Sample records for central muscarinic effects

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. 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.

  7. 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

  8. 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

  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. 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.

  11. 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

  12. 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

  13. 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.

  14. 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

  15. 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

  16. 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.

  17. 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.

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

    NASA Astrophysics Data System (ADS)

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

    1985-05-01

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

  19. 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.

  20. 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.

  1. 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

  2. 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

  3. 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.

  4. 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

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. 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.

  10. 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.

  11. 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

  12. Non-muscarinic therapeutic targets for acute organophosphorus poisoning.

    PubMed

    Rosenbaum, Christopher; Bird, Steven B

    2010-12-01

    Organophosphorus (OP) pesticides are a broad class of acetylcholinesterase inhibitors that are responsible for tremendous morbidity and mortality worldwide, contributing to an estimated 300,000 deaths annually. Current pharmacotherapy for acute OP poisoning includes the use of atropine, an oxime, and benzodiazepines. However, even with such therapy, the mortality from these agents is as high as 40%. It is increasingly recognized that not all OPs are the same. Significant differences exist in their toxicity, lipophilicity, and response to oxime therapy. Other non-muscarinic effects of OP pesticides exist, such as acute and chronic neuromuscular junction failure and central respiratory failure. In part because most of the mortality from these chemicals takes place in the developing world, little National Institutes of Health (NIH) research has been directed towards these agents. However, the similar mechanism of action of OP pesticides and the military nerve agents, along with increasing concerns about chemical terrorism has lead to the formation of the NIH Countermeasures Against Chemical Threats (CounterACT) Program. As part of the CounterACT Program, the NIH has recently designated six OP pesticides as "threat agents". This concept paper describes some of the knowledge gaps related to non-muscarinic effects of OP pesticides and highlights needed areas of further research. Leveraging the current NIH interest in these chemicals to medical necessities in the developing world offers the possibility of delivering new therapeutics where they are needed on a daily basis.

  13. 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.

  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. 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.

  16. 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.

  17. 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

  18. 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

  19. 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.

  20. 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

  1. 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.

  2. 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.

  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. 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. 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

  6. 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

  7. 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.

  8. 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.

  9. Role of muscarinic receptor subtypes in central antinociception.

    PubMed Central

    Bartolini, A.; Ghelardini, C.; Fantetti, L.; Malcangio, M.; Malmberg-Aiello, P.; Giotti, A.

    1992-01-01

    1. The ability to modify the pain threshold by the two M1-muscarinic agonists: McN-A-343 and AF-102B and by the specific M2-agonist arecaidine was examined in mice and rats by using three different noxious stimuli: chemical (writhing test), thermic (hot-plate test) and mechanical (paw pressure test). 2. In the mouse hot-plate test McN-A-343 (20-50 micrograms per mouse i.c.v.) and AF-102B (1-10 mg kg-1 i.p.) produced significant antinociception which was prevented by atropine (1 microgram per mouse i.c.v.) and by the two selective M1 antagonists: pirenzepine (0.01 micrograms per mouse i.c.v.) and dicyclomine (0.08 micrograms per mouse i.c.v. or 10 mg kg-1 i.p.) but not by the specific M2-antagonist AFDX-116 (0.1 micrograms per mouse i.c.v.), naloxone (1 mg kg-1 i.p.) or by the acetylcholine (ACh) depletor hemicholinium-3 (HC-3) (1 micrograms per mouse i.c.v.). McN-A-343 and AF-102B were able to increase the pain threshold also in the mouse acetic acid writhing test and in rat paw pressure test. These antinociceptive effects were completely prevented by dicyclomine (0.08 micrograms per mouse i.c.v. or 10 mg kg-1 i.p.) but not by AFDX-116 (0.1 microgram per mouse or rat i.c.v.). 3. In contrast with the M1-agonists, the M2-agonist arecaidine (0.1-2 micrograms per mouse or rat i.c.v.) did not induce antinociception in all three analgesic tests. However, arecaidine, at the same i.c.v. doses, was able to reduce the pain threshold in the hot-plate and paw pressure tests.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1375858

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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

  17. 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.

  18. 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

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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

  4. 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

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. 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.

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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.

  17. 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.

  18. 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

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. Review of the Pharmacological Properties and Clinical Usefulness of Muscarinic Agonists for Xerostomia in Patients with Sjögren's Syndrome.

    PubMed

    Yasuda, Hiroshi; Niki, Hiroshi

    2002-01-01

    The anti-xerostomia effects of muscarinic agonists (cholinomimetics) are reviewed. Cevimeline (cevimeline monohydrochloride hemihydrate) is a novel muscarinic agonist that stimulates salivary secretion in animals and humans both with normal salivary gland function and with impaired salivary secretion (xerostomia or oral dryness) as effectively as pilocarpine. Other classic and nonselective muscarinic agonists, such as arecoline, carbachol, muscarine and oxotremorine, as well as acetylcholine, failed to exhibit a sufficient salivation effect even at sublethal doses in animals.Oral administration of cevimeline 30mg to humans induces a moderate and lasting increase in salivary flow, and the effect is maintained for at least 4 to 6 hours, longer than with pilocarpine. Mean increases in salivary flow rates after cevimeline treatment were 2-fold higher than after placebo, and no evidence of tolerance of the pharmacological effect has been observed during prolonged administration for up to 12 months.The clinical efficacy of cevimeline in relieving symptoms of xerostomia, including oral dryness and difficulties in chewing, swallowing and speaking, has been demonstrated by placebo-controlled, double-blind, randomised clinical trials in the USA and Japan. In these studies, cevimeline 30mg three times daily increased salivary flow and improved the symptoms of xerostomia in a significantly higher percentage of patients compared with placebo. Some patients receiving cevimeline therapy for xerostomia experienced adverse events such as sweating, gastrointestinal symptoms (nausea, diarrhoea, abdominal pain and vomiting), dizziness and rigors; these effects were related to muscarinic activity and were generally mild and tolerable in comparison with those of pilocarpine.These findings suggest that muscarinic M3 agonists are suitable for the treatment of xerostomia. Cevimeline in particular has a long-lasting salivation effect with fewer adverse events than pilocarpine, and so is

  7. 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

    receptors in nociception also is over-viewed. In fact, muscarinic agonists such as vedaclidine, CMI-936 and CMI-1145 have been demonstrated to have analgesic effects in animal models comparable or more pronounced to those produced by morphine or opiates. Likewise, the crucial role of cholinesterases (acetylcholinesterase and butirylcholinesterase) in neural transmission is discussed, as large number of drugs inhibiting cholinesterase activity have become of increasing relevance particularly for the treatment of neurodegenerative disorders. Herein we summarize the current knowledge of the cholinesterase inhibitors with particular attention to recent patents for Alzheimer's disease drugs.

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. 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

  13. 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

  14. 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

  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. 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.

  17. The Mechanism of Interaction of Oximes with the Muscarinic Cholinergic Complex in the Central Nervous System.

    DTIC Science & Technology

    1985-03-31

    release (40-60%) and the time course of the inhibitory response were similar to those observed with the muscarinic agonist oxotremorine . It thus...to ’At i : ,-,- t he ri, arinik agonist oxotremorine . These results indicate f,~ ~ ~~k xi "/ ’ × (1- sati,iie~i here a1< t ;as T- l , n ,i :a ti t :r...acetylcholine from rat brainstem slices 19 Figure 6. Inhibition of K+-evoked [3H]-acetylcholine release by oxotremorine and its potentiation by

  18. 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

  19. 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

  20. 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

  1. 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.

  2. 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

  3. 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.

  4. 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

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. 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

    receptors in nociception also is over-viewed. In fact, muscarinic agonists such as vedaclidine, CMI-936 and CMI-1145 have been demonstrated to have analgesic effects in animal models comparable or more pronounced to those produced by morphine or opiates. Likewise, the crucial role of cholinesterases (acetylcholinesterase and butirylcholinesterase) in neural transmission is discussed, as large number of drugs inhibiting cholinesterase activity have become of increasing relevance particularly for the treatment of neurodegenerative disorders. Herein we summarize the current knowledge of the cholinesterase inhibitors with particular attention to recent patents for Alzheimer’s disease drugs. PMID:23597304

  13. Analgesic and Antineuropathic Drugs Acting Through Central Cholinergic Mechanisms

    PubMed Central

    Bartolini, Alessandro; Cesare Mannelli, Lorenzo Di; Ghelardini, Carla

    2011-01-01

    The role of muscarinic and nicotinic cholinergic receptors in analgesia and neuropathic pain relief is relatively unknown. This review describes how such drugs induce analgesia or alleviate neuropathic pain by acting on the central cholinergic system. Several pharmacological strategies are discussed which increase synthesis and release of acetylcholine (ACh) from cholinergic neurons. The effects of their acute and chronic administration are described. The pharmacological strategies which facilitate the physiological functions of the cholinergic system without altering the normal modulation of cholinergic signals are highlighted. It is proposed that full agonists of muscarinic or nicotinic receptors should be avoided. Their activation is too intense and un-physiological because neuronal signals are distorted when these receptors are constantly activated. Good results can be achieved by using agents that are able to a) increase ACh synthesis, b) partially inhibit cholinesterase activity c) selectively block the autoreceptor or heteroreceptor feedback mechanisms. Activation of M1 subtype muscarinic receptors induces analgesia. Chronic stimulation of nicotinic (N1) receptors has neuronal protective effects. Recent experimental results indicate a relationship between repeated cholinergic stimulation and neurotrophic activation of the glial derived neurotrophic factor (GDNF) family. At least 9 patents covering novel chemicals for cholinergic system modulation and pain control are discussed. PMID:21585331

  14. Central anticholinergic syndrome: a case report.

    PubMed

    Moos, Daniel D

    2007-10-01

    Postoperative central anticholinergic syndrome (CAS) is caused by anticholinergic medications that cross the blood-brain barrier. Medications with central anticholinergic effects block muscarinic cholinergic receptors, resulting in a wide array of symptoms. Symptoms may range from coma to a highly agitated state. CAS may be underdiagnosed because of its varying presentation and lack of awareness. Differential diagnosis for the patient presenting with abnormal neurological signs and symptoms should include CAS after the exclusion of other potential causes. This case report details the occurrence of CAS in a patient in her 20's. A review of CAS including causes, signs and symptoms, incidence, differential diagnosis, and treatment is discussed.

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. Naloxone Antagonizes Soman-induced Central Respiratory Depression in Rats.

    PubMed

    Škrbić, Ranko; Stojiljković, Miloš P; Ćetković, Slavko S; Dobrić, Silva; Jeremić, Dejan; Vulović, Maja

    2017-06-01

    The influence of naloxone on respiration impaired by the highly toxic organophosphate nerve agent soman in anaesthetized rats was investigated. Soman, administered in a dose that was ineffective in blocking the electrically induced contractions of the phrenic nerve-diaphragm preparation in situ, induced a complete block of the spontaneous respiratory movements of the diaphragm, indicating the domination of central over the peripheral effects. Naloxone dose-dependently antagonized the soman-induced respiratory blockade. Atropine, at a dose that was per se ineffective in counteracting soman-induced respiratory depression, potentiated the protective effects of naloxone and completely restored respiration. Naloxone remained completely ineffective in antagonizing respiratory depression induced by the muscarinic receptor agonist the oxotremorine. It is assumed that naloxone antagonizes soman-induced respiratory inhibition by blocking endogenous opioidergic respiratory control pathways that are independent of the stimulation of muscarinic receptors. © 2016 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

  20. 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

  1. 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.

  2. 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

  3. 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

  4. 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

  5. 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.

  6. 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

  7. 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

  8. 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

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

    DTIC Science & Technology

    1983-09-30

    Pathways; GABAergic Pathway; Atropine; Reserpine; Alphamethylparatyrosine; Oxotremorine ; Feedback 20 ABSTRACT (Continue on reverse side It necessary and...see Preface). The purpose was the compare the regional distribution of the effect of anticholinesterases with oxotremorine ),a selective centrally...hippocampus, differently from oxotremorine which was ineffective. In the other two regions, physostigmine and oxotremorine were equally active. At the

  10. 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.

  11. 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.

  12. 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.

  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. 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).

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. Proceedings of the International Symposium on Subtypes of Muscarinic Receptors (4th), Held in Wiesbaden (Germany, F.R.) on 20-22 July 1989

    DTIC Science & Technology

    1990-06-15

    C. However, primary astrocytes is not stimulated by oxotremorine , a this coupling is much less efficient than that mediated muscarinic agonist which...and oxotremorine -M, possessed suffi- modest improvement in affinity but without the antici- cient intrnsic efficacy to maximally stimulate cortical PI...other cholino- Animal studies mimetics i.e. oxotremorine , in different animal spe- Antinociceptive studies in animals cies s . Centrally administered

  1. 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

  2. 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.

  3. Interactions between scopolamine and muscarinic cholinergic agonists or cholinesterase inhibitors on spatial alternation performance in rats.

    PubMed

    Shannon, H E; Bemis, K G; Hendrix, J C; Ward, J S

    1990-12-01

    The effects on working memory of the muscarinic cholinergic agonists oxotremorine, arecoline, RS86 and pilocarpine, and the cholinesterase inhibitors physostigmine and tetrahydroaminoacadine were investigated in male F344 rats. Working memory was assessed by behavior maintained under a spatial alternation schedule of food presentation in which the interval between trials was varied from 2 to 32 sec. Under control conditions the percentage of correct responses decreased as the retention interval was varied from 2 to 32 sec. Administered alone the cholinergic agonists oxotremorine (0.01-0.1 mg/kg), arecoline (3-30 mg/kg), RS86 (0.3-3 mg/kg) and pilocarpine (0.3-3.0 mg/kg), and the cholinesterase inhibitors physostigmine (0.01-0.1 mg/kg) and tetrahydroaminoacridine (0.3-3.0 mg/kg) either had no effect on or produced dose-related deficits in working memory and decreases in response rates. The muscarinic antagonist scopolamine (0.1 mg/kg) produced retention interval-dependent decreases in the percentage of correct responding and rates of responding. The cholinergic agonists and tetrahydroaminoacridine failed to reverse the effects of scopolamine. However, physostigmine produced a dose-dependent reversal of the working-memory deficits and response-rate decreasing effects of scopolamine. The present results are consistent with the interpretation that drugs which primarily enhance M2 muscarinic cholinergic transmission are ineffective in enhancing working memory or in reversing scopolamine-induced deficits in working memory.

  4. 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.

  5. The effect of indomethacin on the muscarinic induced contractions in the isolated normal guinea pig urinary bladder.

    PubMed

    Rahnama'i, Mohammad S; van Koeveringe, Gommert A; van Kerrebroeck, Philip E V; de Wachter, Stefan G G

    2013-02-07

    To investigate the effect of prostaglandin depletion by means of COX-inhibition on cholinergic enhanced spontaneous contractions. The urethra and bladder of 9 male guinea pigs (weight 270-300 g) were removed and placed in an organ bath with Krebs' solution. A catheter was passed through the urethra through which the intravesical pressure was measured. The muscarinic agonist arecaidine, the non-selective COX inhibitor indomethacin, and PGE2 were subsequently added to the organ bath. The initial average frequency and amplitude of spontaneous contractions in the first 2 minutes after arecaidine application were labelled F(ini) and P(ini), respectively. The steady state frequency (F(steady)) and amplitude (P(steady)) were defined as the average frequency and amplitude during the 5 minutes before the next wash out. Application of 1 μM PGE2 increased the amplitude of spontaneous contractions without affecting frequency. 10 μM of indomethacin reduced amplitude but not frequency.The addition of indomethacin did not alter F(ini) after the first application (p = 0.7665). However, after the second wash, F(ini) was decreased (p = 0.0005). F(steady), P(steady) and P(ini) were not significantly different in any of the conditions. These effects of indomethacin were reversible by PGE2 addition.. Blocking PG synthesis decreased the cholinergically stimulated autonomous contractions in the isolated bladder. This suggests that PG could modify normal cholinergically evoked response. A combination of drugs inhibiting muscarinic receptors and PG function or production can then become an interesting focus of research on a treatment for overactive bladder syndrome.

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. 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

  12. Intranasal scopolamine affects the semicircular canals centrally and peripherally.

    PubMed

    Weerts, Aurélie P; Putcha, Lakshmi; Hoag, Stephen W; Hallgren, Emma; Van Ombergen, Angelique; Van de Heyning, Paul H; Wuyts, Floris L

    2015-08-01

    Space motion sickness (SMS), a condition caused by an intravestibular conflict, remains an important obstacle that astronauts encounter during the first days in space. Promethazine is currently the standard treatment of SMS, but scopolamine is used by some astronauts to prevent SMS. However, the oral and transdermal routes of administration of scopolamine are known to have substantial drawbacks. Intranasal administration of scopolamine ensures a fast absorption and rapid onset of therapeutic effect, which might prove to be suitable for use during spaceflights. The aim of this study was to evaluate the effects of intranasally administered scopolamine (0.4 mg) on the semicircular canals (SCCs) and the otoliths. This double-blind, placebo-controlled study was performed on 19 healthy male subjects. The function of the horizontal SCC and the vestibulo-ocular reflex, as well as the saccular function and utricular function, were evaluated. Scopolamine turned out to affect mainly the SCCs centrally and peripherally but also the utricles to a lesser extent. Centrally, the most probable site of action is the medial vestibular nucleus, where the highest density of muscarinic receptors has been demonstrated and afferent fibers from the SCCs and utricles synapse. Furthermore, our results suggest the presence of muscarinic receptors in the peripheral vestibular system on which scopolamine has a suppressive effect. Given the depressant actions on the SCCs, it is suggested that the pharmacodynamic effect of scopolamine may be attributed to the obliteration of intravestibular conflict that arises during (S)MS. Copyright © 2015 the American Physiological Society.

  13. Hyperpolarizing muscarinic responses of freshly dissociated rat hippocampal CA1 neurones.

    PubMed Central

    Wakamori, M; Hidaka, H; Akaike, N

    1993-01-01

    1. Intracellular mechanisms of the muscarinic acetylcholine (ACh) response were investigated in pyramidal neurones freshly dissociated from the rat hippocampal CA1 region. Current recordings were made in the whole-cell mode using the nystatin 'perforated'-patch technique, by which the muscarinic ACh response can be continuously recorded without so-called 'run-down' phenomenon. The amount of intracellular free Ca2+ ([Ca2+]i) was fluorometrically measured using fura-2. 2. In current clamp conditions, ACh induced a transient hyperpolarization accompanied by a decrease in membrane input resistance. 3. Under voltage clamp conditions at a holding potential (Vh) of -40 mV, ACh induced two types of muscarinic currents observed either alone or together: a transient outward current and a slowly activating sustained inward current. 4. The ACh-induced transient outward current reversed the direction at K+ equilibrium potential (EK), and the reversal potential (EACh) shifted 56.7 mV for a tenfold change of extracellular K+ concentration ([K+]o). 5. The ACh-induced transient outward current increased in a sigmoidal fashion with increase in ACh concentration, where the half-maximal concentration (EC50) and the Hill coefficient (n) were 8 x 10(-7) M and 1.9, respectively. Both muscarine and carbamylcholine mimicked the ACh response, but neither McN-A-343 (M1 agonist) nor oxotremorine (cardiac M2 agonist) induced any current. 6. Muscarinic antagonists reversibly blocked the ACh response in a concentration-dependent manner. The inhibitory potency was in the order of atropine > pirenzepine > AF-DX-116. 7. The ACh-induced transient outward current was never recorded when [Ca2+]i was chelated by the acetoxymethyl ester form of 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA AM). On the other hand, in Ca(2+)-free external solution containing 2 mM EGTA and 10 mM Mg2+, the ACh response was elicited by the first application and successive ACh applications did not induce any

  14. 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

  15. 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

  16. 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.

  17. 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

  18. 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

  19. 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

  20. 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.

  1. 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

  2. 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

  3. 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.

  4. 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

  5. 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

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. 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

  11. 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.

  12. 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.

  13. 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

  14. [Non-neuronal effects of muscarinic antagonists in the prophylaxis of stress].

    PubMed

    Nezhinskaia, G I; Vladykin, A L; Sapronov, N S

    2008-01-01

    We have studied the stress-limiting role of the immune reaction initiated by cholinergic antagonists and the influence of these drugs on the dynamics of antibody formation in the spleen and the blood serum corticosterone level. The protective effect of immune reaction initiated by methacine (muscarinic receptor antagonist) or hexamethonium (nicotinic receptor antagonist) in prevention of stress gastric ulcer in rats (induced by water immersion stress, WIS) was estimated upon administration of the drugs for 5 days (local response) or 14 days (systemic response) prior to WIS. The pharmacological effects of drugs were estimated upon their administration 30 minutes prior to WIS. It is shown that, if cholinergic antagonists affect the systemic immune response the induction of WIS at this level of immune reaction leads to the effective prevention of stress gastric ulcer. The administration of methacine (but not hexamethonium) 14 days prior to WIS effectively reduces gastric lesions up to 1.0 +/- 0.1 arbitrary units in comparison to 3.6 + 0.2 arbitrary units in the control group. Under effective prophylaxis, the number of antibody-forming cells (AFC/10(6) of splenocytes) and corticosterone concentration are close to their basal level, while under stress conditions, these parameters significantly increase up to 870 +/- 21 and 350 +/- 4 vs. 100 +/- 17 and 107 +/- 6 in the control group, accordingly. It is established that both methacine and hexamethonium remain immunologically active for 28 days and more: the maximum amount of AFC upon administration of hexamethonium and methacine was on the 5th day and 14th day, respectively. Thus, determination of the drug influence on the systemic immune response allows one to predict the non-neuronal effects of cholinergic antagonists and, in this way, to affect the pathogenesis of stress gastric ulcer. Estimation of the AFC response and corticosterone level after WIS shows the efficacy ofprophylaxis of the gastric stress lesion.

  15. 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

  16. Using anti-muscarinic drugs in the management of death rattle: evidence-based guidelines for palliative care.

    PubMed

    Bennett, Mike; Lucas, Viv; Brennan, Mary; Hughes, Andrew; O'Donnell, Valerie; Wee, Bee

    2002-09-01

    The management of 'death rattle' was reviewed by a task group on behalf of the Association for Palliative Medicine's Science Committee. Evidence was searched for the effectiveness of various anti-muscarinic drugs in drying oropharyngeal and bronchial secretions in dying patients. Clinical guidelines were constructed based on evidence from volunteer and clinical studies. Death rattle occurs in half of all dying patients and some response occurs in around 80% of treated patients. Clinical studies demonstrate that subcutaneous hyoscine hydrobromide 400 microg is more effective at improving symptoms at 30 min than glycopyrronium 200 microg by the same route. Volunteer studies demonstrate that intramuscular glycopyrronium 400 microg is as effective in drying secretions at 30 min as a dose of 200 microg given intravenously. Duration of response is shortest for hyoscine butylbromide (1 h) and longest for glycopyrronium (more than 6 h). There is insufficient evidence to support the use of one drug over another in a continuous infusion and prescribers should base decisions on different characteristics of each anti-muscarinic drug.

  17. 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.

  18. 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.

  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. 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

  1. 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.

  2. 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

  3. 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.

  4. The character of sleep disturbances produced by multiple administrations of atropine the antagonist of brain muscarinic cholinergic system.

    PubMed

    Maglakelidze, N T; Chkhartishvili, E V; Mchedlidze, O M; Dzadzamiia, Sh Sh; Nachkebiia, N G

    2012-03-01

    Modification of brain muscarinic cholinergic system normal functioning can be considered as an appropriate strategy for the study of its role in sleep-wakefulness cycle basic mechanisms in general and in the course/maintenance of PS in particular. For this aim systemic application of muscarinic cholinoreceptors antagonists is significant because it gives possibility to modify functioning all of known five sub-types of muscarinic cholinoreceptors and to study the character of sleep disturbances in these conditions. Problem is very topical because the question about the intimate aspects of BMChS involvement in PS maintaining mechanisms still remains unsolved. In cats Atropine systemic administration was made once daily at 10:00 a.m. and continuous EEG registration of sleep-wakefulness cycle ultradian structure, lasting for 10 hour daily, was started immediately. In sum each animal received anti-muscarinic drugs for 12 times. Thereafter drug administrations were ceased and EEG registration of sleep-wakefulness cycle ultradian structure was continued during 10 consecutive days. On the basis of results obtained in these conditions we can conclude that brain muscarinic cholinergic system normal functioning is significant for basic mechanisms of sleep-wakefulness cycle. During wakefulness, at the level of neocortex and hippocampus, MChS supports only EEG activation, while it is one of the main factors in PS triggering and maintaining mechanisms.

  5. 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

  6. 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.

  7. 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

  8. 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

  9. 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.

  10. 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

  11. 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.

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

    ERIC Educational Resources Information Center

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

    2004-01-01

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

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. 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.

  1. 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

  2. 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.

  3. Differences in muscarinic acetylcholine receptor subtypes in the central nervous system of long sleep and short sleep mice. [Ethanol effects

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

    Watson, M.; Ming, X.; McArdle, J.J.

