Wess J


Full name : Wess Jurgen

First name : Jurgen

Mail : Molecular Signaling Section and Synthetic Bioactive Molecules Section NIH-NIDDK Bldg 8A - room BIA-05, Bethesda, MD 20892

Zip Code :

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Country : USA

Email : jurgenw@niddk.nih.gov

Phone : 1 301 4023589

Fax : I 301 4024182

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References (112)

Title : M1 muscarinic allosteric modulators slow prion neurodegeneration and restore memory loss - Bradley_2017_J.Clin.Invest_127_487
Author(s) : Bradley SJ , Bourgognon JM , Sanger HE , Verity N , Mogg AJ , White DJ , Butcher AJ , Moreno JA , Molloy C , Macedo-Hatch T , Edwards JM , Wess J , Pawlak R , Read DJ , Sexton PM , Broad LM , Steinert JR , Mallucci GR , Christopoulos A , Felder CC , Tobin AB
Ref : J Clinical Investigation , 127 :487 , 2017
Abstract : The current frontline symptomatic treatment for Alzheimer's disease (AD) is whole-body upregulation of cholinergic transmission via inhibition of acetylcholinesterase. This approach leads to profound dose-related adverse effects. An alternative strategy is to selectively target muscarinic acetylcholine receptors, particularly the M1 muscarinic acetylcholine receptor (M1 mAChR), which was previously shown to have procognitive activity. However, developing M1 mAChR-selective orthosteric ligands has proven challenging. Here, we have shown that mouse prion disease shows many of the hallmarks of human AD, including progressive terminal neurodegeneration and memory deficits due to a disruption of hippocampal cholinergic innervation. The fact that we also show that muscarinic signaling is maintained in both AD and mouse prion disease points to the latter as an excellent model for testing the efficacy of muscarinic pharmacological entities. The memory deficits we observed in mouse prion disease were completely restored by treatment with benzyl quinolone carboxylic acid (BQCA) and benzoquinazoline-12 (BQZ-12), two highly selective positive allosteric modulators (PAMs) of M1 mAChRs. Furthermore, prolonged exposure to BQCA markedly extended the lifespan of diseased mice. Thus, enhancing hippocampal muscarinic signaling using M1 mAChR PAMs restored memory loss and slowed the progression of mouse prion disease, indicating that this ligand type may have clinical benefit in diseases showing defective cholinergic transmission, such as AD.
ESTHER : Bradley_2017_J.Clin.Invest_127_487
PubMedSearch : Bradley_2017_J.Clin.Invest_127_487
PubMedID: 27991860

Title : The M3 muscarinic receptor is required for optimal adaptive immunity to helminth and bacterial infection - Darby_2015_PLoS.Pathog_11_e1004636
Author(s) : Darby M , Schnoeller C , Vira A , Culley FJ , Bobat S , Logan E , Kirstein F , Wess J , Cunningham AF , Brombacher F , Selkirk ME , Horsnell WG
Ref : PLoS Pathog , 11 :e1004636 , 2015
Abstract : Innate immunity is regulated by cholinergic signalling through nicotinic acetylcholine receptors. We show here that signalling through the M3 muscarinic acetylcholine receptor (M3R) plays an important role in adaptive immunity to both Nippostrongylus brasiliensis and Salmonella enterica serovar Typhimurium, as M3R-/- mice were impaired in their ability to resolve infection with either pathogen. CD4 T cell activation and cytokine production were reduced in M3R-/- mice. Immunity to secondary infection with N. brasiliensis was severely impaired, with reduced cytokine responses in M3R-/- mice accompanied by lower numbers of mucus-producing goblet cells and alternatively activated macrophages in the lungs. Ex vivo lymphocyte stimulation of cells from intact BALB/c mice infected with N. brasiliensis and S. typhimurium with muscarinic agonists resulted in enhanced production of IL-13 and IFN-gamma respectively, which was blocked by an M3R-selective antagonist. Our data therefore indicate that cholinergic signalling via the M3R is essential for optimal Th1 and Th2 adaptive immunity to infection.
ESTHER : Darby_2015_PLoS.Pathog_11_e1004636
PubMedSearch : Darby_2015_PLoS.Pathog_11_e1004636
PubMedID: 25629518

Title : Differential regulation of primary afferent input to spinal cord by muscarinic receptor subtypes delineated using knockout mice - Chen_2014_J.Biol.Chem_289_14321
Author(s) : Chen SR , Chen H , Yuan WX , Wess J , Pan HL
Ref : Journal of Biological Chemistry , 289 :14321 , 2014
Abstract : Stimulation of muscarinic acetylcholine receptors (mAChRs) inhibits nociceptive transmission at the spinal level. However, it is unclear how each mAChR subtype regulates excitatory synaptic input from primary afferents. Here we examined excitatory postsynaptic currents (EPSCs) of dorsal horn neurons evoked by dorsal root stimulation in spinal cord slices from wild-type and mAChR subtype knock-out (KO) mice. In wild-type mice, mAChR activation with oxotremorine-M decreased the amplitude of monosynaptic EPSCs in approximately 67% of neurons but increased it in approximately 10% of neurons. The inhibitory effect of oxotremorine-M was attenuated by the M2/M4 antagonist himbacine in the majority of neurons, and the remaining inhibition was abolished by group II/III metabotropic glutamate receptor (mGluR) antagonists in wild-type mice. In M2/M4 double-KO mice, oxotremorine-M inhibited monosynaptic EPSCs in significantly fewer neurons ( approximately 26%) and increased EPSCs in significantly more neurons (33%) compared with wild-type mice. Blocking group II/III mGluRs eliminated the inhibitory effect of oxotremorine-M in M2/M4 double-KO mice. In M2 single-KO and M4 single-KO mice, himbacine still significantly reduced the inhibitory effect of oxotremorine-M. However, the inhibitory and potentiating effects of oxotremorine-M on EPSCs in M3 single-KO and M1/M3 double-KO mice were similar to those in wild-type mice. In M5 single-KO mice, oxotremorine-M failed to potentiate evoked EPSCs, and its inhibitory effect was abolished by himbacine. These findings indicate that activation of presynaptic M2 and M4 subtypes reduces glutamate release from primary afferents. Activation of the M5 subtype either directly increases primary afferent input or inhibits it through indirectly stimulating group II/III mGluRs.
ESTHER : Chen_2014_J.Biol.Chem_289_14321
PubMedSearch : Chen_2014_J.Biol.Chem_289_14321
PubMedID: 24695732

Title : Muscarinic M(3) receptors contribute to allergen-induced airway remodeling in mice - Kistemaker_2014_Am.J.Respir.Cell.Mol.Biol_50_690
Author(s) : Kistemaker LE , Bos ST , Mudde WM , Hylkema MN , Hiemstra PS , Wess J , Meurs H , Kerstjens HA , Gosens R
Ref : American Journal of Respiratory Cellular & Molecular Biology , 50 :690 , 2014
Abstract : Asthma is a chronic obstructive airway disease, characterized by inflammation and remodeling. Acetylcholine contributes to symptoms by inducing bronchoconstriction via the muscarinic M3 receptor. Recent evidence suggests that bronchoconstriction can regulate airway remodeling, and therefore implies a role for the muscarinic M3 receptor. The objective of this work was to study the contribution of the muscarinic M3 receptor to allergen-induced remodeling using muscarinic M3 receptor subtype-deficient (M3R(-/-)) mice. Wild-type (WT), M1R(-/-), and M2R(-/-) mice were used as controls. C57Bl/6 mice were sensitized and challenged with ovalbumin (twice weekly for 4 wk). Control animals were challenged with saline. Allergen exposure induced goblet cell metaplasia, airway smooth muscle thickening (1.7-fold), pulmonary vascular smooth muscle remodeling (1.5-fold), and deposition of collagen I (1.7-fold) and fibronectin (1.6-fold) in the airway wall of WT mice. These effects were absent or markedly lower in M3R(-/-) mice (30-100%), whereas M1R(-/-) and M2R(-/-) mice responded similarly to WT mice. In addition, airway smooth muscle and pulmonary vascular smooth muscle mass were 35-40% lower in saline-challenged M3R(-/-) mice compared with WT mice. Interestingly, allergen-induced airway inflammation, assessed as infiltrated eosinophils and T helper type 2 cytokine expression, was similar or even enhanced in M3R(-/-) mice. Our data indicate that acetylcholine contributes to allergen-induced remodeling and smooth muscle mass via the muscarinic M3 receptor, and not via M1 or M2 receptors. No stimulatory role for muscarinic M3 receptors in allergic inflammation was observed, suggesting that the role of acetylcholine in remodeling is independent of the allergic inflammatory response, and may involve bronchoconstriction.
ESTHER : Kistemaker_2014_Am.J.Respir.Cell.Mol.Biol_50_690
PubMedSearch : Kistemaker_2014_Am.J.Respir.Cell.Mol.Biol_50_690
PubMedID: 24156289

Title : Novel insights into m3 muscarinic acetylcholine receptor physiology and structure - Kruse_2014_J.Mol.Neurosci_53_316
Author(s) : Kruse AC , Li J , Hu J , Kobilka BK , Wess J
Ref : Journal of Molecular Neuroscience , 53 :316 , 2014
Abstract : Recent studies with M3 muscarinic acetylcholine receptor (M3R) mutant mice suggest that drugs selectively targeting this receptor subtype may prove useful for the treatment of various pathophysiological conditions. Moreover, the use of M3R-based designer G protein-coupled receptors (GPCRs) has provided novel insights into how Gq-coupled GPCRs can modulate whole-body glucose homeostasis by acting on specific peripheral cell types. More recently, we succeeded in using X-ray crystallography to determine the structure of the M3R bound to the bronchodilating drug tiotropium, a muscarinic antagonist (inverse agonist). This new structural information should facilitate the development of orthosteric or allosteric M3R-selective drugs that are predicted to have considerable therapeutic potential.
ESTHER : Kruse_2014_J.Mol.Neurosci_53_316
PubMedSearch : Kruse_2014_J.Mol.Neurosci_53_316
PubMedID: 24068573

