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Sample records for muscarinic cholinergic receptor

  1. Muscarinic and dopaminergic receptor subtypes on striatal cholinergic interneurons

    SciTech Connect

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

    1990-12-01

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

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

    SciTech Connect

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

    1988-02-01

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

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

    PubMed

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

    2010-04-01

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

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

    SciTech Connect

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

    1985-07-01

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

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

    PubMed

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

    2015-07-15

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

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

    SciTech Connect

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

    1987-05-01

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

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

    SciTech Connect

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

    1983-12-01

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

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

    SciTech Connect

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

    1987-03-01

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

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

    DTIC Science & Technology

    1983-11-11

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

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

    SciTech Connect

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

    1985-06-01

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

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

    PubMed

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

    2008-07-01

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

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

    PubMed

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

    2013-08-01

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

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

    PubMed

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

    2013-04-01

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

  14. 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.

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

    SciTech Connect

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

    1985-02-01

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

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

    PubMed

    Eglen, R M

    2006-07-01

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

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

    SciTech Connect

    Koide, T.; Matsushita, H.

    1981-03-09

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

  18. 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.

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

    SciTech Connect

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

    1986-10-01

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

  20. Postsynaptic muscarinic m2 receptors at cholinergic and glutamatergic synapses of mouse brainstem motoneurons.

    PubMed

    Csaba, Zsolt; Krejci, Eric; Bernard, Véronique

    2013-06-15

    In many brain areas, few cholinergic synapses are identified. Acetylcholine is released into the extracellular space and acts through diffuse transmission. Motoneurons, however, are contacted by numerous cholinergic terminals, indicating synaptic cholinergic transmission on them. The muscarinic m2 receptor is the major acetylcholine receptor subtype of motoneurons; therefore, we analyzed the localization of the m2 receptor in correlation with synapses by electron microscopic immunohistochemistry in the mouse trigeminal, facial, and hypoglossal motor nuclei. In all nuclei, m2 receptors were localized at the membrane of motoneuronal perikarya and dendrites. The m2 receptors were concentrated at cholinergic synapses located on the perikarya and most proximal dendrites. However, m2 receptors at cholinergic synapses represented only a minority (<10%) of surface m2 receptors. The m2 receptors were also enriched at glutamatergic synapses in both motoneuronal perikarya and dendrites. A relatively large proportion (20-30%) of plasma membrane-associated m2 receptors were located at glutamatergic synapses. In conclusion, the effect of acetylcholine on motoneuron populations might be mediated through a synaptic as well as diffuse type of transmission.

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

    PubMed

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

    2005-06-01

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

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

    PubMed

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

    2013-06-15

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

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

    SciTech Connect

    Scherer, R.W.

    1987-01-01

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

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

    PubMed

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

    1995-01-01

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

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

    SciTech Connect

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

    1991-06-01

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

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

    SciTech Connect

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

    1988-01-01

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

  7. Role of muscarinic and nicotinic cholinergic receptors in an experimental model of epilepsy-induced analgesia.

    PubMed

    de Freitas, Renato Leonardo; de Oliveira, Rithiele Cristina; de Carvalho, Andressa Daiane; Felippotti, Tatiana Tocchini; Bassi, Gabriel Shimizu; Elias-Filho, Daoud Hibrahim; Coimbra, Norberto Cysne

    2004-10-01

    The blockade of GABA-mediated Cl(-) influx with pentylenetetrazol (PTZ) was used in the present work to induce seizures in animals. The neurotransmission in the postictal period has been the focus of many studies, and there is evidence suggesting antinociceptive mechanisms following tonic-clonic seizures in both animals and men. The aim of this work was to study the involvement of acetylcholine in the antinociception induced by convulsions elicited by peripheral administration of PTZ (64 mg/kg). Analgesia was measured by the tail-flick test in eight albino Wistar rats per group. Convulsions were followed by significant increases in tail-flick latencies (TFLs) at least for 120 min of the postictal period. Peripheral administration of atropine (0.25, 1 and 4 mg/kg) caused a significant dose-dependent decrease in the TFL in seizing animals, as compared to controls. These data were corroborated by peripheral administration of mecamylamine, a nicotinic cholinergic receptor blocker, at the same doses (0.25, 1 and 4 mg/kg) used for the muscarinic cholinergic receptor antagonist. The recruitment of the muscarinic receptor was made 10 min postconvulsions and in subsequent periods of postictal analgesia, whereas the involvement of the nicotinic cholinergic receptor was implicated only after 30 min postseizures. The cholinergic antagonists caused a minimal reduction in body temperature, but did not impair baseline TFL, spontaneous exploration or motor coordination in the rotarod test at the maximal dose of 4 mg/kg. These results indicate that acetylcholine may be involved as a neurotransmitter in postictal analgesia.

  8. Alterations in alpha-adrenergic and muscarinic cholinergic receptor binding in rat brain following nonionizing radiation

    SciTech Connect

    Gandhi, V.C.; Ross, D.H.

    1987-01-01

    Microwave radiation produces hyperthermia. The mammalian thermoregulatory system defends against changes in temperature by mobilizing diverse control mechanisms. Neurotransmitters play a major role in eliciting thermoregulatory responses. The involvement of adrenergic and muscarinic cholinergic receptors was investigated in radiation-induced hyperthermia. Rats were subjected to radiation at 700 MHz frequency and 15 mW/cm/sup 2/ power density and the body temperature was raised by 2.5 degrees C. Of six brain regions investigated only the hypothalamus showed significant changes in receptor states, confirming its pivotal role in thermoregulation. Adrenergic receptors, studied by (/sup 3/H)clonidine binding, showed a 36% decrease in binding following radiation after a 2.5 degrees C increase in body temperature, suggesting a mechanism to facilitate norepinephrine release. Norepinephrine may be speculated to maintain thermal homeostasis by activating heat dissipation. Muscarinic cholinergic receptors, studied by (3H)quinuclidinyl benzilate binding, showed a 65% increase in binding at the onset of radiation. This may be attributed to the release of acetylcholine in the hypothalamus in response to heat cumulation. The continued elevated binding during the period of cooling after radiation was shut off may suggest the existence of an extra-hypothalamic heat-loss pathway.

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

    SciTech Connect

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

    1986-05-01

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

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

    PubMed

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

    2013-09-01

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

  11. 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

  12. Cholinergic stimulation of pancreatic amylase release and muscarinic receptors: effect of ionophore A23187

    SciTech Connect

    Larose, L.; Morisset, J.

    1985-07-22

    Dispersed rat pancreatic acini were incubated in 0.5 mM calcium medium with increasing concentrations of carbamylcholine, with or without the ionophore A23187 (10/sup -6/M). Addition of the ionophore reduced maximal amylase release, increased the maximal effective concentration of carbamylcholine and dramatically impaired the agonist's capacity to induce enzyme secretion at low concentration. The ionophore also abolished the inhibition of secretion observed at high carbamylcholine concentrations. These effects of the ionophore on the cholinergic secretory response cannot be explained by interaction at the muscarinic receptor since neither the Bmax, the affinity of the receptor for the (/sup 3/H)QNB nor the binding of carbamylcholine were affected by the ionophore. It is suggested that for the conditions studied, the ionophore can interact with the secretory process at one or several points ulterior to the initial recognition site of carbamylcholine on its receptor. 30 references, 3 figures.

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

    SciTech Connect

    Knapp, F.F. Jr.; McPherson, D.W.; Luo, H.

    1995-06-01

    Interest in the potential use of cerebral SPECT and PET imaging for determination of the density and activity of muscarinic-cholinergic receptors (mAChR) has been stimulated by the changes in these receptors which occur in many neurological diseases. In addition, the important involvement of mAChR in modulating negative inotropic cardiac activity suggests that such receptor ligands may have important applications in evaluation of changes which may occur in cardiac disease. In this paper, the properties of several key muscarinic receptor ligands being developed or which have been used for clinical SPECT and PET are discussed. In addition, the ORNL development of the new iodinated IQNP ligand based on QNB and the results of in vivo biodistribution studies in rats, in vitro competitive binding studies and ex vivo autoradiographic experiments are described. The use of radioiodinated IQNP may offer several advantages in comparison to IQNB because of its easy and high yield preparation and high brain uptake and the potential usefulness of the {open_quotes}partial{close_quotes} subtype selective IONP isomers. We also describe the development of new IQNP-type analogues which offer the opportunity for radiolabeling with positron-emitting radioisotopes (carbon-11, fluorine-18 and bromine-76) for potential use with PET.

  14. Loss of M2 muscarine receptors in the cerebral cortex in Alzheimer's disease and experimental cholinergic denervation.

    PubMed

    Mash, D C; Flynn, D D; Potter, L T

    1985-05-31

    Cerebral cortex samples from patients with Alzheimer's disease and from rats after experimental cholinergic denervation of the cerebral cortex exhibited reductions in the presynaptic marker choline acetyltransferase activity and in the number of M2 muscarine receptors, with no change in the number of M1 receptors. These results are in keeping with evidence that M2 receptors function in cholinergic nerve terminals to regulate the release of acetylcholine, whereas M1 receptors are located on postsynaptic cells and facilitate cellular excitation. New M1-selective agonists and M2-selective antagonists directed at post- or presynaptic sites deserve consideration as potential agents for the treatment of the disease.

  15. Subcellular redistribution of m2 muscarinic acetylcholine receptors in striatal interneurons in vivo after acute cholinergic stimulation.

    PubMed

    Bernard, V; Laribi, O; Levey, A I; Bloch, B

    1998-12-01

    The purpose of our work was to investigate how the cholinergic environment influences the targeting and the intracellular trafficking of the muscarinic receptor m2 (m2R) in vivo. To address this question, we have used immunohistochemical approaches at light and electron microscopic levels to detect the m2R in control rats and rats treated with muscarinic receptor agonists. In control animals, m2Rs were located mostly at postsynaptic sites at the plasma membrane of perikarya and dendrites of cholinergic and NPY-somatostatin interneurons as autoreceptors and heteroreceptors, respectively. Presynaptic receptors were also detected in boutons. The m2Rs were usually detected at extrasynaptic sites, but they could be found rarely in association with symmetrical synapses, suggesting that the cholinergic transmission mediated by m2R occurs via synaptic and nonsynaptic mechanisms. The stimulation of muscarinic receptors with oxotremorine provoked a dramatic alteration of m2R compartmentalization, including endocytosis with a decrease of the density of m2R at the membrane (-63%) and an increase of those associated with endosomes (+86%) in perikarya. The very strong increase of m2R associated with multivesicular bodies (+732%) suggests that oxotremorine activated degradation. The slight increase in the Golgi apparatus (+26%) suggests that the m2R stimulation had an effect on the maturation of m2R. The substance P receptor located at the membrane of the same neurons was unaffected by oxotremorine. Our data demonstrate that cholinergic stimulation dramatically influences the subcellular distribution of m2R in striatal interneurons in vivo. These events may have key roles in controlling abundance and availability of muscarinic receptors via regulation of receptor endocytosis, degradation, and/or neosynthesis. Further, the control of muscarinic receptor trafficking may influence the activity of striatal interneurons, including neurotransmitter release and/or electric activity.

  16. [Propofol-induced myocardial depression: possible role of atrial muscarinic cholinergic receptors].

    PubMed

    Aguero Peña, R E; Pascuzzo-Lima, C; Granado Duque, A E; Bonfante-Cabarcas, R A

    2008-02-01

    To investigate the possible role of muscarinic cholinergic receptors (MCRs) in the depression of myocardial function induced by propofol, an intravenous anesthetic chemically unrelated to other drugs. Although adverse effects are rare, bradycardia has been reported and this can lead to cardiac arrest in some patients. The mechanism behind this effect is still unknown but a possible role for MCRs has been suggested. The interaction of propofol with human atrial MCRs was determined by means of inhibition tests using [3H] quinuclidinyl benzilate ([3H] QNB). The displacement of [3H] QNB binding to human atrial MCRs by propofol was concentration dependent but the observed effect was not consistent with a model of simple competition between propofol and [3H] QNB. Propofol appears to have the ability to modify the activity of human atrial MCRs and this effect may be related to its ability to induce bradycardia.

  17. Local heating of human skin causes hyperemia without mediation by muscarinic cholinergic receptors or prostanoids.

    PubMed

    Golay, Sandrine; Haeberli, Christian; Delachaux, Anne; Liaudet, Lucas; Kucera, Paul; Waeber, Bernard; Feihl, François

    2004-11-01

    Local changes in surface temperature have a powerful influence on the perfusion of human skin. Heating increases local skin blood flow, but the mechanisms and mediators of this response (thermal hyperemia response) are incompletely elucidated. In the present study, we examined the possible dependence of the thermal hyperemia response on stimulation of muscarinic cholinergic receptors and on production of vasodilator prostanoids. In 13 male healthy subjects aged 20-30 yr, a temperature-controlled chamber was positioned on the volar face of one forearm and used to raise surface temperature from 34 to 41 degrees C. The time course of the resulting thermal hyperemia response was recorded with a laser-Doppler imager. In one experiment, each of eight subjects received an intravenous bolus of the antimuscarinic agent glycopyrrolate (4 microg/kg) on one visit and saline on the other. The thermal hyperemia response was determined within the hour after the injections. Glycopyrrolate effectively inhibited the skin vasodilation induced by iontophoresis of acetylcholine but did not influence the thermal hyperemia response. In a second experiment, conducted in five other subjects, 1 g of the cyclooxygenase inhibitor aspirin administered orally totally abolished the vasodilation induced in the skin by anodal current but also failed to modify the thermal hyperemia response. The present study excludes the stimulation of muscarinic receptors and the production of vasodilator prostaglandins as essential and nonredundant mechanisms for the vasodilation induced by local heating in human forearm skin.

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

    SciTech Connect

    Drayer, B.; Jaszczak, R.; Coleman, E.; Storni, A.; Greer, K.; Petry, N.; Lischko, M.; Flanagan, S.

    1982-06-01

    An attempt was made to characterize, in vivo, specific binding to the muscarinic cholinergic receptor in the calf using the radioiodinated ligand quinuclidinyl benzilate (/sup 123/I-OH-QNB) and single photon detection emission computed tomography (SPECT). The supratentorial brain activity was significantly increased after the intravenous infusion of /sup 123/I-OH-QNB as compared to free /sup 123/I. Scopolamine, a muscarinic cholinergic receptor antagonist, decreased the measured brain activity when infused prior to /sup 123/I-OH-QNB consistent with pharmacologic blockade of specific receptor binding. Quantitative in vitro tissue distribution studies obtained following SPECT imaging were consistent with regionally distinct specific receptor binding in the striatum and cortical gray matter, nonspecific binding in the cerebellum, and pharmacologic blockade of specific binding sites with scopolamine. Although /sup 123/I-OH-QNB is not the ideal radioligand, our limited success will hopefully encourage the development of improved binding probes for SPECT imaging and quantitation.

  19. Nipecotic acid ethyl ester: a cholinergic agonist that may differentiate muscarinic receptor subtypes

    SciTech Connect

    Zorn, S.H.; Duman, R.S.; Enna, S.J.; Krogsgaard-Larsen, P.; Micheletti, R.; Giraldo, E.; Giachetti, A.

    1986-03-05

    Reports indicate that nipecotic acid ethyl ester (NAEE) displays cholinomimetic properties in vivo. In the present study a series of physiological and biochemical tests were conducted to characterize this action. NAEE had a negative inotropic effect on the guinea pig atrium, and stimulated contraction of the guinea pig ileum and isolated mouse stomach strip at concentrations similar to bethanechol (BCH). The atrial and ilial effects were reversed by atropine. Unlike BCH, NAEE had no effect on basal acid secretion in the isolated mouse stomach at concentrations < 100 ..mu..M. NAEE was more potent than carbachol (CCH) in displacing /sup 3/H-ONB binding from rat brain membranes. The potency of NAEE to inhibit antagonist binding in rat heart membranes was enhanced by Mg/sup + +/ (Hill coefficient < 1.0) and reduced by Gpp(NH)p. Like CCH, NAEE inhibited GTP-stimulated adenylate cyclase in rat brain striatal membranes. As compared to CCH, NAEE had little effect (< 5%) as a stimulator of inositol phosphate (IP) production in rat brain slices. The results indicate that NAEE is a direct-acting muscarinic receptor agonist. Moreover, its differential effects on acid secretion, IP accumulation, and adenylate cyclase suggest that it may be useful for defining cholinergic receptor subclasses.

  20. Cloning and expression analysis of a muscarinic cholinergic receptor from the brain of ant, Polyrhachis vicina.

    PubMed

    Lü, Shu-Min; Zhao, Zhuo; Li, Ke; Zhang, Ya-Lin; Xi, Geng-Si

    2011-09-01

    Muscarinic acetylcholine receptors (mAchRs) are the predominant cholinergic receptors in the central and peripheral nervous systems of animals. They also have been found in various insect nervous systems. In this article, a full-length cDNA of a pupative mAchR (PmAchR) was obtained from the brains of ant Polyrhachis vicina by homology cloning in combination with rapid amplification of cDNA ends. PmAchR encodes a 599-amino acid protein that exhibits a high degree of homology with other mAchRs. Real-time quantitative RT-PCR analysis showed that PmAchR is differentially expressed in the brains of workers, males, and females. By in situ hybridization, it is revealed that PmAchR is widely expressed in different soma clusters of the brain, including the mushroom bodies, the antennal lobes, as well as the optic lobes (OL), and the most intensely staining is found in Kenyon cells. Nonetheless, there are more positive nerve fibers in the OL of males' brains than in females' and workers' brains.

  1. Selective blockade of central m1 muscarinic cholinergic receptors with pirenzepine impairs cardiovascular and respiratory function in rats with acute hemorrhage.

    PubMed

    Kovalenko, N Ya; Matsievskii, D D

    2006-09-01

    Ultrasound studies showed that selective antagonist of central M1 muscarinic cholinergic receptors pirenzepine (50 mg/kg intravenously) causes transitory hypotension and respiratory depression in anesthetized intact rats. The M1 receptor antagonist had no effect on cardiac output and portal blood flow. Pretreatment with pirenzepine increased the sensitivity of rats with acute massive hemorrhage to circulatory hypoxia. After blockade of central M1 muscarinic cholinergic receptors, the posthemorrhagic period was characterized by primary decompensation of blood pressure, portal blood flow, and respiration and development of low cardiac output syndrome. The animals died over the first minutes after bleeding arrest. Our results indicate that central M1 muscarinic cholinergic receptors act as shock-limiting cholinergic structures under conditions of posthemorrhagic changes in systemic and portal blood flow, as well as during respiratory dysfunction.

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

    SciTech Connect

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

    1985-10-01

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

  3. Dorsal raphe nucleus acetylcholine-mediated neurotransmission modulates post-ictal antinociception: The role of muscarinic and nicotinic cholinergic receptors.

    PubMed

    de Oliveira, Rithiele Cristina; de Oliveira, Ricardo; Biagioni, Audrey Francisco; Falconi-Sobrinho, Luiz Luciano; Coimbra, Norberto Cysne

    2016-01-15

    The dorsal raphe nucleus (DRN) is a key structure of the endogenous pain inhibitory system. Although the DRN is rich in serotoninergic neurons, cholinergic neurons are also found in that nucleus. Both ictal and inter-ictal states are followed by post-ictal analgesia. The present study investigated the role of cholinergic mechanisms in postictal antinociceptive processes using microinjections of atropine and mecamylamine, muscarinic and nicotinic cholinergic receptor antagonists, respectively, in the DRN of rats. Intraperitoneal injection of pentylenetetrazole (PTZ) (at 64mg/kg) caused tonic and tonic-clonic seizures. The convulsive motor reactions were followed by an increase in pain thresholds, a phenomenon known as post-ictal analgesia. Pre-treatment of the DRN with atropine or mecamylamine at 1µg, 3µg and 5µg/0.2µL decreased the post-ictal antinociceptive phenomenon. The present results showed that the post-ictal analgesia was mediated by muscarinic and nicotinic cholinergic receptors in the DRN, a structure crucially involved in the neural network that organises post-ictal hypoalgesia.

  4. Modulation of muscarinic and micotinic cholinergic receptor mediated catecholamine secretion in guinea pig chromaffin cells by phorbol esters

    SciTech Connect

    Figueiredo, J.C.; Fisher, S.K.; Horowitz, M.I.

    1986-05-01

    Isolated guinea pig chromaffin cells possess both nicotinic (nAChR) and muscarinic (mAChR) cholinergic receptors that are positively coupled to catecholamine (CA) release. Sixty to 70% of CA release is mediated by nAChRs and 30-40% by mAChRs. In the absence of added calcium, nAChR mediated CA release was reduced by 65% whereas the muscarinic response was unaffected. The addition of 100nM 12-0-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C (PKC), also resulted in an increased CA release. Temporally and quantitatively, this response resembled that of mAChR activation. Addition of optimal concentrations of nicotine (50..mu..M) and TPA (100nM) induced a synergistic increase in CA release. Addition of muscarine (1mM) and TPA resulted in an additive response despite a 40-60% inhibition of mAChR mediated inositol phosphate release by TPA. Thus, in guinea pig chromaffin cells, it appears that PKC activation alone is a sufficient stimulus for CA release and that activation of both nicotinic and muscarinic receptors may further increase this enzyme's activity.

  5. Purification and characterization of 94kDa and 80kDa forms of the muscarinic cholinergic receptor

    SciTech Connect

    Fracek, S.P. Jr.; Venter, J.C.; Kerlavage, A.R.

    1986-05-01

    Two molecular forms of the muscarinic cholinergic receptor have been consistently observed in a variety of species, albeit in variable amounts. Proteins which are specifically labeled by (/sup 3/H)propylbenzilylcholine mustard ((/sup 3/H)PrBCM) were observed at 94kDa and 80kDa upon SDS-PAGE of membrane proteins prepared from brains and hearts of trout, frog, turtle, chicken, rat, and pig. They have developed a purification procedure which yields each of these proteins in a homogeneous form suitable for structural analysis. The four step procedure involves affinity chromatography on 3-(2'-aminobenzhydryloxy)tropane-sepharose, concentration on hydroxylapatite, preparative SDS-PAGE and extraction of individual bands from the gel. Limited tryptic digestion of purified (/sup 3/H)PrBCM-labeled porcine atrial muscarinic receptor yields (/sup 3/H)-labeled fragments of 75, 65, 52, 40, 35, 30, 25, and 20kDa, in close agreement with results of analogous digestions of muscarinic receptor from other species and tissues. Complete tryptic digestion and subsequent mapping by reverse-phase HPLC yields very similar profiles for (/sup 125/I)-labeled 94kDa and 80kDA receptor forms. Most peaks which elute in the hydrophobic region of the profile overlap for the two proteins while the 94kDa protein contains several additional peaks of apparent low hydrophobicity.

  6. Muscarinic Receptor Antagonists.

    PubMed

    Matera, Maria Gabriella; Cazzola, Mario

    2017-01-01

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

  7. Vitamin D3 restores altered cholinergic and insulin receptor expression in the cerebral cortex and muscarinic M3 receptor expression in pancreatic islets of streptozotocin induced diabetic rats.

    PubMed

    Kumar, Peeyush T; Antony, Sherin; Nandhu, Mohan S; Sadanandan, Jayanarayanan; Naijil, George; Paulose, Chiramadathikudiyil S

    2011-05-01

    Nutritional therapy is a challenging but necessary dimension in the management of diabetes and neurodegenerative changes associated with it. The study evaluates the effect of vitamin D(3) in preventing the altered function of cholinergic, insulin receptors and GLUT3 in the cerebral cortex of diabetic rats. Muscarinic M3 acetylcholine receptors in pancreas control insulin secretion. Vitamin D(3) treatment in M3 receptor regulation in the pancreatic islets was also studied. Radioreceptor binding assays and gene expression was done in the cerebral cortex of male Wistar rats. Immunocytochemistry of muscarinic M3 receptor was studied in the pancreatic islets using specific antibodies. Y-maze was used to evaluate the exploratory and spatial memory. Diabetes induced a decrease in muscarinic M1, insulin and vitamin D receptor expression and an increase in muscarinic M3, α7 nicotinic acetylcholine receptor, acetylcholine esterase and GLUT3 expression. Vitamin D(3) and insulin treatment reversed diabetes-induced alterations to near control. Diabetic rats showed a decreased Y-maze performance while vitamin D(3) supplementation improved the behavioural deficit. In conclusion, vitamin D(3) shows a potential therapeutic effect in normalizing diabetes-induced alterations in cholinergic, insulin and vitamin D receptor and maintains a normal glucose transport and utilisation in the cortex. In addition vitamin D(3) modulated muscarinic M3 receptors activity in pancreas and plays a pivotal role in controlling insulin secretion. Hence our findings proved, vitamin D(3) supplementation as a potential nutritional therapy in ameliorating diabetes mediated cortical dysfunctions and suggest an interaction between vitamin D(3) and muscarinic M3 receptors in regulating insulin secretion from pancreas.

  8. M3 muscarinic acetylcholine receptor expression confers differential cholinergic modulation to neurochemically distinct hippocampal basket cell subtypes

    PubMed Central

    Cea-del Rio, Christian A.; Lawrence, J. Josh; Tricoire, Ludovic; Erdelyi, Ferenc; Szabo, Gabor; McBain, Chris J.

    2010-01-01

    Cholinergic neuromodulation of hippocampal circuitry promotes network oscillations and facilitates learning and memory through cellular actions on both excitatory and inhibitory circuits. Despite widespread recognition that neurochemical content discriminates between functionally distinct interneuron populations, there has been no systematic examination of whether neurochemically distinct interneuron classes undergo differential cholinergic neuromodulation in the hippocampus. Using GFP transgenic mice that enable the visualization of perisomatically targeting parvalbumin-positive (PV+) or cholecystokinin-positive (CCK+) basket cells (BCs), we tested the hypothesis that neurochemically distinct interneuron populations are differentially engaged by muscarinic acetylcholine receptor (mAChR) activation. Cholinergic fiber activation revealed that CCK BCs were more sensitive to synaptic release of ACh than PV BCs. In response to depolarizing current steps, mAChR activation of PV BCs and CCK BCs also elicited distinct cholinergic response profiles, differing in mAChR-induced changes in action potential (AP) waveform, firing frequency, and intrinsic excitability. In contrast to PV BCs, CCK BCs exhibited a mAChR-induced afterdepolarization (mADP) that was frequency and activity-dependent. Pharmacological, molecular, and loss-of-function data converged on the presence of M3 mAChRs in distinguishing CCK BCs from PV BCs. Firing frequency of CCK BCs was controlled through M3 mAChRs but PV BC excitability was altered solely through M1 mAChRs. Finally, upon mAChR activation, glutamatergic transmission enhanced cellular excitability preferentially in CCK BCs but not in PV BCs. Our findings demonstrate that cell-type specific cholinergic specializations are present on neurochemically distinct interneuron subtypes in the hippocampus, revealing an organizing principle that cholinergic neuromodulation depends critically on neurochemical identity. PMID:20427660

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

    SciTech Connect

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

    1985-07-01

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

  10. Changes in contractile activity of rabbit colon under stress conditions and during post-stress period before and after blockade of muscarinic and nicotinic cholinergic receptors.

    PubMed

    Ovsiannikov, V I; Berezina, T P

    2013-06-01

    Psychogenic stress in rabbits induced by fixation of the animal to a frame was accompanied by an increase in contractile activity of the initial portion of the distal colon, which was abolished by blockade of muscarinic and nicotinic cholinergic receptors. Increased contractile activity of the colon was due to centrogenic stimulation of preganglionic neurons of the parasympathetic nervous system followed by the involvement of the effector cholinergic neurons of the enteric nervous system into excitation.

  11. Immunohistochemical localisation of cholinergic muscarinic receptor subtype 1 (M1r) in the guinea pig and human enteric nervous system.

    PubMed

    Harrington, A M; Hutson, J M; Southwell, B R

    2007-07-01

    Little is known regarding the location of cholinergic muscarinic receptor 1 (M1r) in the ENS, even though physiological data suggest that M1rs are central to cholinergic neurotransmission. This study localised M1rs in the ENS of the guinea pig ileum and human colon using fluorescence immunohistochemistry and RT-PCR in human colon. Double labelling using antibodies against neurochemical markers was used to identify neuron subytpes bearing M1r. M1r immunoreactivity (IR) was present on neurons in the myenteric and submucosal ganglia. The two antibodies gave similar M1r-IR patterns and M1r-IR was abolished upon antibody preabsorption. M1r-IR was present on cholinergic and nNOS-IR nerve cell bodies in both guinea pig and human myenteric neurons. Presynaptic M1r-IR was present on NOS-IR and VAChT-IR nerve fibres in the circular muscle in the human colon. In the submucosal ganglia, M1r-IR was present on a population of neurons that contained cChAT-IR, but did not contain NPY-IR or calretinin-IR. M1r-IR was present on endothelial cells of blood vessels in the submucosal plexus. The localisation of M1r-IR in the guinea pig and human ENS shown in this study agrees with physiological studies. M1r-IR in cholinergic and nitrergic neurons and nerve fibres indicate that M1rs have a role in both cholinergic and nitrergic transmission. M1r-IR present in submucosal neurons suggests a role in mediating acetylcholine's effect on submucosal sensory and secretomotor/vasodilator neurons. M1r-IR present on blood vessel endothelial cells suggests that M1rs may also mediate acetylcholine's direct effect on vasoactivation.

  12. Cholinergic modulation of amyloid precursor protein processing with emphasis on M1 muscarinic receptor: perspectives and challenges in treatment of Alzheimer's disease.

    PubMed

    Fisher, Abraham

    2012-01-01

    The prescribed drugs for treatment of cognitive deficits in Alzheimer's disease (AD) patients are regarded as symptomatic drugs. Effective disease modifying therapies are not yet prescribed in AD patients. Three major hallmarks of AD (e.g. cholinergic hypofunction, Aβ and tau neuropathologies) are closely linked raising the expectation that restoring the cholinergic hypofunction to normal, in particular via selective activation of M1 muscarinic receptors, may alter the onset or progression of AD dementia. This review is focused mainly on modulation of amyloid precursor processing and Aβ levels in the brain via cholinergic treatment strategies based on M1 muscarinic agonists versus other cholinergic treatments (e.g. cholinesterase inhibitors prescribed for treatment of AD, M2 antagonists and nicotinic agonists). Advantages and potential drawbacks of these treatment modalities are reviewed versus the notion that due to an elusive etiology of AD, future disease modifiers should address comprehensively most of these AD hallmarks (e.g. Aβ pathology, tau and tangle pathologies, as well as the cholinergic hypofunction and cognitive impairments). This major requirement may be fulfilled with M1-selective muscarinic agonists and less with other reviewed cholinergic treatments.

  13. Neuroanatomical and neuropharmacological approaches to postictal antinociception-related prosencephalic neurons: the role of muscarinic and nicotinic cholinergic receptors

    PubMed Central

    de Freitas, Renato Leonardo; Bolognesi, Luana Iacovelo; Twardowschy, André; Corrêa, Fernando Morgan Aguiar; Sibson, Nicola R; Coimbra, Norberto Cysne

    2013-01-01

    Several studies have suggested the involvement of the hippocampus in the elaboration of epilepsy. There is evidence that suggests the hippocampus plays an important role in the affective and motivational components of nociceptive perception. However, the exact nature of this involvement remains unclear. Therefore, the aim of this study was to determine the role of muscarinic and nicotinic cholinergic receptors in the dorsal hippocampus (dH) in the organization of postictal analgesia. In a neuroanatomical study, afferent connections were found from the somatosensory cortex, the medial septal area, the lateral septal area, the diagonal band of Broca, and the dentate gyrus to the dH; all these areas have been suggested to modulate convulsive activity. Outputs to the dH were also identified from the linear raphe nucleus, the median raphe nucleus (MdRN), the dorsal raphe nucleus, and the locus coeruleus. All these structures comprise the endogenous pain modulatory system and may be involved either in postictal pronociception or antinociception that is commonly reported by epileptic patients. dH-pretreatment with cobalt chloride (1.0 mmol/L CoCl2/0.2 μL) to transiently inhibit local synapses decreased postictal analgesia 10 min after the end of seizures. Pretreatment of the dH with either atropine or mecamylamine (1.0 μg/0.2 μL) attenuated the postictal antinociception 30 min after seizures, while the higher dose (5.0 μg/0.2 μL) decreased postictal analgesia immediately after the end of seizures. These findings suggest that the dH exerts a critical role in the organization of postictal analgesia and that muscarinic and nicotinic cholinergic receptor-mediated mechanisms in the dH are involved in the elaboration of antinociceptive processes induced by generalized tonic-clonic seizures. PMID:23785660

  14. Reduction of [11C](+)3-MPB Binding in Brain of Chronic Fatigue Syndrome with Serum Autoantibody against Muscarinic Cholinergic Receptor

    PubMed Central

    Yamamoto, Shigeyuki; Ouchi, Yasuomi; Nakatsuka, Daisaku; Tahara, Tsuyoshi; Mizuno, Kei; Tajima, Seiki; Onoe, Hirotaka; Yoshikawa, Etsuji; Tsukada, Hideo; Iwase, Masao; Yamaguti, Kouzi; Kuratsune, Hirohiko; Watanabe, Yasuyoshi

    2012-01-01

    Background Numerous associations between brain-reactive antibodies and neurological or psychiatric symptoms have been proposed. Serum autoantibody against the muscarinic cholinergic receptor (mAChR) was increased in some patients with chronic fatigue syndrome (CFS) or psychiatric disease. We examined whether serum autoantibody against mAChR affected the central cholinergic system by measuring brain mAChR binding and acetylcholinesterase activity using positron emission tomography (PET) in CFS patients with positive [CFS(+)] and negative [CFS(−)] autoantibodies. Methodology Five CFS(+) and six CFS(−) patients, as well as 11 normal control subjects underwent a series of PET measurements with N-[11C]methyl-3-piperidyl benzilate [11C](+)3-MPB for the mAChR binding and N-[11C]methyl-4-piperidyl acetate [11C]MP4A for acetylcholinesterase activity. Cognitive function of all subjects was assessed by neuropsychological tests. Although the brain [11C](+)3-MPB binding in CFS(−) patients did not differ from normal controls, CFS(+) patients showed significantly lower [11C](+)3-MPB binding than CFS(−) patients and normal controls. In contrast, the [11C]MP4A index showed no significant differences among these three groups. Neuropsychological measures were similar among groups. Conclusion The present results demonstrate that serum autoantibody against the mAChR can affect the brain mAChR without altering acetylcholinesterase activity and cognitive functions in CFS patients. PMID:23240035

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

    PubMed

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

    2016-02-01

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

  16. 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.

  17. Role of M1, M3, and M5 muscarinic acetylcholine receptors in cholinergic dilation of small arteries studied with gene-targeted mice.

    PubMed

    Gericke, Adrian; Sniatecki, Jan J; Mayer, Veronique G A; Goloborodko, Evgeny; Patzak, Andreas; Wess, Jürgen; Pfeiffer, Norbert

    2011-05-01

    Acetylcholine regulates perfusion of numerous organs via changes in local blood flow involving muscarinic receptor-induced release of vasorelaxing agents from the endothelium. The purpose of the present study was to determine the role of M₁, M₃, and M₅ muscarinic acetylcholine receptors in vasodilation of small arteries using gene-targeted mice deficient in either of the three receptor subtypes (M1R(-/-), M3R(-/-), or M5R(-/-) mice, respectively). Muscarinic receptor gene expression was determined in murine cutaneous, skeletal muscle, and renal interlobar arteries using real-time PCR. Moreover, respective arteries from M1R(-/-), M3R(-/-), M5R(-/-), and wild-type mice were isolated, cannulated with micropipettes, and pressurized. Luminal diameter was measured using video microscopy. mRNA for all five muscarinic receptor subtypes was detected in all three vascular preparations from wild-type mice. However, M(3) receptor mRNA was found to be most abundant. Acetylcholine produced dose-dependent dilation in all three vascular preparations from M1R(-/-), M5R(-/-), and wild-type mice. In contrast, cholinergic dilation was virtually abolished in arteries from M3R(-/-) mice. Deletion of either M₁, M₃, or M₅ receptor genes did not affect responses to nonmuscarinic vasodilators, such as substance P and nitroprusside. These findings provide the first direct evidence that M₃ receptors mediate cholinergic vasodilation in cutaneous, skeletal muscle, and renal interlobar arteries. In contrast, neither M₁ nor M₅ receptors appear to be involved in cholinergic responses of the three vascular preparations tested.

  18. Muscarinic and nicotinic cholinergic agonists: structural analogies and discrepancies.

    PubMed

    Bikádi, Zsolt; Simonyi, Miklós

    2003-12-01

    Acetylcholine, the first identified neurotransmitter acts on both types of cholinergic receptors. Both rigid and flexible derivatives of acetylcholine could either be selective muscarinic or selective nicotinic agonists while some compounds show activity at both receptor subclasses. Earlier structure-activity considerations are revisited. Ligand and receptor based calculations have been applied in the hope to identify characteristic geometrical and steric requirements for the activity on the receptor subtypes. Results are treated critically and applied cautiously for predicting selective structural requirements by the cholinergic receptor subclasses.

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

    PubMed

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

    2003-04-01

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

  20. 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.

  1. A comparison of {beta}-adrenoceptors and muscarinic cholinergic receptors in tissues of brown bullhead catfish (Ameiurus nebulosus) from the Black River and Old Woman Creek, Ohio

    SciTech Connect

    Steevens, J.A.; Baumann, P.C.; Jones, S.B.

    1996-09-01

    {beta}-Adrenoceptors ({beta}ARs) 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 {beta}ARs were found in the gill from Black River fish, possibly indicating a compensatory response subsequent to chemical stress.

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

    DTIC Science & Technology

    1986-04-11

    still unresolved problem connected with the mechanisms by which the anticholinesterases affect cholinergic nerves which should lead to a more thorough...understanding of their most adverse reactions, i.e the generalized cholinergic stimulation, convulsions and neuromuscular paralysis. This may lead to...which the anticholinesterases affect cholinergic nerves which should lead to a more thorough understanding of their most adverse reactions, i.e the

  3. Psychopharmacological evidences for the involvement of muscarinic and nicotinic cholinergic receptors on sweet substance-induced analgesia in Rattus norvegicus.