    Differences in voluntary ethanol consumption have been noted in various inbred strains of mice and pharmacogenetic approaches have been used to study the mechanisms of action of many drugs such as ethanol. Long-sleep (LS) and short-sleep (SS) mice, selectively bred for differences in ethanol induced narcosis, provide a method by which a relationship between the differential responsiveness of these geno-types and muscarinic acetylcholine receptors (mAChR) may be evaluated. Sleep times after injection of 3ml ethanol/kg (i.p.) verified the higher sensitivity of LS vs. SS. Mean body weights of LS (26.5g) vs. SS (22g) were also significantly (p<.01) greater. Binding assaysmore » for ({sup 3}H)(-) quinuclidinylbenzilate (({sup 3}H)(-)QNB), a specific but nonsubtype selective mAChR antagonist, ({sup 3}H)pirenzepine (({sup 3}H)PZ), a specific M1 mAChR antagonist and ({sup 3}H)11-2-((2-((diethylamino) methyl)-1-piperidinyl) acetyl)-5,11-dihydro-6H-pyrido (2,3-b) (1,4) benzodiazepine-6-one, (({sup 3}H)AF-DX 116), an M2 selective antagonist were performed to determine mAChR affinity (K{sub d}) and density (B{sub max}) in CNS regions such as the cerebral cortex, hippocampus, corpus striatum and other areas. Significantly lower (30-40%) ({sup 3}H)(-)QNB binding suggests that SS have fewer mAChR's than LS in many areas. These differences may relate to their differential ethanol sensitivity.« less

  4. 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).

  5. 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

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. Chotosan, a kampo formula, ameliorates chronic cerebral hypoperfusion-induced deficits in object recognition behaviors and central cholinergic systems in mice.

    PubMed

    Zhao, Qi; Murakami, Yukihisa; Tohda, Michihisa; Obi, Ryosuke; Shimada, Yutaka; Matsumoto, Kinzo

    2007-04-01

    We previously demonstrated that the Kampo formula chotosan (CTS) ameliorated spatial cognitive impairment via central cholinergic systems in a chronic cerebral hypoperfusion (P2VO) mouse model. In this study, the object discrimination tasks were used to determine if the ameliorative effects of CTS on P2VO-induced cognitive deficits are a characteristic pharmacological profile of this formula, with the aim of clarifying the mechanisms by which CTS enhances central cholinergic function in P2VO mice. The cholinesterase inhibitor tacrine (THA) and Kampo formula saikokeishito (SKT) were used as controls. P2VO impaired object discrimination performance in the object recognition, location, and context tests. Daily administration of CTS (750 mg/kg, p.o.) and THA (2.5 mg/kg, i.p.) improved the object discrimination deficits, whereas SKT (750 mg/kg, p.o.) did not. In ex vivo assays, tacrine but not CTS or SKT inhibited cortical cholinesterase activity. P2VO reduced the mRNA expression of m(3) and m(5) muscarinic receptors and choline acetyltransferase but not that of other muscarinic receptor subtypes in the cerebral cortex. Daily administration of CTS and THA but not SKT reversed these expression changes. These results suggest that CTS and THA improve P2VO-induced cognitive impairment by normalizing the deficit of central cholinergic systems and that the beneficial effect on P2VO-induced cognitive deficits is a distinctive pharmacological characteristic of CTS.

  11. 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.

  12. Evaluation of levetiracetam effects on pilocarpine-induced seizures: cholinergic muscarinic system involvement.

    PubMed

    Oliveira, A A; Nogueira, C R A; Nascimento, V S; Aguiar, L M V; Freitas, R M; Sousa, F C F; Viana, G S B; Fonteles, M M F

    2005-09-16

    Levetiracetam (LEV) is a new antiepileptic drug effective as adjunctive therapy for partial seizures. It displays a unique pharmacological profile against experimental models of seizures, including pilocarpine-induced seizures in rodents. Aiming to clarify if anticonvulsant activity of LEV occurs due to cholinergic alterations, adult male mice received LEV injections before cholinergic agonists' administration. Pretreatment with LEV (30-200 mg/kg, i.p.) increased the latencies of seizures, but decreased status epilepticus and death on the seizure model induced by pilocarpine, 400 mg/kg, s.c. (P400). LEV (LEV200, 200 mg/kg, i.p.) pretreatment also reduced the intensity of tremors induced by oxotremorine (0.5 mg/kg, i.p). [3H]-N-methylscopolamine-binding assays in mice hippocampus showed that LEV200 pretreatment reverts the downregulation on muscarinic acetylcholine receptors (mAChR), induced by P400 administration, bringing back these density values to control ones (0.9% NaCl, i.p.). However, subtype-specific-binding assays revealed that P400- and LEV-alone treatments result in M1 and M2 subtypes decrease, respectively. The agonist-like behavior of LEV on the inhibitory M2 mAChR subtype, observed in this work, could contribute to explain the reduction on oxotremorine-induced tremors and the delay on pilocarpine-induced seizures, by an increase in the attenuation of neuronal activity mediated by the M1 receptors.

  13. Muscarine- and carbachol-induced aggressions: fear and irritable kinds of aggressions.

    PubMed

    Beleslin, D B; Samardzić, R

    1977-12-28

    In unaneasthetized and unrestrained cats, muscarine and carbachol were injected into the cerebral ventricles. The kind of aggressive behaviour depended on the cholinomimetic drug and was classified as fear and an irritable kind of aggression. Muscarine induced the fear kind of aggression. The aggressive behaviour was usually preceded by attempts to escape and the attack was relevant to the situation. For the attack the presence of some threatening agent was needed. The aggression was accompanied by intense motor but less autonomic activation. On the other hand, carbachol induced an irritable kind of aggression and had the following characteristics: for the attack the presence of some threatening agent was not needed; the attack was not relevant to the situation; the aggression was not preceded by attempts to escape; and the aggressive behaviour was accompanied by intense motor and autonomic activation. It is concluded that cholinoceptive mechanisms are involved in the control of aggressive behaviour.

  14. 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

  15. 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.

  16. 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

  17. 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.

  18. 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...

  19. Effects of oxotremorine on local glucose utilization in the rat cerebral cortex

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

    Dam, M.; Wamsley, J.K.; Rapoport, S.I.

    The (/sup 14/C)2-deoxy-D-glucose technique was used to examine the effects of central muscarinic stimulation on local cerebral glucose utilization (LCGU) in the cerebral cortex of the unanesthetized rat. Systemic administration of the muscarinic agonist oxotremorine (OXO, 0.1 to 1.0 mg/kg, i.p.) increased LCGU in the neocortex, mesocortex, and paleocortex. In the neocortex, OXO was more potent in elevating LCGU of the auditory, frontal, and sensorimotor regions compared with the visual cortex. Within these neocortical regions, OXO effects were greatest in cortical layers IV and V. OXO effects were more dramatic in the neocortex than in the meso- or paleocortex, andmore » no significant effect occurred in the perirhinal and pyriform cortices. OXO-induced LCGU increases were not influenced by methylatropine (1 mg/kg, s.c.) but were antagonized completely by scopolamine (2.5 mg/kg, i.p.). Scopolamine reduced LCGU in layer IV of the auditory cortex and in the retrosplenial cortex. The distribution and magnitude of the cortical LCGU response to OXO apparently were related to the distributions of cholinergic neurochemical markers, especially high affinity muscarinic binding sites.« less

  20. 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

  1. 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…

  2. Participation of non-neuronal muscarinic receptors in the effect of carbachol with paclitaxel on human breast adenocarcinoma cells. Roles of nitric oxide synthase and arginase.

    PubMed

    Español, Alejandro Javier; Salem, Agustina; Rojo, Daniela; Sales, María Elena

    2015-11-01

    Breast cancer is the most common type of cancer in women and represents a major issue in public health. The most frequent methods to treat these tumors are surgery and/or chemotherapy. The latter can exert not only beneficial effects by reducing tumor growth and metastasis, but also toxic actions on normal tissues. Metronomic therapy involves the use of low doses of cytotoxic drugs alone or in combination to improve efficacy and to reduce adverse effects. We have previously reported that breast tumors highly express functional muscarinic acetylcholine receptors (mAChRs) that regulate tumor progression. For this reason, mAChRs could be considered as therapeutic targets in breast cancer. In this paper, we investigated the ability of a combination of the cytotoxic drug paclitaxel plus carbachol, a cholinergic agonist, at low doses, to induce death in breast tumor MCF-7 cells, via mAChR activation, and the role of nitric oxide synthase (NOS) and arginase in this effect. We observed that the combination of carbachol plus paclitaxel at subthreshold doses significantly increased cytotoxicity in tumor cells without affecting MCF-10A cells, derived from human normal mammary gland. This effect was reduced in the presence of the muscarinic antagonist atropine. The combination also increased nitric oxide production by NOS1 and NOS3 via mAChR activation, concomitantly with an up-regulation of NOS3 expression. The latter effects were accompanied by a reduction in arginase II activity. In conclusion, our work demonstrates that mAChRs expressed in breast tumor cells could be considered as candidates to become targets for metronomic therapy in cancer treatment. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Muscarinic agonists and ATP increase the intracellular Ca2+ concentration in chick cochlear hair cells.

    PubMed

    Shigemoto, T; Ohmori, H

    1990-01-01

    , glutamate and bradykinin did not induce Ca2+ responses in the hair cell. 8. ACh induced hyperpolarization of the hair cell membrane under current clamp, most probably by the activation of Ca2+ activated K+ conductance. Therefore, a cholinergic muscarinic receptor may mediate the inhibitory effects of efferent innervation observed in hair cells.

  4. 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

  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. 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.

  7. 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

    of drug binding. 6. Replacement of a pyrrolidino by a piperidino group and increasing the length of the alkylene chain bridging the amino group and the central carbon or silicon atom were associated with either an increase or a decrease of entropy and enthalpy changes of drug binding. However, there was no clear correlation between these structural variations and the thermodynamic effects. 7. Taken together, these results suggest that hydrogen bond-forming OH groups and, to a lesser extent, polarizable phenyl groups contribute significantly to the thermodynamics of interactions between these classes of muscarinic antagonists and M2 muscarinic receptors. PMID:8102927

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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

  14. Effects of pilocarpine hydrochloride and cevimeline on submandibular/sublingual salivation in rat xerostomia model produced by X-ray irradiation.

    PubMed

    Omori, Yasuhiro; Asari, Tetsuya; Maruyama, Kazuyasu; Kusama, Hiroshi; Kojima, Masami; Shibata, Nobuo

    2003-01-01

    The present study was performed to assess the effects of pilocarpine hydrochloride ((3S,4R)-3-ethyl-dihydro-4-[(1-methyl-1H-imidazole-5-yl)methyl]-2(3H)-furanone monohydrochloride, CAS 54-71-7) and cevimeline ((+/-)-cis-2-methylspiro[1,3-oxathiolane-5,3'-quinuclidine] hydrochloride, hemihydrate, CAS 153504-70-2), muscarinic receptor agonists, on salivary secretion from the submandibular/sublingual (SM/SL) glands in normal rats and in rats with xerostomia induced by X-ray (15 Gy) irradiation. To clarify their pharmacological safety profiles, the two drugs were further compared with regard to subtype selectivity for muscarinic receptors (M1, M2, and M3) and central nervous, respiratory, and cardiovascular effects. Pilocarpine hydrochloride (0.1-0.8 mg/kg i.d.) and cevimeline (3-30 mg/kg i.d.) dose-dependently increased salivary flow rate and total salivary volume in a 120-min period from SM/SL glands in both normal and irradiated rats, the minimum effective doses for their sialagogic effects being 0.2 and 10 mg/kg, respectively. Both drugs also increased protein output from SM/SL glands to a degree that depended on the increase in salivary volume in normal and irradiated rats. In a binding study using radiolabeled antagonists, neither pilocarpine hydrochloride nor cevimeline displayed subtype selectivity for muscarinic receptors, indicating non-selective muscarinic agonism. Effects on the central nervous system (CNS) were assessed by monitoring changes in body temperature in conscious normal rats. Pilocarpine hydrochloride (0.4-4 mg/kg p.o.) had no effect on body temperature, but cevimeline (30 and 100 mg/kg p.o.) caused a significant hypothermia. In terms of respiratory and cardiovascular effects in anesthetized normal rats, there was no clear difference in safety margin between pilocarpine hydrochloride and cevimeline, both drugs inducing significant changes in respiratory rate, heart rate, and blood pressure at doses close to those inducing sialagogic effects

  15. 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

  16. Quantitative structure-activity relationships (QSAR) of some 2,2-diphenyl propionate (DPP) derivatives of muscarinic antagonists

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

    Gordon, R.K.; Breuer, E.; Padilla, F.N.

    1987-05-01

    QSAR between biological activities and molecular-chemical properties were investigated to aid in designing more effective and potent antimuscarinic pharmacophores. A molecular modeling program was used to calculate geometrical and topological values of a series of DPP pharmacophores. The newly synthesized pharmacophores were tested for their antagonist activities by: (1) inhibition of (N-methyl-/sup 3/H)scopolamine binding assay to the muscarinic receptors of N4TG1 neuroblastoma cells; (2) blocking of acetylcholine-induced contraction of guinea pig ileum; and (3) inhibition of carbachol-induced ..cap alpha..-amylase release from rat pancreas. The differences in the log of these biological activities were directly and significantly related to the distancesmore » between the carbonyl oxygen of the DPP and the quaternary nitrogen of the modified pharmacophores. The biological activities, while depending on each particular assay, varied between three and four logs of activity. The charge remained the same in all the pharmacophores. There were no QSAR correlations between molecular volume, molecular connectivity, or principle moments and their antagonistic activities, although multivariate QSAR was not employed. Thus, based on distance geometry, potent muscarinic pharmacophores can be predicted.« less

  17. 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.

  18. 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

  19. General pharmacological profile of the novel muscarinic receptor agonist SNI-2011, a drug for xerostomia in Sjögren's syndrome. 4th communication: Effects on gastrointestinal, urinary and reproductive systems and other effects.

    PubMed

    Arisawa, Hirohiko; Fukui, Kenji; Imai, Eiichi; Fujise, Nobuaki; 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 the gastrointestinal, urinary and reproductive systems and other tissues were investigated in mice, rats guinea pigs, rabbits and dogs. 1. Gastrointestinal system: SNI-2011 did not cause any effects on the gastrointestinal system, i.e. the intestinal transport of charcoal meal in mice, the secretion of gastric and bile juices, and the formation of ulcer induced by water immersion restraint in rats. 2. Urinary and reproductive systems: SNI-2011 augmented the spontaneous movement of rat pregnant uterus in vivo at 0.3 mg/kg i.v. or higher, and this effect was not observed in the non-pregnant uterus. SNI-2011 increased the spontaneous movement of isolated guinea pig bladder (3 x 10(-6) mol/l or higher) and increased the in vivo spontaneous movement of rat bladder (0.3 mg/kg i.v. or higher). SNI-2011 caused increases in rat urine volume, pH and urinary excretion of Na+ and Cl- at 30 mg/kg p.o. 3. Others: SNI-2011 had no effect on the vascular permeability in mice, hematological parameters and blood coagulation in rats. SNI-2011 had neither hemolytic nor anti-inflammatory effect. These results suggest that SNI-2011 has muscarinic effects on the gastrointestinal, urinary and reproductive systems and other tissues at the doses approximately 10-fold higher than the doses needed for saliva secretion.

  20. 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.

  1. 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

  2. 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

  3. Muscarinic inhibition of [3H]-noradrenaline release on rabbit iris in vitro: effects of stimulation conditions on intrinsic activity of methacholine and pilocarpine.

    PubMed Central

    Bognar, I. T.; Pallas, S.; Fuder, H.; Muscholl, E.

    1988-01-01

    1. Rabbit isolated irides were loaded with [3H]-noradrenaline and superfused with Tyrode solution. The inhibition by the muscarinic agonists (+/-)-methacholine and pilocarpine of the [3H]-noradrenaline overflow into the superfusate evoked by field stimulation (pulses of 1 ms duration, 75 mA) was measured as an index of activation of presynaptic muscarinic receptors. 2. The fractional rate of release per pulse during the first stimulation period (S1) was low with 360 pulses at 3 Hz, intermediate with 360 pulses at 10 Hz and high with 1200 pulses at 10 Hz. Upon repetitive stimulation (7 periods at 20 min intervals), the fractional rates of release per pulse during S7 no longer differed, suggesting a 'long-term' regulation of [3H]-noradrenaline release depending on the stimulation conditions. 3. The evoked [3H]-noradrenaline overflow was depressed by (+/-)-methacholine in a concentration-dependent manner. The EC50 ranged from 0.29 to 0.42 microM. Methacholine nearly abolished the transmitter release evoked at 3 Hz but reduced that induced at 10 Hz by only 50%. Under the latter condition the methacholine concentration-inhibition curve was bell-shaped and no muscarinic inhibition was observed in the presence of methacholine 30 microM. After washout of methacholine the evoked [3H]-noradrenaline release was temporarily enhanced. 4. Atropine 0.1 microM enhanced the [3H]-noradrenaline overflow (evoked by stimulation with 360 or 1200 pulses at 10 Hz), probably antagonizing a presynaptic inhibition by endogenous acetylcholine. The inhibition by methacholine was competitively antagonized by atropine 0.1 microM (apparent -log KB = 8.5-9.0). 5. Depending on the concentration, pilocarpine reduced the [3H]-noradrenaline overflow evoked by 360 pulses at 3 Hz up to 63%. However, at 10 Hz stimulation frequency the compound was inactive as an agonist but competitively antagonized the presynaptic inhibition induced by methacholine. The KB under the latter condition (0.95 microM) was

  4. 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.

  5. 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

  6. [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

  7. Experiment K-7-18: Effects of Spaceflight in the Muscle Adductor Longus of Rats Flown in the Soviet Biosatellite Cosmos 2044. Part 2; Quantitative Autoradiographic Analysis of Gaba (Benzodiazepine) and Muscarinic (Cholinergic) Receptors in the Forebrain of Rats Flown on Cosmos 2044

    NASA Technical Reports Server (NTRS)

    Wu, L.; Daunton, N. G.; Krasnov, I. B.; DAmelio, F.; Hyde, T. M.; Sigworth, S. K.

    1994-01-01

    Quantitative autoradiographic analysis of receptors for GABA and acetylcholine in the forebrain of rats flown on COSMOS 2044 was undertaken as part of a joint US-Soviet study to determine the effects of microgravity on the central nervous system, and in particular on the sensory and motor portions of the forebrain. Changes in binding of these receptors in tissue from animals exposed to microgravity would provide evidence for possible changes in neural processing as a result of exposure to microgravity. Tritium-labelled diazepam and Quinuclidinyl-benzilate (QNB) were used to visualize GABA (benzodiazepine) and muscarinic (cholinergic) receptors, respectively. The density of tritium-labelled radioligands bound to various regions in the forebrain of both flight and control animals were measured from autoradiograms. Data from rats flown in space and from ground-based control animals that were not exposed to microgravity were compared.

  8. 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.

  9. 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.

  10. 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

  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. 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

  13. 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).

  14. The facilitating effect of systemic administration of Kv7/M channel blocker XE991 on LTP induction in the hippocampal CA1 area independent of muscarinic activation.

    PubMed

    Song, Ming-Ke; Cui, Yong-Yao; Zhang, Wei-Wei; Zhu, Liang; Lu, Yang; Chen, Hong-Zhuan

    2009-09-11

    A large amount of in vitro studies demonstrate suppression of M-current in hippocampal neurons by Kv7/M channel blocker results in depolarization of membrane potential and release of neurotransmitters, such as acetylcholine and glutamate, suggesting that Kv7/M channel may play important roles in regulating synaptic plasticity. In the present study, we examined the in vivo effect of Kv7/M channel inhibition on the long-term potentiation (LTP) induction at basal dendrites in hippocampal CA1 area of urethane-anaesthetized rats. The Kv7/M channel was inhibited by intraperitoneal injection of XE991 (10mg/kg) and the LTP of field excitatory postsynaptic potential (fEPSP) was induced by supra-threshold high frequency stimulation (S1 HFS). A weak protocol which was just below the threshold for evoking LTP was used as sub-threshold high frequency stimulation (S2 HFS). XE991 did not significantly alter the slope of fEPSP and the magnitude of LTP induced by S1 HFS, suggesting that Kv7/M channel inhibition had little or no effect on glutamatergic transmission under basal conditions. However, XE991 could make S2 HFS evoke LTP even after the application of the muscarinic cholinergic (mACh) receptor antagonist scopolamine, suggesting that Kv7/M channel inhibition lowered the threshold for LTP induction and the effect was independent of muscarinic activation. Based on the above findings, we concluded that the facilitating effect of XE991 on LTP induction is not mediated by its ability to enhance the release of acetylcholine; therefore, Kv7/M channel blockers may provide a therapeutic benefit to cholinergic deficiency-related cognitive impairment, e.g., Alzheimer's disease.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  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. 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.

  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. Action of cholinergic poisons on the central nervous system and effectiveness of potential antidotes. Annual report 1 Jul 81-30 Jun 82

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

    Samson, F.; Nelson, S.

    The research aim was to determine the effects of soman, related organophosphate toxins and potential antidotes on brain regional functions in rats: The (/sup 14/C)-2-deoxyglucose procedure (2-DG) was used for mapping brain regional glucose use. Quantitative autoradiography was used for muscarinic and nicotinic cholinergic receptors. The 2-DG procedure gives a quantitative measure of glucose utilization in brain regions and is in index of the 'functional activity' in brain regions and systems. Values were determined in controls, rats with soman induced seizures, seizures induced by convulsants (DFP, strychnine, picrotoxin, pentylenetetrazol, penicillin) and soman pretreated with TAB. Brain regional cholinergic receptor mapsmore » were prepared and some regional muscarinic and nicotinic receptor densities have been quantified. Soman (112 micrograms/kg i.m.) causes strong, continuous seizures and a dramatic (2-6 fold) increase in the rate of glucose use in 10 major brain regions. Most intense increases were in septum, substants nigra reticularis and outer layer of hippcampal dendata gyrus. The overt seizures of rats induced by convulsants DFP, strychnine, picrotoxin, pentylenetetrazol and penicillin (in hippocampus) were strikingly different from that of rats with soman seizures. High doses (2X LD50) of soman in rats protected with TAB caused a 50% depression of glucose use in most brain regions. The effects of repeated soman exposure on muscarinic and nicotinic receptors are under study.« less

  5. Evidence of central cholinergic mechanisms in the appearance of affective aggressive behaviour: dissociation of aggression from autonomic and motor phenomena.

    PubMed

    Beleslin, D B; Samardzić, R

    1979-04-11

    Carbachol, muscarine, eserine and neostigmine injected into the cerebral ventricles of conscious cats evoked emotional behaviour with aggression, autonomic and motor phenomena as well as clonic-tonic convulsions. The main and the most impressive feature of the gross behavioural effects of intraventricular carbachol, muscarine, eserine and neostigmine in conscious cats was the affective type of aggression. However, neostigmine produced aggressive behaviour only in about one-quarter of the experiments. After intraventricular hemicholinium-3 and triethylcholine carbachol, muscarine, eserine and neostigmine elicited autonomic and motor phenomena. In these cats cholinomimetics and anticholinesterases evoked only slight hissing and snarling. Choline administered into the cerebral ventricles of hemicholinium-3 and triethylcholine-treated cats restored the emotional behaviour with aggression, autonomic and motor phenomena as well as clonic-tonic convulsions to intraventricular carbachol, muscarine, eserine and neostigmine. The restored gross behavioural changes to eserine were almost of the same intensity, while those to carbachol and muscarine were of lesser intensity than in control cats. From these experiments it is concluded that cholinergic neurones are involved in the appearance of the affective type of aggression resulting from intraventricular carbachol, muscarine, eserine and neostigmine.

  6. 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.

  7. 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

  8. The discovery of the sub-threshold currents M and Q/H in central neurons.

    PubMed

    Adams, Paul

    2016-08-15

    The history, content and consequences of the highly-cited 1982 Brain Research paper by Halliwell and Adams are summarized. The paper pioneered the use of the single-electrode voltage clamp in mammalian brain slices, described 2 novel sub-threshold voltage-dependent ionic currents, IM and IQ/H, and suggested that cholinergic inputs "enabled" pyramidal cell firing in response to conventional synaptic input, the first example of central neuromodulation. The paper, published in Brain Research to give the first author appropriate importance, heralded an ongoing tidal wave of quantitative electrophysiology in mammalian central neurons. Voltage-clamp analysis of muscarinic excitation in hippocampal neurons Pyramidal cells in the CA1 field of guinea pig hippocampal slices were voltage-clamped using a single microelectrode, at 23-30°C. Small inwardly relaxing currents triggered by step hyperpolarizations from holding potentials of -80 to -40mV were investigated. Inward relaxations occurring for negative steps between -40mV and -70mV resembled M-currents of sympathetic ganglion cells: they were abolished by addition of carbachol, muscarine or bethanechol, as well as by 1mM barium; the relaxations appeared to invert at around -80mV; they became faster at more negative potentials; and the inversion potential was shifted positively by raising external K(+) concentration. Inward relaxations triggered by steps negative to -80mV, in contrast, appeared to reflect passage of another current species, which has been labeled IQ.Thus IQ did not invert negative to -80mV, it was insensitive to muscarinic agonizts or to barium, and it was blocked by 0.5-3mM cesium (which does not block IM). Turn-on of IQ causes the well known droop in the hyperpolarizing electrotonic potential in these cells. The combined effects of IQ and IM make the steady-state current-voltage relation of CA1 cells slightly sigmoidal around rest potential. It is suggested that activation of cholinergic septal inputs to

  9. 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

  10. 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.

  11. Synthesis and functional characterization of novel derivatives related to oxotremorine and oxotremorine-M.

    PubMed

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

    1999-08-01

    Two subseries of nonquaternized (5a-10a) and quaternized derivatives (5b-10b) related to oxotremorine and oxotremorine-M were synthesized and tested. The agonist potency at the muscarinic receptor subtypes of the new compounds was estimated in three classical in vitro functional assays: M1 rabbit vas deferens, M2 guinea pig left atrium and M3 guinea pig ileum. In addition, the occurrence of central muscarinic effects was evaluated as tremorigenic activity after intraperitoneal administration in mice. In in vitro tests a nonselective muscarinic activity was exhibited by all the derivatives with potencies values that, in some instances, surpassed those of the reference compounds (i.e. 8b). Functional selectivity was evidenced only for the oxotremorine-like derivative 9a, which behaved as a mixed M3-agonist/M1-antagonist (pD2 = 5.85; pA2 = 4.76, respectively). In in vivo tests non-quaternary compounds were able to evoke central muscarinic effects, with a potency order parallel to that observed in vitro.