Title : Muscarinic acetylcholine receptor X-ray structures: potential implications for drug development - Kruse_2014_Curr.Opin.Pharmacol_16_24
Author(s) : Kruse AC , Hu J , Kobilka BK , Wess J
Ref : Curr Opin Pharmacol , 16 :24 , 2014
Abstract : 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.
ESTHER : Kruse_2014_Curr.Opin.Pharmacol_16_24
PubMedSearch : Kruse_2014_Curr.Opin.Pharmacol_16_24
PubMedID: 24662799

Title : Muscarinic acetylcholine receptors: novel opportunities for drug development - Kruse_2014_Nat.Rev.Drug.Discov_13_549
Author(s) : Kruse AC , Kobilka BK , Gautam D , Sexton PM , Christopoulos A , Wess J
Ref : Nat Rev Drug Discov , 13 :549 , 2014
Abstract : The muscarinic acetylcholine receptors are a subfamily of G protein-coupled receptors that regulate numerous fundamental functions of the central and peripheral nervous system. The past few years have witnessed unprecedented new insights into muscarinic receptor physiology, pharmacology and structure. These advances include the first structural views of muscarinic receptors in both inactive and active conformations, as well as a better understanding of the molecular underpinnings of muscarinic receptor regulation by allosteric modulators. These recent findings should facilitate the development of new muscarinic receptor subtype-selective ligands that could prove to be useful for the treatment of many severe pathophysiological conditions.
ESTHER : Kruse_2014_Nat.Rev.Drug.Discov_13_549
PubMedSearch : Kruse_2014_Nat.Rev.Drug.Discov_13_549
PubMedID: 24903776

Title : Activation and allosteric modulation of a muscarinic acetylcholine receptor - Kruse_2013_Nature_504_101
Author(s) : Kruse AC , Ring AM , Manglik A , Hu J , Hu K , Eitel K , Hubner H , Pardon E , Valant C , Sexton PM , Christopoulos A , Felder CC , Gmeiner P , Steyaert J , Weis WI , Garcia KC , Wess J , Kobilka BK
Ref : Nature , 504 :101 , 2013
Abstract : Despite recent advances in crystallography and the availability of G-protein-coupled receptor (GPCR) structures, little is known about the mechanism of their activation process, as only the beta2 adrenergic receptor (beta2AR) and rhodopsin have been crystallized in fully active conformations. Here we report the structure of an agonist-bound, active state of the human M2 muscarinic acetylcholine receptor stabilized by a G-protein mimetic camelid antibody fragment isolated by conformational selection using yeast surface display. In addition to the expected changes in the intracellular surface, the structure reveals larger conformational changes in the extracellular region and orthosteric binding site than observed in the active states of the beta2AR and rhodopsin. We also report the structure of the M2 receptor simultaneously bound to the orthosteric agonist iperoxo and the positive allosteric modulator LY2119620. This structure reveals that LY2119620 recognizes a largely pre-formed binding site in the extracellular vestibule of the iperoxo-bound receptor, inducing a slight contraction of this outer binding pocket. These structures offer important insights into the activation mechanism and allosteric modulation of muscarinic receptors.
ESTHER : Kruse_2013_Nature_504_101
PubMedSearch : Kruse_2013_Nature_504_101
PubMedID: 24256733

Title : Muscarinic receptors as model targets and antitargets for structure-based ligand discovery - Kruse_2013_Mol.Pharmacol_84_528
Author(s) : Kruse AC , Weiss DR , Rossi M , Hu J , Hu K , Eitel K , Gmeiner P , Wess J , Kobilka BK , Shoichet BK
Ref : Molecular Pharmacology , 84 :528 , 2013
Abstract : G protein-coupled receptors (GPCRs) regulate virtually all aspects of human physiology and represent an important class of therapeutic drug targets. Many GPCR-targeted drugs resemble endogenous agonists, often resulting in poor selectivity among receptor subtypes and restricted pharmacologic profiles. The muscarinic acetylcholine receptor family exemplifies these problems; thousands of ligands are known, but few are receptor subtype-selective and nearly all are cationic in nature. Using structure-based docking against the M2 and M3 muscarinic receptors, we screened 3.1 million molecules for ligands with new physical properties, chemotypes, and receptor subtype selectivities. Of 19 docking-prioritized molecules tested against the M2 subtype, 11 had substantial activity and 8 represented new chemotypes. Intriguingly, two were uncharged ligands with low micromolar to high nanomolar Ki values, an observation with few precedents among aminergic GPCRs. To exploit a single amino-acid substitution among the binding pockets between the M2 and M3 receptors, we selected molecules predicted by docking to bind to the M3 and but not the M2 receptor. Of 16 molecules tested, 8 bound to the M3 receptor. Whereas selectivity remained modest for most of these, one was a partial agonist at the M3 receptor without measurable M2 agonism. Consistent with this activity, this compound stimulated insulin release from a mouse beta-cell line. These results support the ability of structure-based discovery to identify new ligands with unexplored chemotypes and physical properties, leading to new biologic functions, even in an area as heavily explored as muscarinic pharmacology.
ESTHER : Kruse_2013_Mol.Pharmacol_84_528
PubMedSearch : Kruse_2013_Mol.Pharmacol_84_528
PubMedID: 23887926

Title : Muscarinic receptor subtype-specific effects on cigarette smoke-induced inflammation in mice - Kistemaker_2013_Eur.Respir.J_42_1677
Author(s) : Kistemaker LE , Bos IS , Hylkema MN , Nawijn MC , Hiemstra PS , Wess J , Meurs H , Kerstjens HA , Gosens R
Ref : Eur Respir J , 42 :1677 , 2013
Abstract : Cholinergic tone contributes to airflow obstruction in chronic obstructive pulmonary disease. Accordingly, anticholinergics are effective bronchodilators by blocking the muscarinic M3 receptor on airway smooth muscle. Recent evidence indicates that acetylcholine also contributes to airway inflammation. However, which muscarinic receptor subtype(s) regulates this process is unknown. In this study, the contribution of the M1, M2 and M3 receptor subtypes to cigarette smoke-induced airway inflammation was investigated by exposing muscarinic receptor subtype deficient mice to cigarette smoke for 4 days. In wild-type mice, cigarette smoke induced an increase in macrophages, neutrophils and lymphocytes in bronchoalveolar lavage fluid. Neutrophilic inflammation was higher in M1(-/-) and M2(-/-) mice compared to wild-type mice, but lower in M3(-/-) mice. Accordingly, the release of keratinocyte-derived chemokine (KC), monocyte chemotactic protein-1 and interleukin-6 was higher in M1(-/-) and M2(-/-) mice, and reduced in M3(-/-) mice. Markers of remodelling were not increased after cigarette smoke exposure. However, M3(-/-) mice had reduced expression of transforming growth factor-beta1 and matrix proteins. Cigarette smoke-induced inflammatory cell recruitment and KC release were also prevented by the M3-receptor selective antagonist 1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP) in wild-type mice. Collectively, our data indicate a pro-inflammatory role for the M3 receptor in cigarette smoke-induced neutrophilia and cytokine release, yet an anti-inflammatory role for M1 and M2 receptors.
ESTHER : Kistemaker_2013_Eur.Respir.J_42_1677
PubMedSearch : Kistemaker_2013_Eur.Respir.J_42_1677
PubMedID: 23397297

Title : Muscarinic Acetylcholine Receptor Subtypes as Potential Drug Targets for the Treatment of Schizophrenia, Drug Abuse and Parkinson's Disease - Dencker_2012_ACS.Chem.Neurosci_3_80
Author(s) : Dencker D , Thomsen M , Wortwein G , Weikop P , Cui Y , Jeon J , Wess J , Fink-Jensen A
Ref : ACS Chem Neurosci , 3 :80 , 2012
Abstract : The neurotransmitter dopamine plays important roles in modulating cognitive, affective, and motor functions. Dysregulation of dopaminergic neurotransmission is thought to be involved in the pathophysiology of several psychiatric and neurological disorders, including schizophrenia, Parkinson's disease and drug abuse. Dopaminergic systems are regulated by cholinergic, especially muscarinic, input. Not surprisingly, increasing evidence implicates muscarinic acetylcholine receptor-mediated pathways as potential targets for the treatment of these disorders classically viewed as "dopamine based". There are five known muscarinic receptor subtypes (M(1) to M(5)). Due to their overlapping expression patterns and the lack of receptor subtype-specific ligands, the roles of the individual muscarinic receptors have long remained elusive. During the past decade, studies with knock-out mice lacking specific muscarinic receptor subtypes have greatly advanced our knowledge of the physiological roles of the M(1)-M(5) receptors. Recently, new ligands have been developed that can interact with allosteric sites on different muscarinic receptor subtypes, rather than the conventional (orthosteric) acetylcholine binding site. Such agents may lead to the development of novel classes of drugs useful for the treatment of psychosis, drug abuse and Parkinson's disease. The present review highlights recent studies carried out using muscarinic receptor knock-out mice and new subtype-selective allosteric ligands to assess the roles of M(1), M(4), and M(5) receptors in various central processes that are under strong dopaminergic control. The outcome of these studies opens new perspectives for the use of novel muscarinic drugs for several severe disorders of the CNS.
ESTHER : Dencker_2012_ACS.Chem.Neurosci_3_80
PubMedSearch : Dencker_2012_ACS.Chem.Neurosci_3_80
PubMedID: 22389751