    PubMed

    Irusta, A E; Savoldi, M; Kishi, R; Resende, G C; Freitas, R L; Carvalho, A D; Coimbra, N C

    2001-06-08

    In order to investigate the effects of sweet substance intake on pain modulation, male albino Wistar rats weighing 180-200 g received either tap water or sucrose solutions (250 g/l) for 14 days as their only source of liquid. Each rat consumed an average of 15.6 g sucrose/day. Their tail withdrawal latencies in the tail-flick test (probably a spinal reflex) were measured immediately before and after this treatment. An analgesia index was calculated from the withdrawal latencies before and after treatment. The index (mean +/- SEM, N = 8) for the groups receiving sucrose solution plus saline (NaCl; 0.9%) for 14 days was 0.70 +/- 0.01. Atropine (1 and 2 mg/kg)-treated rats (N = 8) after intake of sucrose exhibited an analgesia index of 0.39 +/- 0.09 and 0.39 +/- 0.08, respectively, while mecamylamine (1 and 2 mg/kg)-treated rats (N = 10) after intake of sucrose had an index of -0.02 +/- 0.07 and 0.03 +/- 0.07, respectively. These results indicate that the effect of sucrose intake on nociceptive thresholds is controlled by neurotransmission of acetylcholine and depends on the nicotinic cholinergic receptors for its major analgesic effect, although muscarinic receptors were also involved in this antinociceptive process.

  4. Cholinergic muscarinic M4 receptor gene polymorphisms: a potential risk factor and pharmacogenomic marker for schizophrenia.

    PubMed

    Scarr, Elizabeth; Um, Jung Yoon; Cowie, Tiffany Frances; Dean, Brian

    2013-05-01

    Although schizophrenia is a widespread disorder of unknown aetiology, we have previously shown that muscarinic M4 receptor (CHRM4) expression is decreased in the hippocampus and caudate-putamen from subjects with the disorder, implicating the receptor in its pathophysiology. These findings led us to determine whether variation in the CHRM4 gene sequence was associated with an altered risk of schizophrenia by sequencing the CHRM4 gene from the brains of 76 people with the disorder and 74 people with no history of psychiatric disorders. In addition, because the CHRM4 is a potential target for antipsychotic drug development, we investigated whether variations in CHRM4 sequence were associated with final recorded doses of, and life-time exposure to, antipsychotic drugs. Gene sequencing identified two single nucleotide polymorphisms (SNPs; rs2067482 and rs72910092) in the CHRM4 gene. For rs2067482, our data suggested that both genotype (1341C/C; p = 0.05) and allele (C; p = 0.03) were associated with an increased risk of schizophrenia. In addition, there was a strong trend (p = 0.08) towards an association between CHRM4 sequence and increased lifetime exposure to antipsychotic drugs. Furthermore, there was a trend for people with the C allele to be prescribed benzodiazepines more frequently (p = 0.06) than those with the T allele. These data, albeit on small cohorts, are consistent with genetic variance at rs2067482 contributing to an altered risk of developing schizophrenia which requires more forceful pharmacotherapy to achieve a clinical response.

  5. Role of various subtypes of muscarinic cholinergic receptors in the development of posthemorrhagic abnormalities in systemic and portal circulation in rats.

    PubMed

    Kovalenko, N Ya; Matsievskii, D D; Reshetnyak, V K

    2012-01-01

    The experiments employing high-frequency ultrasonic technique and selective blockers of M1, M3, and M4 muscarinic cholinergic receptors pirenzepine, 4-DAMP, and tropicamide, respectively, revealed individual roles of these receptors in the development of severe posthemorrhagic hypotension in rats with low or high individual resistance to circulatory hypoxia. The study showed that M1 and M4 muscarinic receptors are involved in shock-limiting and shock-activating processes, respectively, while M3 receptors exert no effect on the course of posthemorrhagic abnormalities in systemic and hepatic portal circulation and on the posthemorrhagic lifespan. Poor resistance of the cardiovascular system to circulatory hypoxia during shock development is considered to be dysregulatory pathology.

  6. Muscarinic cholinergic receptors modulate inhibitory synaptic rhythms in hippocampus and neocortex

    PubMed Central

    Alger, Bradley E.; Nagode, Daniel A.; Tang, Ai-Hui

    2014-01-01

    Activation of muscarinic acetylcholine (ACh) receptors (mAChRs) powerfully affects many neuronal properties as well as numerous cognitive behaviors. Small neuronal circuits constitute an intermediate level of organization between neurons and behaviors, and mAChRs affect interactions among cells that compose these circuits. Circuit activity is often assessed by extracellular recordings of the local field potentials (LFPs), which are analogous to in vivo EEGs, generated by coordinated neuronal interactions. Coherent forms of physiologically relevant circuit activity manifest themselves as rhythmic oscillations in the LFPs. Frequencies of rhythmic oscillations that are most closely associated with animal behavior are in the range of 4–80 Hz, which is subdivided into theta (4–14 Hz), beta (15–29 Hz) and gamma (30–80 Hz) bands. Activation of mAChRs triggers rhythmic oscillations in these bands in the hippocampus and neocortex. Inhibitory responses mediated by GABAergic interneurons constitute a prominent feature of these oscillations, and indeed, appear to be their major underlying factor in many cases. An important issue is which interneurons are involved in rhythm generation. Besides affecting cellular and network properties directly, mAChRs can cause the mobilization of endogenous cannabinoids (endocannabinoids, eCBs) that, by acting on the principal cannabinoid receptor of the brain, CB1R, regulate the release of certain neurotransmitters, including GABA. CB1Rs are heavily expressed on only a subset of interneurons and, at lower density, on glutamatergic neurons. Exogenous cannabinoids typically disrupt oscillations in the theta (θ) and gamma (γ) ranges, which probably contributes to the behavioral effects of these drugs. It is important to understand how neuronal circuit activity is affected by mAChR-driven eCBs, as this information will provide deeper insight into the actions of ACh itself, as well as into the effects of eCBs and exogenous cannabinoids

  7. Axonal transport of muscarinic cholinergic receptors in rat vagus nerve: high and low affinity agonist receptors move in opposite directions and differ in nucleotide sensitivity

    SciTech Connect

    Zarbin, M.A.; Wamsley, J.K.; Kuhar, M.J.

    1982-07-01

    The presence and transport of muscarinic cholinergic binding sites have been detected in the rat vagus nerve. These binding sites accumulate both proximal and distal to ligatures in a time-dependent manner. The results of double ligature and colchicine experiments are compatible with the notion that the anterogradely transported binding sites move by fast transport. Most of the sites accumulating proximal to ligatures bind the agonist carbachol with high affinity, while most of the sites accumulating distally bind carbachol with a low affinity. Also, the receptors transported in the anterograde direction are affected by a guanine nucleotide analogue (GppNHp), while those transported in the retrograde direction are less, or not, affected. The bulk of the sites along the unligated nerve trunk bind carbachol with a low affinity and are less sensitive to GppNHp modulation than the anterogradely transported sites. These results suggest that some receptors in the vagus may undergo axonal transport in association with regulatory proteins and that receptor molecules undergo changes in their binding and regulatory properties during their life cycle. These data also support the notion that the high and low affinity agonist form of the muscarinic receptor represent different modulated forms of a single receptor molecule.

  8. Muscarinic Receptors Are Responsible for the Cholinergic Modulation of Projection Neurons in the Song Production Brain Nucleus RA of Zebra Finches.

    PubMed

    Meng, Wei; Wang, Songhua; Yao, Lihua; Zhang, Nan; Li, Dongfeng

    2017-01-01

    Songbirds are a useful model for the study of learned vocal behavior in vertebrates. The robust nucleus of the arcopallium (RA) is a premotor nucleus in the vocal motor pathway. It receives excitatory synaptic inputs from the anterior forebrain pathway. RA also receives cholinergic inputs from the ventral paleostriatum of the basal forebrain. Our previous study showed that carbachol, a non-selective cholinergic receptor agonist, modulates the electrophysiology of RA projection neurons (PNs), indicating that cholinergic modulation of RA may play an important role in song production. However, the receptor mechanisms underlying these effects are poorly understood. In the present study, we investigated the electrophysiological properties of two acetylcholine receptors on the RA PNs of adult male zebra finches using in vitro whole-cell current clamp. Our results demonstrate that activation of muscarinic acetylcholine receptors (mAChRs) simulate the effects of carbachol. Both carbachol and the mAChR agonist muscarine produced a decrease in the excitability of RA PNs and a hyperpolarization of the membrane potential. The mAChR antagonist atropine blocked the effects of carbachol. Activation of nicotinic acetylcholine receptors (nAChRs) with nAChR agonist nicotine or DMPP had no effect on the excitability of RA PNs, and the nAChR antagonist mecamylamine failed to inhibit the effects of carbachol. These results suggest that mAChRs, but not nAChRs, primarily modulate the effects of carbachol on the activity of RA PNs. Collectively, these findings contribute to our understanding of the mechanism of cholinergic modulation in the vocal nuclei of songbirds.

  9. Muscarinic Receptors Are Responsible for the Cholinergic Modulation of Projection Neurons in the Song Production Brain Nucleus RA of Zebra Finches

    PubMed Central

    Meng, Wei; Wang, Songhua; Yao, Lihua; Zhang, Nan; Li, Dongfeng

    2017-01-01

    Songbirds are a useful model for the study of learned vocal behavior in vertebrates. The robust nucleus of the arcopallium (RA) is a premotor nucleus in the vocal motor pathway. It receives excitatory synaptic inputs from the anterior forebrain pathway. RA also receives cholinergic inputs from the ventral paleostriatum of the basal forebrain. Our previous study showed that carbachol, a non-selective cholinergic receptor agonist, modulates the electrophysiology of RA projection neurons (PNs), indicating that cholinergic modulation of RA may play an important role in song production. However, the receptor mechanisms underlying these effects are poorly understood. In the present study, we investigated the electrophysiological properties of two acetylcholine receptors on the RA PNs of adult male zebra finches using in vitro whole-cell current clamp. Our results demonstrate that activation of muscarinic acetylcholine receptors (mAChRs) simulate the effects of carbachol. Both carbachol and the mAChR agonist muscarine produced a decrease in the excitability of RA PNs and a hyperpolarization of the membrane potential. The mAChR antagonist atropine blocked the effects of carbachol. Activation of nicotinic acetylcholine receptors (nAChRs) with nAChR agonist nicotine or DMPP had no effect on the excitability of RA PNs, and the nAChR antagonist mecamylamine failed to inhibit the effects of carbachol. These results suggest that mAChRs, but not nAChRs, primarily modulate the effects of carbachol on the activity of RA PNs. Collectively, these findings contribute to our understanding of the mechanism of cholinergic modulation in the vocal nuclei of songbirds. PMID:28293176

  10. Knife cuts of entorhinal cortex: effects on development of amygdaloid kindling and seizure-induced decrease of muscarinic cholinergic receptors

    SciTech Connect

    Savage, D.D.; Rigsbee, L.C.; McNamara, J.O.

    1985-02-01

    This report examines the effect of transection of the entorhinal hippocampal projection on amygdaloid kindling. We found that: bilateral knife cuts of entorhinal cortex but not of dorsal neocortex antagonize the development of amygdaloid kindling; and bilateral knife cuts of entorhinal cortex eliminate the seizure-induced decrease in number of muscarinic receptors of dentate granule cells. We suggest the following interpretations of these data: the hippocampal formation circuitry facilitates the development of amygdaloid kindling; and the decline of muscarinic receptors after kindled seizures is due to excessive activation of granule cells by axons from entorhinal cortex, a noncholinergic afferent.

  11. Evidence for cholinergic participation in the control of bird song; acetylcholinesterase distribution and muscarinic receptor autoradiography in the zebra finch brain

    SciTech Connect

    Ryan, S.M.; Arnold, A.P.

    1981-10-20

    Brain regions thought to be involved in the control of song in the zebra finch (Poephila guttata), were examined histochemically using the Karnovsky and Roots direct-coloring method for the detection of acetylcholinesterase (AChE) and the autoradiographic method for the localization of muscarinic cholinergic receptors following injection of tritiated quinuclidinyl benzilate (3H QNB). All presently identified vocal control nuclei in both males and females contain AChE. These nuclei include Area X, magnocellular nucleus of the anterior neostriatum (MAN), nucleus interface (NIF), caudal nucleus of the hyperstriatum ventrale (HVc), intercollicular nucleus (ICo), nucleus uva, robust nucleus of the archistriatum (RA), and tracheosyringeal portion of the hypoglossal nerve nucleus (nXIIts). All nuclei except Area X contain mostly AChE-synthesizing cell bodies. All of these nuclei contain some AChE in the neuropil, with particularly intense staining in Area X, the surrounding LPO, and the dorsomedial portion of ICo. In agreement with this description are very high concentrations of 3H QNB in both Area X and the dorsomedial ICo. HVc also appears specifically labeled. Evidence from these two histological technique suggests that efferent projections of most vocal control area may utilize acetylcholine, and that several of the vocal control nuclei may themselves receive muscarinic cholinergic projection. In Area X, there are sex differences of AChE neuropil staining. This evidence suggesting that sexually dimorphic projections to or within Area X are cholinergic or cholinoceptive.

  12. Arrestin interactions with G protein-coupled receptors. Direct binding studies of wild type and mutant arrestins with rhodopsin, beta 2-adrenergic, and m2 muscarinic cholinergic receptors.

    PubMed

    Gurevich, V V; Dion, S B; Onorato, J J; Ptasienski, J; Kim, C M; Sterne-Marr, R; Hosey, M M; Benovic, J L

    1995-01-13

    Arrestins play an important role in quenching signal transduction initiated by G protein-coupled receptors. To explore the specificity of arrestin-receptor interaction, we have characterized the ability of various wild-type arrestins to bind to rhodopsin, the beta 2-adrenergic receptor (beta 2AR), and the m2 muscarinic cholinergic receptor (m2 mAChR). Visual arrestin was found to be the most selective arrestin since it discriminated best between the three different receptors tested (highest binding to rhodopsin) as well as between the phosphorylation and activation state of the receptor (> 10-fold higher binding to the phosphorylated light-activated form of rhodopsin compared to any other form of rhodopsin). While beta-arrestin and arrestin 3 were also found to preferentially bind to the phosphorylated activated form of a given receptor, they only modestly discriminated among the three receptors tested. To explore the structural characteristics important in arrestin function, we constructed a series of truncated and chimeric arrestins. Analysis of the binding characteristics of the various mutant arrestins suggests a common molecular mechanism involved in determining receptor binding selectivity. Structural elements that contribute to arrestin binding include: 1) a C-terminal acidic region that serves a regulatory role in controlling arrestin binding selectivity toward the phosphorylated and activated form of a receptor, without directly participating in receptor interaction; 2) a basic N-terminal domain that directly participates in receptor interaction and appears to serve a regulatory role via intramolecular interaction with the C-terminal acidic region; and 3) two centrally localized domains that are directly involved in determining receptor binding specificity and selectivity. A comparative structure-function model of all arrestins and a kinetic model of beta-arrestin and arrestin 3 interaction with receptors are proposed.

  13. Activation of midbrain presumed dopaminergic neurones by muscarinic cholinergic receptors: an in vivo electrophysiological study in the rat

    PubMed Central

    Gronier, B; Rasmussen, K

    1998-01-01

    Extracellular single-unit recording and iontophoresis were used to examine the effects of different cholinoceptor agonists and antagonists on the firing rate and firing pattern of A9 and A10 presumed dopaminergic neurones in the anaesthetized rat.Administration of low currents (1–5 nA) of the selective muscarinic agonists oxotremorine M (Oxo M) and muscarine and of the non-selective muscarinic/nicotinic agonist carbamylcholine (CCh) produced a dose-dependent increase in firing rate in most of the A9 and A10 presumed dopaminergic neurones tested. Oxo M-induced activation could be completely blocked by iontophoretic application of the muscarinic antagonist butyl-scopolamine or systemic administration of the muscarinic antagonist scopolamine (300 μg kg−1, i.v.).Iontophoretic application of the selective nicotinic agonist methylcarbamylcholine (MCCh), but not nicotine, induced a consistent increase in firing rate. Surprisingly, the excitatory effect of MCCh was significantly reduced by the selective muscarinic antagonist scopolamine (300 μg kg−1, i.v.), but not by the selective nicotinic antagonist mecamylamine (2.2 mg kg−1, i.v.). Mecamylamine (3 mg kg−1, i.v.) was also ineffective in reducing the CCh-induced activation of presumed dopamine neurones, suggesting that both CCh and MCCh increased the activity of dopamine neurones via an interaction with muscarinic receptors.Iontophoretic application of the endogenous agonist acetylcholine (ACh) had no or little effect on the firing activity of A10 presumed dopaminergic neurones. However, concomitant application of neostigmine, a potent cholinesterase inhibitor, with acetylcholine induced a substantial activation of these neurones. This activation consisted of two components; one, which was prevalent, was scopolamine (300 μg kg−1, i.v.)-sensitive, and the other was mecamylamine (2 mg kg−1, i.v.)-sensitive.In addition to their effect on firing activity, Oxo M, muscarine and

  14. Cholinergic Neurotransmission in the Posterior Insular Cortex Is Altered in Preclinical Models of Neuropathic Pain: Key Role of Muscarinic M2 Receptors in Donepezil-Induced Antinociception

    PubMed Central

    Ferrier, Jérémy; Bayet-Robert, Mathilde; Dalmann, Romain; El Guerrab, Abderrahim; Aissouni, Youssef; Graveron-Demilly, Danielle; Chalus, Maryse; Pinguet, Jérémy; Eschalier, Alain; Richard, Damien; Daulhac, Laurence; Balayssac, David

    2015-01-01

    Neuropathic pain is one of the most debilitating pain conditions, yet no therapeutic strategy has been really effective for its treatment. Hence, a better understanding of its pathophysiological mechanisms is necessary to identify new pharmacological targets. Here, we report important metabolic variations in brain areas involved in pain processing in a rat model of oxaliplatin-induced neuropathy using HRMAS 1H-NMR spectroscopy. An increased concentration of choline has been evidenced in the posterior insular cortex (pIC) of neuropathic animal, which was significantly correlated with animals' pain thresholds. The screening of 34 genes mRNA involved in the pIC cholinergic system showed an increased expression of the high-affinity choline transporter and especially the muscarinic M2 receptors, which was confirmed by Western blot analysis in oxaliplatin-treated rats and the spared nerve injury model (SNI). Furthermore, pharmacological activation of M2 receptors in the pIC using oxotremorine completely reversed oxaliplatin-induced mechanical allodynia. Consistently, systemic treatment with donepezil, a centrally active acetylcholinesterase inhibitor, prevented and reversed oxaliplatin-induced cold and mechanical allodynia as well as social interaction impairment. Intracerebral microdialysis revealed a lower level of acetylcholine in the pIC of oxaliplatin-treated rats, which was significantly increased by donepezil. Finally, the analgesic effect of donepezil was markedly reduced by a microinjection of the M2 antagonist, methoctramine, within the pIC, in both oxaliplatin-treated rats and spared nerve injury rats. These findings highlight the crucial role of cortical cholinergic neurotransmission as a critical mechanism of neuropathic pain, and suggest that targeting insular M2 receptors using central cholinomimetics could be used for neuropathic pain treatment. SIGNIFICANCE STATEMENT Our study describes a decrease in cholinergic neurotransmission in the posterior insular

  15. Balancing Arc synthesis, mRNA decay, and proteasomal degradation: maximal protein expression triggered by rapid eye movement sleep-like bursts of muscarinic cholinergic receptor stimulation.

    PubMed

    Soulé, Jonathan; Alme, Maria; Myrum, Craig; Schubert, Manja; Kanhema, Tambudzai; Bramham, Clive R

    2012-06-22

    Cholinergic signaling induces Arc/Arg3.1, an immediate early gene crucial for synaptic plasticity. However, the molecular mechanisms that dictate Arc mRNA and protein dynamics during and after cholinergic epochs are little understood. Using human SH-SY5Y neuroblastoma cells, we show that muscarinic cholinergic receptor (mAchR) stimulation triggers Arc synthesis, whereas translation-dependent RNA decay and proteasomal degradation strictly limit the amount and duration of Arc expression. Chronic application of the mAchR agonist, carbachol (Cch), induces Arc transcription via ERK signaling and release of calcium from IP(3)-sensitive stores. Arc translation requires ERK activation, but not changes in intracellular calcium. Proteasomal degradation of Arc (half-life ∼37 min) was enhanced by thapsigargin, an inhibitor of the endoplasmic calcium-ATPase pump. Similar mechanisms of Arc protein regulation were observed in cultured rat hippocampal slices. Functionally, we studied the impact of cholinergic epoch duration and temporal pattern on Arc protein expression. Acute Cch treatment (as short as 2 min) induces transient, moderate Arc expression, whereas continuous treatment of more than 30 min induces maximal expression, followed by rapid decline. Cholinergic activity associated with rapid eye movement sleep may function to facilitate long term synaptic plasticity and memory. Employing a paradigm designed to mimic intermittent rapid eye movement sleep epochs, we show that application of Cch in a series of short bursts generates persistent and maximal Arc protein expression. The results demonstrate dynamic, multifaceted control of Arc synthesis during mAchR signaling, and implicate cholinergic epoch duration and repetition as critical determinants of Arc expression and function in synaptic plasticity and behavior.

  16. In vivo biodistribution of two ( sup 18 F)-labelled muscarinic cholinergic receptor ligands: 2-( sup 18 F)- and 4-( sup 18 F)-fluorodexetimide

    SciTech Connect

    Wilson, A.A.; Scheffel, U.A.; Dannals, R.F.; Stathis, M.; Ravert, H.T.; Wagner, H.N. Jr. )

    1991-01-01

    Two ({sup 18}F)-labelled analogues of the potent muscarinic cholinergic receptor (m-AChR) antagonist, dexetimide, were evaluated as potential ligands for imaging m-AChR by positron emission tomography (PET). Intravenous administration of both 2-({sup 18}F)- or 4-({sup 18}F)-fluorodexetimide resulted in high brain uptake of radioactivity in mice. High binding levels were observed in m-AChR rich areas, such as cortex and striatum, with low levels in the receptor-poor cerebellum. Uptake of radioactivity was saturable and could be blocked by pre-administration of dexetimide or atropine. Drugs with different sites of action were ineffective at blocking receptor binding. The results indicate that both radiotracers are promising candidates for use in PET studies.

  17. Simultaneous modulation of retrieval by dopaminergic D(1), beta-noradrenergic, serotonergic-1A and cholinergic muscarinic receptors in cortical structures of the rat.

    PubMed

    Barros, D M; Mello e Souza, T; De David, T; Choi, H; Aguzzoli, A; Madche, C; Ardenghi, P; Medina, J H; Izquierdo, I

    2001-09-28

    Retrieval of inhibitory avoidance has been recently shown to require intact glutamate receptors, protein kinases A and C and mitogen-activated protein kinase in the CA1 region of the rat hippocampus and in the entorhinal, posterior parietal and anterior cingulate cortex. These enzymatic activities are known to be modulated by dopamine D(1), beta-noradrenergic, 5HT1A and cholinergic muscarinic receptors. Here we study the effect on retrieval of this task of well-known agonists and antagonists of these receptors infused in the same brain cortical regions and into the basolateral amygdala, in rats. The drugs used were SKF38393 (D(1) agonist), noradrenaline, 8-HO-DPAT (5HT1A agonist), oxotremorine (muscarinic agonist), SCH23390 (D(1) antagonist), timolol (beta antagonist), NAN-190 (5HT1A antagonist) and scopolamine (muscarinic antagonist). All were studied at two different dose levels. The localised infusion of SKF38393, noradrenaline, NAN-190 and oxotremorine into any of the cortical structures mentioned 10 min prior to a 24-h retention test session of one-trial step-down inhibitory avoidance enhanced retention test performance. SCH2330, timolol, 8-HO-DPAT and scopolamine hindered retention test performance. In the basolateral amygdala only an enhancing effect of noradrenaline and an inhibitory effect of timolol were seen. Three hours after the infusions, retention test performance returned to normal in all cases. None of the treatments affected locomotion or rearing in an open field or behaviour in the elevated plus maze. Therefore, their effects on retention testing can be attributed to an influence on retrieval. In conclusion, memory retrieval of this apparently simple task requires the participation of CA1, entorhinal, posterior parietal and anterior cingulate cortex, and is strongly modulated by, dopaminergic D(1), beta-noradrenergic, muscarinic cholinergic and 5HT1A receptors in the four areas. The first three types of receptor enhance, and the latter inhibits

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

    PubMed Central

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

    1996-01-01

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

  19. Muscarinic Receptor Antagonists: Effects on Pulmonary Function

    PubMed Central

    Buels, Kalmia S.

    2014-01-01

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

  20. Regional development of muscarinic cholinergic binding sites in the prenatal rat brain.

    PubMed

    Schlumpf, M; Palacios, J M; Cortes, R; Lichtensteiger, W

    1991-01-01

    The ontogeny of muscarinic cholinergic binding sites was studied in rat fetal central nervous system by in vitro autoradiographic techniques using [3H]N-methyl scopolamine as ligand (1 nM). Nonspecific binding was determined after the addition of 1 microM atropine. The main findings of this study are the early appearance of muscarinic cholinergic binding sites in fetal rat central nervous system before gestational day 14, their subsequent spread in a caudofrontal direction and the rapid change of patterns within individual brain regions. Muscarinic cholinergic sites are present shortly after cell birth, though the time-lag between cell generation and expression of muscarinic sites differs between neuronal cell populations. High receptor densities are noted in certain brainstem nuclei that are important for early fetal and neonatal behaviors.

  1. Muscarinic and nicotinic cholinergic receptor antagonists differentially mediate acquisition of fructose-conditioned flavor preference and quinine-conditioned flavor avoidance in rats.

    PubMed

    Rotella, Francis M; Olsson, Kerstin; Vig, Vishal; Yenko, Ira; Pagirsky, Jeremy; Kohen, Ilanna; Aminov, Alon; Dindyal, Trisha; Bodnar, Richard J

    2015-09-01

    Rats display both conditioned flavor preference (CFP) for fructose, and conditioned flavor avoidance (CFA) following sweet adulteration with quinine. Previous pharmacological analyses revealed that fructose-CFP expression was significantly reduced by dopamine (DA) D1 or D2 antagonists, but not NMDA or opioid antagonists. Fructose-CFP acquisition was significantly reduced by DA D1, DA D2 or NMDA antagonists, but not opioid antagonists. Quinine-CFA acquisition was significantly enhanced and prolonged by DA D1, NMDA or opioid, but not DA D2 antagonists. Cholinergic interneurons and projections interact with DA systems in the nucleus accumbens and ventral tegmental area. Further, both muscarinic and nicotinic cholinergic receptor signaling have been implicated in sweet intake and development of food-related preferences. Therefore, the present study examined whether systemic administration of muscarinic (scopolamine: SCOP) or nicotinic (mecamylamine: MEC) cholinergic receptor antagonists mediated fructose-CFP expression, fructose-CFP acquisition and quinine-CFA acquisition. For fructose-CFP expression, rats were trained over 10 sessions with a CS+ flavor in 8% fructose and 0.2% saccharin and a CS- flavor in 0.2% saccharin. Two-bottle choice tests with CS+ and CS- flavors mixed in 0.2% saccharin occurred following vehicle, SCOP (0.1-10mg/kg) and MEC (1-8mg/kg). For fructose-CFP acquisition, six groups of rats received vehicle, SCOP (1 or 2.5mg/kg), MEC (4 or 6mg/kg) or a limited intake vehicle control 0.5h prior to 10 CS+ and CS- training sessions followed by six 2-bottle CS+ and CS- choice tests in 0.2% saccharin. For quinine-CFA acquisition, five groups of rats received vehicle, SCOP (1 or 2.5mg/kg) or MEC (4 or 6mg/kg) 0.5h prior to 8 one-bottle CS- (8% fructose+0.2% saccharin: FS) and CS+ (fructose+saccharin+quinine (0.030%: FSQ) training sessions followed by six 2-bottle CS- and CS+ choice tests in fructose-saccharin solutions. Fructose-CFP expression was

  2. Striatal muscarinic receptors promote activity-dependence of dopamine transmission via distinct receptor subtypes on cholinergic interneurons in ventral versus dorsal striatum

    PubMed Central

    Threlfell, Sarah; Clements, Michael A.; Khodai, Tansi; Pienaar, Ilse S.; Exley, Richard; Wess, Jürgen; Cragg, Stephanie J.

    2010-01-01

    Striatal dopamine (DA) and acetylcholine (ACh) regulate motivated behaviors and striatal plasticity. Interactions between these neurotransmitters may be important, through synchronous changes in parent neuron activities and reciprocal presynaptic regulation of release. How DA signalling is regulated by striatal muscarinic receptors (mAChRs) is unresolved; contradictory reports indicate suppression or facilitation, implicating several mAChR-subtypes on various neurons. We investigated whether mAChR regulation of DA signaling varies with presynaptic activity, and identified the mAChRs responsible in sensorimotor- versus limbic-associated striatum. We detected DA in real-time at carbon-fiber microelectrodes in mouse striatal slices. Broad-spectrum mAChR agonists (oxotremorine-M, APET) decreased DA release evoked by low-frequency stimuli (1–10 Hz, 4 pulses) but increased the sensitivity of DA release to presynaptic activity, even enhancing release by high frequencies (e.g. >25 Hz for 4 pulses). These bidirectional effects depended upon ACh input to striatal nicotinic receptors (nAChRs) on DA axons but not GABA- or glutamate-input. In caudate-putamen (CPu), knockout of M2- or M4-mAChRs (not M5) prevented mAChR control of DA indicating that M2- and M4-mAChRs are required. In nucleus accumbens (NAc) core or shell, mAChR function was prevented in M4-knockouts, but not M2- or M5-knockouts. These data indicate that striatal mAChRs, by inhibiting ACh release from cholinergic interneurons and thus modifying nAChR activity, offer variable control of DA release probability that promotes how DA release reflects activation of dopaminergic axons. Furthermore, different coupling of striatal M2-/M4-mAChRs to the control of DA release in CPu versus NAc suggests targets to influence DA/ACh function differentially between striatal domains. PMID:20203199

  3. 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.

  4. Pharmacological doses of Zn2+ induce a muscarinic cholinergic supersensitivity.

    PubMed

    Bonfante-Cabarcas, R; Bravo, I; Nello, C; Gutiérrez-Reyes, E; Loureiro Dos Santos, N E; Moreno-Yanes, J A

    2002-01-01

    The goal of this study was to evaluate the effect of chronic Zn2+ administration (1 mg/kg/day for 1 month) in Sprague-Dawley rats (n = 11) on motility and rearing behaviors (number of events/10 min measured in motility cage), on memory (percentage of failures using a foot-shock double T maze), on the number of muscarinic receptors (using [(3)H]-QNB as a marker) and on the cholinacetyltransferase (Chat) activity (determined by Fonnun's method) in various brain areas (striatum, hippocampus and frontal cortex), as compared with saline-treated rats (n = 10). Our results showed that Zn2+ induced a decrease in rearing (control: 24.6 +/- 3; Zn2+: 15.91 +/- 2.19) and in locomotor activity (control: 37 +/- 3.79; Zn2+: 25 +/- 4.37), a decrease in failures during memory trials (control: 26.12 +/- 5.6; Zn2+: 5.33 +/- 2.71) and an increase in muscarinic receptor density (fmol/mg) in the striatum (control: 539 +/- 6.18; Zn2+: 720 +/- 14.69), hippocampus (control: 396 +/- 7.41; Zn 2+: 458 +/- 5.05) and frontal cortex (control: 506 +/- 10.28; Zn2+: 716 +/- 16.54). Chat activity (pmol/mg/min) was decreased only in the striatum (control: 4240 +/- 158; Zn2+: 2311 +/- 69). We conclude that Zn 2+ induces a cholinergic functional supersensitivity which is related to receptor upregulation. Copyright 2002 National Science Council, ROC and S. Karger AG, Basel

  5. Electron microscopic localization of M2-muscarinic receptors in cholinergic and noncholinergic neurons of the laterodorsal tegmental and pedunculopontine nuclei of the rat mesopontine tegmentum.

    PubMed

    Garzón, Miguel; Pickel, Virginia M

    2016-10-15

    Muscarinic m2 receptors (M2Rs) are implicated in autoregulatory control of cholinergic output neurons located within the pedunculopontine (PPT) and laterodorsal tegmental (LTD) nuclei of the mesopontine tegmentum (MPT). However, these nuclei contain many noncholinergic neurons in which activation of M2R heteroceptors may contribute significantly to the decisive role of the LTD and PPT in sleep-wakefulness. We examined the electron microscopic dual immunolabeling of M2Rs and the vesicular acetylcholine transporter (VAchT) in the MPT of rat brain to identify the potential sites for M2R activation. M2R immunogold labeling was predominately seen in somatodendritic profiles throughout the PPT/LTD complex. In somata, M2R immunogold particles were often associated with Golgi lamellae and cytoplasmic endomembrannes, but were rarely in contact with the plasma membrane, as was commonly seen in dendrites. Approximately 36% of the M2R-labeled somata and 16% of the more numerous M2R-labeled dendrites coexpressed VAchT. M2R and M2R/VAchT-labeled dendritic profiles received synapses from inhibitory- and excitatory-type axon terminals, over 88% of which were unlabeled and others contained exclusively M2R or VAchT immunoreactivity. In axonal profiles M2R immunogold was localized to plasmalemmal and cytoplasmic regions and showed a similar distribution in many VAchT-negative glial profiles. These results provide ultrastructural evidence suggestive of somatic endomembrane trafficking of M2Rs, whose activation serves to regulate the postsynaptic excitatory and inhibitory responses in dendrites of cholinergic and noncholinergic neurons in the MPT. They also suggest the possibility that M2Rs in this brain region mediate the effects of acetylcholine on the release of other neurotransmitters and on glial signaling. J. Comp. Neurol. 524:3084-3103, 2016. © 2016 Wiley Periodicals, Inc.

  6. Sexual dimorphism in the volume of song control nuclei in European starlings: assessment by a Nissl stain and autoradiography for muscarinic cholinergic receptors.

    PubMed

    Bernard, D J; Casto, J M; Ball, G F

    1993-08-22

    Previous studies have found that the volume of several song control nuclei is larger in male songbirds than in female songbirds. The degree of this volumetric sex difference within a given species appears to be systematically related to the degree of the behavioral sex difference. The largest volumetric differences have been reported in species in which the male sings and the female sings little, if at all, and the smallest sex differences in volume have been reported in species in which males and females both sing in nearly equal amounts. We compared the volume of three song control nuclei in male and female European starlings (Sturnus vulgaris), a species in which females are known to sing, though at a much lower rate than males. We investigated the volume of hyperstriatum ventrale, pars caudale, nucleus robustus archistriatalis, and area X of the lobus parolfactorius as defined with the use of a Nissl stain. In addition, we measured the volume of area X as defined by the density of muscarinic cholinergic receptors visualized by in vitro receptor autoradiographic methods. The volumes of all three of the song nuclei, as defined by Nissl staining, are significantly larger in males than in females. For area X, Nissl staining and receptor autoradiography indicate the same significant volumetric sex difference. The three nuclei are approximately one and one half to two times larger in males than in females, a degree of dimorphism that is intermediate to those reported for other species. Previous investigations of sex differences in the avian vocal control system have used only Nissl stains to define nuclear volumes. We demonstrate in this paper that receptor autoradiography can be used to assess dimorphisms in nuclear volume. Broad application of this approach to a number of neurotransmitter receptor systems will better characterize the dimorphisms in the song system, and therefore will provide greater insight into the neuroanatomical and neurochemical control of

  7. Activation of Muscarinic Acetylcholine Receptor Subtype 4 Is Essential for Cholinergic Stimulation of Gastric Acid Secretion: Relation to D Cell/Somatostatin

    PubMed Central

    Takeuchi, Koji; Endoh, Takuya; Hayashi, Shusaku; Aihara, Takeshi

    2016-01-01

    Background/Aim: Muscarinic acetylcholine receptors exist in five subtypes (M1∼M5), and they are widely expressed in various tissues to mediate diverse autonomic functions, including gastric secretion. In the present study, we demonstrated, using M1∼M5 KO mice, the importance of M4 receptors in carbachol (CCh) stimulation of acid secretion and investigated how the secretion is modulated by the activation of M4 receptors. Methods: C57BL/6J mice of wild-type (WT) and M1–M5 KO were used. Under urethane anesthesia, acid secretion was measured in the stomach equipped with an acute fistula. CCh (30 μg/kg) was given subcutaneously (s.c.) to stimulate acid secretion. Atropine or octreotide (a somatostatin analog) was given s.c. 20 min before the administration of CCh. CYN154806 (a somatostatin SST2 receptor antagonist) was given i.p. 20 min before the administration of octreotide or CCh. Results: CCh caused an increase of acid secretion in WT mice, and the effect was totally inhibited by prior administration of atropine. The effect of CCh was similarly observed in the animals lacking M1, M2 or M5 receptors but significantly decreased in M3 or M4 KO mice. CYN154806, the SST2 receptor antagonist, dose-dependently and significantly reversed the decreased acid response to CCh in M4 but not M3 KO mice. Octreotide, the somatostatin analog, inhibited the secretion of acid under CCh-stimulated conditions in WT mice. The immunohistochemical study showed the localization of M4 receptors on D cells in the stomach. Serum somatostatin levels in M4 KO mice were higher than WT mice under basal conditions, while those in WT mice were significantly decreased in response to CCh. Conclusions: These results suggest that under cholinergic stimulation the acid secretion is directly mediated by M3 receptors and indirectly modified by M4 receptors. It is assumed that the activation of M4 receptors inhibits the release of somatostatin from D cells and minimizes the acid inhibitory effect of

  8. 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.

  9. Cadmium-induced cell death of basal forebrain cholinergic neurons mediated by muscarinic M1 receptor blockade, increase in GSK-3β enzyme, β-amyloid and tau protein levels.

    PubMed

    Del Pino, Javier; Zeballos, Gabriela; Anadón, María José; Moyano, Paula; Díaz, María Jesús; García, José Manuel; Frejo, María Teresa

    2016-05-01

    Cadmium is a neurotoxic compound which induces cognitive alterations similar to those produced by Alzheimer's disease (AD). However, the mechanism through which cadmium induces this effect remains unknown. In this regard, we described in a previous work that cadmium blocks cholinergic transmission and induces a more pronounced cell death on cholinergic neurons from basal forebrain which is partially mediated by AChE overexpression. Degeneration of basal forebrain cholinergic neurons, as happens in AD, results in memory deficits attributable to the loss of cholinergic modulation of hippocampal synaptic circuits. Moreover, cadmium has been described to activate GSK-3β, induce Aβ protein production and tau filament formation, which have been related to a selective loss of basal forebrain cholinergic neurons and development of AD. The present study is aimed at researching the mechanisms of cell death induced by cadmium on basal forebrain cholinergic neurons. For this purpose, we evaluated, in SN56 cholinergic mourine septal cell line from basal forebrain region, the cadmium toxic effects on neuronal viability through muscarinic M1 receptor, AChE splice variants, GSK-3β enzyme, Aβ and tau proteins. This study proves that cadmium induces cell death on cholinergic neurons through blockade of M1 receptor, overexpression of AChE-S and GSK-3β, down-regulation of AChE-R and increase in Aβ and total and phosphorylated tau protein levels. Our present results provide new understanding of the mechanisms contributing to the harmful effects of cadmium on cholinergic neurons and suggest that cadmium could mediate these mechanisms by M1R blockade through AChE splices altered expression.