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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

  4. 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.

  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. 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

  7. 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

  8. Effects of subcutaneous and intracerebroventricular injection of physostigmine on the acute corneal nociception in rats.

    PubMed

    Tamaddonfard, Esmaeal; Hamzeh-Gooshchi, Nasrin

    2010-01-01

    The present study investigated the effects of subcutaneous (sc) and intracerebroventricular (icv) injections of physostigmine (a cholinesterase inhibitor), atropine (an antagonist of muscarinic cholinergic receptors) and hexamethonium (an antagonist of nicotinic cholinergic receptors) on the acute corneal nociception in rats. Local application of 5 M NaCl solution on the corneal surface of the eye produced a significant nociceptive behavior, characterized by eye wiping. The number of eye wipes was counted during the first 30 s. The sc (0.25, 0.5 and 1 mg/kg) and icv (1.25, 2.5, 5 and 10 μg) injections of physostigmine significantly (p < 0.05) decreased the number of eye wipes. Atropine and hexamethonium at (2 mg/kg, sc and 20 μg, icv) had no effects when used alone, however, atropine, but not hexamethonium prevented the antinociception induced by physostigmine (sc and icv). The results of this study indicate that the central muscarinic, but not nicotinic receptors might be involved in the antinociceptive effect of physostigmine in the acute corneal model of pain in rats.

  9. 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.

  10. 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

  11. 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.

  12. Effects of presynaptic muscarinic cholinoreceptor blockade on neuromuscular transmission as assessed by the train-of-four and the tetanic fade response to rocuronium.

    PubMed

    Kim, Yong Beom; Lee, Sangseok; Lee, Kyeong Chun; Kim, Ha Jung; Ro, Young Jin; Yang, Hong-Seuk

    2017-07-01

    This study investigated the effect of muscarinic M 1 and M 2 receptor antagonists on the rocuronium-induced train of four (TOF) fade and tetanic fade, respectively. Ex-vivo phrenic nerves and diaphragms were obtained from adult Sprague-Dawley rats and stabilized in Krebs buffer; the nerve-stimulated muscle TOF fade was observed at 20 s intervals. For the TOF study, phrenic nerves and diaphragms were incubated with pirenzepine (an M 1 blocker) at concentrations of 0 nmol L -1 (control), 10 nmol L -1 (PZP10), or 100 nmol L -1 (PZP100). Rocuronium was then administered incrementally until the first twitch tension had depressed by >95% during TOF stimulation. The mean TOF ratios were compared when the first twitch tensions were depressed by 40%-50%. For the tetanic fade study, 50 Hz/5 s tetani was applied initially, 30 min after the administration of a loading dose of rocuronium and methoctramine (an M 2 receptor blocker, loaded at 0 μmol L -1 [control], 1 μmol L -1 [MET1], or 10 μmol L -1 [MET10]). The EC 95 of rocuronium was significantly lower in the PZP10 group than in the control group. In the PZP10 group, the TOF ratios at 50% and first twitch tension depression were significantly lower than those in the control group (P=.02). During tetanic stimulation, the tetanic fade was significantly enhanced in the MET10 group compared to the other groups. This study shows that antagonists of muscarinic M 1 and M 2 receptors affect the rocuronium-induced neuromuscular block as demonstrated by the reduced EC 95 and TOF ratios (M 1 antagonist, pirenzepine) or the enhanced 50-Hz tetanic fade (M 2 antagonist, methoctramine). © 2017 The Authors. Clinical and Experimental Pharmacology and Physiology Published by John Wiley & Sons Australia, Ltd.

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. 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

    had no effect on the A9 L cells. 6. Fura-2 measurements revealed ACh-induced increases in Cai2+. The Ca2+ responses were abolished by atropine showing that they were muscarinic in nature. Removal of extracellular Ca2+ did not affect the initial ACh-induced increase in Cai2+ but subsequent Cai2+ responses to ACh were depressed, suggesting depletion of Ca2+ intracellular stores. Residual though small responses continued to be elicited by ACh. Barium (5 mM) had little effect and cobalt slightly reduced the ACh-induced Ca2+ response. 7. The ACh-induced currents recorded at -50 mV were unaffected by removal of extracellular Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS) Images Fig. 9 Fig. 10 PMID:1693402

  18. 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.

  19. 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

  20. 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.

  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. 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

  3. Disruption of trace conditioning of the nictitating membrane response in rabbits by central cholinergic blockade.

    PubMed

    Kaneko, T; Thompson, R F

    1997-05-01

    Central muscarinic cholinergic involvement in classical conditioning of eyeblink responses was determined in trace and delay paradigms. Rabbits were trained on a trace procedure in which a 250-ms tone conditioned stimulus (CS) and a 100-ms air-puff unconditioned stimulus (UCS) were presented with a 500-ms trace interval. Each training session day consisted of ten tone alone, ten air-puff alone and 80 paired CS-UCS trials. Scopolamine hydrochloride at doses of 0.03 and 0.1 mg/0.5 ml per kg, s.c. dose-dependently disrupted acquisition of conditioned responses. Rabbits that were treated with scopolamine and failed to learn showed a gradual increase in conditioned responses during an additional training period with saline injections and no transfer from earlier training. Scopolamine methyl bromide, which does not appreciably cross the blood-brain barrier, showed no effects in the trace conditioning paradigm at a dose of 0.1 mg/kg, s.c., indicating central cholinergic blockade is responsible for the suppressive effect of scopolamine. Scopolamine hydrochloride at a dose of 0.1 mg/kg, s.c. did not block acquisition in the delay procedure with a 250-ms inter-stimulus interval, although the rate of acquisition was somewhat reduced by the drug. These data are the first to demonstrate that classical conditioning of the eyeblink response in the trace procedure is highly sensitive to central cholinergic deficits.

  4. 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...

  5. Cerebellum cholinergic muscarinic receptor (subtype-2 and -3) and cytoarchitecture after developmental exposure to methylmercury: an immunohistochemical study in rat.

    PubMed

    Roda, Elisa; Coccini, Teresa; Acerbi, Davide; Castoldi, Anna; Bernocchi, Graziella; Manzo, Luigi

    2008-05-01

    The developing central nervous system (CNS) is a target of the environmental toxicant methylmercury (MeHg), and the cerebellum seems the most susceptible tissue in response to this neurotoxicant. The cholinergic system is essential for brain development, acting as a modulator of neuronal proliferation, migration and differentiation processes; its muscarinic receptors (MRs) play pivotal roles in regulating important basic physiologic functions. By immunohistochemistry, we investigated the effects of perinatal (GD7-PD21) MeHg (0.5 mg/kg bw/day in drinking water) administration on cerebellum of mature (PD36) and immature (PD21) rats, evaluating the: (i) M2- and M3-MR expression; (ii) presence of gliosis; (iii) cytoarchitecture alterations. Regarding to M2-MRs, we showed that: at PD21, MeHg-treated animals did not display any differences compared to controls, while, at PD36 there was a significant increase of M2-immunopositive Bergmann cells in the molecular layer (ML), suggesting a MeHg-related cytotoxic effect. Similarly to M2-MRs, at PD21 the M3-MRs were not affected by MeHg, while, at PD36 a lacking immunoreactivity of the granular layer (IGL) was observed after MeHg treatment. In MeHg-treated rats, at both developmental points, we showed reactive gliosis, e.g. a significant increase in Bergmann glia of the ML and astrocytes of the IGL, identified by their expression of glial fibrillar acidic protein. No MeHg-related effects on Purkinje cells were detected neither at weaning nor at puberty. These findings suggest: (i) a delayed MeHg exposure-related effect on M2- and M3-MRs, (ii) an overt MeHg-related cytotoxic effect on cerebellar oligodendroglia, e.g. reactive gliosis, (iii) a selective vulnerability of granule cells and Purkinje neurons to MeHg, with the latter that remain unharmed.

  6. 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

  7. 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

  8. Medications influencing central cholinergic pathways affect fixation stability, saccadic response time and associated eye movement dynamics during a temporally-cued visual reaction time task.

    PubMed

    Naicker, Preshanta; Anoopkumar-Dukie, Shailendra; Grant, Gary D; Modenese, Luca; Kavanagh, Justin J

    2017-02-01

    Anticholinergic medications largely exert their effects due to actions on the muscarinic receptor, which mediates the functions of acetylcholine in the peripheral and central nervous systems. In the central nervous system, acetylcholine plays an important role in the modulation of movement. This study investigated the effects of over-the-counter medications with varying degrees of central anticholinergic properties on fixation stability, saccadic response time and the dynamics associated with this eye movement during a temporally-cued visual reaction time task, in order to establish the significance of central cholinergic pathways in influencing eye movements during reaction time tasks. Twenty-two participants were recruited into the placebo-controlled, human double-blind, four-way crossover investigation. Eye tracking technology recorded eye movements while participants reacted to visual stimuli following temporally informative and uninformative cues. The task was performed pre-ingestion as well as 0.5 and 2 h post-ingestion of promethazine hydrochloride (strong centrally acting anticholinergic), hyoscine hydrobromide (moderate centrally acting anticholinergic), hyoscine butylbromide (anticholinergic devoid of central properties) and a placebo. Promethazine decreased fixation stability during the reaction time task. In addition, promethazine was the only drug to increase saccadic response time during temporally informative and uninformative cued trials, whereby effects on response time were more pronounced following temporally informative cues. Promethazine also decreased saccadic amplitude and increased saccadic duration during the temporally-cued reaction time task. Collectively, the results of the study highlight the significant role that central cholinergic pathways play in the control of eye movements during tasks that involve stimulus identification and motor responses following temporal cues.

  9. Discovery of novel quaternary ammonium derivatives of (3R)-quinuclidinyl amides as potent and long acting muscarinic antagonists.

    PubMed

    Prat, Maria; Buil, María Antonia; Fernández, Maria Dolors; Tort, Laia; Monleón, Juan Manuel; Casals, Gaspar; Ferrer, Manuel; Castro, Jordi; Gavaldà, Amadeu; Miralpeix, Montserrat; Ramos, Israel; Vilella, Dolors; Huerta, Josep Maria; Espinosa, Sònia; Hernández, Begoña; Segarra, Victor; Córdoba, Mònica

    2015-04-15

    Novel quaternary ammonium derivatives of (3R)-quinuclidinyl amides have been identified as potent M3 muscarinic antagonists with a long duration of action in an in vivo model of bronchoconstriction. The synthesis, structure-activity relationships and biological evaluation of this series of compounds are reported. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. 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.

  11. 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

  12. 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.

  13. 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

  14. 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.

  15. General pharmacological profile of the novel muscarinic receptor agonist SNI-2011, a drug for xerostomia in Sjögren's syndrome. 3rd communication: effects on respiratory and cardiovascular systems.

    PubMed

    Arisawa, Hirohiko; 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 the respiratory and cardiovascular systems were investigated in guinea pigs and dogs. SNI-2011 reduced the contractile force and beating rate of isolated right guinea pig atrium at 1 x 10(-6) mol/l or higher and 3 x 10(-6) mol/l or higher, respectively. SNI-2011 reduced the contractile force of isolated left atrium induced by electric stimulation at 1 x 10(-6) mol/l or higher. In anesthetized dogs, SNI-2011 caused a transient decrease in blood pressure, tachycardia and an increase in femoral arterial blood flow at 0.01 mg/kg i.v. or higher. At 1 mg/kg it caused continuous bradycardia, a decrease in femoral arterial blood flow and an increase in respiration rate in addition to the changes observed immediately after injection. A transient negative T-wave was observed as the only change in the ECG immediately after injection at 1 mg/kg. However, when SNI-2011 was injected intraduodenally, a decrease in femoral arterial blood flow, bradycardia and a tendency to increase respiration rate were observed at doses of 1 to 3 mg/kg. All these events in dogs were antagonized by atropine. These results suggest that oral administration of SNI-2011, that is the clinical administration route, can distinctly reduce the muscarinic effects on the respiratory and cardiovascular systems compared to intravenous administration.

  16. Sympathetic sprouting drives hippocampal cholinergic reinnervation that prevents loss of a muscarinic receptor-dependent long-term depression at CA3-CA1 synapses.

    PubMed

    Scheiderer, Cary L; McCutchen, Eve; Thacker, Erin E; Kolasa, Krystyna; Ward, Matthew K; Parsons, Dee; Harrell, Lindy E; Dobrunz, Lynn E; McMahon, Lori L

    2006-04-05

    Degeneration of septohippocampal cholinergic neurons results in memory deficits attributable to loss of cholinergic modulation of hippocampal synaptic circuits. A remarkable consequence of cholinergic degeneration is the sprouting of noradrenergic sympathetic fibers from the superior cervical ganglia into hippocampus. The functional impact of sympathetic ingrowth on synaptic physiology has never been investigated. Here, we report that, at CA3-CA1 synapses, a Hebbian form of long-term depression (LTD) induced by muscarinic M1 receptor activation (mLTD) is lost after medial septal lesion. Unexpectedly, expression of mLTD is rescued by sympathetic sprouting. These effects are specific because LTP and other forms of LTD are unaffected. The rescue of mLTD expression is coupled temporally with the reappearance of cholinergic fibers in hippocampus, as assessed by the immunostaining of fibers for VAChT (vesicular acetylcholine transporter). Both the cholinergic reinnervation and mLTD rescue are prevented by bilateral superior cervical ganglionectomy, which also prevents the noradrenergic sympathetic sprouting. The new cholinergic fibers likely originate from the superior cervical ganglia because unilateral ganglionectomy, performed when cholinergic reinnervation is well established, removes the reinnervation on the ipsilateral side. Thus, the temporal coupling of the cholinergic reinnervation with mLTD rescue, together with the absence of reinnervation and mLTD expression after ganglionectomy, demonstrate that the autonomic-driven cholinergic reinnervation is essential for maintaining mLTD after central cholinergic cell death. We have discovered a novel phenomenon whereby the autonomic and central nervous systems experience structural rearrangement to replace lost cholinergic innervation in hippocampus, with the consequence of preserving a form of LTD that would otherwise be lost as a result of cholinergic degeneration.

  17. Locomotor activating effects of cocaine and scopolamine combinations in rats: isobolographic analysis

    PubMed Central

    Thomsen, Morgane

    2014-01-01

    Muscarinic cholinergic receptors are receiving renewed interest as viable targets for treating various psychiatric disorders. Dopaminergic and muscarinic systems interact in complex ways. The goal of this study was to quantify the interaction of a systemically administered psychomotor stimulant and muscarinic antagonist at the behavioral level. Using isobolographic analysis of locomotor activity data, we assessed the effects of three cocaine/scopolamine mixtures in terms of deviation from simple dose addition (additivity), at four effect levels. All three mixtures produced some more-than-additive (synergistic) effects, as lower doses were needed to produce given effects relative to the calculated effect of additive doses. A mixture with comparable contributions from cocaine and scopolamine produced significantly more-than-additive effects at all but the lowest effect level examined. A mostly-cocaine mixture was more-than-additive at low effect levels only, while a mostly-scopolamine mixture produced effects more consistent with additivity, with only the highest effect level barely reaching significant synergism. Our study confirms and quantifies previous findings that suggested synergistic effects of stimulants and muscarinic antagonists. The synergism implies that cocaine and scopolamine stimulate locomotor activity through non-identical pathways, and was most pronounced for a mixture containing cocaine and scopolamine in comparable proportions. PMID:24769455

  18. A centrally mediated prolonged hypotension produced by oxotremorine or pilocarpine

    PubMed Central

    Dage, R.C.

    1979-01-01

    1 Oxotremorine, methyloxotremorine, pilocarpine or arecoline were given intravenously to anaesthetized cats, dogs or rats, and intraperitoneally to conscious normotensive and spontaneously hypertensive rats, pretreated with doses of methylatropine that completely blocked peripheral muscarinic receptors, to ascertain their effects on blood pressure and heart rate. 2 Oxotremorine but not methyloxotremorine produced a prolonged hypotension in cats and dogs but not in rats. Heart rate was not changed. Pilocarpine, although less potent, produced an identical effect, whereas the effect of arecoline was short by comparison. The hypotensive effect of these drugs was reversed by atropine. 3 In dogs, oxotremorine produced a prolonged hypotension with no change in heart rate or cardiac output. 4 A decrease in spontaneous sympathetic nerve activity accompanied the hypotension in cats. Both effects were reversed by atropine but could be reinvoked by large doses of oxotremorine. 5 The oxotremorine-induced hypotension in cats was not altered by decerebration but was abolished by high cervical spinal section. 6 The results indicate that the prolonged hypotension elicited by oxotremorine is mediated by an action at muscarinic receptors in the brain stem resulting in a decrease in sympathetic nerve activity and peripheral resistance but not heart rate or cardiac output. PMID:760887

  19. 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.

  20. 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.

  1. 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.

  2. 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

  3. Accentuated antagonism in vagal heart rate control mediated through muscarinic potassium channels.

    PubMed

    Mizuno, Masaki; Kamiya, Atsunori; Kawada, Toru; Miyamoto, Tadayoshi; Shimizu, Shuji; Shishido, Toshiaki; Sugimachi, Masaru

    2008-12-01

    Although muscarinic K(+) (K(ACh)) channels contribute to a rapid heart rate (HR) response to vagal stimulation, whether background sympathetic tone affects the HR control via the K(ACh)channels remains to be elucidated. In seven anesthetized rabbits with sinoaortic denervation and vagotomy, we estimated the dynamic transfer function of the HR response by using random binary vagal stimulation (0-10 Hz). Tertiapin, a selective K(ACh) channel blocker, decreased the dynamic gain (to 2.3+/- 0.9 beats.min(-1).Hz(-1), from 4.6+/- 1.1, P < 0.01, mean+/- SD) and the corner frequency (to 0.05+/- 0.01 Hz, from 0.26+/- 0.04, P < 0.01). Under 5 Hz tonic cardiac sympathetic stimulation (CSS), tertiapin decreased the dynamic gain (to 3.6+/- 1.0 beats.min(-1).Hz(-1), from 7.3+/- 1.1, P < 0.01) and the corner frequency (to 0.06+/- 0.02 Hz, from 0.23+/- 0.06, P < 0.01). Two-way analysis of variance indicated significant interaction between the tertiapin and CSS effects on the dynamic gain. In contrast, no significant interactions were observed between the tertiapin and CSS effects on the corner frequency and the lag time. In conclusion, although a cyclic AMP-dependent mechanism has been well established, an accentuated antagonism also occurred in the direct effect of ACh via the K(ACh) channels. The rapidity of the HR response obtained by the K(ACh) channel pathway was robust during the accentuated antagonism.

  4. 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.

  5. 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.

  6. 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.

  7. Muscarinic Long-Term Enhancement of Tonic and Phasic GABAA Inhibition in Rat CA1 Pyramidal Neurons

    PubMed Central

    Domínguez, Soledad; Fernández de Sevilla, David; Buño, Washington

    2016-01-01

    Acetylcholine (ACh) regulates network operation in the hippocampus by controlling excitation and inhibition in rat CA1 pyramidal neurons (PCs), the latter through gamma-aminobutyric acid type-A receptors (GABAARs). Although, the enhancing effects of ACh on GABAARs have been reported (Dominguez et al., 2014, 2015), its role in regulating tonic GABAA inhibition has not been explored in depth. Therefore, we aimed at determining the effects of the activation of ACh receptors on responses mediated by synaptic and extrasynaptic GABAARs. Here, we show that under blockade of ionotropic glutamate receptors ACh, acting through muscarinic type 1 receptors, paired with post-synaptic depolarization induced a long-term enhancement of tonic GABAA currents (tGABAA) and puff-evoked GABAA currents (pGABAA). ACh combined with depolarization also potentiated IPSCs (i.e., phasic inhibition) in the same PCs, without signs of interactions of synaptic responses with pGABAA and tGABAA, suggesting the contribution of two different GABAA receptor pools. The long-term enhancement of GABAA currents and IPSCs reduced the excitability of PCs, possibly regulating plasticity and learning in behaving animals. PMID:27833531

  8. Characterization of a new muscarinic toxin from the venom of the Brazilian coral snake Micrurus lemniscatus in rat hippocampus.

    PubMed

    da Silva, Daniel Coelho; de Medeiros, Wyara Aparecida Araújo; Batista, Isabel de Fátima Correia; Pimenta, Daniel Carvalho; Lebrun, Ivo; Abdalla, Fernando Maurício Francis; Sandoval, Maria Regina Lopes

    2011-12-19

    We have isolated a new muscarinic protein (MT-Mlα) from the venom of the Brazilian coral snake Micrurus lemniscatus. This small protein, which had a molecular mass of 7,048Da, shared high sequence homology with three-finger proteins that act on cholinergic receptors. The first 12 amino acid residues of the N-terminal sequence were determined to be: Leu-Ile-Cys-Phe-Ile-Cys-Phe-Ser-Pro-Thr-Ala-His. The MT-Mlα was able to displace the [(3)H]QNB binding in the hippocampus of rats. The binding curve in competition experiments with MT-Mlα was indicative of two types of [(3)H]QNB-binding site with pK(i) values of 9.08±0.67 and 6.17±0.19, n=4, suggesting that various muscarinic acetylcholine receptor (mAChR) subtypes may be the target proteins of MT-Mlα. The MT-Mlα and the M(1) antagonist pirenzepine caused a dose-dependent block on total [(3)H]inositol phosphate accumulation induced by carbachol. The IC(50) values for MT-Mlα and pirenzepine were, respectively, 33.1 and 2.26 nM. Taken together, these studies indicate that the MT-Mlα has antagonist effect on mAChRs in rat hippocampus. The results of the present study show, for the first time, that mAChRs function is drastically affected by MT-Mlα since it not only has affinity for mAChRs but also has the ability to inhibit mAChRs. Copyright © 2011 Elsevier Inc. All rights reserved.

  9. 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

  10. 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.

  11. 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

  12. 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

  13. 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.

  14. 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.

  15. Inhibition of muscarinic-stimulated polyphosphoinositide hydrolysis and Ca2+ mobilization in cat iris sphincter smooth muscle cells by cAMP-elevating agents.

    PubMed

    Ding, K H; Husain, S; Akhtar, R A; Isales, C M; Abdel-Latif, A A

    1997-09-01

    The effects of carbachol (CCh) on inositol 1,4,5-trisphosphate (IP3) production and intracellular calcium ([Ca2+]i) mobilization, and their regulation by cAMP-elevating agents were investigated in SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells. CCh produced time- and dose-dependent increases in IP3 production; the t1/2 and EC50 values were 68 s and 0.5 microM, respectively. The muscarinic agonist provoked a transient increase in [Ca2+]i which reached maximum within 77 s, and increased [Ca2+]i mobilization in a concentration-dependent manner with an EC50 of 1.4 microM. Thapsigargin, a Ca(2+)-pump inhibitor, caused a rapid rise in [Ca2+]i and subsequent addition of CCh was without effect. Both CCh-induced IP3 production and CCh-induced [Ca2+]i mobilization were more potently antagonized by 4-DAMP, an M3 muscarinic receptor antagonist, than by pirenzepine, an M1 receptor antagonist, suggesting that both responses are mediated through the M3 receptor subtype. Treatment of the cells with U73122, a phospholipase C (PLC) inhibitor, resulted in a concentration-dependent decrease in both CCh-stimulated IP3 production and [Ca2+]i mobilization. These data indicate close correlation between enhanced IP3 production and [Ca2+]i mobilization in these smooth muscle cells and suggest that the CCh-stimulated increase in [Ca2+]i could be mediated through increased IP3 production. Isoproterenol (ISO) inhibited CCh-induced IP3 production (IC50 = 80 nM) and [Ca2+]i mobilization (IC50 = 0.17 microM) in a concentration-dependent manner. Microsomal fractions isolated from SV-CISM-2 cells contained phospholipase C (PLC) which was stimulated by CCh (10 microM) and GTP gamma S (0.1 microM). Pretreatment of the cells with ISO or forskolin, 5 microM each, produced membrane fractions in which CCh-stimulated PLC activity was significantly attenuated. Furthermore, when microsomal fractions isolated from SV-CISM-2 cells were phosphorylated with Protein kinase A (PKA), the CCh

  16. 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.