Title : An allosteric enhancer of M(4) muscarinic acetylcholine receptor function inhibits behavioral and neurochemical effects of cocaine - Dencker_2012_Psychopharmacology.(Berl)_224_277
Author(s) : Dencker D , Weikop P , Sorensen G , Woldbye DP , Wortwein G , Wess J , Fink-Jensen A
Ref : Psychopharmacology (Berl) , 224 :277 , 2012
Abstract : RATIONALE: The mesostriatal dopamine system plays a key role in mediating the reinforcing effects of psychostimulant drugs like cocaine. The muscarinic M(4) acetylcholine receptor subtype is centrally involved in the regulation of dopamine release in striatal areas. Consequently, striatal M(4) receptors could be a novel target for modulating psychostimulant effects of cocaine. OBJECTIVES: For the first time, we here addressed this issue by investigating the effects of a novel selective positive allosteric modulator of M(4) receptors, VU0152100, on cocaine-induced behavioral and neurochemical effects in mice.
METHODS: To investigate the effect of VU0152100 on the acute reinforcing effects of cocaine, we use an acute cocaine self-administration model. We used in vivo microdialysis to investigate whether the effects of VU0152100 in the behavioral studies were mediated via effects on dopaminergic neurotransmission. In addition, the effect of VU0152100 on cocaine-induced hyperactivity and rotarod performance was evaluated.
RESULTS: We found that VU0152100 caused a prominent reduction in cocaine self-administration, cocaine-induced hyperlocomotion, and cocaine-induced striatal dopamine increase, without affecting motor performance. Consistent with these effects of VU0152100 being mediated via M(4) receptors, its inhibitory effects on cocaine-induced increases in striatal dopamine were abolished in M(4) receptor knockout mice. Furthermore, selective deletion of the M(4) receptor gene in dopamine D(1) receptor-expressing neurons resulted in a partial reduction of the VU0152100 effect, indicating that VU0152100 partly regulates dopaminergic neurotransmission via M(4) receptors co-localized with D(1) receptors.
CONCLUSIONS: These results show that positive allosteric modulators of the M(4) receptor deserve attention as agents in the future treatment of cocaine abuse.
ESTHER : Dencker_2012_Psychopharmacology.(Berl)_224_277
PubMedSearch : Dencker_2012_Psychopharmacology.(Berl)_224_277
PubMedID: 22648127

Title : Structure and dynamics of the M3 muscarinic acetylcholine receptor - Kruse_2012_Nature_482_552
Author(s) : Kruse AC , Hu J , Pan AC , Arlow DH , Rosenbaum DM , Rosemond E , Green HF , Liu T , Chae PS , Dror RO , Shaw DE , Weis WI , Wess J , Kobilka BK
Ref : Nature , 482 :552 , 2012
Abstract : 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(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(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.
ESTHER : Kruse_2012_Nature_482_552
PubMedSearch : Kruse_2012_Nature_482_552
PubMedID: 22358844

Title : Novel muscarinic receptor mutant mouse models - Wess_2012_Handb.Exp.Pharmacol_208_95
Author(s) : Wess J
Ref : Handbook of Experimental Pharmacology , :95 , 2012
Abstract : Muscarinic acetylcholine (ACh) receptors (mAChRs; M(1)-M(5)) regulate the activity of an extraordinarily large number of important physiological processes. During the past 10-15 years, studies with whole-body M(1)-M(5) mAChR knockout mice have provided many new insights into the physiological and pathophysiological roles of the individual mAChR subtypes. This review will focus on the characterization of a novel generation of mAChR mutant mice, including mice in which distinct mAChR genes have been excised in a tissue- or cell type-specific fashion, various transgenic mouse lines that overexpress wild-type or different mutant M(3) mAChRs in certain tissues or cells only, as well as a novel M(3) mAChR knockin mouse strain deficient in agonist-induced M(3) mAChR phosphorylation. Phenotypic analysis of these new animal models has greatly advanced our understanding of the physiological roles of the various mAChR subtypes and has identified potential targets for the treatment of type 2 diabetes, schizophrenia, Parkinson's disease, drug addiction, cognitive disorders, and several other pathophysiological conditions.
ESTHER : Wess_2012_Handb.Exp.Pharmacol_208_95
PubMedSearch : Wess_2012_Handb.Exp.Pharmacol_208_95
PubMedID: 22222697

Title : Presynaptic muscarinic M(2) receptors modulate glutamatergic transmission in the bed nucleus of the stria terminalis - Guo_2012_Neuropharmacol_62_1671
Author(s) : Guo JD , Hazra R , Dabrowska J , Muly EC , Wess J , Rainnie DG
Ref : Neuropharmacology , 62 :1671 , 2012
Abstract : The anterolateral cell group of the bed nucleus of the stria terminalis (BNST(ALG)) serves as an important relay station in stress circuitry. Limbic inputs to the BNST(ALG) are primarily glutamatergic and activity-dependent changes in this input have been implicated in abnormal behaviors associated with chronic stress and addiction. Significantly, local infusion of acetylcholine (ACh) receptor agonists into the BNST trigger stress-like cardiovascular responses, however, little is known about the effects of these agents on glutamatergic transmission in the BNST(ALG). Here, we show that glutamate- and ACh-containing fibers are found in close association in the BNST(ALG). Moreover, in the presence of the acetylcholinesterase inhibitor, eserine, endogenous ACh release evoked a long-lasting reduction of the amplitude of stimulus-evoked EPSCs. This effect was mimicked by exogenous application of the ACh analog, carbachol, which caused a reversible, dose-dependent, reduction of the evoked EPSC amplitude, and an increase in both the paired-pulse ratio and coefficient of variation, suggesting a presynaptic site of action. Uncoupling of postsynaptic G-proteins with intracellular GDP-beta-S, or application of the nicotinic receptor antagonist, tubocurarine, failed to block the carbachol effect. In contrast, the carbachol effect was blocked by prior application of atropine or M(2) receptor-preferring antagonists, and was absent in M(2)/M(4) receptor knockout mice, suggesting that presynaptic M(2) receptors mediate the effect of ACh. Immunoelectron microscopy studies further revealed the presence of M(2) receptors on axon terminals that formed asymmetric synapses with BNST neurons. Our findings suggest that presynaptic M(2) receptors might be an important modulator of the stress circuit and hence a novel target for drug development.
ESTHER : Guo_2012_Neuropharmacol_62_1671
PubMedSearch : Guo_2012_Neuropharmacol_62_1671
PubMedID: 22166222

Title : Critical metabolic roles of beta-cell M3 muscarinic acetylcholine receptors - Ruiz_2012_Life.Sci_91_986
Author(s) : Ruiz de Azua I , Gautam D , Jain S , Guettier JM , Wess J
Ref : Life Sciences , 91 :986 , 2012
Abstract : Muscarinic acetylcholine (ACh) receptors (mAChRs; M(1)-M(5)) regulate the activity of an extraordinarily large number of important physiological processes. We and others previously demonstrated that pancreatic beta-cells are endowed with M(3) mAChRs which are linked to G proteins of the G(q) family. The activation of these receptors by ACh or other muscarinic agonists leads to the augmentation of glucose-induced insulin release via multiple mechanisms. Interestingly, in humans, ACh acting on human beta-cell mAChRs is released from adjacent alpha-cells which express both choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (vAChT), indicative of the presence of a non-neuronal cholinergic system in human pancreatic islets. In order to shed light on the physiological roles of beta-cell M(3) receptors, we recently generated and analyzed various mutant mouse models. Specifically, we carried out studies with mice which overexpressed M(3) receptors or mutant M(3) receptors in pancreatic beta-cells or which selectively lacked M(3) receptors or M(3)-receptor-associated proteins in pancreatic beta-cells. Our findings indicate that beta-cell M(3) receptors play a key role in maintaining proper insulin release and whole body glucose homeostasis and that strategies aimed at enhancing signaling through beta-cell M(3) receptors may prove useful to improve beta-cell function for the treatment of type 2 diabetes (T2D).
ESTHER : Ruiz_2012_Life.Sci_91_986
PubMedSearch : Ruiz_2012_Life.Sci_91_986
PubMedID: 22525375

Title : Regulation of M(3) muscarinic receptor expression and function by transmembrane protein 147 - Rosemond_2011_Mol.Pharmacol_79_251
Author(s) : Rosemond E , Rossi M , McMillin SM , Scarselli M , Donaldson JG , Wess J
Ref : Molecular Pharmacology , 79 :251 , 2011
Abstract : The M(3) muscarinic acetylcholine receptor (M3R) regulates many fundamental physiological functions. To identify novel M3R-interacting proteins, we used a recently developed yeast two-hybrid screen (split ubiquitin method) to detect interactions among membrane proteins. This screen led to the identification of many novel M3R-associated proteins, including the putative membrane protein transmembrane protein 147 (Tmem147). The amino acid sequence of Tmem147 is highly conserved among mammals, but its physiological roles are unknown at present. We initially demonstrated that Tmem147 could be coimmunoprecipitated with M3Rs in cotransfected mammalian cells (COS-7 cells). Confocal imaging studies showed that Tmem147 was localized to endoplasmic reticulum (ER) membranes and that the Tmem147/M3R interaction occurred in the ER of cotransfected COS-7 cells, resulting in impaired trafficking of the M3R to the cell surface. To study the role of Tmem147 in modulating M3R function in a more physiologically relevant setting, we carried out studies with H508 human colon cancer cells that endogenously express M3Rs and Tmem147. Treatment of H508 cells with carbachol, a hydrolytically stable acetylcholine analog, promoted H508 cell proliferation and activation of the mitogenic kinase, p90RSK. Small interfering RNA-mediated knockdown of Tmem147 expression significantly augmented the stimulatory effects of carbachol on H508 cell proliferation and p90RSK activation. These effects were associated with an increase in the density of cell surface M3Rs. Our data clearly indicate that Tmem147 represents a potent negative regulator of M3R function, most likely by interacting with M3Rs in an intracellular compartment (ER). These findings may lead to new strategies aimed at modulating M3R activity for therapeutic purposes.
ESTHER : Rosemond_2011_Mol.Pharmacol_79_251
PubMedSearch : Rosemond_2011_Mol.Pharmacol_79_251
PubMedID: 21056967