  10. Effects of adjunct galantamine to risperidone, or haloperidol, in animal models of antipsychotic activity and extrapyramidal side-effect liability: involvement of the cholinergic muscarinic receptor.

    PubMed

    Wadenberg, Marie-Louise G; Fjällström, Ann-Kristin; Federley, Malin; Persson, Pernilla; Stenqvist, Pia

    2011-06-01

    The acetylcholine esterase inhibitor/cholinergic nicotinic receptor (nAChR) allosteric modulator galantamine (Gal) is used against cognitive impairment in Alzheimer's disease. Negative/cognitive and psychotic symptom improvement in schizophrenia by adjunct Gal to antipsychotic drugs (APDs) has been reported. Cognitive symptoms in schizophrenia may involve brain prefrontal hypo-dopaminergia. Experimental data by others indicate nAChR involvement in animal pro-cognitive effects of Gal. The role of nAChRs in antipsychotic effects by Gal has, however, not been elucidated. Using the conditioned avoidance response (CAR) and the catalepsy tests for antipsychotic activity and extrapyramidal side-effect (EPS) liability, respectively, we here investigated the effects of adjunct Gal (1.25 mg/kg) to the typical APD haloperidol (Hal) (0.05 mg/kg), or the atypical APD risperidone (Ris) (0.2 mg/kg), in rats. Adjunct Gal significantly enhanced APD-like effects by low doses of Hal or Ris, but showed a safe EPS liability profile only in combination with Ris. Pretreatment with the muscarinic receptor (mAChR) antagonist scopolamine, but not the nAChR antagonist mecamylamine, completely reversed the enhancing effects of adjunct Gal to Hal treatment, in the CAR test. While the nAChR-modulating properties of Gal probably contribute to pro-cognitive activity, as shown by others, the present data suggest that any contribution to antipsychotic activity by Gal is mediated primarily via mAChRs. This property combination of Gal may offer a unique, favourable therapeutic profile for schizophrenia treatment.

  11. Short-term desensitization of muscarinic cholinergic receptors in mouse neuroblastoma cells: selective loss of agonist low-affinity and pirenzepine high-affinity binding sites

    SciTech Connect

    Cioffi, C.L.; el-Fakahany, E.E.

    1986-09-01

    The effects of brief incubation with carbamylcholine on subsequent binding of (/sup 3/H)N-methylscopolamine were investigated in mouse neuroblastoma cells (clone N1E-115). This treatment demonstrated that the muscarinic receptors in this neuronal clone can be divided into two types; one which is readily susceptible to regulation by receptor agonists, whereas the other is resistant in this regard. In control cells, both pirenzepine and carbamylcholine interacted with high- and low-affinity subsets of muscarinic receptors. Computer-assisted analysis of the competition between pirenzepine and carbamylcholine with (/sup 3/H)N-methylscopolamine showed that the receptor sites remaining upon desensitization are composed mainly of pirenzepine low-affinity and agonist high-affinity binding sites. Furthermore, there was an excellent correlation between the ability of various muscarinic receptor agonists to induce a decrease in consequent (/sup 3/H)N-methylscopolamine binding and their efficacy in stimulating cyclic GMP synthesis in these cells. Thus, only the agonists that are known to recognize the receptor's low-affinity conformation in order to elicit increases in cyclic GMP levels were capable of diminishing ligand binding. Taken together, our present results suggest that the receptor population that is sensitive to regulation by agonists includes both the pirenzepine high-affinity and the agonist low-affinity receptor binding states. In addition, the sensitivity of these receptor subsets to rapid regulation by agonists further implicates their involvement in desensitization of muscarinic receptor-mediated cyclic GMP formation.

  12. (/sup 3/H)AF-DX 116 labels subsets of muscarinic cholinergic receptors in rat brain and heart

    SciTech Connect

    Wang, J.X.; Roeske, W.R.; Gulya, K.; Wang, W.; Yamamura, H.I.

    1987-10-05

    The in vitro binding properties of the novel muscarinic antagonist (/sup 3/H)AF-DX 116 were studied using a rapid filtration technique. Association and dissociation rates of (/sup 3/H)AF-DX 116 binding were rapid at 25/sup 0/C but 20-40 times slower at 0-4/sup 0/C. Kinetic dissociation constants (K/sub d/s) were estimated to be 31.8 nM and 30.9 nM at 25/sup 0/C; 23.1 nM and 22.9 nM at 0-4/sup 0/C for the cerebral cortex and heart, respectively. In saturation studies, (/sup 3/H)AF-DX 116 labeled 29% of the total (/sup 3/H)(-)QNB binding sites in the cerebral cortical membranes and 87% in the cardiac membranes, with K/sub d/ values of 28.9 nM and 17.9 nM, respectively. Muscarinic antagonists inhibited (/sup 3/H)AF-DX 116 binding in a rank order of potency of atropine > dexetimide > AF-DX 116 > PZ > levetimide in both tissues. Except for PZ/(/sup 3/H)AF-DX 116 and AF-DX 116/(/sup 3/H)AF-DX 116 in the cerebral cortex, all the antagonist competition curves had Hill coefficients close to one. 19 references, 2 figures, 4 tables.

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

    PubMed

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

    2012-01-01

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

  14. Functional and laminar dissociations between muscarinic and nicotinic cholinergic neuromodulation in the tree shrew primary visual cortex.

    PubMed

    Bhattacharyya, Anwesha; Bießmann, Felix; Veit, Julia; Kretz, Robert; Rainer, Gregor

    2012-04-01

    Acetylcholine is an important neuromodulator involved in cognitive function. The impact of cholinergic neuromodulation on computations within the cortical microcircuit is not well understood. Here we investigate the effects of layer-specific cholinergic drug application in the tree shrew primary visual cortex during visual stimulation with drifting grating stimuli of varying contrast and orientation. We describe differences between muscarinic and nicotinic cholinergic effects in terms of both the layer of cortex and the attribute of visual representation. Nicotinic receptor activation enhanced the contrast response in the granular input layer of the cortex, while tending to reduce neural selectivity for orientation across all cortical layers. Muscarinic activation modestly enhanced the contrast response across cortical layers, and tended to improve orientation tuning. This resulted in highest orientation selectivity in the supra- and infragranular layers, where orientation selectivity was already greatest in the absence of pharmacological stimulation. Our results indicate that laminar position plays a crucial part in functional consequences of cholinergic stimulation, consistent with the differential distribution of cholinergic receptors. Nicotinic receptors function to enhance sensory representations arriving in the cortex, whereas muscarinic receptors act to boost the cortical computation of orientation tuning. Our findings suggest close homology between cholinergic mechanisms in tree shrew and primate visual cortices.

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

    PubMed Central

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

    2008-01-01

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

  16. Synthesis of four stereoisomers of 1-azabiocyclo[2.2.2]OCT-3-YL-{alpha}-fluoroalkyl-{alpha}-hydroxy-{alpha}-phenylacetate (FQNPe): Potential imaging ligands for the muscarinic-cholinergic receptor (m-AChR) by PET

    SciTech Connect

    Luo, H.; McPherson, D.W.; Knapp, F.F. Jr.

    1996-10-01

    Earlier studies with the racemic 1-azabiocyclo[2.2.2]oct-3-yl {alpha}-fluoroalkyl-{alpha}-hydroxy-{alpha}-phenylacetate (FQNPe) mixture had demonstrated high in vitro binding affinity for the muscarinic-cholinergic receptor (m-AChR). Pre-treatment of rats with this new agent significantly blocked receptor localization of subsequently injected [I-131]-Z-(-,-)-IQNP, which is an established high affinity m-AChR ligand. Syntheses and characterization of the four FQNPe stereoisomers: (-)(-) FQNPe, (-)(+) FQNPe, (+)(-) FQNPe, and (+)(+) FQNPe will be presented. The interesting NMR spectra of the diastereomeric salts formed in the resolution of racemic {alpha}-(1-chloropent-5-yl)-{alpha}-hydroxy {alpha}-phenylacetic acid will also be discussed.

  17. 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…

  18. Basic and modern concepts on cholinergic receptor: A review

    PubMed Central

    Tiwari, Prashant; Dwivedi, Shubhangi; Singh, Mukesh Pratap; Mishra, Rahul; Chandy, Anish

    2013-01-01

    Cholinergic system is an important system and a branch of the autonomic nervous system which plays an important role in memory, digestion, control of heart beat, blood pressure, movement and many other functions. This article serves as both structural and functional sources of information regarding cholinergic receptors and provides a detailed understanding of the determinants governing specificity of muscarinic and nicotinic receptor to researchers. The study helps to give overall information about the fundamentals of the cholinergic system, its receptors and ongoing research in this field.

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

    PubMed

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

    2010-05-18

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

  20. 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

  1. Characterization of muscarinic receptor subtypes in human tissues

    SciTech Connect

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

    1988-01-01

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

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

    PubMed

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

    2014-08-20

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

  3. Effects of beta-amyloid protein on M1 and M2 subtypes of muscarinic acetylcholine receptors in the medial septum-diagonal band complex of the rat: relationship with cholinergic, GABAergic, and calcium-binding protein perikarya.

    PubMed

    González, Iván; Arévalo-Serrano, Juan; Sanz-Anquela, José Miguel; Gonzalo-Ruiz, Alicia

    2007-06-01

    Cortical cholinergic dysfunction has been correlated with the expression and processing of beta-amyloid precursor protein. However, it remains unclear as to how cholinergic dysfunction and beta-amyloid (Abeta) formation and deposition might be related to one another. Since the M1- and M2 subtypes of muscarinic acetylcholine receptors (mAChRs) are considered key molecules that transduce the cholinergic message, the purpose of the present study was to assess the effects of the injected Abeta peptide on the number of M1mAchR- and M2mAChR-immunoreactive cells in the medial septum-diagonal band (MS-nDBB) complex of the rat. Injections of Abeta protein into the retrosplenial cortex resulted in a decrease in M1mAChR and M2mAChR immunoreactivity in the MS-nDBB complex. Quantitative analysis revealed a significant reduction in the number of M1mAChR- and M2mAChR-immunoreactive cells in the medial septum nucleus (MS) and in the horizontal nucleus of the diagonal band of Broca (HDB) as compared to the corresponding hemisphere in control animals and with that seen in the contralateral hemisphere, which corresponds to the PBS-injected side. Co-localization studies showed that the M1mAChR protein is localized in GABA-immunoreactive cells of the MS-nDBB complex, in particular those of the MS nucleus, while M2mAChR protein is localized in both the cholinergic and GABAergic cells. Moreover, GABAergic cells containing M2mAChR are mainly localized in the MS nucleus, while cholinergic cells containing M2mAChR are localized in the MS and the HDB nuclei. Our findings suggest that Abeta induces a reduction in M1mAChR- and M2mAChR-containing cells, which may contribute to impairments of cholinergic and GABAergic transmission in the MS-nDBB complex.

  4. Comparison of (/sup 3/H)pirenzepine and (/sup 3/H)quinuclidinylbenzilate binding to muscarinic cholinergic receptors in rat brain

    SciTech Connect

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

    1984-03-01

    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. 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.

  5. Pharmacological characteristics of Sho-seiryu-to, an antiallergic Kampo medicine without effects on histamine H1 receptors and muscarinic cholinergic system in the brain.

    PubMed

    Sakaguchi, M; Iizuka, A; Yuzurihara, M; Ishige, A; Komatsu, Y; Matsumiya, T; Takeda, H

    1996-01-01

    The pharmacological characteristics of Sho-seiryu-to, an antiallergic Kampo medicine, were investigated. Forty-eight-hour passive cutaneous anaphylactic (PCA) reaction was significantly inhibited in rats orally administered Sho-seiryu-to (1000 mg/kg). Sho-seiryu-to significantly inhibited increase in vascular permeability induced by histamine. These data confirm previous findings that Sho-seiryu-to has antiallergic activity in animals and suggest that the antagonism of histamine may be an antiallergic mechanism of Sho-seiryu-to. Sho-seiryu-to did not affect locomotor activity or motor coordination in mice. Although ketotifen prolonged sleeping time induced by pentobarbital, Sho-seiryu-to had no such effect. Nor was there any effect on oxotremorine-induced tremor and [3H]-mepyramine binding to histamine H1 receptors in rat brain. Thus, Sho-seiryu-to appears to be useful for treating type I allergy, with relatively few side effects such as sedation and drowsiness due mainly to blockade of histamine H1 and muscarinic receptors in the brain.

  6. A muscarinic cholinergic mechanism underlies activation of the central pattern generator for locust flight.

    PubMed

    Buhl, Edgar; Schildberger, Klaus; Stevenson, Paul A

    2008-07-01

    A central question in behavioural control is how central pattern generators (CPGs) for locomotion are activated. This paper disputes the key role generally accredited to octopamine in activating the CPG for insect flight. In deafferented locusts, fictive flight was initiated by bath application of the muscarinic agonist pilocarpine, the acetylcholine analogue carbachol, and the acetylcholinesterase blocker eserine, but not by nicotine. Furthermore, in addition to octopamine, various other amines including dopamine, tyramine and histamine all induced fictive flight, but not serotonin or the amine-precursor amino acid tyrosine. However, flight initiation was not reversibly blocked by aminergic antagonists, and was still readily elicited by both natural stimulation (wind) and pilocarpine in reserpinized, amine-depleted locusts. By contrast, the muscarinic antagonists atropine and scopolamine reversibly blocked flight initiated by wind, cholinergic agonists, octopamine, and by selective stimulation of a flight-initiating interneurone (TCG). The short delay from TCG stimulation to flight onset suggests that TCG acts directly on the flight CPG, and accordingly that TCG, or its follower cell within the flight generating circuit, is cholinergic. We conclude that acetylcholine acting via muscarinic receptors is the key neurotransmitter in the mechanism underlying the natural activation of the locust flight CPG. Amines are not essential for this, but must be considered as potential neuromodulators for facilitating flight release and tuning the motor pattern. We speculate that muscarinic activation coupled to aminergic facilitation may be a general feature of behavioural control in insects for ensuring conditional recruitment of individual motor programs in accordance with momentary adaptive requirements.

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

    SciTech Connect

    Dawson, V.L.

    1989-01-01

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

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

    PubMed

    De Angelis, Federica; Tata, Ada Maria

    2016-01-01

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

  9. Heterogeneity of muscarinic receptor subtypes in cerebral blood vessels

    SciTech Connect

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

    1991-07-01

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

  10. Biochemical and immunological studies of the Muscarinic acetylcholine receptor

    SciTech Connect

    Gainer, M.W.

    1985-01-01

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

  11. Effects of imidafenacin (KRP-197/ONO-8025), a new anti-cholinergic agent, on muscarinic acetylcholine receptors. High affinities for M3 and M1 receptor subtypes and selectivity for urinary bladder over salivary gland.

    PubMed

    Kobayashi, Fumiyoshi; Yageta, Yuichi; Segawa, Mitsuru; Matsuzawa, Shigeki

    2007-01-01

    Imidafenacin (CAS 170105-16-5, KRP-197, ONO-8025) is an antagonist for the muscarinic acetylcholine (ACh) receptor currently under development for the treatment of overactive bladder. Affinities of imidafenacin and other drugs for muscarinic ACh receptor subtypes were investigated by examining inhibitory effects on ACh release in the rat urinary bladder and K+ efflux in the rat salivary gland in functional and binding assays. In the functional assay, imidafenacin had higher affnities for M3 and M1 receptors than for the M2 receptor. In contrast, metabolites of imidafenacin (M-2, M-4 and M-9) had low affinities for muscarinic ACh receptor subtypes. Darifenacin had selectivity for the M3 receptor, while propiverine, tolterodine and oxybutynin had no selectivity for muscarinic ACh receptors. In carbamylcholine (CCh)-induced contraction in the urinary bladder, imidafenacin, propiverine, tolterodine and oxybutynin had affinities similar to those for the M3 receptor in the ileum. In the binding assay for human muscarinic ACh receptor subtypes, imidafenacin had higher affinities for m3 and m1 receptors than for m2 receptor, but tolterodine had no selectivity for m1, m2 and m3 receptors. In ACh release in the urinary bladder, inhibitory effects of imidafenacin, tolterodine, oxybutynin and darifenacin seemed to be partially mediated by the M1 receptor. In ACh-induced and electrical stimulation-induced K+ efflux from the salivary gland, inhibitory effects (IC50) of imidafenacin, propiverine, tolterodine, oxybutynin and darifenacin might be closely related to those for the M3 receptor in the ileum. These results suggest that imidafenacin more strongly antagonizes cholinomimetics on M3 and M1 receptors than on the M2 receptor. Moreover, imidafenacin seems to inhibit the contraction of the bladder smooth muscle by mediating antagonism to the M3 receptor and to regulate ACh release by mediating prejunctional facilitatory M1 receptor. Imidafenacin also inhibited K+ efflux from

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

    PubMed

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

    2000-01-20

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

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

    SciTech Connect

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

    1987-12-01

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

  14. [Probable mechanism of recognition of cholinergic ligands by acetylcholine receptors].

    PubMed

    Demushkin, V P; Kotelevtsev, Iu V; Pliashkevich, Iu G; Khramtsov, N V

    1982-01-01

    Dryding's models were used for the conformational analysis of compounds affecting muscarin-specific acetylcholine receptor and nicotin-specific acetylcholine receptor. Ammonium group and ether oxygen (3.6 A apart from the ammonium group) specifically oriented to each other were shown to be necessary structural elements to reveal muscarin-type cholinergic activity. Ammonium group along with carbonyl oxygen or its substituent (5 A distance) are the necessary structural units providing nicotin-type cholinergic activity. The presence of two hydrophobic substituents (one in the ammonium area and the other neighbouring the second active grouping) is the additional factor. The developed principles were justified by the use of a series of synthetic samples. The compounds were obtained likely favouring affinitive modification of acetylcholine receptor (dissociation constants of acetylcholine receptor complexes equalling to 10(-4)--10(-7) M-1).

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

    PubMed

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

    2012-01-01

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

  16. Presence of a non-neuronal cholinergic system and occurrence of up- and down-regulation in expression of M2 muscarinic acetylcholine receptors: new aspects of importance regarding Achilles tendon tendinosis (tendinopathy).

    PubMed

    Bjur, Dennis; Danielson, Patrik; Alfredson, Håkan; Forsgren, Sture

    2008-02-01

    Limited information is available concerning the existence of a cholinergic system in the human Achilles tendon. We have studied pain-free normal Achilles tendons and chronically painful Achilles tendinosis tendons with regard to immunohistochemical expression patterns of the M(2) muscarinic acetylcholine receptor (M(2)R), choline acetyltransferase (ChAT), and vesicular acetylcholine transporter (VAChT). M(2)R immunoreactivity was detected in the walls of blood vessels. As evidenced via parallel staining for CD31 and alpha-smooth muscle actin, most M(2)R immunoreactivity was present in the endothelium. M(2)R immunoreactivity also occured in tenocytes, which regularly immunoreact for vimentin. The degree of M(2)R immunoreactivity was highly variable, tendinosis tendons that exhibit hypercellularity and hypervascularity showing the highest levels of immunostaining. Immunoreaction for ChAT and VAChT was detected in tenocytes in tendinosis specimens, particularly in aberrant cells. In situ hybridization revealed that mRNA for ChAT is present in tenocytes in tendinosis specimens. Our results suggest that autocrine/paracrine effects occur concerning the tenocytes in tendinosis. Up-regulation/down-regulation in the levels of M(2)R immunoreactivity possibly take place in tenocytes and blood vessel cells during the various stages of tendinosis. The presumed local production of acetylcholine (ACh), as evidenced by immunoreactivity for ChAT and VAChT and the detection of ChAT mRNA, appears to evolve in response to tendinosis. These observations are of importance because of the well-known vasoactive, trophic, and pain-modulating effects that ACh is known to have and do unexpectedly establish the presence of a non-neuronal cholinergic system in the Achilles tendon.

  17. Involvement of HCN Channel in Muscarinic Inhibitory Action on Tonic Firing of Dorsolateral Striatal Cholinergic Interneurons

    PubMed Central

    Zhao, Zhe; Zhang, Kang; Liu, Xiaoyan; Yan, Haitao; Ma, Xiaoyun; Zhang, Shuzhuo; Zheng, Jianquan; Wang, Liyun; Wei, Xiaoli

    2016-01-01

    The striatum is the most prominent nucleus in the basal ganglia and plays an important role in motor movement regulation. The cholinergic interneurons (ChIs) in striatum are involved in the motion regulation by releasing acetylcholine (ACh) and modulating the output of striatal projection neurons. Here, we report that muscarinic ACh receptor (M receptor) agonists, ACh and Oxotremorine (OXO-M), decreased the firing frequency of ChIs by blocking the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Scopolamine (SCO), a nonselective antagonist of M receptors, abolished the inhibition. OXO-M exerted its function by activating the Gi/o cAMP signaling cascade. The single-cell reverse transcription polymerase chain reaction (scRT-PCR) revealed that all the five subtypes of M receptors and four subtypes of HCN channels were expressed on ChIs. Among them, M2 receptors and HCN2 channels were the most dominant ones and expressed in every single studied cholinergic interneuron (ChI).Our results suggest that ACh regulates not only the output of striatal projection neurons, but also the firing activity of ChIs themselves by activating presynaptic M receptors in the dorsal striatum. The activation of M2 receptors and blockage of HCN2 channels may play an important role in ACh inhibition on the excitability of ChIs. This finding adds a new G-protein coupled receptor mediated regulation on ChIs and provides a cellular mechanism for control of cholinergic activity and ACh release in the dorsal striatum. PMID:27047336

  18. Dextran sodium sulphate-induced colitis perturbs muscarinic cholinergic control of colonic epithelial ion transport

    PubMed Central

    Sayer, Brooke; Lu, Jun; Green, Christina; Söderholm, Johan D; Akhtar, Mahmood; McKay, Derek M

    2002-01-01

    Neuronal cholinergic input is an important regulator of epithelial electrolyte transport and hence water movement in the gut. In this study, colitis was induced by treating mice with 4% (w v−1) dextran sodium-sulphate (DSS)-water for 5 days followed by 3 days of normal water. Mid-colonic segments were mounted in Ussing chambers and short-circuit current (Isc, indicates net ion movement) responses to the cholinergic agonist, carbachol (CCh; 10−4 M)±tetrodotoxin, atropine (ATR), hexamethonium (HEX), naloxone or phenoxybenzamine were assessed. Tissues from mice with DSS-induced colitis displayed a drop in Isc in response to CCh (−11.3±3.3 μA/cm2), while those from control mice showed a transient increase in Isc (76.3±13.0 μA/cm2). The ΔIsc in colon from DSS-treated mice was tetrodotoxin-sensitive, atropine-insensitive and was reversed by hexamethonium (HEX+CCh=16.7±7.8 μA/cm2), indicating involvement of a nicotinic receptor. CCh induced a drop in Isc in tissues from controls only when they were pretreated with the cholinergic muscarinic receptor blocker, atropine: ATR+CCh=−21.3±7.0 μA/cm2. Nicotine elicited a drop in Isc in Ussing-chambered colon from both control and DSS-treated mice that was TTX-sensitive. The drop in Isc evoked by CCh challenge of colonic tissue from DSS-treated mice or ATR+CCh challenge of control tissue was not significantly affected by blockade of opiate or α-adrenergic receptors by naloxone or phenoxybenzamine, respectively. The data indicate that DSS-colitis reveals a nicotinic receptor that becomes important in cholinergic regulation of ion transport. PMID:11934821

  19. Muscarinic M1 receptor partially modulates higher sensitivity to cadmium-induced cell death in primary basal forebrain cholinergic neurons: A cholinesterase variants dependent mechanism.

    PubMed

    Del Pino, Javier; Zeballos, Gabriela; Anadon, María José; Díaz, María Jesús; Moyano, Paula; Díaz, Gloria Gómez; García, Jimena; Lobo, Margarita; Frejo, María Teresa

    2016-06-15

    Cadmium is a toxic compound reported to produce cognitive dysfunctions, though the mechanisms involved are unknown. In a previous work we described how cadmium blocks cholinergic transmission and induces greater cell death in primary cholinergic neurons from the basal forebrain. It also induces cell death in SN56 cholinergic neurons from the basal forebrain through M1R blockage, alterations in the expression of AChE variants and GSK-3β, and an increase in Aβ and total and phosphorylated Tau protein levels. It was observed that the silencing or blockage of M1R altered ChAT activity, GSK-3β, AChE splice variants gene expression, and Aβ and Tau protein formation. Furthermore, AChE-S variants were associated with the same actions modulated by M1R. Accordingly, we hypothesized that cholinergic transmission blockage and higher sensitivity to cadmium-induced cell death of primary basal forebrain cholinergic neurons is mediated by M1R blockage, which triggers this effect through alteration of the expression of AChE variants. To prove this hypothesis, we evaluated, in primary culture from the basal forebrain region, whether M1R silencing induces greater cell death in cholinergic neurons than cadmium does, and whether in SN56 cells M1R mediates the mechanisms described so as to play a part in the cadmium induction of cholinergic transmission blockage and cell death in this cell line through alteration of the expression of AChE variants. Our results prove that M1R silencing by cadmium partially mediates the greater cell death observed on basal forebrain cholinergic neurons. Moreover, all previously described mechanisms for blocking cholinergic transmission and inducing cell death on SN56 cells after cadmium exposure are partially mediated by M1R through the alteration of AChE expression. Thus, our results may explain cognitive dysfunctions observed in cadmium toxicity. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  20. Endosulfan and cholinergic (muscarinic) transmission: effect on electroencephalograms and (/sup 3/H)quinuclidinyl benzilate in pigeon brain

    SciTech Connect

    Anand, M.; Agrawal, A.K.; Gopal, K.; Sur, R.N.; Seth, P.K.

    1986-08-01

    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. The results of this behavioral and biochemical study indicate the involvement of a cholinergic (muscarinic) transmitter system in endosulfan-induced neurotoxicity.

  1. Lymphocyte muscarinic cholinergic activity and PGE2 involvement in experimental Trypanosoma cruzi infection.

    PubMed

    Sterin-Borda, L; Gorelik, G; Goren, N; Cappa, S G; Celentano, A M; Borda, E

    1996-11-01

    In this paper, we demonstrated that the production of PGE2 by CD8+ T lymphocytes through muscarinic cholinergic receptor (mAChR) activation of lymphocytes from mice acutely infected with nonlethal Trypanosoma cruzi CA-1 strain could enhance resistance to infection. Treatment in vivo with either atropine or cyclooxygenase inhibitors enhanced mortality rates and parasitemia of mice infected with T. cruzi CA-1 strain. The mechanism by which CD8+ T lymphocytes released PGE2 appears to involve the activation of the cells by circulating IgG present in mice infected with T. cruzi CA-1 strain. Binding of these antibodies to mAChR on CD8+ T lymphocytes triggered the release of large amounts of PGE2. The results point to a role of serum antibodies against mAChR in the protection of T. cruzi infection. The prostanoid acting as an immunomodulator contributed to the maintenance of the chronic course of experimental Chagas disease.

  2. Action of AF64A on rat brain muscarinic receptors

    SciTech Connect

    Eva, C.; Costa, E.

    1986-03-01

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

  3. 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.

  4. 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.

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

    PubMed

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

    2017-02-01

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

  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 and ligands in cancer

    PubMed Central

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

    2009-01-01

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

  8. Muscarinic Receptor Agonists and Antagonists: Effects on Cancer

    PubMed Central

    2012-01-01

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

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

    PubMed

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

    1999-01-01

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

  10. Muscarinic receptor heterogeneity in follicle-enclosed Xenopus oocytes

    PubMed Central

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

    1999-01-01

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

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

    PubMed

    Degroot, Aldemar; Nomikos, George G

    2006-02-15

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

  12. Spin labeled acetylcholine analogs: studies of cholinergic receptor.

    PubMed

    Rosen, G M; Abou-Donia, M B; Yeh, J Z; Menzel, D B

    1975-10-01

    Some spin-labeled acetylcholine analogs, in which the number of methylene groups between the quaternary nitrogen and the ether oxygen ranged between 1-5, were synthesized to study drug interacitons with acetylcholine receptors. None of the compounds tested, with the exception of the one that contained 2 methylene groups (SL-2) had any cholinergic activity. SL-2 was not capable of producing any nicotinic cholinomimetic activity. On the other hand it proved to have a very weak nicotinic cholinolytic activity on the receptors of the frog satorius muscle. This compound exhibited strong antagonism against muscarinic receptors of the isolated frog heart. The muscarinic cholinolytic action of the spin-label ACh analog is discussed in terms of the molecular perturbation theory of drug action.

  13. HIGH AFFINITY ACYLATING ANTAGONISTS FOR MUSCARINIC RECEPTORS

    PubMed Central

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

    2012-01-01

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

  14. Chronic ethanol (EtOH) feeding increases muscarinic receptor (mAChR) density in esophagus without parallel change in dose response (D-R) to cholinergic agonists

    SciTech Connect

    Keshavarzian, A.; Gordon, J.H.; Urban, G.; Fields, J.Z. VA Hospital, Hines, IL )

    1991-03-11

    The mAChR/effector pathway for signal transduction is important in the physiology of esophagus and mAChR alterations are involved in EtOH induced changes in several organs. To see if EtOH-induced increases in lower esophageal sphincter pressure (LESP) are due to upregulation of mAChR, the authors evaluated mAChR binding and D-R curves for bethanechol (IV) induced increases in LESP, and compared these values to changes in LESP after acute and chronic EtOH. EtOH was given to cats acutely or chronically. The number of mAChR sites (Bmax) in esophagus was lowered by acute EtOH, withdrawal from chronic EtOH raised Bmax. Acute injection of EtOH to cats in withdrawal reversed this increase in mAChR density. These changes correlated with the earlier data on EtOH-induced changes in LESP. In contrast, the D-R curve for bethanechol shifted to the right. Thus, the withdrawal-associated increase in Bmax is more likely to be a compensatory response to deficits distal to the receptor recognition site than to proximal deficits and doesn't cause LESP hyperactivity. Also, receptor binding changes do not necessarily translate into physiological changes.

  15. 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.

  16. 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.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-03-11

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

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

    PubMed Central

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

    2009-01-01

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

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

    PubMed

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

    2013-10-01

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    1994-03-01

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

  4. The cholinergic system, sigma-1 receptors and cognition.

    PubMed

    van Waarde, Aren; Ramakrishnan, Nisha K; Rybczynska, Anna A; Elsinga, Philip H; Ishiwata, Kiichi; Nijholt, Ingrid M; Luiten, Paul G M; Dierckx, Rudi A

    2011-08-10

    This article provides an overview of present knowledge regarding the relationship between the cholinergic system and sigma-1 receptors, and discusses potential applications of sigma-1 receptor agonists in the treatment of memory deficits and cognitive disorders. Sigma-1 receptors, initially considered as a subtype of the opioid family, are unique ligand-regulated molecular chaperones in the endoplasmatic reticulum playing a modulatory role in intracellular calcium signaling and in the activity of several neurotransmitter systems, particularly the cholinergic and glutamatergic pathways. Several central nervous system (CNS) drugs show high to moderate affinities for sigma-1 receptors, including acetylcholinesterase inhibitors (donepezil), antipsychotics (haloperidol, rimcazole), selective serotonin reuptake inhibitors (fluvoxamine, sertraline) and monoamine oxidase inhibitors (clorgyline). These compounds can influence cognitive functions both via their primary targets and by activating sigma-1 receptors in the CNS. Sigma-1 agonists show powerful anti-amnesic and neuroprotective effects in a large variety of animal models of cognitive dysfunction involving, among others (i) pharmacologic target blockade (with muscarinic or NMDA receptor antagonists or p-chloroamphetamine); (ii) selective lesioning of cholinergic neurons; (iii) CNS administration of β-amyloid peptides; (iv) aging-induced memory loss, both in normal and senescent-accelerated rodents; (v) neurodegeneration induced by toxic compounds (CO, trimethyltin, cocaine), and (vi) prenatal restraint stress.

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

    SciTech Connect

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

    1987-09-01

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

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

    PubMed

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

    2013-10-01

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

  7. Muscarinic receptors: their roles in disorders of the central nervous system and potential as therapeutic targets.

    PubMed

    Scarr, Elizabeth

    2012-05-01

    Phylogenetically, acetylcholine is an ancient neurochemical. Therefore, it is not surprising that cholinergic neurons project extensively throughout the central nervous system, innervating a wide range of structures within the brain. In fact, acetylcholine is involved in processes that underpin some of our most basic central functions. Both muscarinic and nicotinic receptor families, which mediate cholinergic transmission, have been implicated in the pathophysiology of psychiatric and neurological disorders. The question that remains to be definitively answered is whether or not these receptors are viable targets for the development of future therapeutic agents.

  8. 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

  9. 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.

  10. Cholinergic receptor activation on epithelia protects against cytokine-induced barrier dysfunction.

    PubMed

    Dhawan, S; Hiemstra, I H; Verseijden, C; Hilbers, F W; Te Velde, A A; Willemsen, L E M; Stap, J; den Haan, J M; de Jonge, W J

    2015-04-01

    Various types of cholinergic receptors are expressed on intestinal epithelia. Their function is not completely understood. We hypothesize that cholinergic receptor activation on epithelium may serve a protective function in cytokine-induced barrier dysfunction. The effect of cholinergic receptor activation on cellular barrier function in epithelial cells was assessed by measuring electrical impedance, and by determining para-cellular transport in transwell experiments. Cell lysates treated with cytokine and/or cholinergic agonists were analysed for cyto- and chemokine production, and tight junction (TJ) protein rearrangement was assessed. Primary colonic epithelial cells were isolated from surgically resected colon tissue of patients with inflammatory bowel disease. IL-1β induced production of chemokines (CXCL-1, CXCL-10, IL-8, CCL-7) and led to a rearrangement of TJ proteins (occludin and ZO-1). This response was inhibited by pre-treatment with muscarinic, rather than nicotinic, acetylcholine receptor agonists. Treatment with IL-1β enhanced paracellular permeability (4kD dextran) and reduced impedance across the monolayer, which was counteracted by pre-incubation with acetylcholine, or muscarinic receptor agonist bethanechol. The protective effect of acetylcholine was antagonized by atropine, underscoring muscarinic receptor involvement. IL-1β induced transcription of myosin light chain kinase and phosphorylation of myosin light chain, and this cytokine-induced phosphorylation of MLC was inhibited by muscarinic receptor agonists. Furthermore, in epithelial cells from resection material of patients with Crohn's disease and ulcerative colitis, high expression of CXCL-8 was associated with a reduced choline acetyl transferase expression, suggesting an aberrant epithelial production of ACh in inflammatory context. Acetylcholine acts on muscarinic receptors on epithelial cells to maintain epithelial barrier function under inflammatory conditions. © 2015

  11. External imaging of cerebral muscarinic acetylcholine receptors

    SciTech Connect

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

    1984-01-20

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

  12. External Imaging of Cerebral Muscarinic Acetylcholine Receptors

    NASA Astrophysics Data System (ADS)

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

    1984-01-01

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

  13. Differential effects of M1 muscarinic receptor blockade and nicotinic receptor blockade in the dorsomedial striatum on response reversal learning

    PubMed Central

    Tzavos, Arianna; Jih, Jane; Ragozzino, Michael E.

    2011-01-01

    The present studies determined whether blockade of M1-like muscarinic or nicotinic cholinergic receptors in the dorsomedial striatum affects acquisition or reversal learning of a response discrimination. Testing occurred in a modified cross-maze across two consecutive sessions. In the acquisition phase, a rat learned to turn to the left or to the right. In the reversal learning phase, a rat learned to turn in the opposite direction as required during acquisition. Experiment 1 investigated the effects of the M1-like muscarinic receptor antagonist, pirenzepine infused into the dorsomedial striatum on acquisition and reversal learning. Experiment 2 examined the effects of the nicotinic cholinergic antagonist, mecamylamine injected into the dorsomedial striatum on acquisition and reversal learning. Bilateral injections of pirenzepine at 10 µg, but not 1 µg, selectively impaired reversal learning. Analysis of the errors indicated that pirenzepine treatment did not impair the initial shift, but increased reversions back to the original response choice following the initial shift. Bilateral injections of mecamylamine, 6 or 18 µg, did not affect acquisition or reversal learning. The results suggest that activation of M1 muscarinic cholinergic receptors, but not nicotinic cholinergic receptors, in the dorsomedial striatum is important for facilitating the flexible shifting of response patterns. PMID:15302131

  14. Homology modeling of human muscarinic acetylcholine receptors.

    PubMed

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

    2014-01-27

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

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

    PubMed

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

    2011-11-16

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

  16. The Role of Muscarinic Receptors in the Pathophysiology of Mood Disorders: A Potential Novel Treatment?

    PubMed Central

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

    2015-01-01

    The central cholinergic system has been implicated in the pathophysiology of mood disorders. An imbalance in central cholinergic neurotransmitter activity has been proposed to contribute to the manic and depressive episodes typical of these disorders. Neuropharmacological studies into the effects of cholinergic agonists and antagonists on mood state have provided considerable support for this hypothesis. Furthermore, recent clinical studies have shown that the pan-CHRM antagonist, scopolamine, produces rapid-acting antidepressant effects in individuals with either major depressive disorder (MDD) or bipolar disorder (BPD), such as bipolar depression, contrasting the delayed therapeutic response of conventional mood stabilisers and antidepressants. This review presents recent data from neuroimaging, post-mortem and genetic studies supporting the involvement of muscarinic cholinergic receptors (CHRMs), particularly CHRM2, in the pathophysiology of MDD and BPD. Thus, novel drugs that selectively target CHRMs with negligible effects in the peripheral nervous system might produce more rapid and robust clinical improvement in patients with BPD and MDD. PMID:26630954

  17. Allosteric binding sites on muscarinic acetylcholine receptors.

    PubMed

    Wess, Jürgen

    2005-12-01

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

  18. 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.