  17. M1 muscarinic agonists can modulate some of the hallmarks in Alzheimer's disease: implications in future therapy.

    PubMed

    Fisher, Abraham; Pittel, Zipora; Haring, Rachel; Bar-Ner, Nira; Kliger-Spatz, Michal; Natan, Niva; Egozi, Inbal; Sonego, Hagar; Marcovitch, Itzhak; Brandeis, Rachel

    2003-01-01

    M1 muscarinic receptors (M1 mAChRs) play a role in an apparent linkage of three major hallmarks of Alzheimer's disease (AD): beta-amyloid (Abeta) peptide; tau hyperphosphorylation and paired helical filaments (PHFs); and loss of cholinergic function conducive to cognitive impairments. We evaluated the M1 muscarinic agonists AF102B (Cevimeline, EVOXAC trade mark : prescribed for Sjøgren's syndrome), AF150(S), and AF267B on some of these hallmarks of AD. Activation of M1 mAChRs with these agonists leads, inter alia, to enhanced secretion of amyloid precursor protein (alpha-APP), (via alpha-secretase activation), to decreased Abeta (via gamma-secretase inhibition), and to inhibition of Abeta- and/or oxidative stress-induced cell death. In several animal models mimicking different aspects of AD, these drugs restored cognitive impairments, and in select cases induced a decrease in brain Abeta elevation, with a high safety margin, following po administration. Notably, in mice with small hippocampi, unlike rivastigmine and nicotine, AF150(S) and AF267B restored cognitive impairments also on escape latency in a Morris water maze paradigm, in reversal learning. Studies from other labs showed that AF102B and talsaclidine (another M1 agonist) decreased cerbrospinal fluid (CSF) Abeta in AD patients following chronic treatment, being the first reported drugs with such a profile. The clinical significance of these studies remains to be elucidated, yet based on in vivo (rabbits) and in vitro studies (cell cultures), our M1 agonists can decrease brain Abeta, owing to a novel and dual complementary effect (e.g., inhibition of gamma-secretase and activation of alpha-secretase). Remarkably, although M1 agonists can decrease CSF Abeta in AD patients, an increased AD-type pathology in Parkinson's disease was recently been associated with chronic antimuscarinic treatment. In another aspect, these agonists decreased tau hyperphosphorylation in vitro and in vivo. Notably, nicotinic

  18. 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

  19. 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

  20. 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

  1. 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

  2. Performance of conventional pigs and Göttingen miniature pigs in a spatial holeboard task: effects of the putative muscarinic cognition impairer Biperiden

    PubMed Central

    2013-01-01

    Background The pig is emerging as a model species that bridges the gap between rodents and humans in research. In particular, the miniature pig (referred to hereafter as the minipig) is increasingly being used as non-rodent species in pharmacological and toxicological studies. However, there is as yet a lack of validated behavioral tests for pigs, although there is evidence that the spatial holeboard task can be used to assess the working and reference memory of pigs. In the present study, we compared the learning performance of commercial pigs and Göttingen minipigs in a holeboard task. Methods Biperiden, a muscarinic M1 receptor blocker, is used to induce impairments in cognitive function in animal research. The two groups of pigs were treated orally with increasing doses of biperiden (0.05 – 20 mg.kg-1) after they had reached asymptotic performance in the holeboard task. Results Both the conventional pigs and the Göttingen minipigs learned the holeboard task, reaching nearly errorless asymptotic working and reference memory performance within approximately 100 acquisition trials. Biperiden treatment affected reference, but not working, memory, increasing trial duration and the latency to first hole visit at doses ≥ 5 mg.kg-1. Conclusion Both pig breeds learned the holeboard task and had a comparable performance. Biperiden had only a minor effect on holeboard performance overall, and mainly on reference memory performance. The effectiveness needs to be evaluated further before definitive conclusions can be drawn about the ability of this potential cognition impairer in pigs. PMID:23305134

  3. 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

  4. 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.

  5. 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.

  6. Presynaptic muscarinic receptors, calcium channels, and protein kinase C modulate the functional disconnection of weak inputs at polyinnervated neonatal neuromuscular synapses.

    PubMed

    Santafe, M M; Garcia, N; Lanuza, M A; Tomàs, M; Besalduch, N; Tomàs, J

    2009-04-01

    We studied the relation among calcium inflows, voltage-dependent calcium channels (VDCC), presynaptic muscarinic acetylcholine receptors (mAChRs), and protein kinase C (PKC) activity in the modulation of synapse elimination. We used intracellular recording to determine the synaptic efficacy in dually innervated endplates of the levator auris longus muscle of newborn rats during axonal competition in the postnatal synaptic elimination period. In these dual junctions, the weak nerve terminal was potentiated by partially reducing calcium entry (P/Q-, N-, or L-type VDCC-specific block or 500 muM magnesium ions), M1- or M4-type selective mAChR block, or PKC block. Moreover, reducing calcium entry or blocking PKC or mAChRs results in unmasking functionally silent nerve endings that now recover neurotransmitter release. Our results show interactions between these molecules and indicate that there is a release inhibition mechanism based on an mAChR-PKC-VDCC intracellular cascade. When it is fully active in certain weak motor axons, it can depress ACh release and even disconnect synapses. We suggest that this mechanism plays a central role in the elimination of redundant neonatal synapses, because functional axonal withdrawal can indeed be reversed by mAChRs, VDCCs, or PKC block.

  7. 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.

  8. 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.

  9. 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

  10. Effects of (-)-S-2,8-dimethyl-3-methylene-1-oxa-8-azaspiro[4,5]decane L-tartrate monohydrate (YM796), a novel muscarinic agonist, on disturbance of passive avoidance learning behavior in drug-treated and senescence-accelerated mice.

    PubMed

    Suzuki, M; Yamaguchi, T; Ozawa, Y; Ohyama, M; Yamamoto, M

    1995-11-01

    Effects of YM796 (-)-S-2,8-dimethyl-3-methylene-1-oxa-8-azaspiro[4,5]decane L-tartrate monohydrate; a novel muscarinic agonist, were observed on disturbance of passive avoidance learning behavior in drug- (protein synthesis inhibitor and anticholinergic drugs) treated and senescence-accelerated mice in comparison with those of a muscarinic agonist (AF102B) and acetylcholinesterase inhibitors (E2020 (1-benzyl-4-[(5,6-dimethoxy-1-indanone-2-yl) methyl] piperidene hydrochloride), NIK247 [9-amino-2,3,5,6,7,8-hexahydro-1H-cyclopenta(b)-quinoline monohydrate hydrochloride], THA (9-amino-1,2,3,4-tetrahydroacridine) and physostigmine). All tested drugs administered before training significantly prolonged the shortened latency of step-through induced by the protein synthesis inhibitor cycloheximide (150 mg/kg s.c.). This shortened latency was also significantly prolonged when YM796 was administered immediately after training, but not when administered before the test trial. The ameliorating effect of YM796 on the impairment in learning behavior by cycloheximide was significantly suppressed by pirenzepine (0.1 micrograms/mouse i.c.v.). When administered before training, all test drugs prolonged the shortened latency of step-through induced by treatment with the anticholinergic drugs [scopolamine (1 mg/kg s.c.) and hemicholinium-3 (0.3 microgram/mouse i.c.v.)], suggesting that they ameliorated the impairment of learning behavior. This shortened latency in scopolamine-treated mice was also significantly prolonged by YM796, AF102B, E2020, NIK247 and physostigmine when administered immediately after training, but not when administered before the test trial. The pharmacological actions of YM796 administered immediately after training and before the test trial in hemicholinium-3-treated mice were similar to those in scopolamine-treated mice.(ABSTRACT TRUNCATED AT 250 WORDS)

  11. Central obscuration effects on optical synthetic aperture imaging

    NASA Astrophysics Data System (ADS)

    Wang, Xue-wen; Luo, Xiao; Zheng, Li-gong; Zhang, Xue-jun

    2014-02-01

    Due to the central obscuration problem exists in most optical synthetic aperture systems, it is necessary to analyze its effects on their image performance. Based on the incoherent diffraction limited imaging theory, a Golay-3 type synthetic aperture system was used to study the central obscuration effects on the point spread function (PSF) and the modulation transfer function (MTF). It was found that the central obscuration does not affect the width of the central peak of the PSF and the cutoff spatial frequency of the MTF, but attenuate the first sidelobe of the PSF and the midfrequency of the MTF. The imaging simulation of a Golay-3 type synthetic aperture system with central obscuration proved this conclusion. At last, a Wiener Filter restoration algorithm was used to restore the image of this system, the images were obviously better.

  12. 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

  13. 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.

  14. Simultaneous determination of mushroom toxins α-amanitin, β-amanitin and muscarine in human urine by solid-phase extraction and ultra-high-performance liquid chromatography coupled with ultra-high-resolution TOF mass spectrometry.

    PubMed

    Tomková, Jana; Ondra, Peter; Válka, Ivo

    2015-06-01

    This paper presents a method for the simultaneous determination of α-amanitin, β-amanitin and muscarine in human urine by solid-phase extraction (SPE) and ultra-high-performance liquid chromatography coupled with ultra-high-resolution TOF mass spectrometry. The method can be used for a diagnostics of mushroom poisonings. Different SPE cartridges were tested for sample preparation, namely hydrophilic modified reversed-phase (Oasis HLB) and polymeric weak cation phase (Strata X-CW). The latter gave better results and therefore it was chosen for the subsequent method optimization and partial validation. In the course of validation, limits of detection, linearity, intraday and interday precisions and recoveries were evaluated. The obtained LOD values of α-amanitin and β-amanitin were 1ng/mL and of muscarine 0.09ng/mL. The intraday and interday precisions of human urine spiked with α-amanitin (10ng/mL), β-amanitin (10ng/mL) and muscarine (1ng/mL) ranged from 6% to 10% and from 7% to 13%, respectively. The developed method was proved to be a relevant tool for the simultaneous determination of the studied mushroom toxins in human urine after mushroom poisoning. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  15. 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

  16. Memory Enhancement Induced by Post-Training Intrabasolateral Amygdala Infusions of [beta]-Adrenergic or Muscarinic Agonists Requires Activation of Dopamine Receptors: Involvement of Right, but Not Left, Basolateral Amygdala

    ERIC Educational Resources Information Center

    LaLumiere, Ryan T.; McGaugh, James L.

    2005-01-01

    Previous findings indicate that the noradrenergic, dopaminergic, and cholinergic innervations of the basolateral amygdala (BLA) modulate memory consolidation. The current study investigated whether memory enhancement induced by post-training intra-BLA infusions of a [beta]-adrenergic or muscarinic cholinergic agonist requires concurrent activation…

  17. 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

  18. Analogues of the muscarinic agent 2'-methylspiro[1-azabicyclo[2.2.2]octane-3,4'-[1,3]dioxolane]: synthesis and pharmacology.

    PubMed

    Nordvall, G; Sundquist, S; Glas, G; Gogoll, A; Nilvebrant, L; Hacksell, U

    1992-05-01

    A number of tetrahydrofuran analogues of 2'-methylspiro[1-azabicyclo[2.2.2]octane-3,4'-[1,3]dioxolane] (1) have been prepared with the aim to obtain information about the relative importance of each of the oxygens in 1 for efficacy and for selectivity. In addition, the dimethyl and desmethyl analogues of 1 were prepared. The new compounds were compared to cis- and trans-1 with regard to their ability to displace (-)-[3H]-3-quinuclidinyl benzilate ((-)-[3H]QNB) from muscarinic receptors in cerebral cortex, heart, parotid gland, and urinary bladder from guinea pigs. Functional studies were made on isolated guinea pig bladder and ileum. The new compounds exhibited both lower affinity and efficacy than cis-1. A conformational study was performed, and the effects of steric and electronic factors on the biological activity of the compounds are discussed.

  19. 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.

  20. 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

  1. The Central Nervous System and Bone Metabolism: An Evolving Story.

    PubMed

    Dimitri, Paul; Rosen, Cliff

    2017-05-01

    Our understanding of the control of skeletal metabolism has undergone a dynamic shift in the last two decades, primarily driven by our understanding of energy metabolism. Evidence demonstrating that leptin not only influences bone cells directly, but that it also plays a pivotal role in controlling bone mass centrally, opened up an investigative process that has changed the way in which skeletal metabolism is now perceived. Other central regulators of bone metabolism have since been identified including neuropeptide Y (NPY), serotonin, endocannabinoids, cocaine- and amphetamine-regulated transcript (CART), adiponectin, melatonin and neuromedin U, controlling osteoblast and osteoclast differentiation, proliferation and function. The sympathetic nervous system was originally identified as the predominant efferent pathway mediating central signalling to control skeleton metabolism, in part regulated through circadian genes. More recent evidence points to a role of the parasympathetic nervous system in the control of skeletal metabolism either through muscarinic influence of sympathetic nerves in the brain or directly via nicotinic receptors on osteoclasts, thus providing evidence for broader autonomic skeletal regulation. Sensory innervation of bone has also received focus again widening our understanding of the complex neuronal regulation of bone mass. Whilst scientific advance in this field of bone metabolism has been rapid, progress is still required to understand how these model systems work in relation to the multiple confounders influencing skeletal metabolism, and the relative balance in these neuronal systems required for skeletal growth and development in childhood and maintaining skeletal integrity in adulthood.

  2. The Beneficial Effect of Fesoterodine, a Competitive Muscarinic Receptor Antagonist on Erectile Dysfunction in Streptozotocin-induced Diabetic Rats.

    PubMed

    Yilmaz-Oral, Didem; Bayatli, Nur; Gur, Serap

    2017-09-01

    To investigate the possible role of fesoterodine (a competitive muscarinic receptor antagonist) on erectile dysfunction in streptozotocin-induced diabetic rats. A total of 16 adult male Sprague-Dawley rats were equally divided into control and diabetic groups. Diabetes was induced by a single intravenous injection of streptozotocin (25-35 mg/kg). In vivo erectile responses were evaluated by the stimulation of cavernosal nerves, and measurements were repeated after the intracavernosal injection of fesoterodine (1 µM) in rats. The relaxation responses to fesoterodine were examined via incubation with various inhibitors. The relaxant responses of corpus cavernosum (CC) strips were observed in the presence or the absence of fesoterodine (10 µM). Intracavernous administration of fesoterodine restored in vivo erectile response at 5.0- and 7.5-V levels, except for 2.5 V in diabetic rats. Basal intracavernosal pressure (5.4 ± 0.9 mm Hg) in diabetic rats was markedly increased after injection of fesoterodine (33.9 ± 7.9 mm Hg, P <.001). In bath studies, fesoterodine resulted in a relaxation of CC in a concentration-dependent manner, which was reduced in diabetic rats. Nifedipine (l-type Ca 2+ channel blocker) inhibited maximum fesoterodine-induced relaxation by 58%. The nonselective K + channel blocker tetraethylammonium and glibenclamide incubation did not change the relaxant response to fesoterodine. The relaxant responses to acetylcholine (10 µM), electrical field stimulation (10 Hz), and sodium nitroprusside (0.01 µM) in diabetic rats were increased after incubation with fesoterodine (10 µM). Fesoterodine improved erectile function and relaxation of isolated strips of rat CC. The underlying mechanism of fesoterodine is likely due to the blocking of l-type calcium channels independent of the nitric oxide-cyclic guanosine monophosphate pathway. Further investigations are warranted to fully elucidate the restorative effects of

  3. Muscarinic receptors, nitric oxide formation and cyclooxygenase pathway involved in tracheal smooth muscle relaxant effect of hydro-ethanolic extract of Lavandula angustifolia flowers.

    PubMed

    Naghdi, Farzaneh; Gholamnezhad, Zahra; Boskabady, Mohammad Hossein; Bakhshesh, Morteza

    2018-06-01

    L. angustifolia that was lower than the effect of theophylline. The possible mechanisms of relaxant effect of this plant on tracheal smooth muscle are muscarinic receptors blockade, inhibition of cyclooxygenase pathways and/or involvement of nitric oxide production. Its clinical applications should be investigated in further studies. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  4. [The effect of atropine on the ultrastructural postsynaptic plasticity of the associative type in the rat neocortex].

    PubMed

    Khludova, G G; Gusev, P A

    1998-01-01

    The effect of muscarinic antagonist atropine on thickness of postsynaptic density of axodendritic synapses was studied in the sensorimotor region of the brain cortex of rats during paired repeated microapplication of glutamate and acetylcholine. In the applied conditioning paradigm atropine significantly decreased morphological dimensions of the postsynaptic density, however, the control values were not reached. This finding testifies to participation of both muscarinic and nicotinic cholinoreceptors in associative postsynaptic plasticity.

  5. 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.

  6. 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

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. Curcumin restores diabetes induced neurochemical changes in the brain stem of Wistar rats.

    PubMed

    Kumar, Peeyush T; George, Naijil; Antony, Sherin; Paulose, Cheramadathikudiyil Skaria

    2013-02-28

    Diabetes mellitus, when poorly controlled, leads to debilitating central nervous system (CNS) complications including cognitive deficits, somatosensory and motor dysfunction. The present study investigated curcumin's potential in modulating diabetes induced neurochemical changes in brainstem. Expression analysis of cholinergic, insulin receptor and GLUT-3 in the brainstem of streptozotocin (STZ) induced diabetic rats were studied. Radioreceptor binding assays, gene expression studies and immunohistochemical analysis were done in the brainstem of male Wistar rats. Our result showed that Bmax of total muscarinic and muscarinic M3 receptors were increased and muscarinic M1 receptor was decreased in diabetic rats compared to control. mRNA level of muscarinic M3, α7-nicotinic acetylcholine, insulin receptors, acetylcholine esterase, choline acetyltransferase and GLUT-3 significantly increased and M1 receptor decreased in the brainstem of diabetic rats. Curcumin and insulin treatment restored the alterations and maintained all parameters to near control. The results show that diabetes is associated with significant reduction in brainstem function coupled with altered cholinergic, insulin receptor and GLUT-3 gene expression. The present study indicates beneficial effect of curcumin in diabetic rats by regulating the cholinergic, insulin receptor and GLUT-3 in the brainstem similar to the responses obtained with insulin therapy. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. Muscarinic agonists and phorbol esters increase tyrosine phosphorylation of a 40-kilodalton protein in hippocampal slices

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

    Stratton, K.R.; Worley, P.F.; Huganir, R.L.

    The authors have used the hippocampal slice preparation to investigate the regulation of protein tyrosine phosphorylation in brain. After pharmacological treatment of intact slices, proteins were separated by electrophoresis, and levels of protein tyrosine phosphorylation were assessed by immunoblotting with specific anti-phosphotyrosine antibodies. Phorbol esters, activators of the serine- and threonine-phosphorylating enzyme protein kinase C, selectively increase tyrosine phosphorylation of a soluble protein with an apparent molecular mass of approximately 40 kilodaltons. Muscarinic agonists such as carbachol and oxotremorine M that strongly activate the inositol phospholipid system also increase tyrosine phosphorylation of this protein. Neurotransmitter activation of the inositol phospholipidmore » system and protein kinase C appears to trigger a cascade leading to increased tyrosine phosphorylation.« less

  13. 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

  14. 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.

  15. The effects of oxotremorine, epibatidine, atropine, mecamylamine and naloxone in the tail-flick, hot-plate, and formalin tests in the naked mole-rat (Heterocephalus glaber).

    PubMed

    Dulu, Thomas D; Kanui, Titus I; Towett, Philemon K; Maloiy, Geoffrey M; Abelson, Klas S P

    2014-01-01

    The naked mole-rat (Heterocephalus glaber) is a promising animal model for the study of pain mechanisms, therefore a thorough characterization of this species is essential. The aim of the present study was to establish the naked mole-rat as a model for studying the cholinergic receptor system in antinociception by investigating the involvement of muscarinic, nicotinic and opioid receptors in nociceptive tests in this species. The effects of systemic administration of the muscarinic receptor agonist oxotremorine and the nicotinic receptor agonist epibatidine were investigated in the tail-flick, the hot-plate, and the formalin tests. The effects of co-administration of the muscarinic receptor antagonist atropine, the nicotinic receptor antagonist mecamylamine, and the opioid receptor antagonist naloxone were also investigated. Oxotremorine and epibatidine induced a significant, dose-dependent antinociceptive effect in the tail-flick, hot-plate, and formalin tests, respectively. The effects of oxotremorine and epibatidine were blocked by atropine and mecamylamine, respectively. In all three nociceptive tests, naloxone in combination with oxotremorine or epibatidine enhanced the antinociceptive effects of the drugs. The present study demonstrated that stimulation of muscarinic and nicotinic receptors produces antinociceptive effects in the naked-mole rat. The reversal effect of atropine and mecamylamine suggests that this effect is mediated by cholinergic receptors. As naloxone increases the antinociceptive effects of cholinergic agonists, it is suggested that the cholinergic antinociception acts via a gateway facilitated by opioid receptor blockage; however, the precise interaction between these receptor systems needs further investigation.

  16. Alteration in behavioral sensitivity to amphetamine after treatment with oxotremorine. Effect of dose and test environment.

    PubMed

    Gralewicz, Sławomir; Lutz, Piotr; Wiaderna, Dorota; Tomas, Tadeusz

    2003-12-17

    Our earlier experiment revealed that rats pretreated once with an anticholinesterase develop hyposensitivity to amphetamine (AMPH). One of the likely causes of this effect might be a transient hyperexcitation of the central muscarinic receptors. It has appeared, however, that rats pretreated with oxotremorine (OX), a muscarinic agonist, show an augmented behavioral response to AMPH weeks later. The present experiments were performed in order to obtain more information on the relationship between the OX-induced sensitization to AMPH and the OX dose and dosing regime (single or repeated), and to find out whether the environment associated with the acute effects of OX could affect the response to AMPH. In experiment 1, adult male rats were given a single i.p. injection of OX in home cages at a moderate (0.5 mg/kg) or high (1.0 mg/kg) dose. In experiment 2, the rats received eight 1.0 mg/kg doses of OX in the course of three days. After each injection, some animals returned to their home cages, and some were placed in the test cages for 30 min. In both experiments, the response to AMPH was assessed on day 21 after the treatment. The obtained results indicate that: (i) a single i.p. exposure to OX results in an increase of the rat's behavioral sensitivity to AMPH but the moderate dose is more effective in inducing this effect; (ii) repeated exposure to OX at high doses, in a regime enabling development of tolerance to the acute OX effects, does not alter the rat sensitivity to AMPH, and (iii) expression of the AMPH response is suppressed in environment which has been associated with acute effects of OX.

  17. Age-Related Decrements in the Muscarinic Enhancement of K(+)-Evoked Release of Endogenous Striatal Dopamine: An Indicator of Altered Cholinergic-Dopaminergic Reciprocal Inhibitory Control in Senescence

    DTIC Science & Technology

    1988-01-01

    oxotremorine , pilocarpine, carbachol or bethanecol) or nicotinic (nicotine) agonistt In some experiments DA autoreceptor function was assessed...muscarinic (e.g. oxotremorine , carbachol, be- studies using the ligand, [3H](-)-quinuclidinyl benzi- thanecol) or nicotinic (e.g. nicotine) agonists can... oxotremorine MATERIALS AND METHODS r or carbachol in striatal broken cell preparations pre- pared from young rats (6 months), these agents were Procedure

  18. Central command does not suppress baroreflex control of cardiac sympathetic nerve activity at the onset of spontaneous motor activity in the decerebrate cat.

    PubMed

    Matsukawa, Kanji; Ishii, Kei; Asahara, Ryota; Idesako, Mitsuhiro

    2016-10-01

    Our laboratory has reported that central command blunts the sensitivity of the aortic baroreceptor-heart rate (HR) reflex at the onset of voluntary static exercise in animals. We have examined whether baroreflex control of cardiac sympathetic nerve activity (CSNA) and/or cardiovagal baroreflex sensitivity are altered at the onset of spontaneously occurring motor behavior, which was monitored with tibial nerve activity in paralyzed, decerebrate cats. CSNA exhibited a peak increase (126 ± 17%) immediately after exercise onset, followed by increases in HR and mean arterial pressure (MAP). With development of the pressor response, CSNA and HR decreased near baseline, although spontaneous motor activity was not terminated. Atropine methyl nitrate (0.1-0.2 mg/kg iv) with little central influence delayed the initial increase in HR but did not alter the response magnitudes of HR and CSNA, while atropine augmented the pressor response. The baroreflex-induced decreases in CSNA and HR elicited by brief occlusion of the abdominal aorta were challenged at the onset of spontaneous motor activity. Spontaneous motor activity blunted the baroreflex reduction in HR by aortic occlusion but did not alter the baroreflex inhibition of CSNA. Similarly, atropine abolished the baroreflex reduction in HR but did not influence the baroreflex inhibition of CSNA. Thus it is likely that central command increases CSNA and decreases cardiac vagal outflow at the onset of spontaneous motor activity while preserving baroreflex control of CSNA. Accordingly, central command must attenuate cardiovagal baroreflex sensitivity against an excess rise in MAP as estimated from the effect of muscarinic blockade. Copyright © 2016 the American Physiological Society.

  19. Presence of central nervous system, cardiovascular and overall co-morbidity burden in patients with overactive bladder disorder in a real-world setting.