Title : Identification of the muscarinic acetylcholine receptor subtype mediating cholinergic vasodilation in murine retinal arterioles - Gericke_2011_Invest.Ophthalmol.Vis.Sci_52_7479
Author(s) : Gericke A , Sniatecki JJ , Goloborodko E , Steege A , Zavaritskaya O , Vetter JM , Grus FH , Patzak A , Wess J , Pfeiffer N
Ref : Invest Ophthalmol Vis Sci , 52 :7479 , 2011
Abstract : PURPOSE: To identify the muscarinic acetylcholine receptor subtype that mediates cholinergic vasodilation in murine retinal arterioles. METHODS: Muscarinic receptor gene expression was determined in murine retinal arterioles using real-time PCR. To assess the functional relevance of muscarinic receptors for mediating vascular responses, retinal vascular preparations from muscarinic receptor-deficient mice were studied in vitro. Changes in luminal arteriole diameter in response to muscarinic and nonmuscarinic vasoactive substances were measured by video microscopy. RESULTS: Only mRNA for the M(3) receptor was detected in retinal arterioles. Thus, M(3) receptor-deficient mice (M3R(-/-)) and respective wild-type controls were used for functional studies. Acetylcholine concentration-dependently dilated retinal arterioles from wild-type mice. In contrast, vasodilation to acetylcholine was almost completely abolished in retinal arterioles from M3R(-/-) mice, whereas responses to the nitric oxide (NO) donor nitroprusside were retained. Carbachol, an acetylcholinesterase-resistant analog of acetylcholine, also evoked dilation in retinal arterioles from wild-type, but not from M3R(-/-), mice. Vasodilation responses from wild-type mice to acetylcholine were negligible after incubation with the non-subtype-selective muscarinic receptor blocker atropine or the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester, and were even reversed to contraction after endothelial damage with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. CONCLUSIONS: These findings provide evidence that endothelial M(3) receptors mediate cholinergic vasodilation in murine retinal arterioles via activation of NO synthase.
ESTHER : Gericke_2011_Invest.Ophthalmol.Vis.Sci_52_7479
PubMedSearch : Gericke_2011_Invest.Ophthalmol.Vis.Sci_52_7479
PubMedID: 21873683

Title : Involvement of a subpopulation of neuronal M4 muscarinic acetylcholine receptors in the antipsychotic-like effects of the M1\/M4 preferring muscarinic receptor agonist xanomeline - Dencker_2011_J.Neurosci_31_5905
Author(s) : Dencker D , Wortwein G , Weikop P , Jeon J , Thomsen M , Sager TN , Mork A , Woldbye DP , Wess J , Fink-Jensen A
Ref : Journal of Neuroscience , 31 :5905 , 2011
Abstract : Disturbances in central dopaminergic neurotransmission are believed to be centrally involved in the pathogenesis of schizophrenia. Central dopaminergic and cholinergic systems interact and the cholinergic muscarinic agonist xanomeline has shown antipsychotic effects in clinical studies. Preclinical studies indicate that the M(4) muscarinic cholinergic receptor subtype (mAChR) modulates the activity of the dopaminergic system and that this specific mAChR subtype is involved in mediating the antipsychotic-like effects of xanomeline. A specific neuronal subpopulation that expresses M(4) mAChRs together with D(1) dopamine receptors seems to be especially important in modulating dopamine-dependent behaviors. Using mutant mice that lack the M(4) mAChR only in D(1) dopamine receptor-expressing cells (D1-M4-KO), we investigated the role of this neuronal population in the antipsychotic-like effects of xanomeline in amphetamine-induced hyperactivity and apomorphine-induced climbing. Interestingly, the antipsychotic-like effects of xanomeline in the two models were almost completely abolished in D1-M4-KO mice, suggesting that M(4) mAChRs colocalized with D(1) dopamine receptors are centrally involved in mediating the antipsychotic-like effects of xanomeline. This is consistent with the hypothesis that activation of the M(4) mAChR represents a potential target for the future medical treatment of psychosis.
ESTHER : Dencker_2011_J.Neurosci_31_5905
PubMedSearch : Dencker_2011_J.Neurosci_31_5905
PubMedID: 21508215

Title : Antipsychotic-induced catalepsy is attenuated in mice lacking the M4 muscarinic acetylcholine receptor - Fink-Jensen_2011_Eur.J.Pharmacol_656_39
Author(s) : Fink-Jensen A , Schmidt LS , Dencker D , Schulein C , Wess J , Wortwein G , Woldbye DP
Ref : European Journal of Pharmacology , 656 :39 , 2011
Abstract : A delicate balance exists between the central dopaminergic and cholinergic neurotransmitter systems with respect to motor function. An imbalance can result in motor dysfunction as observed in Parkinson's disease patients and in patients treated with antipsychotic compounds. Cholinergic receptor antagonists can alleviate extrapyramidal symptoms in Parkinson's disease and motor side effects induced by antipsychotics. The effects of anticholinergics are mediated by muscarinic receptors of which five subtypes (M(1)-M(5)) exist. Muscarinic M(4) receptors are found at high concentrations in motor parts of the striatum, suggesting a role for muscarinic M(4) receptors in the motor side effects of antipsychotics, and in the alleviation of these side effects by anticholinergics. Here we investigated the potential role of the muscarinic M(4) receptor in catalepsy induced by antipsychotics (haloperidol and risperidone) as well as the anti-cataleptic effects of the non-selective anticholinergic drug scopolamine in fully backcrossed muscarinic M(4) receptor knockout mice. The drug-induced catalepsy was strongly attenuated, but not abolished, in M(4) knockout mice as compared to wild-type controls. Scopolamine further attenuated the cataleptic response in M(4) knockout mice, suggesting that non-M(4) muscarinic receptors also participate in the anti-cataleptic effects. In conclusion, these data indicate an important role for M(4) receptors in antipsychotic-induced motor side effects and suggest that M(4) receptors could be a target for future pharmacological treatment of antipsychotic-induced as well as idiopathic parkinsonism.
ESTHER : Fink-Jensen_2011_Eur.J.Pharmacol_656_39
PubMedSearch : Fink-Jensen_2011_Eur.J.Pharmacol_656_39
PubMedID: 21269601

Title : A novel fast mechanism for GPCR-mediated signal transduction--control of neurotransmitter release - Kupchik_2011_J.Cell.Biol_192_137
Author(s) : Kupchik YM , Barchad-Avitzur O , Wess J , Ben-Chaim Y , Parnas I , Parnas H
Ref : Journal of Cell Biology , 192 :137 , 2011
Abstract : Reliable neuronal communication depends on accurate temporal correlation between the action potential and neurotransmitter release. Although a requirement for Ca(2+) in neurotransmitter release is amply documented, recent studies have shown that voltage-sensitive G protein-coupled receptors (GPCRs) are also involved in this process. However, how slow-acting GPCRs control fast neurotransmitter release is an unsolved question. Here we examine whether the recently discovered fast depolarization-induced charge movement in the M(2)-muscarinic receptor (M(2)R) is responsible for M(2)R-mediated control of acetylcholine release. We show that inhibition of the M(2)R charge movement in Xenopus oocytes correlated well with inhibition of acetylcholine release at the mouse neuromuscular junction. Our results suggest that, in addition to Ca(2+) influx, charge movement in GPCRs is also necessary for release control.
ESTHER : Kupchik_2011_J.Cell.Biol_192_137
PubMedSearch : Kupchik_2011_J.Cell.Biol_192_137
PubMedID: 21200029

Title : Increased cocaine self-administration in M4 muscarinic acetylcholine receptor knockout mice - Schmidt_2011_Psychopharmacology.(Berl)_216_367
Author(s) : Schmidt LS , Thomsen M , Weikop P , Dencker D , Wess J , Woldbye DP , Wortwein G , Fink-Jensen A
Ref : Psychopharmacology (Berl) , 216 :367 , 2011
Abstract : RATIONALE: The reinforcing effects of cocaine are mediated by the mesolimbic dopamine system. Behavioral and neurochemical studies have shown that the cholinergic muscarinic M(4) receptor subtype plays an important role in regulation of dopaminergic neurotransmission. OBJECTIVES: Here we investigated for the first time the involvement of M(4) receptors in the reinforcing effects of cocaine using chronic intravenous cocaine self-administration in extensively backcrossed M(4) receptor knockout (M(4) (-/-)) mice.
METHODS: We evaluated acquisition of cocaine self-administration in experimentally naive mice. Both cocaine self-administration and food-maintained operant behavior were evaluated under fixed ratio 1 (FR 1) and progressive ratio (PR) schedules of reinforcement. In addition, cocaine-induced dopamine release and cocaine-induced hyperactivity were evaluated.
RESULTS: M(4) (-/-) mice earned significantly more cocaine reinforcers and reached higher breaking points than their wild-type littermates (M(4) (+/+)) at intermediate doses of cocaine under both FR 1 and PR schedules of reinforcement. Under the PR schedule, M(4) (-/-) mice exhibited significantly higher response rates at the lowest liquid food concentration. In accordance with these results, cocaine-induced dopamine efflux in the nucleus accumbens and hyperlocomotion were increased in M(4) (-/-) mice compared to M(4) (+/+) mice.
CONCLUSIONS: Our data suggest that M(4) receptors play an important role in regulation of the reward circuitry and may serve as a new target in the medical treatment of drug addiction.
ESTHER : Schmidt_2011_Psychopharmacology.(Berl)_216_367
PubMedSearch : Schmidt_2011_Psychopharmacology.(Berl)_216_367
PubMedID: 21373792