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

    PubMed

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

    2008-04-23

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

  20. 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.

  1. Identification of a family of muscarinic acetylcholine receptor genes

    SciTech Connect

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

    1987-07-31

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

  2. Binding of /sup 3/H-acetylcholine to cholinergic receptors in bovine cerebral arteries

    SciTech Connect

    Shimohama, S.; Tsukahara, T.; Taniguchi, T.; Fujiwara, M.

    1985-11-18

    Cholinergic receptor sites in bovine cerebral arteries were analyzed using radioligand binding techniques with the cholinergic agonist, /sup 3/H-acetylcholine (ACh), as the ligand. Specific binding of /sup 3/H-ACh to membrane preparations of bovine cerebral arteries was saturable, of two binding sites, with dissociation constant (K/sub D/) values of 0.32 and 23.7 nM, and maximum binding capacity (Bmax) values of 67 and 252 fmol/mg protein, respectively. Specific binding of /sup 3/H-ACh was displaced effectively by muscarinic cholinergic agents and less effectively by nicotinic cholinergic agents. IC/sub 50/ values of cholinergic drugs for /sup 3/H-ACh binding were as follows: atropine, 38.5 nM; ACh, 59.8 nM; oxotremorine, 293 nM; scopolamine 474 nM; carbamylcholine, 990 nM. IC/sub 50/ values of nicotinic cholinergic agents such as nicotine, cytisine and ..cap alpha..-bungarotoxin exceeded 50 ..mu..M. Choline acetyltransferase activity was 1.09 nmol/mg protein/hour in the cerebral arteries. These findings suggest that the cholinergic nerves innervate the bovine cerebral arteries and that there are at least two classes of ACh binding sites of different affinities on muscarinic reporters in these arteries. 18 references, 2 figures, 2 tables.

  3. Pharmacological characteristics of catalepsy induced by intracerebroventricular administration of histamine in mice: the importance of muscarinic step in central cholinergic neurons.

    PubMed

    Onodera, K; Shinoda, H

    1991-05-01

    Histamine-induced catalepsy was antagonized potently by scopolamine, an antimuscarinic drug, and partially blocked by sparteine. Neither methylatropine nor antinicotinic drugs could reverse histamine-induced catalepsy. These results indicate the greater importance of muscarinic receptors rather than their nicotinic counterparts in histamine-induced catalepsy. Various antiparkinson drugs, i.e. biperiden and trihexyphenidyl, which have antimuscarinic activity or dopamine agonists, i.e. L-dopa, amantadine and bromocriptine, could antagonize the histamine-induced catalepsy to various degrees. Thus, catalepsy induced by icv histamine can be evoked not only by an activation of the histamine receptor, but also indirectly due to cholinergic and dopaminergic imbalance.

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

    PubMed Central

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

    2004-01-01

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

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

    USDA-ARS?s Scientific Manuscript database

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

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

    PubMed

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

    1994-09-01

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

  7. Muscarinic receptor activation tunes mouse stratum oriens interneurones to amplify spike reliability

    PubMed Central

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

    2006-01-01

    Cholinergic activation of hippocampal targets can initiate and sustain network oscillations in vivo and in vitro, yet the impact of cholinergic modulation on the oscillatory properties of interneurones remains virtually unexplored. Using whole cell current clamp recordings in acute hippocampal slices, we investigated the influence of muscarinic receptor (mAChR) activation on the oscillatory properties of CA1 stratum oriens (SO) interneurones in vitro. In response to suprathreshold oscillatory input, mAChR activation increased spike reliability and precision, and extended the bandwidth that interneurone firing phase-locked. These suprathreshold effects were largest at theta frequencies, indicating that mAChR activation tunes active conductances to enhance firing reliability and precision to theta frequency input. Muscarinic tuning of the intrinsic oscillatory properties of interneurones is a novel mechanism that may be crucial for the genesis of the theta rhythm. PMID:16439425

  8. Central Muscarinic Cholinergic Activation Alters Interaction between Splenic Dendritic Cell and CD4+CD25- T Cells in Experimental Colitis

    PubMed Central

    Pavlov, Valentin A.; Tracey, Kevin J.; Khafipour, Ehsan; Ghia, Jean-Eric

    2014-01-01

    Background The cholinergic anti-inflammatory pathway (CAP) is based on vagus nerve (VN) activity that regulates macrophage and dendritic cell responses in the spleen through alpha-7 nicotinic acetylcholine receptor (a7nAChR) signaling. Inflammatory bowel disease (IBD) patients present dysautonomia with decreased vagus nerve activity, dendritic cell and T cell over-activation. The aim of this study was to investigate whether central activation of the CAP alters the function of dendritic cells (DCs) and sequential CD4+/CD25−T cell activation in the context of experimental colitis. Methods The dinitrobenzene sulfonic acid model of experimental colitis in C57BL/6 mice was used. Central, intracerebroventricular infusion of the M1 muscarinic acetylcholine receptor agonist McN-A-343 was used to activate CAP and vagus nerve and/or splenic nerve transection were performed. In addition, the role of α7nAChR signaling and the NF-kB pathway was studied. Serum amyloid protein (SAP)-A, colonic tissue cytokines, IL-12p70 and IL-23 in isolated splenic DCs, and cytokines levels in DC-CD4+CD25−T cell co-culture were determined. Results McN-A-343 treatment reduced colonic inflammation associated with decreased pro-inflammatory Th1/Th17 colonic and splenic cytokine secretion. Splenic DCs cytokine release was modulated through α7nAChR and the NF-kB signaling pathways. Cholinergic activation resulted in decreased CD4+CD25−T cell priming. The anti-inflammatory efficacy of central cholinergic activation was abolished in mice with vagotomy or splenic neurectomy. Conclusions Suppression of splenic immune cell activation and altered interaction between DCs and T cells are important aspects of the beneficial effect of brain activation of the CAP in experimental colitis. These findings may lead to improved therapeutic strategies in the treatment of IBD. PMID:25295619

  9. The modulatory role of M2 muscarinic receptor on apomorphine-induced yawning and genital grooming.

    PubMed

    Gamberini, Maria Thereza; Bolognesi, Maria Laura; Nasello, Antonia Gladys

    2012-12-07

    The interaction between dopaminergic and cholinergic pathways in the induction of behavioral responses has been previously established. In the brain, M2 receptors are found predominantly in presynaptic cholinergic neurons as autoreceptors, and in dopaminergic neurons as heteroceptors, suggesting a control role of acetylcholine and dopamine release, respectively. Our aim was to investigate the role of M2 receptors on the yawning and genital grooming of rats induced by apomorphine, a dopaminergic receptor agonist, focusing on the interaction between cholinergic and dopaminergic pathways. Initially, the effect of atropine, a non-selective muscarinic antagonist, on yawning and genital grooming induced by apomorphine (100 μg/kg s.c.) was analyzed. Atropine doses of 0.5, 1 and 2 mg/kg i.p. were administered to Wistar rats 30 min before induction of the behavioral responses by apomorphine. Number of yawns and time spent genital grooming were quantified over a 60 min period. Apomorphine-induced yawning was increased by low dose (0.5 mg/kg i.p.) but not by high doses (1 and 2 mg/kg, i.p.) of atropine. Genital grooming was antagonized by 2 mg/kg i.p. of atropine and showed no changes at the other doses tested. Tripitramine, a selective M2 cholinergic antagonist, was used as a tool for distinguishing between M2 and all other muscarinic receptor subtypes in yawning and genital grooming. Tripitramine doses of 0.01, 0.02 and 0.04 μmol/kg i.p. were administered to Wistar rats 30 min before apomorphine (100 μg/kg s.c.). Number of yawns and time spent genital grooming were also quantified over a 60 min period. Tripitramine 0.01 μmol/kg increased all parameters. Higher doses, which possibly block all subtypes of muscarinic receptor, did not modify the response of apomorphine, suggesting a non-selective effect of tripitramine at these doses. Given that low doses of tripitramine increased the behavioral responses induced by apomorphine and that the main distribution of the M2

  10. 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.

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

    PubMed

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

    2003-03-28

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    SciTech Connect

    Messer, W.S.

    1986-01-01

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

  15. Evidence of paired M2 muscarinic receptors

    SciTech Connect

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

    1991-02-01

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

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

    PubMed

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

    2011-02-15

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

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

    PubMed

    Oki, Tomomi; Takeuchi, Chihiro; Yamada, Shizuo

    2006-07-01

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

  18. An autoradiographic analysis of cholinergic receptors in mouse brain after chronic nicotine treatment

    SciTech Connect

    Pauly, J.R.; Marks, M.J.; Gross, S.D.; Collins, A.C. )

    1991-09-01

    Quantitative autoradiographic procedures were used to examine the effects of chronic nicotine infusion on the number of central nervous system nicotinic cholinergic receptors. Female DBA mice were implanted with jugular cannulas and infused with saline or various doses of nicotine (0.25, 0.5, 1.0 or 2.0 mg/kg/hr) for 10 days. The animals were then sacrificed and the brains were removed and frozen in isopentane. Cryostat sections were collected and prepared for autoradiographic procedures as previously described. Nicotinic cholinergic receptors were labeled with L-(3H)nicotine or alpha-(125I)bungarotoxin; (3H)quinuclidinyl benzilate was used to measure muscarinic cholinergic receptor binding. Chronic nicotine infusion increased the number of sites labeled by (3H)nicotine in most brain areas. However, the extent of the increase in binding as well as the dose-response curves for the increase were widely different among brain regions. After the highest treatment dose, binding was increased in 67 of 86 regions measured. Septal and thalamic regions were most resistant to change. Nicotinic binding measured by alpha-(125I)bungarotoxin also increased after chronic treatment, but in a less robust fashion. At the highest treatment dose, only 26 of 80 regions were significantly changes. Muscarinic binding was not altered after chronic nicotine treatment. These data suggest that brain regions are not equivalent in the mechanisms that regulate alterations in nicotinic cholinergic receptor binding after chronic nicotine treatment.

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

    PubMed

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

    2012-01-01

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

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

    SciTech Connect

    Monsma, F.J.

    1987-01-01

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

  1. Comparative peptide mapping of adrenergic and cholinergic neutrotransmitter receptors by reverse-HPLC

    SciTech Connect

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

    1986-05-01

    The authors have developed a methodology for unambiguously identifying neutrotransmitter receptor proteins and comparing structural features of related receptors as well as those in different classes. These techniques have been applied to the study of the ..cap alpha..- and ..beta..-adrenergic receptors as well as the muscarinic and nicotinic cholinergic receptors. The method involves comparative peptide mapping of total proteolytic digests of receptor proteins by microbore reverse-phase HPLC in conjunction with covalent modification by specific receptor ligands or (/sup 125/I)-labeling. Femtomole amounts of receptor can be analyzed. The maps of all the (/sup 125/I)-labeled receptors contain between 20 and 25 peaks and each receptor has a unique profile although all are similar in the very hydrophobic region of the map. The ..cap alpha../sub 2/-adrenergic receptor from human platelets has a higher total percentage of hydrophilic peaks than either the guinea pig lung ..beta../sub 2/-adrenergic receptor or the porcine atria muscarinic receptor. Two forms of the muscarinic receptor have very similar but clearly distinct profiles. The nicotinic receptor subunits show a higher degree of homology by this method than was revealed by previous mapping studies which utilized SDS-PAGE or thin-layer techniques.

  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.

  3. 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.

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

    PubMed

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

    1997-12-01

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

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

    SciTech Connect

    Lee, W.

    1989-01-01

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

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

    PubMed

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

    2000-12-01

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

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

    DTIC Science & Technology

    1983-11-03

    suggest that bisquaternary oximes are allosteric inhibitors of the scarinic receptors, and may induce irreversible effect on the muscarinic inding sites...muscarinic ACh receptor, and are capable of distin- guishing between receptor states and inducing specific irreversible effects . They also point out...membranes, however, Co2+ could no longer induce its effect (Fig. 17B, Table VI), although the apparent affinity of carbamylcholine was not changed . (Fig

  8. 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.

  9. Pharmacological identification of cholinergic receptor subtypes on Drosophila melanogaster larval heart.

    PubMed

    Malloy, Cole A; Ritter, Kyle; Robinson, Jonathan; English, Connor; Cooper, Robin L

    2016-01-01

    The Drosophila melanogaster heart is a popular model in which to study cardiac physiology and development. Progress has been made in understanding the role of endogenous compounds in regulating cardiac function in this model. It is well characterized that common neurotransmitters act on many peripheral and non-neuronal tissues as they flow through the hemolymph of insects. Many of these neuromodulators, including acetylcholine (ACh), have been shown to act directly on the D. melanogaster larval heart. ACh is a primary neurotransmitter in the central nervous system (CNS) of vertebrates and at the neuromuscular junctions on skeletal and cardiac tissue. In insects, ACh is the primary excitatory neurotransmitter of sensory neurons and is also prominent in the CNS. A full understanding regarding the regulation of the Drosophila cardiac physiology by the cholinergic system remains poorly understood. Here we use semi-intact D. melanogaster larvae to study the pharmacological profile of cholinergic receptor subtypes, nicotinic acetylcholine receptors (nAChRs) and muscarinic acetylcholine receptors (mAChRs), in modulating heart rate (HR). Cholinergic receptor agonists, nicotine and muscarine both increase HR, while nAChR agonist clothianidin exhibits no significant effect when exposed to an open preparation at concentrations as low as 100 nM. In addition, both nAChR and mAChR antagonists increase HR as well but also display capabilities of blocking agonist actions. These results provide evidence that both of these receptor subtypes display functional significance in regulating the larval heart's pacemaker activity.

  10. 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.

  11. Circadian activity of corticosterone in an animal model of depression: response to muscarinic cholinergic stimulation.

    PubMed

    Bonilla-Jaime, H; Retana-Márquez, S; Arteaga-Silva, M; Hernández-González, M; Vázquez-Palacios, G

    2010-06-16

    Neonatal treatment with clomipramine (CMI) in rats induces multiple behavioral alterations during adulthood that resemble certain symptoms of human depression, such as impairments of pleasure-seeking behaviors. CMI may also induce permanent changes in the reactivity of the hypothalamic-pituitary-adrenocortical axis (HPA) to different stimuli; however, the endocrinal changes induced by this treatment are still a matter of debate. In the present study, we evaluated the levels of corticosterone in rats treated in the neonatal period with CMI in basal conditions (0, 6, 12 and 18 h after lights on) and after treatment with the antidepressant fluoxetine (FLX; 5mg/kg for 14 days). To evaluate the response of the HPA axis to a cholinergic agonist, we analyzed the effect of oxotremorine administration (OXO; 0.4, 0.8 mg/kg) on plasma levels of corticosterone. Administration of OXO took place at the beginning of each one of the two phases of the light-dark cycle (time points 0 and 12h, respectively). Results showed an increase in basal plasma levels of corticosterone in CMI-treated rats at time point zero and at 6h after the onset of the light period. While treatment with FLX reversed the increase in corticosterone plasma levels in CMI-treated rats, the results regarding cholinergic stimulation indicate that those rats do not respond to the administration of a low dose of OXO (0.4 mg/kg) at the onset of the dark phase (time point 12h). In conclusion, this study supports the hypothesis that neonatal treatment with CMI induces a hypersecretion of corticosterone in adulthood that was reversed through treatment with the antidepressant FLX. The CMI-treated rats showed a hyporesponse to cholinergic stimulation with OXO at low doses and at the beginning of the dark phase. Thus, the present results do not support the assumption that an increased sensitivity of the muscarinic cholinergic system is one of the possible correlates of the behavioral alterations seen in CMI-treated rats

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

    PubMed

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

    2011-05-02

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

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

    PubMed

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

    2002-11-01

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

  14. Cholinergic and GABAergic receptor functional deficit in the hippocampus of insulin-induced hypoglycemic and streptozotocin-induced diabetic rats.

    PubMed

    Sherin, A; Anu, J; Peeyush, K T; Smijin, S; Anitha, M; Roshni, B T; Paulose, C S

    2012-01-27

    Neurotransmitter receptor functional regulation plays an important role in controlling the excitability and responsiveness of hippocampal neurons. Deregulation of its function is associated with seizure generation, motor deficits, and memory impairment. In the present study we investigated the changes in hippocampal cholinergic and GABA receptor binding and gene expression in insulin-induced hypoglycemic and streptozotocin-induced diabetic rats. Expression of cholinergic enzymes; acetylcholine esterase (AChE) and choline acetyltransferase (ChAT) upregulated and downregulated, respectively, in diabetic group, which was further exacerbated by hypoglycemia. Total muscarinic receptor, muscarinic M1, and GABA maximal binding (B(max)) significantly decreased in hypoglycemic and diabetic rats. In hypoglycemic group, the B(max) showed further decline compared with diabetes. Muscarinic M3 receptor B(max) and gene expression upregulated in hypoglycemic and diabetic group. Alpha7 nicotinic acetylcholine receptor (α7 nAChR) expression significantly downregulated in hypoglycemic and diabetic rats. Gene expression of glutamate decarboxylase (GAD), GABAAα1, and GABAB in hypoglycemic and diabetic rats downregulated, with more significant decrease in hypoglycemic group. Present findings show altered cholinergic, muscarinic, nicotinic receptor expression and thereby function. Decreased GABA receptor expression is associated with decline in GABAergic neurotransmission. Thus cholinergic receptor dysfunction and decreased GABAergic neuroprotective inhibitory function in the hippocampus of hypoglycemic and diabetic rats account for the increased vulnerability of hippocampus predisposing to neuronal damage, which is suggested to contribute to cognitive impairment and memory deficit reported in hypoglycemia and diabetes. Also, recurrent hypoglycemia in diabetes exacerbates the hippocampal dysfunction induced by diabetes, which has clinical significance in diabetes therapy.

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

    PubMed Central

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

    2015-01-01

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

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

    SciTech Connect

    Murray, T.F.; Mpitsos, G.J.; Siebenaller, J.F.; Barker, D.L.

    1985-12-01

    The muscarinic antagonist L-(/sup 3/H)quinuclidinyl benzilate (L-(/sup 3/H)QNB) binds with a high affinity (Kd = 0.77 nM) to a single population of specific sites (Bmax = 47 fmol/mg of protein) in nervous tissue of the gastropod mollusc, Aplysia. The specific L-(/sup 3/H)QNB binding is displaced stereoselectively by the enantiomers of benzetimide, dexetimide, and levetimide. The pharmacologically active enantiomer, dexetimide, is more potent than levetimide as an inhibitor of L-(/sup 3/H)QNB binding. Moreover, the muscarinic cholinergic ligands, scopolamine, atropine, oxotremorine, and pilocarpine are effective inhibitors of the specific L-(/sup 3/H)QNB binding, whereas nicotinic receptor antagonists, decamethonium and d-tubocurarine, are considerably less effective. These pharmacological characteristics of the L-(/sup 3/H)QNB-binding site provide evidence for classical muscarinic receptors in Aplysia nervous tissue. The physiological relevance of the dexetimide-displaceable L-(/sup 3/H)QNB-binding site was supported by the demonstration of the sensitivity of the specific binding to thermal denaturation. Specific binding of L-(/sup 3/H)QNB was also detected in nervous tissue of another marine gastropod, Pleurobranchaea californica. The characteristics of the Aplysia L-(/sup 3/H)QNB-binding site are in accordance with studies of numerous vertebrate and invertebrate tissues indicating that the muscarinic cholinergic receptor site has been highly conserved through evolution.

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

    PubMed Central

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

    2009-01-01

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

  18. Muscarinic M1 receptors modulate endotoxemia-induced loss of synaptic plasticity.

    PubMed

    Zivkovic, Aleksandar R; Sedlaczek, Oliver; von Haken, Rebecca; Schmidt, Karsten; Brenner, Thorsten; Weigand, Markus A; Bading, Hilmar; Bengtson, C Peter; Hofer, Stefan

    2015-11-04

    Septic encephalopathy is associated with rapid deterioration of cortical functions. Using magnetic resonance imaging (MRI) we detected functional abnormalities in the hippocampal formation of patients with septic delirium. Hippocampal dysfunction was further investigated in an animal model for sepsis using lipopolysaccharide (LPS) injections to induce endotoxemia in rats, followed by electrophysiological recordings in brain slices. Endotoxemia induced a deficit in long term potentiation which was completely reversed by apamin, a blocker of small conductance calcium-activated potassium (SK) channels, and partly restored by treatment with physostigmine (eserine), an acetylcholinesterase inhibitor, or TBPB, a selective M1 muscarinic acetylcholine receptor agonist. These results suggest a novel role for SK channels in the etiology of endotoxemia and explain why boosting cholinergic function restores deficits in synaptic plasticity. Drugs which enhance cholinergic or M1 activity in the brain may prove beneficial in treatment of septic delirium in the intensive care unit.

  19. Current excitement from insect muscarinic receptors.

    PubMed

    Trimmer, B A

    1995-02-01

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

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

    PubMed

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

    2009-08-01

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

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

    PubMed

    Carden, W B; Bickford, M E

    1999-12-10

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

  2. The M2-Muscarinic Receptor Inhibits the Development of Streptozotocin-Induced Neuropathy in Mouse Urinary Bladder

    PubMed Central

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

    2010-01-01

    We investigate the role of M2-muscarinic receptors in maintaining neurogenic bladder contraction during hyperglycemia. Mice were injected with a single dose of streptozotocin (125 mg/kg), and neurogenic contraction of urinary bladder from wild type and M2-muscarinic receptor knockout (M2 KO) mice was measured at 8 to 24 weeks after treatment. In wild-type bladder lacking urothelium, the summation of the cholinergic (64%) and purinergic (56%) components of the electrical-field-stimulated response exceeded 100%, indicating a reserve capacity. Although the cholinergic component was slightly less in the M2 KO mouse, the total electrical-field-stimulated contraction was the same as wild type. The cholinergic and purinergic components of contraction in wild-type bladder were minimally affected by streptozotocin treatment. In M2 KO bladder, streptozotocin treatment reduced both the cholinergic (after 8–9 and 20–24 weeks) and purinergic (after 20–24 weeks only) components. The loss of function was approximately 50 to 70%. Similar results were observed in bladder with intact urothelium. M2 KO bladder was more sensitive to the relaxant effect of isoproterenol compared with wild type, and this difference significantly increased at the early and late time points after streptozotocin treatment. In the presence of urothelium, however, this difference in isoproterenol sensitivity was smaller with streptozotocin treatment, but this trend reversed over time. Our results show that M2 receptors oppose urinary bladder distension in wild-type bladder and inhibit streptozotocin-induced neuropathy. PMID:20624991

  3. Muscarinic Acetylcholine Receptor Localization and Activation Effects on Ganglion Response Properties

    PubMed Central

    Renna, Jordan M.; Amthor, Franklin R.; Keyser, Kent T.

    2010-01-01

    Purpose. The activation and blockade of muscarinic acetylcholine receptors (mAChRs) affects retinal ganglion cell light responses and firing rates. This study was undertaken to identify the full complement of mAChRs expressed in the rabbit retina and to assess mAChR distribution and the functional effects of mAChR activation and blockade on retinal response properties. Methods. RT-PCR, Western blot analysis, and immunohistochemistry were used to identify the complement and distribution of mAChRs in the rabbit retina. Extracellular electrophysiology was used to determine the effects of the activation or blockade of mAChRs on ganglion cell response properties. Results. RT-PCR of whole neural retina resulted in the amplification of mRNA transcripts for the m1 to m5 mAChR subtypes. Western blot and immunohistochemical analyses confirmed that all five mAChR subtypes were expressed by subpopulations of bipolar, amacrine, and ganglion cells in the rabbit retina, including subsets of cells in cholinergic and glycinergic circuits. Nonspecific muscarinic activation and blockade resulted in the class-specific modulation of maintained ganglion cell firing rates and light responses. Conclusions. The expression of mAChR subtypes on subsets of bipolar, amacrine, and ganglion cells provides a substrate for both enhancement and suppression of retinal responses via activation by cholinergic agents. Thus, the muscarinic cholinergic system in the retina may contribute to the modulation of complex stimuli. Understanding the distribution and function of mAChRs in the retina has the potential to provide important insights into the visual changes that are caused by decreased ACh in the retinas of Alzheimer's patients and the potential visual effects of anticholinergic treatments for ocular diseases. PMID:20042645

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

    PubMed

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

    2011-10-01

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

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

    PubMed Central

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

    2012-01-01

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

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

    SciTech Connect

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

    1986-07-01

    Regulation of muscarinic receptors in cultured guinea pig pancreatic acini was investigated by assessing the effects of cholinergic agonists on binding of (N-methyl-TH)scopolamine ((TH)NMS) and on amylase release. Freshly dispersed acini bound (TH)NMS with a K/sub d/ of 74 pM and a maximal binding level (B/sub max/) of 908 fmol/mg DNA. Carbachol (CCh) stimulated amylase secretion and inhibited (TH)NMS binding. Incubation of acini for 30 min with 0.1 mM CCh decreased the subsequent efficacy of CCh in stimulating amylase release by threefold but had no effect on its potency. In contrast, amylase release in response to cholecystokinin octapeptide (CCK-8) was not altered by CCh preincubation. (TH)NMS binding to acini was decreased only 15-20% after 30-min incubation with CCh. However, culture of acini with 0.1 mM CCh decreased (TH)NMS binding by 50% at 3-4 h and by 85-90% at 24 h. This decrease was attributable primarily to a reduction in B/sub max/ (TH)NMS binding also was decreased to a similar extent by the cholinergic agonists bethanechol and methacholine but not by other secretagogues. The decrease in antagonist binding induced by CCh was dose dependent, with the IC50, 5.8 M, approximating the EC50 for amylase release, 4.3 M. Cultured of acini for 24 h with CCh abolished subsequent amylase release in response to CCh but not to CCK-8. The results indicate that muscarinic receptor turnover in the pancreatic acinus is regulated by receptor activation and that both a decease in receptor numbers and sensitivity to agonists follows prolonged cholinergic agonist exposure.

  7. A role for muscarinic receptors in neutrophil extracellular trap formation and levamisole-induced autoimmunity

    PubMed Central

    Carmona-Rivera, Carmelo; Purmalek, Monica M.; Moore, Erica; Waldman, Meryl; Walter, Peter J.; Garraffo, H. Martin; Phillips, Karran A.; Preston, Kenzie L.; Graf, Jonathan; Grayson, Peter C.

    2017-01-01

    Levamisole, an anthelmintic drug with cholinergic properties, has been implicated in cases of drug-induced vasculitis when added to cocaine for profit purposes. Neutrophil extracellular trap (NET) formation is a cell death mechanism characterized by extrusion of chromatin decorated with granule proteins. Aberrant NET formation and degradation have been implicated in idiopathic autoimmune diseases that share features with levamisole-induced autoimmunity as well as in drug-induced autoimmunity. This study’s objective was to determine how levamisole modulates neutrophil biology and its putative effects on the vasculature. Murine and human neutrophils exposed to levamisole demonstrated enhanced NET formation through engagement of muscarinic subtype 3 receptor. Levamisole-induced NETosis required activation of Akt and the RAF/MEK/ERK pathway, ROS induction through the nicotinamide adenine dinucleotide phosphate oxidase, and peptidylarginine deiminase activation. Sera from two cohorts of patients actively using levamisole-adulterated cocaine displayed autoantibodies against NET components. Cutaneous biopsy material obtained from individuals exposed to levamisole suggests that neutrophils produce NETs in areas of vasculitic inflammation and thrombosis. NETs generated by levamisole were toxic to endothelial cells and impaired endothelium-dependent vasorelaxation. Stimulation of muscarinic receptors on neutrophils by cholinergic agonists may contribute to the pathophysiology observed in drug-induced autoimmunity through the induction of inflammatory responses and neutrophil-induced vascular damage. PMID:28194438

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

    PubMed Central

    Nathanson, Neil M.

    2008-01-01

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

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

    PubMed

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

    1995-07-01

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

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

    SciTech Connect

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

    1985-07-25

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

  11. Cellular and molecular basis of cholinergic function

    SciTech Connect

    Dowdall, M.J.; Hawthorne, J.N.

    1987-01-01

    This book contains 105 selections. Some of the titles are: Functional correlates of brain nicotine receptors; Muscarinic receptor subclasses; Cholinergic innervation and levels of nerve growth factor and its mRNA in the central nervous system; Developmentally regulated neurontrophic activities of Torpedo electric organ tissue; and Association of a regulatory peptide with cholinergic neurons.

  12. Characteristics of muscarinic receptors that selectively couple to inhibition of adenylate cyclase or stimulation of phospholipase C on NG108-15 and 1321N1 cells

    SciTech Connect

    Liang, M.

    1988-01-01

    The purpose of this dissertation was to establish whether different muscarinic receptor proteins selectively couple to different second messenger response system. Although both second messenger response systems are fully functional in both cell lines, activation of muscarinic cholinergic receptors only results in inhibition of adenylate cyclase in NG108-15 neuroblastoma {times} glioma cells and stimulation of phosphoinositide hydrolysis in 1321N1 human astrocytoma cells. Muscarinic receptors on both cell types were covalently labeled with ({sup 3}H)Propylbenzilylcholine mustard (({sup 3}H)PBCM) and the mobilities of the ({sup 3}H)PBCM-labelled species of both cells were compared by SDS-PAGE. 1321N1 and NG108-15 cells each primarily expressed a single ({sup 3}H)PBCM-labelled species with an apparent size of approximately 92,000 and 66,000 Da, respectively. ({sup 3}H)PBCM labelling was completely inhibited by 1 {mu}M atropine or by down-regulation of muscarinic receptors by an overnight incubation with carbachol. The apparent size of the ({sup 3}H)PBCM-labelled species of both cell lines was not altered by treatment with a series of protease inhibitors or by treatment with dithiothreitol and iodoacetamide. Another approach for determining differences in the muscarinic receptors of 2 cells lines was to study agonist-induced alteration of muscarinic receptor number. Exposure of both cell types to agonists resulted in rapid loss of muscarinic receptors from cell surface without change of total cellular muscarinic receptors followed by subsequently loss of receptors from cells. Muscarinic receptors on both cell lines were regulated by agonist with similar properties.

  13. Effects of a centipede venom fraction on insect nervous system, a native Xenopus oocyte receptor and on an expressed Drosophila muscarinic receptor.

    PubMed

    Stankiewicz, M; Hamon, A; Benkhalifa, R; Kadziela, W; Hue, B; Lucas, S; Mebs, D; Pelhate, M

    1999-10-01

    Centipede venoms are complex protein mixtures; very few is known about their pharmacological actions. Application of a Scolopendra sp. venom fraction (SC1) on the cockroach giant axon induced an increase in the leak current correlated with a decrease in the membrane resistance, suggesting the presence in SC1 of components opening non-specific pores in the axonal membrane. On a cockroach central cholinergic synapse, microinjection of SC1 induced a small transient depolarization of the postsynaptic membrane, followed by a slow stable depolarization and a drastic decrease in the evoked subthreshold excitatory postsynaptic potential amplitude. A pretreatment of the ganglion with atropine or scopolamine reduced the amplitude of the SC1-induced depolarizing wave, suggesting a possible cholinergic muscarinic target. On control Xenopus oocytes, SC1 induced an inward oscillatory Ca2(+)-dependent Cl- current mediated through the activation of native lysophosphatidic acid receptors (LPAr). Indeed, pretreatment of oocytes with 1 microM N-palmitoyl-tyrosine phosphoric acid, a selective competitive antagonist of LPAr, decreased responses to SC1 by 70%. Application of SC1 to oocytes expressing a cloned Drosophila muscarinic receptor (Dml) induced a biphasic response comprising: (1) a large fast Cl- current that was abolished by pretreatment with atropine and scopolamine and (2) a slow and small oscillating Cl- current corresponding to the response observed in control oocytes. These observations confirm the presence of muscarinic agonists in SCI and reveal their direct action on an insect muscarinic receptor subtype homologous to mammalian M1-M3 receptors.

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

    PubMed

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

    2008-06-26

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

  15. [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

  16. Cholinergic and ghrelinergic receptors and KCNQ channels in the medial PFC regulate the expression of palatability

    PubMed Central

    Parent, Marc A.; Amarante, Linda M.; Swanson, Kyra; Laubach, Mark

    2015-01-01

    The medial prefrontal cortex (mPFC) is a key brain region for the control of consummatory behavior. Neuronal activity in this area is modulated when rats initiate consummatory licking and reversible inactivations eliminate reward contrast effects and reduce a measure of palatability, the duration of licking bouts. Together, these data suggest the hypothesis that rhythmic neuronal activity in the mPFC is crucial for the control of consummatory behavior. The muscarinic cholinergic system is known to regulate membrane excitability and control low-frequency rhythmic activity in the mPFC. Muscarinic receptors (mAChRs) act through KCNQ (Kv7) potassium channels, which have recently been linked to the orexigenic peptide ghrelin. To understand if drugs that act on KCNQ channels within the mPFC have effects on consummatory behavior, we made infusions of several muscarinic drugs (scopolamine, oxotremorine, physostigmine), the KCNQ channel blocker XE-991, and ghrelin into the mPFC and evaluated their effects on consummatory behavior. A consistent finding across all drugs was an effect on the duration of licking bouts when animals consume solutions with a relatively high concentration of sucrose. The muscarinic antagonist scopolamine reduced bout durations, both systemically and intra-cortically. By contrast, the muscarinic agonist oxotremorine, the cholinesterase inhibitor physostigmine, the KCNQ channel blocker XE-991, and ghrelin all increased the durations of licking bouts when infused into the mPFC. Our findings suggest that cholinergic and ghrelinergic signaling in the mPFC, acting through KCNQ channels, regulates the expression of palatability. PMID:26578914

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

    SciTech Connect

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

    1986-03-05

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

  18. Flurbiprofen, a cyclooxygenase inhibitor, reduces the brain arachidonic acid signal in response to the cholinergic muscarinic agonist, arecoline, in awake rats.

    PubMed

    Basselin, Mireille; Villacreses, Nelly E; Lee, Ho-Joo; Bell, Jane M; Rapoport, Stanley I

    2007-11-01

    Cholinergic muscarinic receptors, when stimulated by arecoline, can activate cytosolic phospholipase A(2) (cPLA(2)) to release arachidonic acid (AA) from membrane phospholipid. This signal can be imaged in the brain in vivo using quantitative autoradiography following the intravenous injection of radiolabeled AA, as an increment in a regional brain AA incorporation coefficient k*. Arecoline increases k* significantly in brain regions having muscarinic M(1,3,5) receptors in wild-type but not in cyclooxygenase (COX)-2 knockout mice. To further clarify the roles of COX enzymes in the AA signal, in this paper we imaged k* following arecoline (5 mg/kg i.p.) or saline in each of 81 brain regions of unanesthetized rats pretreated 6 h earlier with the non-selective COX inhibitor flurbiprofen (FB, 60 mg/kg s.c.) or with vehicle. Baseline values of k* were unaffected by FB treatment, which however reduced by 80% baseline brain concentrations of prostaglandin E(2) (PGE(2)) and thromboxane B(2) (TXB(2)), eicosanoids preferentially derived from AA via COX-2 and COX-1, respectively. In vehicle-pretreated rats, arecoline increased the brain PGE(2) but not TXB(2) concentration, as well as values for k* in 77 of the 81 brain regions. FB-pretreatment prevented these arecoline-provoked changes. These results and those reported in COX-2 knockout mice suggest that the AA released in brain following muscarinic receptor-mediated activation is lost via COX-2 to PGE(2) but not via COX-1 to TXB(2), and that increments in k* following arecoline largely represent replacement by unesterified plasma AA of this loss.

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

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

    2009-01-01

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

  1. Activations of muscarinic M1 receptors in the anterior cingulate cortex contribute to the antinociceptive effect via GABAergic transmission

    PubMed Central

    Matsuzaki, Yu; Honda, Kenji; Eto, Fumihiro; Furukawa, Tomonori; Migita, Keisuke; Irie, Keiichi; Mishima, Kenichi; Ueno, Shinya

    2017-01-01

    Background Cholinergic systems regulate the synaptic transmission resulting in the contribution of the nociceptive behaviors. Anterior cingulate cortex is a key cortical area to play roles in nociception and chronic pain. However, the effect of the activation of cholinergic system for nociception is still unknown in the cortical area. Here, we tested whether the activation of cholinergic receptors can regulate nociceptive behaviors in adult rat anterior cingulate cortex by integrative methods including behavior, immunohistochemical, and electrophysiological methods. Results We found that muscarinic M1 receptors were clearly expressed in the anterior cingulate cortex. Using behavioral tests, we identified that microinjection of a selective muscarinic M1 receptors agonist McN-A-343 into the anterior cingulate cortex dose dependently increased the mechanical threshold. In contrast, the local injection of McN-A-343 into the anterior cingulate cortex showed normal motor function. The microinjection of a selective M1 receptors antagonist pirenzepine blocked the McN-A-343-induced antinociceptive effect. Pirenzepine alone into the anterior cingulate cortex decreased the mechanical thresholds. The local injection of the GABAA receptors antagonist bicuculline into the anterior cingulate cortex also inhibited the McN-A-343-induced antinociceptive effect and decreased the mechanical threshold. Finally, we further tested whether the activation of M1 receptors could regulate GABAergic transmission using whole-cell patch-clamp recordings. The activation of M1 receptors enhanced the frequency of spontaneous and miniature inhibitory postsynaptic currents as well as the amplitude of spontaneous inhibitory postsynaptic currents in the anterior cingulate cortex. Conclusions These results suggest that the activation of muscarinic M1 receptors in part increased the mechanical threshold by increasing GABAergic transmitter release and facilitating GABAergic transmission in the anterior

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

    SciTech Connect

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

    1990-05-01

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

  3. Stress and re-stress increases conditioned taste aversion learning in rats: possible frontal cortical and hippocampal muscarinic receptor involvement.

    PubMed

    Brand, Linda; Groenewald, Ilse; Stein, Dan J; Wegener, Gregers; Harvey, Brian H

    2008-05-31

    Symptoms of posttraumatic stress disorder are often precipitated by sensory cues in the form of visual, auditory, olfactory and gustatory "flashbacks" resulting in enhanced fear-memory consolidation and the characteristic symptoms of re-experiencing, avoidance and hyper-arousal. Single prolonged stress with and without re-stress have been used to explore the neurobiology of this disorder, particularly with respect to contextual conditioning and spatial memory impairment. However, less work has been done regarding associative sensory-related memories linked to aversive events. Although growing evidence supports a role for cholinergic pathways in stress, this has not been studied in the above animal models. We studied the effects of single prolonged stress with and without re-stress on conditioned taste aversion learning in rats, together with differential analysis of frontal cortical and hippocampal [3H]-quinuclidinyl benzylate ([3H]-QNB) muscarinic receptor binding. Single prolonged stress with and without re-stress both enhanced associative sensory aversion learning 7 days after stressor-taste pairing, although re-stress did not strengthen this response. Increased cortical and hippocampal muscarinic receptor density (Bmax) was found 7 days after single prolonged stress with re-stress, although receptor affinity remained unaltered. Frontal cortical and hippocampal muscarinic receptor changes may thus underlie conditioned taste aversion learning in rats exposed to stress and re-stress. These data suggest that it may be useful to study the role of cholinergic pathways in mediating associative memory in psychiatric disorders such as posttraumatic stress disorder.

