179018

  11. Expression of cholinergic, insulin, vitamin D receptors and GLUT 3 in the brainstem of streptozotocin induced diabetic rats: effect of treatment with vitamin D₃.

    PubMed

    Peeyush Kumar, T; Paul, Jes; Antony, Sherin; Paulose, C S

    2011-11-01

    Complications arising from diabetes mellitus include cognitive deficits, neurophysiological and structural changes in the brain. The current study investigated the expression of cholinergic, insulin, Vitamin D receptor and GLUT 3 in the brainstem of streptozotocin-induced diabetic rats. Radioreceptor binding assays and gene expression were done in the brainstem of male Wistar rats. Our results showed that B(max) of total muscarinic, muscarinic M3 receptors was increased and muscarinic M1 receptor was decreased in diabetic rats compared to control. A significant increase in gene expression of muscarinic M3, α7 nicotinic acetylcholine, insulin, Vitamin D₃ receptors, acetylcholine esterase, choline acetyl transferase and GLUT 3 were observed in the brainstem of diabetic rats. Immunohistochemistry studies of muscarinic M1, M3 and α7 nicotinic acetylcholine receptors confirmed the gene expression at protein level. Vitamin D₃ and insulin treatment reversed diabetes-induced alterations to near control. This study provides an evidence that diabetes can alter the expression of cholinergic, insulin, Vitamin D receptors and GLUT 3 in brainstem. We found that Vitamin D₃ treatment could modulate the Vitamin D receptors and plays a pivotal role in maintaining the glucose transport and expressional level of cholinergic receptors in the brainstem of diabetic rats. Thus, our results suggest a therapeutic role of Vitamin D₃ in managing neurological disorders associated with diabetes.

  12. Expression of m1-type muscarinic acetylcholine receptors by parvalbumin-immunoreactive neurons in the primary visual cortex: a comparative study of rat, guinea pig, ferret, macaque, and human.

    PubMed

    Disney, Anita A; Reynolds, John H

    2014-04-01

    Cholinergic neuromodulation is a candidate mechanism for aspects of arousal and attention in mammals. We have reported previously that cholinergic modulation in the primary visual cortex (V1) of the macaque monkey is strongly targeted toward GABAergic interneurons, and in particular that the vast majority of parvalbumin-immunoreactive (PV) neurons in macaque V1 express the m1-type (pirenzepine-sensitive, Gq-coupled) muscarinic ACh receptor (m1AChR). In contrast, previous physiological data indicates that PV neurons in rats rarely express pirenzepine-sensitive muscarinic AChRs. To examine further this apparent species difference in the cholinergic effectors for the primary visual cortex, we have conducted a comparative study of the expression of m1AChRs by PV neurons in V1 of rats, guinea pigs, ferrets, macaques, and humans. We visualize PV- and mAChR-immunoreactive somata by dual-immunofluorescence confocal microscopy and find that the species differences are profound; the vast majority (>75%) of PV-ir neurons in macaques, humans, and guinea pigs express m1AChRs. In contrast, in rats only ∼25% of the PV population is immunoreactive for m1AChRs. Our data reveal that while they do so much less frequently than in primates, PV neurons in rats do express Gq-coupled muscarinic AChRs, which appear to have gone undetected in the previous in vitro studies. Data such as these are critical in determining the species that represent adequate models for the capacity of the cholinergic system to modulate inhibition in the primate cortex. Copyright © 2013 Wiley Periodicals, Inc.

  13. Role of cholinergic receptors in locomotion induced by scopolamine and oxotremorine-M.

    PubMed

    Chintoh, Araba; Fulton, James; Koziel, Nicole; Aziz, Mariam; Sud, Manu; Yeomans, John S

    2003-08-01

    Mesopontine cholinergic neurons activate dopamine neurons important for reward-seeking and locomotor activity. The present studies tested whether cholinergic receptor blockade in the ventral tegmental area (VTA) altered locomotion induced by scopolamine (3 mg/kg i.p.) or by oxotremorine-M (0.1 microg bilaterally in the VTA). It was predicted that cholinergic blockers in the VTA would attenuate these cholinergic-induced locomotor increases. Locomotor activity was increased by scopolamine and oxotremorine-M administration in all treatments. When dihydro-beta-erythroidine (DHBE), a nicotinic receptor antagonist, was applied in VTA prior to oxotremorine-M, locomotion was reduced to slightly above saline baseline levels, but atropine, a muscarinic antagonist, had no effect. This suggests that the locomotor effect of oxotremorine-M at this dose was mediated mainly via nicotinic, not muscarinic, receptors. Intra-VTA injections of DHBE, however, did not attenuate scopolamine-induced locomotion indicating that scopolamine-induced locomotion is not mediated mainly via VTA cholinergic receptors. In mutant mice with a deletion in the M5 muscarinic receptor gene, scopolamine-induced locomotion was increased versus wild type mice after scopolamine injection. This suggests that the M5 receptor has an inhibitory effect on scopolamine-induced locomotion.

  14. Immunocytochemical localization of choline acetyltransferase and muscarinic ACh receptors in the antenna during development of the sphinx moth Manduca sexta.

    PubMed

    Clark, Julie; Meisner, Shannon; Torkkeli, Päivi H

    2005-04-01

    Immunocytochemistry with monoclonal antibodies was used to investigate the locations of muscarinic acetylcholine receptors (mAChR) and choline acetyltransferase (ChAT) in sections of the developing antennae of the moth Manduca sexta. The results were correlated with a previous morphological investigation in the developing antennae which allowed us to locate different cell types at various stages of development. Our findings indicated that the muscarinic cholinergic system was not restricted to the sensory neurons but was also present in glial and epidermal cells. By day 4-5 of adult development, immunoreactivity against both antibodies was present in the axons of the antennal nerve, and more intense labeling was present in sections from older pupae. At days 4-9, the cell bodies of the sensory neurons in the basal part of the epidermis were also intensely immunolabeled by the anti-mAChR antibody. In mature flagella, large numbers of cells, some with processes into hairs, were strongly labeled by both antibodies. Antennal glial cells were intensely immunolabeled with both antibodies by days 4-5, but in later stages, it was not possible to discriminate between glial and neural staining. At days 4-9, we observed a distinctly labeled layer of epidermal cells close to the developing cuticle. The expression of both ChAT and mAChRs by neurons in moth antennae may allow the regulation of excitability by endogenous ACh. Cholinergic communication between neurons and glia may be part of the system that guides axon elongation during development. The cholinergic system in the apical part of the developing epidermis could be involved in cuticle formation.

  15. Dual-Color Luciferase Complementation for Chemokine Receptor Signaling.

    PubMed

    Luker, Kathryn E; Luker, Gary D

    2016-01-01

    Chemokine receptors may share common ligands, setting up potential competition for ligand binding, and association of activated receptors with downstream signaling molecules such as β-arrestin. Determining the "winner" of competition for shared effector molecules is essential for understanding integrated functions of chemokine receptor signaling in normal physiology, disease, and response to therapy. We describe a dual-color click beetle luciferase complementation assay for cell-based analysis of interactions of two different chemokine receptors, CXCR4 and ACKR3, with the intracellular scaffolding protein β-arrestin 2. This assay provides real-time quantification of receptor activation and signaling in response to chemokine CXCL12. More broadly, this general imaging strategy can be applied to quantify interactions of any set of two proteins that interact with a common binding partner. © 2016 Elsevier Inc. All rights reserved.

  16. Cocaine Inhibits Dopamine D2 Receptor Signaling via Sigma-1-D2 Receptor Heteromers

    PubMed Central

    Navarro, Gemma; Moreno, Estefania; Bonaventura, Jordi; Brugarolas, Marc; Farré, Daniel; Aguinaga, David; Mallol, Josefa; Cortés, Antoni; Casadó, Vicent; Lluís, Carmen; Ferre, Sergi

    2013-01-01

    Under normal conditions the brain maintains a delicate balance between inputs of reward seeking controlled by neurons containing the D1-like family of dopamine receptors and inputs of aversion coming from neurons containing the D2-like family of dopamine receptors. Cocaine is able to subvert these balanced inputs by altering the cell signaling of these two pathways such that D1 reward seeking pathway dominates. Here, we provide an explanation at the cellular and biochemical level how cocaine may achieve this. Exploring the effect of cocaine on dopamine D2 receptors function, we present evidence of σ1 receptor molecular and functional interaction with dopamine D2 receptors. Using biophysical, biochemical, and cell biology approaches, we discovered that D2 receptors (the long isoform of the D2 receptor) can complex with σ1 receptors, a result that is specific to D2 receptors, as D3 and D4 receptors did not form heteromers. We demonstrate that the σ1-D2 receptor heteromers consist of higher order oligomers, are found in mouse striatum and that cocaine, by binding to σ1 -D2 receptor heteromers, inhibits downstream signaling in both cultured cells and in mouse striatum. In contrast, in striatum from σ1 knockout animals these complexes are not found and this inhibition is not seen. Taken together, these data illuminate the mechanism by which the initial exposure to cocaine can inhibit signaling via D2 receptor containing neurons, destabilizing the delicate signaling balance influencing drug seeking that emanates from the D1 and D2 receptor containing neurons in the brain. PMID:23637801

  17. Homeostatic Changes in GABA and Acetylcholine Muscarinic Receptors on GABAergic Neurons in the Mesencephalic Reticular Formation following Sleep Deprivation

    PubMed Central

    2017-01-01

    Abstract We have examined whether GABAergic neurons in the mesencephalic reticular formation (RFMes), which are believed to inhibit the neurons in the pons that generate paradoxical sleep (PS or REMS), are submitted to homeostatic regulation under conditions of sleep deprivation (SD) by enforced waking during the day in mice. Using immunofluorescence, we investigated first, by staining for c-Fos, whether GABAergic RFMes neurons are active during SD and then, by staining for receptors, whether their activity is associated with homeostatic changes in GABAA or acetylcholine muscarinic type 2 (AChM2) receptors (Rs), which evoke inhibition. We found that a significantly greater proportion of the GABAergic neurons were positively stained for c-Fos after SD (∼27%) as compared to sleep control (SC; ∼1%) and sleep recovery (SR; ∼6%), suggesting that they were more active during waking with SD and less active or inactive during sleep with SC and SR. The density of GABAARs and AChM2Rs on the plasma membrane of the GABAergic neurons was significantly increased after SD and restored to control levels after SR. We conclude that the density of these receptors is increased on RFMes GABAergic neurons during presumed enhanced activity with SD and is restored to control levels during presumed lesser or inactivity with SR. Such increases in GABAAR and AChM2R with sleep deficits would be associated with increased susceptibility of the wake-active GABAergic neurons to inhibition from GABAergic and cholinergic sleep-active neurons and to thus permitting the onset of sleep and PS with muscle atonia. PMID:29302615

  18. Free energy landscape of G-protein coupled receptors, explored by accelerated molecular dynamics.

    PubMed

    Miao, Yinglong; Nichols, Sara E; McCammon, J Andrew

    2014-04-14

    G-protein coupled receptors (GPCRs) mediate cellular responses to various hormones and neurotransmitters and are important targets for treating a wide spectrum of diseases. They are known to adopt multiple conformational states (e.g., inactive, intermediate and active) during their modulation of various cell signaling pathways. Here, the free energy landscape of GPCRs is explored using accelerated molecular dynamics (aMD) simulations as demonstrated on the M2 muscarinic receptor, a key GPCR that regulates human heart rate and contractile forces of cardiomyocytes. Free energy profiles of important structural motifs that undergo conformational transitions upon GPCR activation and allosteric signaling are analyzed in detail, including the Arg(3.50)-Glu(6.30) ionic lock, the Trp(6.48) toggle switch and the hydrogen interactions between Tyr(5.58)-Tyr(7.53).

  19. Muscarinic Receptor-Dependent Long Term Depression in the Perirhinal Cortex and Recognition Memory are Impaired in the rTg4510 Mouse Model of Tauopathy.

    PubMed

    Scullion, Sarah E; Barker, Gareth R I; Warburton, E Clea; Randall, Andrew D; Brown, Jonathan T

    2018-02-26

    Neurodegenerative diseases affecting cognitive dysfunction, such as Alzheimer's disease and fronto-temporal dementia, are often associated impairments in the visual recognition memory system. Recent evidence suggests that synaptic plasticity, in particular long term depression (LTD), in the perirhinal cortex (PRh) is a critical cellular mechanism underlying recognition memory. In this study, we have examined novel object recognition and PRh LTD in rTg4510 mice, which transgenically overexpress tau P301L . We found that 8-9 month old rTg4510 mice had significant deficits in long- but not short-term novel object recognition memory. Furthermore, we also established that PRh slices prepared from rTg4510 mice, unlike those prepared from wildtype littermates, could not support a muscarinic acetylcholine receptor-dependent form of LTD, induced by a 5 Hz stimulation protocol. In contrast, bath application of the muscarinic agonist carbachol induced a form of chemical LTD in both WT and rTg4510 slices. Finally, when rTg4510 slices were preincubated with the acetylcholinesterase inhibitor donepezil, the 5 Hz stimulation protocol was capable of inducing significant levels of LTD. These data suggest that dysfunctional cholinergic innervation of the PRh of rTg4510 mice, results in deficits in synaptic LTD which may contribute to aberrant recognition memory in this rodent model of tauopathy.

  20. Nuclear Receptor Signaling Atlas: Opening Access to the Biology of Nuclear Receptor Signaling Pathways.

    PubMed

    Becnel, Lauren B; Darlington, Yolanda F; Ochsner, Scott A; Easton-Marks, Jeremy R; Watkins, Christopher M; McOwiti, Apollo; Kankanamge, Wasula H; Wise, Michael W; DeHart, Michael; Margolis, Ronald N; McKenna, Neil J

    2015-01-01

    Signaling pathways involving nuclear receptors (NRs), their ligands and coregulators, regulate tissue-specific transcriptomes in diverse processes, including development, metabolism, reproduction, the immune response and neuronal function, as well as in their associated pathologies. The Nuclear Receptor Signaling Atlas (NURSA) is a Consortium focused around a Hub website (www.nursa.org) that annotates and integrates diverse 'omics datasets originating from the published literature and NURSA-funded Data Source Projects (NDSPs). These datasets are then exposed to the scientific community on an Open Access basis through user-friendly data browsing and search interfaces. Here, we describe the redesign of the Hub, version 3.0, to deploy "Web 2.0" technologies and add richer, more diverse content. The Molecule Pages, which aggregate information relevant to NR signaling pathways from myriad external databases, have been enhanced to include resources for basic scientists, such as post-translational modification sites and targeting miRNAs, and for clinicians, such as clinical trials. A portal to NURSA's Open Access, PubMed-indexed journal Nuclear Receptor Signaling has been added to facilitate manuscript submissions. Datasets and information on reagents generated by NDSPs are available, as is information concerning periodic new NDSP funding solicitations. Finally, the new website integrates the Transcriptomine analysis tool, which allows for mining of millions of richly annotated public transcriptomic data points in the field, providing an environment for dataset re-use and citation, bench data validation and hypothesis generation. We anticipate that this new release of the NURSA database will have tangible, long term benefits for both basic and clinical research in this field.

  1. Mechanisms of signal transduction by ethylene: overlapping and non-overlapping signalling roles in a receptor family

    PubMed Central

    Shakeel, Samina N.; Wang, Xiaomin; Binder, Brad M.; Schaller, G. Eric

    2013-01-01

    The plant hormone ethylene regulates growth and development as well as responses to biotic and abiotic stresses. Over the last few decades, key elements involved in ethylene signal transduction have been identified through genetic approaches, these elements defining a pathway that extends from initial ethylene perception at the endoplasmic reticulum to changes in transcriptional regulation within the nucleus. Here, we present our current understanding of ethylene signal transduction, focusing on recent developments that support a model with overlapping and non-overlapping roles for members of the ethylene receptor family. We consider the evidence supporting this model for sub-functionalization within the receptor family, and then discuss mechanisms by which such a sub-functionalization may occur. To this end, we consider the importance of receptor interactions in modulating their signal output and how such interactions vary in the receptor family. In addition, we consider evidence indicating that ethylene signal output by the receptors involves both phosphorylation-dependent and phosphorylation-independent mechanisms. We conclude with a current model for signalling by the ethylene receptors placed within the overall context of ethylene signal transduction. PMID:23543258

  2. Nuclear Receptor Signaling Atlas: Opening Access to the Biology of Nuclear Receptor Signaling Pathways

    PubMed Central

    Becnel, Lauren B.; Darlington, Yolanda F.; Ochsner, Scott A.; Easton-Marks, Jeremy R.; Watkins, Christopher M.; McOwiti, Apollo; Kankanamge, Wasula H.; Wise, Michael W.; DeHart, Michael; Margolis, Ronald N.; McKenna, Neil J.