Title : Striatal muscarinic receptors promote activity dependence of dopamine transmission via distinct receptor subtypes on cholinergic interneurons in ventral versus dorsal striatum - Threlfell_2010_J.Neurosci_30_3398
Author(s) : Threlfell S , Clements MA , Khodai T , Pienaar IS , Exley R , Wess J , Cragg SJ
Ref : Journal of Neuroscience , 30 :3398 , 2010
Abstract : 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 signaling 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 (arecaidine propargyl ester tosylate)] decreased DA release evoked by low-frequency stimuli (1-10 Hz, four pulses) but increased the sensitivity of DA release to presynaptic activity, even enhancing release by high frequencies (e.g., >25 Hz for four pulses). These bidirectional effects depended on ACh input to striatal nicotinic receptors (nAChRs) on DA axons but not GABA or glutamate input. In caudate-putamen (CPu), knock-out of M(2)- or M(4)-mAChRs (not M(5)) prevented mAChR control of DA, indicating that M(2)- and M(4)-mAChRs are required. In nucleus accumbens (NAc) core or shell, mAChR function was prevented in M(4)-knock-outs, but not M(2)- or M(5)-knock-outs. 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 M(2)/M(4)-mAChRs to the control of DA release in CPu versus NAc suggests targets to influence DA/ACh function differentially between striatal domains.
ESTHER : Threlfell_2010_J.Neurosci_30_3398
PubMedSearch : Threlfell_2010_J.Neurosci_30_3398
PubMedID: 20203199

Title : M3-muscarinic receptor promotes insulin release via receptor phosphorylation\/arrestin-dependent activation of protein kinase D1 - Kong_2010_Proc.Natl.Acad.Sci.U.S.A_107_21181
Author(s) : Kong KC , Butcher AJ , McWilliams P , Jones D , Wess J , Hamdan FF , Werry T , Rosethorne EM , Charlton SJ , Munson SE , Cragg HA , Smart AD , Tobin AB
Ref : Proc Natl Acad Sci U S A , 107 :21181 , 2010
Abstract : The activity of G protein-coupled receptors is regulated via hyper-phosphorylation following agonist stimulation. Despite the universal nature of this regulatory process, the physiological impact of receptor phosphorylation remains poorly studied. To address this question, we have generated a knock-in mouse strain that expresses a phosphorylation-deficient mutant of the M(3)-muscarinic receptor, a prototypical G(q/11)-coupled receptor. This mutant mouse strain was used here to investigate the role of M(3)-muscarinic receptor phosphorylation in the regulation of insulin secretion from pancreatic islets. Importantly, the phosphorylation deficient receptor coupled to G(q/11)-signaling pathways but was uncoupled from phosphorylation-dependent processes, such as receptor internalization and beta-arrestin recruitment. The knock-in mice showed impaired glucose tolerance and insulin secretion, indicating that M(3)-muscarinic receptors expressed on pancreatic islets regulate glucose homeostasis via receptor phosphorylation-/arrestin-dependent signaling. The mechanism centers on the activation of protein kinase D1, which operates downstream of the recruitment of beta-arrestin to the phosphorylated M(3)-muscarinic receptor. In conclusion, our findings support the unique concept that M(3)-muscarinic receptor-mediated augmentation of sustained insulin release is largely independent of G protein-coupling but involves phosphorylation-/arrestin-dependent coupling of the receptor to protein kinase D1.
ESTHER : Kong_2010_Proc.Natl.Acad.Sci.U.S.A_107_21181
PubMedSearch : Kong_2010_Proc.Natl.Acad.Sci.U.S.A_107_21181
PubMedID: 21078968

Title : Muscarinic signaling in the cochlea: presynaptic and postsynaptic effects on efferent feedback and afferent excitability - Maison_2010_J.Neurosci_30_6751
Author(s) : Maison SF , Liu XP , Vetter DE , Eatock RA , Nathanson NM , Wess J , Liberman MC
Ref : Journal of Neuroscience , 30 :6751 , 2010
Abstract : Acetylcholine is the major neurotransmitter of the olivocochlear efferent system, which provides feedback to cochlear hair cells and sensory neurons. To study the role of cochlear muscarinic receptors, we studied receptor localization with immunohistochemistry and reverse transcription-PCR and measured olivocochlear function, cochlear responses, and histopathology in mice with targeted deletion of each of the five receptor subtypes. M2, M4, and M5 were detected in microdissected immature (postnatal days 10-13) inner hair cells and spiral ganglion cells but not outer hair cells. In the adult (6 weeks), the same transcripts were found in microdissected organ of Corti and spiral ganglion samples. M2 protein was found, by immunohistochemistry, in olivocochlear fibers in both outer and inner hair cell areas. M3 mRNA was amplified only from whole cochleas, and M1 message was never seen in wild-type ears. Auditory brainstem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs) were unaffected by loss of Gq-coupled receptors (M1, M3, or M5), as were shock-evoked olivocochlear effects and vulnerability to acoustic injury. In contrast, loss of Gi-coupled receptors (M2 and/or M4) decreased neural responses without affecting DPOAEs (at low frequencies). This phenotype and the expression pattern are consistent with excitatory muscarinic signaling in cochlear sensory neurons. At high frequencies, both ABRs and DPOAEs were attenuated by loss of M2 and/or M4, and the vulnerability to acoustic injury was dramatically decreased. This aspect of the phenotype and the expression pattern are consistent with a presynaptic role for muscarinic autoreceptors in decreasing ACh release from olivocochlear terminals during high-level acoustic stimulation and suggest that muscarinic antagonists could enhance the resistance of the inner ear to noise-induced hearing loss.
ESTHER : Maison_2010_J.Neurosci_30_6751
PubMedSearch : Maison_2010_J.Neurosci_30_6751
PubMedID: 20463237

Title : A subpopulation of neuronal M4 muscarinic acetylcholine receptors plays a critical role in modulating dopamine-dependent behaviors - Jeon_2010_J.Neurosci_30_2396
Author(s) : Jeon J , Dencker D , Wortwein G , Woldbye DP , Cui Y , Davis AA , Levey AI , Schutz G , Sager TN , Mork A , Li C , Deng CX , Fink-Jensen A , Wess J
Ref : Journal of Neuroscience , 30 :2396 , 2010
Abstract : Acetylcholine (ACh) regulates many key functions of the CNS by activating cell surface receptors referred to as muscarinic ACh receptors (M(1)-M(5) mAChRs). Like other mAChR subtypes, the M(4) mAChR is widely expressed in different regions of the forebrain. Interestingly, M(4) mAChRs are coexpressed with D(1) dopamine receptors in a specific subset of striatal projection neurons. To investigate the physiological relevance of this M(4) mAChR subpopulation in modulating dopamine-dependent behaviors, we used Cre/loxP technology to generate mutant mice that lack M(4) mAChRs only in D(1) dopamine receptor-expressing cells. The newly generated mutant mice displayed several striking behavioral phenotypes, including enhanced hyperlocomotor activity and increased behavioral sensitization following treatment with psychostimulants. These behavioral changes were accompanied by a lack of muscarinic inhibition of D(1) dopamine receptor-mediated cAMP stimulation in the striatum and an increase in dopamine efflux in the nucleus accumbens. These novel findings demonstrate that a distinct subpopulation of neuronal M(4) mAChRs plays a critical role in modulating several important dopamine-dependent behaviors. Since enhanced central dopaminergic neurotransmission is a hallmark of several severe disorders of the CNS, including schizophrenia and drug addiction, our findings have substantial clinical relevance.
ESTHER : Jeon_2010_J.Neurosci_30_2396
PubMedSearch : Jeon_2010_J.Neurosci_30_2396
PubMedID: 20147565

Title : Attenuation of cocaine's reinforcing and discriminative stimulus effects via muscarinic M1 acetylcholine receptor stimulation - Thomsen_2010_J.Pharmacol.Exp.Ther_332_959
Author(s) : Thomsen M , Conn PJ , Lindsley C , Wess J , Boon JY , Fulton BS , Fink-Jensen A , Caine SB
Ref : Journal of Pharmacology & Experimental Therapeutics , 332 :959 , 2010
Abstract : Muscarinic cholinergic receptors modulate dopaminergic function in brain pathways thought to mediate cocaine's abuse-related effects. Here, we sought to confirm and extend in the mouse species findings that nonselective muscarinic receptor antagonists can enhance cocaine's discriminative stimulus. More importantly, we tested the hypothesis that muscarinic receptor agonists with varied receptor subtype selectivity can blunt cocaine's discriminative stimulus and reinforcing effects; we hypothesized a critical role for the M(1) and/or M(4) receptor subtypes in this modulation. Mice were trained to discriminate cocaine from saline, or to self-administer intravenous cocaine chronically. The nonselective muscarinic antagonists scopolamine and methylscopolamine, the nonselective muscarinic agonists oxotremorine and pilocarpine, the M(1)/M(4)-preferring agonist xanomeline, the putative M(1)-selective agonist (4-hydroxy-2-butynyl)-1-trimethylammonium-3-chlorocarbanilate chloride (McN-A-343), and the novel M(1)-selective agonist 1-(1-2-methylbenzyl)-1,4-bipiperidin-4-yl)-1H benzo[d]imidazol-2(3H)-one (TBPB) were tested as substitution and/or pretreatment to cocaine. Both muscarinic antagonists partially substituted for cocaine and enhanced its discriminative stimulus. Conversely, muscarinic agonists blunted cocaine discrimination and abolished cocaine self-administration with varying effects on food-maintained behavior. Specifically, increasing selectivity for the M(1) subtype (oxotremorine < xanomeline < TBPB) conferred lesser nonspecific rate-suppressing effects, with no rate suppression for TBPB. In mutant mice lacking M(1) and M(4) receptors, xanomeline failed to diminish cocaine discrimination while rate-decreasing effects were intact. Our data suggest that central M(1) receptor activation attenuates cocaine's abuse-related effects, whereas non-M(1)/M(4) receptors probably contribute to undesirable effects of muscarinic stimulation. These data provide the first demonstration of anticocaine effects of systemically applied, M(1) receptor agonists and suggest the possibility of a new approach to pharmacotherapy for cocaine addiction.
ESTHER : Thomsen_2010_J.Pharmacol.Exp.Ther_332_959
PubMedSearch : Thomsen_2010_J.Pharmacol.Exp.Ther_332_959
PubMedID: 19996296