    PubMed

    Asche, Carl V; Kim, Jaewhan; Kulkarni, Amit S; Chakravarti, Paula; Andersson, Karl-Erik

    2012-02-01

    •  To determine the proportion of patients with overactive bladder (OAB) potentially at risk for adverse events by assessing their pre-existing central nervous system (CNS), cardiovascular (CV) and other co-morbidities. •  The GE Centricity Electronic Medical Record database was utilized to identify patients with a diagnosis of OAB using International Classification of Diseases, Ninth Revision (ICD-9) codes or a prescription between 1 January 1996 and 30 March 2007 for an OAB anti-muscarinic agent. •  Matched non-OAB patients were assigned the same index date as the corresponding OAB patient. Based on the presence of ≥ one pharmacy claim for an OAB anti-muscarinic agent, the OAB cohort was stratified as treated or untreated. A random sample of age- and gender-matched patients formed a non-OAB control cohort. •  An additional and separate analysis focusing on all co-morbidities was performed examining non-OAB patients who were matched to OAB patients on 1:1 propensity score matching, based on age, body mass index (BMI) and gender at baseline. •  Charlson Comorbidity Index (CCI), using ICD-9 codes, and the Chronic Disease Score (CDS), using prescribed drugs, were calculated. •  When compared with non-OAB patients (N= 77,272; 83.2% women; median age 64 years), OAB patients (N= 41,440; 83.6% women; median age 65 years) had more overall CNS co-morbidities (45.4 vs 29.0%; P < 0.001). •  In addition, OAB patients had a higher use of medications with anti-muscarinic effects (39.6 vs 25.4%; P < 0.001). OAB patients were also more likely to have CV co-morbidities (57.6 vs 44.6%; P < 0.001). •  CNS co-morbidities were slightly more common in untreated (n= 8 106) than in treated (n= 33 334) OAB patients (47.2 vs 45.0%; P < 0.001). CV co-morbidities were higher in treated OAB patients (58.8 vs 53.7%; P < 0.001). •  In the additional separate analysis, which focused on all co-morbidities, patients with OAB had higher mean CCI and CDS scores

  20. 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

  1. Nonmuscarinic Neurotoxicity of Oxotremorine,

    DTIC Science & Technology

    The ability of various treatments to prevent peripheral parasympathetic actions, central effects and lethality of the muscarinic agonist oxotremorine ...was studied in rats. The percentage of animals exhibiting effects of oxotremorine was dose and time dependent. Doses of oxotremorine in excess of 5...salivation were not present in atropine-treated rats. In the presence of 40 mg/ kg of atropine, ED50 values for oxotremorine were shifted more that 12

  2. Neurophysiological effects of mistletoe (Viscum album L.) on isolated rat intestines.

    PubMed

    Radenkovic, M; Ivetic, V; Popovic, M; Mimica-Dukic, N; Veljkovic, S

    2006-05-01

    Mistletoe (Viscum album L.) is well known as a medicine from ancient times and the earliest notes. Today it is used as a remedy. The aim of this research was to examine the effects of mistletoe extracts and their components on some neurophysiological parameters in rat intestines. The tonus and contractile responses of isolated intestinal segments (duodenum, ileum and distal colon) were analysed. The experiment was carried out in three groups. In the first group (control group) different concentrations of acetylcholine were added into the organ bath (10-50 nmol/L). In the second group, mistletoe extracts were added into the organ bath with increasing concentrations and in the third group, atropine, a non-selective muscarinic receptor antagonist, was added into the organ bath (concentration 10(-7) mol/L) and after atropine plant extracts were administered. The results obtained suggest that extracts from different parts of mistletoe have neurophysiological effects and change intestinal contractions. The results also suggest that the effects of mistletoe extracts on intestinal contractility act via cholinergic pathways, activating muscarinic receptors in the intestines. However, in order to establish the subtype of receptors, further investigations are necessary where selective antagonists of muscarinic cholinergic receptors should be used. Copyright 2006 John Wiley & Sons, Ltd.

  3. 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.

  4. 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

  5. [Modulation of the cholinergic system during inflammation].

    PubMed

    Nezhinskaia, G I; Vladykin, A L; Sapronov, N S

    2008-01-01

    This review describes the effects of realization of the central and peripheral "cholinergic antiinflammatory pathway" in a model of endotoxic and anaphylactic shock. Under endotoxic shock conditions, a pharmacological correction by means of the central m-cholinomimetic action (electrical stimulation of the distal ends of nervus vagus after bilateral cervical vagotomy, surgical implantation of the stimulant devise, activation of efferent vagal neurons by means of muscarinic agonist) is directed toward the elimination of LPS-induced hypotension. During the anaphylaxis, peripheral effects of the cholinergic system induced by blocking m-AChR on the target cells (neuronal and non-neuronal lung cells) and acetylcholinesterase inhibition are related to suppression of the bronchoconstrictor response. The role of immune system in the pathogenesis of endotoxic shock is associated with the production of proinflammatory cytokines by macrophages, increase in IgM concentration, and complement activation, while the role in the pathogenesis of anaphylactic shock is associated with IgE, IgG1 augmentation. Effects of B cell stimulation may be important in hypoxia and in the prophylaxis of stress ulcers and other diseases. Plasma proteins can influence the effects of the muscarinic antagonist methacine: IgG enhance its action while albumin and CRP abolish it.

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

    PubMed

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

    2016-10-04

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

  7. 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.

  8. Muscarinic Receptor Occupancy and Cognitive Impairment: A PET Study with [11C](+)3-MPB and Scopolamine in Conscious Monkeys

    PubMed Central

    Yamamoto, Shigeyuki; Nishiyama, Shingo; Kawamata, Masahiro; Ohba, Hiroyuki; Wakuda, Tomoyasu; Takei, Nori; Tsukada, Hideo; Domino, Edward F

    2011-01-01

    The muscarinic cholinergic receptor (mAChR) antagonist scopolamine was used to induce transient cognitive impairment in monkeys trained in a delayed matching to sample task. The temporal relationship between the occupancy level of central mAChRs and cognitive impairment was determined. Three conscious monkeys (Macaca mulatta) were subjected to positron emission tomography (PET) scans with the mAChR radioligand N-[11C]methyl-3-piperidyl benzilate ([11C](+)3-MPB). The scan sequence was pre-, 2, 6, 24, and 48 h post-intramuscular administration of scopolamine in doses of 0.01 and 0.03 mg/kg. Occupancy levels of mAChR were maximal 2 h post-scopolamine in cortical regions innervated primarily by the basal forebrain, thalamus, and brainstem, showing that mAChR occupancy levels were 43–59 and 65–89% in doses of 0.01 and 0.03 mg/kg, respectively. In addition, dose-dependent impairment of working memory performance was measured 2 h after scopolamine. A positive correlation between the mAChR occupancy and cognitive impairment 2 and 6 h post-scopolamine was the greatest in the brainstem (P<0.00001). Although cognitive impairment was not observed 24 h post-scopolamine, sustained mAChR occupancy (11–24%) was found with both doses in the basal forebrain and thalamus, but not in the brainstem. These results indicate that a significant degree of mAChRs occupancy is needed to produce cognitive impairment by scopolamine. Furthermore, the importance of the brainstem cholinergic system in working memory in monkey is described. PMID:21430646

  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. 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.

  11. Effects of a neurotensin analogue (PD149163) and antagonist (SR142948A) on the scopolamine-induced deficits in a novel object discrimination task.

    PubMed

    Azmi, Norazrina; Norman, Christine; Spicer, Clare H; Bennett, Geoffrey W

    2006-06-01

    Various lines of evidence suggest a role in cognition for the endogenous neuropeptide, neurotensin, involving an interaction with the central nervous system cholinergic pathways. A preliminary study has shown that central administration of neurotensin enhances spatial and nonspatial working memory in the presence of scopolamine, a muscarinic receptor antagonist which induces memory deficits. Utilizing similar methods, the present study employed a two-trial novel object discrimination task to determine the acute effect of a neurotensin peptide analogue with improved metabolic stability, PD149163, on recognition memory in Lister hooded rats. Consistent with previous findings with neurotensin, animals receiving an intracerebroventricular injection of PD149163 (3 microg) significantly discriminated the novel from familiar object during the choice trial. In addition, a similar dose of PD149163 restored the scopolamine-induced deficit in novelty recognition. The restoration effect on scopolamine-induced amnesia produced by PD149163 was blocked by SR142948A, a nonselective neurotensin receptor antagonist, at a dose of 1 mg/kg (intraperitonial) but not at 0.1 mg/kg. In conclusion, the present results confirm a role for neurotensin in mediating memory processes, possibly via central cholinergic mechanisms.

  12. Dual autonomic inhibitory action of central Apelin on gastric motor functions in rats.

    PubMed

    Bülbül, Mehmet; Sinen, Osman

    2018-07-01

    Centrally administered apelin has been shown to inhibit gastric emptying (GE) in rodents, however, the relevant mechanism has been investigated incompletely. Using male Wistar rats, we investigated the efferent pathways involved in gastroinhibitory action of central apelin. Stereotaxic intracerebroventricular (icv) cannulation, subdiaphragmatic vagotomy (VGX) and/or celiac ganglionectomy (CGX) were performed 7 days prior to the experiments. Apelin-13 was administered (30 nmol, icv) 90 min prior to GE measurement. Nitric oxide synthase inhibitor L-NAME (100 mg/kg), sympatholytic agent guanethidine (5 mg/kg) and/or muscarinic receptor agonist bethanechol (1 mg/kg) were administered intraperitoneally 30 min prior to the central apelin-13 injection. Two strain gages were implanted serosally onto antrum and pylorus to monitor gastric postprandial motility. Heart rate variability (HRV) analysis was performed before and after central vehicle or apelin-13 administration. Apelin-13 delayed solid GE significantly by disturbing coordinated antral and pyloric postprandial contractions. The apelin-induced delayed GE was attenuated partially by CGX or VGX, whereas it was restored completely in rats underwent both CGX and VGX. L-NAME did not change the apelin-induced alterations. Guanethidine or bethanechol restored the apelin-induced gastroinhibition partially, while it was abolished completely in rats received both agents. Apelin-13 decreased the HRV spectral activity in high-frequency range by increasing low-frequency component and the ratio of LF:HF. The present data suggest that (1) both vagal parasympathetic and sympathetic pathways play a role in apelin-induced gastroinhibition, (2) central apelin attenuates vagal cholinergic pathway rather than activating nonadrenergic-noncholinergic pathway. Apelin/APJ receptor system might be candidate for the treatment of autonomic dysfunction and gastrointestinal motor disorders. Copyright © 2018 Elsevier B.V. All

  13. 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.

  14. 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.

  15. Reversal of androgen inhibition of estrogen-activated sexual behavior by cholinergic agents.

    PubMed

    Dohanich, G P; Cada, D A

    1989-12-01

    Androgens have been found to inhibit lordosis activated by estrogen treatment of ovariectomized female rats. In the present experiments, dihydrotestosterone propionate (200 micrograms for 3 days) inhibited the incidence of lordosis in ovariectomized females treated with estradiol benzoate (1 microgram for 3 days). This inhibition of lordosis was reversed 15 min after bilateral intraventricular infusion of physostigmine (10 micrograms/cannula), an acetylcholinesterase inhibitor, or carbachol (0.5 microgram/cannula), a cholinergic receptor agonist. This reversal of inhibition appears to be mediated by cholinergic muscarinic receptors since pretreatment with scopolamine (4 mg/kg, ip), a muscarinic receptor blocker, prevented the reversal of androgen inhibition by physostigmine. These results indicate that androgens may inhibit estrogen-activated lordosis through interference with central cholinergic muscarinic mechanisms.

  16. 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

  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. 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

  19. 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.

  20. 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.

  1. 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.

  2. 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

  3. 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

  4. Central tendency effects in time interval reproduction in autism

    PubMed Central

    Karaminis, Themelis; Cicchini, Guido Marco; Neil, Louise; Cappagli, Giulia; Aagten-Murphy, David; Burr, David; Pellicano, Elizabeth

    2016-01-01

    Central tendency, the tendency of judgements of quantities (lengths, durations etc.) to gravitate towards their mean, is one of the most robust perceptual effects. A Bayesian account has recently suggested that central tendency reflects the integration of noisy sensory estimates with prior knowledge representations of a mean stimulus, serving to improve performance. The process is flexible, so prior knowledge is weighted more heavily when sensory estimates are imprecise, requiring more integration to reduce noise. In this study we measure central tendency in autism to evaluate a recent theoretical hypothesis suggesting that autistic perception relies less on prior knowledge representations than typical perception. If true, autistic children should show reduced central tendency than theoretically predicted from their temporal resolution. We tested autistic and age- and ability-matched typical children in two child-friendly tasks: (1) a time interval reproduction task, measuring central tendency in the temporal domain; and (2) a time discrimination task, assessing temporal resolution. Central tendency reduced with age in typical development, while temporal resolution improved. Autistic children performed far worse in temporal discrimination than the matched controls. Computational simulations suggested that central tendency was much less in autistic children than predicted by theoretical modelling, given their poor temporal resolution. PMID:27349722

  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. 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. 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

  8. Acute Effects of Anticholinesterase Agents on Pupillary Function.

    DTIC Science & Technology

    1984-09-15

    stimulated by ACh or oxotremorine results in release inhibition. We assume that DFP via increased ACh levels, acts at this receptor to inhibit ACh release...muscarinic ligands. The following muscarinic ligands will be employed: 1) (N-methyl- 3H) oxotremorine M acetate 2) l-quinaclidinyl[phenyl-4-3H]bensylate 3) 1

  9. Effects of Brazilian scorpion venoms on the central nervous system.

    PubMed

    Nencioni, Ana Leonor Abrahão; Neto, Emidio Beraldo; de Freitas, Lucas Alves; Dorce, Valquiria Abrão Coronado

    2018-01-01

    In Brazil, the scorpion species responsible for most severe incidents belong to the Tityus genus and, among this group, T. serrulatus , T. bahiensis , T. stigmurus and T. obscurus are the most dangerous ones. Other species such as T. metuendus , T. silvestres, T. brazilae , T. confluens , T. costatus , T. fasciolatus and T. neglectus are also found in the country, but the incidence and severity of accidents caused by them are lower. The main effects caused by scorpion venoms - such as myocardial damage, cardiac arrhythmias, pulmonary edema and shock - are mainly due to the release of mediators from the autonomic nervous system. On the other hand, some evidence show the participation of the central nervous system and inflammatory response in the process. The participation of the central nervous system in envenoming has always been questioned. Some authors claim that the central effects would be a consequence of peripheral stimulation and would be the result, not the cause, of the envenoming process. Because, they say, at least in adult individuals, the venom would be unable to cross the blood-brain barrier. In contrast, there is some evidence showing the direct participation of the central nervous system in the envenoming process. This review summarizes the major findings on the effects of Brazilian scorpion venoms on the central nervous system, both clinically and experimentally. Most of the studies have been performed with T. serrulatus and T. bahiensis . Little information is available regarding the other Brazilian Tityus species.

  10. Muscarinic receptor stimulation of D-aspartate uptake into human SH-SY5Y neuroblastoma cells is attenuated by hypoosmolarity.

    PubMed

    Foster, Daniel J; Heacock, Anne M; Fisher, Stephen K

    2010-04-01

    In addition to its function as an excitatory neurotransmitter, glutamate plays a major role as an osmolyte within the central nervous system (CNS). Accordingly, mechanisms that regulate glutamate release and uptake are of physiological importance not only during conditions in which cell volume remains constant but also when cells are subjected to hypoosmotic stress. In the present study, the ability of muscarinic cholinergic receptors (mAChRs) to regulate the uptake of glutamate (monitored as D-aspartate) into human SH-SY5Y neuroblastoma cells under isotonic or hypotonic conditions has been examined. In isotonic media, agonist activation of mAChRs resulted in a significant increase (250-300% of control) in the uptake of D-aspartate and, concurrently, a cellular redistribution of the excitatory amino acid transporter 3 (EAAT3) to the plasma membrane. mAChR-mediated increases in d-aspartate uptake were potently blocked by the EAAT3 inhibitor l-beta-threo-benzyl-aspartate. In hypotonic media, the ability of mAChR activation to facilitate D-aspartate uptake was significantly attenuated (40-50%), and the cellular distribution of EAAT3 was disrupted. Reduction of mAChR-stimulated D-aspartate uptake under hypoosmotic conditions could be fully reversed upon re-exposure of the cells to isotonic media. Under both isotonic and hypotonic conditions, mAChR-mediated increases in D-aspartate uptake depended on cytoskeletal integrity, protein kinase C and phosphatidylinositol 3-kinase activities, and the availability of intracellular Ca2+. In contrast, dependence on extracellular Ca2+ was observed only under isotonic conditions. The results suggest that, although the uptake of D-aspartate into SH-SY5Y cells is enhanced after mAChR activation, this process is markedly attenuated by hypoosmolarity.

  11. In vitro effects of bethanechol on specimens of intestinal smooth muscle obtained from the duodenum and jejunum of healthy dairy cows

    PubMed Central

    Pfeiffer, Julia B. R.; Mevissen, Meike; Steiner, Adrian; Portier, Christopher J.; Meylan, Mireille

    2009-01-01

    Objective To describe the in vitro effects of bethanechol on contractility of smooth muscle preparations from the small intestines of healthy cows and define the muscarinic receptor subtypes involved in mediating contraction. Sample Population Tissue samples from the duodenum and jejunum collected immediately after slaughter of 40 healthy cows. Procedures Cumulative concentration-response curves were determined for the muscarinic receptor agonist bethanechol with or without prior incubation with subtype-specific receptor antagonists in an organ bath. Effects of bethanechol and antagonists and the influence of intestinal location on basal tone, maximal amplitude (Amax), and area under the curve (AUC) were evaluated. Results Bethanechol induced a significant, concentration-dependent increase in all preparations and variables. The effect of bethanechol was more pronounced in jejunal than in duodenal samples and in circular than in longitudinal preparations. Significant inhibition of the effects of bethanechol was observed after prior incubation with muscarinic receptor subtype M3 antagonists (more commonly for basal tone than for Amax and AUC). The M2 receptor antagonists partly inhibited the response to bethanechol, especially for basal tone. The M3 receptor antagonists were generally more potent than the M2 receptor antagonists. In a protection experiment, an M3 receptor antagonist was less potent than when used in combination with an M2 receptor antagonist. Receptor antagonists for M1 and M4 did not affect contractility variables. Conclusions and Clinical Relevance Bethanechol acting on muscarinic receptor subtypes M2 and M3 may be of clinical use as a prokinetic drug for motility disorders of the duodenum and jejunum in dairy cows. PMID:17331022

  12. Distinct effects of cevimeline and pilocarpine on salivary mechanisms, cardiovascular response and thirst sensation in rats.

    PubMed

    Ono, K; Inagaki, T; Iida, T; Wakasugi-Sato, N; Hosokawa, R; Inenaga, K

    2012-04-01

    Cevimeline and pilocarpine (muscarinic receptor agonists) are used as sialogogues in xerostomia treatment. It is important to know the different effects on their salivary mechanisms and the side effects. The aim of the present study was to clarify and compare the comprehensive effects of cevimeline to pilocarpine on salivary, cardiovascular and central mechanisms in rats. Under anaesthesia, whole saliva secretion, parotid blood flow and blood pressure were measured following intra-peritoneal administrations of the sialogogues. In digested parotid cells, intracellular Ca(2+) concentrations were measured after the sialogogue application. In the conscious condition, changes in angiotensin II-induced water intake were observed after cevimeline administration. In the subfornical organ, which is a thirst-related central nucleus, the effect of cevimeline on the neuronal activity was electrophysiologically investigated. Cevimeline at 80μmolkg(-1) showed slowly increasing and lasting salivation, a similar blood flow increment in the parotid gland and higher pressor response when compared to pilocarpine at 4μmolkg(-1). In parotid cells, cevimeline increased the intracellular Ca(2+) concentration in a similar manner to pilocarpine, but at a higher concentration than pilocarpine. Cevimeline inhibited angiotensin II-induced water intake and neuronal activity in the subfornical organ, which is in contrast to reported effects of pilocarpine. Cevimeline activates common salivary mechanism with pilocarpine but has a slower onset of activation, longer duration of salivation and an increased pressor response at higher doses. The anti-dipsogenic effect of cevimeline is due to the inhibitory neuronal effect on the thirst-related central nuclei. Copyright © 2011 Elsevier Ltd. All rights reserved.

  13. Biphasic effects of oxotremorine-M on turning behavior induced by caffeine in 6-OHDA-lesioned rats.

    PubMed

    Núñez-Taltavull, Juan Francisco; Prat, Gemma; Rubio, Antonia; Robledo, Patricia; Casas, Miguel

    2004-12-03

    This work studied the interactions between cholinergic and adenosine systems in the denervated striatum. For that purpose, we evaluated the effects of an intrastriatal administration of the muscarincic receptor agonist, oxotremorine-M on turning behavior induced by systemic caffeine in unilaterally 6-hydroxydopamine-lesioned rats. Low doses of oxotremorine-M (0.1 ng/microl) enhanced, whereas high doses (100 ng/microl) attenuated contralateral turning induced by caffeine. These results support a functional link between muscarinic and adenosinergic systems in the denervated striatum and suggest opposite actions of muscarinic M2 and M1 receptors on caffeine-induced turning behavior.

  14. New antimuscarinic agents for improved treatment of poisoning by cholinesterase inhibitors. Annual report, 1 November 1983-1 August 1984

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

    Stubbins, J.F.

    The object of this project is to find a more effective antimuscarinic agent than atropine for use as an antidote for poisoning by organophosphate cholinesterase inhibitors. To start this search, 30 structurally diverse antimuscarinic agents have been selected for initial testing. These compounds are to be evaluated for peripheral and central antimuscarinic activity in a variety of in vitro and in vivo tests in addition to determining their effectiveness as antidotes (in combination with an oxime reactivator) for poisoning by soman. Twenty-two of the compounds have now been evaluated for their ability to block acetylcholine-induced contractions in guinea pig intestinalmore » smooth muscle when compared to atropine. Ability to displace radiolabeled quinuclidinyl benzilate from muscarinic receptors of mouse brain homogenate has been determined for atropine, hyoscine and 26 of the compounds. Only triflupromazine appeared to have a distinctly greater affinity for brain receptors than muscle receptors to atropine. Intestinal smooth muscle blockade; oxotremorine tremor inhibition; muscarinic receptor subtypes.« less

  15. Age-dependent effects of brain stimulation on network centrality.

    PubMed

    Antonenko, Daria; Nierhaus, Till; Meinzer, Marcus; Prehn, Kristin; Thielscher, Axel; Ittermann, Bernd; Flöel, Agnes

    2018-04-18

    Functional magnetic resonance imaging (fMRI) studies have suggested that advanced age may mediate the effects of transcranial direct current stimulation (tDCS) on brain function. However, studies directly comparing neural tDCS effects between young and older adults are scarce and limited to task-related imaging paradigms. Resting-state (rs-) fMRI, that is independent of age-related differences in performance, is well suited to investigate age-associated differential neural tDCS effects. Three "online" tDCS conditions (anodal, cathodal, sham) were compared in a cross-over, within-subject design, in 30 young and 30 older adults. Active stimulation targeted the left sensorimotor network (active electrode over left sensorimotor cortex with right supraorbital reference electrode). A graph-based rs-fMRI data analysis approach (eigenvector centrality mapping) and complementary seed-based analyses characterized neural tDCS effects. An interaction between anodal tDCS and age group was observed. Specifically, centrality in bilateral paracentral and posterior regions (precuneus, superior parietal cortex) was increased in young, but decreased in older adults. Seed-based analyses revealed that these opposing patterns of tDCS-induced centrality modulation originated from differential effects of tDCS on functional coupling of the stimulated left paracentral lobule. Cathodal tDCS did not show significant effects. Our study provides first evidence for differential tDCS effects on neural network organization in young and older adults. Anodal stimulation mainly affected coupling of sensorimotor with ventromedial prefrontal areas in young and decoupling with posteromedial areas in older adults. Copyright © 2018 Elsevier Inc. All rights reserved.

  16. Effect of fenoterol-induced constitutive beta(2)-adrenoceptor activity on contractile receptor function in airway smooth muscle.

    PubMed

    de Vries, B; Roffel, A F; Zaagsma, J; Meurs, H

    2001-11-23

    In the present study, we investigated the effect of fenoterol-induced constitutive beta(2)-adrenoceptor activity on muscarinic receptor agonist- and histamine-induced bovine tracheal smooth muscle contractions. Bovine tracheal smooth muscle strips were incubated with 10 microM fenoterol or vehicle for various periods of time (5, 30 min, 18 h) at 37 degrees C. After extensive washout (3 h, 37 degrees C), isometric contractions were measured to the full muscarinic receptor agonist methacholine, the partial muscarinic receptor agonist 4-(m-chlorophenyl-carbamoyloxy)-2-butynyltrimethylammonium (McN-A-343) and histamine. Fenoterol treatment significantly reduced the sensitivity (pEC(50)) to methacholine in a time-dependent manner, without affecting maximal contraction (E(max)). Fenoterol treatment similarly reduced the pEC(50) of McN-A-343 and histamine; however, E(max) values were also reduced, to approximately 70% of control after 18-h treatment. The inverse agonist timolol, having no effect on control preparations, consistently restored the reduced pEC(50) and E(max) values of the contractile agonists. Remarkably, in the presence of timolol the pEC(50) values of McN-A-343 and histamine in fenoterol-treated airways were significantly enhanced compared to controls. In conclusion, fenoterol-induced constitutive beta(2)-adrenoceptor activity reduces muscarinic receptor agonist- and histamine-induced contractions of bovine tracheal smooth muscle, which can be reversed by the inverse agonist timolol. Moreover, after beta(2)-adrenoceptor agonist treatment, inverse agonism by beta-adrenoceptor antagonists may cause enhanced airway reactivity to contractile mediators.

  17. 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

  18. 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.

  19. 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

  20. Central effects of acetylsalicylic acid on trigeminal-nociceptive stimuli

    PubMed Central

    2014-01-01

    Background Acetylsalicylic acid is one of the most used analgesics to treat an acute migraine attack. Next to the inhibitory effects on peripheral prostaglandin synthesis, central mechanisms of action have also been discussed. Methods Using a standardized model for trigeminal-nociceptive stimulation during fMRI scanning, we investigated the effect of acetylsalicylic acid on acute pain compared to saline in 22 healthy volunteers in a double-blind within-subject design. Painful stimulation was applied using gaseous ammonia and presented in a pseudo-randomized order with several control stimuli. All participants were instructed to rate the intensity and unpleasantness of every stimulus on a VAS scale. Based on previous results, we hypothesized to find an effect of ASA on central pain processing structures like the ACC, SI and SII as well as the trigeminal nuclei and the hypothalamus. Results Even though we did not find any differences in pain ratings between saline and ASA, we observed decreased BOLD signal changes in response to trigemino-nociceptive stimulation in the ACC and SII after administration of ASA compared to saline. This finding is in line with earlier imaging results investigating the effect of ASA on acute pain. Contrary to earlier findings from animal studies, we could not find an effect of ASA on the trigeminal nuclei in the brainstem or within the hypothalamic area. Conclusion Taken together our study replicates earlier findings of an attenuating effect of ASA on pain processing structures, which adds further evidence to a possibly central mechanism of action of ASA. PMID:25201152

  1. Effect of natural products on diabetes associated neurological disorders.

    PubMed

    Patel, Sita Sharan; Udayabanu, Malairaman

    2017-04-01

    Diabetes mellitus, a metabolic disorder, is associated with neurological complications such as depression, anxiety, hypolocomotion, cognitive dysfunction, phobias, anorexia, stroke, pain, etc. Traditional system of medicine is long known for its efficient management of diabetes. The current review discusses the scope of some common medicinal herbs as well as secondary metabolites with a special focus on diabetes-mediated central nervous system complications. Literatures suggest that natural products reduce diabetes-mediated neurological complications partly by reducing oxidative stress and/or inflammation or apoptosis in certain brain regions. Natural products are known to modulate diabetes-mediated alterations in the level of acetylcholinesterase, choline acetyltransferase, monoamine oxidase, serotonin receptors, muscarinic receptors, insulin receptor, nerve growth factor, brain-derived neurotrophic factor, and neuropeptide in brain. Further, there are several natural products reported to manage diabetic complications with unknown mechanism. In conclusion, medicinal plants or their secondary metabolites have a wide scope and possess therapeutic potential to effectively manage neurological complications associated with chronic diabetes.