    2015-01-01

    Signaling pathways involving nuclear receptors (NRs), their ligands and coregulators, regulate tissue-specific transcriptomes in diverse processes, including development, metabolism, reproduction, the immune response and neuronal function, as well as in their associated pathologies. The Nuclear Receptor Signaling Atlas (NURSA) is a Consortium focused around a Hub website (www.nursa.org) that annotates and integrates diverse ‘omics datasets originating from the published literature and NURSA-funded Data Source Projects (NDSPs). These datasets are then exposed to the scientific community on an Open Access basis through user-friendly data browsing and search interfaces. Here, we describe the redesign of the Hub, version 3.0, to deploy “Web 2.0” technologies and add richer, more diverse content. The Molecule Pages, which aggregate information relevant to NR signaling pathways from myriad external databases, have been enhanced to include resources for basic scientists, such as post-translational modification sites and targeting miRNAs, and for clinicians, such as clinical trials. A portal to NURSA’s Open Access, PubMed-indexed journal Nuclear Receptor Signaling has been added to facilitate manuscript submissions. Datasets and information on reagents generated by NDSPs are available, as is information concerning periodic new NDSP funding solicitations. Finally, the new website integrates the Transcriptomine analysis tool, which allows for mining of millions of richly annotated public transcriptomic data points in the field, providing an environment for dataset re-use and citation, bench data validation and hypothesis generation. We anticipate that this new release of the NURSA database will have tangible, long term benefits for both basic and clinical research in this field. PMID:26325041

  3. G Protein-coupled Receptor Kinases of the GRK4 Protein Subfamily Phosphorylate Inactive G Protein-coupled Receptors (GPCRs).

    PubMed

    Li, Lingyong; Homan, Kristoff T; Vishnivetskiy, Sergey A; Manglik, Aashish; Tesmer, John J G; Gurevich, Vsevolod V; Gurevich, Eugenia V

    2015-04-24

    G protein-coupled receptor (GPCR) kinases (GRKs) play a key role in homologous desensitization of GPCRs. It is widely assumed that most GRKs selectively phosphorylate only active GPCRs. Here, we show that although this seems to be the case for the GRK2/3 subfamily, GRK5/6 effectively phosphorylate inactive forms of several GPCRs, including β2-adrenergic and M2 muscarinic receptors, which are commonly used as representative models for GPCRs. Agonist-independent GPCR phosphorylation cannot be explained by constitutive activity of the receptor or membrane association of the GRK, suggesting that it is an inherent ability of GRK5/6. Importantly, phosphorylation of the inactive β2-adrenergic receptor enhanced its interactions with arrestins. Arrestin-3 was able to discriminate between phosphorylation of the same receptor by GRK2 and GRK5, demonstrating preference for the latter. Arrestin recruitment to inactive phosphorylated GPCRs suggests that not only agonist activation but also the complement of GRKs in the cell regulate formation of the arrestin-receptor complex and thereby G protein-independent signaling. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Control of glycinergic input to spinal dorsal horn neurons by distinct muscarinic receptor subtypes revealed using knockout mice.

    PubMed

    Zhang, Hong-Mei; Zhou, Hong-Yi; Chen, Shao-Rui; Gautam, Dinesh; Wess, Jürgen; Pan, Hui-Lin

    2007-12-01

    Muscarinic acetylcholine receptors (mAChRs) play an important role in the tonic regulation of nociceptive transmission in the spinal cord. However, how mAChR subtypes contribute to the regulation of synaptic glycine release is unknown. To determine their role, glycinergic spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded in lamina II neurons by using whole-cell recordings in spinal cord slices of wild-type (WT) and mAChR subtype knockout (KO) mice. In WT mice, the mAChR agonist oxotremorine-M dose-dependently decreased the frequency of sIPSCs in most neurons, but it had variable effects in other neurons. In contrast, in M3-KO mice, oxotremorine-M consistently decreased the glycinergic sIPSC frequency in all neurons tested, and in M2/M4 double-KO mice, it always increased the sIPSC frequency. In M2/M4 double-KO mice, the potentiating effect of oxotremorine-M was attenuated by higher concentrations in some neurons through activation of GABA(B) receptors. In pertussis toxin-treated WT mice, oxotremorine-M also consistently increased the sIPSC frequency. In M2-KO and M4-KO mice, the effect of oxotremorine-M on sIPSCs was divergent because of the opposing functions of the M3 subtype and the M2 and M4 subtypes. This study demonstrates that stimulation of the M2 and M4 subtypes inhibits glycinergic inputs to spinal dorsal horn neurons of mice, whereas stimulation of the M3 subtype potentiates synaptic glycine release. Furthermore, GABA(B) receptors are involved in the feedback regulation of glycinergic synaptic transmission in the spinal cord. This study revealed distinct functions of mAChR subtypes in controlling glycinergic input to spinal dorsal horn neurons.

  5. Differential Effects of Systemic Cholinergic Receptor Blockade on Pavlovian Incentive Motivation and Goal-Directed Action Selection

    PubMed Central

    Ostlund, Sean B; Kosheleff, Alisa R; Maidment, Nigel T

    2014-01-01

    Reward-seeking actions can be guided by external cues that signal reward availability. For instance, when confronted with a stimulus that signals sugar, rats will prefer an action that produces sugar over a second action that produces grain pellets. Action selection is also sensitive to changes in the incentive value of potential rewards. Thus, rats that have been prefed a large meal of sucrose will prefer a grain-seeking action to a sucrose-seeking action. The current study investigated the dependence of these different aspects of action selection on cholinergic transmission. Hungry rats were given differential training with two unique stimulus-outcome (S1-O1 and S2-O2) and action-outcome (A1-O1 and A2-O2) contingencies during separate training phases. Rats were then given a series of Pavlovian-to-instrumental transfer tests, an assay of cue-triggered responding. Before each test, rats were injected with scopolamine (0, 0.03, or 0.1 mg/kg, intraperitoneally), a muscarinic receptor antagonist, or mecamylamine (0, 0.75, or 2.25 mg/kg, intraperitoneally), a nicotinic receptor antagonist. Although the reward-paired cues were capable of biasing action selection when rats were tested off-drug, both anticholinergic treatments were effective in disrupting this effect. During a subsequent round of outcome devaluation testing—used to assess the sensitivity of action selection to a change in reward value—we found no effect of either scopolamine or mecamylamine. These results reveal that cholinergic signaling at both muscarinic and nicotinic receptors mediates action selection based on Pavlovian reward expectations, but is not critical for flexibly selecting actions using current reward values. PMID:24370780

  6. Homeostatic Changes in GABA and Acetylcholine Muscarinic Receptors on GABAergic Neurons in the Mesencephalic Reticular Formation following Sleep Deprivation.

    PubMed

    Toossi, Hanieh; Del Cid-Pellitero, Esther; Jones, Barbara E

    2017-01-01

    We have examined whether GABAergic neurons in the mesencephalic reticular formation (RFMes), which are believed to inhibit the neurons in the pons that generate paradoxical sleep (PS or REMS), are submitted to homeostatic regulation under conditions of sleep deprivation (SD) by enforced waking during the day in mice. Using immunofluorescence, we investigated first, by staining for c-Fos, whether GABAergic RFMes neurons are active during SD and then, by staining for receptors, whether their activity is associated with homeostatic changes in GABA A or acetylcholine muscarinic type 2 (AChM2) receptors (Rs), which evoke inhibition. We found that a significantly greater proportion of the GABAergic neurons were positively stained for c-Fos after SD (∼27%) as compared to sleep control (SC; ∼1%) and sleep recovery (SR; ∼6%), suggesting that they were more active during waking with SD and less active or inactive during sleep with SC and SR. The density of GABA A Rs and AChM2Rs on the plasma membrane of the GABAergic neurons was significantly increased after SD and restored to control levels after SR. We conclude that the density of these receptors is increased on RFMes GABAergic neurons during presumed enhanced activity with SD and is restored to control levels during presumed lesser or inactivity with SR. Such increases in GABA A R and AChM2R with sleep deficits would be associated with increased susceptibility of the wake-active GABAergic neurons to inhibition from GABAergic and cholinergic sleep-active neurons and to thus permitting the onset of sleep and PS with muscle atonia.

  7. Presynaptic muscarinic control of glutamatergic synaptic transmission.

    PubMed

    Buño, W; Cabezas, C; Fernández de Sevilla, D

    2006-01-01

    The hippocampus receives cholinergic projections from the medial septal nucleus and Broca's diagonal band that terminate in the CA1, CA3, and dentate gyrus regions (Frotscher and Leranth, 1985). Glutamatergic synapses between CA3 and CA1 pyramidal neurons are presynaptically inhibited by acetylcholine (ACh), via activation of muscarinic ACh receptors (mAChRs) at the terminals of Schaffer collaterals (SCs) (Hounsgaard, 1978; Fernández de Sevilla et al., 2002, 2003). There are two types of SC-CA1 pyramidal neuron synapses. One type, called functional synapse, shows postsynaptic alpha- amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-receptor mediated currents at resting potential (Vm) and both AMPA and N-methyl-D-aspartate receptor (NMDAR)-mediated currents when depolarized. The other type, termed silent synapse, only displays postsynaptic NMDAR-mediated currents at depolarized Vms, but does not respond at the resting Vm (Isaac et al., 1995). Using hippocampal slices obtained from young Wistar rats, we examined the effects of activation of cholinergic afferents at the stratum oriens/alveus on excitatory postsynaptic currents (EPSCs) evoked in CA1 pyramidal neurons by stimulation of SCs. We also tested the action of the nonhydrolyzable cholinergic agonist carbamylcholine chloride (CCh) on EPSCs evoked by minimal stimulation of SCs (which activates a single or very few synapses) in functional and silent synapses.

  8. B Cell Antigen Receptor Signaling and Internalization Are Mutually Exclusive Events

    PubMed Central

    Hou, Ping; Araujo, Elizabeth; Zhao, Tong; Zhang, Miao; Massenburg, Don; Veselits, Margaret; Doyle, Colleen; Dinner, Aaron R; Clark, Marcus R

    2006-01-01

    Engagement of the B cell antigen receptor initiates two concurrent processes, signaling and receptor internalization. While both are required for normal humoral immune responses, the relationship between these two processes is unknown. Herein, we demonstrate that following receptor ligation, a small subpopulation of B cell antigen receptors are inductively phosphorylated and selectively retained at the cell surface where they can serve as scaffolds for the assembly of signaling molecules. In contrast, the larger population of non-phosphorylated receptors is rapidly endocytosed. Each receptor can undergo only one of two mutually exclusive fates because the tyrosine-based motifs that mediate signaling when phosphorylated mediate internalization when not phosphorylated. Mathematical modeling indicates that the observed competition between receptor phosphorylation and internalization enhances signaling responses to low avidity ligands. PMID:16719564

  9. TAM Receptors in Leukemia: Expression, Signaling, and Therapeutic Implications

    PubMed Central

    Brandão, Luis; Migdall-Wilson, Justine; Eisenman, Kristen; Graham, Douglas K.

    2016-01-01

    In the past 30 years there has been remarkable progress in the treatment of leukemia and lymphoma. However, current treatments are largely ineffective against relapsed leukemia and, in the case of pediatric patients, are often associated with severe long-term toxicities. Thus, there continues to be a critical need for the development of effective biologically targeted therapies. The TAM family of receptor tyrosine kinases—Tyro3, Axl, and Mer—plays an important role in normal hematopoiesis, including natural killer cell maturation, macrophage function, and platelet activation and signaling. Furthermore, TAM receptor activation leads to upregulation of pro-survival and proliferation signaling pathways, and aberrant TAM receptor expression contributes to cancer development, including myeloid and lymphoid leukemia. This review summarizes the role of TAM receptors in leukemia. We outline TAM receptor expression patterns in different forms of leukemia, describe potential mechanisms leading to their overexpression, and delineate the signaling pathways downstream of receptor activation that have been implicated in leukemogenesis. Finally, we discuss the current research focused on inhibitors against these receptors in an effort to develop new therapeutic strategies for leukemia. PMID:22150307

  10. Urothelial Signaling

    PubMed Central

    Andersson, Karl-Erik

    2013-01-01

    The urothelium, which lines the inner surface of the renal pelvis, the ureters, and the urinary bladder, not only forms a high-resistance barrier to ion, solute and water flux, and pathogens, but also functions as an integral part of a sensory web which receives, amplifies, and transmits information about its external milieu. Urothelial cells have the ability to sense changes in their extracellular environment, and respond to chemical, mechanical and thermal stimuli by releasing various factors such as ATP, nitric oxide, and acetylcholine. They express a variety of receptors and ion channels, including P2X3 purinergic receptors, nicotinic and muscarinic receptors, and TRP channels, which all have been implicated in urothelial-neuronal interactions, and involved in signals that via components in the underlying lamina propria, such as interstitial cells, can be amplified and conveyed to nerves, detrusor muscle cells, and ultimately the central nervous system. The specialized anatomy of the urothelium and underlying structures, and the possible communication mechanisms from urothelial cells to various cell types within the bladder wall are described. Changes in the urothelium/lamina propria (“mucosa”) produced by different bladder disorders are discussed, as well as the mucosa as a target for therapeutic interventions. PMID:23589830

  11. The orphan receptor ERRα interferes with steroid signaling

    PubMed Central

    Teyssier, Catherine; Bianco, Stéphanie; Lanvin, Olivia; Vanacker, Jean-Marc

    2008-01-01

    The estrogen receptor-related receptor α (ERRα) is an orphan member of the nuclear receptor superfamily that has been shown to interfere with the estrogen-signaling pathway. In this report, we demonstrate that ERRα also cross-talks with signaling driven by other steroid hormones. Treatment of human prostatic cells with a specific ERRα inverse agonist reduces the expression of several androgen-responsive genes, in a manner that does not involve perturbation of androgen receptor expression or activity. Furthermore, ERRα activates the expression of androgen response elements (ARE)-containing promoters, such as that of the prostate cancer marker PSA, in an ARE-dependent manner. In addition, promoters containing a steroid response element can be activated by all members of the ERR orphan receptor subfamily, and this, even in the presence of antisteroid compounds. PMID:18697814

  12. Receptor signaling: when dimerization is not enough.

    PubMed

    Jiang, G; Hunter, T

    Activation of receptors that signal via tyrosine kinase domains has been thought to involve receptor dimerization and transphosphorylation of juxtaposed catalytic domains. Recent results suggest things might be more complex - specific intersubunit conformational changes within a dimer can also be important.

  13. Neutrophil cell surface receptors and their intracellular signal transduction pathways☆

    PubMed Central

    Futosi, Krisztina; Fodor, Szabina; Mócsai, Attila

    2013-01-01

    Neutrophils play a critical role in the host defense against bacterial and fungal infections, but their inappropriate activation also contributes to tissue damage during autoimmune and inflammatory diseases. Neutrophils express a large number of cell surface receptors for the recognition of pathogen invasion and the inflammatory environment. Those include G-protein-coupled chemokine and chemoattractant receptors, Fc-receptors, adhesion receptors such as selectins/selectin ligands and integrins, various cytokine receptors, as well as innate immune receptors such as Toll-like receptors and C-type lectins. The various cell surface receptors trigger very diverse signal transduction pathways including activation of heterotrimeric and monomeric G-proteins, receptor-induced and store-operated Ca2 + signals, protein and lipid kinases, adapter proteins and cytoskeletal rearrangement. Here we provide an overview of the receptors involved in neutrophil activation and the intracellular signal transduction processes they trigger. This knowledge is crucial for understanding how neutrophils participate in antimicrobial host defense and inflammatory tissue damage and may also point to possible future targets of the pharmacological therapy of neutrophil-mediated autoimmune or inflammatory diseases. PMID:23994464

  14. Novel Insights on Thyroid-Stimulating Hormone Receptor Signal Transduction

    PubMed Central

    Neumann, Susanne; Grüters, Annette; Krude, Heiko

    2013-01-01

    The TSH receptor (TSHR) is a member of the glycoprotein hormone receptors, a subfamily of family A G protein-coupled receptors. The TSHR is of great importance for the growth and function of the thyroid gland. The TSHR and its endogenous ligand TSH are pivotal proteins with respect to a variety of physiological functions and malfunctions. The molecular events of TSHR regulation can be summarized as a process of signal transduction, including signal reception, conversion, and amplification. The steps during signal transduction from the extra- to the intracellular sites of the cell are not yet comprehensively understood. However, essential new insights have been achieved in recent years on the interrelated mechanisms at the extracellular region, the transmembrane domain, and intracellular components. This review contains a critical summary of available knowledge of the molecular mechanisms of signal transduction at the TSHR, for example, the key amino acids involved in hormone binding or in the structural conformational changes that lead to G protein activation or signaling regulation. Aspects of TSHR oligomerization, signaling promiscuity, signaling selectivity, phenotypes of genetic variations, and potential extrathyroidal receptor activity are also considered, because these are relevant to an understanding of the overall function of the TSHR, including physiological, pathophysiological, and pharmacological perspectives. Directions for future research are discussed. PMID:23645907

  15. Receptor-mediated signalling in plants: molecular patterns and programmes

    PubMed Central

    Tör, Mahmut; Lotze, Michael T.; Holton, Nicholas

    2009-01-01

    A highly evolved surveillance system in plants is able to detect a broad range of signals originating from pathogens, damaged tissues, or altered developmental processes, initiating sophisticated molecular mechanisms that result in defence, wound healing, and development. Microbe-associated molecular pattern molecules (MAMPs), damage-associated molecular pattern molecules (DAMPs), virulence factors, secreted proteins, and processed peptides can be recognized directly or indirectly by this surveillance system. Nucleotide binding-leucine rich repeat proteins (NB-LRR) are intracellular receptors and have been targeted by breeders for decades to elicit resistance to crop pathogens in the field. Receptor-like kinases (RLKs) or receptor like proteins (RLPs) are membrane bound signalling molecules with an extracellular receptor domain. They provide an early warning system for the presence of potential pathogens and activate protective immune signalling in plants. In addition, they act as a signal amplifier in the case of tissue damage, establishing symbiotic relationships and effecting developmental processes. The identification of several important ligands for the RLK-type receptors provided an opportunity to understand how plants differentiate, how they distinguish beneficial and detrimental stimuli, and how they co-ordinate the role of various types of receptors under varying environmental conditions. The diverse roles of extra-and intracellular plant receptors are examined here and the recent findings on how they promote defence and development is reviewed. PMID:19628572

  16. Involvement of the basolateral amygdala in muscarinic cholinergic modulation of extinction memory consolidation

    PubMed Central

    Boccia, Mariano M.; Blake, Mariano G.; Baratti, Carlos M.; McGaugh, James L.