Title : The M3-muscarinic receptor regulates learning and memory in a receptor phosphorylation\/arrestin-dependent manner - Poulin_2010_Proc.Natl.Acad.Sci.U.S.A_107_9440
Author(s) : Poulin B , Butcher A , McWilliams P , Bourgognon JM , Pawlak R , Kong KC , Bottrill A , Mistry S , Wess J , Rosethorne EM , Charlton SJ , Tobin AB
Ref : Proc Natl Acad Sci U S A , 107 :9440 , 2010
Abstract : 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.
ESTHER : Poulin_2010_Proc.Natl.Acad.Sci.U.S.A_107_9440
PubMedSearch : Poulin_2010_Proc.Natl.Acad.Sci.U.S.A_107_9440
PubMedID: 20439723

Title : A chemical-genetic approach to study G protein regulation of beta cell function in vivo - Guettier_2009_Proc.Natl.Acad.Sci.U.S.A_106_19197
Author(s) : Guettier JM , Gautam D , Scarselli M , Ruiz de Azua I , Li JH , Rosemond E , Ma X , Gonzalez FJ , Armbruster BN , Lu H , Roth BL , Wess J
Ref : Proc Natl Acad Sci U S A , 106 :19197 , 2009
Abstract : Impaired functioning of pancreatic beta cells is a key hallmark of type 2 diabetes. beta cell function is modulated by the actions of different classes of heterotrimeric G proteins. The functional consequences of activating specific beta cell G protein signaling pathways in vivo are not well understood at present, primarily due to the fact that beta cell G protein-coupled receptors (GPCRs) are also expressed by many other tissues. To circumvent these difficulties, we developed a chemical-genetic approach that allows for the conditional and selective activation of specific beta cell G proteins in intact animals. Specifically, we created two lines of transgenic mice each of which expressed a specific designer GPCR in beta cells only. Importantly, the two designer receptors differed in their G protein-coupling properties (G(q/11) versus G(s)). They were unable to bind endogenous ligand(s), but could be efficiently activated by an otherwise pharmacologically inert compound (clozapine-N-oxide), leading to the conditional activation of either beta cell G(q/11) or G(s) G proteins. Here we report the findings that conditional and selective activation of beta cell G(q/11) signaling in vivo leads to striking increases in both first- and second-phase insulin release, greatly improved glucose tolerance in obese, insulin-resistant mice, and elevated beta cell mass, associated with pathway-specific alterations in islet gene expression levels. Selective stimulation of beta cell G(s) triggered qualitatively similar in vivo metabolic effects. Thus, this developed chemical-genetic strategy represents a powerful approach to study G protein regulation of beta cell function in vivo.
ESTHER : Guettier_2009_Proc.Natl.Acad.Sci.U.S.A_106_19197
PubMedSearch : Guettier_2009_Proc.Natl.Acad.Sci.U.S.A_106_19197
PubMedID: 19858481

Title : Multiple muscarinic pathways mediate the suppression of voltage-gated Ca2+ channels in mouse intestinal smooth muscle cells - Tanahashi_2009_Br.J.Pharmacol_158_1874
Author(s) : Tanahashi Y , Unno T , Matsuyama H , Ishii T , Yamada M , Wess J , Komori S
Ref : British Journal of Pharmacology , 158 :1874 , 2009
Abstract : BACKGROUND AND PURPOSE: Stimulation of muscarinic receptors in intestinal smooth muscle cells results in suppression of voltage-gated Ca2+ channel currents (I(Ca)). However, little is known about which receptor subtype(s) mediate this effect. EXPERIMENTAL APPROACH: The effect of carbachol on I(Ca) was studied in single intestinal myocytes from M2 or M3 muscarinic receptor knockout (KO) and wild-type (WT) mice. KEY
RESULTS: In M2KO cells, carbachol (100 microM) induced a sustained I(Ca) suppression as seen in WT cells. However, this suppression was significantly smaller than that seen in WT cells. Carbachol also suppressed I(Ca) in M3KO cells, but with a phasic time course. In M2/M3-double KO cells, carbachol had no effect on I(Ca). The extent of the suppression in WT cells was greater than the sum of the I(Ca) suppressions in M2KO and M3KO cells, indicating that it is not a simple mixture of M2 and M3 receptor responses. The G(i/o) inhibitor, Pertussis toxin, abolished the I(Ca) suppression in M3KO cells, but not in M2KO cells. In contrast, the G(q/11) inhibitor YM-254890 strongly inhibited only the I(Ca) suppression in M2KO cells. Suppression of I(Ca) in WT cells was markedly reduced by either Pertussis toxin or YM-254890. CONCLUSION AND IMPLICATIONS: In intestinal myocytes, M2 receptors mediate a phasic I(Ca) suppression via G(i/o) proteins, while M3 receptors mediate a sustained I(Ca) suppression via G(q/11) proteins. In addition, another pathway that requires both M2/G(i/o) and M3/G(q/11) systems may be operative in inducing a sustained I(Ca) suppression.
ESTHER : Tanahashi_2009_Br.J.Pharmacol_158_1874
PubMedSearch : Tanahashi_2009_Br.J.Pharmacol_158_1874
PubMedID: 20050185

Title : Structural basis of the selectivity of the beta(2)-adrenergic receptor for fluorinated catecholamines - Pooput_2009_Bioorg.Med.Chem_17_7987
Author(s) : Pooput C , Rosemond E , Karpiak J , Deflorian F , Vilar S , Costanzi S , Wess J , Kirk KL
Ref : Bioorganic & Medicinal Chemistry , 17 :7987 , 2009
Abstract : The important and diverse biological functions of adrenergic receptors, a subclass of G protein-coupled receptors (GPCRs), have made the search for compounds that selectively stimulate or inhibit the activity of different adrenergic receptor subtypes an important area of medicinal chemistry. We previously synthesized 2-, 5-, and 6-fluoronorepinehprine (FNE) and 2-, 5-, and 6-fluoroepinephrine (FEPI) and found that 2FNE and 2FEPI were selective beta-adrenergic agonists and that 6FNE and 6FEPI were selective alpha-adrenergic agonists, while 5FNE and 5FEPI were unselective. Agonist potencies correlated well with receptor binding affinities. Here, through a combination of molecular modeling and site-directed mutagenesis, we have identified N293 in the beta(2)-adrenergic receptor as a crucial residue for the selectivity of the receptor for catecholamines fluorinated at different positions.
ESTHER : Pooput_2009_Bioorg.Med.Chem_17_7987
PubMedSearch : Pooput_2009_Bioorg.Med.Chem_17_7987
PubMedID: 19857969

Title : An allosteric potentiator of M4 mAChR modulates hippocampal synaptic transmission - Shirey_2008_Nat.Chem.Biol_4_42
Author(s) : Shirey JK , Xiang Z , Orton D , Brady AE , Johnson KA , Williams R , Ayala JE , Rodriguez AL , Wess J , Weaver D , Niswender CM , Conn PJ
Ref : Nat Chemical Biology , 4 :42 , 2008
Abstract : Muscarinic acetylcholine receptors (mAChRs) provide viable targets for the treatment of multiple central nervous system disorders. We have used cheminformatics and medicinal chemistry to develop new, highly selective M4 allosteric potentiators. VU10010, the lead compound, potentiates the M4 response to acetylcholine 47-fold while having no activity at other mAChR subtypes. This compound binds to an allosteric site on the receptor and increases affinity for acetylcholine and coupling to G proteins. Whole-cell patch clamp recordings revealed that selective potentiation of M4 with VU10010 increases carbachol-induced depression of transmission at excitatory but not inhibitory synapses in the hippocampus. The effect was not mimicked by an inactive analog of VU10010 and was absent in M4 knockout mice. Selective regulation of excitatory transmission by M4 suggests that targeting of individual mAChR subtypes could be used to differentially regulate specific aspects of mAChR modulation of function in this important forebrain structure.
ESTHER : Shirey_2008_Nat.Chem.Biol_4_42
PubMedSearch : Shirey_2008_Nat.Chem.Biol_4_42
PubMedID: 18059262

Title : Analysis of CD8+ T cell-mediated anti-viral responses in mice with targeted deletions of the M1 or M5 muscarinic cholinergic receptors - Vezys_2007_Life.Sci_80_2330
Author(s) : Vezys V , Masopust D , Desmarets M , Wess J , Zimring JC
Ref : Life Sciences , 80 :2330 , 2007
Abstract : A number of studies have demonstrated that non-neuronal acetylcholine can play a role in the regulation of T cell function. Recently, we reported that CD8(+) T cells, from mice with a targeted deletion of the M(1) muscarinic receptor, had a defect in differentiating into cytolytic T lymphocytes when stimulated in vitro. In the current report, we analyze the in vivo function of CD8(+) T cells from mice with targeted deletions of either M(1) or M(5) muscarinic receptors. M(1) or M(5) knockout mice were infected with either lymphocytic choriomeningitis virus or vesicular stomatitis virus. Expansion of anti-viral CD8(+) T cells was monitored by staining with tetramer reagents specific for the immunodominant peptides of the viruses. No defect in expansion of CD8(+) T cells was observed in either M(1) or M(5) knockout mice. The extent to which one can draw a generalized conclusion that M(1) and M(5) are not involved in anti-viral immunity depends upon issues of antigen strength, genetic background, induction of redundant receptors, and the potential for qualitative defects in the expanded CD8(+) T cells.
ESTHER : Vezys_2007_Life.Sci_80_2330
PubMedSearch : Vezys_2007_Life.Sci_80_2330
PubMedID: 17286988