  4. Co-expression of alpha7 and beta2 nicotinic acetylcholine receptor subunit mRNAs within rat brain cholinergic neurons.

    PubMed

    Azam, L; Winzer-Serhan, U; Leslie, F M

    2003-01-01

    Nicotine enhances cognitive and attentional processes through stimulation of the basal forebrain cholinergic system. Although muscarinic cholinergic autoreceptors have been well characterized, pharmacological characterization of nicotinic autoreceptors has proven more difficult. The present study used double-labeling in situ hybridization to determine expression of nicotinic acetylcholine receptor (nAChR) subunit mRNAs within basal forebrain cholinergic neurons in order to gain information about possible nAChR autoreceptor properties. Cholinergic cells of the mesopontine tegmentum and striatal interneurons were also examined, as were septohippocampal GABAergic neurons that interact with cholinergic neurons to regulate hippocampal activity. alpha7 and beta2 nAChR mRNAs were found to be co-expressed in almost all cholinergic cells and in the majority of GABAergic neurons examined. alpha4 nAChR mRNA expression was restricted to cholinergic cells of the nucleus basalis magnocellularis, and to non-cholinergic cells of the medial septum and mesopontine tegmentum. These data suggest possible regional differences in the pharmacological properties of nicotinic autoreceptors on cholinergic cells. Whereas most cholinergic cells express rapidly desensitizing alpha7 homomers or alpha7beta2 heteromers, cortical projection neurons may also express a pharmacologically distinct alpha4beta2 nAChR subtype. There may also be differential nAChR regulation of cholinergic and non-cholinergic cells within the mesopontine tegmentum that are implicated in acquisition of nicotine self-administration.

  5. Muscarinic receptors of the albino rabbit ciliary process.

    PubMed

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

    1989-04-01

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

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

    SciTech Connect

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

    1987-03-01

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

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

    SciTech Connect

    Lee, Haesung; Yee, S.; Geddes, J.; Choi, Byung, H. Univ. of California, Irvine )

    1991-03-11

    Methylmercury (MeHg) is reported to inhibit several stages of cholinergic neurotransmission in brain tissue in-vitro and in-vivo. To examine whether or not behavioral disturbances and/or selective vulnerability of specific neuronal groups in MeHg poisoning may be related to MeHg effects on cholinergic receptors in specific regions of the brain, the density and distribution of muscarinic receptors in the brains of C57BL/6J mice were determined following repeated injections of 5 mg/kg of methylmercuric chloride (MMC). The receptor densities in six cortical laminae of seven cerebral cortical regions, hippocampus and striatum were quantitated by computer-assisted imaging system following in-vitro labeling with ({sup 3}H)-pirenzepine (M1) and ({sup 3}H)N-methyl scopolamine (M2). The results showed heterogeneous distribution of M1 and M2 sites in different regions of the brain, and significant reduction in the density of both receptor subtypes following MeHg poisoning in many cortical and subcortical regions. However, the changes in the density were variable in different laminae even in the same cortical regions. Prominent reductions in M1 densities were noted in the temporal and entorhinal cortices, CA3 and hilar regions of the hippocampus as compared to control, whereas the reduction in M2 receptor density was most prominently noted in the frontal, perirhinal and entorhinal cortices, and CA1 and hilar regions of the hippocampus. Thus, it is apparent that MeHg significantly affects muscarinic receptors in the mouse brain, and that these data when used in conjunction with immunocytochemical and other morphological studies would provide further insights into the mechanisms of neurotoxic effects of MeHg.

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

    PubMed

    Abraham, Getu

    2016-02-01

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

  9. GABAA receptors are located in cholinergic terminals in the nucleus pontis oralis of the rat: implications for REM sleep control.

    PubMed

    Liang, Chang-Lin; Marks, Gerald A

    2014-01-16

    The oral pontine reticular formation (PnO) of rat is one region identified in the brainstem as a rapid eye movement (REM) sleep induction zone. Microinjection of GABA(A) receptor antagonists into PnO induces a long lasting increase in REM sleep, which is similar to that produced by cholinergic agonists. We previously showed that this REM sleep-induction can be completely blocked by a muscarinic antagonist, indicating that the REM sleep-inducing effect of GABA(A) receptor antagonism is dependent upon the local cholinergic system. Consistent with these findings, it has been reported that GABA(A) receptor antagonists microdialyzed into PnO resulted in increased levels of acetylcholine. We hypothesize that GABA(A) receptors located on cholinergic boutons in the PnO are responsible for the REM sleep induction by GABA(A) receptor antagonists through blocking GABA inhibition of acetylcholine release. Cholinergic, varicose axon fibers were studied in the PnO by immunofluorescence and confocal, laser scanning microscopy. Immunoreactive cholinergic boutons were found to be colocalized with GABA(A) receptor subunit protein γ2. This finding implicates a specific subtype and location of GABA(A) receptors in PnO of rat in the control of REM sleep. © 2013 Published by Elsevier B.V.

  10. Long-term effects of methamphetamine exposure on cognitive function and muscarinic acetylcholine receptor levels in mice.

    PubMed

    Siegel, Jessica A; Craytor, Michael J; Raber, Jacob

    2010-10-01

    Exposure to methamphetamine during brain development impairs cognition in humans and rodents. In mice, these impairments are more severe in females than males. Genetic factors, such as apolipoprotein E genotype, may modulate the cognitive effects of methamphetamine. Methamphetamine-induced alterations in the brain acetylcholine system may contribute to the cognitive effects of methamphetamine and may also be modulated by apolipoprotein E isoform. We assessed the long-term effects of methamphetamine exposure during brain development on cognitive function and muscarinic acetylcholine receptors in mice, and whether apolipoprotein E isoform modulates these effects. Mice expressing human apolipoprotein E3 or E4 were exposed to methamphetamine (5 mg/kg) or saline once a day from postnatal days 11-20 and behaviorally tested in adulthood. Muscarinic acetylcholine receptor binding was measured in the hippocampus and cortex. Methamphetamine exposure impaired novel location recognition in female, but not male, mice. Methamphetamine-exposed male and female mice showed impaired novel object recognition and increased number of muscarinic acetylcholine receptors in the hippocampus. The cognitive and cholinergic effects of methamphetamine were similar in apolipoprotein E3 and E4 mice. Thus, the cholinergic system, but not apolipoprotein E isoform, might play an important role in the long-term methamphetamine-induced cognitive deficits in adulthood.

  11. Long-term effects of methamphetamine exposure on cognitive function and muscarinic acetylcholine receptor levels in mice

    PubMed Central

    Siegel, Jessica A.; Craytor, Michael J.; Raber, Jacob

    2010-01-01

    Exposure to methamphetamine during brain development impairs cognition in humans and rodents. In mice, these impairments are greater in females than males. Genetic factors, such as apolipoprotein E genotype, may modulate the cognitive effects of methamphetamine. Methamphetamine-induced alterations in the brain acetylcholine system may contribute to the cognitive effects of methamphetamine and may also be modulated by apolipoprotein E isoform. We assessed the long-term effects of methamphetamine exposure during brain development on cognitive function and muscarinic acetylcholine receptors in mice, and whether apolipoprotein E isoform modulates these effects. Mice expressing human apolipoprotein E3 or E4 were exposed to methamphetamine (5 mg/kg) or saline once a day from postnatal day 11-20 and behaviorally tested in adulthood. Muscarinic acetylcholine receptor binding was measured in the hippocampus and cortex. Methamphetamine exposure impaired novel location recognition in female, but not male, mice. Methamphetamine-exposed male and female mice showed impaired novel object recognition and increased number of muscarinic acetylcholine receptors in the hippocampus. The cognitive and cholinergic effects of methamphetamine were similar in apolipoprotein E3 and E4 mice. Thus, the cholinergic system, but not apolipoprotein E isoform, might play an important role in the long-term methamphetamine-induced cognitive deficits in adulthood. PMID:20729719

  12. 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.

  13. Functional characterization of muscarinic receptors in murine airways.

    PubMed Central

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

    1993-01-01

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

  14. A cholinergic receptor gene (CHRM2) affects event-related oscillations.

    PubMed

    Jones, Kevin A; Porjesz, Bernice; Almasy, Laura; Bierut, Laura; Dick, Danielle; Goate, Alison; Hinrichs, Anthony; Rice, John P; Wang, Jen C; Bauer, Lance O; Crowe, Raymond; Foroud, Tatiana; Hesselbrock, Victor; Kuperman, Samuel; Nurnberger, John; O'Connor, Sean J; Rohrbaugh, John; Schuckit, Marc A; Tischfield, Jay; Edenberg, Howard J; Begleiter, Henri

    2006-09-01

    We report genetic linkage and association findings which implicate the gene encoding the muscarinic acetylcholine receptor M2 (CHRM2) in the modulation of a scalp-recorded electrophysiological phenotype. The P3 (P300) response was evoked using a three-stimulus visual oddball paradigm and a phenotype that relates to the energy in the theta band (4-5 Hz) was analyzed. Studies have shown that similar electrophysiological measures represent cognitive correlates of attention, working memory, and response selection; a role has been suggested for the ascending cholinergic pathway in the same functions. The results of our genetic association tests, combined with knowledge regarding the presence of presynaptic cholinergic M2 autoreceptors in the basal forebrain, indicate that the cognitive processes required by the experiment may in part be mediated by inhibitory neural networks. These findings underscore the utility of electrophysiology and neurogenetics in the understanding of cognitive function and the study of brain-related disorders.

  15. Cardiac Muscarinic Receptor Overexpression in Sudden Infant Death Syndrome

    PubMed Central

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

    2010-01-01

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

  16. Muscarinic receptors in perirhinal cortex control trace conditioning.

    PubMed

    Bang, Sun Jung; Brown, Thomas H

    2009-04-08

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

  17. Ionic mechanism of the slow afterdepolarization induced by muscarinic receptor activation in rat prefrontal cortex.

    PubMed

    Haj-Dahmane, S; Andrade, R

    1998-09-01

    The mammalian prefrontal cortex receives a dense cholinergic innervation from subcortical regions. We previously have shown that cholinergic stimulation of layer V pyramidal neurons of the rat prefrontal cortex results in a depolarization and the appearance of a slow afterdepolarization (sADP). In the current report we examine the mechanism underlying the sADP with the use of sharp microelectrode and whole cell recording techniques in in vitro brain slices. The ability of acetylcholine (ACh) and carbachol to induce the appearance of an sADP in pyramidal cells of layer V of prefrontal cortex is antagonized in a surmountable manner by atropine and is mimicked by application of muscarine or oxotremorine. These results indicate that ACh acts on muscarinic receptors to induce the sADP. In many cell types afterpotentials are triggered by calcium influx into the cell. Therefore we examined the possibility that calcium influx might be the trigger for the generation of the sADP. Consistent with this possibility, buffering intracellular calcium reduced or abolished the sADP but had little effect on the direct muscarinic receptor-induced depolarization also seen in these cells. These results, coupled to the previous observation that calcium channel blockers inhibit the sADP, indicated that the sADP results from a rise in intracellular calcium secondary to calcium influx into the cell. The ionic basis for the current underlying the sADP (IsADP) was examined with the use of ion substitution experiments. The amplitude of IsADP was found to be reduced in a graded fashion by replacement of extracellular sodium with N-methyl-D-glucamine (NMDG). In contrast no clear evidence for the involvement of potassium or chloride channels in the generation of the sADP or IsADP could be found. This result indicated that IsADP is carried by sodium ions flowing into the cell. However, the dependence of IsADP on extracellular sodium was less pronounced than expected for a pure sodium current. We

  18. A model of the human M2 muscarinic acetylcholine receptor

    NASA Astrophysics Data System (ADS)

    Jöhren, Kirstin; Höltje, Hans-Dieter

    2002-11-01

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

  19. Mice Lacking M1 and M3 Muscarinic Acetylcholine Receptors Have Impaired Odor Discrimination and Learning

    PubMed Central

    Chan, Wilson; Singh, Sanmeet; Keshav, Taj; Dewan, Ramita; Eberly, Christian; Maurer, Robert; Nunez-Parra, Alexia; Araneda, Ricardo C.

    2017-01-01

    The cholinergic system has extensive projections to the olfactory bulb (OB) where it produces a state-dependent regulation of sensory gating. Previous work has shown a prominent role of muscarinic acetylcholine (ACh) receptors (mAChRs) in regulating the excitability of OB neurons, in particular the M1 receptor. Here, we examined the contribution of M1 and M3 mAChR subtypes to olfactory processing using mice with a genetic deletion of these receptors, the M1−/− and the M1/M3−/− knockout (KO) mice. Genetic ablation of the M1 and M3 mAChRs resulted in a significant deficit in odor discrimination of closely related molecules, including stereoisomers. However, the discrimination of dissimilar molecules, social odors (e.g., urine) and novel object recognition was not affected. In addition the KO mice showed impaired learning in an associative odor-learning task, learning to discriminate odors at a slower rate, indicating that both short and long-term memory is disrupted by mAChR dysfunction. Interestingly, the KO mice exhibited decreased olfactory neurogenesis at younger ages, a deficit that was not maintained in older animals. In older animals, the olfactory deficit could be restored by increasing the number of new born neurons integrated into the OB after exposing them to an olfactory enriched environment, suggesting that muscarinic modulation and adult neurogenesis could be two different mechanism used by the olfactory system to improve olfactory processing. PMID:28210219

  20. Nicotinic cholinergic receptors in rat brain. Annual report No. 3, 1 May 85-30 Apr 86

    SciTech Connect

    Kellar, K.J.

    1986-05-01

    We have compared the characteristics of the recognition sites for 3(H)acetylcholine and 3H(-)nicotine in rat brain and found that the pharmacology, distribution, disulfide bond requirement, and regulation by chronic administration of nicotine and soman are identical. From these studies we conclude that 3Hacetylcholine and 3H(-)nicotine recognize the same recognition site which has the characteristics expected of a nicotinic cholinergic receptor. We have also determined that 3Hacetylcholine of high specific radioactivity (80 Ci/mmol) is an excellent ligand with which to study muscarinic receptors that have high affinity for agonists. These receptors may represent a subtype of muscarinic receptors found in brain, heart, glands, an some smooth muscle. (JS)

  1. Ethanol impairs muscarinic receptor-induced neuritogenesis in rat hippocampal slices: role of astrocytes and extracellular matrix proteins

    PubMed Central

    Giordano, Gennaro; Guizzetti, Marina; Dao, Khoi; Mattison, Hayley A.; Costa, Lucio G.

    2011-01-01

    In an in vitro co-culture system of astrocytes and neurons, stimulation of cholinergic muscarinic receptors in astrocytes had been shown to cause neuritogenesis in hippocampal neurons, and this effect was inhibited by ethanol. The present study sought to confirm these earlier findings in a more complex system, in vitro rat hippocampal slices in culture. Exposure of hippocampal slices to the cholinergic agonist carbachol (1 mM for 24 h) induced neurite outgrowth in hippocampal pyramidal neurons, which was mediated by activation of muscarinic M3 receptors. Specifically, carbachol induced a >4-fold increase in the length of the longest neurite, and a 4-fold increase in the length of minor neurites and in the number of branches. Co-incubation of carbachol with ethanol (50 mM) resulted in significant inhibition of the effects induced by carbachol on all parameters measured. Neurite outgrowth in CNS neurons is dependent on various permissive factors that are produced and released by glial cells. In hippocampal slices carbachol increased the levels of two extracellular matrix protein, fibronectin and laminin-1, by 1.6-fold, as measured by Western blot. Co-incubation of carbachol with ethanol significantly inhibited these increases. Carbachol-induced increases in levels of extracellular matrix proteins were antagonized by a M3 muscarinic receptor antagonist. Furthermore, function-blocking fibronectin or laminin-1 antibodies antagonized the effect of carbachol on neurite outgrowth. These results indicate that in hippocampal slices stimulation of muscarinic M3 receptors induces neurite outgrowth, which is mediated by fibronectin and laminin-1, two extracellular matrix proteins released by astrocytes. By decreasing fibronectin and laminin levels ethanol prevents carbachol-induced neuritogenesis. These findings highlight the importance of glial-neuronal interactions as important targets in the developmental neurotoxicity of alcohol. PMID:21884684

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

    PubMed

    Cameron, H L; Perdue, M H

    2007-01-01

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

  3. Using cholinergic M1 receptor positive allosteric modulators to improve memory via enhancement of brain cholinergic communication.

    PubMed

    Chambon, Caroline; Jatzke, Claudia; Wegener, Nico; Gravius, Andreas; Danysz, Wojciech

    2012-12-15

    Benzylquinolone carboxylic acid (BQCA) is a recently described cholinergic muscarinic M(1) receptor positive allosteric modulator having potential as cognitive enhancer in dementia. The present study focused on the characterisation of BQCA's mode of action in relation to positive effects on memory and side-effects in an animal model. To get insight into this mode of action, in vitro receptor potency/left shift experiments in cells stably expressing the rat's M(1) receptor were performed. They revealed an inflection point value of BQCA corresponding to 306nM, and potentiation of the agonist response up to 47-fold in presence of 10μM of BQCA. In vivo, brain microdialysis showed a maximal brain level of 270nM, 40min after i.p. administration at 10mg/kg. Based on in vitro data obtained with this dose, it can be concluded that BQCA reaches brain levels which should potentiate the agonist response about 4-fold. Behavioural data confirmed that BQCA used at 10mg/kg attenuated scopolamine-induced memory deficit in a spontaneous alternation task. Moreover, BQCA showed no side effect at 10mg/kg and above in spontaneous locomotion and salivation tests. The profile of BQCA observed in the present study displays a clear advantage over the M(1)-M(3) agonist cevimeline. The present data show the therapeutic potential of the M(1) receptor positive allosteric modulator BQCA for the treatment of memory deficits observed in Alzheimer's disease.

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

    SciTech Connect

    McLeskey, S.W.

    1989-01-01

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

  5. Naltrexone pretreatment blocks microwave-induced changes in central cholinergic receptors

    SciTech Connect

    Lai, H.; Carino, M.A.; Wen, Y.F.; Horita, A.; Guy, A.W. )

    1991-01-01

    Repeated exposure of rats to pulsed, circularly polarized microwaves (2,450-MHz, 2-microseconds pulses at 500 pps, power density 1 mW/cm2, at an averaged, whole-body SAR of 0.6 W/kg) induced biphasic changes in the concentration of muscarinic cholinergic receptors in the central nervous system. An increase in receptor concentration occurred in the hippocampus of rats subjected to ten 45-min sessions of microwave exposure, whereas a decrease in concentration was observed in the frontal cortex and hippocampus of rats exposed to ten 20-min sessions. These findings, which confirm earlier work in the authors' laboratory, were extended to include pretreatment of rats with the narcotic antagonist naltrexone (1 mg/kg, IP) before each session of exposure. The drug treatment blocked the microwave-induced changes in cholinergic receptors in the brain. These data further support the authors' hypothesis that endogenous opioids play a role in the effects of microwaves on central cholinergic systems.

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

    SciTech Connect

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

    1984-03-05

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

  7. Organophosphorus Pesticides Decrease M2 Muscarinic Receptor Function in Guinea Pig Airway Nerves via Indirect Mechanisms

    PubMed Central

    Proskocil, Becky J.; Bruun, Donald A.; Thompson, Charles M.; Fryer, Allison D.; Lein, Pamela J.

    2010-01-01

    Background Epidemiological studies link organophosphorus pesticide (OP) exposures to asthma, and we have shown that the OPs chlorpyrifos, diazinon and parathion cause airway hyperreactivity in guinea pigs 24 hr after a single subcutaneous injection. OP-induced airway hyperreactivity involves M2 muscarinic receptor dysfunction on airway nerves independent of acetylcholinesterase (AChE) inhibition, but how OPs inhibit neuronal M2 receptors in airways is not known. In the central nervous system, OPs interact directly with neurons to alter muscarinic receptor function or expression; therefore, in this study we tested whether the OP parathion or its oxon metabolite, paraoxon, might decrease M2 receptor function on peripheral neurons via similar direct mechanisms. Methodology/Principal Findings Intravenous administration of paraoxon, but not parathion, caused acute frequency-dependent potentiation of vagally-induced bronchoconstriction and increased electrical field stimulation (EFS)-induced contractions in isolated trachea independent of AChE inhibition. However, paraoxon had no effect on vagally-induced bradycardia in intact guinea pigs or EFS-induced contractions in isolated ileum, suggesting mechanisms other than pharmacologic antagonism of M2 receptors. Paraoxon did not alter M2 receptor expression in cultured cells at the mRNA or protein level as determined by quantitative RT-PCR and radio-ligand binding assays, respectively. Additionally, a biotin-labeled fluorophosphonate, which was used as a probe to identify molecular targets phosphorylated by OPs, did not phosphorylate proteins in guinea pig cardiac membranes that were recognized by M2 receptor antibodies. Conclusions/Significance These data indicate that neither direct pharmacologic antagonism nor downregulated expression of M2 receptors contributes to OP inhibition of M2 function in airway nerves, adding to the growing evidence of non-cholinergic mechanisms of OP neurotoxicity. PMID:20479945

  8. Muscarinic acetylcholine receptors regulating cell cycle progression are expressed in human gingival keratinocytes.

    PubMed

    Arredondo, J; Hall, L L; Ndoye, A; Chernyavsky, A I; Jolkovsky, D L; Grando, S A

    2003-02-01

    We have previously reported the presence in human gingival keratinocytes (GKC) of choline acetyltransferase, the acetylcholine (ACh) synthesizing enzyme, acetylcholinesterase, the ACh degrading enzyme, and alpha 3, alpha 5, alpha 7, beta 2 as well as alpha 9 nicotinic ACh receptor subunits. To expand the knowledge about the role of ACh in oral biology, we investigated the presence of the muscarinic ACh receptor (mAChR) subtypes in GKC. RT-PCR demonstrated the presence of m2, m3, m4, and m5 mRNA transcripts. Synthesis of the respective proteins was verified by immunoblotting with the subtype-specific antibodies that revealed receptor bands at the expected molecular weights. The antibodies mapped mAChR subtypes in the epithelium of human attached gingiva and also visualized them on the cell membrane of cultured GKC. The whole cell radioligand binding assay revealed that GKC have specific binding sites for the muscarinic ligand [3H]quinuclidinyl benzilate, Bmax = 222.9 fmol/106 cells with a Kd of 62.95 pM. The downstream coupling of the mAChRs to regulation of cell cycle progression in GKC was studied using quantitative RT-PCR and immunoblotting assays. Incubation of GKC for 24 h with 10 micro m muscarine increased relative amounts of Ki-67, PCNA and p53 mRNAs and PCNA, cyclin D1, p21 and p53 proteins. These effects were abolished in the presence of 50 micro m atropine. The finding in GKC of mAChRs coupled to regulation of the cell cycle progression demonstrate further the structure/function of the non-neuronal cholinergic system operating in human oral epithelium. The results obtained in this study help clarify the role for keratinocyte ACh axis in the physiologic control of oral gingival homeostasis.

  9. Primary cultures of corticostriatal cells from newborn rats: a model to study muscarinic receptor subtypes regulation and function.

    PubMed

    Eva, C; Bovolin, P; Balzac, F; Botta, C; Gamalero, S R; Vaccarino, F M

    1990-01-01

    In the present work we characterized both the presynaptic and postsynaptic components of cholinergic transmission in a primary culture of corticostriatal neurons prepared from newborn rat brain. This culture preparation contains a small population of choline acetyltransferase (ChAT) immunoreactive neurons, corresponding to approximately 3% of the total cell number, and synthesizes increasing amounts of acetylcholine (ACh) from the third day in vitro (DIV), which reaches a plateau around the 10 day of culture. Muscarinic cholinergic receptors (mAChR), measured by the binding of the muscarinic antagonist [3H]quinuclidinyl benzilate ([3H]QNB), are detectable from the fifth DIV and increase linearly during the time of culture. At the twelfth DIV, the density of mAChRs (approximately 600 fmol/mg protein) is comparable to the density of mAChR in adult rat cortex. These receptors are coupled to second messenger systems, since muscarinic agonists inhibit adenylate cyclase activity and stimulate phosphoinositide breakdown with efficacies and potencies similar to those found in adult rat cortex. Moreover, by using the reverse transcriptase-polymerase chain reaction (RT-PCR) technique, we were able to demonstrate the presence of the m1, m3, and m4 mAChR subtype mRNAs in this neuronal culture at 12 DIV. Our data suggest that corticostriatal neuronal cultures develop in vitro ACh-synthesizing neurons and functionally active cholinergic receptors. This therefore makes them ideally suited to study the development and properties of brain mAChR subtypes.

  10. Changes in acetylcholine content, release and muscarinic receptors in rat hippocampus under cold stress

    SciTech Connect

    Fatranska, M.; Budai, D.; Gulya, K; Kvetnansky, R.

    1989-01-01

    The aim was to study the mechanism of the previously established decrease in acetylcholine (ACh) concentration in the rat hippocampus under cold stress. Male rats were exposed for 14 days to cold (5/degree/C) or kept (controls) at room temperature (24/degree/C). Acetylcholine content, release and muscarinic receptor binding were investigated in the hippocampus. Cold exposure resulted in a decrease of ACh concentration in the dorsal hippocampus. Moreover, the potassium-evoked release of ACh from hippocampal slices was increased and an increase of maximal binding capacity of (/sup 3/H)(-) quinuclidinyl benzilate in the dorsal hippocampus of cold exposed animals was also observed. Thus the decrease of hippocampal ACh concentration under cold exposure is probably due to its increased release. On balance then, our results demonstrate that cold stress in the rat induces significant activation of the hippocampal cholinergic system.

  11. M1 muscarinic acetylcholine receptor agonism alters sleep without affecting memory consolidation.

    PubMed

    Nissen, Christoph; Power, Ann E; Nofzinger, Eric A; Feige, Bernd; Voderholzer, Ulrich; Kloepfer, Corinna; Waldheim, Bernhard; Radosa, Marc-Philipp; Berger, Mathias; Riemann, Dieter

    2006-11-01

    Preclinical studies have implicated cholinergic neurotransmission, specifically M1 muscarinic acetylcholine receptor (mAChR) activation, in sleep-associated memory consolidation. In the present study, we investigated the effects of administering the direct M1 mAChR agonist RS-86 on pre-post sleep memory consolidation. Twenty healthy human participants were tested in a declarative word-list task and a procedural mirror-tracing task. RS-86 significantly reduced rapid eye movement (REM) sleep latency and slow wave sleep (SWS) duration in comparison with placebo. Presleep acquisition and postsleep recall rates were within the expected ranges. However, recall rates in both tasks were almost identical for the RS-86 and placebo conditions. These results indicate that selective M1 mAChR activation in healthy humans has no clinically relevant effect on pre-post sleep consolidation of declarative or procedural memories at a dose that reduces REM sleep latency and SWS duration.

  12. Striatal Input- and Rate-Dependent Effects of Muscarinic Receptors on Pallidal Firing

    PubMed Central

    Querejeta, Enrique; Alatorre, Alberto; Ríos, Alain; Barrientos, Rafael; Oviedo-Chávez, Aldo; Bobadilla-Lugo, Rosa Amalia; Delgado, Alfonso

    2012-01-01

    The globus pallidus (GP) plays a key role in the overall basal ganglia (BG) activity. Despite evidence of cholinergic inputs to GP, their role in the spiking activity of GP neurons has not received attention. We examine the effect of local activation and blockade of muscarinic receptors (MRs) in the spontaneous firing of GP neurons both in normal and ipsilateral striatum-lesioned rats. We found that activation of MRs produces heterogeneous responses in both normal and ipsilateral striatum-lesioned rats: in normal rats the response evoked by MRs depends on the predrug basal firing rate; the inhibition evoked by MRs is higher in normal rats than in striatum-lesioned rats; the number of neurons that undergo inhibition is lower in striatum-lesioned rats than in normal rats. Our data suggest that modulation of MRs in the GP depends on the firing rate before their activation and on the integrity of the striato-pallidal pathway. PMID:22654627

  13. M1 muscarinic receptors are necessary for retrieval of remote context fear memory.

    PubMed

    Patricio, Rafael Rodisanski; Soares, Juliana Carlota Kramer; Oliveira, Maria Gabriela Menezes

    2017-02-01

    Several studies have investigated the transition of consolidation of recent memory to remote memory in aversively motivated tasks, such as contextual fear conditioning (CFC) and inhibitory avoidance (IA). However, the mechanisms that serve the retrieval of remote memories, has not yet been fully understood. Some evidences suggest that the central cholinergic system appears be involved in the modulation of these processes. Therefore, the present study aimed to investigate the effects of a pre-test administration of dicyclomine, a high-affinity M1 muscarinic receptor antagonist, on the retrieval of remote memories in fear conditioning and IA tasks. Male Wistar rats were trained, and after 1 or 28days, the rats received dicyclomine (16 or 32mg/kg, intraperitoneally, i.p.) and were tested in CFC, tone fear conditioning (TFC) and IA tasks. At both time intervals, 32mg/kg dicyclomine induced impairment of CFC. In TFC task only the performance of the rats 28days after training was impaired. The IA task was not affected in any of the studied intervals. These findings suggest a differential contribution of muscarinic receptors on recent and remote memories retrieval revealing a more generalized role in remote memory.

  14. Muscarinic receptors: evidence for a nonuniform distribution in tracheal smooth muscle and exocrine glands

    SciTech Connect

    Basbaum, C.B.; Grillo, M.A.; Widdicombe, J.H.

    1984-02-01

    Muscarinic receptor distribution in smooth muscle, exocrine glands, and epithelium of the ferret trachea was determined using (3H)propylbenzilylcholine mustard ((3H)PrBCM) binding and autoradiography. Specific, atropine-sensitive (3H)PrBCM binding was quantified autoradiographically in the trachealis muscle (approximately 21 binding sites/microns2), surface epithelium (approximately 6 binding sites/microns2), and submucosal glands (approximately 5 binding sites/microns2). Serous and mucous cells in the glands did not differ in receptor density. Binding sites on gland and epithelial cells were associated with basolateral membranes. In the trachealis muscle, a gradient in receptor density was observed, with outer layers of muscle containing 3 to 10 times more receptors per unit area than inner layers. Receptor distribution in both glands and muscle paralleled the distribution of cholinergic axons. However, at the light microscope level, there was no evidence for the presence of receptor ''hot spots'' related to the position of individual axons. The parallelism in the distribution of axons and receptors suggests the possibility of neural control of the genesis and/or maintenance of receptor distribution in these tissues.

  15. Distinct primary structures, ligand-binding properties and tissue-specific expression of four human muscarinic acetylcholine receptors.

    PubMed Central

    Peralta, E G; Ashkenazi, A; Winslow, J W; Smith, D H; Ramachandran, J; Capon, D J

    1987-01-01

    To investigate the molecular basis for the diversity in muscarinic cholinergic function, we have isolated the genes encoding the human M1 and M2 muscarinic receptors (mAChR) as well as two previously undiscovered mAChR subtypes, designated HM3 and HM4. The amino acid sequence of each subtype reflects a structure consisting of seven, highly conserved transmembrane segments and a large intracellular region unique to each subtype, which may constitute the ligand-binding and effector-coupling domains respectively. Significant differences in affinity for muscarinic ligands were detected in individual mAChR subtypes produced by transfection of mammalian cells. Each subtype exhibited multiple affinity states for agonists; differences among subtypes in the affinities and proportions of such sites suggest the capacity of mAChR subtypes to interact differentially with the cellular effector-coupling apparatus. Subtype-specific mRNA expression was observed in the heart, pancreas and a neuronal cell line, indicating that the regulation of mAChR gene expression contributes to the differentiation of cholinergic activity. Images Fig. 3. PMID:3443095

  16. 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.

  17. Is behavioral sensitization to 3,4-methylenedioxymethamphetamine (MDMA) mediated in part by cholinergic receptors?

    PubMed

    Lettfuss, Nadine Y; Seeger-Armbruster, Sonja; von Ameln-Mayerhofer, Andreas

    2013-05-01

    Behavioral sensitization to the repeated administration of a psychostimulant presumably plays a key role in the pathogenesis of addiction and schizophrenia. Among other psychostimulants, 3,4-methylenedioxymethamphetamine (MDMA) is known to produce behavioral sensitization, too, but its mechanism of action is still not fully understood. Along with the strong release of catecholamines and serotonin, MDMA exerts actions at additional transmitter systems, including acetylcholine (ACh). To identify the cholinergic involvement in the development and expression of MDMA-induced sensitization, rats were treated daily with MDMA (5.0 mg/kg), MDMA plus the muscarinic antagonist atropine (4.28 mg/kg), or MDMA plus the nicotinic antagonist mecamylamine (1.0 mg/kg) for 13 consecutive days. The results show that atropine co-treatment was able to block the development of behavioral sensitization to MDMA, measured as horizontal activity and rearing, whereas mecamylamine did not. Pharmacological challenge with MDMA alone increased the locomotion in all substance pretreated groups with the MDMA plus atropine group showing the lowest values. The second challenge with MDMA plus atropine showed a decrease in locomotor behavior in the MDMA- and an increase in the MDMA plus atropine pretreated groups, resulting in similar levels of activity for both groups. A control experiment revealed no change in horizontal activity and rearing when only the cholinergic antagonists (atropine; mecamylamine) were administered. This is the first study that shows a substantial role of muscarinic receptors for the development of behavioral sensitization to MDMA.

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

    PubMed Central

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

    1993-01-01

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

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

    PubMed

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

    2014-06-01

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

  20. Muscarinic receptor size on smooth muscle cells and membranes

    SciTech Connect

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

    1986-08-01

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

  1. 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

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

    SciTech Connect

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

    1987-08-01

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

  3. Muscarinic presynaptic modulation in GABAergic pallidal synapses of the rat.

    PubMed

    Hernández-Martínez, Ricardo; Aceves, José J; Rueda-Orozco, Pavel E; Hernández-Flores, Teresa; Hernández-González, Omar; Tapia, Dagoberto; Galarraga, Elvira; Bargas, José

    2015-02-01

    The external globus pallidus (GPe) is central for basal ganglia processing. It expresses muscarinic cholinergic receptors and receives cholinergic afferents from the pedunculopontine nuclei (PPN) and other regions. The role of these receptors and afferents is unknown. Muscarinic M1-type receptors are expressed by synapses from striatal projection neurons (SPNs). Because axons from SPNs project to the GPe, one hypothesis is that striatopallidal GABAergic terminals may be modulated by M1 receptors. Alternatively, some M1 receptors may be postsynaptic in some pallidal neurons. Evidence of muscarinic modulation in any of these elements would suggest that cholinergic afferents from the PPN, or other sources, could modulate the function of the GPe. In this study, we show this evidence using striatopallidal slice preparations: after field stimulation in the striatum, the cholinergic muscarinic receptor agonist muscarine significantly reduced the amplitude of inhibitory postsynaptic currents (IPSCs) from synapses that exhibited short-term synaptic facilitation. This inhibition was associated with significant increases in paired-pulse facilitation, and quantal content was proportional to IPSC amplitude. These actions were blocked by atropine, pirenzepine, and mamba toxin-7, suggesting that receptors involved were M1. In addition, we found that some pallidal neurons have functional postsynaptic M1 receptors. Moreover, some evoked IPSCs exhibited short-term depression and a different kind of modulation: they were indirectly modulated by muscarine via the activation of presynaptic cannabinoid CB1 receptors. Thus pallidal synapses presenting distinct forms of short-term plasticity were modulated differently. Copyright © 2015 the American Physiological Society.

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed Central

    Hegde, Sharath S

    2006-01-01

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

  6. Pharmacology, Distribution and Development of Muscarinic Acetylcholine Receptor Subtypes in the Optic Tectum of Rana Pipiens

    PubMed Central

    Butt, C. M.; Pauly, J. R.; Wilkins, L. H.; Dwoskin, L. P.; Debski, E. A.

    2008-01-01

    Visually evoked behaviors mediated by the frog optic tectum require cholinergic activity, but the receptor subtypes through which acetylcholine acts are not yet identified. Using quantitative autoradiography and scintillation spectrometry, we examined the binding of [3H]pirenzepine and [3H]AF-DX 384 in the laminated optic tectum of the frog. In mammalian systems, these substances bind excitatory (m1 and m3 subtypes) and inhibitory (m2 and m4 subtypes) muscarinic acetylcholine receptors, respectively. Pharmacological analyses, including the use of specific muscarinic toxins, confirmed the subtype selectivity of the radioligands in the frog brain. Binding sites for [3H]pirenzepine were distinct from those for [3H]AF-DX 384. In the adult tectum, [3H]pirenzepine demonstrated specific binding in tectal layers 5–9. [3H]Pirenzepine binding was also present in tadpoles as young as stage V, but all sampled stages of tadpole tectum had significantly less binding when compared to adults. Lesioning of the optic nerve had no effect on [3H]pirenzepine binding. Specific [3H]AF-DX 384 binding was found in all layers of the adult tectum. All sampled tadpole stages exhibited binding sites for [3H]AF-DX 384, but the densities of these sites were also significantly higher in adults than they were in developing stages. Short-term lesions of the optic nerve reduced [3H]AF-DX 384 binding in all tectal layers of the deafferented lobe when compared to the afferented one. Long-term lesions decreased [3H]AF-DX 384 sites in both lobes. These results indicate that multiple muscarinic acetylcholine receptor binding sites reside in the frog optic tectum at all stages of development, and their pharmacology resembles that of mammalian m1/m3, m2 and m4 subtypes. Our data indicate that few, if any, of these receptors are likely to be located on retinal ganglion cell terminals. Furthermore, the expression of inhibitory muscarinic subtypes seems to be regulated by different mechanisms than that for

  7. Muscarinic cholinergic inhibition of beta-adrenergic stimulation of phospholamban phosphorylation and CaS transport in guinea pig ventricles

    SciTech Connect

    Lindemann, J.P.; Watanabe, A.M.