    2009-01-01

    Previous studies have reported that drugs affecting neuromodulatory systems within the basolateral amygdala (BLA), including drugs affecting muscarinic cholinergic receptors, modulate the consolidation of many kinds of training, including contextual fear conditioning (CFC). The present experiments investigated the involvement of muscarinic cholinergic influences within the BLA in modulating the consolidation of CFC extinction memory. Male Sprague Dawley rats implanted with unilateral cannula aimed at the BLA were trained on a CFC task, using footshock stimulation, and 24 and 48 h later were given extinction training by replacing them in the apparatus without footshock. Following each extinction session they received intra-BLA infusions of the cholinergic agonist oxotremorine (10 ng). Immediate post-extinction BLA infusions significantly enhanced extinction but infusions administered 180 min after extinction training did not influence extinction. Thus the oxotremorine effects were time-dependent and not attributable to non-specific effects on retention performance. These findings provide evidence that, as previously found with original CFC learning, cholinergic activation within the BLA modulates the consolidation of CFC extinction. PMID:18706510

  17. The LDL receptor gene family: signaling functions during development.

    PubMed

    Howell, B W; Herz, J

    2001-02-01

    The traditional views regarding the biological functions of the low-density lipoprotein (LDL) receptor gene family have been revisited recently with new evidence that at least some of the members of this receptor family act as signal-transduction molecules. Known for their role in endocytosis, particularly of their namesake the LDLs, and for their role in the prevention of atherosclerosis, these receptors belong to an ancient family with numerous ligands, effector molecules and functions. Recent evidence implicates this family of receptors in diverse signaling pathways, long-term potentiation and neuronal degeneration.

  18. Receptor mechanisms in fish chromatophores--VII. Muscarinic cholinoceptors and alpha adrenoceptors, both mediating pigment aggregation, strangely coexist in Corydoras melanophores.

    PubMed

    Kasukawa, H; Fujii, R

    1985-01-01

    Both acetylcholine and catecholamines showed melanin-aggregating action within melanophores on an isolated bony plate of the mailed catfish Corydoras paleatus. Chromatic nervous stimulation either by an electrical field or by an elevation of [K+]0 brought about melanosome aggregation. Alpha adrenolytic agents antagonized the melanin-aggregating effects either of catecholamines or of nervous stimuli. Muscarinic cholinolytics interfered with the action of acetylcholine, but did not have any effect on the responses to nervous stimuli. In addition to the alpha adrenoceptors which participate in sympathetic-melanophore transmission, muscarinic cholinoceptors of unknown functional significance, which also mediate melanosome aggregation in the cell, exist in Corydoras melanophores.

  19. Scopolamine and amphetamine produce similar decision-making deficits on a rat gambling task via independent pathways.

    PubMed

    Silveira, Mason M; Malcolm, Emma; Shoaib, Mohammed; Winstanley, Catharine A

    2015-03-15

    Disorders characterized by disturbed cholinergic signaling, such as schizophrenia, exhibit impaired performance on measures of real-world cost/benefit decision-making. Whether the cholinergic system contributes to the choice deficits observed is currently unknown. We therefore determined the effects of broad-acting agonists and antagonists at the nicotinic and muscarinic receptor on decision making, as measured by the rodent gambling task (rGT). Given the anatomical and functional connectivity of the cholinergic and dopaminergic systems, we also sought to modulate amphetamine's previously reported effect on rGT performance via the cholinergic system. Male rats were trained on the rGT, during which animals chose from four different options. The optimal strategy on the rGT is to favor options associated with smaller immediate rewards and less punishment/loss. Impulsive action was also measured by recording the number of premature responses made. Performance on the rGT was assessed following acute treatment with the muscarinic receptor agonist oxotremorine, the muscarinic receptor antagonist scopolamine, nicotine, and the nicotinic receptor antagonist mecamylamine. Similar to the effect produced by amphetamine, muscarinic receptor antagonism with scopolamine (0.1mg/kg) impaired decision making, albeit to a lesser degree. Prior muscarinic agonism with oxotremorine was unable to attenuate amphetamine's effects on rGT performance. Oxotremorine, nicotine, and mecamylamine did not affect the choice profile. We therefore conclude that modulation of the muscarinic, but not nicotinic, receptor system can affect decision making under conditions of risk and uncertainty. Such findings contribute to a broader understanding of the cognitive deficits observed in disorders in which cholinergic signaling is compromised. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Urothelial effects of oral agents for overactive bladder.

    PubMed

    Andersson, Karl-Erik; Fullhase, Claudius; Soler, Roberto

    2008-11-01

    The cholinergic system of the bladder includes muscarinic receptors distributed to detrusor myocytes and structures within mucosa including bladder afferent (sensory) nerves. The receptors have been shown to be involved in afferent signaling from the bladder, but it has not been established to what extent effects on this mucosal signaling pathway contribute to the therapeutic efficacy of the clinically used antimuscarinics. Mucosa can be influenced by antimuscarinics via the bloodstream. However, some antimuscarinics and their active metabolites are excreted in urine in amounts that may affect the mucosal muscarinic receptors from the luminal side. This has not yet been demonstrated to imply superior clinical efficacy. Nevertheless, mucosal afferent signaling pathways are therapeutically interesting targets that should be further explored.

  1. Endosulfan and cholinergic (muscarinic) transmission: effect on electroencephalograms and (/sup 3/H)quinuclidinyl benzilate in pigeon brain

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Anand, M.; Agrawal, A.K.; Gopal, K.

    Single exposure of endosulfan (5 mg/kg) to pigeons (Columbia livia) caused neuronal hyperexcitability as evidence by spike discharges of 200-500 ..mu..V in the electroencephalograms (EEG) from the telencephalon and hyperstriatum, but there was not effect on the ectostriatal area. Cholinergic (muscarinic) receptor binding study using (/sup 3/H)quinuclidinyl benzilate ((/sup 3/H)QNB) as a specific ligand indicated that a single exposure to 5 mg/kg of endosulfan caused a significant increase in (/sup 3/H)QNB binding to the striatal membrane. Behavior study further indicated that a single dose of 200 ..mu..g/kg of oxotremorine produced a significant induction in the tremor in endosulfan-pretreated pigeons. Themore » results of this behavioral and biochemical study indicate the involvement of a cholinergic (muscarinic) transmitter system in endosulfan-induced neurotoxicity.« less

  2. Evolution of neuronal signalling: transmitters and receptors.

    PubMed

    Hoyle, Charles H V

    2011-11-16

    Evolution is a dynamic process during which the genome should not be regarded as a static entity. Molecular and morphological information yield insights into the evolution of species and their phylogenetic relationships, and molecular information in particular provides information into the evolution of signalling processes. Many signalling systems have their origin in primitive, even unicellular, organisms. Through time, and as organismal complexity increased, certain molecules were employed as intercellular signal molecules. In the autonomic nervous system the basic unit of chemical transmission is a ligand and its cognate receptor. The general mechanisms underlying evolution of signal molecules and their cognate receptors have their basis in the alteration of the genome. In the past this has occurred in large-scale events, represented by two or more doublings of the whole genome, or large segments of the genome, early in the deuterostome lineage, after the emergence of urochordates and cephalochordates, and before the emergence of vertebrates. These duplications were followed by extensive remodelling involving subsequent small-scale changes, ranging from point mutations to exon duplication. Concurrent with these processes was multiple gene loss so that the modern genome contains roughly the same number of genes as in early deuterostomes despite the large-scale genomic duplications. In this review, the principles that underlie evolution that have led to large and small families of autonomic neurotransmitters and their receptors are discussed, with emphasis on G protein-coupled receptors. Copyright © 2010 Elsevier B.V. All rights reserved.

  3. Evolution of the toxins muscarine and psilocybin in a family of mushroom-forming fungi.

    PubMed

    Kosentka, Pawel; Sprague, Sarah L; Ryberg, Martin; Gartz, Jochen; May, Amanda L; Campagna, Shawn R; Matheny, P Brandon

    2013-01-01

    Mushroom-forming fungi produce a wide array of toxic alkaloids. However, evolutionary analyses aimed at exploring the evolution of muscarine, a toxin that stimulates the parasympathetic nervous system, and psilocybin, a hallucinogen, have never been performed. The known taxonomic distribution of muscarine within the Inocybaceae is limited, based only on assays of species from temperate regions of the northern hemisphere. Here, we present a review of muscarine and psilocybin assays performed on species of Inocybaceae during the last fifty years. To supplement these results, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine whether muscarine was present in 30 new samples of Inocybaceae, the majority of which have not been previously assayed or that originated from either the tropics or temperate regions of the southern hemisphere. Our main objective is to test the hypothesis that the presence of muscarine is a shared ancestral feature of the Inocybaceae. In addition, we also test whether species of Inocyabceae that produce psilocybin are monophyletic. Our findings suggest otherwise. Muscarine has evolved independently on several occasions, together with several losses. We also detect at least two independent transitions of muscarine-free lineages to psilocybin-producing states. Although not ancestral for the family as a whole, muscarine is a shared derived trait for an inclusive clade containing three of the seven major lineages of Inocybaceae (the Inocybe, Nothocybe, and Pseudosperma clades), the common ancestor of which may have evolved ca. 60 million years ago. Thus, muscarine represents a conserved trait followed by several recent losses. Transitions to psilocybin from muscarine-producing ancestors occurred more recently between 10-20 million years ago after muscarine loss in two separate lineages. Statistical analyses firmly reject a single origin of muscarine-producing taxa.

  4. Evolution of the Toxins Muscarine and Psilocybin in a Family of Mushroom-Forming Fungi

    PubMed Central

    Kosentka, Pawel; Sprague, Sarah L.; Ryberg, Martin; Gartz, Jochen; May, Amanda L.; Campagna, Shawn R.; Matheny, P. Brandon

    2013-01-01

    Mushroom-forming fungi produce a wide array of toxic alkaloids. However, evolutionary analyses aimed at exploring the evolution of muscarine, a toxin that stimulates the parasympathetic nervous system, and psilocybin, a hallucinogen, have never been performed. The known taxonomic distribution of muscarine within the Inocybaceae is limited, based only on assays of species from temperate regions of the northern hemisphere. Here, we present a review of muscarine and psilocybin assays performed on species of Inocybaceae during the last fifty years. To supplement these results, we used liquid chromatography–tandem mass spectrometry (LC–MS/MS) to determine whether muscarine was present in 30 new samples of Inocybaceae, the majority of which have not been previously assayed or that originated from either the tropics or temperate regions of the southern hemisphere. Our main objective is to test the hypothesis that the presence of muscarine is a shared ancestral feature of the Inocybaceae. In addition, we also test whether species of Inocyabceae that produce psilocybin are monophyletic. Our findings suggest otherwise. Muscarine has evolved independently on several occasions, together with several losses. We also detect at least two independent transitions of muscarine-free lineages to psilocybin-producing states. Although not ancestral for the family as a whole, muscarine is a shared derived trait for an inclusive clade containing three of the seven major lineages of Inocybaceae (the Inocybe, Nothocybe, and Pseudosperma clades), the common ancestor of which may have evolved ca. 60 million years ago. Thus, muscarine represents a conserved trait followed by several recent losses. Transitions to psilocybin from muscarine-producing ancestors occurred more recently between 10–20 million years ago after muscarine loss in two separate lineages. Statistical analyses firmly reject a single origin of muscarine-producing taxa. PMID:23717644

  5. The selective M1 muscarinic cholinergic agonist CDD-0102A enhances working memory and cognitive flexibility.

    PubMed

    Ragozzino, Michael E; Artis, Sonja; Singh, Amritha; Twose, Trevor M; Beck, Joseph E; Messer, William S

    2012-03-01

    Various neurodegenerative diseases and psychiatric disorders are marked by alterations in brain cholinergic function and cognitive deficits. Efforts to alleviate such deficits have been limited by a lack of selective M(1) muscarinic agonists. 5-(3-Ethyl-1,2,4-oxadiazol-5-yl)-1,4,5,6-tetrahydropyrimidine hydrochloride (CDD-0102A) is a partial agonist at M(1) muscarinic receptors with limited activity at other muscarinic receptor subtypes. The present studies investigated the effects of CDD-0102A on working memory and strategy shifting in rats. CDD-0102A administered intraperitoneally 30 min before testing at 0.1, 0.3, and 1 mg/kg significantly enhanced delayed spontaneous alternation performance in a four-arm cross maze, suggesting improvement in working memory. In separate experiments, CDD-0102A had potent enhancing effects on learning and switching between a place and visual cue discrimination. Treatment with CDD-0102A did not affect acquisition of either a place or visual cue discrimination. In contrast, CDD-0102A at 0.03 and 0.1 mg/kg significantly enhanced a shift between a place and visual cue discrimination. Analysis of the errors in the shift to the place or shift to the visual cue strategy revealed that in both cases CDD-0102A significantly increased the ability to initially inhibit a previously relevant strategy and maintain a new, relevant strategy once selected. In anesthetized rats, the minimum dose required to induce salivation was approximately 0.3 mg/kg i.p. Salivation increased with dose, and the estimated ED(50) was 2.0 mg/kg. The data suggest that CDD-0102A has unique memory and cognitive enhancing properties that might be useful in the treatment of neurological disorders at doses that do not produce adverse effects such as salivation.

  6. Pharmacokinetics and metabolism of the novel muscarinic receptor agonist SNI-2011 in rats and dogs.

    PubMed

    Washio, Takuo; Kohsaka, Kazuhiro; Arisawa, Hirohiko; Masunaga, Hiroaki

    2003-01-01

    In this study, the pharmacokinetics of SNI-2011 ((+/-)-cis-2-methylspiro[1,3-oxathiolane-5,3'-quinuclidine]monohydrochloride hemihydrate, cevimeline, CAS 153504-70-2), a novel muscarinic acetylcholine receptor agonist developed for the treatment of Sjögren's syndrome, in rats and dogs were determined following intravenous or oral administration using liquid chromatography/mass spectrometry (LC/MS). The in vitro metabolism of SNI-2011 was also evaluated with rat and dog liver microsomes. After oral administration, plasma concentrations of SNI-2011 reached to Cmax within 1 h in both species, suggesting that SNI-2011 was quickly absorbed, and then decreased with a t1/2 of 0.4-1.1 h. The bioavailability was approximately 50% and 30% in rats and dogs, respectively. Major metabolites in plasma were both S- and N-oxidized metabolites in rats and only N-oxidized metabolite in dogs, indicating that a large species difference was observed in the metabolism of SNI-2011. Sex difference was also observed in the pharmacokinetics of SNI-2011 in rats, but not in dogs. In the in vitro study, chemical inhibition and pH-dependent studies revealed that the sulf-oxidation and N-oxidation of SNI-2011 were mediated by cytochrome P450 (CYP) and flavin-containing monooxygenase (FMO), respectively, in both species. In addition, CYP2D and CYP3A were mainly responsible for the sulfoxidation in rat liver microsomes.