  2. 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.

  3. Effect of Artificial Gravity: Central Nervous System Neurochemical Studies

    NASA Technical Reports Server (NTRS)

    Fox, Robert A.; D'Amelio, Fernando; Eng, Lawrence F.

    1997-01-01

    The major objective of this project was to assess chemical and morphological modifications occurring in muscle receptors and the central nervous system of animals subjected to altered gravity (2 x Earth gravity produced by centrifugation and simulated micro gravity produced by hindlimb suspension). The underlying hypothesis for the studies was that afferent (sensory) information sent to the central nervous system by muscle receptors would be changed in conditions of altered gravity and that these changes, in turn, would instigate a process of adaptation involving altered chemical activity of neurons and glial cells of the projection areas of the cerebral cortex that are related to inputs from those muscle receptors (e.g., cells in the limb projection areas). The central objective of this research was to expand understanding of how chronic exposure to altered gravity, through effects on the vestibular system, influences neuromuscular systems that control posture and gait. The project used an approach in which molecular changes in the neuromuscular system were related to the development of effective motor control by characterizing neurochemical changes in sensory and motor systems and relating those changes to motor behavior as animals adapted to altered gravity. Thus, the objective was to identify changes in central and peripheral neuromuscular mechanisms that are associated with the re-establishment of motor control which is disrupted by chronic exposure to altered gravity.

  4. 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.

  5. Direct renal tubular effects of choline on electrolyte excretion in the chicken.

    PubMed

    Besseghir, K; Rennick, B

    1981-03-01

    Direct local effects of choline on electrolyte effects did not reappear. Acetylcholine was more potent than choline in producing the electrolyte effects. These results suggest that choline-induced changes in renal electrolyte excretion are mediated by a muscarinic receptor completely separate from the choline transport system. These effects imply that choline is not an "inert" cation.

  6. 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

  7. 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

  8. CENTRAL REINFORCING EFFECTS OF ETHANOL ARE BLOCKED BY CATALASE INHIBITION

    PubMed Central

    Nizhnikov, Michael Edward; Molina, Juan Carlos; Spear, Norman

    2007-01-01

    Recent studies have systematically indicated that newborn rats are highly sensitive to ethanol’s positive reinforcing effects. Central administrations of ethanol (25–200 mg %) associated with an olfactory conditioned stimulus (CS) promote subsequent conditioned approach to the CS as evaluated through the newborn’s response to a surrogate nipple scented with the CS. It has been shown that ethanol’s first metabolite, acetaldehyde, exerts significant reinforcing effects in the central nervous system. A significant amount of acetaldehyde is derived from ethanol metabolism via the catalase system. In newborn rats catalase levels are particularly high in several brain structures. The present study tested the effect of catalase inhibition on central ethanol reinforcement. In the first experiment, pups experienced lemon odor either paired or unpaired with intracisternal (i.c.) administrations of 100 mg% ethanol. Half of the animals corresponding to each learning condition were pretreated with i.c. administrations of either physiological saline or a catalase inhibitor (sodium-azide). Catalase inhibition completely suppressed ethanol reinforcement in paired groups without affecting responsiveness to the CS during conditioning or responding by unpaired control groups. A second experiment tested whether these effects were specific to ethanol reinforcement or due instead to general impairment in learning and expression capabilities. Central administration of an endogenous kappa opioid receptor agonist (dynorphin A-13) was employed as an alternative source of reinforcement. Inhibition of the catalase system had no effect on the reinforcing properties of dynorphin. The present results support the hypothesis that ethanol metabolism regulated by the catalase system plays a critical role in determination of ethanol reinforcement in newborn rats. PMID:17980789

  9. Changes in EEG power spectra and behavioral states in rats exposed to the acetylcholinesterase inhibitor chlorpyrifos and muscarinic agonist oxotremorine.

    PubMed

    Timofeeva, O A; Gordon, C J

    2001-03-02

    Organophosphates (OPs) inhibit acetylcholinesterase (AChE) activity causing cholinergic stimulation in the central nervous system (CNS). Cholinergic systems are crucial in electroencephalogram (EEG) generation and regulation of behavior; however, little is known about how OP exposure affects the EEG and behavioral states. We recorded EEG, core temperature and motor activity before and after exposure to the OP pesticide chlorpyrifos (CHP) in adult female rats implanted with telemetric transmitters. The recording and reference electrodes were placed in the occipital and frontal bones, respectively. The animals received CHP, 25 mg/kg, p.o., or oxotremorine (OX), 0.2 mg/kg, s.c. CHP led to a significant increase in delta (0.1-3.5 Hz), slow theta (4-6.5 Hz), gamma 2 (35.5-50 Hz), reduction in fast theta (7-8.5 Hz), alpha/sigma (9-14 Hz), beta 1 (14.5-24 Hz), beta 2 (24.5-30 Hz) and gamma 1 (30.5-35 Hz) powers, slowing of peak frequencies in 1-9 Hz range, hypothermia and decrease in motor activity. The drop in 7-14 Hz was associated with cholinergic suppression of sleep spindles. Changes in behavioral state were characterized by dramatic diminution of sleep postures and exploring activity and prolongation of quiet waking. There was recovery in all bands in spite of continued inhibition of AChE activity [44,45] in rats exposed to CHP. OX-induced EEG and behavioral alterations were similar to CHP except there was no increase in delta and the onset and recovery were more rapid. We did not find a correlation between the EEG and core temperature alterations. Overall, changes in EEG (except in delta band) and behavior following CHP were attributable to muscarinic stimulation. Cortical arousal together with increased quiet waking and decreased sleep after CHP occurred independently from inhibition of motor activity and lowering of core temperature.

  10. 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

  11. Anxiolytic effects of muscarinic acetylcholine receptors agonist oxotremorine in chronically stressed rats and related changes in BDNF and FGF2 levels in the hippocampus and prefrontal cortex.

    PubMed

    Di Liberto, Valentina; Frinchi, Monica; Verdi, Vincenzo; Vitale, Angela; Plescia, Fulvio; Cannizzaro, Carla; Massenti, Maria F; Belluardo, Natale; Mudò, Giuseppa

    2017-02-01

    In depressive disorders, one of the mechanisms proposed for antidepressant drugs is the enhancement of synaptic plasticity in the hippocampus and cerebral cortex. Previously, we showed that the muscarinic acetylcholine receptor (mAChR) agonist oxotremorine (Oxo) increases neuronal plasticity in hippocampal neurons via FGFR1 transactivation. Here, we aimed to explore (a) whether Oxo exerts anxiolytic effect in the rat model of anxiety-depression-like behavior induced by chronic restraint stress (CRS), and (b) if the anxiolytic effect of Oxo is associated with the modulation of neurotrophic factors, brain-derived neurotrophic factor (BDNF) and fibroblast growth factor-2 (FGF2), and phosphorylated Erk1/2 (p-Erk1/2) levels in the dorsal or ventral hippocampus and in the medial prefrontal cortex. The rats were randomly divided into four groups: control unstressed, CRS group, CRS group treated with 0.2 mg/kg Oxo, and unstressed group treated with Oxo. After 21 days of CRS, the groups were treated for 10 days with Oxo or saline. The anxiolytic role of Oxo was tested by using the following: forced swimming test, novelty suppressed feeding test, elevated plus maze test, and light/dark box test. The hippocampi and prefrontal cortex were used to evaluate BDNF and FGF2 protein levels and p-Erk1/2 levels. Oxo treatment significantly attenuated anxiety induced by CRS. Moreover, Oxo treatment counteracted the CRS-induced reduction of BDNF and FGF2 levels in the ventral hippocampus and medial prefrontal cerebral cortex CONCLUSIONS: The present study showed that Oxo treatment ameliorates the stress-induced anxiety-like behavior and rescues FGF2 and BDNF levels in two brain regions involved in CRS-induced anxiety, ventral hippocampal formation, and medial prefrontal cortex.

  12. IN-VITRO METHODS FOR EVALUATING SIDE EFFECTS OF PESTICIDES AND TOXIC SUBSTANCES

    EPA Science Inventory

    Several skeletal muscle and smooth muscle preparations have been examined for their usefulness in evaluating the toxic effects of a variety of insecticides. The following preparations were found satisfactory for such test: guinea pig ileum for muscarinic receptors, guinea pig hea...

  13. Opposing actions of dibutyryl cyclic AMP and GMP on temperature in conscious guinea-pigs

    NASA Technical Reports Server (NTRS)

    Kandasamy, S. B.; Williaes, B. A.

    1983-01-01

    It is shown that the intracerebroventricular administration of dibutyryl cyclic AMP (Db-cAMP) induced hyperthermia in guinea pigs which was not mediated through prostaglandins or norepinephrine since a prostaglandin synthesis inhibitor and an alpha-adrenergic receptor blocking agent did not antagonize the hyperthermia. However, the hyperthermic response to Db-cAMP was attenuated by the central administration of a beta-adrenergic receptor antagonist, which indicates that cAMP may be involved, through beta-adrenergic receptors, in the central regulation of heat production and conservation. The central administration of Db-cGMP produced hypothermia which was not mediated via histamine H1 or H2 receptors and serotonin. The antagonism of hypothermia induced by Db-cGMP and acetylcholine + physostigmine by central administration of a cholinergic muscarine receptor antagonist and not by a cholinergic nicotinic receptor antagonist suggests that cholinoceptive neurons and endogenous cGMP may regulate heat loss through cholinergic muscarine receptors. It is concluded that these results indicate a regulatory role in thermoregulation provided by a balance between opposing actions of cAMP and cGMP in guinea pigs.

  14. Hippocampal place cell dysfunction and the effects of muscarinic M1 receptor agonism in a rat model of Alzheimer's disease.

    PubMed

    Galloway, Claire R; Ravipati, Kaushik; Singh, Suyashi; Lebois, Evan P; Cohen, Robert M; Levey, Allan I; Manns, Joseph R

    2018-05-09

    Alzheimer's disease (AD) is a neurodegenerative disease that disproportionately impacts memory and the hippocampus. However, it is unclear how AD pathology influences the activity of surviving neurons in the hippocampus to contribute to the memory symptoms in AD. One well-understood connection between spatial memory and neuronal activity in healthy brains is the activity of place cells, neurons in the hippocampus that fire preferentially in a specific location of a given environment (the place field of the place cell). In the present study, place cells were recorded from the hippocampus in a recently-developed rat model of AD (Tg-F344 AD) at an age (12-20 months) at which the AD rats showed marked spatial memory deficits. Place cells in the CA2 and CA3 pyramidal regions of the hippocampus in AD rats showed sharply reduced spatial fidelity relative to wild-type (WT) rats. In contrast, spiking activity of place cells recorded in region CA1 in AD rats showed good spatial fidelity that was similar to CA1 place cells in WT rats. Oral administration of the M 1 muscarinic acetylcholine receptor agonist VU0364572 impacted place cell firing rates in CA1 and CA2/3 hippocampal regions but did not improve the spatial fidelity of CA2/3 hippocampal place cells in AD rats. The results indicated that, to the extent the spatial memory impairment in AD rats was attributable to hippocampal dysfunction, the memory impairment was more attributable to dysfunction in hippocampal regions CA2 and CA3 rather than CA1. This article is protected by copyright. All rights reserved. © 2018 Wiley Periodicals, Inc.

  15. Differential regulation of primary afferent input to spinal cord by muscarinic receptor subtypes delineated using knockout mice.

    PubMed

    Chen, Shao-Rui; Chen, Hong; Yuan, Wei-Xiu; Wess, Jürgen; Pan, Hui-Lin

    2014-05-16

    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. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. 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.

  17. 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.

  18. 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

  19. Cholinergic agonists increase intracellular calcium concentration in frog vestibular hair cells.

    PubMed

    Ohtani, M; Devau, G; Lehouelleur, J; Sans, A

    1994-11-01

    Acetylcholine (ACh) is usually considered to be the neurotransmitter of the efferent vestibular system. The nature and the localization of cholinergic receptors have been investigated on frog isolated vestibular hair cells (VHCs), by measuring variations of intracellular calcium concentration ([Ca2+]i), using calcium sensitive dye fura-2. Focal iontophoretic ACh (1 M, 300 nA.40 ms) application induced a rapid increase in [Ca2+]i, reaching a peak in 20 s and representing about 5-fold the resting level (from 61 +/- 6 to 320 +/- 26 nM). Applications of muscarinic agonists as methacholine and carbachol induced weaker calcium responses (from 78 +/- 25 to 238 +/- 53 nM) than the one obtained with ACh applications. These muscarinic agonists were efficient only in precise zones. Desensitization of muscarinic receptors to successive stimulations was significant. Perfusion of nicotine or 1,1-dimethyl-4-phenyl-piperazinium (DMPP), a nicotinic agonist, induced an increase in [Ca2+]i only in some cells (4/28 with DMPP). These results indicated the presence of cholinergic receptors on frog VHCs: muscarinic receptors were more responsive than nicotinic receptors. Presence of muscarinic and nicotinic receptors in the membrane of VHCs could indicate different modulations of VHCs activity mediated by [Ca2+]i and involving an efferent control which represents a central regulation of the vestibular afferent message.

  20. Intra- and intersegmental influences among central pattern generating networks in the walking system of the stick insect.

    PubMed

    Mantziaris, Charalampos; Bockemühl, Till; Holmes, Philip; Borgmann, Anke; Daun, Silvia; Büschges, Ansgar

    2017-10-01

    To efficiently move around, animals need to coordinate their limbs. Proper, context-dependent coupling among the neural networks underlying leg movement is necessary for generating intersegmental coordination. In the slow-walking stick insect, local sensory information is very important for shaping coordination. However, central coupling mechanisms among segmental central pattern generators (CPGs) may also contribute to this. Here, we analyzed the interactions between contralateral networks that drive the depressor trochanteris muscle of the legs in both isolated and interconnected deafferented thoracic ganglia of the stick insect on application of pilocarpine, a muscarinic acetylcholine receptor agonist. Our results show that depressor CPG activity is only weakly coupled between all segments. Intrasegmental phase relationships differ between the three isolated ganglia, and they are modified and stabilized when ganglia are interconnected. However, the coordination patterns that emerge do not resemble those observed during walking. Our findings are in line with recent studies and highlight the influence of sensory input on coordination in slowly walking insects. Finally, as a direct interaction between depressor CPG networks and contralateral motoneurons could not be observed, we hypothesize that coupling is based on interactions at the level of CPG interneurons. NEW & NOTEWORTHY Maintaining functional interleg coordination is vitally important as animals locomote through changing environments. The relative importance of central mechanisms vs. sensory feedback in this process is not well understood. We analyzed coordination among the neural networks generating leg movements in stick insect preparations lacking phasic sensory feedback. Under these conditions, the networks governing different legs were only weakly coupled. In stick insect, central connections alone are thus insufficient to produce the leg coordination observed behaviorally. Copyright © 2017 the

  1. 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

  2. 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.

  3. Essential role of UCP1 modulating the central effects of thyroid hormones on energy balance

    PubMed Central

    Alvarez-Crespo, Mayte; Csikasz, Robert I.; Martínez-Sánchez, Noelia; Diéguez, Carlos; Cannon, Barbara; Nedergaard, Jan; López, Miguel

    2016-01-01

    Objective Classically, metabolic effects of thyroid hormones (THs) have been considered to be peripherally mediated, i.e. different tissues in the body respond directly to thyroid hormones with an increased metabolism. An alternative view is that the metabolic effects are centrally regulated. We have examined here the degree to which prolonged, centrally infused triiodothyronine (T3) could in itself induce total body metabolic effects and the degree to which brown adipose tissue (BAT) thermogenesis was essential for such effects, by examining uncoupling protein 1 (UCP1) KO mice. Methods Wildtype and UPC1 KO mice were centrally-treated with T3 by using minipumps. Metabolic measurements were analyzed by indirect calorimetry and expression analysis by RT-PCR or western blot. BAT morphology and histology were studied by immunohistochemistry. Results We found that central T3-treatment led to reduced levels of hypothalamic AMP-activated protein kinase (AMPK) and elevated body temperature (0.7 °C). UCP1 was essential for the T3-induced increased rate of energy expenditure, which was only observable at thermoneutrality and notably only during the active phase, for the increased body weight loss, for the increased hypothalamic levels of neuropeptide Y (NPY) and agouti-related peptide (AgRP) and for the increased food intake induced by central T3-treatment. Prolonged central T3-treatment also led to recruitment of BAT and britening/beiging (“browning”) of inguinal white adipose tissue (iWAT). Conclusions We conclude that UCP1 is essential for mediation of the central effects of thyroid hormones on energy balance, and we suggest that similar UCP1-dependent effects may underlie central energy balance effects of other agents. PMID:27069867

  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. 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

  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. 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.

  9. Central effects of humanin on hepatic triglyceride secretion.

    PubMed

    Gong, Zhenwei; Su, Kai; Cui, Lingguang; Tas, Emir; Zhang, Ting; Dong, H Henry; Yakar, Shoshana; Muzumdar, Radhika H

    2015-08-01

    Humanin (HN) is an endogenous mitochondria-associated peptide that has been shown to protect against various Alzheimer's disease-associated insults, myocardial ischemia-reperfusion injury, and reactive oxygen species-induced cell death. We have shown previously that HN improves whole body glucose homeostasis by improving insulin sensitivity and increasing glucose-stimulated insulin secretion (GSIS) from the β-cells. Here, we report that intraperitoneal treatment with one of HN analogs, HNG, decreases body weight gain, visceral fat, and hepatic triglyceride (TG) accumulation in high-fat diet-fed mice. The decrease in hepatic TG accumulation is due to increased activity of hepatic microsomal triglyceride transfer protein (MTTP) and increased hepatic TG secretion. Both intravenous (iv) and intracerebroventricular (icv) infusion of HNG acutely increase TG secretion from the liver. Vagotomy blocks the effect on both iv and icv HNG on TG secretion, suggesting that the effects of HNG on hepatic TG flux are centrally mediated. Our data suggest that HN is a new player in central regulation of peripheral lipid metabolism. Copyright © 2015 the American Physiological Society.

  10. 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.

  11. 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.

  12. Muscarinic subtypes profile modulation within a series of new antagonists, bridged bicyclic derivatives of 2,2-diphenyl-[1,3]-dioxolan-4-ylmethyl-dimethylamine.

    PubMed

    Piergentili, Alessandro; Gentili, Francesco; Ghelfi, Francesca; Marucci, Gabriella; Pigini, Maria; Quaglia, Wilma; Giannella, Mario

    2003-09-01

    A set of new muscarinic antagonists, bridged bicyclic derivatives of 2,2-diphenyl-[1,3]-dioxolan-4-ylmethyl-dimethylamine (1), was synthesized and tested to evaluate their affinity and selectivity for M(1), M(2), M(3) and M(4) receptor subtypes. The conformational constraint of 1 in a bicyclic structure, and the variation in distance and stereochemistry of the active functions allowed us to modulate the selectivity of interaction with the M(1)-M(3) receptor subtypes. The most interesting compound was (cis,trans)-2-(2,2-diphenylethyl)-5-methyl-tetrahydro-[1,3]dioxolo[4,5-c]pyrrole oxalate (6), which is equipotent with Pirenzepine on rabbit vas deferens (M(1)-putative) but shows a better selectivity profile.

  13. Sarin (GB, O-isopropyl methylphosphonofluoridate) neurotoxicity: critical review

    PubMed Central

    Abou-Donia, Mohamed B.; Siracuse, Briana; Gupta, Natasha; Sokol, Ashly Sobel

    2017-01-01

    Sarin (GB, O-isopropyl methylphosphonofluoridate) is a potent organophosphorus (OP) nerve agent that inhibits acetylcholinesterase (AChE) irreversibly. The subsequent build-up of acetylcholine (ACh) in the central nervous system (CNS) provokes seizures and, at sufficient doses, centrally-mediated respiratory arrest. Accumulation of ACh at peripheral autonomic synapses leads to peripheral signs of intoxication and overstimulation of the muscarinic and nicotinic receptors, which is described as “cholinergic crisis” (i.e. diarrhea, sweating, salivation, miosis, bronchoconstriction). Exposure to high doses of sarin can result in tremors, seizures, and hypothermia. More seriously, build-up of ACh at neuromuscular junctions also can cause paralysis and ultimately peripherally-mediated respiratory arrest which can lead to death via respiratory failure. In addition to its primary action on the cholinergic system, sarin possesses other indirect effects. These involve the activation of several neurotransmitters including gamma-amino-butyric acid (GABA) and the alteration of other signaling systems such as ion channels, cell adhesion molecules, and inflammatory regulators. Sarin exposure is associated with symptoms of organophosphate-induced delayed neurotoxicity (OPIDN) and organophosphate-induced chronic neurotoxicity (OPICN). Moreover, sarin has been involved in toxic and immunotoxic effects as well as organophosphate-induced endocrine disruption (OPIED). The standard treatment for sarin-like nerve agent exposure is post-exposure injection of atropine, a muscarinic receptor antagonist, accompanied by an oxime, an AChE reactivator, and diazepam. PMID:27705071

  14. 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.

  15. Transient cardiac effects in a child with acute cholinergic syndrome due to rivastigmine poisoning.

    PubMed

    Raucci, Umberto; Vanacore, Nicola; Cecchetti, Corrado; Russo, Mario Salvatore; Rossi, Rossella; Pirozzi, Nicola

    2014-07-01

    We report a case of rivastigmine poisoning resulting in a full cholinergic syndrome with nicotinic, muscarinic, and central effects requiring supportive or intensive care in a pediatric patient. A 3-year-old girl was admitted to the Emergency Department suspected of having ingested one or two pills of rivastigmine. The child was hyporeactive, with symptoms of altered mental status, sialorrhea, sweating, and diarrhea. Subsequently, she started showing signs of respiratory failure, severe tracheobronchial involvement, and gastric and abdominal distension. An electrocardiogram recorded frequent monomorphic ventricular ectopic beats with bigeminy and trigeminy. Long-term follow-up showed a transient dysrhythmia. Poisoning with rivastigmine can be a life-threatening condition. Timely identification and appropriate management of the toxic effects of this drug are essential and often life-saving. This is particularly true in cases of cholinergic syndrome subsequent to drug poisoning. Patients with cholinergic syndrome should also be assessed for possible cardiac complications such as dysrhythmias. The main factors predisposing to the development of such complications are autonomic disorder, hypoxemia, acidosis, and electrolyte imbalance. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. RORγt antagonist suppresses M3 muscarinic acetylcholine receptor-induced Sjögren's syndrome-like sialadenitis.