    1985-10-25

    The effects of muscarinic cholinergic stimulation on beta-adrenergic induced increases in phospholamban phosphorylation and CaS transport were studied in intact myocardium. Isolated guinea pig ventricles were perfused via the coronary arteries with TSPi, after which membrane vesicles were isolated from individual hearts. Isoproterenol produced reversible increases in TSP incorporation into phospholamban. Associated with the increases in TSP incorporation were increases in the initial rate of phosphate-facilitated CaS uptake measured in aliquots of the same membrane vesicles isolated from the perfused hearts. The increases in TSP incorporation and calcium transport were significantly attenuated by the simultaneous administration of acetylcholine. Acetylcholine also attenuated increases in phospholamban phosphorylation and CaS uptake produced by the phosphodiesterase inhibitor isobutylmethylxanthine and forskolin. The contractile effects of all agents which increased cAMP levels (increased contractility and a reduction in the t1/2 of relaxation) were also attenuated by acetylcholine. The inhibitory effects of acetylcholine were associated with attenuation of the increases in cAMP levels produced by isoproterenol and isobutylmethylxanthine but not by forskolin. Acetylcholine also increased the rate of reversal of the functional and biochemical effects of isoproterenol by propranolol without affecting cAMP levels. These results suggest that cholinergic agonists inhibit the functional effects of beta-adrenergic stimulation in part by inhibition of phospholamban phosphorylation. This inhibition may be mediated by two potential mechanisms: inhibition of beta-adrenergic activation of adenylate cyclase and stimulation of dephosphorylation.

  8. Cholinergic treatments with emphasis on m1 muscarinic agonists as potential disease-modifying agents for Alzheimer's disease.

    PubMed

    Fisher, Abraham

    2008-07-01

    The only prescribed drugs for treatment of Alzheimer's disease (AD) are acetylcholinesterase inhibitors (e.g., donepezil, rivastigmine, galantamine, and tacrine) and memantine, an NMDA antagonist. These drugs ameliorate mainly the symptoms of AD, such as cognitive impairments, rather than halting or preventing the causal neuropathology. There is currently no cure for AD and there is no way to stop its progression, yet there are numerous therapeutic approaches directed against various pathological hallmarks of AD that are extensively being pursued. In this context, the three major hallmark characteristics of AD (i.e., the CNS cholinergic hypofunction, formation of beta-amyloid plaques, and tangles containing hyperphosphorylated tau proteins) are apparently linked. Such linkages may have therapeutic implications, and this review is an attempt to analyze these versus the advantages and drawbacks of some cholinergic compounds, such as acetylcholinesterase inhibitors, M1 muscarinic agonists, M2 antagonists, and nicotinic agonists. Among the reviewed treatments, M1 selective agonists emerge, in particular, as potential disease modifiers.

  9. Selectivity of oxomemazine for the M1 muscarinic receptors.

    PubMed

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

    1994-12-01

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

  10. Muscarinic receptor subtype selectivity of novel heterocyclic QNB analogues

    SciTech Connect

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

    1991-01-01

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

  11. Muscarinic cholinergic regulation of cardiac myocyte ICa-L is absent in mice with targeted disruption of endothelial nitric oxide synthase

    PubMed Central

    Han, Xinqiang; Kubota, Isao; Feron, Olivier; Opel, Douglas J.; Arstall, Margaret A.; Zhao, You-Yang; Huang, Paul; Fishman, Mark C.; Michel, Thomas; Kelly, Ralph A.

    1998-01-01

    Cardiac myocytes have been shown to express constitutively endothelial nitric oxide synthase (eNOS) (nitric oxide synthase 3), the activation of which has been implicated in the regulation of myocyte L-type voltage-sensitive calcium channel current (ICa-L) and myocyte contractile responsiveness to parasympathetic nervous system signaling, although this implication remains controversial. Therefore, we examined the effect of the muscarinic cholinergic agonist carbachol (CCh) on ICa-L and contractile amplitude in isoproterenol (ISO)-prestimulated ventricular myocytes isolated from adult mice, designated eNOSnull mice, with targeted disruption of the eNOS gene. Although both eNOSnull and wild-type (WT) ventricular myocytes exhibited similar increases in ICa-L in response to ISO, there was no measurable suppression of ICa-L by CCh in cells from eNOSnull mice, in contrast to cells from WT mice. These results were reflected in the absence of an effect of CCh on the positive inotropic effect of ISO in eNOSnull myocytes. Also, unlike myocytes from WT animals, eNOSnull myocytes failed to exhibit an increase in cGMP content in response to CCh. Nevertheless, the pharmacologic nitric oxide donors 3-morpholino-sydnonimine and S-nitroso-acetyl-cystein increased cGMP generation and suppressed ISO-augmented ICa-L in eNOSnull cells, suggesting that the signal transduction pathway(s) downstream of eNOS remained intact. Of importance, activation of the acetylcholine-activated K+ channel by CCh was unaffected in atrial and ventricular eNOSnull myocytes. These results confirm the obligatory role of eNOS in coupling muscarinic receptor activation to cGMP-dependent control of ICa-L in cardiac myocytes. PMID:9600997

  12. Cholinergic receptor blockade by scopolamine and mecamylamine exacerbates global cerebral ischemia induced memory dysfunction in C57BL/6J mice.

    PubMed

    Ray, R S; Rai, S; Katyal, A

    2014-12-01

    Global cerebral ischemia/reperfusion (GCI/R) injury encompasses complex pathophysiological sequalae, inducing loss of hippocampal neurons and behavioural deficits. Progressive neuronal death and memory dysfunctions culminate from several different mechanisms like oxidative stress, excitotoxicity, neuroinflammation and cholinergic hypofunction. Experimental evidences point to the beneficial effects of cholinomimetic agents such as rivastigmine and galantamine in improving memory outcomes following GCI/R injury. However, the direct implications of muscarinic and nicotinic receptor blockade during global cerebral ischemia/reperfusion injury have not been investigated. Therefore, we evaluated the relative involvement of muscarinic and nicotinic receptors in spatial/associative memory functions and neuronal damage during global cerebral ischemia reperfusion injury. The outcomes of present study support the idea that preservation of both muscarinic and nicotinic receptor functions is essential to alleviate hippocampal neuronal death in CA1 region following global cerebral ischemia/reperfusion injury.

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

    PubMed Central

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

    2014-01-01

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

  14. Role of acetylcholine and muscarinic receptors in serotonin-induced bronchoconstriction in the mouse.

    PubMed

    Kummer, Wolfgang; Wiegand, Silke; Akinci, Sibel; Schinkel, Alfred H; Wess, Jürgen; Koepsell, Hermann; Haberberger, Rainer Viktor; Lips, Katrin Susanne

    2006-01-01

    For the murine trachea, it has been reported that constriction evoked by serotonin (5-HT) is largely dependent on acetylcholine (ACh) released from the epithelium, owing to the sensitivity of the 5-HT response to epithelium removal, sensitivity to atropine, and insensitivity to tetrodotoxin (Moffatt et al., 2003). Consistent with this assumption, the respiratory epithelium contains ACh, its synthesizing enzyme, and the high-affinity choline transporter CHT1 (Reinheimer et al., 1996; Pfeil et al., 2003; Proskocil et al., 2004). Recently, we demonstrated that ACh can be released from non-neuronal cells by corticosteroid-sensitive polyspecific organic cation transporters (OCTs), which are also expressed by airway epithelial cells (Lips et al., 2005). Hence, we proposed that 5-HT evokes release of ACh from epithelial cells via OCTs and that this epithelial-derived ACh induces bronchoconstriction. We tested this hypothesis in a well-established model of videomorphometric analysis of bronchial diameter in precision-cut murine lung slices utilizing epithelium removal to assess the role of the epithelium, OCT mouse knockout (KO) strains to assess the role of OCT isoforms, and muscarinic receptor M2/M3 double-KO mice to assess the cholinergic component of 5-HT induced bronchoconstriction, as bronchi of this strain are entirely unresponsive to cholinergic stimulation(Struckmann et al., 2003).

  15. Impaired off-line consolidation of motor memories after combined blockade of cholinergic receptors during REM sleep-rich sleep.

    PubMed

    Rasch, Björn; Gais, Steffen; Born, Jan

    2009-06-01

    Rapid eye movement (REM) sleep has been considered important for the consolidation of memories, particularly of procedural skills. REM sleep, in contrast to slow-wave sleep (SWS), is hallmarked by the high, wake-like activity of the neurotransmitter acetylcholine (ACh), which promotes certain synaptic plastic processes underlying the formation of memories. Here, we show in healthy young men that off-line consolidation of a motor skill during a period of late sleep with high amounts of REM sleep depends essentially on high cholinergic activity. After a 3-h sleep period during the early night to satisfy the need for SWS, subjects learned a procedural finger sequence tapping task and a declarative word-pair learning task. After learning, they received either placebo or a combination of the muscarinic receptor antagonist scopolamine (4 microg/kg bodyweight, intravenously) and the nicotinic receptor antagonist mecamylamine (5 mg, orally), and then slept for another 3 h, ie, the late nocturnal sleep period, which is dominated by REM sleep. Retrieval was tested the following evening. Combined cholinergic receptor blockade significantly impaired motor skill consolidation, whereas word-pair memory remained unaffected. Additional data show that the impairing effect of cholinergic receptor blockade is specific to sleep-dependent consolidation of motor skill and does not occur during a wake-retention interval. Taken together, these results identify high cholinergic activity during late, REM sleep-rich sleep as an essential factor promoting sleep-dependent consolidation of motor skills.

  16. Phase II drugs that target cholinergic receptors for the treatment of overactive bladder.

    PubMed

    Zacche, Martino Maria; Giarenis, Ilias; Cardozo, Linda

    2014-10-01

    Overactive bladder (OAB) is a term used to describe the symptom syndrome of urgency, with or without urgency incontinence, usually associated with frequency and nocturia. Antimuscarinics are the most widely prescribed class of drugs for OAB, although their systemic adverse effects limit their use in clinical practice as compliance. This has led to developments in the field. In this review, the authors describe Phase II drugs that target cholinergic receptors. First, the authors present the new antimuscarinics (tarafenacin and afacifenacin). This is followed by reports on a combination drug (tolenix) containing a muscarinic antagonist (tolterodine) associated with a muscarinic agonist (pilocarpine). Further, the authors discuss the trials of well-known drugs in either new combination therapy (solifenacin and mirabegron) or with new routes of delivery (oxybutynin vaginal ring). Finally, the authors examine the option of targeting nicotinic acetylcholine receptors (dexmecamylamine). Different strategies have been adopted to improve the efficacy and tolerability of therapeutics for OAB. Nicotinic receptors represent a novel therapeutic target; however, it is unlikely that antimuscarinic agents will be replaced as standard first-line therapy in the near future.

  17. A role for M(2) and M(3) muscarinic receptors in the contraction of rat and human small airways.

    PubMed

    Brown, Stephanie M; Koarai, Akira; Sturton, Richard G; Nicholson, Andrew G; Barnes, Peter J; Donnelly, Louise E

    2013-02-28

    Large airway bronchoconstriction acts mainly through cholinergic pathways via muscarinic M3 receptors with some contribution from M2 receptors. By contrast, the mechanisms of small airway contraction are largely unknown. This study used precision cut lung slices to examine the role of muscarinic M2 and M3 receptors in the contractile response of rat and human small airways. In rat small airways, the M3 preferential antagonist, 4-DAMP, inhibited carbachol-mediated contraction (1×10(-6) M) more than that of the M2 selective antagonist, AF-DX116 (pIC50 values: 8.85±0.18 and 6.31±0.19, n=6-8 respectively). Tiotropium, inhibited the contractile response to carbachol with (pIC50: 9.86±0.07, n=6), but could not distinguish between M2 and M3 mediated effects. Similar experiments using human small airways with tiotropium and AF-DX116, gave a pIC50 of 10.35±0.05 and a pKB of 6.37±0.13, n=5 respectively. Therefore, M3 receptors play a key role in muscarinic contraction of small airways in both rats and humans but the effect of M2 receptors cannot be excluded. To investigate the role of M2 receptors, carbachol-induced contraction of small airways was performed in the presence and absence of a β2-agonist in order to elevate intracellular cAMP levels prior to contraction. Isoproterenol-induced relaxation was significantly increased by AF-DX116 (P<0.001) in rat small airways and by AF-DX116 (P<0.01), gallamine (P<0.05) and pertussis toxin (P<0.05) in human small airways. Taken together, these data suggest that cholinergic antagonism of muscarinic receptors in human and rat small airways inhibits airway contraction via direct inhibition of contraction through M3 receptors, and by M2 receptor mediated inhibition of relaxation. Copyright © 2013 Elsevier B.V. All rights reserved.

  18. Muscarinic receptor modulation of GABA-mediated giant depolarizing potentials in the neonatal rat hippocampus

    PubMed Central

    Avignone, Elena; Cherubini, Enrico

    1999-01-01

    The whole-cell patch clamp technique was used to study the role of muscarinic receptors in regulating the frequency of giant depolarizing potentials (GDPs) in CA3 hippocampal neurones in slices from postnatal (P) P1-P8 rats. Atropine (1 μM) reduced the frequency of GDPs by 64·2 ± 2·9%. The acetylcholinesterase inhibitor edrophonium (20 μM) increased the frequency of GDPs in a developmentally regulated way. This effect was antagonized by the M1 muscarinic receptor antagonist pirenzepine. In the presence of edrophonium, tetanic stimulation of cholinergic fibres induced either an enhancement of GDP frequency (179 ± 79%) or a membrane depolarization (27 ± 16 mV) associated with an increase in synaptic noise. These effects were prevented by atropine. Application of carbachol (3 μM) produced an increase in GDP frequency that at P5-P6 was associated with a membrane depolarization and an increase in synaptic noise. These effects were prevented by atropine, pirenzepine (3 μM) and bicuculline (10 μM). In the presence of pirenzepine, carbachol reduced GDP frequency by 50 ± 4%. Conversely, in the presence of methoctramine (3 μM), carbachol enhanced GDP frequency by 117 ± 4%. It is concluded that endogenous acetylcholine, through the activation of M1 receptors, enhances the release of γ-aminobutyric acid (GABA), in a developmentally regulated way. On the other hand, carbachol exerts both an up- and downregulation of GABA release through the activation of M1 and M2 receptors, respectively. PMID:10373692

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

    SciTech Connect

    Hunter, D.D.

    1986-01-01

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

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

    PubMed Central

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

    1980-01-01

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

  1. The Role of Hippocampal NMDA Receptors in Long-Term Emotional Responses following Muscarinic Receptor Activation.

    PubMed

    Hoeller, Alexandre A; Costa, Ana Paula R; Bicca, Maíra A; Matheus, Filipe C; Lach, Gilliard; Spiga, Francesca; Lightman, Stafford L; Walz, Roger; Collingridge, Graham L; Bortolotto, Zuner A; de Lima, Thereza C M

    2016-01-01

    Extensive evidence indicates the influence of the cholinergic system on emotional processing. Previous findings provided new insights into the underlying mechanisms of long-term anxiety, showing that rats injected with a single systemic dose of pilocarpine--a muscarinic receptor (mAChR) agonist--displayed persistent anxiogenic-like responses when evaluated in different behavioral tests and time-points (24 h up to 3 months later). Herein, we investigated whether the pilocarpine-induced long-term anxiogenesis modulates the HPA axis function and the putative involvement of NMDA receptors (NMDARs) following mAChRs activation. Accordingly, adult male Wistar rats presented anxiogenic-like behavior in the elevated plus-maze (EPM) after 24 h or 1 month of pilocarpine injection (150 mg/kg, i.p.). In these animals, mAChR activation disrupted HPA axis function inducing a long-term increase of corticosterone release associated with a reduced expression of hippocampal GRs, as well as consistently decreased NMDAR subunits expression. Furthermore, in another group of rats injected with memantine--an NMDARs antagonist (4 mg/kg, i.p.)--prior to pilocarpine, we found inhibition of anxiogenic-like behaviors in the EPM but no further alterations in the pilocarpine-induced NMDARs downregulation. Our data provide evidence that behavioral anxiogenesis induced by mAChR activation effectively yields short- and long-term alterations in hippocampal NMDARs expression associated with impairment of hippocampal inhibitory regulation of HPA axis activity. This is a novel mechanism associated with anxiety-like responses in rats, which comprise a putative target to future translational studies.

  2. Characterization and photoaffinity labeling of the muscarinic acetylcholine receptor

    SciTech Connect

    Cremo, C.R.

    1983-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Hedlund, Britta; Abens, Janis; Bartfai, Tamas

    1983-04-01

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

  4. Reduction in choline acetyltransferase immunoreactivity but not muscarinic-m2 receptor immunoreactivity in the brainstem of SIDS infants.

    PubMed

    Mallard, C; Tolcos, M; Leditschke, J; Campbell, P; Rees, S

    1999-03-01

    The cholinergic neurotransmitter system is vital for several brainstem functions including cardiorespiratory control and central chemosensitivity. This study has examined aspects of the cholinergic neurotransmitter system in the brainstem of sudden infant death syndrome (SIDS) and control infants. The cellular localisation and the optical density of the immunoreactivity of the cholinergic enzyme choline acetyltransferase (CHAT-IR) and the muscarinic acetylcholine receptor m2 (m2-IR) in the medulla was described in 14 SIDS and 9 control cases. There was a reduction in the number of CHAT-IR neurons in the hypoglossal nucleus (control: 71.2+/-8.3% vs SIDS: 46.1+/-5.3%) and the dorsal motor nucleus of the vagus (DMV) (control: 77.2+/-5.0% vs SIDS: 52.5+/-7.4%) and reduced optical density of CHAT-IR in the hypoglossal nucleus (control: 0.20+/-0.01 vs SIDS; 0.14+/-0.02) in SIDS infants. In contrast there were no changes in the optical density of m2-IR in the hypoglossal nucleus, the DMV, or the arcuate nucleus. Hypoplasia of the arcuate nucleus was observed in one SIDS infant. These results suggest that there is a specific defect in some cholinergic motor neurons in the medulla of SIDS infants. This could lead to abnormal control of cardiovascular and respiratory function and airway patency and may be one of the contributing factors in the etiology of SIDS.

  5. Decreased binding capacity (Bmax) of muscarinic acetylcholine receptors in fibroblasts from boys with attention-deficit/hyperactivity disorder (ADHD).

    PubMed

    Johansson, Jessica; Landgren, Magnus; Fernell, Elisabeth; Lewander, Tommy; Venizelos, Nikolaos

    2013-09-01

    Monoaminergic dysregulation is implicated in attention-deficit/hyperactivity disorder (ADHD), and methylphenidate and amphetamines are the most frequently prescribed pharmacological agents for treating ADHD. However, it has recently been proposed that the core symptoms of the disorder might be due to an imbalance between monoaminergic and cholinergic systems. In this study, we used fibroblast cell homogenates from boys with and without ADHD as an extraneural cell model to examine the cholinergic receptor density, that is, muscarinic acetylcholine receptors (mAChRs). We found that the binding capacity (Bmax) of [³H] Quinuclidinyl benzilate (³H-QNB) to mAChRs was decreased by almost 50 % in the children with ADHD (mean = 30.6 fmol/mg protein, SD = 25.6) in comparison with controls [mean = 63.1 fmol/mg protein, SD = 20.5, p ≤ 0.01 (Student's unpaired t test)]. The decreased Bmax indicates a reduced cholinergic receptor density, which might constitute a biomarker for ADHD. However, these preliminary findings need to be replicated in larger ADHD and comparison cohorts.

  6. Different muscarinic receptor subtypes modulate proliferation of primary human detrusor smooth muscle cells via Akt/PI3K and map kinases.

    PubMed

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

    2013-08-01

    While acetylcholine (ACh) and muscarinic receptors in the bladder are mainly known for their role in the regulation of smooth muscle contractility, in other tissues they are involved in tissue remodelling and promote cell growth and proliferation. In the present study we have used primary cultures of human detrusor smooth muscle cells (HDSMCs), in order to investigate the role of muscarinic receptors in HDSMC proliferation. Samples were obtained as discarded tissue from men >65 years undergoing radical cystectomy for bladder cancer and cut in pieces that were either immediately frozen or placed in culture medium for the cell culture establishment. HDSMCs were isolated from samples, propagated and maintained in culture. [(3)H]-QNB radioligand binding on biopsies revealed the presence of muscarinic receptors, with a Kd of 0.10±0.02nM and a Bmax of 72.8±0.1fmol/mg protein. The relative expression of muscarinic receptor subtypes, based on Q-RT-PCR, was similar in biopsies and HDSMC with a rank order of M2≥M3>M1>M4>M5. The cholinergic agonist carbachol (CCh, 1-100μM) concentration-dependently increased [(3)H]-thymidine incorporation (up to 46±4%). This was concentration-dependently inhibited by the general muscarinic receptor antagonist atropine and by subtype-preferring antagonists with an order of potency of darifenacin >4-DAMP>AF-DX 116. The CCh-induced cell proliferation was blocked by selective PI-3 kinase and ERK activation inhibitors, strongly suggesting that these intracellular pathways mediate, at least in part, the muscarinic receptor-mediated cell proliferation. This work shows that M2 and M3 receptors can mediate not only HDSM contraction but also proliferation; they may also contribute bladder remodelling including detrusor hypertrophy.

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

    PubMed

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

    2013-02-01

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

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

    PubMed Central

    Szelenyi, I.

    1982-01-01

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

  9. Up-regulation of M1 muscarinic receptors expressed in CHOm1 cells by panaxynol via cAMP pathway.

    PubMed

    Hao, Wang; Xing-Jun, Wu; Yong-Yao, Cui; Liang, Zhu; Yang, Lu; Hong-Zhuan, Chen

    Loss of cholinergic neurons along with muscarinic acetylcholine receptors (mAChRs) in cerebral cortex and hippocampus is closely associated with Alzheimer's disease (AD). Recent drug development for AD treatment focuses heavily on identifying M(1) receptor agonists. However, mAChRs undergo down-regulation in response to agonist-induced sustained activation. Therefore, therapeutic effectiveness wanes during continuous use. Thus, another potentially effective approach, which overcomes this drawback is to develop compounds, which instead up-regulate M(1) receptor expression. In the present study, we took this alternative approach and contrasted in Chinese hamster ovary cells transfected with human m(1) subtype gene (CHOm(1) cells) changes of M(1) receptor expression levels caused by muscarinic agonists and upregulators of its expression. The muscarinic agonists carbachol and pilocarpine reduced M(1) receptor number in CHOm(1) cells by 29 and 46%, respectively, at 100muM, whereas panaxynol, a polyacetylene compound isolated from the lipophilic fraction of Panax notoginseng, concentration-dependently up-regulated the M(1) receptor number after pre-incubation with CHOm(1) cells for 48 h, reaching a plateau at 1 microM, and was accompanied by enhanced M(1) mRNA levels. Moreover, the protein kinase A (PKA) inhibitor RP-adenosine-3',5'-cyclic mono-phosphoro-thioate triethylamine salt (RP-cAMPs) 5 microM completely prevented panaxynol-induced up-regulation of M(1) receptors. Panaxynol (1muM) caused a significant and consistent stimulation of cAMP accumulation (27% increase above basal at 40 min). These results suggest that in CHOm(1) cells panaxynol up-regulates M(1) receptor number through cAMP pathway-mediated stimulation of gene transcription.

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

    Ockenga, Wymke; Tikkanen, Ritva

    2015-05-12

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

  12. Revisiting the Endocytosis of the M2 Muscarinic Acetylcholine Receptor

    PubMed Central

    Ockenga, Wymke; Tikkanen, Ritva

    2015-01-01

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

  13. Autoradiographic visualization of muscarinic receptors in human bronchi

    SciTech Connect

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

    1988-02-01

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

  14. 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.

  15. Comparative distribution of binding of the muscarinic receptor ligands pirenzepine, AF-DX 384, (R,R)-I-QNB and (R,S)-I-QNB to human brain.

    PubMed

    Piggott, Margaret; Owens, Jonathan; O'Brien, John; Paling, Sean; Wyper, David; Fenwick, John; Johnson, Mary; Perry, Robert; Perry, Elaine

    2002-09-01

    Quinuclidinyl benzilate (QNB) and its derivatives are being developed to investigate muscarinic receptor changes in vivo in Alzheimer's disease and dementia with Lewy bodies. This is the first study of [125I]-(R,R)-I-QNB and [125I]-(R,S)-I-QNB binding in vitro in human brain. We have compared the in vitro binding of the muscarinic ligands [3H]pirenzepine and [3H]AF-DX 384, which have selectivity for the M1 and M2/M4 receptor subtypes, respectively, to the binding of [125I]-(R,R)-I-QNB and [125I]-(R,S)-I-QNB. This will provide a guide to the interpretation of in vivo SPET images generated with [123I]-(R,R)-I-QNB and [123I]-(R,S)-I-QNB. Binding was investigated in striatum, globus pallidus, thalamus and cerebellum, and cingulate, insula, temporal and occipital cortical areas, which show different proportions of muscarinic receptor subtypes, in post-mortem brain from normal individuals. M1 receptors are of high density in cortex and striatum and are relatively low in the thalamus and cerebellum, while M4 receptors are mainly expressed in the striatum, and M2 receptors are most evident in the cerebellum and thalamus. [125I]-(R,R)-I-QNB and [125I]-(R,S)-I-QNB density distribution patterns were consistent with binding to both M1 and M4 receptors, with [125I]-(R,R)-I-QNB additionally binding to a non-cholinergic site not displaceable by atropine. This distribution can be exploited by in vivo imaging, developing ligands for both SPET and PET, to reveal muscarinic receptor changes in Alzheimer's disease and dementia with Lewy bodies during the disease process and following cholinergic therapy.

  16. Cholinergic Receptor Substrates of Neuronal Plasticity and Learning

    DTIC Science & Technology

    1992-01-29

    M2, GABAA, L2, P1, 5-HT1 , Mp opoid, delta opiod and neurotensin receptors. Additional assays of nor-epinephrine (NE), serotonin (5-HT) and of...specific to GABAA and opiod receptors, are in preparation. b. OXO Binding in Limbic Thalamus. OXO binding increased significantly during training, in the...battery of behavioral treatments followed by assay for M2 GABAA, and opiod receptors. 5. Cholinergic deafferentation of cingulate cortex induced by

  17. Molecular mechanics calculations on muscarinic agonists

    NASA Astrophysics Data System (ADS)

    Kooijman, Huub; Kanters, Jan A.; Kroon, Jan

    1990-10-01

    Molecular mechanics calculations have been performed on the conformation freedom with respect to the torsion angles OCCN and COCC of acetylcholine, α( R-methylacetylcholine,β( S)-methylacetylcholine, α( R),β( S)-diemthylacetylcholine and muscarine, in order to obtain information about the active conformation and its interaction with the muscarinic cholinergic receptor. Muscarine has a rather flexible ring system, which makes modelling of the receptor site on the active conformation of this particular ligand a difficult problem. A common minimum for these compounds was found at {+ gauche,anti}), which is identified with the active conformation. However, OCCN angles of up to 120° can be accommodated in the receptor site. The reduced cholinergic activity of the α-methyl derivatives is probably caused by unfavourable interactions between the α-methyl group and the receptor site. The apparent contradictory high activity of the 2-acetyloxycyclopropylammonium ion can be explained by the distorted geometry of α substitution.

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

    PubMed

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

    1994-09-01

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

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

    PubMed

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

    2015-01-01

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

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

    SciTech Connect

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

    1990-01-01

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

  1. M2 muscarinic acetylcholine receptors regulate long-term potentiation at hippocampal CA3 pyramidal cell synapses in an input-specific fashion.

    PubMed

    Zheng, Fang; Wess, Jürgen; Alzheimer, Christian

    2012-07-01

    Muscarinic receptors have long been known as crucial players in hippocampus-dependent learning and memory, but our understanding of the cellular underpinnings and the receptor subtypes involved lags well behind. This holds in particular for the hippocampal CA3 region, where the mechanisms of synaptic plasticity depend on the type of afferent input. Williams and Johnston (Williams S, Johnston D. Science 242: 84-87, 1988; Williams S, Johnston D. J Neurophysiol 64: 1089-1097, 1990) demonstrated muscarinic depression of mossy fiber (MF) long-term potentiation (LTP) through a presynaptic site of action and Maeda et al. (Maeda T, Kaneko S, Satoh M. Brain Res 619: 324-330, 1993) proposed a bidirectional modulation of MF LTP by muscarinic receptor subtypes. Since then, this issue, as well as muscarinic regulation of plasticity at associational/commissural (A/C) fiber-CA3 synapses has remained largely neglected, not least because of the lack of highly selective ligands for the different muscarinic receptor subtypes. In the present study, we performed field potential and whole cell recordings from the hippocampal CA3 region of M(2) receptor knockout mice to determine the role of M(2) receptors in short-term and long-term plasticity at A/C and MF inputs to CA3 pyramidal cells. At the A/C synapse, M(2) receptors promoted short-term facilitation and LTP. Unexpectedly, M(2) receptors mediated the opposite effect on LTP at the MF synapse, which was significantly reduced, most likely involving a depressant effect of M(2) receptors on adenylyl cyclase activity in MF terminals. Our data demonstrate that cholinergic projections recruit M(2) receptors to redistribute the gain of LTP in CA3 pyramidal cells in an input-specific manner.

  2. Stress, chemical defense agents, and cholinergic receptors. Midterm report, 1 November 1987-31 July 1989

    SciTech Connect

    Lane, J.D.

    1989-11-30

    This project is assessing the affects of exposure to a chemical defense agent on anxiety and stress, by using rat models of anxiety (conditioned emotional response (CER); conditioned suppression) and unconditioned non-specific stres (exposure to footshock). The specific experiments determined the plasticity of muscarinic cholinergic binding sites in the central nervous system. The neuroanatomical locus and neuropharmacological profile of changes in binding sites were assessed in brain areas enriched in cholinergic markers. Acetylcholine turnover was measured to determine if the receptor response is compensatory or independent. The effects of acute exposure to doses of a chemical defense agent (soman--XGD) on lethality and behaviors were examined. The experiments involved training and conditioning adult rats to CER using standard operant/respondent techniques. The binding of radiolabelled ligand was studied in vitro using brain membranes and tissue sections (autoradiography). The major findings are that CER produces increases in acetylcholine turnover in brain areas involved in anxiety, and that primarily post-synaptic M1 receptors compensatorly decrease in response. These neurochemical phenomena are directly correlated with several behaviors, including onset and extinction of CER and non-specific stress. Followup experiments have been designed to test the interaction of CER, XGD and neurochemistry.

  3. Expression of adrenergic and cholinergic receptors in murine renal intercalated cells.

    PubMed

    Jun, Jin-Gon; Maeda, Seishi; Kuwahara-Otani, Sachi; Tanaka, Koichi; Hayakawa, Tetsu; Seki, Makoto

    2014-11-01

    Neurons influence renal function and help to regulate fluid homeostasis, blood pressure and ion excretion. Intercalated cells (ICCs) are distributed throughout the renal collecting ducts and help regulate acid/base equilibration. Because ICCs are located among principal cells, it has been difficult to determine the effects that efferent nerve fibers have on this cell population. In this study, we examined the expression of neurotransmitter receptors on the murine renal epithelial M-1 cell line. We found that M-1 cells express a2 and b2 adrenergic receptor mRNA and the b2 receptor protein. Further, b2 receptor-positive cells in the murine cortical collecting ducts also express AQP6, indicating that these cells are ICCs. M-1 cells were found to express m1, m4 and m5 muscarinic receptor mRNAs and the m1 receptor protein. Cells in the collecting ducts also express the m1 receptor protein, and some m1-positive cells express AQP6. Acetylcholinesterase was detected in cortical collecting duct cells. Interestingly, acetylcholinesterase-positive cells neighbored AQP6-positive cells, suggesting that principal cells may regulate the availability of acetylcholine. In conclusion, our data suggest that ICCs in murine renal collecting ducts may be regulated by the adrenergic and cholinergic systems.

  4. Expression of Adrenergic and Cholinergic Receptors in Murine Renal Intercalated Cells

    PubMed Central

    JUN, Jin-Gon; MAEDA, Seishi; KUWAHARA-OTANI, Sachi; TANAKA, Koichi; HAYAKAWA, Tetsu; SEKI, Makoto

    2014-01-01

    ABSTRACT Neurons influence renal function and help to regulate fluid homeostasis, blood pressure and ion excretion. Intercalated cells (ICCs) are distributed throughout the renal collecting ducts and help regulate acid/base equilibration. Because ICCs are located among principal cells, it has been difficult to determine the effects that efferent nerve fibers have on this cell population. In this study, we examined the expression of neurotransmitter receptors on the murine renal epithelial M-1 cell line. We found that M-1 cells express a2 and b2 adrenergic receptor mRNA and the b2 receptor protein. Further, b2 receptor-positive cells in the murine cortical collecting ducts also express AQP6, indicating that these cells are ICCs. M-1 cells were found to express m1, m4 and m5 muscarinic receptor mRNAs and the m1 receptor protein. Cells in the collecting ducts also express the m1 receptor protein, and some m1-positive cells express AQP6. Acetylcholinesterase was detected in cortical collecting duct cells. Interestingly, acetylcholinesterase-positive cells neighbored AQP6-positive cells, suggesting that principal cells may regulate the availability of acetylcholine. In conclusion, our data suggest that ICCs in murine renal collecting ducts may be regulated by the adrenergic and cholinergic systems. PMID:25069412

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

    PubMed

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

    2017-01-15

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

  6. Endogenous Inhibition of the Trigeminally Evoked Neurotransmission to Cardiac Vagal Neurons by Muscarinic Acetylcholine Receptors

    PubMed Central

    Gorini, C.; Philbin, K.; Bateman, R.

    2010-01-01

    Stimulation of the nasal mucosa by airborne irritants or water evokes a pronounced bradycardia accompanied by peripheral vasoconstriction and apnea. The dive response, which includes the trigeminocardiac reflex, is among the most powerful autonomic responses. These responses slow the heart rate and reduce myocardial oxygen consumption. Although normally cardioprotective, exaggeration of this reflex can be detrimental and has been implicated in cardiorespiratory diseases, including sudden infant death syndrome (SIDS). An essential component of the diving response and trigeminocardiac reflex is activation of the parasympathetic cardiac vagal neurons (CVNs) in the nucleus ambiguus that control heart rate. This study examined the involvement of cholinergic receptors in trigeminally evoked excitatory postsynaptic currents in CVNs in an in vitro preparation from rats. CVNs were identified using a retrograde tracer injected into the fat pads at the base of the heart. Application of the acetylcholinesterase inhibitor neostigmine significantly decreased the amplitude of glutamatergic neurotransmission to CVNs on stimulation of trigeminal fibers. Whereas nicotine did not have any effect on the glutamatergic responses, the muscarinic acetylcholine receptor (mAChR) agonist bethanechol significantly decreased the excitatory neurotransmission. Atropine, an mAChR antagonist, facilitated these responses indicating this trigeminally evoked brain stem pathway in vitro is endogenously inhibited by mAChRs. Tropicamide, an m4 mAChR antagonist, prevented the inhibitory action of the muscarinic agonist bethanechol. These results indicate that the glutamatergic synaptic neurotransmission in the trigeminally evoked pathway to CVNs is endogenously inhibited in vitro by m4 mAChRs. PMID:20719927

  7. Ethanol inhibits neuritogenesis induced by astrocyte muscarinic receptors.

    PubMed

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

    2010-09-01

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

  8. Activation and dynamic network of the M2 muscarinic receptor

    PubMed Central

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

    2013-01-01

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

  9. beta-Adrenergic and cholinergic receptors in hypertension-induced hypertrophy

    SciTech Connect

    Vatner, D.E.; Kirby, D.A.; Homcy, C.J.; Vatner, S.F.

    1985-05-01

    Perinephritic hypertension was produced in dogs by wrapping one kidney with silk and removing the contralateral kidney 1 week later. Mean arterial pressure rose from 104 +/- 3 to 156 +/- 11 mm Hg, while left ventricular free wall weight, normalized for body weight, was increased by 49%. Muscarinic, cholinergic receptor density measured with (/sup 3/H)-quinuclidinyl benzilate, fell in hypertensive left ventricles (181 +/- 19 fmol/mg, n = 6; p less than 0.01) as compared with that found in normal left ventricles (272 +/- 16 fmol/mg, n = 8), while receptor affinity was not changed. The beta-adrenergic receptor density, measured by binding studies with (/sup 3/H)-dihydroalprenolol, rose in the hypertensive left ventricles (108 +/- 10 fmol/mg, n = 7; p less than 0.01) as compared with that found in normal left ventricles (68.6 +/- 5.2 fmol/mg, n = 15), while beta-adrenergic receptor affinity decreased in the hypertensive left ventricles (10.4 +/- 1.2 nM) compared with that found in the normal left ventricles (5.0 +/- 0.7 nM). Plasma norepinephrine levels were similar in the two groups, but myocardial norepinephrine levels were depressed (p less than 0.05) in dogs with hypertension. Moderate left ventricular hypertrophy induced by long-term aortic banding in dogs resulted in elevations in beta-adrenergic receptor density (115 +/- 14 fmol/mg) and decreases in affinity (10.4 +/- 2.2 nM) similar to those observed in the dogs with left ventricular hypertrophy induced by hypertension. Thus, these results suggest that perinephritic hypertension in the dog induces divergent effects on cholinergic and beta-adrenergic receptor density. The increased beta-adrenergic receptor density and decreased affinity may be a characteristic of left ventricular hypertrophy rather than hypertension.

  10. Expression of the M3 Muscarinic Receptor on Orexin Neurons that Project to the Rostral Ventrolateral Medulla.

    PubMed

    Dai, Yu-Wen E; Lee, Yen-Hsien; Chen, Jennifer Y S; Lin, Yen-Kuang; Hwang, Ling-Ling

    2016-05-01

    Activation of central cholinergic receptors causes a pressor response in rats, and the hypothalamus is important for this response. Projections from hypothalamic orexin neurons to the rostral ventrolateral medulla (RVLM) are involved in sympatho-excitation of the cardiovascular system. A small population of orexin neurons is regulated by cholinergic inputs through M3 muscarinic acetylcholine receptor (M3 R). To elucidate whether the M3 R on orexin neurons is involved in cardiosympathetic regulation through the RVLM, we examined the presence of the M3 R on retrograde-labeled RVLM-projecting orexin neurons. The retrograde tracer was unilaterally injected into the RVLM. Within the hypothalamus, retrograde-labeled neurons were located predominantly ipsilateral to the injection side. In the anterior hypothalamus (-1.5 to -2.3 mm to the bregma), retrograde-labeled neurons were densely distributed in the paraventricular nuclei and scattered in the retrochiasmatic area. At -2.3 to -3.5 mm from the bregma, labeled neurons were located in the regions where orexin neurons were situated, that is, the tuberal lateral hypothalamic area, perifornical area, and dorsomedial nuclei. Very few retrograde-labeled neurons were observed in the hypothalamus at -3.5 to -4.5 mm from the bregma. About 19.5% ± 1.6% of RVLM-projecting neurons in the tuberal hypothalamus were orexinergic. The M3 R was present on 18.7% ± 3.0% of RVLM-projecting orexin neurons. Injection of a muscarinic agonist, oxotremorine, in the perifornical area resulted in a pressor response, which was attenuated by a pretreatment of atropine. We conclude that cholinergic inputs to orexin neurons may be involved in cardiosympathetic regulation through the M3 R on the orexin neurons that directly project to the RVLM.