  7. Acute food deprivation reverses morphine-induced locomotion deficits in M5 muscarinic receptor knockout mice.

    PubMed

    Steidl, Stephan; Lee, Esther; Wasserman, David; Yeomans, John S

    2013-09-01

    Lesions of the pedunculopontine tegmental nucleus (PPT), one of two sources of cholinergic input to the ventral tegmental area (VTA), block conditioned place preference (CPP) for morphine in drug-naïve rats. M5 muscarinic cholinergic receptors, expressed by midbrain dopamine neurons, are critical for the ability of morphine to increase nucleus accumbens dopamine levels and locomotion, and for morphine CPP. This suggests that M5-mediated PPT cholinergic inputs to VTA dopamine neurons critically contribute to morphine-induced dopamine activation, reward and locomotion. In the current study we tested whether food deprivation, which reduces PPT contribution to morphine CPP in rats, could also reduce M5 contributions to morphine-induced locomotion in mice. Acute 18-h food deprivation reversed the phenotypic differences usually seen between non-deprived wild-type and M5 knockout mice. That is, food deprivation increased morphine-induced locomotion in M5 knockout mice but reduced morphine-induced locomotion in wild-type mice. Food deprivation increased saline-induced locomotion equally in wild-type and M5 knockout mice. Based on these findings, we suggest that food deprivation reduces the contribution of M5-mediated PPT cholinergic inputs to the VTA in morphine-induced locomotion and increases the contribution of a PPT-independent pathway. The contributions of cholinergic, dopaminergic and GABAergic neurons to the effects of acute food deprivation are discussed. Copyright © 2013 Elsevier B.V. All rights reserved.

  8. Sweet Taste Receptor Signaling Network: Possible Implication for Cognitive Functioning

    PubMed Central

    Welcome, Menizibeya O.; Mastorakis, Nikos E.; Pereverzev, Vladimir A.

    2015-01-01

    Sweet taste receptors are transmembrane protein network specialized in the transmission of information from special “sweet” molecules into the intracellular domain. These receptors can sense the taste of a range of molecules and transmit the information downstream to several acceptors, modulate cell specific functions and metabolism, and mediate cell-to-cell coupling through paracrine mechanism. Recent reports indicate that sweet taste receptors are widely distributed in the body and serves specific function relative to their localization. Due to their pleiotropic signaling properties and multisubstrate ligand affinity, sweet taste receptors are able to cooperatively bind multiple substances and mediate signaling by other receptors. Based on increasing evidence about the role of these receptors in the initiation and control of absorption and metabolism, and the pivotal role of metabolic (glucose) regulation in the central nervous system functioning, we propose a possible implication of sweet taste receptor signaling in modulating cognitive functioning. PMID:25653876

  9. The cannabinoid receptor CB1 modulates the signaling properties of the lysophosphatidylinositol receptor GPR55.

    PubMed

    Kargl, Julia; Balenga, Nariman; Parzmair, Gerald P; Brown, Andrew J; Heinemann, Akos; Waldhoer, Maria

    2012-12-28

    The G protein-coupled receptor (GPCR) 55 (GPR55) and the cannabinoid receptor 1 (CB1R) are co-expressed in many tissues, predominantly in the central nervous system. Seven transmembrane spanning (7TM) receptors/GPCRs can form homo- and heteromers and initiate distinct signaling pathways. Recently, several synthetic CB1 receptor inverse agonists/antagonists, such as SR141716A, AM251, and AM281, were reported to activate GPR55. Of these, SR141716A was marketed as a promising anti-obesity drug, but was withdrawn from the market because of severe side effects. Here, we tested whether GPR55 and CB1 receptors are capable of (i) forming heteromers and (ii) whether such heteromers could exhibit novel signaling patterns. We show that GPR55 and CB1 receptors alter each others signaling properties in human embryonic kidney (HEK293) cells. We demonstrate that the co-expression of FLAG-CB1 receptors in cells stably expressing HA-GPR55 specifically inhibits GPR55-mediated transcription factor activation, such as nuclear factor of activated T-cells and serum response element, as well as extracellular signal-regulated kinases (ERK1/2) activation. GPR55 and CB1 receptors can form heteromers, but the internalization of both receptors is not affected. In addition, we observe that the presence of GPR55 enhances CB1R-mediated ERK1/2 and nuclear factor of activated T-cell activation. Our data provide the first evidence that GPR55 can form heteromers with another 7TM/GPCR and that this interaction with the CB1 receptor has functional consequences in vitro. The GPR55-CB1R heteromer may play an important physiological and/or pathophysiological role in tissues endogenously co-expressing both receptors.

  10. The Cannabinoid Receptor CB1 Modulates the Signaling Properties of the Lysophosphatidylinositol Receptor GPR55*

    PubMed Central

    Kargl, Julia; Balenga, Nariman; Parzmair, Gerald P.; Brown, Andrew J.; Heinemann, Akos; Waldhoer, Maria

    2012-01-01

    The G protein-coupled receptor (GPCR) 55 (GPR55) and the cannabinoid receptor 1 (CB1R) are co-expressed in many tissues, predominantly in the central nervous system. Seven transmembrane spanning (7TM) receptors/GPCRs can form homo- and heteromers and initiate distinct signaling pathways. Recently, several synthetic CB1 receptor inverse agonists/antagonists, such as SR141716A, AM251, and AM281, were reported to activate GPR55. Of these, SR141716A was marketed as a promising anti-obesity drug, but was withdrawn from the market because of severe side effects. Here, we tested whether GPR55 and CB1 receptors are capable of (i) forming heteromers and (ii) whether such heteromers could exhibit novel signaling patterns. We show that GPR55 and CB1 receptors alter each others signaling properties in human embryonic kidney (HEK293) cells. We demonstrate that the co-expression of FLAG-CB1 receptors in cells stably expressing HA-GPR55 specifically inhibits GPR55-mediated transcription factor activation, such as nuclear factor of activated T-cells and serum response element, as well as extracellular signal-regulated kinases (ERK1/2) activation. GPR55 and CB1 receptors can form heteromers, but the internalization of both receptors is not affected. In addition, we observe that the presence of GPR55 enhances CB1R-mediated ERK1/2 and nuclear factor of activated T-cell activation. Our data provide the first evidence that GPR55 can form heteromers with another 7TM/GPCR and that this interaction with the CB1 receptor has functional consequences in vitro. The GPR55-CB1R heteromer may play an important physiological and/or pathophysiological role in tissues endogenously co-expressing both receptors. PMID:23161546

  11. Cholinergic and ghrelinergic receptors and KCNQ channels in the medial PFC regulate the expression of palatability.

    PubMed

    Parent, Marc A; Amarante, Linda M; Swanson, Kyra; Laubach, Mark

    2015-01-01

    The medial prefrontal cortex (mPFC) is a key brain region for the control of consummatory behavior. Neuronal activity in this area is modulated when rats initiate consummatory licking and reversible inactivations eliminate reward contrast effects and reduce a measure of palatability, the duration of licking bouts. Together, these data suggest the hypothesis that rhythmic neuronal activity in the mPFC is crucial for the control of consummatory behavior. The muscarinic cholinergic system is known to regulate membrane excitability and control low-frequency rhythmic activity in the mPFC. Muscarinic receptors (mAChRs) act through KCNQ (Kv7) potassium channels, which have recently been linked to the orexigenic peptide ghrelin. To understand if drugs that act on KCNQ channels within the mPFC have effects on consummatory behavior, we made infusions of several muscarinic drugs (scopolamine, oxotremorine, physostigmine), the KCNQ channel blocker XE-991, and ghrelin into the mPFC and evaluated their effects on consummatory behavior. A consistent finding across all drugs was an effect on the duration of licking bouts when animals consume solutions with a relatively high concentration of sucrose. The muscarinic antagonist scopolamine reduced bout durations, both systemically and intra-cortically. By contrast, the muscarinic agonist oxotremorine, the cholinesterase inhibitor physostigmine, the KCNQ channel blocker XE-991, and ghrelin all increased the durations of licking bouts when infused into the mPFC. Our findings suggest that cholinergic and ghrelinergic signaling in the mPFC, acting through KCNQ channels, regulates the expression of palatability.

  12. Signaling through G protein coupled receptors.

    PubMed

    Tuteja, Narendra

    2009-10-01

    Heterotrimeric G proteins (Galpha, Gbeta/Ggamma subunits) constitute one of the most important components of cell signaling cascade. G Protein Coupled Receptors (GPCRs) perceive many extracellular signals and transduce them to heterotrimeric G proteins, which further transduce these signals intracellular to appropriate downstream effectors and thereby play an important role in various signaling pathways. GPCRs exist as a superfamily of integral membrane protein receptors that contain seven transmembrane alpha-helical regions, which bind to a wide range of ligands. Upon activation by a ligand, the GPCR undergoes a conformational change and then activate the G proteins by promoting the exchange of GDP/GTP associated with the Galpha subunit. This leads to the dissociation of Gbeta/Ggamma dimer from Galpha. Both these moieties then become free to act upon their downstream effectors and thereby initiate unique intracellular signaling responses. After the signal propagation, the GTP of Galpha-GTP is hydrolyzed to GDP and Galpha becomes inactive (Galpha-GDP), which leads to its re-association with the Gbeta/Ggamma dimer to form the inactive heterotrimeric complex. The GPCR can also transduce the signal through G protein independent pathway. GPCRs also regulate cell cycle progression. Till to date thousands of GPCRs are known from animal kingdom with little homology among them, but only single GPCR has been identified in plant system. The Arabidopsis GPCR was reported to be cell cycle regulated and also involved in ABA and in stress signaling. Here I have described a general mechanism of signal transduction through GPCR/G proteins, structure of GPCRs, family of GPCRs and plant GPCR and its role.

  13. Signal Diversity of Receptor for Advanced Glycation End Products.

    PubMed

    Sakaguchi, Masakiyo; Kinoshita, Rie; Putranto, Endy Widya; Ruma, I Made Winarsa; Sumardika, I Wayan; Youyi, Chen; Tomonobu, Naoko; Yamamoto, Ken-Ichi; Murata, Hitoshi

    2017-12-01

    The receptor for advanced glycation end products (RAGE) is involved in inflammatory pathogenesis. It functions as a receptor to multiple ligands such as AGEs, HMGB1 and S100 proteins, activating multiple intracellular signaling pathways with each ligand binding. The molecular events by which ligand-activated RAGE controls diverse signaling are not well understood, but some progress was made recently. Accumulating evidence revealed that RAGE has multiple binding partners within the cytoplasm and on the plasma membrane. It was first pointed out in 2008 that RAGE's cytoplasmic tail is able to recruit Diaphanous-1 (Dia-1), resulting in the acquisition of increased cellular motility through Rac1/Cdc42 activation. We also observed that within the cytosol, RAGE's cytoplasmic tail behaves similarly to a Toll-like receptor (TLR4)-TIR domain, interacting with TIRAP and MyD88 adaptor molecules that in turn activate multiple downstream signals. Subsequent studies demonstrated the presence of an alternative adaptor molecule, DAP10, on the plasma membrane. The coupling of RAGE with DAP10 is critical for enhancing the RAGE-mediated survival signal. Interestingly, RAGE interaction on the membrane was not restricted to DAP10 alone. The chemotactic G-protein-coupled receptors (GPCRs) formyl peptide receptors1 and 2 (FPR1 and FPR2) also interacted with RAGE on the plasma membrane. Binding interaction between leukotriene B4 receptor 1 (BLT1) and RAGE was also demonstrated. All of the interactions affected the RAGE signal polarity. These findings indicate that functional interactions between RAGE and various molecules within the cytoplasmic area or on the membrane area coordinately regulate multiple ligand-mediated RAGE responses, leading to typical cellular phenotypes in several pathological settings. Here we review RAGE's signaling diversity, to contribute to the understanding of the elaborate functions of RAGE in physiological and pathological contexts.

  14. Dopamine D2-like receptor signaling suppresses human osteoclastogenesis.

    PubMed

    Hanami, Kentaro; Nakano, Kazuhisa; Saito, Kazuyoshi; Okada, Yosuke; Yamaoka, Kunihiro; Kubo, Satoshi; Kondo, Masahiro; Tanaka, Yoshiya

    2013-09-01

    Dopamine, a major neurotransmitter, transmits signals via five different seven-transmembrane G protein-coupled receptors termed D1 to D5. Although the relevance of neuroendocrine system to bone metabolism has been emerging, the precise effects of dopaminergic signaling upon osteoclastogenesis remain unknown. Here, we demonstrate that human monocyte-derived osteoclast precursor cells express all dopamine-receptor subtypes. Dopamine and dopamine D2-like receptor agonists such as pramipexole and quinpirole reduced the formation of TRAP-positive multi-nucleated cells, cathepsin K mRNA expression, and pit formation area in vitro. These inhibitory effects were reversed by pre-treatment with a D2-like receptor antagonist haloperidol or a Gαi inhibitor pertussis toxin, but not with the D1-like receptor antagonist SCH-23390. Dopamine and dopamine D2-like receptor agonists, but not a D1-like receptor agonist, suppressed intracellular cAMP concentration as well as RANKL-meditated induction of c-Fos and NFATc1 mRNA expression in human osteoclast precursor cells. Finally, the dopamine D2-like receptor agonist suppressed LPS-induced osteoclast formation in murine bone marrow culture ex vivo. These findings indicate that dopaminergic signaling plays an important role in bone homeostasis via direct effects upon osteoclast differentiation and further suggest that the clinical use of neuroleptics is likely to affect bone mass. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. Biased signaling of the proton-sensing receptor OGR1 by benzodiazepines.

    PubMed

    Pera, Tonio; Deshpande, Deepak A; Ippolito, Michael; Wang, Bin; Gavrila, Adelina; Michael, James V; Nayak, Ajay P; Tompkins, Eric; Farrell, Eleni; Kroeze, Wesley K; Roth, Bryan L; Panettieri, Reynold A; Benovic, Jeffrey L; An, Steven S; Dulin, Nickolai O; Penn, Raymond B

    2018-02-01

    GPCRs have diverse signaling capabilities, based on their ability to assume various conformations. Moreover, it is now appreciated that certain ligands can promote distinct receptor conformations and thereby bias signaling toward a specific pathway to differentially affect cell function. The recently deorphanized G protein-coupled receptor OGR1 [ovarian cancer G protein-coupled receptor 1 ( GPR68)] exhibits diverse signaling events when stimulated by reductions in extracellular pH. We recently demonstrated airway smooth muscle cells transduce multiple signaling events, reflecting a diverse capacity to couple to multiple G proteins. Moreover, we recently discovered that the benzodiazepine lorazepam, more commonly recognized as an agonist of the γ-aminobutyric acid A (GABA A ) receptor, can function as an allosteric modulator of OGR1 and, similarly, can promote multiple signaling events. In this study, we demonstrated that different benzodiazepines exhibit a range of biases for OGR1, with sulazepam selectively activating the canonical Gs of the G protein signaling pathway, in heterologous expression systems, as well as in several primary cell types. These findings highlight the potential power of biased ligand pharmacology for manipulating receptor signaling qualitatively, to preferentially activate pathways that are therapeutically beneficial.-Pera, T., Deshpande, D. A., Ippolito, M., Wang, B., Gavrila, A., Michael, J. V., Nayak, A. P., Tompkins, E., Farrell, E., Kroeze, W. K., Roth, B. L., Panettieri, R. A. Jr Benovic, J. L., An, S. S., Dulin, N. O., Penn, R. B. Biased signaling of the proton-sensing receptor OGR1 by benzodiazepines.

  16. Receptor Heteromerization Expands the Repertoire of Cannabinoid Signaling in Rodent Neurons

    PubMed Central

    Rozenfeld, Raphael; Bushlin, Ittai; Gomes, Ivone; Tzavaras, Nikos; Gupta, Achla; Neves, Susana; Battini, Lorenzo; Gusella, G. Luca; Lachmann, Alexander; Ma'ayan, Avi; Blitzer, Robert D.; Devi, Lakshmi A.

    2012-01-01

    A fundamental question in G protein coupled receptor biology is how a single ligand acting at a specific receptor is able to induce a range of signaling that results in a variety of physiological responses. We focused on Type 1 cannabinoid receptor (CB1R) as a model GPCR involved in a variety of processes spanning from analgesia and euphoria to neuronal development, survival and differentiation. We examined receptor dimerization as a possible mechanism underlying expanded signaling responses by a single ligand and focused on interactions between CB1R and delta opioid receptor (DOR). Using co-immunoprecipitation assays as well as analysis of changes in receptor subcellular localization upon co-expression, we show that CB1R and DOR form receptor heteromers. We find that heteromerization affects receptor signaling since the potency of the CB1R ligand to stimulate G-protein activity is increased in the absence of DOR, suggesting that the decrease in CB1R activity in the presence of DOR could, at least in part, be due to heteromerization. We also find that the decrease in activity is associated with enhanced PLC-dependent recruitment of arrestin3 to the CB1R-DOR complex, suggesting that interaction with DOR enhances arrestin-mediated CB1R desensitization. Additionally, presence of DOR facilitates signaling via a new CB1R-mediated anti-apoptotic pathway leading to enhanced neuronal survival. Taken together, these results support a role for CB1R-DOR heteromerization in diversification of endocannabinoid signaling and highlight the importance of heteromer-directed signal trafficking in enhancing the repertoire of GPCR signaling. PMID:22235275

  17. Asparagine, valine, and threonine in the third extracellular loop of muscarinic receptor have essential roles in the positive cooperativity of strychnine-like allosteric modulators.