    PubMed

    Tahara, M; Tsuboi, H; Segawa, S; Asashima, H; Iizuka-Koga, M; Hirota, T; Takahashi, H; Kondo, Y; Matsui, M; Matsumoto, I; Sumida, T

    2017-02-01

    We showed recently that M3 muscarinic acetylcholine receptor (M3R)-reactive CD3 + T cells play a pathogenic role in the development of murine autoimmune sialadenitis (MIS), which mimics Sjögren's syndrome (SS). The aim of this study was to determine the effectiveness and mechanism of action of retinoic acid-related orphan receptor-gamma t (RORγt) antagonist (A213) in MIS. Splenocytes from M3R knockout (M3R -/- ) mice immunized with murine M3R peptide mixture were inoculated into recombination-activating gene 1 knockout (Rag-1 -/- ) mice (M3R -/- →Rag-1 -/- ) with MIS. Immunized M3R -/- mice (pretransfer treatment) and M3R -/- →Rag-1 -/- mice (post-transfer treatment) were treated with A213 every 3 days. Salivary volume, severity of sialadenitis and cytokine production from M3R peptide-stimulated splenocytes and lymph node cells were examined. Effects of A213 on cytokine production were analysed by enzyme-linked immunosorbent assay (ELISA) and on T helper type 1 (Th1), Th17 and Th2 differentiation from CD4 + T cells by flow cytometry. Pretransfer A213 treatment maintained salivary volume, improved MIS and reduced interferon (IFN)-γ and interleukin (IL)-17 production significantly compared with phosphate-buffered saline (PBS) (P < 0·05). These suppressive effects involved CD4 + T cells rather than CD11c + cells. Post-transfer treatment with A213 increased salivary volume (P < 0·05), suppressed MIS (P < 0·005) and reduced IFN-γ and IL-17 production (P < 0·05). In vitro, A213 suppressed IFN-γ and IL-17 production from M3R-stimulated splenocytes and CD4 + T cells of immunized M3R -/- mice (P < 0·05). In contrast with M3R specific responses, A213 suppressed only IL-17 production from Th17 differentiated CD4 + T cells without any effect on Th1 and Th2 differentiation in vitro. Our findings suggested that RORγt antagonism is potentially suitable treatment strategy for SS-like sialadenitis through suppression of IL-17 and IFN-γ production

  17. Central effects of thyronamines on glucose metabolism in rats.

    PubMed

    Klieverik, Lars P; Foppen, Ewout; Ackermans, Mariëtte T; Serlie, Mireille J; Sauerwein, Hans P; Scanlan, Thomas S; Grandy, David K; Fliers, Eric; Kalsbeek, Andries

    2009-06-01

    Thyronamines are naturally occurring, chemical relatives of thyroid hormone. Systemic administration of synthetic 3-iodothyronamine (T(1)AM) and - to a lesser extent - thyronamine (T(0)AM), leads to acute bradycardia, hypothermia, decreased metabolic rate, and hyperglycemia. This profile led us to hypothesize that the central nervous system is among the principal targets of thyronamines. We investigated whether a low dose i.c.v. infusion of synthetic thyronamines recapitulates the changes in glucose metabolism that occur following i.p. thyronamine administration. Plasma glucose, glucoregulatory hormones, and endogenous glucose production (EGP) using stable isotope dilution were monitored in rats before and 120 min after an i.p. (50 mg/kg) or i.c.v. (0.5 mg/kg) bolus infusion of T(1)AM, T(0)AM, or vehicle. To identify the peripheral effects of centrally administered thyronamines, drug-naive rats were also infused intravenously with low dose (0.5 mg/kg) thyronamines. Systemic T(1)AM rapidly increased EGP and plasma glucose, increased plasma glucagon, and corticosterone, but failed to change plasma insulin. Compared with i.p.-administered T(1)AM, a 100-fold lower dose administered centrally induced a more pronounced acute EGP increase and hyperglucagonemia while plasma insulin tended to decrease. Both systemic and central infusions of T(0)AM caused smaller increases in EGP, plasma glucose, and glucagon compared with T(1)AM. Neither T(1)AM nor T(0)AM influenced any of these parameters upon low dose i.v. administration. We conclude that central administration of low-dose thyronamines suffices to induce the acute alterations in glucoregulatory hormones and glucose metabolism following systemic thyronamine infusion. Our data indicate that thyronamines can act centrally to modulate glucose metabolism.

  18. Atmospheric chemistry effects of the 1998 Mexican/Central American fires measured in central New Mexico USA.

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

    Popp, C. J.

    1998-12-16

    Atmospheric effects from large fires have received a great deal of interest recently, especially when the fires have the potential to effect human health when the plumes are transported long distances over areas of high population density. Examples are the recent large fires in Southeast Asia in 1997 (1) and the wildfires occurring in southern Mexico and Central America that were manifested in decreased visibility and high aerosol concentrations in the United States at distances of 2500-4000 km from the fires. In addition to fine aerosols, these biomass fires have the potential to produce and transport large quantities of oxygenatedmore » organic species such as aldehydes, ketones and carboxylic acids, hydrocarbons, and sulfate and nitrate species. Most of the literature reports dealing with products of biomass burning have been related to fireplace and wood burning stove emissions (2,3) and with local effects from forest fires(4). The recent super-large fires occurring in Indonesia and Mexico/Central America also bring about the issue of atmospheric reactivity because long-range transport affords long reaction times for photochemical reactions, wet and dry deposition and surface reactions on the aerosol particles. The smoke/haze conditions prompted considerable concern among the general population in New Mexico regarding health hazards and a large number of calls to the Albuquerque, NM Air Quality Division which reported the PM{sub 10} samples collected showed no significant increase in mass(5). The conclusion was that the particles were very fine and therefore had considerable influence on the visibility but did not violate health standards. In this study, organic and inorganic chemical species in the gaseous and aerosol phases have been identified and quantified under non-smoky and smoky conditions in Central New Mexico approximately 3000 km from the source of the fires.« less

  19. Central and peripheral effects of the non-neural substances on respiration before and after vagotomy.

    PubMed

    Sahin, G; Oruç, T; Simşek, G; Güner, I

    1997-08-01

    The central effects of capsaicin, veratrine, histamine and bradykinin were studied by injecting them directly into the oerebrospinal fluid and their peripheral effects were examined by injecting into femoral vein. Our experiments were performed in Na-pentobarbital-anaesthetized dogs. Tidal volume (VT), respiratory frequency (f/min), systemic arterial pressure (BP) were recorded. A significant increase in f, and an initial apnea or hypoventilation followed by a significant increase in VT were observed with central and peripheral capsaicin. Vagotomy removed the peripheral VT response, but not the central one. While central capsaicin administration increased BP, peripheral administration decreased. After vagotomy, a significant increase was observed in BP for both administrations. Respiratory responses to central and peripheral administrations of veratrine were similar to those of capsaicin. Significant increases were observed in f and VT of the intact group in response to central and peripheral administration of histamine. Response to peripheral administration disappeared after vagotomy. While central and peripheral bradykinin increased VT significantly, there was no significant change in f. Vagotomy only removed the increase in VT in response to peripheral administration. In conclusion, respiratory responses to central administration of capsaicin and veratrine are due to direct effects of these substances on respiratory neurons. In peripheral administration, disappearance of the responses after vagotomy indicate that the responses are brought about by stimulation of the lung receptors.

  20. Muscarinic-agonist and guanine nucleotide stimulation of myo-inositol trisphosphate formation in membranes isolated from bovine iris sphincter smooth muscle: effects of short-term cholinergic desensitization.

    PubMed

    Honkanen, R E; Abdel-Latif, A A

    1989-01-01

    The effect of short-term cholinergic desensitization on muscarinic acetylcholine receptor (mAChR)-mediated activation of phospholipase C was investigated in membranes isolated from the bovine iris sphincter smooth muscle. Membranes prepared from normal or desensitized muscles, prelabeled with either [3H]myo-inositol or 32P from [gamma-32P]ATP, were incubated with a hydrolysis-resistant analogue of GTP, GTP gamma S, or GTP gamma S plus carbachol (CCh), and the production of [3H]myo-inositol 1,4,5-trisphosphate (IP3) and the breakdown of polyphosphoinositides were assessed. In normal membranes, GTP (greater than or equal to 1 mM), GTP gamma S (greater than 10 microM) and GTP gamma S (1 microM) plus CCh (10 microM), but not GDP or GDP beta S, increased phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and IP3 production. GTP gamma S increased IP3 accumulation in a time- and dose-dependent manner, and CCh, which had no effect on phospholipase C activity in the absence of GTP gamma S, potentiated the effects of GTP gamma S. The effect of CCh plus GTP gamma S on IP3 production was inhibited by atropine, had an absolute requirement for nM amounts of Ca2+ and was not affected by pertussis toxin. At higher concentrations (greater than 1 microM), Ca2+ alone induced PIP2 hydrolysis. Short-term exposure (less than 60 min) of the muscle to CCh (100 microM) did not affect the total number (Bmax) of mAChRs nor their affinity (KD) for [3H]-N-methylscopolamine. Desensitization did, however, result in: (1) a loss of the CCh-high affinity binding state of the sphincter mAChRs in a manner analogous to that produced by GTP gamma S; (2) a loss of the ability of GTP gamma S to affect CCh binding to the receptors; and (3) an attenuation of the GTP gamma S plus CCh-stimulated PIP2 hydrolysis. In conclusion, the data presented suggest that, in the iris smooth muscle, G-proteins are involved in the coupling of mAChRs to phospholipase C and that short-term cholinergic desensitization

  1. Effects of Static Stretching Exercise on Lumbar Flexibility and Central Arterial Stiffness.

    PubMed

    Logan, Jeongok G; Kim, Suk-Sun; Lee, Mijung; Byon, Ha Do; Yeo, SeonAe

    Previous studies have demonstrated that arterial stiffness is associated with lumbar flexibility (LF). Stretching exercise targeted to improve LF may have a beneficial effect on reducing arterial stiffness. We examined the effects of a single bout of a structured, static stretching exercise on arterial stiffness, LF, peripheral and central blood pressure (BP), and heart rate (HR) and tested the association between LF and central arterial stiffness. The study had a pretest-posttest design without a control group. Thirty healthy women followed a video demonstration of a 30-minute whole-body stretching exercise. Carotid-femoral pulse wave velocity (cf-PWV), augmentation index, LF, peripheral and central BP, and HR were measured before and after the stretching exercise. One bout of a static stretching exercise significantly reduced cf-PWV (t29 = 2.708, P = .011) and HR (t29 = 7.160, P = .000) and increased LF (t29 = 12.248, P < .000). Augmentation index and peripheral and central BP also decreased but did not reach statistical significance. Despite no association found between cf-PWV and LF, the larger increase in LF the subjects had, the larger decrease in cf-PWV they had after exercise (r = 0.500, P = .005). Study findings highlight the potential benefit of a static stretching exercise on central arterial stiffness, an independent predictor of cardiovascular morbidity. Static stretching exercise conducted in the sitting position may be used as an effective intervention to reduce cardiovascular risk after a cardiac event or for patients whose sympathetic function should not be overly activated or whose gaits are not stable.

  2. Severe peri-ictal respiratory dysfunction is common in Dravet syndrome

    PubMed Central

    Kim, YuJaung; Bravo, Eduardo; Thirnbeck, Caitlin K.; Smith-Mellecker, Lori A.; Kim, Se Hee; Gehlbach, Brian K.; Laux, Linda C.; Zhou, Xiuqiong; Nordli, Douglas R.

    2018-01-01

    Dravet syndrome (DS) is a severe childhood-onset epilepsy commonly due to mutations of the sodium channel gene SCN1A. Patients with DS have a high risk of sudden unexplained death in epilepsy (SUDEP), widely believed to be due to cardiac mechanisms. Here we show that patients with DS commonly have peri-ictal respiratory dysfunction. One patient had severe and prolonged postictal hypoventilation during video EEG monitoring and died later of SUDEP. Mice with an Scn1aR1407X/+ loss-of-function mutation were monitored and died after spontaneous and heat-induced seizures due to central apnea followed by progressive bradycardia. Death could be prevented with mechanical ventilation after seizures were induced by hyperthermia or maximal electroshock. Muscarinic receptor antagonists did not prevent bradycardia or death when given at doses selective for peripheral parasympathetic blockade, whereas apnea, bradycardia, and death were prevented by the same drugs given at doses high enough to cross the blood-brain barrier. When given via intracerebroventricular infusion at a very low dose, a muscarinic receptor antagonist prevented apnea, bradycardia, and death. We conclude that SUDEP in patients with DS can result from primary central apnea, which can cause bradycardia, presumably via a direct effect of hypoxemia on cardiac muscle. PMID:29329111

  3. 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.

  4. Impaired muscarinic endothelium-dependent relaxation and cyclic guanosine 5'-monophosphate formation in atherosclerotic human coronary artery and rabbit aorta.

    PubMed Central

    Bossaller, C; Habib, G B; Yamamoto, H; Williams, C; Wells, S; Henry, P D

    1987-01-01

    The dependence of vascular relaxation on an intact endothelium and the relationship between relaxation and cyclic GMP accumulation were determined in coronary arteries isolated from cardiac transplantation patients with or without coronary atherosclerosis. In nonatherosclerotic arteries, the endothelium-dependent agent acetylcholine produced concentration-related relaxations. In atherosclerotic arteries, endothelium-dependent relaxations were abolished with acetylcholine, partly suppressed with substance P and histamine, and completely preserved with the ionophore A23187. In these arteries, the endothelium-independent agent nitroglycerin remained fully active. Accumulation of cyclic GMP in atherosclerotic strips was suppressed with acetylcholine but unattenuated with A23187 and nitroglycerin. In aortas from rabbits with diet-induced atherosclerosis, there was likewise an impaired cholinergic relaxation and cyclic GMP accumulation in the presence of preserved responses to A23187 and nitroglycerin. The results demonstrate that impaired cholinergic responses in atherosclerotic arteries reflect a muscarinic defect and not an inability of endothelium to release endothelial factor or smooth muscle to respond to it. PMID:2432088

  5. Stevia Nonsweetener Fraction Displays an Insulinotropic Effect Involving Neurotransmission in Pancreatic Islets.

    PubMed

    Piovan, Silvano; Pavanello, Audrei; Peixoto, Giuliana Maria Ledesma; Matiusso, Camila Cristina Ianoni; de Moraes, Ana Maria Praxedes; Martins, Isabela Peixoto; Malta, Ananda; Palma-Rigo, Kesia; da Silva Franco, Claudinéia Conationi; Milani, Paula Gimenez; Dacome, Antonio Sérgio; da Costa, Silvio Claudio; de Freitas Mathias, Paulo Cezar; Mareze-Costa, Cecília Edna

    2018-01-01

    Stevia rebaudiana (Bert.) Bertoni besides being a source of noncaloric sweeteners is also an important source of bioactive molecules. Many plant extracts, mostly obtained with ethyl acetate solvent, are rich in polyphenol compounds that present insulinotropic effects. To investigate whether the nonsweetener fraction, which is rich in phenolic compounds isolated from Stevia rebaudiana with the solvent ethyl acetate (EAF), has an insulinotropic effect, including interference at the terminals of the autonomic nervous system of the pancreatic islets of rats. Pancreatic islets were isolated from Wistar rats and incubated with EAF and inhibitory or stimulatory substances of insulin secretion, including cholinergic and adrenergic agonists and antagonists. EAF potentiates glucose-stimulated insulin secretion (GSIS) only in the presence of high glucose and calcium-dependent concentrations. EAF increased muscarinic insulinotropic effects in pancreatic islets, interfering with the muscarinic receptor subfamily M 3 . Adrenergic inhibitory effects on GSIS were attenuated in the presence of EAF, which interfered with the adrenergic α 2 receptor. Results suggest that EAF isolated from stevia leaves is a potential therapy for treating type 2 diabetes mellitus by stimulating insulin secretion only in high glucose concentrations, enhancing parasympathetic signal transduction and inhibiting sympathetic signal transduction in beta cells.

  6. Muscarinic acetylcholine receptors are expressed by most parvalbumin-immunoreactive neurons in area MT of the macaque

    PubMed Central

    Disney, Anita A; Alasady, Hussein A; Reynolds, John H

    2014-01-01

    Background In the mammalian neocortex, cells that express parvalbumin (PV neurons) comprise a dominant class of inhibitory neuron that substantially overlaps with the fast/narrow-spiking physiological phenotype. Attention has pronounced effects on narrow-spiking neurons in the extrastriate cortex of macaques, and more consistently so than on their broad-spiking neighbors. Cortical neuromodulation by acetylcholine (ACh) is a candidate mechanism for aspects of attention and in the primary visual cortex (V1) of the macaque, receptors for ACh (AChRs) are strongly expressed by inhibitory neurons. In particular, most PV neurons in macaque V1 express m1 muscarinic AChRs and exogenously applied ACh can cause the release of γ-aminobutyric acid. In contrast, few PV neurons in rat V1 express m1 AChRs. While this could be a species difference, it has also been argued that macaque V1 is anatomically unique when compared with other cortical areas in macaques. Aims The aim of this study was to better understand the extent to which V1 offers a suitable model circuit for cholinergic anatomy in the macaque occipital lobe, and to explore cholinergic modulation as a biological basis for the changes in circuit behavior seen with attention. Materials and methods We compared expression of m1 AChRs by PV neurons between area V1 and the middle temporal visual area (MT) in macaque monkeys using dual-immunofluorescence confocal microscopy. Results and conclusion We find that, as in V1, most PV neurons in MT express m1 AChRs but, unlike in V1, it appears that so do most excitatory neurons. This provides support for V1 as a model of cholinergic modulation of inhibition in macaque visual cortex, but not of cholinergic modulation of visual cortical circuits in general. We also propose that ACh acting via m1 AChRs is a candidate underlying mechanism for the strong effects of attention on narrow-spiking neurons observed in behaving animals. PMID:24944872

  7. Muscarinic acetylcholine receptors are expressed by most parvalbumin-immunoreactive neurons in area MT of the macaque.

    PubMed

    Disney, Anita A; Alasady, Hussein A; Reynolds, John H

    2014-05-01

    In the mammalian neocortex, cells that express parvalbumin (PV neurons) comprise a dominant class of inhibitory neuron that substantially overlaps with the fast/narrow-spiking physiological phenotype. Attention has pronounced effects on narrow-spiking neurons in the extrastriate cortex of macaques, and more consistently so than on their broad-spiking neighbors. Cortical neuromodulation by acetylcholine (ACh) is a candidate mechanism for aspects of attention and in the primary visual cortex (V1) of the macaque, receptors for ACh (AChRs) are strongly expressed by inhibitory neurons. In particular, most PV neurons in macaque V1 express m1 muscarinic AChRs and exogenously applied ACh can cause the release of γ-aminobutyric acid. In contrast, few PV neurons in rat V1 express m1 AChRs. While this could be a species difference, it has also been argued that macaque V1 is anatomically unique when compared with other cortical areas in macaques. The aim of this study was to better understand the extent to which V1 offers a suitable model circuit for cholinergic anatomy in the macaque occipital lobe, and to explore cholinergic modulation as a biological basis for the changes in circuit behavior seen with attention. We compared expression of m1 AChRs by PV neurons between area V1 and the middle temporal visual area (MT) in macaque monkeys using dual-immunofluorescence confocal microscopy. We find that, as in V1, most PV neurons in MT express m1 AChRs but, unlike in V1, it appears that so do most excitatory neurons. This provides support for V1 as a model of cholinergic modulation of inhibition in macaque visual cortex, but not of cholinergic modulation of visual cortical circuits in general. We also propose that ACh acting via m1 AChRs is a candidate underlying mechanism for the strong effects of attention on narrow-spiking neurons observed in behaving animals.

  8. 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

  9. Cholinergic Blockade Reduces Theta-Gamma Phase Amplitude Coupling and Speed Modulation of Theta Frequency Consistent with Behavioral Effects on Encoding

    PubMed Central

    Gillet, Shea N.; Climer, Jason R.; Hasselmo, Michael E.

    2013-01-01

    Large-scale neural activation dynamics in the hippocampal-entorhinal circuit local field potential, observable as theta and gamma rhythms and coupling between these rhythms, is predictive of encoding success. Behavioral studies show that systemic administration of muscarinic acetylcholine receptor antagonists selectively impairs encoding, suggesting that they may also disrupt the coupling between the theta and gamma bands. Here, we tested the hypothesis that muscarinic antagonists selectively disrupt coupling between theta and gamma. Specifically, we characterized the effects of systemically administered scopolamine on movement-induced theta and gamma rhythms recorded in the superficial layers of the medial entorhinal cortex (MEC) of freely moving rats. We report the novel result that gamma power at the peak of theta was most reduced following muscarinic blockade, significantly shifting the phase of maximal gamma power to occur at later phases of theta. We also characterize the existence of multiple distinct gamma bands in the superficial layers of the MEC. Further, we observed that theta frequency was significantly less modulated by movement speed following muscarinic blockade. Finally, the slope relating speed to theta frequency, a correlate of familiarity with a testing enclosure, increased significantly less between the preinjection and recovery trials when scopolamine was administered during the intervening injection session than when saline was administered, suggesting that scopolamine reduced encoding of the testing enclosure. These data are consistent with computational models suggesting that encoding and retrieval occur during the peak and trough of theta, respectively, and support the theory that acetylcholine regulates the balance between encoding versus retrieval. PMID:24336727

  10. Higher order net-proton number cumulants dependence on the centrality definition and other spurious effects

    NASA Astrophysics Data System (ADS)

    Sombun, S.; Steinheimer, J.; Herold, C.; Limphirat, A.; Yan, Y.; Bleicher, M.

    2018-02-01

    We study the dependence of the normalized moments of the net-proton multiplicity distributions on the definition of centrality in relativistic nuclear collisions at a beam energy of \\sqrt{{s}{NN}}=7.7 {GeV}. Using the ultra relativistic quantum molecular dynamics model as event generator we find that the centrality definition has a large effect on the extracted cumulant ratios. Furthermore we find that the finite efficiency for the determination of the centrality introduces an additional systematic uncertainty. Finally, we quantitatively investigate the effects of event-pile up and other possible spurious effects which may change the measured proton number. We find that pile-up alone is not sufficient to describe the data and show that a random double counting of events, adding significantly to the measured proton number, affects mainly the higher order cumulants in most central collisions.

  11. In vivo antimuscarinic actions of the third generation antihistaminergic agent, desloratadine.

    PubMed

    Howell, G; West, L; Jenkins, C; Lineberry, B; Yokum, D; Rockhold, R

    2005-08-18

    Muscarinic receptor mediated adverse effects, such as sedation and xerostomia, significantly hinder the therapeutic usefulness of first generation antihistamines. Therefore, second and third generation antihistamines which effectively antagonize the H1 receptor without significant affinity for muscarinic receptors have been developed. However, both in vitro and in vivo experimentation indicates that the third generation antihistamine, desloratadine, antagonizes muscarinic receptors. To fully examine the in vivo antimuscarinic efficacy of desloratadine, two murine and two rat models were utilized. The murine models sought to determine the efficacy of desloratadine to antagonize muscarinic agonist induced salivation, lacrimation, and tremor. Desloratadine's effect on the cardiovascular system was explored in both rodent models. In the pithed rat, both desloratadine (1.0 mg/kg, i.v.) and the muscarinic M2 selective antagonist, methoctramine (0.5 mg/kg, i.v.), inhibited negative inotropic (left ventricular dP/dt) effects caused by oxotremorine, a nonselective muscarinic agonist (p < 0.05). Negative chronotropic effects caused by oxotremorine were inhibited by desloratadine, methoctramine, and the muscarinic M3 selective antagonist, 4-DAMP (1.0 mg/kg, i.v.). A late positive inotropic event observed after the initial decrease was inhibited by all three test compounds with desloratadine and 4-DAMP being the most efficacious. In the conscious animal, inhibition of baroreflex-mediated bradycardia was evaluated. Unlike atropine (0.5 mg/kg, i.v.), desloratadine did not alter this bradycardia. The antimuscarinic action of desloratadine on salivation, lacrimation, and tremor was also explored. In urethane-anesthetized (1.5 g/kg, i.p.) male ICR mice (25-35 g) desloratadine (1.0, 5.0 mg/kg) did not inhibit oxotremorine-induced (0.5 mg/kg, s.c.) salivation, unlike atropine (0.5 mg/kg) and 4-DAMP (1.0 mg/kg). In conscious mice, desloratadine failed to inhibit oxotremorine

  12. Verapamil Blocks Scopolamine Enhancement Effect on Memory Consolidation in Passive Avoidance Task in Rats

    PubMed Central

    Giménez De Béjar, Verónica; Caballero Bleda, María; Popović, Natalija; Popović, Miroljub

    2017-01-01

    Our recent data have indicated that scopolamine, a non-selective muscarinic receptor antagonist, improves memory consolidation, in a passive avoidance task, tested in rats. It has been found that verapamil, a phenylalkylamine class of the L-type voltage-dependent calcium channel antagonist, inhibits [3H] N-methyl scopolamine binding to M1 muscarinic receptors. However, there are no data about the effect of verapamil on memory consolidation in the passive avoidance task, in rats. The purpose of the present study was to examine the effects of verapamil (0.5, 1.0, 2.5, 5.0, 10, or 20 mg/kg i.p.) as well as the interaction between scopolamine and verapamil on memory consolidation in the step-through passive avoidance task, in Wistar rats. Our results showed that verapamil (1.0 and 2.5 mg/kg) administered immediately after the acquisition task significantly increased the latency of the passive avoidance response, on the 48 h retested trial, improving memory consolidation. On the other hand, verapamil in a dose of 5 mg/kg, that per se does not affect memory consolidation, significantly reversed the memory consolidation improvement induced by scopolamine (1 mg/kg, i.p., administered immediately after verapamil treatment) but did not change the passive avoidance response in rats treated by an ineffective dose of scopolamine (30 mg/kg). In conclusion, the present data suggest that (1) the post-training administration of verapamil, dose-dependently, improves the passive avoidance response; (2) verapamil, in ineffective dose, abolished the improvement of memory consolidation effect of scopolamine; and (3) exists interaction between cholinergic muscarinic receptors and calcium homeostasis-related mechanisms in the consolidation of emotional memory. PMID:28878678

  13. [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

  14. Flumazenil antagonizes the central effects of zolpidem, an imidazopyridine hypnotic.

    PubMed

    Patat, A; Naef, M M; van Gessel, E; Forster, A; Dubruc, C; Rosenzweig, P

    1994-10-01

    Zolpidem is a new imidazopyridine-hypnotic that selectively binds to the central omega 1-receptor subtype. A double-blind, randomized, three-way, crossover placebo-controlled study was carried out in nine healthy male volunteers to assess the possible antagonism of central nervous system--depressant effects of zolpidem by flumazenil. Subjects received zolpidem (0.21 mg/kg) or placebo, intravenously, followed 17 minutes later by flumazenil (0.04 mg/kg) or placebo. Vigilance and performance were assessed by a trained anesthetist with use of ciliary reflex, response to a verbal instruction, subjective sedation, a tracking task, and a free recall task. Zolpidem produced a clinically relevant hypnotic effect in five subjects and significantly impaired performance in all nine subjects up to 90 minutes after dosing. Flumazenil rapidly antagonized clinical sedation in the five subjects who were asleep and significantly reversed the performance decrement within 3 minutes, without any escape phenomenon. Flumazenil did not change zolpidem plasma concentrations, confirming the pharmacodynamic nature of the interaction. Flumazenil may thus be a safe and effective antidote in patients with zolpidem overdosage.