Title : Expression of muscarinic and nicotinic acetylcholine receptors in the mouse urothelium - Zarghooni_2007_Life.Sci_80_2308
Author(s) : Zarghooni S , Wunsch J , Bodenbenner M , Bruggmann D , Grando SA , Schwantes U , Wess J , Kummer W , Lips KS
Ref : Life Sciences , 80 :2308 , 2007
Abstract : Acetylcholine (ACh) and its receptors play a crucial role in bladder physiology. Here, we investigated the presence of muscarinic receptor subtypes (MR) and nicotinic acetylcholine receptor (nAChR) alpha-subunits in the mouse urothelium by RT-PCR and immunohistochemistry. With RT-PCR, we detected mRNAs coding for all of the five different MR subtypes and for the nicotinic receptor subunits alpha2, alpha4, alpha5, alpha6, alpha7, alpha9 and alpha10, whereas the alpha3-subunit was not expressed. Using immunohistochemistry, we localised a panel of acetylcholine receptors in the different layers of the murine bladder urothelium, with predominant appearance in the basal plasma membrane of the basal cell layer and in the apical membrane of the umbrella cells. M2R and subunit alpha9 were observed exclusively in the umbrella cells, whereas the MR subtypes 3-5 and the nAChR subunits alpha4, alpha7 and alpha10 were also detected in the intermediate and basal cell layers. The subunit alpha5 was localised only in the basal cell layer. In conclusion, the murine urothelium expresses multiple cholinergic receptors, including several subtypes of both MR and nAChR, which are differentially distributed among the urothelial cell types. Since these receptors have different electrophysiological and pharmacological properties, and therefore are considered to be responsible for different cellular responses to ACh, this differential distribution is expected to confer cell type-specificity of cholinergic regulation in the bladder urothelium.
ESTHER : Zarghooni_2007_Life.Sci_80_2308
PubMedSearch : Zarghooni_2007_Life.Sci_80_2308
PubMedID: 17337281

Title : Decreased prepulse inhibition and increased sensitivity to muscarinic, but not dopaminergic drugs in M5 muscarinic acetylcholine receptor knockout mice - Thomsen_2007_Psychopharmacology.(Berl)_192_97
Author(s) : Thomsen M , Wortwein G , Fink-Jensen A , Woldbye DP , Wess J , Caine SB
Ref : Psychopharmacology (Berl) , 192 :97 , 2007
Abstract : RATIONALE: Schizophrenic patients show decreased measures of sensorimotor gating, such as prepulse inhibition of startle (PPI). In preclinical models, these measures may be used to predict antipsychotic activity. While current antipsychotic drugs act largely at dopamine receptors, the muscarinic acetylcholine receptors offer promising novel pharmacotherapy targets. Of these, the M(5) receptor gene was recently implicated in susceptibility to schizophrenia. Due to the lack of selective ligands, muscarinic receptor knockout mice have been generated to elucidate the roles of the five receptor subtypes (M(1)-M(5)). OBJECTIVES: Here, we used M(5) receptor knockout (M(5)-/-) mice to investigate the involvement of M(5) receptors in behavioral measures pertinent to schizophrenia. We tested the hypothesis that disruption of M(5) receptors affected PPI or the effects of muscarinic or dopaminergic agents in PPI or psychomotor stimulation. MATERIALS AND
METHODS: We measured PPI in M(5)-/-, heterozygous and wild-type mice without drugs, and with clozapine (0.56-3.2 mg/kg) or haloperidol (0.32-3.2 mg/kg) alone, and as pretreatment to D: -amphetamine. In addition, we evaluated locomotor stimulation by the muscarinic antagonist trihexyphenidyl (0.56-56 mg/kg) and by cocaine (3.2-56 mg/kg).
RESULTS: The M(5)-/- mice showed decreased PPI relative to wild-type mice, and clozapine appeared to reduce this difference, while haloperidol increased PPI regardless of genotype. The M(5)-/- mice also showed more locomotor stimulation by trihexyphenidyl than wild-type mice, while cocaine had similar effects between genotypes.
CONCLUSIONS: These data suggest that disruption of the M(5) receptor gene affected sensorimotor gating mechanisms, increased sensitivity to clozapine and to the psychostimulant effects of muscarinic antagonists without modifying the effect of dopaminergic drugs.
ESTHER : Thomsen_2007_Psychopharmacology.(Berl)_192_97
PubMedSearch : Thomsen_2007_Psychopharmacology.(Berl)_192_97
PubMedID: 17310388

Title : Role of acetylcholine and polyspecific cation transporters in serotonin-induced bronchoconstriction in the mouse - Kummer_2006_Respir.Res_7_65
Author(s) : Kummer W , Wiegand S , Akinci S , Wessler I , Schinkel AH , Wess J , Koepsell H , Haberberger RV , Lips KS
Ref : Respir Res , 7 :65 , 2006
Abstract : BACKGROUND: It has been proposed that serotonin (5-HT)-mediated constriction of the murine trachea is largely dependent on acetylcholine (ACh) released from the epithelium. We recently 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. Hence, the hypothesis emerged that 5-HT evokes bronchoconstriction by inducing release of ACh from epithelial cells via OCTs.
METHODS: We tested this hypothesis by analysing bronchoconstriction in precision-cut murine lung slices using OCT and muscarinic ACh receptor knockout mouse strains. Epithelial ACh content was measured by HPLC, and the tissue distribution of OCT isoforms was determined by immunohistochemistry.
RESULTS: Epithelial ACh content was significantly higher in OCT1/2 double-knockout mice (42 +/- 10 % of the content of the epithelium-denuded trachea, n = 9) than in wild-type mice (16.8 +/- 3.6 %, n = 11). In wild-type mice, 5-HT (1 microM) caused a bronchoconstriction that slightly exceeded that evoked by muscarine (1 microM) in intact bronchi but amounted to only 66% of the response to muscarine after epithelium removal. 5-HT-induced bronchoconstriction was undiminished in M2/M3 muscarinic ACh receptor double-knockout mice which were entirely unresponsive to muscarine. Corticosterone (1 microM) significantly reduced 5-HT-induced bronchoconstriction in wild-type and OCT1/2 double-knockout mice, but not in OCT3 knockout mice. This effect persisted after removal of the bronchial epithelium. Immunohistochemistry localized OCT3 to the bronchial smooth muscle. CONCLUSION: The doubling of airway epithelial ACh content in OCT1/2-/- mice is consistent with the concept that OCT1 and/or 2 mediate ACh release from the respiratory epithelium. This effect, however, does not contribute to 5-HT-induced constriction of murine intrapulmonary bronchi. Instead, this activity involves 1) a non-cholinergic epithelium-dependent component, and 2) direct stimulation of bronchial smooth muscle cells, a response which is partly sensitive to acutely administered corticosterone acting on OCT3. These data provide new insights into the mechanisms involved in 5-HT-induced bronchoconstriction, including novel information about non-genomic, acute effects of corticosteroids on bronchoconstriction.
ESTHER : Kummer_2006_Respir.Res_7_65
PubMedSearch : Kummer_2006_Respir.Res_7_65
PubMedID: 16608531

Title : Role of acetylcholine and muscarinic receptors in serotonin-induced bronchoconstriction in the mouse - Kummer_2006_J.Mol.Neurosci_30_67
Author(s) : Kummer W , Wiegand S , Akinci S , Schinkel AH , Wess J , Koepsell H , Haberberger RV , Lips KS
Ref : Journal of Molecular Neuroscience , 30 :67 , 2006
Abstract : 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).
ESTHER : Kummer_2006_J.Mol.Neurosci_30_67
PubMedSearch : Kummer_2006_J.Mol.Neurosci_30_67
PubMedID: 17192631

Title : M1-M3 muscarinic acetylcholine receptor-deficient mice: novel phenotypes - Gautam_2006_J.Mol.Neurosci_30_157
Author(s) : Gautam D , Duttaroy A , Cui Y , Han SJ , Deng C , Seeger T , Alzheimer C , Wess J
Ref : Journal of Molecular Neuroscience , 30 :157 , 2006
Abstract : The five muscarinic acetylcholine receptors (M1-M5 mAChRs) mediate a very large number of important physiological functions (Caulfield, 1993; Caulfield and Birdsall, 1998; Wess, 2004). Because of the lack of small molecule ligands endowed with a high degree of receptor subtype selectivity and the fact that most tissues or cell types express two or more mAChR subtypes, identification of the physiological and pathophysiological roles of the individual mAChR subtypes has proved to be a challenging task. To overcome these difficulties, we recently generated mutant mouse lines deficient in each of the five mAChR genes (M1R-/- mice, M2R-/- mice, M3R-/- mice, etc. [Wess, 2004]). Phenotyping studies showed that each of the five mutant mouse lines displayed characteristic physiological, pharmacological, behavioral, biochemical, or neurochemical deficits (Wess, 2004). This chapter summarizes recent findings dealing with the importance of the M2mAChR for cognitive processes and the roles of the M1 and M3 mAChRs in mediating stimulation of glandular secretion.
ESTHER : Gautam_2006_J.Mol.Neurosci_30_157
PubMedSearch : Gautam_2006_J.Mol.Neurosci_30_157
PubMedID: 17192665

Title : Acetylcholine modulates cortical synaptic transmission via different muscarinic receptors, as studied with receptor knockout mice - Kuczewski_2005_J.Physiol_566_907
Author(s) : Kuczewski N , Aztiria E , Gautam D , Wess J , Domenici L
Ref : Journal de Physiologie , 566 :907 , 2005
Abstract : The central cholinergic system plays a crucial role in synaptic plasticity and spatial attention; however, the roles of the individual cholinergic receptors involved in these activities are not well understood at present. In the present study, we show that acetylcholine (ACh) can facilitate or depress synaptic transmission in occipital slices of mouse visual cortex. The precise nature of the ACh effects depends on the ACh concentration, and is input specific, as shown by stimulating different synaptic pathways. Pharmacological blockade of muscarinic receptor (mAChR) subtypes and the use of M1-M5 mAChR-deficient mice showed that specific mAChR subtypes, together with the activity of the cholinesterases (ChEs), mediate facilitation or depression of synaptic transmission. The present data suggest that local ACh, acting through mAChRs, regulates the cortical dynamics making cortical circuits respond to specific stimuli.
ESTHER : Kuczewski_2005_J.Physiol_566_907
PubMedSearch : Kuczewski_2005_J.Physiol_566_907
PubMedID: 15919709