    PubMed

    Jakubík, J; Krejcí, A; Dolezal, V

    2005-05-01

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

  18. Human GH Receptor-IGF-1 Receptor Interaction: Implications for GH Signaling

    PubMed Central

    Gan, Yujun; Buckels, Ashiya; Liu, Ying; Zhang, Yue; Paterson, Andrew J.; Jiang, Jing; Zinn, Kurt R.

    2014-01-01

    GH signaling yields multiple anabolic and metabolic effects. GH binds the transmembrane GH receptor (GHR) to activate the intracellular GHR-associated tyrosine kinase, Janus kinase 2 (JAK2), and downstream signals, including signal transducer and activator of transcription 5 (STAT5) activation and IGF-1 gene expression. Some GH effects are partly mediated by GH-induced IGF-1 via IGF-1 receptor (IGF-1R), a tyrosine kinase receptor. We previously demonstrated in non-human cells that GH causes formation of a GHR-JAK2-IGF-1R complex and that presence of IGF-1R (even without IGF-1 binding) augments proximal GH signaling. In this study, we use human LNCaP prostate cancer cells as a model system to further study the IGF-1R's role in GH signaling. GH promoted JAK2 and GHR tyrosine phosphorylation and STAT5 activation in LNCaP cells. By coimmunoprecipitation and a new split luciferase complementation assay, we find that GH augments GHR/IGF-1R complex formation, which is inhibited by a Fab of an antagonistic anti-GHR monoclonal antibody. Short hairpin RNA-mediated IGF-1R silencing in LNCaP cells reduced GH-induced GHR, JAK2, and STAT5 phosphorylation. Similarly, a soluble IGF-1R extracellular domain fragment (sol IGF-1R) interacts with GHR in response to GH and blunts GH signaling. Sol IGF-1R also markedly inhibits GH-induced IGF-1 gene expression in both LNCaP cells and mouse primary osteoblast cells. On the basis of these and other findings, we propose a model in which IGF-1R augments GH signaling by allowing a putative IGF-1R-associated molecule that regulates GH signaling to access the activated GHR/JAK2 complex and envision sol IGF-1R as a dominant-negative inhibitor of this IGF-1R-mediated augmentation. Physiological implications of this new model are discussed. PMID:25211187

  19. The Growth Hormone Receptor: Mechanism of Receptor Activation, Cell Signaling, and Physiological Aspects

    PubMed Central

    Dehkhoda, Farhad; Lee, Christine M. M.; Medina, Johan; Brooks, Andrew J.

    2018-01-01

    The growth hormone receptor (GHR), although most well known for regulating growth, has many other important biological functions including regulating metabolism and controlling physiological processes related to the hepatobiliary, cardiovascular, renal, gastrointestinal, and reproductive systems. In addition, growth hormone signaling is an important regulator of aging and plays a significant role in cancer development. Growth hormone activates the Janus kinase (JAK)–signal transducer and activator of transcription (STAT) signaling pathway, and recent studies have provided a new understanding of the mechanism of JAK2 activation by growth hormone binding to its receptor. JAK2 activation is required for growth hormone-mediated activation of STAT1, STAT3, and STAT5, and the negative regulation of JAK–STAT signaling comprises an important step in the control of this signaling pathway. The GHR also activates the Src family kinase signaling pathway independent of JAK2. This review covers the molecular mechanisms of GHR activation and signal transduction as well as the physiological consequences of growth hormone signaling. PMID:29487568

  20. Glutamate Delta-1 Receptor Regulates Metabotropic Glutamate Receptor 5 Signaling in the Hippocampus.

    PubMed

    Suryavanshi, Pratyush S; Gupta, Subhash C; Yadav, Roopali; Kesherwani, Varun; Liu, Jinxu; Dravid, Shashank M

    2016-08-01

    The delta family of ionotropic glutamate receptors consists of glutamate delta-1 (GluD1) and glutamate delta-2 receptors. We have previously shown that GluD1 knockout mice exhibit features of developmental delay, including impaired spine pruning and switch in the N-methyl-D-aspartate receptor subunit, which are relevant to autism and other neurodevelopmental disorders. Here, we identified a novel role of GluD1 in regulating metabotropic glutamate receptor 5 (mGlu5) signaling in the hippocampus. Immunohistochemical analysis demonstrated colocalization of mGlu5 with GluD1 punctas in the hippocampus. Additionally, GluD1 protein coimmunoprecipitated with mGlu5 in the hippocampal membrane fraction, as well as when overexpressed in human embryonic kidney 293 cells, demonstrating that GluD1 and mGlu5 may cooperate in a signaling complex. The interaction of mGlu5 with scaffold protein effector Homer, which regulates mechanistic target of rapamycin (mTOR) signaling, was abnormal both under basal conditions and in response to mGlu1/5 agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) in GluD1 knockout mice. The basal levels of phosphorylated mTOR and protein kinase B, the signaling proteins downstream of mGlu5 activation, were higher in GluD1 knockout mice, and no further increase was induced by DHPG. We also observed higher basal protein translation and an absence of DHPG-induced increase in GluD1 knockout mice. In accordance with a role of mGlu5-mediated mTOR signaling in synaptic plasticity, DHPG-induced internalization of surface α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunits was impaired in the GluD1 knockout mice. These results demonstrate that GluD1 interacts with mGlu5, and loss of GluD1 impairs normal mGlu5 signaling potentially by dysregulating coupling to its effector. These studies identify a novel role of the enigmatic GluD1 subunit in hippocampal function. Copyright © 2016 by The American Society for Pharmacology and Experimental

  1. Differential Regulation of Primary Afferent Input to Spinal Cord by Muscarinic Receptor Subtypes Delineated Using Knockout Mice*

    PubMed Central

    Chen, Shao-Rui; Chen, Hong; Yuan, Wei-Xiu; Wess, Jürgen; Pan, Hui-Lin

    2014-01-01

    Stimulation of muscarinic acetylcholine receptors (mAChRs) inhibits nociceptive transmission at the spinal level. However, it is unclear how each mAChR subtype regulates excitatory synaptic input from primary afferents. Here we examined excitatory postsynaptic currents (EPSCs) of dorsal horn neurons evoked by dorsal root stimulation in spinal cord slices from wild-type and mAChR subtype knock-out (KO) mice. In wild-type mice, mAChR activation with oxotremorine-M decreased the amplitude of monosynaptic EPSCs in ∼67% of neurons but increased it in ∼10% of neurons. The inhibitory effect of oxotremorine-M was attenuated by the M2/M4 antagonist himbacine in the majority of neurons, and the remaining inhibition was abolished by group II/III metabotropic glutamate receptor (mGluR) antagonists in wild-type mice. In M2/M4 double-KO mice, oxotremorine-M inhibited monosynaptic EPSCs in significantly fewer neurons (∼26%) and increased EPSCs in significantly more neurons (33%) compared with wild-type mice. Blocking group II/III mGluRs eliminated the inhibitory effect of oxotremorine-M in M2/M4 double-KO mice. In M2 single-KO and M4 single-KO mice, himbacine still significantly reduced the inhibitory effect of oxotremorine-M. However, the inhibitory and potentiating effects of oxotremorine-M on EPSCs in M3 single-KO and M1/M3 double-KO mice were similar to those in wild-type mice. In M5 single-KO mice, oxotremorine-M failed to potentiate evoked EPSCs, and its inhibitory effect was abolished by himbacine. These findings indicate that activation of presynaptic M2 and M4 subtypes reduces glutamate release from primary afferents. Activation of the M5 subtype either directly increases primary afferent input or inhibits it through indirectly stimulating group II/III mGluRs. PMID:24695732

  2. Mitogenic signaling of urokinase receptor-deficient kidney fibroblasts: actions of an alternative urokinase receptor and LDL receptor-related protein.

    PubMed

    Zhang, Guoqiang; Cai, Xiaohe; López-Guisa, Jesús M; Collins, Sarah J; Eddy, Allison A

    2004-08-01

    The urokinase receptor (uPAR) attenuates myofibroblast recruitment and fibrosis in the kidney. This study examined the role of uPAR and its co-receptor LDL receptor-related protein (LRP) in the regulation of kidney fibroblast proliferation and extracellular signal-regulated kinase (ERK) signaling. Compared with uPAR+/+ cells, uPAR-/- kidney fibroblasts were hyperproliferative. UPAR-/- fibroblast proliferation was 60% inhibited by an ERK kinase inhibitor. LRP protein was reduced and extracellular accumulation of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) proteins were greater in uPAR-/- cultures. Addition of functional uPA protein or LRP antisense RNA significantly increased ERK signaling and cell mitosis in both genotypes. Enhanced uPAR-/- fibroblast proliferation was reversed by a recombinant nonfunctional uPA peptide. The density of cell-bound fluor-uPA was similar between uPAR-/- and uPAR+/+ fibroblasts (78 +/- 6 versus 92 +/- 16 units). These data suggest that uPAR-deficient kidney fibroblasts express lower levels of its scavenger co-receptor LRP, resulting in greater extracellular accumulation of uPA and PAI-1. Enhanced proliferation of uPAR-/- fibroblasts seems to be mediated by uPA-dependent ERK signaling via an alternative urokinase receptor.

  3. The M sub 1 muscarinic receptor and its second messenger coupling in human neuroblastoma cells and transfected murine fibroblast cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mei, Lin.

    1989-01-01

    The data of this study indicate that pirenzepine (PZ)-high affinity muscarinic receptors (mAChRs) are coupled to the hydrolysis of inositol lipids and not to the adenylate cyclase system in human neuroblastoma SH-SY5Y cells. The maximal carbachol(CCh)-stimulated ({sup 3}H)IP{sub 1} accumulation in the SH-SY5Y cells was decreased in the presence of 1{mu}g/ml pertussis toxin, suggesting that a pertussis toxin sensitive G-protein may be involved in the coupling. Several cell clones which express only M{sub 1} mAChR were generated by transfecting the murine fibroblast B82 cells with the cloned rat genomic m{sub 1} gene. The transfected B82 cells (cTB10) showed specific ({supmore » 3}H)(-)QNB binding activity. The mAChRs in these cells are of the M{sub 1} type defined by their high affinity for PZ and low affinity for AF-DX 116 and coupled to hydrolysis of inositol lipids, possibly via a pertussis toxin sensitive G protein. The relationship between the M{sub 1} mAChR density and the receptor-mediated hydrolysis of inositol lipids was studied in 7 clones. The M{sub 1} mAChR densities in these cells characterized by ({sup 3}H)(-)MQNB binding ranged from 12 fmol/10{sup 6} cells in LK3-1 cells to 260 fmol/10{sup 6} cells in the LK3-8 cells.« less

  4. Role of the nicotinic acetylcholine receptor in Alzheimer's disease pathology and treatment.

    PubMed

    Lombardo, Sylvia; Maskos, Uwe

    2015-09-01

    Alzheimer's Disease (AD) is the major form of senile dementia, characterized by neuronal loss, extracellular deposits, and neurofibrillary tangles. It is accompanied by a loss of cholinergic tone, and acetylcholine (ACh) levels in the brain, which were hypothesized to be responsible for the cognitive decline observed in AD. Current medication is restricted to enhancing cholinergic signalling for symptomatic treatment of AD patients. The nicotinic acetylcholine receptor family (nAChR) and the muscarinic acetylcholine receptor family (mAChR) are the target of ACh in the brain. Both families of receptors are affected in AD. It was demonstrated that amyloid beta (Aβ) interacts with nAChRs. Here we discuss how Aβ activates or inhibits nAChRs, and how this interaction contributes to AD pathology. We will discuss the potential role of nAChRs as therapeutic targets. This article is part of the Special Issue entitled 'The Nicotinic Acetylcholine Receptor: From Molecular Biology to Cognition'. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. Distinct interneuron types express m2 muscarinic receptor immunoreactivity on their dendrites or axon terminals in the hippocampus.

    PubMed

    Hájos, N; Papp, E C; Acsády, L; Levey, A I; Freund, T F

    1998-01-01

    In previous studies m2 muscarinic acetylcholine receptor-immunoreactive interneurons and various types of m2-positive axon terminals have been described in the hippocampal formation. The aim of the present study was to identify the types of interneurons expressing m2 receptor and to examine whether the somadendritic and axonal m2 immunostaining labels the same or distinct cell populations. In the CA1 subfield, neurons immunoreactive for m2 have horizontal dendrites, they are located at the stratum oriens/alveus border and have an axon that project to the dendritic region of pyramidal cells. In the CA3 subfield and the hilus, m2-positive neurons are multipolar and are scattered in all layers except stratum lacunosum-moleculare. In stratum pyramidale of the CA1 and CA3 regions, striking axon terminal staining for m2 was observed, surrounding the somata and axon initial segments of pyramidal cells in a basket-like manner. The co-localization of m2 with neurochemical markers and GABA was studied using the "mirror" technique and fluorescent double-immunostaining at the light microscopic level and with double-labelling using colloidal gold-conjugated antisera and immunoperoxidase reaction (diaminobenzidine) at the electron microscopic level. GABA was shown to be present in the somata of most m2-immunoreactive interneurons, as well as in the majority of m2-positive terminals in all layers. The calcium-binding protein parvalbumin was absent from practically all m2-immunoreactive cell bodies and dendrites. In contrast, many of the terminals synapsing on pyramidal cell somata and axon initial segments co-localized parvalbumin and m2, suggesting a differential distribution of m2 receptor immunoreactivity on the axonal and somadendritic membrane of parvalbumin-containing basket and axo-axonic cells. The co-existence of m2 receptors with the calcium-binding protein calbindin and the neuropeptides cholecystokinin and vasoactive intestinal polypeptide was rare throughout the

  6. Differential effects of m1 and m2 receptor antagonists in perirhinal cortex on visual recognition memory in monkeys

    PubMed Central

    Wu, Wei; Saunders, Richard C.; Mishkin, Mortimer; Turchi, Janita

    2012-01-01

    Microinfusions of the nonselective muscarinic antagonist scopolamine into perirhinal cortex impairs performance on visual recognition tasks, indicating that muscarinic receptors in this region play a pivotal role in recognition memory. To assess the mnemonic effects of selective blockade in perirhinal cortex of muscarinic receptor subtypes, we locally infused either the m1-selective antagonist pirenzepine or the m2-selective antagonist methoctramine in animals performing one-trial visual recognition, and compared these scores with those following infusions of equivalent volumes of saline. Compared to these control infusions, injections of pirenzepine, but not of methoctramine, significantly impaired recognition accuracy. Further, similar doses of scopolamine and pirenzepine yielded similar deficits, suggesting that the deficits obtained earlier with scopolamine were due mainly, if not exclusively, to blockade of m1 receptors. The present findings indicate that m1 and m2 receptors have functionally dissociable roles, and that the formation of new visual memories is critically dependent on the cholinergic activation of m1 receptors located on perirhinal cells. PMID:22561485

  7. Differential effects of m1 and m2 receptor antagonists in perirhinal cortex on visual recognition memory in monkeys.

    PubMed

    Wu, Wei; Saunders, Richard C; Mishkin, Mortimer; Turchi, Janita

    2012-07-01

    Microinfusions of the nonselective muscarinic antagonist scopolamine into perirhinal cortex impairs performance on visual recognition tasks, indicating that muscarinic receptors in this region play a pivotal role in recognition memory. To assess the mnemonic effects of selective blockade in perirhinal cortex of muscarinic receptor subtypes, we locally infused either the m1-selective antagonist pirenzepine or the m2-selective antagonist methoctramine in animals performing one-trial visual recognition, and compared these scores with those following infusions of equivalent volumes of saline. Compared to these control infusions, injections of pirenzepine, but not of methoctramine, significantly impaired recognition accuracy. Further, similar doses of scopolamine and pirenzepine yielded similar deficits, suggesting that the deficits obtained earlier with scopolamine were due mainly, if not exclusively, to blockade of m1 receptors. The present findings indicate that m1 and m2 receptors have functionally dissociable roles, and that the formation of new visual memories is critically dependent on the cholinergic activation of m1 receptors located on perirhinal cells. Published by Elsevier Inc.

  8. Co-Expression of Regulator of G Protein Signaling 4 (RGS4) and the MU Opioid Receptor in Regions of Rat Brain: Evidence That RGS4 Attenuates MU Opioid Receptor Signaling

    DTIC Science & Technology

    2003-01-01

    coupled receptor signal transduction proposes that agonist-induced conformational changes in the receptor result in an enhanced release of GDP...Regulators of G protein Signalling (RGS) proteins influence G protein-coupled receptor signal transduction by enhancing the intrinsic GTPase activity...of G proteins. The RGS- enhanced GTPase activity of G proteins may be responsible for the desensitization of certain G protein-coupled receptors

  9. Receptor density balances signal stimulation and attenuation in membrane-assembled complexes of bacterial chemotaxis signaling proteins

    PubMed Central

    Besschetnova, Tatiana Y.; Montefusco, David J.; Asinas, Abdalin E.; Shrout, Anthony L.; Antommattei, Frances M.; Weis, Robert M.