  11. Activation of muscarinic receptors in porcine airway smooth muscle elicits a transient increase in phospholipase D activity.

    PubMed

    Mamoon, A M; Smith, J; Baker, R C; Farley, J M

    1999-01-01

    Phospholipase D (PLD) is a phosphodiesterase that catalyses hydrolysis of phosphatidylcholine to produce phosphatidic acid and choline. In the presence of ethanol, PLD also catalyses the formation of phosphatidylethanol, which is a unique characteristic of this enzyme. Muscarinic receptor-induced changes in the activity of PLD were investigated in porcine tracheal smooth muscle by measuring the formation of [3H]phosphatidic acid ([3H]PA) and [3H]phosphatidylethanol ([3H]PEth) after labeling the muscle strips with [3H]palmitic acid. The cholinergic receptor agonist acetylcholine (Ach) significantly but transiently increased formation of both [3H]PA and [3H]PEth in a concentration-dependent manner (>105-400% vs. controls in the presence of 10(-6) to 10(-4) M Ach) when pretreated with 100 mM ethanol. The Ach receptor-mediated increase in PLD activity was inhibited by atropine (10(-6) M), indicating that activation of PLD occurred via muscarinic receptors. Activation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate (PMA) increased PLD activity that was effectively blocked by the PKC inhibitors calphostin C (10(-8) to 10(-6) M) and GFX (10(-8) to 10(-6) M). Ach-induced increases in PLD activity were also significantly, but incompletely, inhibited by both GFX and calphostin C. From the present data, we conclude that in tracheal smooth muscle, muscarinic acetylcholine receptor-induced PLD activation is transient in nature and coupled to these receptors via PKC. However, PKC activation is not solely responsible for Ach-induced activation of PLD in porcine tracheal smooth muscle.

  12. Cholinergic Targets in Lung Cancer.

    PubMed

    Spindel, Eliot R

    2016-01-01

    Lung cancers express an autocrine cholinergic loop in which secreted acetylcholine can stimulate tumor growth through both nicotinic and muscarinic receptors. Because activation of mAChR and nAChR stimulates growth; tumor growth can be stimulated by both locally synthesized acetylcholine as well as acetylcholine from distal sources and from nicotine in the high percentage of lung cancer patients who are smokers. The stimulation of lung cancer growth by cholinergic agonists offers many potential new targets for lung cancer therapy. Cholinergic signaling can be targeted at the level of choline transport; acetylcholine synthesis, secretion and degradation; and nicotinic and muscarinic receptors. In addition, the newly describe family of ly-6 allosteric modulators of nicotinic signaling such as lynx1 and lynx2 offers yet another new approach to novel lung cancer therapeutics. Each of these targets has their potential advantages and disadvantages for the development of new lung cancer therapies which are discussed in this review.

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

    SciTech Connect

    Klangkalya, B.; Chan, A.

    1988-01-01

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

  14. Stress, Chemical Defense Agents and Cholinergic Receptors

    DTIC Science & Technology

    1989-11-30

    permitted to avoid a comparable 1 -mA scrambled footshock in the chamber by reaching the safe platform within 10 sec of being placed in the apparatus. For...rate constant h was compared for the incorporation into and decline in specl.ic 3ctlvities of choline and ACh (see Smith et al., 1984a; tac’gni et al...to detect cholinergic function was assessed (Table 2). When compared with controls (no CS presentation), rats which had been exposed to the CS

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    1992-06-09

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

  17. 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

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

    PubMed

    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.

  19. Lipid rafts of mouse liver contain nonextended and extended acetylcholinesterase variants along with M3 muscarinic receptors.

    PubMed

    Montenegro, María Fernanda; Cabezas-Herrera, Juan; Campoy, F Javier; Muñoz-Delgado, Encarnación; Vidal, Cecilio J

    2017-02-01

    The observation of acetylcholinesterase (AChE) type H (AChEH), which is the predominant AChE variant in visceral organs and immune cells, in lipid rafts of muscle supports functional reasons for the raft targeting of glypiated AChEH The search for these reasons revealed that liver AChE activity is mostly confined to rafts and that the liver is able to make N-extended AChE variants and target them to rafts. These results prompted us to test whether AChE and muscarinic receptors existed in the same raft. Isolation of flotillin-2-rich raft fractions by their buoyancy in sucrose gradients, followed by immunoadsorption and matrix-assisted laser desorption ionization-time of flight-mass spectrometry application, gave the following results: 1) most hepatic AChE activity emanates from AChE-H mRNA, and its product, glypiated AChEH, accumulates in rafts; 2) N-extended N-AChE readthrough variant, nonglypiated N-AChEH, and N-AChE tailed variant were all identified in liver rafts; and 3) M3 AChRs were observed in rafts, and coprecipitation of raft-confined N-AChE and M3 receptors by using anti-M3 antibodies showed that enzyme and receptor reside in the same raft unit. A raft domain that harbors tightly packed muscarinic receptor and AChE may represent a molecular device that, by means of which, the intensity and duration of cholinergic inputs are regulated.-Montenegro, M. F., Cabezas-Herrera, J., Campoy, F. J., Muñoz-Delgado, E., Vidal, C. J. Lipid rafts of mouse liver contain nonextended and extended acetylcholinesterase variants along with M3 muscarinic receptors.

  20. Cholinergic modulation of the medial prefrontal cortex: the role of nicotinic receptors in attention and regulation of neuronal activity

    PubMed Central

    Bloem, Bernard; Poorthuis, Rogier B.; Mansvelder, Huibert D.

    2014-01-01

    Acetylcholine (ACh) release in the medial prefrontal cortex (mPFC) is crucial for normal cognitive performance. Despite the fact that many have studied how ACh affects neuronal processing in the mPFC and thereby influences attention behavior, there is still a lot unknown about how this occurs. Here we will review the evidence that cholinergic modulation of the mPFC plays a role in attention and we will summarize the current knowledge about the role between ACh receptors (AChRs) and behavior and how ACh receptor activation changes processing in the cortical microcircuitry. Recent evidence implicates fast phasic release of ACh in cue detection and attention. This review will focus mainly on the fast ionotropic nicotinic receptors and less on the metabotropic muscarinic receptors. Finally, we will review limitations of the existing studies and address how innovative technologies might push the field forward in order to gain understanding into the relation between ACh, neuronal activity and behavior. PMID:24653678

  1. Role of muscarinic receptors in the contraction of jejunal smooth muscle in the horse: An in vitro study.

    PubMed

    Menozzi, Alessandro; Pozzoli, Cristina; Poli, Enzo; Bontempi, Giada; Serventi, Paolo; Meucci, Valentina; Intorre, Luigi; Bertini, Simone

    2017-07-11

    Nonselective antimuscarinic drugs are clinically useful in several pathologic conditions of horses, but, blocking all muscarinic receptor (MR) subtypes, may cause several side effects. The availability of selective antimuscarinic drugs could improve therapeutic efficacy and safety. We aimed to enlighten the role of different MR subtypes by evaluating the effects of nonselective, and selective M1, M2 and M3 MR antagonists on the contractions of horse jejunum. Segments of circular muscle of equine jejunum, were put into organ baths, connected to isotonic transducers, and the effects on ACh concentration-response curves, and on electrical field stimulation (EFS)-evoked contractions of intestinal preparations, induced by nonselective or selective MR antagonists, compared to pre-drug level, were studied. Atropine (nonselective MR antagonist), pirenzepine (selective M1 antagonist), and p-FHHSiD (selective M3 antagonist) competitively antagonized ACh (pA2=9.78±0.21; 7.14±0.25 and 7.56±0.17, respectively). Methoctramine (selective M2 antagonist) antagonized ACh in a concentration-unrelated fashion; however, it competitively antagonized carbachol, a nonselective muscarinic agonist (pA2=6.42±0.23). Atropine dose-dependently reduced EFS-evoked contractions, reaching a maximal effect of -45.64±6.54%; the simultaneous block of neurokinin receptors, almost completely abolished the atropine-insensitive contractions. p-FHHSiD dose-dependently reduced EFS-induced contractions, while pirenzepine caused a minor decrease. Methoctramine, ineffective up to 10(-7)M, enhanced the contractions at 10(-6)M; the block of neurokinin receptors abolished the increase of contraction. Cholinergic contractions of horse jejunum are mainly mediated by M3 receptors; M2 selective antagonists seem to scarcely affect cholinergic, and to enhance neurokininergic contractions of equine jejunum, thus their use entails a lower risk of causing intestinal hypomotility, compared to nonselective drugs

  2. 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

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

    NASA Technical Reports Server (NTRS)

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

    1990-01-01

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

  4. Cellular localization of cerebellar muscarinic receptors: an autoradiographic analysis of weaver, reeler, Purkinje cell degeneration and staggerer mice

    SciTech Connect

    Neustadt, A.; Frostholm, A.; Rotter, A.

    1988-02-01

    Light microscopic autoradiography of (/sup 3/H)quinuclidinyl benzilate binding sites was used to study the distribution of muscarinic cholinergic receptors in mouse mutants which have abnormalities affecting specific cerebellar cell types. In the normal C57BL/6J mouse, binding sites were distributed throughout the cerebellar cortex, with the highest levels in the granule cell layer and deep cerebellar nuclei. Normal binding site density was observed in the cerebellum of the weaver mutant in which the majority of granule cells had degenerated. The density of (/sup 3/H)quinuclidinyl benzilate binding sites was elevated in the cortex of the reeler, despite a reduction in the number of granule cells. The concentration of binding sites was also high over the Purkinje cell masses where granule cells were largely absent. No significant reduction in cortical (/sup 3/H)quinuclidinyl benzilate binding site density was detected in the Purkinje cell degeneration mutant, in which essentially all Purkinje cells had degenerated. In contrast, receptor binding in the deep cerebellar nuclei of this mutant was significantly increased. A substantial increase in labeling was observed in the cortex and deep nuclei of the staggerer cerebellum in which a large fraction of Golgi II cells, Purkinje cells, granule cells and mossy fibers have degenerated. We discuss the possibility that the persistence of (/sup 3/H)quinuclidinyl benzilate binding sites in all four mutants may imply a non-neuronal localization for a large proportion of muscarinic receptors in the mouse cerebellar cortex.

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

    PubMed Central

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

    2011-01-01

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

  6. 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

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

    PubMed Central

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

    2015-01-01

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

  8. Central muscarinic cholinergic involvement in serial pattern learning: Atropine impairs acquisition and retention in a serial multiple choice (SMC) task in rats.

    PubMed

    Chenoweth, Amber M; Fountain, Stephen B

    2015-09-01

    Atropine sulfate is a muscarinic cholinergic antagonist which impairs acquisition and retention performance on a variety of cognitive tasks. The present study examined the effects of atropine on acquisition and retention of a highly-structured serial pattern in a serial multiple choice (SMC) task. Rats were given daily intraperitoneal injections of either saline or atropine sulfate (50mg/kg) and trained in an octagonal operant chamber equipped with a lever on each wall. They learned to press the levers in a particular order (the serial pattern) for brain-stimulation reward in a discrete-trial procedure with correction. The two groups learned a pattern composed of eight 3-element chunks ending with a violation element: 123-234-345-456-567-678-781-818 where the digits represent the clock-wise positions of levers in the chamber, dashes indicate 3-s pauses, and other intertrial intervals were 1s. Central muscarinic cholinergic blockade by atropine caused profound impairments during acquisition, specifically in the encoding of chunk-boundary elements (the first element of chunks) and the violation element of the pattern, but had a significant but negligible effect on the encoding of within-chunk elements relative to saline-injected rats. These effects persisted when atropine was removed, and similar impairments were also observed in retention performance. The results indicate that intact central muscarinic cholinergic systems are necessary for learning and producing appropriate responses at places in sequences where pattern structure changes. The results also provide further evidence that multiple cognitive systems are recruited to learn and perform within-chunk, chunk-boundary, and violation elements of a serial pattern.

  9. Structure and dynamics of the M3 muscarinic acetylcholine receptor

    SciTech Connect

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

    2012-03-01

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

  10. Relationship between action potential sodium channels and muscarinic receptors in mouse brain

    SciTech Connect

    Mack, J.E.

    1986-01-01

    Cholinergic agonists and antagonists were tested for their ability to influence stimulated and unstimulated /sup 22/Na uptake in preparations of forebrain and hindbrain in mice in vitro. In mouse forebrain, atropine and pirenzepine decreased stimulated sodium uptake. Dicyclomine decreased stimulated uptake in both the forebrain and hindbrain. McN-A-343 decreased stimulated sodium uptake in the forebrain. The effects of sodium channel ligands on muscarinic receptors was investigated in forebrain and hindbrain preparations. In the forebrain, veratridine and aconitine appeared to inhibit the binding of (/sup 3/H)QNB in a competitive manner. Tetrodotoxin alone had not effect on binding, but enhanced the inhibition by veratridine, with no effect on aconitine inhibition. In the hindbrain, veratridine appeared to inhibit (/sup 3/H)QNB binding non-competitively and competitively. The addition of magnesium increased the K/sub i/ value in the veratridine inhibition. GTP enhanced the inhibition by veratridine. Tetrodotoxin increased the K/sub i/ value of the veratridine inhibition curve. Tetrodotoxin alone also inhibited (/sup 3/H)QNB binding. Tetrodotoxin inhibited QNB binding in both a non-competitive and uncompetitive manner.

  11. 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

  12. Pilocarpine protects cobalt chloride-induced apoptosis of RGC-5 cells: involvement of muscarinic receptors and HIF-1 alpha pathway.

    PubMed

    Zhu, Xu; Zhou, Wei; Cui, Yongyao; Zhu, Liang; Li, Juan; Feng, Xuemei; Shao, Biyun; Qi, Hong; Zheng, Jun; Wang, Hao; Chen, Hongzhuan

    2010-04-01

    The retina is the most metabolically active tissue in the human body and hypoxia-induced retinal ganglion cell (RGC) death has been implicated in glaucomatous optic neuropathy. The aim of this study is to determine whether muscarinic receptor agonist pilocarpine, a classic antiglaucoma drug, possesses neuroprotection against cobalt chloride (CoCl(2))-mimetic hypoxia-induced apoptosis of rat retinal ganglion cells (RGC-5 cells) and its underlying mechanisms. Cell viability was determined by Cell Counting Kit-8 assay and apoptosis was examined by annexin V and mitochondrial membrane potential (MMP) assays. Expressions of hypoxia-induced factor-1 alpha (HIF-1 alpha), p53, and BNIP3 were investigated by quantitative real-time PCR and western blot analysis. After treatment of 200 microM CoCl(2) for 24 h, RGC-5 cells showed a marked decrease of cell viability by approximately 30%, increased apoptosis rate and obvious decline in MMP, which could largely be reversed by the pretreatment of 1 microM pilocarpine mainly via the activation of muscarinic receptors. Meanwhile, pretreatment of 1 microM pilocarpine could significantly prevent CoCl(2)-induced HIF-1 alpha translocation from cytoplasm to nucleus and down-regulate the expression of HIF-1 alpha, p53, and BNIP3. These studies demonstrated that pilocarpine had effective protection against hypoxia-induced apoptosis in RGCs via muscarinic receptors and HIF-1 alpha pathway. The findings suggest that HIF-1 alpha pathway as a "master switch" may be used as a therapeutic target in the cholinergic treatment of glaucoma.

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

    DTIC Science & Technology

    1990-12-14

    Because of its nonsjmaptic nature , the latency to peak of the antidromic spike was usually less than 2 msec. Analysis of changes in the amplitude...population spike produced by both D(-)APV and CPP, so we performed a series of experiments designed to elucidate the nature of this depression. Fig 8 shows... nature of the voltage-dependent Mg^ block of the NMDA receptor at resting membrane potential, the question arose as to 55 Fig 8. NMDA receptor

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

    PubMed Central

    Moulton, Bart C; Fryer, Allison D

    2011-01-01

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

  15. 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

  16. Muscarinic receptor plasticity in the brain of senescent rats: down-regulation after repeated administration of diisopropyl fluorophosphate

    SciTech Connect

    Pintor, A.; Fortuna, S.; Volpe, M.T.; Michalek, H.

    1988-01-01

    Potential age-related differences in the response of Fischer 344 rats to subchronic treatment with diisopropylfluorophosphate (DFP) were evaluated in terms of brain cholinesterase (ChE) inhibition and muscarinic receptor sites. Male 3- and 24-month old rats were sc injected with sublethal doses of DFP for 2 weeks and killed 48 hrs after the last treatment. In the cerebral cortex, hippocampus and striatum of control rats a significant age-related reduction of ChE and of maximum number of /sup 3/H-QNB binding sites (Bmax) was observed. The administration of DFP to senescent rats resulted in more pronounced and longer lasting syndrome of cholinergic stimulation, with marked body weight loss and 60% mortality. The percentage inhibition of brain ChE induced by DFP did not differ between young and senescent rats. As expected, in young rats DFP caused a significant decrease of Bmax, which in the cerebral cortex reached about 40%. In the surviving senescent rats, the percentage decrease of Bmax due to DFP with respect to age-matched controls was very similar to that of young animals, especially in the cerebral cortex. Thus there is great variability in the response of aged rats to DFP treatment, from total failure of adaptive mechanisms resulting in death to considerable muscarinic receptor plasticity.

  17. Depression Case Control (DeCC) Study fails to support involvement of the muscarinic acetylcholine receptor M2 (CHRM2) gene in recurrent major depressive disorder.

    PubMed

    Cohen-Woods, Sarah; Gaysina, Daria; Craddock, Nick; Farmer, Anne; Gray, Joanna; Gunasinghe, Cerisse; Hoda, Farzana; Jones, Lisa; Knight, Jo; Korszun, Ania; Owen, Michael J; Sterne, Abram; Craig, Ian W; McGuffin, Peter

    2009-04-15

    It has been suggested that alteration in the muscarinic-cholinergic system is involved in modulation of mood. Three studies have reported linkage on chromosome 7 with major depressive disorder (MDD) in or close to a region containing the muscarinic receptor CHRM2 gene. A haplotype of SNPs located in CHRM2 (rs1824024-rs2061174-rs324650) has been significantly associated with MDD in a previous study. We report the first study investigating this gene in a large, adequately powered, clinical depression case-control sample (n = 1420 cases, 1624 controls). Our data fail to support association with the CHRM2 polymorphisms previously implicated in the genetic aetiology of depression. It is possible our failure to replicate may be a consequence of differences in definition of the MDD phenotype and/or ethnic differences.

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

    SciTech Connect

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

    1987-05-01

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

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

    PubMed

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

    2014-05-27

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

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

    PubMed Central

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

    2014-01-01

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

  1. [F-18]-(-,-)-FQNPe - an attractive ligand for evaluation of muscarinic-cholinergic neuron activity by PET

    SciTech Connect

    Luo, H.; McPherson, D.W.; Beets, A.L.; Knapp, F.F. Jr.

    1997-05-01

    The stereoisomers of 1-azabicyclo[2.2.2]oct-3-yl {alpha}-{alpha}-(1-fluoropentan-5-yl)-{alpha}-hydroxy-{alpha}-phenylacetate ({open_quotes}FQNPe{close_quotes}) have been resolved. (-,-)- receptors (K{sub i}, nM; ml, 0.3; m2, 0.1). [F-18]-(-,-)-FQNPe demonstrated high cerebral and myocardial uptake in rats in vivo. We now report significant blocking of [F-18]-(-.-)-FQNPe uptake in receptor-rich tissues in rats in vivo after (R)-QNB pretreatment and the absence of any TLC detectable FQNPe metabolites in tissue extracts. Rats were injected with (R)-QNB (3 mg/kg) 1 h prior to [F-18]-FQNPe injection (370-629 KBq). After 1 h, rats were sacrificed and tissues removed and counted. (R)-QNB significantly decreased FQNPe uptake in heart and all receptor-rich regions but not blood (Table; Mean % ID/g, n=5); C, control; Q, (R)-QNB; Hrt, heart; Cer, cerebellum; Pon, pons; Med, medulla; Cor, cortex; Stri, striatum; Hip, hippocampus; Th, thallamus; SuC, superior colliculi; InC, inferior colliculi. Tissues from untreated rats were Folch-extracted and 71-77% of activity was in organic extracts from brain and heart. TLC of organic extracts indicated a single radioactive component with R{sub f} of FQNPe. These combined results demonstrate that [F-18]-(-,-)-FQNPe does not appear to be metabolized in heart and brain, shows good receptor localization and is thus an attractive ligand for evaluation as a potential imaging agent by PET.

  2. Characterization of the muscarinic cholinoceptors in the human detrusor

    SciTech Connect

    Nilvebrant, L.; Andersson, K.E.; Mattiasson, A.

    1985-08-01

    Contractions of the human detrusor are thought to be mediated mainly via cholinergic muscarinic receptors. In the present study, the authors used a receptor-binding technique with 1-quinuclidinyl(phenyl 4-/sup 3/H)benzilate ((-)/sup 3/H-QNB) as radioligand to directly demonstrate the presence of muscarinic receptors in homogenates of the human detrusor. The binding of (-)/sup 3/H-QNB was of high affinity (KD = (1.2 +/- 0.1) X 10(-10) M), saturable (Ro = 160 +/- 15 fmol./mg. protein) and possessed the pharmacological specificity expected of an interaction with muscarinic receptors. Muscarinic receptor antagonists were bound to a virtually uniform population of sites, whereas muscarinic receptor agonists recognized more than one population of muscarinic binding sites. The affinities of a series of antimuscarinic drugs, determined in competition experiments with (-)/sup 3/H-QNB, were found to correlate with the capacity to inhibit carbachol-induced contractions in isolated human bladder muscle. Binding data together with the functional data indicated that the human detrusor does not contain any significant number of muscarinic spare receptors. The results suggest that a selective effect on the muscarinic receptors of human bladder is not possible to obtain with presently available antimuscarinic agents.

  3. Endocannabinoids Mediate Muscarinic Acetylcholine Receptor-Dependent Long-Term Depression in the Adult Medial Prefrontal Cortex.

    PubMed

    Martin, Henry G S; Bernabeu, Axel; Lassalle, Olivier; Bouille, Clément; Beurrier, Corinne; Pelissier-Alicot, Anne-Laure; Manzoni, Olivier J

    2015-01-01

    Cholinergic inputs into the prefrontal cortex (PFC) are associated with attention and cognition; however there is evidence that acetylcholine also has a role in PFC dependent learning and memory. Muscarinic acetylcholine receptors (mAChR) in the PFC can induce synaptic plasticity, but the underlying mechanisms remain either opaque or unresolved. We have characterized a form of mAChR mediated long-term depression (LTD) at glutamatergic synapses of layer 5 principal neurons in the adult medial PFC. This mAChR LTD is induced with the mAChR agonist carbachol and inhibited by selective M1 mAChR antagonists. In contrast to other cortical regions, we find that this M1 mAChR mediated LTD is coupled to endogenous cannabinoid (eCB) signaling. Inhibition of the principal eCB CB1 receptor blocked carbachol induced LTD in both rats and mice. Furthermore, when challenged with a sub-threshold carbachol application, LTD was induced in slices pretreated with the monoacylglycerol lipase (MAGL) inhibitor JZL184, suggesting that the eCB 2-arachidonylglyerol (2-AG) mediates M1 mAChR LTD. Yet, when endogenous acetylcholine was released from local cholinergic afferents in the PFC using optogenetics, it failed to trigger eCB-LTD. However coupling patterned optical and electrical stimulation to generate local synaptic signaling allowed the reliable induction of LTD. The light-electrical pairing induced LTD was M1 mAChR and CB1 receptor mediated. This shows for the first time that connecting excitatory synaptic activity with coincident endogenously released acetylcholine controls synaptic gain via eCB signaling. Together these results shed new light on the mechanisms of synaptic plasticity in the adult PFC and expand on the actions of endogenous cholinergic signaling.

  4. Endocannabinoids Mediate Muscarinic Acetylcholine Receptor-Dependent Long-Term Depression in the Adult Medial Prefrontal Cortex

    PubMed Central

    Martin, Henry G. S.; Bernabeu, Axel; Lassalle, Olivier; Bouille, Clément; Beurrier, Corinne; Pelissier-Alicot, Anne-Laure; Manzoni, Olivier J.

    2015-01-01

    Cholinergic inputs into the prefrontal cortex (PFC) are associated with attention and cognition; however there is evidence that acetylcholine also has a role in PFC dependent learning and memory. Muscarinic acetylcholine receptors (mAChR) in the PFC can induce synaptic plasticity, but the underlying mechanisms remain either opaque or unresolved. We have characterized a form of mAChR mediated long-term depression (LTD) at glutamatergic synapses of layer 5 principal neurons in the adult medial PFC. This mAChR LTD is induced with the mAChR agonist carbachol and inhibited by selective M1 mAChR antagonists. In contrast to other cortical regions, we find that this M1 mAChR mediated LTD is coupled to endogenous cannabinoid (eCB) signaling. Inhibition of the principal eCB CB1 receptor blocked carbachol induced LTD in both rats and mice. Furthermore, when challenged with a sub-threshold carbachol application, LTD was induced in slices pretreated with the monoacylglycerol lipase (MAGL) inhibitor JZL184, suggesting that the eCB 2-arachidonylglyerol (2-AG) mediates M1 mAChR LTD. Yet, when endogenous acetylcholine was released from local cholinergic afferents in the PFC using optogenetics, it failed to trigger eCB-LTD. However coupling patterned optical and electrical stimulation to generate local synaptic signaling allowed the reliable induction of LTD. The light—electrical pairing induced LTD was M1 mAChR and CB1 receptor mediated. This shows for the first time that connecting excitatory synaptic activity with coincident endogenously released acetylcholine controls synaptic gain via eCB signaling. Together these results shed new light on the mechanisms of synaptic plasticity in the adult PFC and expand on the actions of endogenous cholinergic signaling. PMID:26648844

  5. 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

  6. Cholinergic regulation of fear learning and extinction.

    PubMed

    Wilson, Marlene A; Fadel, Jim R

    2017-03-01

    Cholinergic activation regulates cognitive function, particularly long-term memory consolidation. This Review presents an overview of the anatomical, neurochemical, and pharmacological evidence supporting the cholinergic regulation of Pavlovian contextual and cue-conditioned fear learning and extinction. Basal forebrain cholinergic neurons provide inputs to neocortical regions and subcortical limbic structures such as the hippocampus and amygdala. Pharmacological manipulations of muscarinic and nicotinic receptors support the role of cholinergic processes in the amygdala, hippocampus, and prefrontal cortex in modulating the learning and extinction of contexts or cues associated with threat. Additional evidence from lesion studies and analysis of in vivo acetylcholine release with microdialysis similarly support a critical role of cholinergic neurotransmission in corticoamygdalar or corticohippocampal circuits during acquisition of fear extinction. Although a few studies have suggested a complex role of cholinergic neurotransmission in the cellular plasticity essential for extinction learning, more work is required to elucidate the exact cholinergic mechanisms and physiological role of muscarinic and nicotinic receptors in these fear circuits. Such studies are important for elucidating the role of cholinergic neurotransmission in disorders such as posttraumatic stress disorder that involve deficits in extinction learning as well as for developing novel therapeutic approaches for such disorders. © 2016 Wiley Periodicals, Inc.

  7. Cholinergic Partition Cells and Lamina X Neurons Induce a Muscarinic-Dependent Short-Term Potentiation of Commissural Glutamatergic Inputs in Lumbar Motoneurons

    PubMed Central

    Bertrand, Sandrine S.; Cazalets, Jean-René

    2011-01-01

    Acetylcholine and the activation of muscarinic receptors influence the activity of neural networks generating locomotor behavior in the mammalian spinal cord. Using electrical stimulations of the ventral commissure, we show that commissural muscarinic (CM) depolarizations could be induced in lumbar motoneurons. We provide a detailed electrophysiological characterization of the muscarinic receptors and the membrane conductance involved in these responses. Activation of the CM terminals, originating from lamina X neurons and partition cells, induced a pathway-specific short-term potentiation (STP) of commissural glutamatergic inputs in motoneurons. This STP is occluded in the presence of the muscarinic antagonist atropine. During fictive locomotion, the activation of the commissural pathways transiently enhanced the motor output in a muscarinic-dependent manner. This study describes for the first time a novel regulatory mechanism of synaptic strength in spinal locomotor networks. Such cellular mechanisms would endow the locomotor central pattern generators with adaptive processes needed to generate appropriate synaptic inputs to motoneurons during different motor tasks. PMID:22069380

  8. Dose-dependent effect of donepezil administration on long-term enhancement of visually evoked potentials and cholinergic receptor overexpression in rat visual cortex.

    PubMed

    Chamoun, Mira; Groleau, Marianne; Bhat, Menakshi; Vaucher, Elvire

    2016-09-01

    Stimulation of the cholinergic system tightly coupled with periods of visual stimulation boosts the processing of specific visual stimuli via muscarinic and nicotinic receptors in terms of intensity, priority and long-term effect. However, it is not known whether more diffuse pharmacological stimulation with donepezil, a cholinesterase inhibitor, is an efficient tool for enhancing visual processing and perception. The goal of the present study was to potentiate cholinergic transmission with donepezil treatment (0.5 and 1mg/kg) during a 2-week visual training to examine the effect on visually evoked potentials and to profile the expression of cholinergic receptor subtypes. The visual training was performed daily, 10min a day, for 2weeks. One week after the last training session, visual evoked potentials were recorded, or the mRNA expression level of muscarinic (M1-5) and nicotinic (α/β) receptors subunits was determined by quantitative RT-PCR. The visual stimulation coupled with any of the two doses of donepezil produced significant amplitude enhancement of cortical evoked potentials compared to pre-training values. The enhancement induced by the 1mg/kg dose of donepezil was spread to neighboring spatial frequencies, suggesting a better sensitivity near the visual detection threshold. The M3, M4, M5 and α7 receptors mRNA were upregulated in the visual cortex for the higher dose of donepezil but not the lower one, and the receptors expression was stable in the somatosensory (non-visual control) cortex. Therefore, higher levels of acetylcholine within the cortex sustain the increased intensity of the cortical response and trigger the upregulation of cholinergic receptors.

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

    PubMed Central

    2009-01-01

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

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

    SciTech Connect

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

    1988-04-01

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

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

    PubMed

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

    2008-12-03

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

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

    SciTech Connect

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

    1990-06-01

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

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

    PubMed

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

    2016-07-05

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

  14. Inhibitory role of cholinergic system mediated via alpha7 nicotinic acetylcholine receptor in LPS-induced neuro-inflammation.

    PubMed

    Tyagi, Ethika; Agrawal, Rahul; Nath, Chandishwar; Shukla, Rakesh

    2010-02-01

    This study investigated the influence of the cholinergic system on neuro-inflammation using nicotinic and muscarinic receptor agonists and antagonists. Intracerebroventricular (ICV) injection of lipopolysaccharide (LPS, 50 microg) was used to induce neuro-inflammation in rats and estimations of pro-inflammatory cytokines, alpha7 nicotinic acetylcholine receptor (nAChR) mRNA expression were done in striatum, cerebral cortex, hippocampus and hypothalamus at 24 h after LPS injection. Nicotine (0.2, 0.4 and 0.8 mg/kg, i.p.) or oxotremorine (0.2, 0.4 and 0.8 mg/kg, i.p.) were administered 2 h prior to sacrifice. We found that only nicotine was able to block the proinflammatory cytokines induced by LPS whereas, oxotremorine was found ineffective. Methyllycaconitine (MLA; 1.25, 2.5 and 5 mg/kg, i.p.), an alpha7 nAChR antagonist or dihydro-beta-erythroidine (DHbetaE; 1.25, 2.5 and 5 mg/kg, i.p.), an alpha4beta2 nAChR antagonist, was given 20 min prior to nicotine in LPS-treated rats. Methyllycaconitine antagonized the anti-inflammatory effect of nicotine whereas DHbetaE showed no effect demonstrating that alpha7 nAChR is responsible for attenuation of LPS-induced pro-inflammatory cytokines. This study suggests that the inhibitory role of the central cholinergic system on neuro-inflammation is mediated via alpha7 nicotinic acetylcholine receptor and muscarinic receptors are not involved.

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

    PubMed

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

    2013-02-01

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

  16. Muscarinic agonists and antagonists: effects on the urinary bladder.

    PubMed

    Sellers, Donna J; Chess-Williams, Russ

    2012-01-01

    Voiding of the bladder is the result of a parasympathetic muscarinic receptor activation of the detrusor smooth muscle. However, the maintenance of continence and a normal bladder micturition cycle involves a complex interaction of cholinergic, adrenergic, nitrergic and peptidergic systems that is currently little understood. The cholinergic component of bladder control involves two systems, acetylcholine (ACh) released from parasympathetic nerves and ACh from non-neuronal cells within the urothelium. The actions of ACh on the bladder depend on the presence of muscarinic receptors that are located on the detrusor smooth muscle, where they cause direct (M₃) and indirect (M₂) contraction; pre-junctional nerve terminals where they increase (M₁) or decrease (M₄) the release of ACh and noradrenaline (NA); sensory nerves where they influence afferent nerve activity; umbrella cells in the urothelium where they stimulate the release of ATP and NO; suburothelial interstitial cells with unknown function; and finally, other unidentified sites in the urothelium from where prostaglandins and inhibitory/relaxatory factors are released. Thus, the actions of muscarinic receptor agonists and antagonists on the bladder may be very complex even when considering only local muscarinic actions. Clinically, muscarinic antagonists remain the mainstay of treatment for the overactive bladder (OAB), while muscarinic agonists have been used to treat hypoactive bladder. The antagonists are effective in treating OAB, but their precise mechanisms and sites of action (detrusor, urothelium, and nerves) have yet to be established. Potentially more selective agents may be developed when the cholinergic systems within the bladder are more fully understood.

  17. Differential involvement of cortical muscarinic and NMDA receptors in short- and long-term taste aversion memory.

    PubMed

    Ferreira, G; Gutiérrez, R; De La Cruz, V; Bermúdez-Rattoni, F

    2002-09-01

    In conditioned taste aversion, an animal avoids a taste previously associated with toxic effects, and this aversive memory formation requires an intact insular cortex. In this paper, we investigated the possible differential involvement of cholinergic and glutamatergic receptors in the insular cortex in short-term memory (STM) and long-term memory (LTM) of taste aversion in rats. Taste aversion was induced by intraperitoneal administration of lithium chloride (a malaise-inducing drug) 15 min after experience with an unfamiliar taste. In order to test STM and LTM of taste aversion, taste stimulus was again presented 4 h and 72 h after lithium injection, respectively. During the acquisition, microinjection of the muscarinic antagonist, scopolamine, in the insular cortex before, but not after, the presentation of the new taste, abolished STM as well as LTM. Blockade of the NMDA receptor, in the insular cortex, by AP5 before, but not after, the presentation of the taste stimulus, impaired LTM but left STM intact. Moreover, when injected 1 h after malaise induction (i.e., during taste-illness association), AP5 disrupted both STM and LTM. These results suggest that activation of muscarinic receptors in the insular cortex is involved in the acquisition of taste memory, whereas NMDA receptors participate in taste memory consolidation. These data demonstrate that different neurochemical mechanisms subserve different memory phases. NMDA receptors are also probably involved in processing the visceral input, thus allowing subsequent taste-illness association. This indicates that in the same cortical area the same neurotransmitter system can be involved in distinct processes: taste memory consolidation vs. taste-illness association.

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

    PubMed Central

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

    2011-01-01

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

  19. Muscarinic receptors in amygdala control trace fear conditioning.

    PubMed

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

    2012-01-01

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

  20. Muscarinic Receptors in Amygdala Control Trace Fear Conditioning

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

    Maclagan, J.; Faulkner, D.

    1989-01-01

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

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

    PubMed Central

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

    2006-01-01

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

  3. Adenosine receptor expression and function in rat striatal cholinergic interneurons.

    PubMed

    Preston, Z; Lee, K; Widdowson, L; Freeman, T C; Dixon, A K; Richardson, P J

    2000-06-01

    Cholinergic neurons were identified in rat striatal slices by their size, membrane properties, sensitivity to the NK(1) receptor agonist (Sar(9), Met(O(2))(11)) Substance P, and expression of choline acetyltransferase mRNA. A(1) receptor mRNA was detected in 60% of the neurons analysed, and A(2A) receptor mRNA in 67% (n=15). The A(1) receptor agonist R-N(6)-(2-phenylisopropyl)adenosine (R-PIA) hyperpolarized cholinergic neurons in a concentration dependent manner sensitive to the A(1) antagonist 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX, 100 nM). In dual stimulus experiments, the A(2A) receptor antagonist 8-(3-chlorostyryl)caffeine (CSC, 500 nM) decreased release of [(3)H]-acetylcholine from striatal slices (S2/S1 0.78+/-0.07 versus 0.95+/-0.05 in control), as did adenosine deaminase (S2/S1 ratio 0.69+/-0.05), whereas the A(1) receptor antagonist DPCPX (100 nM) had no effect (S2/S1 1.05+/-0.14). In the presence of adenosine deaminase the adenosine A(2A) receptor agonist 2-p-((carboxyethyl)phenylethylamino)-5'-N-ethylcarboxamidoadeno sin e (CGS21680, 10 nM) increased release (S2/S1 ratio 1.03+/-0.05 versus 0.88+/-0.05 in control), an effect blocked by the antagonist CSC (500 nM, S2/S1 0.68+/-0.05, versus 0.73+/-0.08 with CSC alone). The combined superfusion of bicuculline (10 microM), saclofen (1 microM) and naloxone (10 microM) had no effect on the stimulation by CGS21680 (S2/S1 ratio 0.99+/-0.04). The A(1) receptor agonist R-PIA (100 nM) inhibited the release of [(3)H]-acetylcholine (S2/S1 ratio 0.70+/-0.03), an effect blocked by DPCPX (S2/S1 ratio 1.06+/-0.07). It is concluded that both A(1) and A(2A) receptors are expressed on striatal cholinergic neurons where they are functionally active.