  15. An assessment and comparison of the effects of oxotremorine, D-cycloserine, and bicuculline on delayed matching-to-sample performance in rats.

    PubMed

    Harper, D N

    2000-05-01

    The effects of a muscarinic antagonist (scopolamine), a muscarinic agonist (oxotremorine), an agonist at the N-methyl-D-aspartate receptor site (D-cycloserine), and a GABAa antagonist (bicuculline) on working memory were compared using rats performing a delayed matching-to-sample task. When administered on their own, oxotremorine, D-cycloserine, and bicuculline had no effect on performance in the current task. When administered concurrently with scopolamine, oxotremorine (at 1 dose) and bicuculline (at 2 doses) improved accuracy (in terms of percentage correct) by ameliorating the scopolamine-induced increase in response bias. None of the drugs, however, were successful in ameliorating the scopolamine-induced impairment in bias-free recognition performance per se (as measured by Log d). Therefore, none of the drugs examined were able to fully ameliorate all aspects of the memory impairment caused by scopolamine.

  16. 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

  17. Impaired muscarinic type 3 (M3) receptor/PKC and PKA pathways in islets from MSG-obese rats.

    PubMed

    Ribeiro, Rosane Aparecida; Balbo, Sandra Lucinei; Roma, Letícia Prates; Camargo, Rafael Ludemann; Barella, Luiz Felipe; Vanzela, Emerielle Cristine; de Freitas Mathias, Paulo Cesar; Carneiro, Everardo Magalhães; Boschero, Antonio Carlos; Bonfleur, Maria Lúcia

    2013-07-01

    Monosodium glutamate-obese rats are glucose intolerant and insulin resistant. Their pancreatic islets secrete more insulin at increasing glucose concentrations, despite the possible imbalance in the autonomic nervous system of these rats. Here, we investigate the involvement of the cholinergic/protein kinase (PK)-C and PKA pathways in MSG β-cell function. Male newborn Wistar rats received a subcutaneous injection of MSG (4 g/kg body weight (BW)) or hyperosmotic saline solution during the first 5 days of life. At 90 days of life, plasma parameters, islet static insulin secretion and protein expression were analyzed. Monosodium glutamate rats presented lower body weight and decreased nasoanal length, but had higher body fat depots, glucose intolerance, hyperinsulinemia and hypertrigliceridemia. Their pancreatic islets secreted more insulin in the presence of increasing glucose concentrations with no modifications in the islet-protein content of the glucose-sensing proteins: the glucose transporter (GLUT)-2 and glycokinase. However, MSG islets presented a lower secretory capacity at 40 mM K(+) (P < 0.05). The MSG group also released less insulin in response to 100 μM carbachol, 10 μM forskolin and 1 mM 3-isobutyl-1-methyl-xantine (P < 0.05, P < 0.0001 and P < 0.01). These effects may be associated with a the decrease of 46 % in the acetylcholine muscarinic type 3 (M3) receptor, and a reduction of 64 % in PKCα and 36 % in PKAα protein expressions in MSG islets. Our data suggest that MSG islets, whilst showing a compensatory increase in glucose-induced insulin release, demonstrate decreased islet M3/PKC and adenylate cyclase/PKA activation, possibly predisposing these prediabetic rodents to the early development of β-cell dysfunction.

  18. Species differences in the negative inotropic effect of acetylcholine and soman in rat, guinea pig, and rabbit hearts. (Reannouncement with new availability information)

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

    Maxwell, D.M.; Thomsen, R.H.; Baskin, S.I.

    1991-12-31

    Acetylcholine reduced atrial contractions by 82.5% in guinea pig, 50.8% in rat, and 41.5% in rabbit. 2. The EC50, values for the negative inotropic effect of acetylcholine were 3.3 x 10(-7) M in rat and guinea pig atria and 4.1 x 10(-6) M in rabbit atria. 3. There was no correlation between the species differences in the negative inotropic effect of acetylcholine in atria and the density or affinity of acetylcholinesterase or muscarinic receptors. 4. Inhibition of atrial acetylcholinesterase with soman reduced the EC50 of acetylcholine three-fold in all species, but did not change the maximal inotropic effect of acetylcholine.more » 5. Species differences in the negative inotropic effect of acetylcholine may be caused by differences in the coupling between myocardial muscarinic receptors and the ion channels that mediate negative inotropy. Acetylcholine, cardiovascular response, species variation negative inotropic response.« less

  19. Selective expression of muscarinic acetylcholine receptor subtype M3 by mouse type III taste bud cells.

    PubMed

    Mori, Yusuke; Eguchi, Kohgaku; Yoshii, Kiyonori; Ohtubo, Yoshitaka

    2016-11-01

    Each taste bud cell (TBC) type responds to a different taste. Previously, we showed that an unidentified cell type(s) functionally expresses a muscarinic acetylcholine (ACh) receptor subtype, M3, and we suggested the ACh-dependent modification of its taste responsiveness. In this study, we found that M3 is expressed by type III TBCs, which is the only cell type that possesses synaptic contacts with taste nerve fibers in taste buds. The application of ACh to the basolateral membrane of mouse fungiform TBCs in situ increased the intracellular Ca 2+ concentration in 2.4 ± 1.4 cells per taste bud (mean ± SD, n = 14). After Ca 2+ imaging, we supravitally labeled type II cells (phospholipase C β2 [PLCβ2]-immunoreactive cells) with Lucifer yellow CH (LY), a fluorescent dye and investigated the positional relationship between ACh-responding cells and LY-labeled cells. After fixation, the TBCs were immunohistostained to investigate the positional relationships between immunohistochemically classified cells and LY-labeled cells. The overlay of the two positional relationships obtained by superimposing the LY-labeled cells showed that all of the ACh-responding cells were type III cells (synaptosomal-associated protein 25 [SNAP-25]-immunoreactive cells). The ACh responses required no added Ca 2+ in the bathing solution. The addition of 1 μM U73122, a phospholipase C inhibitor, decreased the magnitude of the ACh response, whereas that of 1 μM U73343, a negative control, had no effect. These results suggest that type III cells respond to ACh and release Ca 2+ from intracellular stores. We also discuss the underlying mechanism of the Ca 2+ response and the role of M3 in type III cells.

  20. Molecular modeling, theoretical calculations and property evaluation of three muscarinic agonists. X-ray structures of LU 25-109 and WAL 2014

    NASA Astrophysics Data System (ADS)

    Dolmella, A.; Bandoli, G.; Cavallin, M.

    2000-08-01

    LU 25-109 ( II) and WAL 2014 (talsaclidine, III) are two M1 muscarinic agonists chemically related to the natural substance arecoline ( I). All these compounds have beneficial effects on memory and cognition in animals and humans, and they have been proposed in the treatment of Alzheimer's disease, but only III will likely find a place in therapy. In this work we have investigated the solid state structures of II and III, and the X-ray structures of the two molecules and of the parent compound I have been used to input a series of computational chemistry efforts. In particular, the X-ray geometries have been manipulated to model 20 molecular structures ( 1- 20) which have been submitted to ab initio, semiempirical quantum mechanics and molecular mechanics calculations. The conformational space accessible to the 20 structures has been assessed by means of potential energy maps. The reactivities of 1- 20 have been estimated by examining at the graphics terminal the composition and the extension of the frontier orbitals (HOMOs and LUMOs) and of the molecular electrostatic potential. The information obtained has been interpreted to explain the different degrees of activity shown by I- III. Our data indicate that III has better in vivo activity for its intermediate size, less polar surface, conformational rigidity and orientation of reactive domains.

  1. A Review of Chronic Musculoskeletal Pain: Central and Peripheral Effects of Diclofenac.

    PubMed

    Atzeni, Fabiola; Masala, Ignazio Francesco; Sarzi-Puttini, Piercarlo

    2018-06-05

    Diclofenac is widely used to manage chronic inflammatory and degenerative joint diseases such as osteoarthritis (OA), rheumatoid arthritis (RA), ankylosing spondylitis, and extra-articular rheumatism. Its various mechanisms of action make it particularly effective in treating nociceptive pain, but it is also an alternative for treating spinal and chronic central pain. Osteoarthritis and rheumatoid arthritis are the most frequently encountered arthritic conditions in adults. The management of nociceptive pain requires a sequential hierarchical approach, with the initial NSAID treatment being characterized by the replacement of one drug with another, or complete discontinuation usually because of insufficient pain control. OA- and RA-related pain is complex and multifactorial, and due to physiological interactions between the signaling of the central and peripheral nervous systems. The mechanisms of action of diclofenac make it particularly effective in treating both nociceptive pain and chronic central pain. This review underlines the mechanisms of diclofenac involved in chronic and acute joint pain, the most relevant adverse events.

  2. Cholinergic regulation of the evoked quantal release at frog neuromuscular junction

    PubMed Central

    Nikolsky, Eugeny E; Vyskočil, František; Bukharaeva, Ella A; Samigullin, Dmitry; Magazanik, Lev G

    2004-01-01

    The effects of cholinergic drugs on the quantal contents of the nerve-evoked endplate currents (EPCs) and the parameters of the time course of quantal release (minimal synaptic latency, main modal value of latency histogram and variability of synaptic latencies) were studied at proximal, central and distal regions of the frog neuromuscular synapse. Acetylcholine (ACh, 5 × 10−4 m), carbachol (CCh, 1 × 10−5 m) or nicotine (5 × 10−6 m) increased the numbers of EPCs with long release latencies mainly in the distal region of the endplate (90–120 μm from the last node of Ranvier), where the synchronization of transmitter release was the most pronounced. The parameters of focally recorded motor nerve action potentials were not changed by either ACh or CCh. The effects of CCh and nicotine on quantal dispersion were reduced substantially by 5 × 10−7 m (+)tubocurarine (TC). The muscarinic agonists, oxotremorine and the propargyl ester of arecaidine, as well as antagonists such as pirenzepine, AF-DX 116 and methoctramine, alone or in combination, did not affect the dispersion of the release. Muscarinic antagonists did not block the dispersion action of CCh. Cholinergic drugs either decreased the quantal content mo (muscarinic agonist, oxotremorine M, and nicotinic antagonist, TC), or decreased mo and dispersed the release (ACh, CCh and nicotine). The effects on mo were not related either to the endplate region or to the initial level of release dispersion. It follows that the mechanisms regulating the amount and the time course of transmitter release are different and that, among other factors, they are altered by presynaptic nicotinic receptors. PMID:15254150

  3. Hawthorn (Crataegus monogyna Jacq.) extract exhibits atropine-sensitive activity in a cultured cardiomyocyte assay.

    PubMed

    Salehi, Satin; Long, Shannon R; Proteau, Philip J; Filtz, Theresa M

    2009-01-01

    Hawthorn (Crataegus spp.) plant extract is used as a herbal alternative medicine for the prevention and treatment of various cardiovascular diseases. Recently, it was shown that hawthorn extract preparations caused negative chronotropic effects in a cultured neonatal murine cardiomyocyte assay, independent of beta-adrenergic receptor blockade. The aim of this study was to further characterize the effect of hawthorn extract to decrease the contraction rate of cultured cardiomyocytes. To test the hypothesis that hawthorn is acting via muscarinic receptors, the effect of hawthorn extract on atrial versus ventricular cardiomyocytes in culture was evaluated. As would be expected for activation of muscarinic receptors, hawthorn extract had a greater effect in atrial cells. Atrial and/or ventricular cardiomyocytes were then treated with hawthorn extract in the presence of atropine or himbacine. Changes in the contraction rate of cultured cardiomyocytes revealed that both muscarinic antagonists significantly attenuated the negative chronotropic activity of hawthorn extract. Using quinuclidinyl benzilate, L-[benzylic-4,4'-(3)H] ([(3)H]-QNB) as a radioligand antagonist, the effect of a partially purified hawthorn extract fraction to inhibit muscarinic receptor binding was quantified. Hawthorn extract fraction 3 dose-dependently inhibited [(3)H]-QNB binding to mouse heart membranes. Taken together, these findings suggest that decreased contraction frequency by hawthorn extracts in neonatal murine cardiomyocytes may be mediated via muscarinic receptor activation.

  4. 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

  5. 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

  6. The Central Effects of Androgenic-anabolic Steroid Use.

    PubMed

    Mędraś, Marek; Brona, Anna; Jóźków, Paweł

    : Millions of men use androgenic-anabolic steroids (AAS) to stimulate muscle growth and improve physical appearance. Although 1 out of 3 people who uses androgenic-anabolic steroids develops a steroid use disorder, the effects of the drugs on the central nervous system and the psyche are still not well understood. Although most addictive substances improve mood immediately after administration, AAS exert less pronounced euphoric effects. Instead, they are primarily taken for the delayed gratification of increased muscle mass. Withdrawal from AAS may lead to a range of somatic and psychiatric symptoms, and, in many cases, comprehensive treatment supervised by an endocrinologist and a psychiatrist is required.

  7. 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.

  8. 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

  9. Venomic and antivenomic analyses of the Central American coral snake, Micrurus nigrocinctus (Elapidae).

    PubMed

    Fernández, Julián; Alape-Girón, Alberto; Angulo, Yamileth; Sanz, Libia; Gutiérrez, José María; Calvete, Juan J; Lomonte, Bruno

    2011-04-01

    The proteome of the venom of Micrurus nigrocinctus (Central American coral snake) was analyzed by a "venomics" approach. Nearly 50 venom peaks were resolved by RP-HPLC, revealing a complex protein composition. Comparative analyses of venoms from individual specimens revealed that such complexity is an intrinsic feature of this species, rather than the sum of variable individual patterns of simpler composition. Proteins related to eight distinct families were identified by MS/MS de novo peptide sequencing or N-terminal sequencing: phospholipase A(2) (PLA(2)), three-finger toxin (3FTx), l-amino acid oxidase, C-type lectin/lectin-like, metalloproteinase, serine proteinase, ohanin, and nucleotidase. PLA(2)s and 3FTxs are predominant, representing 48 and 38% of the venom proteins, respectively. Within 3FTxs, several isoforms of short-chain α-neurotoxins as well as muscarinic-like toxins and proteins with similarity to long-chain κ-2 bungarotoxin were identified. PLA(2)s are also highly diverse, and a toxicity screening showed that they mainly exert myotoxicity, although some are lethal and may contribute to the known presynaptic neurotoxicity of this venom. An antivenomic characterization of a therapeutic monospecific M. nigrocinctus equine antivenom revealed differences in immunorecognition of venom proteins that correlate with their molecular mass, with the weakest recognition observed toward 3FTxs.

  10. 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

  11. Combining benefits of an adrenergic and a muscarinic blocker in a single formulation - a pharmacokinetic evaluation.

    PubMed

    Nazarudheen, Shabana; Dey, Surajit; Kandhwal, Kirti; Arora, Rachna; Reyar, Simrit; Khuroo, Arshad H; Monif, Tausif; Madan, Sumit; Arora, Vinod

    2013-11-01

    A pharmacokinetic bioequivalence study was conducted in Asian subjects, to compare a fixed dose combination capsule single oral dose of alpha adrenoceptor blocker-Alfuzosin hydrochloride 10mg extended release and muscarinic antagonists-Solifenacin succinate 5mg against individually administered Xatral XL 10mg tablets (Alfuzosin) of Sanofi Synthelabo Limited, United Kingdom (UK) and Vesicare 5mg tablets (Solifenacin) of Astellas Pharma Limited, UK under fed conditions. Blood samples were collected pre-dose up to 72 h post dose for determination of plasma Alfuzosin and Solifenacin concentrations and calculation of the pharmacokinetic parameters. ANOVA was performed on the log (natural)-transformed pharmacokinetic parameters. A 90% confidence interval for the ratios of the test and reference product averages (least square means) were calculated for alfuzosin and solifenacin. The 90% confidence intervals obtained for alfuzosin for Cmax, AUC0-t and AUC0-∞ were 102.74-122.75%, 95.84-116.96% and 95.82-116.76%, respectively. The 90% confidence intervals obtained for Solifenacin for Cmax, and AUC0-72 were 89.55-97.91% and 90.47-99.38%, respectively. Based on the results, the fixed dose combination was concluded to be bioequivalent to individually administered products. Copyright © 2013 Elsevier Inc. All rights reserved.

  12. 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

  13. 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.

  14. Urothelium muscarinic activation phosphorylates CBSSer227 via cGMP/PKG pathway causing human bladder relaxation through H2S production

    PubMed Central

    d’Emmanuele di Villa Bianca, Roberta; Mitidieri, Emma; Fusco, Ferdinando; Russo, Annapina; Pagliara, Valentina; Tramontano, Teresa; Donnarumma, Erminia; Mirone, Vincenzo; Cirino, Giuseppe; Russo, Giulia; Sorrentino, Raffaella

    2016-01-01

    The urothelium modulates detrusor activity through releasing factors whose nature has not been clearly defined. Here we have investigated the involvement of H2S as possible mediator released downstream following muscarinic (M) activation, by using human bladder and urothelial T24 cell line. Carbachol stimulation enhances H2S production and in turn cGMP in human urothelium or in T24 cells. This effect is reversed by cysthationine-β-synthase (CBS) inhibition. The blockade of M1 and M3 receptors reverses the increase in H2S production in human urothelium. In T24 cells, the blockade of M1 receptor significantly reduces carbachol-induced H2S production. In the functional studies, the urothelium removal from human bladder strips leads to an increase in carbachol-induced contraction that is mimicked by CBS inhibition. Instead, the CSE blockade does not significantly affect carbachol-induced contraction. The increase in H2S production and in turn of cGMP is driven by CBS-cGMP/PKG-dependent phosphorylation at Ser227 following carbachol stimulation. The finding of the presence of this crosstalk between the cGMP/PKG and H2S pathway downstream to the M1/M3 receptor in the human urothelium further implies a key role for H2S in bladder physiopathology. Thus, the modulation of the H2S pathway can represent a feasible therapeutic target to develop drugs for bladder disorders. PMID:27509878

  15. Evidence for the gastric cytoprotective effect of centrally injected agmatine.

    PubMed

    Zádori, Zoltán S; Tóth, Viktória E; Fehér, Ágnes; Philipp, Kirsch; Németh, József; Gyires, Klára

    2014-09-01

    Agmatine (decarboxylated arginine) exerts cytoprotective action in several tissues, such as in the brain, heart or kidneys, but there is still controversy over the effects of agmatine on the gastric mucosa. The aim of the present study was to reveal the potential gastroprotective action of agmatine by using an acid-independent ulcer model to clarify which receptors and peripheral factors are involved in it. Gastric mucosal damage was induced by acidified ethanol. Mucosal levels of calcitonin gene-related peptide (CGRP) and somatostatin were determined by radioimmunoassay. For analysis of gastric motor activity the rubber balloon method was used. It was found that agmatine given intracerebroventricularly (i.c.v., 0.044-220 nmol/rat) significantly inhibited the development of ethanol-induced mucosal damage, while in the case of intraperitoneal injection (0.001-50mg/kg i.p.) it had only a minor effect. The central gastroprotective action of agmatine was completely antagonized by mixed alpha2-adrenoceptor and imidazoline I1 receptor antagonists (idazoxan, efaroxan), but only partially by yohimbine (selective alpha2-adrenoceptor antagonist) and AGN 192403 (selective I1 receptor ligand, putative antagonist). It was also inhibited by the non-selective opioid-receptor antagonist naloxone and the selective δ-opioid receptor antagonist naltrindole, but not by β-funaltrexamine and nor-Binaltorphimine (selective μ- and κ-opioid receptor antagonists, respectively). Furthermore, the effect of agmatine was antagonized by bilateral cervical vagotomy and by pretreatment with indomethacin and NG-nitro-l-arginine. Agmatine also reversed the ethanol-induced reduction of gastric mucosal CGRP and somatostatin content, but did not have any significant effect on gastric motor activity. These results indicate that agmatine given centrally induces gastric cytoprotection, which is mediated by central imidazoline I1 receptors, alpha2-adrenoceptors and δ-opioid receptors. Activation of

  16. Assessment Drives Learning: The Effect of Central Exit Exams on Curricular Knowledge and Mathematical Literacy

    ERIC Educational Resources Information Center

    Jurges, Hendrik; Schneider, Kerstin; Senkbeil, Martin; Carstensen, Claus H.

    2012-01-01

    In this paper, we use data from the German PISA 2003 sample to study the effects of central exit examinations on student performance and student attitudes. Unlike earlier studies we use (i) a value-added measure to pin down the effect of central exit exams on learning in the last year before the exam and (ii) separate test scores for mathematical…

  17. Effects of antihypertensive drugs on central blood pressure in humans: a preliminary observation.

    PubMed

    Agnoletti, Davide; Zhang, Yi; Borghi, Claudio; Blacher, Jacques; Safar, Michel E

    2013-08-01

    Central blood pressure (BP) is considered a better predictor of cardiovascular events than brachial BP. Modifications of central, beyond brachial BP, can be assessed by pressure amplification, a potential new cardiovascular risk factor. Comparison between drugs' effect on central hemodynamics has been poorly studied. Our aim was to assess the hemodynamic effect of a 12-week treatment with amlodipine 5mg, or candesartan 8mg, or indapamide sustained-release 1.5mg, in comparison with placebo. We analyzed 145 out-patients with essential hypertension in primary prevention enrolled in the Natrilix SR Versus Candesartan and Amlodipine in the Reduction of Systolic Blood Pressure in Hypertensive Patients (X-CELLENT) study, a multicenter, randomized, double-blinded, placebo-controlled trial. Arterial stiffness, central BP, pressure amplification, and wave reflection were measured by applanation tonometry. Baseline characteristics of patients were homogeneous between groups. After treatment, we found that active drugs produced similar reduction of both central and peripheral BPs, with no significant interdrug differences (all P < 0.05; excluded peripheral pulse pressure, compared with placebo). Second, amlodipine (1.9% ± 15.3%), candesartan (3.0% ± 14.6%) and indapamide (4.1% ± 14.4%) all increased pulse pressure amplification, but only indapamide was statistically different from placebo (P = 0.02). Finally, no significant changes were observed on pulse wave velocity, heart rate, and augmentation index. The 3 antihypertensive drugs similarly reduced peripheral and central BP, as compared with placebo, but a significant increase in pulse pressure amplification was obtained only with indapamide, independently of arterial stiffness modifications. 3283161 by BIOPHARMA.

  18. GABA interneurons mediate the rapid antidepressant-like effects of scopolamine

    PubMed Central

    Wohleb, Eric S.; Wu, Min; Gerhard, Danielle M.; Taylor, Seth R.; Picciotto, Marina R.; Alreja, Meenakshi; Duman, Ronald S.

    2016-01-01

    Major depressive disorder (MDD) is a recurring psychiatric illness that causes substantial health and socioeconomic burdens. Clinical reports have revealed that scopolamine, a nonselective muscarinic acetylcholine receptor antagonist, produces rapid antidepressant effects in individuals with MDD. Preclinical models suggest that these rapid antidepressant effects can be recapitulated with blockade of M1-type muscarinic acetylcholine receptors (M1-AChR); however, the cellular mechanisms underlying activity-dependent synaptic and behavioral responses to scopolamine have not been determined. Here, we demonstrate that the antidepressant-like effects of scopolamine are mediated by GABA interneurons in the medial prefrontal cortex (mPFC). Both GABAergic (GAD67+) interneurons and glutamatergic (CaMKII+) interneurons in the mPFC expressed M1-AChR. In mice, viral-mediated knockdown of M1-AChR specifically in GABAergic neurons, but not glutamatergic neurons, in the mPFC attenuated the antidepressant-like effects of scopolamine. Immunohistology and electrophysiology showed that somatostatin (SST) interneurons in the mPFC express M1-AChR at higher levels than parvalbumin interneurons. Moreover, knockdown of M1-AChR in SST interneurons in the mPFC demonstrated that M1-AChR expression in these neurons is required for the rapid antidepressant-like effects of scopolamine. These data indicate that SST interneurons in the mPFC are a promising pharmacological target for developing rapid-acting antidepressant therapies. PMID:27270172

  19. GABA interneurons mediate the rapid antidepressant-like effects of scopolamine.

    PubMed

    Wohleb, Eric S; Wu, Min; Gerhard, Danielle M; Taylor, Seth R; Picciotto, Marina R; Alreja, Meenakshi; Duman, Ronald S

    2016-07-01

    Major depressive disorder (MDD) is a recurring psychiatric illness that causes substantial health and socioeconomic burdens. Clinical reports have revealed that scopolamine, a nonselective muscarinic acetylcholine receptor antagonist, produces rapid antidepressant effects in individuals with MDD. Preclinical models suggest that these rapid antidepressant effects can be recapitulated with blockade of M1-type muscarinic acetylcholine receptors (M1-AChR); however, the cellular mechanisms underlying activity-dependent synaptic and behavioral responses to scopolamine have not been determined. Here, we demonstrate that the antidepressant-like effects of scopolamine are mediated by GABA interneurons in the medial prefrontal cortex (mPFC). Both GABAergic (GAD67+) interneurons and glutamatergic (CaMKII+) interneurons in the mPFC expressed M1-AChR. In mice, viral-mediated knockdown of M1-AChR specifically in GABAergic neurons, but not glutamatergic neurons, in the mPFC attenuated the antidepressant-like effects of scopolamine. Immunohistology and electrophysiology showed that somatostatin (SST) interneurons in the mPFC express M1-AChR at higher levels than parvalbumin interneurons. Moreover, knockdown of M1-AChR in SST interneurons in the mPFC demonstrated that M1-AChR expression in these neurons is required for the rapid antidepressant-like effects of scopolamine. These data indicate that SST interneurons in the mPFC are a promising pharmacological target for developing rapid-acting antidepressant therapies.

  20. 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.