    2008-01-01

    All cells possess transmembrane signaling systems that function in the environment of the lipid bilayer. In the Escherichia coli chemotaxis pathway, the binding of attractants to a two-dimensional array of receptors and signaling proteins simultaneously inhibits an associated kinase and stimulates receptor methylation—a slower process that restores kinase activity. These two opposing effects lead to robust adaptation toward stimuli through a physical mechanism that is not understood. Here, we provide evidence of a counterbalancing influence exerted by receptor density on kinase stimulation and receptor methylation. Receptor signaling complexes were reconstituted over a range of defined surface concentrations by using a template-directed assembly method, and the kinase and receptor methylation activities were measured. Kinase activity and methylation rates were both found to vary significantly with surface concentration—yet in opposite ways: samples prepared at high surface densities stimulated kinase activity more effectively than low-density samples, whereas lower surface densities produced greater methylation rates than higher densities. FRET experiments demonstrated that the cooperative change in kinase activity coincided with a change in the arrangement of the membrane-associated receptor domains. The counterbalancing influence of density on receptor methylation and kinase stimulation leads naturally to a model for signal regulation that is compatible with the known logic of the E. coli pathway. Density-dependent mechanisms are likely to be general and may operate when two or more membrane-related processes are influenced differently by the two-dimensional concentration of pathway elements. PMID:18711126

  10. Generation and functional analysis of monoclonal antibodies against the second extracellular loop of human M3 muscarinic acetylcholine receptor.

    PubMed

    Tsuboi, Hiroto; Nakamura, Yumi; Iizuka, Mana; Matsuo, Naomi; Matsumoto, Isao; Sumida, Takayuki

    2012-04-01

    The M3 muscarinic acetylcholine receptor (M3R) plays a crucial role in the activation of salivary and lachrymal glands. The M3R contains four extracellular domains (the N-terminal, and the first, second, and third extracellular loops), and we recently detected antibodies against each of these four domains in a subgroup of patients with Sjögren's syndrome (SS). Functional analysis indicated that the influence of such anti-M3R antibodies on salivary secretion might differ based on the epitopes to which they bind. To clarify the relationship between B-cell epitopes on the M3R and its function, we generated two hybridomas producing anti-M3R monoclonal antibodies against the second extracellular loop of M3R (anti-M3R(2nd) mAbs) and analyzed their function by Ca(2+)-influx assays, using a human salivary gland (HSG) cell line. These two anti-M3R(2nd) mAbs suppressed Ca(2+)-influx in the HSG cells induced by cevimeline stimulation, suggesting that autoantibodies against the second extracellular loop of M3R could be involved in salivary dysfunction in patients with SS.

  11. Decreased microRNA levels lead to deleterious increases in neuronal M2 muscarinic receptors in Spinal Muscular Atrophy models

    PubMed Central

    O'Hern, Patrick J; do Carmo G. Gonçalves, Inês; Brecht, Johanna; López Soto, Eduardo Javier; Simon, Jonah; Chapkis, Natalie; Lipscombe, Diane; Kye, Min Jeong; Hart, Anne C

    2017-01-01

    Spinal Muscular Atrophy (SMA) is caused by diminished Survival of Motor Neuron (SMN) protein, leading to neuromuscular junction (NMJ) dysfunction and spinal motor neuron (MN) loss. Here, we report that reduced SMN function impacts the action of a pertinent microRNA and its mRNA target in MNs. Loss of the C. elegans SMN ortholog, SMN-1, causes NMJ defects. We found that increased levels of the C. elegans Gemin3 ortholog, MEL-46, ameliorates these defects. Increased MEL-46 levels also restored perturbed microRNA (miR-2) function in smn-1(lf) animals. We determined that miR-2 regulates expression of the C. elegans M2 muscarinic receptor (m2R) ortholog, GAR-2. GAR-2 loss ameliorated smn-1(lf) and mel-46(lf) synaptic defects. In an SMA mouse model, m2R levels were increased and pharmacological inhibition of m2R rescued MN process defects. Collectively, these results suggest decreased SMN leads to defective microRNA function via MEL-46 misregulation, followed by increased m2R expression, and neuronal dysfunction in SMA. DOI: http://dx.doi.org/10.7554/eLife.20752.001 PMID:28463115

  12. Role of M2 and M3 muscarinic acetylcholine receptor subtypes in activation of bladder afferent pathways in spinal cord injured rats.

    PubMed

    Matsumoto, Yoshihiro; Miyazato, Minoru; Yokoyama, Hitoshi; Kita, Masafumi; Hirao, Yoshihiko; Chancellor, Michael B; Yoshimura, Naoki

    2012-05-01

    To evaluate the role of M2 and M3 muscarinic acetylcholine receptor (mAChR) subtypes in the activation of bladder afferent pathways in rats with chronic spinal cord injury (SCI). Adult female Sprague-Dawley rats were spinalized at the T9 level. Continuous cystometry was performed under awake conditions 2 or 4 weeks after SCI. The effects of intravesical administration of an mAChR agonist (oxotremorine-methiodide), a nonselective antagonist (atropine), an M2-selective antagonist (methoctramine), and an M3-selective antagonist (darifenacin) were examined. After cystometry, the bladder was removed and separated into the mucosa and detrusor, and the M2 and M3 mAChR mRNA expression in the mucosa was determined using real-time quantitative polymerase chain reaction. At 2 and 4 weeks after SCI, intravesical administration of a nonselective mAChR agonist (25 μM oxotremorine-methiodide) increased the area under the curve of nonvoiding contractions, although the intercontraction interval of voiding contractions and maximal voiding pressure did not change. This effect was blocked by atropine and methoctramine (10 μM) but not by darifenacin (50 μM). However, mAChR antagonists alone (10-50 μM) had no effect on cystometric parameters. M2 mAChR mRNA expression was increased in the mucosa of SCI rats compared with that in normal rats. Our results suggest that the M2 mAChR subtype plays an important role in bladder afferent activation that enhances detrusor overactivity in SCI rats. However, because mAChR antagonists alone did not affect any cystometric parameters, the muscarinic mechanism controlling bladder afferent activity might not be involved in the emergence of detrusor overactivity in SCI. Copyright © 2012 Elsevier Inc. All rights reserved.

  13. Muscarinic receptor binding increases in anterior thalamus and cingulate cortex during discriminative avoidance learning

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Vogt, B.A.; Gabriel, M.; Vogt, L.J.

    Training-induced neuronal activity develops in the mammalian limbic system during discriminative avoidance conditioning. This study explores behaviorally relevant changes in muscarinic ACh receptor binding in 52 rabbits that were trained to one of five stages of conditioned response acquisition. Sixteen naive and 10 animals yoked to criterion performance served as control cases. Upon reaching a particular stage of training, the brains were removed and autoradiographically assayed for 3H-oxotremorine-M binding with 50 nM pirenzepine (OxO-M/PZ) or for 3H-pirenzepine binding in nine limbic thalamic nuclei and cingulate cortex. Specific OxO-M/PZ binding increased in the parvocellular division of the anterodorsal nucleus early inmore » training when the animals were first exposed to pairing of the conditional and unconditional stimuli. Elevated binding in this nucleus was maintained throughout subsequent training. In the parvocellular division of the anteroventral nucleus (AVp), OxO-M/PZ binding progressively increased throughout training, reached a peak at the criterion stage of performance, and returned to control values during extinction sessions. Peak OxO-M/PZ binding in AVp was significantly elevated over that for cases yoked to criterion performance. In the magnocellular division of the anteroventral nucleus (AVm), OxO-M/PZ binding was elevated only during criterion performance of the task, and it was unaltered in any other limbic thalamic nuclei. Specific OxO-M/PZ binding was also elevated in most layers in rostral area 29c when subjects first performed a significant behavioral discrimination. Training-induced alterations in OxO-M/PZ binding in AVp and layer Ia of area 29c were similar and highly correlated.« less

  14. Structural insights into μ-opioid receptor activation

    PubMed Central

    Huang, Weijiao; Manglik, Aashish; Venkatakrishnan, A. J.; Laeremans, Toon; Feinberg, Evan N.; Sanborn, Adrian L.; Kato, Hideaki E.; Livingston, Kathryn E.; Thorsen, Thor S.; Kling, Ralf; Granier, Sébastien; Gmeiner, Peter; Husbands, Stephen M.; Traynor, John R.; Weis, William I.; Steyaert, Jan; Dror, Ron O.; Kobilka, Brian K.

    2015-01-01

    Summary Activation of the μ-opioid receptor (μOR) is responsible for the efficacy of the most effective analgesics. To understand the structural basis for μOR activation, we obtained a 2.1 Å X-ray crystal structure of the μOR bound to the morphinan agonist BU72 and stabilized by a G protein-mimetic camelid-antibody fragment. The BU72-stabilized changes in the μOR binding pocket are subtle and differ from those observed for agonist-bound structures of the β2 adrenergic receptor (β2AR) and the M2 muscarinic receptor (M2R). Comparison with active β2AR reveals a common rearrangement in the packing of three conserved amino acids in the core of the μOR, and molecular dynamics simulations illustrate how the ligand-binding pocket is conformationally linked to this conserved triad. Additionally, an extensive polar network between the ligand-binding pocket and the cytoplasmic domains appears to play a similar role in signal propagation for all three GPCRs. PMID:26245379

  15. Regulation and signaling of human bombesin receptors and their biological effects.

    PubMed

    Weber, H Christian

    2009-02-01

    This review will highlight recent advances in the understanding of molecular mechanisms by which mammalian bombesin receptors are regulated and which intracellular signaling pathways have been characterized to mediate agonist-dependent receptor biological effects. Mammalian bombesin receptors have been demonstrated to be involved in a larger array of physiological and pathophysiological conditions than previously reported. Pharmacological experiments in vitro and in vivo as well as utilization of animals genetically deficient of the gastrin-releasing peptide receptor demonstrated roles in memory and fear behavior, lung development and injury, small intestinal cell repair, autocrine tumor growth, and mediating signals for pruritus and penile reflexes. Intracellular signaling studies predominantly of the gastrin-releasing peptide receptor owing to its frequent overexpression in some human malignancies showed that PI3 kinase activation is an important mechanism of cell proliferation. Tumor cell treatment including gastrin-releasing peptide receptor antagonists combined with inhibition of epidermal growth factor receptor resulted in an additive effect on blocking cell proliferation. Novel molecular mechanisms of the orphan bombesin receptor subtype-3 and gastrin-releasing peptide receptor gene regulation have been elucidated. Inhibition of gastrin-releasing peptide receptor signaling in human malignancies represents an attractive target for pharmacological treatment. Novel functions of bombesin related peptides have been identified including processes in the central nervous system, lung and intestinal tract.

  16. Tie2 and Eph Receptor Tyrosine Kinase Activation and Signaling

    PubMed Central

    Barton, William A.; Dalton, Annamarie C.; Seegar, Tom C.M.; Himanen, Juha P.

    2014-01-01

    The Eph and Tie cell surface receptors mediate a variety of signaling events during development and in the adult organism. As other receptor tyrosine kinases, they are activated on binding of extracellular ligands and their catalytic activity is tightly regulated on multiple levels. The Eph and Tie receptors display some unique characteristics, including the requirement of ligand-induced receptor clustering for efficient signaling. Interestingly, both Ephs and Ties can mediate different, even opposite, biological effects depending on the specific ligand eliciting the response and on the cellular context. Here we discuss the structural features of these receptors, their interactions with various ligands, as well as functional implications for downstream signaling initiation. The Eph/ephrin structures are already well reviewed and we only provide a brief overview on the initial binding events. We go into more detail discussing the Tie-angiopoietin structures and recognition. PMID:24478383

  17. Presynaptic membrane receptors in acetylcholine release modulation in the neuromuscular synapse.

    PubMed

    Tomàs, Josep; Santafé, Manel M; Garcia, Neus; Lanuza, Maria A; Tomàs, Marta; Besalduch, Núria; Obis, Teresa; Priego, Mercedes; Hurtado, Erica

    2014-05-01

    Over the past few years, we have studied, in the mammalian neuromuscular junction (NMJ), the local involvement in transmitter release of the presynaptic muscarinic ACh autoreceptors (mAChRs), purinergic adenosine autoreceptors (P1Rs), and trophic factor receptors (TFRs; for neurotrophins and trophic cytokines) during development and in the adult. At any given moment, the way in which a synapse works is largely the logical outcome of the confluence of these (and other) metabotropic signalling pathways on intracellular kinases, which phosphorylate protein targets and materialize adaptive changes. We propose an integrated interpretation of the complementary function of these receptors in the adult NMJ. The activity of a given receptor group can modulate a given combination of spontaneous, evoked, and activity-dependent release characteristics. For instance, P1Rs can conserve resources by limiting spontaneous quantal leak of ACh (an A1 R action) and protect synapse function, because stimulation with adenosine reduces the magnitude of depression during repetitive activity. The overall outcome of the mAChRs seems to contribute to upkeep of spontaneous quantal output of ACh, save synapse function by decreasing the extent of evoked release (mainly an M2 action), and reduce depression. We have also identified several links among P1Rs, mAChRs, and TFRs. We found a close dependence between mAChR and some TFRs and observed that the muscarinic group has to operate correctly if the tropomyosin-related kinase B receptor (trkB) is also to operate correctly, and vice versa. Likewise, the functional integrity of mAChRs depends on P1Rs operating normally. Copyright © 2014 Wiley Periodicals, Inc.

  18. RNA sequencing to determine the contribution of kinase receptor transactivation to G protein coupled receptor signalling in vascular smooth muscle cells.

    PubMed

    Kamato, Danielle; Bhaskarala, Venkata Vijayanand; Mantri, Nitin; Oh, Tae Gyu; Ling, Dora; Janke, Reearna; Zheng, Wenhua; Little, Peter J; Osman, Narin

    2017-01-01

    G protein coupled receptor (GPCR) signalling covers three major mechanisms. GPCR agonist engagement allows for the G proteins to bind to the receptor leading to a classical downstream signalling cascade. The second mechanism is via the utilization of the β-arrestin signalling molecule and thirdly via transactivation dependent signalling. GPCRs can transactivate protein tyrosine kinase receptors (PTKR) to activate respective downstream signalling intermediates. In the past decade GPCR transactivation dependent signalling was expanded to show transactivation of serine/threonine kinase receptors (S/TKR). Kinase receptor transactivation enormously broadens the GPCR signalling paradigm. This work utilizes next generation RNA-sequencing to study the contribution of transactivation dependent signalling to total protease activated receptor (PAR)-1 signalling. Transactivation, assessed as gene expression, accounted for 50 percent of the total genes regulated by thrombin acting through PAR-1 in human coronary artery smooth muscle cells. GPCR transactivation of PTKRs is approximately equally important as the transactivation of the S/TKR with 209 and 177 genes regulated respectively, via either signalling pathway. This work shows that genome wide studies can provide powerful insights into GPCR mediated signalling pathways.

  19. Impairment of skin barrier function via cholinergic signal transduction in a dextran sulphate sodium-induced colitis mouse model.

    PubMed

    Yokoyama, Satoshi; Hiramoto, Keiichi; Koyama, Mayu; Ooi, Kazuya

    2015-10-01

    Dry skin has been clinically associated with visceral diseases, including liver disease, as well as for our previously reported small intestinal injury mouse model, which have abnormalities in skin barrier function. To clarify this disease-induced skin disruption, we used a dextran sulphate sodium (DSS)-induced colitis mouse model. Following treatment with DSS, damage to the colon and skin was monitored using histological and protein analysis methods as well as the detection of inflammatory mediators in the plasma. Notably, transepidermal water loss was higher, and skin hydration was lower in DSS-treated mice compared to controls. Tumor necrosis factor-alpha (TNF-α), interleukin 6 and NO2-/NO3- levels were also upregulated in the plasma, and a decrease in body weight and colon length was observed in DSS-treated mice. However, when administered TNF-α antibody or an iNOS inhibitor, no change in skin condition was observed, indicating that another signalling mechanism is utilized. Interestingly, the number of tryptase-expressing mast cells, known for their role in immune function via cholinergic signal transduction, was elevated. To evaluate the function of cholinergic signalling in this context, atropine (a muscarinic cholinoceptor antagonist) or hexamethonium (a nicotinic cholinergic ganglion-blocking agent) was administered to DSS-treated mice. Our data indicate that muscarinic acetylcholine receptors (mAChRs) are the primary receptors functioning in colon-to-skin signal transduction, as DSS-induced skin disruption was suppressed by atropine. Thus, skin disruption is likely associated with DSS-induced colitis, and the activation of mast cells via mAChRs is critical to this association. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  20. Scopolamine rapidly increases mTORC1 signaling, synaptogenesis, and antidepressant behavioral responses

    PubMed Central

    Voleti, Bhavya; Navarria, Andrea; Liu, Rong-Jian; Banasr, Mounira; Li, Nanxin; Terwilliger, Rose; Sanacora, Gerard; Eid, Tore; Aghajanian, George; Duman, Ronald S.