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

    SciTech Connect

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

    1985-08-12

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

  5. Pharmacological effects of turmeric on learning, memory and expression of muscarinic receptor genes (M1, M3 and M5) in stress-induced mouse model.

    PubMed

    Khalid¥, Aliya; Shakeel¥, Rabia; Justin, Saira; Iqbal, Ghazala; Shah, Syed Adnan Ali; Zahid, Saadia; Ahmed, Touqeer

    2017-03-15

    Stress is involved in memory impairment by multiple mechanisms including activation of Hypothalamic-Pituitary Axis, which in turn activates release of corticosterone in blood. Cholinergic system blockade by muscarinic antagonist like scopolamine, also impairs memory.

  6. Influence of urothelial or suburothelial cholinergic receptors on bladder reflexes in chronic spinal cord injured cats.

    PubMed

    Ungerer, Timothy D; Kim, Kyoungeun A; Daugherty, Stephanie L; Roppolo, James R; Tai, Changfeng; de Groat, William C

    2016-11-01

    The effects of intravesical administration of a muscarinic receptor agonist (oxotremorine-M, OXO-M) and antagonist (atropine methyl nitrate, AMN) and of a nicotinic receptor agonist (nicotine) and antagonist (hexamethonium, C6) on reflex bladder activity were investigated in conscious female chronic spinal cord injured (SCI) cats using cystometry. OXO-M (50μM) decreased bladder capacity (BC) for triggering micturition contractions, increased maximal micturition pressure (MMP), increased frequency and area under the curve of pre-micturition contractions (PMC-AUC). Nicotine (250μM) decreased BC, increased MMP, but did not alter PMC-AUC. The effects of OXO-M on BC and PMC-AUC were suppressed by intravesical administration of AMN (50-100μM), and the effects of nicotine were blocked by hexamethonium (1mM). Antagonists infused intravesically alone did not alter reflex bladder activity. However, AMN (0.2mg/kg, subcutaneously) decreased PMC-AUC. 8-OH-DPAT (0.5mg/kg, s.c.), a 5-HT1A receptor agonist, suppressed the OXO-M-induced decrease in BC but not the enhancement of PMC-AUC. These results indicate that activation of cholinergic receptors located near the lumenal surface of the bladder modulates two types of reflex bladder activity (i.e., micturition and pre-micturition contractions). The effects may be mediated by activation of receptors on suburothelial afferent nerves or receptors on urothelial cells which release transmitters that can in turn alter afferent excitability. The selective action of nicotine on BC, while OXO-M affects both BC and PMC-AUC, suggests that micturition reflexes and PMCs are activated by different populations of afferent nerves. The selective suppression of the OXO-M effect on BC by 8-OH-DPAT without altering the effect on PMCs supports this hypothesis. The failure of intravesical administration of either AMN or hexamethonium alone to alter bladder activity indicates that cholinergic receptors located near the lumenal surface do not

  7. Activation of muscarinic receptors by ACh release in hippocampal CA1 depolarizes VIP but has varying effects on parvalbumin-expressing basket cells

    PubMed Central

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

    2015-01-01

    We investigated the effect of acetylcholine release on mouse hippocampal CA1 perisomatically projecting interneurons. Acetylcholine was optogenetically released in hippocampal slices by expressing the excitatory optogenetic protein oChIEF-tdTomato in medial septum/diagonal band of Broca cholinergic neurons using Cre recombinase-dependent adeno-associated virally mediated transfection. The effect of optogenetically released acetylcholine was assessed on interneurons expressing Cre recombinase in vasoactive intestinal peptide (VIP) or parvalbumin (PV) interneurons using whole cell patch clamp methods. Acetylcholine released onto VIP interneurons that innervate pyramidal neuron perisomatic regions (basket cells, BCs) were depolarized by muscarinic receptors. Although PV BCs were also excited by muscarinic receptor activation, they more frequently responded with hyperpolarizing or biphasic responses. Muscarinic receptor activation resulting from ACh release increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in downstream hippocampal CA1 pyramidal neurons with peak instantaneous frequencies occurring in both the gamma and theta bandwidths. Both PV and VIP BCs contributed to the increased sIPSC frequency in pyramidal neurons and optogenetic suppression of PV or VIP BCs inhibited sIPSCs occurring in the gamma range. Therefore, we propose acetylcholine release in CA1 has a complex effect on CA1 pyramidal neuron output through varying effects on perisomatically projecting interneurons. PMID:25556796

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

    PubMed

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

    2009-08-01

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

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

    PubMed

    Chapple, Christopher R

    2004-11-01

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

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

    PubMed

    Busch, Lucila; Borda, Enri

    2003-05-01

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

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

    PubMed Central

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

    2012-01-01

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

  12. Muscarinic receptor subtypes differentially control synaptic input and excitability of cerebellum-projecting medial vestibular nucleus neurons.

    PubMed

    Zhu, Yun; Chen, Shao-Rui; Pan, Hui-Lin

    2016-04-01

    found that activation of pre-synaptic M2 muscarinic receptors inhibit glutamatergic input from vestibular primary afferents, whereas stimulation of post-synaptic M3 muscarinic receptors increases the firing activity of cerebellum-projecting MVN neurons. This new information advances our understanding of the cholinergic mechanism regulating the vestibular system. © 2016 International Society for Neurochemistry.

  13. Nematode cholinergic pharmacology

    SciTech Connect

    Segerberg, M.A.

    1989-01-01

    Nematode acetylcholine (ACh) receptors were characterized using both biochemical and electrophysiological techniques, including: (1) receptor binding studies in crude homogenates of the free-living nematode Caenorhabditis elegans and the parasitic nematode Ascaris lumbricoides with the high-affinity probe ({sup 3}H)N-methylscopolamine (({sup 3}H)NMS) which binds to muscarinic receptors in many vertebrate and invertebrate tissues (2) measurement of depolarization and contraction induced by a variety of cholinergic agents, including N-methylscopolamine (NMS), in an innervated dorsal muscle strip preparation of Ascaris; (3) examination of the antagonistic actions of d-tubocurarine (dTC) and NMS at dorsal neuromuscular junction; (4) measurement of input resistance changes in Ascaris commissural motorneurons induced by ACh, dTC, NMS, pilocarpine and other cholinergic drugs.

  14. Cholinergic modulation of hippocampal cells and circuits

    PubMed Central

    Cobb, Stuart R; Davies, Ceri H

    2005-01-01

    Septo-hippocampal cholinergic fibres ramify extensively throughout the hippocampal formation to release acetylcholine upon a diverse range of muscarinic and nicotinic acetylcholine receptors that are differentially expressed by distinct populations of neurones. The resultant modulation of cellular excitability and synaptic transmission within hippocampal circuits underlies the ability of acetylcholine to influence the dynamic properties of the hippocampal network and results in the emergence of a range of stable oscillatory network states. Recent findings suggest a multitude of actions contribute to the oscillogenic properties of acetylcholine which are principally induced by activation of muscarinic receptors but also regulated through activation of nicotinic receptor subtypes. PMID:15528238

  15. Differential effects of systemic cholinergic receptor blockade on Pavlovian incentive motivation and goal-directed action selection.

    PubMed

    Ostlund, Sean B; Kosheleff, Alisa R; Maidment, Nigel T

    2014-05-01

    Reward-seeking actions can be guided by external cues that signal reward availability. For instance, when confronted with a stimulus that signals sugar, rats will prefer an action that produces sugar over a second action that produces grain pellets. Action selection is also sensitive to changes in the incentive value of potential rewards. Thus, rats that have been prefed a large meal of sucrose will prefer a grain-seeking action to a sucrose-seeking action. The current study investigated the dependence of these different aspects of action selection on cholinergic transmission. Hungry rats were given differential training with two unique stimulus-outcome (S1-O1 and S2-O2) and action-outcome (A1-O1 and A2-O2) contingencies during separate training phases. Rats were then given a series of Pavlovian-to-instrumental transfer tests, an assay of cue-triggered responding. Before each test, rats were injected with scopolamine (0, 0.03, or 0.1 mg/kg, intraperitoneally), a muscarinic receptor antagonist, or mecamylamine (0, 0.75, or 2.25 mg/kg, intraperitoneally), a nicotinic receptor antagonist. Although the reward-paired cues were capable of biasing action selection when rats were tested off-drug, both anticholinergic treatments were effective in disrupting this effect. During a subsequent round of outcome devaluation testing-used to assess the sensitivity of action selection to a change in reward value--we found no effect of either scopolamine or mecamylamine. These results reveal that cholinergic signaling at both muscarinic and nicotinic receptors mediates action selection based on Pavlovian reward expectations, but is not critical for flexibly selecting actions using current reward values.

  16. 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).

  17. Muscarinic, but not nicotinic, acetylcholine receptor blockade in the ventral tegmental area attenuates cue-induced sucrose-seeking

    PubMed Central

    Addy, Nii A.; Nunes, Eric J.; Wickham, Robert J.

    2015-01-01

    The mesolimbic dopamine (DA) system is known to play a role in cue-mediated reward-seeking for natural rewards and drugs of abuse. Specifically, cholinergic and glutamatergic receptors in the ventral tegmental area (VTA) have been shown to regulate cue-induced drug-seeking. However, the potential role of these VTA receptors in regulating cue-induced reward seeking for natural rewards is unknown. Here, we examined whether blockade of VTA acetylcholine receptors (AChRs) and N-methyl-D-aspartate receptors (NMDARs) would alter cue-induced sucrose seeking in male Sprague-Dawley rats. Subjects underwent 10 days of sucrose self-administration training (fixed ratio 1 schedule) followed by 7 days of forced abstinence. On withdrawal day 7, rats received bilateral VTA infusion of vehicle, the muscarinic AChR antagonist scopolamine (2.4 or 24 μg/side), the nicotinic AChR antagonist mecamylamine (3 or 30 μg/side), or the NMDAR antagonist AP-5 (0.1 or 1 μg/side) immediately prior to examination of cue-induced sucrose-seeking. Scopolamine infusion led to robust attenuation, but did not completely block, sucrose-seeking behavior. In contrast, VTA administration of mecamylamine or AP-5 did not alter cue-induced sucrose-seeking. Together, the data suggest that VTA muscarinic AChRs, but not nicotinic AChRs nor NMDARs, facilitate the ability of food-associated cues to drive seeking behavior for a food reward. PMID:26026787

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

    SciTech Connect

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

    2011-11-18

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

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

    SciTech Connect

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

    1987-11-09

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

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

    SciTech Connect

    Hoover, R.K.

    1989-01-01

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

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

    PubMed Central

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

    1982-01-01

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

  2. Muscarinic receptor antagonism in the basolateral amygdala blocks acquisition of cocaine-stimulus association in a model of relapse to cocaine-seeking behavior in rats.

    PubMed

    See, R E; McLaughlin, J; Fuchs, R A

    2003-01-01

    Recent evidence has demonstrated a critical role for the basolateral amygdala complex in the reinstatement of extinguished drug-seeking behavior produced by drug-paired cues. In the current study, we utilized a model of the acquisition and expression of cocaine-stimulus associative pairing in order to study the role of cholinergic input to the basolateral amygdala in mediating conditioned-cued reinstatement. Male, Sprague-Dawley rats were first trained daily to self-administer i.v. cocaine on a fixed ratio 1 schedule of reinforcement. The muscarinic acetylcholine receptor antagonist, scopolamine, was directly infused into the basolateral amygdala prior to: a) classically conditioned pairing of a tone+light stimulus with cocaine infusions (acquisition), or b) testing of conditioned-cued reinstatement following a period of withdrawal from cocaine and extinction of cocaine-paired lever responding. Infusion of scopolamine just prior to the classical conditioning trial produced a dose-dependent disruption of cocaine-seeking behavior maintained by cocaine-paired cues during the reinstatement test. In contrast, infusion of scopolamine prior to the reinstatement test had no effect on conditioned-cued reinstatement of cocaine-seeking behavior. These results indicate a crucial role for cholinergic innervation of muscarinic acetylcholine receptors in the basolateral amygdala during the formation, but not the expression, of stimulus-reward associations that mediate cue-induced cocaine-seeking behavior.

  3. Individual and combined manipulation of muscarinic, NMDA, and benzodiazepine receptor activity in the water maze task: implications for a rat model of Alzheimer dementia.

    PubMed

    Cain, D P; Ighanian, K; Boon, F

    2000-06-15

    Recent evidence indicates that Alzheimer disease typically involves different degrees of impairment in a variety of neurotransmitter systems, behaviors, and cognitive abilities in different patients. To investigate the relations between neurotransmitter system, behavioral, and cognitive impairments in an animal model of Alzheimer disease we studied spatial learning in a Morris water maze in male Long-Evans rats given neurochemical agents that targeted muscarinic cholinergic, NMDA, or benzodiazepine systems. Naive rats given a single agent or a combination of agents were severely impaired in place responding and had behavioral strategy impairments. Rats made familiar with the required water maze behavioral strategies by non-spatial pretraining performed as well as controls if given a single agent. Non-spatially pretrained rats with manipulation of both muscarinic cholinergic and NMDA or muscarinic cholinergic and benzodiazepine systems had a specific place response impairment but no behavioral strategy impairments. The results suggest that impairment of both muscarinic cholinergic and NMDA, or muscarinic cholinergic and benzodiazepine systems may model some aspects of human Alzheimer disease (impairments in navigation in familiar environments), but not other aspects of this disorder (global dementia leading to general loss of adaptive behavior). Previous research suggests that impairment of both muscarinic cholinergic and serotonergic systems may provide a better model of global dementia. The water maze testing and detailed behavioral analysis techniques used here appear to provide a means of investigating the contributions of various combinations of neurotransmitter system impairments to an animal model of Alzheimer disease.

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

    PubMed

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

    2012-01-25

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

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

    PubMed

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

    1988-01-01

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

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

    SciTech Connect

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

    2012-03-15

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

  7. 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.

  8. Modes and Models of Forebrain Cholinergic Neuromodulation of Cognition

    PubMed Central

    Hasselmo, Michael E; Sarter, Martin

    2011-01-01

    As indicated by the profound cognitive impairments caused by cholinergic receptor antagonists, cholinergic neurotransmission has a vital role in cognitive function, specifically attention and memory encoding. Abnormally regulated cholinergic neurotransmission has been hypothesized to contribute to the cognitive symptoms of neuropsychiatric disorders. Loss of cholinergic neurons enhances the severity of the symptoms of dementia. Cholinergic receptor agonists and acetylcholinesterase inhibitors have been investigated for the treatment of cognitive dysfunction. Evidence from experiments using new techniques for measuring rapid changes in cholinergic neurotransmission provides a novel perspective on the cholinergic regulation of cognitive processes. This evidence indicates that changes in cholinergic modulation on a timescale of seconds is triggered by sensory input cues and serves to facilitate cue detection and attentional performance. Furthermore, the evidence indicates cholinergic induction of evoked intrinsic, persistent spiking mechanisms for active maintenance of sensory input, and planned responses. Models have been developed to describe the neuronal mechanisms underlying the transient modulation of cortical target circuits by cholinergic activity. These models postulate specific locations and roles of nicotinic and muscarinic acetylcholine receptors and that cholinergic neurotransmission is controlled in part by (cortical) target circuits. The available evidence and these models point to new principles governing the development of the next generation of cholinergic treatments for cognitive disorders. PMID:20668433

  9. Association of a variant in the muscarinic acetylcholine receptor 2 gene (CHRM2) with nicotine addiction.

    PubMed

    Mobascher, A; Rujescu, D; Mittelstraß, K; Giegling, I; Lamina, C; Nitz, B; Brenner, H; Fehr, C; Breitling, L P; Gallinat, J; Rothenbacher, D; Raum, E; Müller, H; Ruppert, A; Hartmann, A M; Möller, H J; Gal, A; Gieger, Ch; Wichmann, H E; Illig, T; Dahmen, N; Winterer, G

    2010-03-05

    Genetic factors contribute to the overall risk of developing nicotine addiction, which is the major cause of preventable deaths in western countries. However, knowledge regarding specific polymorphisms influencing smoking phenotypes remains scarce. In the present study we provide evidence that a common single nucleotide polymorphism (SNP) in the 5' untranslated region of CHRM2, the gene coding for the muscarinic acetylcholine receptor 2 is associated with nicotine addiction. CHRM2 was defined as a candidate gene for nicotine addiction based on previous evidence that linked variations in CHRM2 to alcohol and drug dependence. A total of more than 5,500 subjects representative of the German population were genotyped and assessed regarding their smoking habits. The impact of three SNPs in CHRM2 on smoking behavior/nicotine addiction was investigated using logistic regression models or a quasi-Poisson regression model, respectively. We found the T allele of SNP rs324650 to be associated with an increased risk of smoking/nicotine dependence according to three different models, the recessive models of regular or heavy smokers vs. never-smokers (odds ratio 1.17 in both analyses) and according to the Fagerström index of nicotine addiction. In the analysis stratified by gender this association was only found in females. Our data provide further evidence that variations in CHRM2 may be associated with the genetic risk of addiction in general or with certain personality traits that predispose to the development of addiction. Alternatively, variations in CHRM2 could modulate presynaptic auto-regulation in cholinergic systems and may thereby affect an individual's response to nicotine more specifically. (c) 2009 Wiley-Liss, Inc.

  10. Antagonism of nucleus accumbens M(2) muscarinic receptors disrupts operant responding for sucrose under a progressive ratio reinforcement schedule.

    PubMed

    Cousens, Graham A; Beckley, Jacob T

    2007-07-19

    Diverse cholinergic signaling mechanisms regulate the excitability of striatal principal neurons and modulate striatal-dependent behavior. These effects are mediated, in part, by action at muscarinic receptors (mAChR), subtypes of which exhibit distinct patterns of expression across striatal neuronal populations. Non-selective mAChR blockade within the nucleus accumbens (NAc) has been shown to disrupt operant responding for food and to inhibit food consumption. However, the specific receptor subtypes mediating these effects are not known. Thus, we evaluated effects of intra-NAc infusions of pirenzepine and methoctramine, mAChR antagonisits with distinct binding affinity profiles, on operant responding for sucrose reward under a progressive ratio (PR) reinforcement schedule. Moderate to high doses of methoctramine disrupted operant responding and reduced behavioral breakpoint. In contrast, pirenzepine failed to impact operant performance at any dose tested. Methoctramine failed to affect latencies to complete appetitive-consummatory response sequences or to impact measures of acoustic startle, suggesting that its' disruptive effects on operant behavior were not consequent to gross motor impairment. Since methoctramine has a greater affinity for M(2) receptors compared to pirenzepine, which has a greater relative affinity for M(1) and M(3) receptors, these findings suggest that M(2) mAChRs within the NAc regulate behavioral processes underling the acquisition of reward.

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

    SciTech Connect

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

    1988-01-12

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

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

    SciTech Connect

    Entzeroth, M.; Mayer, N. )

    1991-01-01

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

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

    PubMed

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

    2014-03-15

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

  14. Advantages for transdermal over oral oxybutynin to treat overactive bladder: Muscarinic receptor binding, plasma drug concentration, and salivary secretion.

    PubMed

    Oki, Tomomi; Toma-Okura, Ayako; Yamada, Shizuo

    2006-03-01

    To clarify pharmacological usefulness of transdermal oxybutynin in the therapy of overactive bladder, we have characterized muscarinic receptor binding in rat tissues with measurement of plasma concentrations of oxybutynin and its metabolite N-desethyl-oxybutynin (DEOB) and salivation after transdermal oxybutynin compared with oral route. At 1 and 3 h after oral administration of oxybutynin, there was a significant increase in apparent dissociation constant (Kd) for specific [N-methyl-3H]scopolamine ([3H]NMS) binding in the rat bladder, submaxillary gland, heart, and colon compared with control values. Concomitantly, submaxillary gland and heart showed a significant decrease in maximal number of binding sites (Bmax) for [3H]NMS binding, which lasted until 24 h. Transdermal application of oxybutynin caused dose-dependent increases in Kd values for specific [3H]NMS binding in rat tissues. The increment of Kd values by transdermal oxybutynin was dependent on the application time. Plasma concentrations of oxybutynin and DEOB peaked at 1 h after oral oxybutynin. In contrast, plasma concentrations of oxybutynin increased slowly, depending on the transdermal application time of this drug until 12 h. Suppression of pilocarpine-induced salivation in rats due to transdermal oxybutynin was significantly weaker and more reversible than that by oral oxybutynin, which abolished salivary secretion. The present study has shown that transdermal oxybutynin binds significantly to rat bladder muscarinic receptors without producing both long-lasting occupation of exocrine receptors and cessation of cholinergic salivation evoked by oral oxybutynin. Thus, the present study provides further pharmacological basis for advantage of transdermal over oral oxybutynin in the therapy of overactive bladder.

  15. Role of various kinases in muscarinic M3 receptor-mediated contraction of longitudinal muscle of rat colon.

    PubMed

    Anderson, Charles D; Kendig, Derek M; Al-Qudah, Mohammad; Mahavadi, Sunila; Murthy, Karnam S; Grider, John R

    2014-01-01

    The longitudinal muscle layer in gut is the functional opponent to the circular muscle layer during peristalsis. Differences in innervation of the layers allow for the contraction of one layer concurrently with the relaxation of the other, enabling the passage of gut contents in a controlled fashion. Differences in development have given the cells of the two layers differences in receptor populations, membrane lipid handling, and calcium handling profiles/behaviors. The contractile activity of the longitudinal muscle is largely mediated by cholinergic neural input from myenteric plexus. Activation of muscarinic receptors leads to rapid activation of several kinases including MLC kinase, ERK1/2, CaMKII and Rho kinase. Phosphorylation of myosin light chain (MLC20) by MLC kinase (MLCK) is a prerequisite for contraction in both circular and longitudinal muscle cells. In rat colonic longitudinal muscle strips, we measured muscarinic receptor-mediated contraction following incubation with kinase inhibitors. Basal tension was differentially regulated by Rho kinase, ERK1/2, CaMKII and CaMKK. Selective inhibitors of Rho kinase, ERK1/2, CaMKK/AMPK, and CaMKII each reduced carbachol-induced contraction in the innervated muscle strips. These inhibitors had no direct effect on MLCK activity. Thus unlike previously reported for isolated muscle cells where CaMKII and ERK1/2 are not involved in contraction, we conclude that the regulation of carbachol-induced contraction in innervated longitudinal muscle strips involves the interplay of Rho kinase, ERK1/2, CaMKK/AMPK, and CAMKII.

  16. Beyond Acetylcholinesterase Inhibitors: Novel Cholinergic Treatments for Alzheimer's Disease.

    PubMed

    Kamkwalala, Asante R; Newhouse, Paul A

    2017-01-01

    The major components of the cholinergic receptor system of the human brain include projections from the basal forebrain nuclei, and utilize the two types of receptors that they synapse on, nicotinic and muscarinic acetylcholine receptors. With the widespread cortical and subcortical projections of the basal forebrain, activity of these two receptor systems provide modulation of neurotransmitter activity underlying normal cognitive processes, such as attention, episodic memory, and working memory. Alzheimer's disease (AD) targets and damages cholinergic neurons in the basal forebrain, and as these projections are lost, cognitive performance progressively declines. Currently, the most widely prescribed treatment for AD is acetylcholinesterase inhibitor medications, which work by partially blocking the degradation of acetylcholine in the synapse and enabling more of the neurotransmitter to reach and activate cholinergic receptors. However since these medications have limited effectiveness, alternate treatments that focus on augmenting the activity of the receptors themselves, independent of acetylcholinesterase inhibition, are being explored. This review will discuss: 1) the role of the cholinergic system in modulating cognition, 2) novel cholinergic treatment strategies for AD-related cognitive decline, in particular treatments intended to increase cholinergic system activity by selectively targeting muscarinic and nicotinic acetylcholinergic receptors to improve cognitive performance, 3) risks, and additional considerations for cholinergic cognitive treatments for AD.

  17. Angiotensinergic and cholinergic receptors of the subfornical organ mediate sodium intake induced by GABAergic activation of the lateral parabrachial nucleus.

    PubMed

    Roncari, C F; David, R B; Johnson, R F; De Paula, P M; Colombari, D S A; De Luca, L A; Johnson, A K; Colombari, E; Menani, J V

    2014-03-14

    Bilateral injections of the GABA(A) agonist muscimol into the lateral parabrachial nucleus (LPBN) induce 0.3 M NaCl and water intake in satiated and normovolemic rats, a response reduced by intracerebroventricular (icv) administration of losartan or atropine (angiotensinergic type 1 (AT₁) and cholinergic muscarinic receptor antagonists, respectively). In the present study, we investigated the effects of the injections of losartan or atropine into the subfornical organ (SFO) on 0.3M NaCl and water intake induced by injections of muscimol into the LPBN. In addition, using intracellular calcium measurement, we also tested the sensitivity of SFO-cultured cells to angiotensin II (ANG II) and carbachol (cholinergic agonist). In male Holtzman rats with cannulas implanted bilaterally into the LPBN and into the SFO, injections of losartan (1 μg/0.1 μl) or atropine (2 nmol/0.1 μl) into the SFO almost abolished 0.3M NaCl and water intake induced by muscimol (0.5 nmol/0.2 μl) injected into the LPBN. In about 30% of the cultured cells of the SFO, carbachol and ANG II increased intracellular calcium concentration ([Ca²⁺](i)). Three distinct cell populations were found in the SFO, i.e., cells activated by either ANG II (25%) or carbachol (2.6%) or by both stimuli (2.3%). The results suggest that the activation of angiotensinergic and cholinergic mechanisms in the SFO is important for NaCl and water intake induced by the deactivation of LPBN inhibitory mechanisms with muscimol injections. They also show that there are cells in the SFO activated by both angiotensinergic and cholinergic stimuli, perhaps those involved in the responses to muscimol into the LPBN. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

  18. Angiotensinergic and cholinergic receptors of the subfornical organ mediate sodium intake induced by GABAergic activation of the lateral parabrachial nucleus

    PubMed Central

    Roncari, Camila F.; David, Richard B.; Johnson, Ralph F.; De Paula, Patrícia M.; Colombari, Débora S. A.; De Luca, Laurival A.; Johnson, Alan Kim; Colombari, Eduardo; Menani, José V.

    2015-01-01

    Bilateral injections of the GABAA agonist muscimol into the lateral parabrachial nucleus (LPBN) induce 0.3 M NaCl and water intake in satiated and normovolemic rats, a response reduced by intracerebroventricular (icv) administration of losartan or atropine (angiotensinergic type 1 (AT1) and cholinergic muscarinic receptor antagonists, respectively). In the present study, we investigated the effects of the injections of losartan or atropine into the subfornical organ (SFO) on 0.3 M NaCl and water intake induced by injections of muscimol into the LPBN. In addition, using intracellular calcium measurement, we also tested the sensitivity of SFO-cultured cells to angiotensin II (ANG II) and carbachol (cholinergic agonist). In male Holtzman rats with cannulas implanted bilaterally into the LPBN and into the SFO, injections of losartan (1 μg/0.1 μl) or atropine (2 nmol/0.1 μl) into the SFO almost abolished 0.3 M NaCl and water intake induced by muscimol (0.5 nmol/0.2 μl) injected into the LPBN. In about 30% of the cultured cells of the SFO, carbachol and ANG II increased intracellular calcium concentration ([Ca2+]i). Three distinct cell populations were found in the SFO, i.e., cells activated by either ANG II (25%) or carbachol (2.6%) or by both stimuli (2.3%). The results suggest that the activation of angiotensinergic and cholinergic mechanisms in the SFO is important for NaCl and water intake induced by the deactivation of LPBN inhibitory mechanisms with muscimol injections. They also show that there are cells in the SFO activated by both angiotensinergic and cholinergic stimuli, perhaps those involved in the responses to muscimol into the LPBN. PMID:24374079

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

    PubMed Central

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

    2014-01-01

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

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

    SciTech Connect

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

    1994-09-01

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

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

    PubMed Central

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

    1997-01-01

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

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

    SciTech Connect

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

    1985-04-01

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

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

    SciTech Connect

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

    1990-01-01

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

  4. Cholinergic interneurons control the excitatory input to the striatum.

    PubMed

    Pakhotin, Pavel; Bracci, Enrico

    2007-01-10

    How the extent and time course of presynaptic inhibition depend on the action potentials of the neuron controlling the terminals is unknown. We investigated this issue in the striatum using paired recordings from cholinergic interneurons and projection neurons. Glutamatergic EPSCs were evoked in projection neurons and cholinergic interneurons by stimulation of afferent fibers in the cortex and the striatum, respectively. A single spike in a cholinergic interneuron caused significant depression of the evoked glutamatergic EPSC in 34% of projection neurons located within 100 microm and 41% of cholinergic interneurons located within 200 microm. The time course of these effects was similar in the two cases, with EPSC inhibition peaking 20-30 ms after the spike and disappearing after 40-80 ms. Maximal depression of EPSC amplitude was up to 27% in projection neurons and to 19% in cholinergic interneurons. These effects were reversibly blocked by muscarinic receptor antagonists (atropine or methoctramine), which also significantly increased baseline EPSC (evoked without a preceding spike in the cholinergic interneuron), suggesting that some tonic cholinergic presynaptic inhibition was present. This was confirmed by the fact that lowering extracellular potassium, which silenced spontaneously active cholinergic interneurons, also increased baseline EPSC amplitude, and these effects were occluded by previous application of muscarinic receptor antagonists. Collectively, these results show that a single spike in a cholinergic interneuron exerts a fast and powerful inhibitory control over the glutamatergic input to striatal neurons.

  5. Involvement of Cholinergic and Adrenergic Receptors in Pathogenesis and Inflammatory Response Induced by Alpha-Neurotoxin Bot III of Scorpion Venom.

    PubMed

    Nakib, Imene; Martin-Eauclaire, Marie-France; Laraba-Djebari, Fatima

    2016-10-01

    Bot III neurotoxin is the most lethal α neurotoxin purified from Buthus occitanus tunetanus scorpion venom. This toxin binds to the voltage-gated sodium channel of excitable cells and blocks its inactivation, inducing an increased release of neurotransmitters (acetylcholine and catecholamines). This study aims to elucidate the involvement of cholinergic and adrenergic receptors in pathogenesis and inflammatory response triggered by this toxin. Injection of Bot III to animals induces an increase of peroxidase activities, an imbalance of oxidative status, tissue damages in lung parenchyma, and myocardium correlated with metabolic disorders. The pretreatment with nicotine (nicotinic receptor agonist) or atropine (muscarinic receptor antagonist) protected the animals from almost all disorders caused by Bot III toxin, especially the immunological alterations. Bisoprolol administration (selective β1 adrenergic receptor antagonist) was also efficient in the protection of animals, mainly on tissue damage. Propranolol (non-selective adrenergic receptor antagonist) showed less effect. These results suggest that both cholinergic and adrenergic receptors are activated in the cardiopulmonary manifestations induced by Bot III. Indeed, the muscarinic receptor appears to be more involved than the nicotinic one, and the β1 adrenergic receptor seems to dominate the β2 receptor. These results showed also that the activation of nicotinic receptor leads to a significant protection of animals against Bot III toxin effect. These findings supply a supplementary data leading to better understanding of the mechanism triggered by scorpionic neurotoxins and suggest the use of drugs targeting these receptors, especially the nicotinic one in order to counteract the inflammatory response observed in scorpion envenomation.

  6. Neonatal hypoxic insult-mediated cholinergic disturbances in the brain stem: effect of glucose, oxygen and epinephrine resuscitation.

    PubMed

    Anju, T R; Naijil, G; Shilpa, J; Roshni, T; Paulose, C S

    2013-03-01

    Molecular processes regulating cholinergic functions play an important role in the control of respiration under neonatal hypoxia. The present study evaluates neonatal hypoxic insult-mediated cholinergic alterations and the protective role of glucose, oxygen and epinephrine resuscitation. The changes in total muscarinic, muscarinic M1, M2, M3 receptors and the enzymes involved in acetylcholine metabolism--cholineacetyl transferase and acetylcholine easterase in the brain stem were analyzed. Hypoxic stress decreased total muscarinic receptors along with a reduction in muscarinic M1, M2 and M3 receptor genes in the brain stem. The reduction in acetylcholine metabolism is indicated by the down regulated cholineacetyl transferase and up regulated acetylcholine easterase expression. These cholinergic disturbances in the brain stem were reversed by glucose resuscitation to hypoxic neonates. The adverse effects of immediate oxygenation and epinephrine administration were also reported. This has immense clinical significance in establishing a proper resuscitation for the management of neonatal hypoxia.

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

    PubMed

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

    2014-12-01

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

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

    SciTech Connect

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

    1985-06-01

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

  9. Adenosine induces a cholinergic tracheal reflex contraction in guinea pigs in vivo via an adenosine A1 receptor-dependent mechanism.

    PubMed

    Reynolds, Sandra M; Docherty, Reginald; Robbins, Jon; Spina, Domenico; Page, Clive P

    2008-07-01

    Adenosine induces dyspnea, cough, and airways obstruction in asthma, a phenomenon that also occurs in various sensitized animal models in which a neuronal involvement has been implicated. Although adenosine has been suggested to activate cholinergic nerves, the precise mechanism has not been established. In the present study, the adenosine A(1) receptor agonist N(6)-cyclopentyladenosine (CPA) induced a cholinergic reflex, causing tracheal smooth muscle contraction that was significantly inhibited by the adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 100 microg/kg) (P < 0.05) in anesthetized animals. Furthermore, the adenosine A(2) agonist 2-p-(2-carboxyethyl) phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680) induced a small reflex, whereas the A(3) selective agonist N(6)-(3-iodobenzyl)-5'-N-methylcarbamoyladenosine (IB-MECA) was without effect. The tracheal reflex induced by CPA was also inhibited by recurrent nerve ligation or muscarinic receptor blockade (P < 0.001), indicating that a cholinergic neuronal mechanism of action accounted for this response. The cholinergic reflex in response to aerosolized CPA was significantly greater in passively sensitized compared with naive guinea pigs (P < 0.01). Chronic capsaicin treatment, which inhibited sensory nerve function, failed to inhibit CPA-induced reflex tracheal contractions in passively sensitized guinea pigs, although the local anesthetic lidocaine inhibited CPA-induced tracheal contractions. The effects of CPA on the reflex response was not dependent on the release of histamine from tissue mast cells or endogenous prostaglandins as shown by the lack of effect of the histamine H(1) receptor antagonist pyrilamine (1 mg/kg) or the cyclooxygenase inhibitor meclofenamic acid (3 mg/kg), respectively. In conclusion, activation of pulmonary adenosine A(1) receptors can stimulate cholinergic reflexes, and these reflexes are increased in allergic guinea pigs.

  10. 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

  11. 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.

  12. 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.

  13. Systemic administration of arecoline reduces ethanol-induced sleeping through activation of central muscarinic receptor in mice.

    PubMed

    Sun, Yan-Ping; Liu, Qing; Luo, Juan; Guo, Ping; Chen, Feng; Lawrence, Andrew J; Liang, Jian-Hui

    2010-01-01

    Epidemiological evidence of co-use of alcohol and areca nuts suggests a potential central interaction between arecoline, a major alkaloid of areca and a muscarinic receptor agonist, and ethanol. Moreover, the central cholinergic system plays an important role in the depressant action of ethanol and barbiturates. The purpose of this study was to investigate the effects of arecoline on pentobarbital- and ethanol-induced hypnosis in mice. Male ICR mice were tested for locomotor activity following acute systemic administration of ethanol alone, arecoline alone, or ethanol plus arecoline. For the loss of the righting reflex (LORR) induced by pentobarbital and ethanol, sleep latency and sleeping duration were evaluated in mice treated with arecoline alone or the combination of arecoline and scopolamine or methscopolamine. Ethanol (1.0 to 3.0 g/kg, i.p.) reduced locomotor activity significantly and a declining trend was observed after treatment with arecoline (0.25 to 1.0 mg/kg, i.p.), but there were no synergistic effects of ethanol and arecoline on locomotor activity. The experiments on LORR demonstrated that arecoline (0.125 to 1.0 mg/kg, s.c.) shortened the duration of sleeping induced by ethanol (4.0 g/kg, i.p.), but not pentobarbital (45 mg/kg, i.p.). In addition, alterations of sleep latency were not obvious in both pentobarbital- and ethanol-induced LORR. Statistical analyses revealed that scopolamine (centrally acting), but not methscopolamine (peripherally acting), could antagonize the effect of arecoline on the duration of ethanol-induced LORR in mice. These results suggest that central muscarinic receptor is a pharmacological target for the action of arecoline to modulate ethanol-induced hypnosis.

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

    PubMed Central

    Brunner, F.; Kukovetz, W. R.

    1991-01-01

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

  15. [Comparison of distribution of cholinergic nerves and M2 receptors between rat atria and ventricles].

    PubMed

    Xu, Xiao-li; Zang, Wei-jin; Kang, Xin-qin; Li, Ming; Yu, Xiao-jiang; Chen, Li-na; Luo, Hong-li

    2006-08-01

    To investigate the general pattern of cholinergic nerve distribution and M(2) receptors in adult rat heart. Karnovsky-Roots histochemical staining combining point counting method and immunochemical SABC method with image analysis were used to identify the cholinergic nerves and M(2) receptors, respectively, in adult rat heart. Positive staining of cholinergic nerves and M(2) receptors was found in all regions of the rat heart, and the point count of cholinergic nerves in the atria was 4.6 times as much as that in ventricles, and the area of immunoreactive substance for M(2) receptors two-fold higher in the atria than in the ventricles. The point counts of the cholinergic nerves in the medial-layer myocardium were fewer than that in subepicardial and endocardial tissues of the left ventricular free wall. However, M(2) receptors were comparable among the 3 layers of the left free ventricular wall. Cholinergic nerves and M(2) receptors are located in both rat atria and ventricles, but their density is much higher in the atria than in the ventricles. Transmural heterogeneity characterizes cholinergic nerve innervation in the left ventricular free wall without significant differences in M(2) receptor density.

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

    PubMed

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

    2014-10-15

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    2009-01-01

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

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

    PubMed

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

    2009-07-17

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

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

    PubMed

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

    2000-04-01

    Increasing evidence has shown that some neurotransmitters act as growth-regulatory signals during brain development. Here we report a role for the classical neurotransmitter acetylcholine (ACh) to stimulate proliferation of neural stem cells and stem cell-derived progenitor cells during neural cell lineage progression in vitro. Neuroepithelial