    2013-01-01

    Background Clinical studies report that scopolamine, an acetylcholine muscarinic receptor antagonist, produces rapid antidepressant effects in depressed patients, but the mechanisms underlying the therapeutic response have not been determined. The present study examines the role of the mammalian target of rapamycin complex 1 (mTORC1) and synaptogenesis, which have been implicated in the rapid actions of NMDA receptor antagonists. Methods The influence of scopolamine on mTORC1 signaling was determined by analysis of the phosphorylated and activated forms of mTORC1 signaling proteins in the prefrontal cortex (PFC). The numbers and function of spine synapses were analyzed by whole cell patch clamp recording and 2-photon image analysis of PFC neurons. The actions of scopolamine were examined in the forced swim test in the absence or presence of selective mTORC1 and AMPA receptor inhibitors. Results The results demonstrate that a single, low dose of scopolamine rapidly increases mTORC1 signaling and the number and function of spine synapses in layer V pyramidal neurons in the PFC. Scopolamine administration also produces an antidepressant response in the forced swim test that is blocked by pretreatment with the mTORC1 inhibitor or by a glutamate AMPA receptor antagonist. Conclusions Taken together, the results demonstrate that the antidepressant actions of scopolamine require mTORC1 signaling and are associated with increased glutamate transmission, and synaptogenesis, similar to NMDA receptor antagonists. These findings provide novel targets for safer and more efficacious rapid acting antidepressant agents. PMID:23751205

  1. [Effects of dexamethasone on the expression of muscarinic receptor mRNA in asthmatic guinea pig airway smooth muscle and eosinophil infiltration in bronchoalveolar lavage fluid].

    PubMed

    Shi, Liang; Luo, Ya-ling; Lai, Wen-yan; Luo, Liang

    2005-08-01

    To investigate the effect of dexamethasone on the expression of muscarinic receptor (MR) mRNA in smooth muscle and infiltration of eosinophils (Eos) in the airway of asthmatic guinea pigs. Thirty healthy guinea pigs were randomized into 3 equal groups, the control group, asthmatic group and dexamethasone therapy group. Asthma was induced in the latter 2 groups with the asthma-inducing agents and received treatments as indicated. Bronchial alveolar lavage fluid(BALF) were collected subsequently from the guinea pigs for examining the total cell number and cell classification, and histopathologic examination of the lung tissue was performed. Semi-quantitative analysis with reverse transcriptional- polymerase chain reaction (RT-PCR) was performed for M(2) and M(3) receptor mRNA in airway smooth muscle. Compared with the control and the asthmatic group, the number of Eos in the BALF of dexamethasone therapy group was significantly lower (P<0.01). In spite of the presence of hyperemia and edema in the lung tissues of the dexamethasone therapy group, Eos infiltration was less severe than that in the asthmatic group. As found by RT-PCR, the quantity of M(2) receptor mRNA in the airway smooth muscle of the dexamethasone therapy group was significantly higher than those in both the control and asthmatic groups (P<0.01), and the quantity of M(3) receptor mRNA in the airway smooth muscle of dexamethasone therapy group was significantly higher than that in the asthmatic group, but did not significantly differ from that in the control group. The quantities of M(2) and M(3) receptor mRNAs in the control group were both significantly higher than that in asthmatic group (P<0.01). The expression of M(2) receptor is increased in antigen- challenged guinea pigs, and that of M(3) receptor decreased. Dexamethasone can treat asthma by inhibiting inflammatory action involving Eos infiltration, regulating the expressions of M(2) and M(3) receptors and restoring the function of M(2

  2. Functional autoantibodies targeting G protein-coupled receptors in rheumatic diseases.

    PubMed

    Cabral-Marques, Otavio; Riemekasten, Gabriela

    2017-11-01

    G protein-coupled receptors (GPCRs) comprise the largest and most diverse family of integral membrane proteins that participate in different physiological processes such as the regulation of the nervous and immune systems. Besides the endogenous ligands of GPCRs, functional autoantibodies are also able to bind GPCRs to trigger or block intracellular signalling pathways, resulting in agonistic or antagonistic effects, respectively. In this Review, the effects of functional GPCR-targeting autoantibodies on the pathogenesis of autoimmune diseases, including rheumatic diseases, are discussed. Autoantibodies targeting β1 and β2 adrenergic receptors, which are expressed by cardiac and airway smooth muscle cells, respectively, have an important role in the development of asthma and cardiovascular diseases. In addition, high levels of autoantibodies against the muscarinic acetylcholine receptor M3 as well as those targeting endothelin receptor type A and type 1 angiotensin II receptor have several implications in the pathogenesis of rheumatic diseases such as Sjögren syndrome and systemic sclerosis. Expanding the knowledge of the pathophysiological roles of autoantibodies against GPCRs will shed light on the biology of these receptors and open avenues for new therapeutic approaches.

  3. Location-Dependent Signaling of the Group 1 Metabotropic Glutamate Receptor mGlu5

    PubMed Central

    Jong, Yuh-Jiin I.; Sergin, Ismail; Purgert, Carolyn A.

    2014-01-01

    Although G protein–coupled receptors are primarily known for converting extracellular signals into intracellular responses, some receptors, such as the group 1 metabotropic glutamate receptor, mGlu5, are also localized on intracellular membranes where they can mediate both overlapping and unique signaling effects. Thus, besides “ligand bias,” whereby a receptor’s signaling modality can shift from G protein dependence to independence, canonical mGlu5 receptor signaling can also be influenced by “location bias” (i.e., the particular membrane and/or cell type from which it signals). Because mGlu5 receptors play important roles in both normal development and in disorders such as Fragile X syndrome, autism, epilepsy, addiction, anxiety, schizophrenia, pain, dyskinesias, and melanoma, a large number of drugs are being developed to allosterically target this receptor. Therefore, it is critical to understand how such drugs might be affecting mGlu5 receptor function on different membranes and in different brain regions. Further elucidation of the site(s) of action of these drugs may determine which signal pathways mediate therapeutic efficacy. PMID:25326002

  4. Prostaglandin E2 Inhibits Histamine-Evoked Ca2+ Release in Human Aortic Smooth Muscle Cells through Hyperactive cAMP Signaling Junctions and Protein Kinase A

    PubMed Central

    Taylor, Emily J. A.; Pantazaka, Evangelia; Shelley, Kathryn L.

    2017-01-01

    In human aortic smooth muscle cells, prostaglandin E2 (PGE2) stimulates adenylyl cyclase (AC) and attenuates the increase in intracellular free Ca2+ concentration evoked by activation of histamine H1 receptors. The mechanisms are not resolved. We show that cAMP mediates inhibition of histamine-evoked Ca2+ signals by PGE2. Exchange proteins activated by cAMP were not required, but the effects were attenuated by inhibition of cAMP-dependent protein kinase (PKA). PGE2 had no effect on the Ca2+ signals evoked by protease-activated receptors, heterologously expressed muscarinic M3 receptors, or by direct activation of inositol 1,4,5-trisphosphate (IP3) receptors by photolysis of caged IP3. The rate of Ca2+ removal from the cytosol was unaffected by PGE2, but PGE2 attenuated histamine-evoked IP3 accumulation. Substantial inhibition of AC had no effect on the concentration-dependent inhibition of Ca2+ signals by PGE2 or butaprost (to activate EP2 receptors selectively), but it modestly attenuated responses to EP4 receptors, activation of which generated less cAMP than EP2 receptors. We conclude that inhibition of histamine-evoked Ca2+ signals by PGE2 occurs through “hyperactive signaling junctions,” wherein cAMP is locally delivered to PKA at supersaturating concentrations to cause uncoupling of H1 receptors from phospholipase C. This sequence allows digital signaling from PGE2 receptors, through cAMP and PKA, to histamine-evoked Ca2+ signals. PMID:28877931

  5. Inhibition of allergen-induced basophil activation by ASM-024, a nicotinic receptor ligand.

    PubMed

    Watson, Brittany M; Oliveria, John Paul; Nusca, Graeme M; Smith, Steven G; Beaudin, Sue; Dua, Benny; Watson, Rick M; Assayag, Evelynne Israël; Cormier, Yvon F; Sehmi, Roma; Gauvreau, Gail M

    2014-01-01

    Nicotinic acetylcholine receptors (nAChRs) were identified on eosinophils and shown to regulate inflammatory responses, but nAChR expression on basophils has not been explored yet. We investigated surface receptor expression of nAChR α4, α7 and α1/α3/α5 subunits on basophils. Furthermore, we examined the effects of ASM-024, a synthetic nicotinic ligand, on in vitro anti-IgE and in vivo allergen-induced basophil activation. Basophils were enriched from the peripheral blood of allergic donors and the expression of nAChR subunits and muscarinic receptors was determined. Purified basophils were stimulated with anti-IgE in the presence of ASM-024 with or without muscarinic or nicotinic antagonists for the measurement of CD203c expression and histamine release. The effect of 9 days of treatment with 50 and 200 mg ASM-024 on basophil CD203c expression was examined in the blood of mild allergic asthmatics before and after allergen inhalation challenge. nAChR α4, α7 and α1/α3/α5 receptor subunit expression was detected on basophils. Stimulation of basophils with anti-IgE increased CD203c expression and histamine release, which was inhibited by ASM-024 (10(-5) to 10(-)(3) M, p < 0.05). The effect of ASM-024 was reversed in the presence of muscarinic and nicotinic antagonists. In subjects with mild asthma, ASM-024 inhalation significantly inhibited basophil CD203c expression measured 24 h after allergen challenge (p = 0.03). This study shows that ASM-024 inhibits IgE- and allergen-induced basophil activation through both nicotinic and muscarinic receptors, and suggests that ASM-024 may be an efficacious agent for modulating allergic asthma responses. © 2015 S. Karger AG, Basel.

  6. Research Resource: A Reference Transcriptome for Constitutive Androstane Receptor and Pregnane X Receptor Xenobiotic Signaling

    PubMed Central

    Ochsner, Scott A.; Tsimelzon, Anna; Dong, Jianrong; Coarfa, Cristian

    2016-01-01

    The pregnane X receptor (PXR) (PXR/NR1I3) and constitutive androstane receptor (CAR) (CAR/NR1I2) members of the nuclear receptor (NR) superfamily of ligand-regulated transcription factors are well-characterized mediators of xenobiotic and endocrine-disrupting chemical signaling. The Nuclear Receptor Signaling Atlas maintains a growing library of transcriptomic datasets involving perturbations of NR signaling pathways, many of which involve perturbations relevant to PXR and CAR xenobiotic signaling. Here, we generated a reference transcriptome based on the frequency of differential expression of genes across 159 experiments compiled from 22 datasets involving perturbations of CAR and PXR signaling pathways. In addition to the anticipated overrepresentation in the reference transcriptome of genes encoding components of the xenobiotic stress response, the ranking of genes involved in carbohydrate metabolism and gonadotropin action sheds mechanistic light on the suspected role of xenobiotics in metabolic syndrome and reproductive disorders. Gene Set Enrichment Analysis showed that although acetaminophen, chlorpromazine, and phenobarbital impacted many similar gene sets, differences in direction of regulation were evident in a variety of processes. Strikingly, gene sets representing genes linked to Parkinson's, Huntington's, and Alzheimer's diseases were enriched in all 3 transcriptomes. The reference xenobiotic transcriptome will be supplemented with additional future datasets to provide the community with a continually updated reference transcriptomic dataset for CAR- and PXR-mediated xenobiotic signaling. Our study demonstrates how aggregating and annotating transcriptomic datasets, and making them available for routine data mining, facilitates research into the mechanisms by which xenobiotics and endocrine-disrupting chemicals subvert conventional NR signaling modalities. PMID:27409825

  7. Research Resource: A Reference Transcriptome for Constitutive Androstane Receptor and Pregnane X Receptor Xenobiotic Signaling.

    PubMed

    Ochsner, Scott A; Tsimelzon, Anna; Dong, Jianrong; Coarfa, Cristian; McKenna, Neil J

    2016-08-01

    The pregnane X receptor (PXR) (PXR/NR1I3) and constitutive androstane receptor (CAR) (CAR/NR1I2) members of the nuclear receptor (NR) superfamily of ligand-regulated transcription factors are well-characterized mediators of xenobiotic and endocrine-disrupting chemical signaling. The Nuclear Receptor Signaling Atlas maintains a growing library of transcriptomic datasets involving perturbations of NR signaling pathways, many of which involve perturbations relevant to PXR and CAR xenobiotic signaling. Here, we generated a reference transcriptome based on the frequency of differential expression of genes across 159 experiments compiled from 22 datasets involving perturbations of CAR and PXR signaling pathways. In addition to the anticipated overrepresentation in the reference transcriptome of genes encoding components of the xenobiotic stress response, the ranking of genes involved in carbohydrate metabolism and gonadotropin action sheds mechanistic light on the suspected role of xenobiotics in metabolic syndrome and reproductive disorders. Gene Set Enrichment Analysis showed that although acetaminophen, chlorpromazine, and phenobarbital impacted many similar gene sets, differences in direction of regulation were evident in a variety of processes. Strikingly, gene sets representing genes linked to Parkinson's, Huntington's, and Alzheimer's diseases were enriched in all 3 transcriptomes. The reference xenobiotic transcriptome will be supplemented with additional future datasets to provide the community with a continually updated reference transcriptomic dataset for CAR- and PXR-mediated xenobiotic signaling. Our study demonstrates how aggregating and annotating transcriptomic datasets, and making them available for routine data mining, facilitates research into the mechanisms by which xenobiotics and endocrine-disrupting chemicals subvert conventional NR signaling modalities.

  8. An anti-nicotinic cognitive challenge model using mecamylamine in comparison with the anti-muscarinic cognitive challenge using scopolamine.

    PubMed

    Baakman, Anne Catrien; Alvarez-Jimenez, Ricardo; Rissmann, Robert; Klaassen, Erica S; Stevens, Jasper; Goulooze, Sebastiaan C; den Burger, Jeroen C G; Swart, Eleonora L; van Gerven, Joop M A; Groeneveld, Geert Jan

    2017-08-01

    The muscarinic acetylcholine receptor antagonist scopolamine is often used for proof-of-pharmacology studies with pro-cognitive compounds. From a pharmacological point of view, it would seem more rational to use a nicotinic rather than a muscarinic anticholinergic challenge to prove pharmacology of a nicotinic acetylcholine receptor agonist. This study aims to characterize a nicotinic anticholinergic challenge model using mecamylamine and to compare it to the scopolamine model. In this double-blind, placebo-controlled, four-way cross-over trial, 12 healthy male subjects received oral mecamylamine 10 and 20 mg, intravenous scopolamine 0.5 mg and placebo. Pharmacokinetics were analysed using non-compartmental analysis. Pharmacodynamic effects were measured with a multidimensional test battery that includes neurophysiological, subjective, (visuo)motor and cognitive measurements. All treatments were safe and well tolerated. Mecamylamine had a t max of 2.5 h and a C max of 64.5 ng ml -1 for the 20 mg dose. Mecamylamine had a dose-dependent effect decreasing the adaptive tracking performance and VAS alertness, and increasing the finger tapping and visual verbal learning task performance time and errors. Scopolamine significantly affected almost all pharmacodynamic tests. This study demonstrated that mecamylamine causes nicotinic receptor specific temporary decline in cognitive functioning. Compared with the scopolamine model, pharmacodynamic effects were less pronounced at the dose levels tested; however, mecamylamine caused less sedation. The cognitive effects of scopolamine might at least partly be caused by sedation. Whether the mecamylamine model can be used for proof-of-pharmacology of nicotinic acetylcholine receptor agonists remains to be established. © 2017 The British Pharmacological Society.

  9. Down-regulation of muscarinic receptors and the m3 subtype in white-footed mice by dietary exposure to parathion

    USGS Publications Warehouse

    Jett, David A.; Hill, E.F.; Fernando, J.C.; Eldefrawi, M.E.; Eldefrawi, A.T.

    1993-01-01

    The effect of ad libitum dietary exposure (as occurs in the field) to parathion for 14 d was investigated on the muscarinic acetylcholine receptor (mAChR) in brains and submaxillary glands of adults of a field species, the white-footed mouse Peromyscus leucopus. Immunoprecipitation using subtype selective antibodies revealed that the relative ratios of the m1-m5 mAChR subtypes in Peromyscus brain were similar to those in rat brain. There was little variability in acetylcholinesterase (AChE) activity in control mice brains but large variability in 39 exposed mice, resulting from differences in food ingestion and parathion metabolism. Accordingly, data on radioligand binding to mAChRs in each mouse brain were correlated with brain AChE activity in the same mouse, and AChE inhibition served as a biomarker of exposure reflecting in situ paraoxon concentrations. Exposure to parathion for 14 d reduced maximal binding (Bmax) of [3H]quinuclidinyl benzilate ([3H]QNB), [3H]-N-methylscopolamine ([3H]NMS), and [3H]-4-diphenylacetoxy-N-methylpiperidine methiodide ([3H]-4-DAMP) by up to approximately 58% without affecting receptor affinities for these ligands. Maximal reduction in Bmax of [3H]QNB and [3H]-4-DAMP binding occurred in mice with highest AChE inhibition, while equivalent maximal reduction in Bmax of [3H]NMS occurred in mice with only approximately 10% AChE inhibition, without further change at higher parathion doses. This is believed to be due to the hydrophilicity of [3H]NMS, which limits its accessibility to internalized desensitized receptors. In submaxillary glands (mAChRs are predominantly m3 subtype), there were significant dose-dependent reductions in [3H]QNB binding and m3 mRNA levels in exposed mice, revealed by Northern blot analyses. The reduction in m3 receptors is suggested to result mostly from reduced synthesis at the transcription level, rather than from translational or posttranslational events. The data suggest that down-regulation of mAChRs occurs

  10. Insights into the structural biology of G-protein coupled receptors impacts drug design for central nervous system neurodegenerative processes

    PubMed Central

    Dalet, Farfán-García Eunice; Guadalupe, Trujillo-Ferrara José; María del Carmen, Castillo-Hernández; Humberto, Guerra-Araiza Christian; Antonio, Soriano-Ursúa Marvin

    2013-01-01

    In the last few years, there have been important new insights into the structural biology of G-protein coupled receptors. It is now known that allosteric binding sites are involved in the affinity and selectivity of ligands for G-protein coupled receptors, and that signaling by these receptors involves both G-protein dependent and independent pathways. The present review outlines the physiological and pharmacological implications of this perspective for the design of new drugs to treat disorders of the central nervous system. Specifically, new possibilities are explored in relation to allosteric and orthosteric binding sites on dopamine receptors for the treatment of Parkinson's disease, and on muscarinic receptors for Alzheimer's disease. Future research can seek to identify ligands that can bind to more than one site on the same receptor, or simultaneously bind to two receptors and form a dimer. For example, the design of bivalent drugs that can reach homo/hetero-dimers of D2 dopamine receptor holds promise as a relevant therapeutic strategy for Parkinson's disease. Regarding the treatment of Alzheimer's disease, the design of dualsteric ligands for mono-oligomeric rinic receptors could increase therapeutic effectiveness by generating potent compounds that could activate more than one signaling pathway. PMID:25206539

  11. Immune signaling by RIG-I-like receptors

    PubMed Central

    Loo, Yueh-Ming; Gale, Michael

    2011-01-01

    The RIG-I-like receptors (RLRs) RIG-I, MDA5, and LGP2 play a major role in pathogen sensing of RNA virus infection to initiate and modulate antiviral immunity. The RLRs detect viral RNA ligands or processed self RNA in the cytoplasm to triggers innate immunity and inflammation and to impart gene expression that serves to control infection. Importantly, RLRs cooperate in signaling crosstalk networks with Toll-like receptors and other factors to impart innate immunity and to modulate the adaptive immune response. RLR regulation occurs at a variety of levels ranging from autoregulation to ligand and co-factor interactions and post-translational modifications. Abberant RLR signaling or dysregulation of RLR expression is now implicated in the development of autoimmune diseases. Understanding the processes of RLR signaling and response will provide insights to guide RLR-targeted therapeutics for antiviral and immune modifying applications. PMID:21616437

  12. Testin, a novel binding partner of the calcium-sensing receptor, enhances receptor-mediated Rho-kinase signalling

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Magno, Aaron L.; Department of Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Hospital Avenue, Nedlands, Western Australia 6009; Ingley, Evan

    Highlights: {yields} A yeast two-hybrid screen revealed testin bound to the calcium-sensing receptor. {yields} The second zinc finger of LIM domain 1 of testin is critical for interaction. {yields} Testin bound to a region of the receptor tail important for cell signalling. {yields} Testin and receptor interaction was confirmed in mammalian (HEK293) cells. {yields} Overexpression of testin enhanced receptor-mediated Rho signalling in HEK293 cells. -- Abstract: The calcium-sensing receptor (CaR) plays an integral role in calcium homeostasis and the regulation of other cellular functions including cell proliferation and cytoskeletal organisation. The multifunctional nature of the CaR is manifested through ligand-dependentmore » stimulation of different signalling pathways that are also regulated by partner binding proteins. Following a yeast two-hybrid library screen using the intracellular tail of the CaR as bait, we identified several novel binding partners including the focal adhesion protein, testin. Testin has not previously been shown to interact with cell surface receptors. The sites of interaction between the CaR and testin were mapped to the membrane proximal region of the receptor tail and the second zinc-finger of LIM domain 1 of testin, the integrity of which was found to be critical for the CaR-testin interaction. The CaR-testin association was confirmed in HEK293 cells by coimmunoprecipitation and confocal microscopy studies. Ectopic expression of testin in HEK293 cells stably expressing the CaR enhanced CaR-stimulated Rho activity but had no effect on CaR-stimulated ERK signalling. These results suggest an interplay between the CaR and testin in the regulation of CaR-mediated Rho signalling with possible effects on the cytoskeleton.« less

  13. Role of CRF Receptor Signaling in Stress Vulnerability, Anxiety, and Depression

    PubMed Central

    Hauger, Richard L.; Risbrough, Victoria; Oakley, Robert H.; Olivares-Reyes, J. Alberto; Dautzenberg, Frank M.

    2011-01-01

    Markers of hyperactive central corticotropin releasing factor (CRF) systems and CRF-related single nucleotide polymorphisms (SNPs) have been identified in patients with anxiety and depressive disorders. Designing more effective antagonists may now be guided by data showing that small molecules bind to transmembrane domains. Specifically, CRF1 receptor antagonists have been developed as novel anxiolytic and antidepressant treatments. Because CRF1 receptors become rapidly desensitized by G protein-coupled receptor kinase (GRK) and β-arrestin mechanisms in the presence of high agonist concentrations, neuronal hypersecretion of synaptic CRF alone may be insufficient to account for excessive central CRF neurotransmission in stress-induced affective pathophysiology. In addition to desensitizing receptor function, GRK phosphorylation and β-arrestin binding can shift a G protein-coupled receptor (GPCR) to signal selectively via the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK-MAPK) or Akt pathways independent of G proteins. Also, Epac-dependent CRF1 receptor signaling via the ERK-MAPK pathway has been found to potentiate brain-derived neurotrophic factor (BDNF)-stimulated TrkB signaling. Thus, genetic or acquired abnormalities in GRK and β-arrestin function may be involved in the pathophysiology of stress-induced anxiety and depression. PMID:19906236

  14. Behavioral Consequences of Neurotransmitter Receptor Regulation.

    DTIC Science & Technology

    1987-10-13

    with the agonist, oxotremorine . This agent produced a reduction in muscarinic receptors in cortex and hippocampus and receptors did not return to...control levels for at least 4-8 days after cessation of treatment. Tolerance to oxotremorine developed as a result of chronic treatment. At 24 hrs after...challenge dose of oxotremorine was lost by 48 hrs after cessation of treatment. Likewise, animals that began acquisition training in the spatial

  15. RGS6, but not RGS4, is the dominant regulator of G protein signaling (RGS) modulator of the parasympathetic regulation of mouse heart rate.

    PubMed

    Wydeven, Nicole; Posokhova, Ekaterina; Xia, Zhilian; Martemyanov, Kirill A; Wickman, Kevin

    2014-01-24

    Parasympathetic activity decreases heart rate (HR) by inhibiting pacemaker cells in the sinoatrial node (SAN). Dysregulation of parasympathetic influence has been linked to sinus node dysfunction and arrhythmia. RGS (regulator of G protein signaling) proteins are negative modulators of the parasympathetic regulation of HR and the prototypical M2 muscarinic receptor (M2R)-dependent signaling pathway in the SAN that involves the muscarinic-gated atrial K(+) channel IKACh. Both RGS4 and RGS6-Gβ5 have been implicated in these processes. Here, we used Rgs4(-/-), Rgs6(-/-), and Rgs4(-/-):Rgs6(-/-) mice to compare the relative influence of RGS4 and RGS6 on parasympathetic regulation of HR and M2R-IKACh-dependent signaling in the SAN. In retrogradely perfused hearts, ablation of RGS6, but not RGS4, correlated with decreased resting HR, increased heart rate variability, and enhanced sensitivity to the negative chronotropic effects of the muscarinic agonist carbachol. Similarly, loss of RGS6, but not RGS4, correlated with enhanced sensitivity of the M2R-IKACh signaling pathway in SAN cells to carbachol and a significant slowing of M2R-IKACh deactivation rate. Surprisingly, concurrent genetic ablation of RGS4 partially rescued some deficits observed in Rgs6(-/-) mice. These findings, together with those from an acute pharmacologic approach in SAN cells from Rgs6(-/-) and Gβ5(-/-) mice, suggest that the partial rescue of phenotypes in Rgs4(-/-):Rgs6(-/-) mice is attributable to another R7 RGS protein whose influence on M2R-IKACh signaling is masked by RGS4. Thus, RGS6-Gβ5, but not RGS4, is the primary RGS modulator of parasympathetic HR regulation and SAN M2R-IKACh signaling in mice.

  16. TAM receptor signaling in development.

    PubMed

    Burstyn-Cohen, Tal

    2017-01-01

    TYRO3, AXL and MERTK comprise the TAM family of receptor protein tyrosine kinases. Activated by their ligands, protein S (PROS1) and growth-arrest-specific 6 (GAS6), they mediate numerous cellular functions throughout development and adulthood. Expressed by a myriad of cell types and tissues, they have been implicated in homeostatic regulation of the immune, nervous, vascular, bone and reproductive systems. The loss-of-function of TAM signaling in adult tissues culminates in the destruction of tissue homeostasis and diseased states, while TAM gain-of-function in various tumors promotes cancer phenotypes. Combinatorial ligand-receptor interactions may elicit different molecular and cellular responses. Many of the TAM regulatory functions are essentially developmental, taking place both during embryogenesis and postnatally. This review highlights current knowledge on the role of TAM receptors and their ligands during these developmental processes in the immune, nervous, vascular and reproductive systems.

  17. High cell surface death receptor expression determines type I versus type II signaling.

    PubMed

    Meng, Xue Wei; Peterson, Kevin L; Dai, Haiming; Schneider, Paula; Lee, Sun-Hee; Zhang, Jin-San; Koenig, Alexander; Bronk, Steve; Billadeau, Daniel D; Gores, Gregory J; Kaufmann, Scott H

    2011-10-14

    Previous studies have suggested that there are two signaling pathways leading from ligation of the Fas receptor to induction of apoptosis. Type I signaling involves Fas ligand-induced recruitment of large amounts of FADD (FAS-associated death domain protein) and procaspase 8, leading to direct activation of caspase 3, whereas type II signaling involves Bid-mediated mitochondrial perturbation to amplify a more modest death receptor-initiated signal. The biochemical basis for this dichotomy has previously been unclear. Here we show that type I cells have a longer half-life for Fas message and express higher amounts of cell surface Fas, explaining the increased recruitment of FADD and subsequent signaling. Moreover, we demonstrate that cells with type II Fas signaling (Jurkat or HCT-15) can signal through a type I pathway upon forced receptor overexpression and that shRNA-mediated Fas down-regulation converts cells with type I signaling (A498) to type II signaling. Importantly, the same cells can exhibit type I signaling for Fas and type II signaling for TRAIL (TNF-α-related apoptosis-inducing ligand), indicating that the choice of signaling pathway is related to the specific receptor, not some other cellular feature. Additional experiments revealed that up-regulation of cell surface death receptor 5 levels by treatment with 7-ethyl-10-hydroxy-camptothecin converted TRAIL signaling in HCT116 cells from type II to type I. Collectively, these results suggest that the type I/type II dichotomy reflects differences in cell surface death receptor expression.

  18. Activation of RIG-I-like Receptor Signal Transduction

    PubMed Central

    Bruns, Annie; Horvath, Curt M.

    2011-01-01

    Mammalian cells have the ability to recognize virus infection and mount a powerful antiviral response. Pattern recognition receptor proteins detect molecular signatures of virus infection and activate antiviral signaling cascades. The RIG-I-like receptors are cytoplasmic DExD/H box proteins that can specifically recognize virus-derived RNA species as a molecular feature discriminating the pathogen from the host. The RIG-I-like receptor family is composed of three homologous proteins, RIG-I, MDA5, and LGP2. All of these proteins can bind double-stranded RNA species with varying affinities via their conserved DExD/H box RNA helicase domains and C-terminal regulatory domains. The recognition of foreign RNA by the RLRs activates enzymatic functions and initiates signal transduction pathways resulting in the production of antiviral cytokines and the establishment of a broadly effective cellular antiviral state that protects neighboring cells from infection and triggers innate and adaptive immune systems. The propagation of this signal via the interferon antiviral system has been studied extensively, while the precise roles for enzymatic activities of the RNA helicase domain in antiviral responses are only beginning to be elucidated. Here, current models for RLR ligand recognition and signaling are reviewed. PMID:22066529

  19. Preclinical to Human Translational Pharmacology of the Novel M1 Positive Allosteric Modulator MK-7622.

    PubMed

    Uslaner, Jason M; Kuduk, Scott D; Wittmann, Marion; Lange, Henry S; Fox, Steve V; Min, Chris; Pajkovic, Natasa; Harris, Dawn; Cilissen, Caroline; Mahon, Chantal; Mostoller, Kate; Warrington, Steve; Beshore, Douglas C

    2018-06-01

    The current standard of care for treating Alzheimer's disease is acetylcholinesterase inhibitors, which nonselectively increase cholinergic signaling by indirectly enhancing activity of nicotinic and muscarinic receptors. These drugs improve cognitive function in patients, but also produce unwanted side effects that limit their efficacy. In an effort to selectively improve cognition and avoid the cholinergic side effects associated with the standard of care, various efforts have been aimed at developing selective M 1 muscarinic receptor activators. In this work, we describe the preclinical and clinical pharmacodynamic effects of the M 1 muscarinic receptor-positive allosteric modulator, MK-7622. MK-7622 attenuated the cognitive-impairing effects of the muscarinic receptor antagonist scopolamine and altered quantitative electroencephalography (qEEG) in both rhesus macaque and human. For both scopolamine reversal and qEEG, the effective exposures were similar between species. However, across species the minimum effective exposures to attenuate the scopolamine impairment were lower than for qEEG. Additionally, there were differences in the spectral power changes produced by MK-7622 in rhesus versus human. In sum, these results are the first to demonstrate translation of preclinical cognition and target modulation to clinical effects in humans for a selective M 1 muscarinic receptor-positive allosteric modulator. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

  20. Differential Modulation of Beta-Adrenergic Receptor Signaling by Trace Amine-Associated Receptor 1 Agonists

    PubMed Central

    Nürnberg, Daniela; Grüters, Annette; Führer-Sakel, Dagmar; Krude, Heiko; Köhrle, Josef; Schöneberg, Torsten; Biebermann, Heike

    2011-01-01

    Trace amine-associated receptors (TAAR) are rhodopsin-like G-protein-coupled receptors (GPCR). TAAR are involved in modulation of neuronal, cardiac and vascular functions and they are potentially linked with neurological disorders like schizophrenia and Parkinson's disease. Subtype TAAR1, the best characterized TAAR so far, is promiscuous for a wide set of ligands and is activated by trace amines tyramine (TYR), phenylethylamine (PEA), octopamine (OA), but also by thyronamines, dopamine, and psycho-active drugs. Unfortunately, effects of trace amines on signaling of the two homologous β-adrenergic receptors 1 (ADRB1) and 2 (ADRB2) have not been clarified yet in detail. We, therefore, tested TAAR1 agonists TYR, PEA and OA regarding their effects on ADRB1/2 signaling by co-stimulation studies. Surprisingly, trace amines TYR and PEA are partial allosteric antagonists at ADRB1/2, whereas OA is a partial orthosteric ADRB2-antagonist and ADRB1-agonist. To specify molecular reasons for TAAR1 ligand promiscuity and for observed differences in signaling effects on particular aminergic receptors we compared TAAR, tyramine (TAR) octopamine (OAR), ADRB1/2 and dopamine receptors at the structural level. We found especially for TAAR1 that the remarkable ligand promiscuity is likely based on high amino acid similarity in the ligand-binding region compared with further aminergic receptors. On the other hand few TAAR specific properties in the ligand-binding site might determine differences in ligand-induced effects compared to ADRB1/2. Taken together, this study points to molecular details of TAAR1-ligand promiscuity and identified specific trace amines as allosteric or orthosteric ligands of particular β-adrenergic receptor subtypes. PMID:22073124