Title : Allosteric binding sites on muscarinic acetylcholine receptors - Wess_2005_Mol.Pharmacol_68_1506
Author(s) : Wess J
Ref : Molecular Pharmacology , 68 :1506 , 2005
Abstract : In this issue of Molecular Pharmacology, Trankle 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-pyridi nium 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, Trankle 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 Trankle et al. should be of considerable general interest.
ESTHER : Wess_2005_Mol.Pharmacol_68_1506
PubMedSearch : Wess_2005_Mol.Pharmacol_68_1506
PubMedID: 16183853

Title : Reduced cocaine self-administration in muscarinic M5 acetylcholine receptor-deficient mice - Thomsen_2005_J.Neurosci_25_8141
Author(s) : Thomsen M , Woldbye DP , Wortwein G , Fink-Jensen A , Wess J , Caine SB
Ref : Journal of Neuroscience , 25 :8141 , 2005
Abstract : The reinforcing effects of cocaine have been related to increased extracellular concentrations of dopamine in the ventral striatum. Several studies suggest that M5 muscarinic receptors facilitate striatal dopamine release. We tested the hypothesis that the reinforcing effects of cocaine are decreased in M5 receptor-deficient mice using chronic intravenous cocaine self-administration in extensively backcrossed mice. We also assessed whether operant performance generally, rather than cocaine self-administration specifically, was altered in the mutant mice. To this end, we evaluated both food-maintained operant behavior and cocaine self-administration under a fixed ratio 1 and a progressive ratio (PR) schedule of reinforcement. We also evaluated acquisition of self-administration in experimentally naive mice using several doses of cocaine. M5 receptor deletion decreased self-administration of low to moderate doses of cocaine under a PR schedule of reinforcement and diminished acquisition of self-administration of a low dose in experimentally naive mice. We found no differences between genotypes in food-maintained behavior. The present study extends our previous findings using backcrossed mice and covering various experimental conditions. Our results indicate that M5 receptor deletion diminished the reinforcing effects of low doses of cocaine and identified specific conditions under which this may be observed.
ESTHER : Thomsen_2005_J.Neurosci_25_8141
PubMedSearch : Thomsen_2005_J.Neurosci_25_8141
PubMedID: 16148222

Title : Depolarization initiates phasic acetylcholine release by relief of a tonic block imposed by presynaptic M2 muscarinic receptors - Parnas_2005_J.Neurophysiol_93_3257
Author(s) : Parnas H , Slutsky I , Rashkovan G , Silman I , Wess J , Parnas I
Ref : Journal of Neurophysiology , 93 :3257 , 2005
Abstract : The role of presynaptic muscarinic autoreceptors in the initiation of phasic acetylcholine (ACh) release at frog and mouse neuromuscular junctions was studied by measuring the dependency of the amount (m) of ACh release on the level of presynaptic depolarization. Addition of methoctramine (a blocker of M2 muscarinic receptors), or of acetylcholinesterase (AChE), increased release in a voltage-dependent manner; enhancement of release declined as the depolarizing pulse amplitude increased. In frogs and wild-type mice the slope of log m/log pulse amplitude (PA) was reduced from about 7 in the control to about 4 in the presence of methoctramine or AChE. In M2 muscarinic receptor knockout mice, the slope of log m/log PA was much smaller (about 4) and was not further reduced by addition of either methoctramine or AChE. The effect of a brief (0.1 ms), but strong (-1.2 microA) depolarizing prepulse on the dependency of m on PA was also studied. The depolarizing prepulse had effects similar to those of methoctramine and AChE. In particular, it enhanced release of test pulses in a voltage-dependent manner and reduced the slope of log m/log PA from about 7 to about 4. Methoctramine + AChE occluded the prepulse effects. In knockout mice, the depolarizing prepulse had no effects. The cumulative results suggest that initiation of phasic ACh release is achieved by depolarization-mediated relief of a tonic block imposed by presynaptic M2 muscarinic receptors.
ESTHER : Parnas_2005_J.Neurophysiol_93_3257
PubMedSearch : Parnas_2005_J.Neurophysiol_93_3257
PubMedID: 15703226

Title : M2 muscarinic acetylcholine receptor knock-out mice show deficits in behavioral flexibility, working memory, and hippocampal plasticity - Seeger_2004_J.Neurosci_24_10117
Author(s) : Seeger T , Fedorova I , Zheng F , Miyakawa T , Koustova E , Gomeza J , Basile AS , Alzheimer C , Wess J
Ref : Journal of Neuroscience , 24 :10117 , 2004
Abstract : Muscarinic acetylcholine receptors are known to play key roles in facilitating cognitive processes. However, the specific roles of the individual muscarinic receptor subtypes (M1-M5) in learning and memory are not well understood at present. In the present study, we used wild-type (M2+/+) and M2 receptor-deficient (M2-/-) mice to examine the potential role of M2 receptors in learning and memory and hippocampal synaptic plasticity. M2-/- mice showed significant deficits in behavioral flexibility and working memory in the Barnes circular maze and the T-maze delayed alternation tests, respectively. The behavioral deficits of M2-/- mice were associated with profound changes in neuronal plasticity studied at the Schaffer-CA1 synapse of hippocampal slices. Strikingly, short-term potentiation (STP) was abolished, and long-term potentiation (LTP) was drastically reduced after high-frequency stimulation of M2-/- hippocampi. Treatment of M2-/- hippocampal slices with the GABA(A) receptor antagonist, bicuculline, restored STP and significantly increased LTP. Whole-cell recordings from CA1 pyramidal cells demonstrated a much stronger disinhibition of GABAergic than glutamatergic transmission in M2-/- hippocampi, which was particularly prominent during stimulus trains. Increased strength of GABAergic inhibition is thus a likely mechanism underlying the impaired synaptic plasticity observed with M2-/- hippocampi. Moreover, the persistent enhancement of excitatory synaptic transmission in CA1 pyramidal cells induced by the transient application of a low concentration of a muscarinic agonist (referred to as LTP(m)) was totally abolished in M2-/- mice. Because impaired muscarinic cholinergic neurotransmission is associated with Alzheimer's disease and normal aging processes, these findings should be of considerable therapeutic relevance.
ESTHER : Seeger_2004_J.Neurosci_24_10117
PubMedSearch : Seeger_2004_J.Neurosci_24_10117
PubMedID: 15537882

Title : Synergistic control of keratinocyte adhesion through muscarinic and nicotinic acetylcholine receptor subtypes - Nguyen_2004_Exp.Cell.Res_294_534
Author(s) : Nguyen VT , Chernyavsky AI , Arredondo J , Bercovich D , Orr-Urtreger A , Vetter DE , Wess J , Beaudet AL , Kitajima Y , Grando SA
Ref : Experimental Cell Research , 294 :534 , 2004
Abstract : The biological mechanisms involved in initiating, coordinating, and ultimately terminating cell-cell adhesion in the stratified epithelium are not well understood at present. This study was designed to elucidate the roles of the muscarinic M3, the nicotinic alpha3, and the mixed muscarinic-nicotinic alpha9 acetylcholine receptors in physiologic control of keratinocyte adhesion. Both muscarinic and nicotinic antagonists caused keratinocyte detachment and reversibly increased the permeability of keratinocyte monolayers, indicative of the involvement of both muscarinic and nicotinic pathways in the cholinergic control of keratinocyte adhesion. Since phosphorylation of adhesion proteins plays an important role in rapid assembly and disassembly of intercellular junctions, we measured muscarinic and nicotinic effects on phosphorylation of keratinocyte adhesion molecules. The phosphorylation levels of E-cadherin, beta-catenin, and gamma-catenin increased following pharmacological blockage of muscarinic receptors. Long-term blocking of alpha3, alpha9, and M3 receptor signaling pathways with antisense oligonucleotides resulted in cell-cell detachment and changes in the expression levels of E-cadherin, beta-catenin, and gamma-catenin in cultured human keratinocytes. Simultaneous inhibition of several receptor subtypes with a mixture of antisense oligonucleotides produced intensified abnormalities with cell adhesion. Moreover, altered cell-cell adhesion was found in the stratified epithelium of alpha3, alpha9, and M3 receptor knockout mice. Keratinocytes from these mice exhibited abnormal expression of adhesion molecules at both the protein and the mRNA levels. Thus, our data indicate that the alpha3, alpha9, and M3 acetylcholine receptors play key roles in regulating in a synergistic mode keratinocyte adhesion, most probably by modulating cadherin and catenin levels and activities. These findings may aid in the development of novel methods useful for the treatment of skin adhesion diseases and tumor metastasis.
ESTHER : Nguyen_2004_Exp.Cell.Res_294_534
PubMedSearch : Nguyen_2004_Exp.Cell.Res_294_534
PubMedID: 15023540

Title : Generation and analysis of muscarinic acetylcholine receptor knockout mice. -
Author(s) : Duttaroy A , Yamada M , Gomeza J , Zhang W , Miyakawa T , Makita R , Bymaster FP , Felder CC , Deng CX , Wess J
Ref : Cholinergic Mechanisms, CRC Press :63 , 2004

Title : Novel signaling pathways mediating reciprocal control of keratinocyte migration and wound epithelialization through M3 and M4 muscarinic receptors - Chernyavsky_2004_J.Cell.Biol_166_261
Author(s) : Chernyavsky AI , Arredondo J , Wess J , Karlsson E , Grando SA
Ref : Journal of Cell Biology , 166 :261 , 2004
Abstract : To test the hypothesis that keratinocyte (KC) migration is modulated by distinct muscarinic acetylcholine (ACh) receptor subtypes, we inactivated signaling through specific receptors in in vitro and in vivo models of reepithelialization by subtype-selective antagonists, small interfering RNA, and gene knockout in mice. KC migration and wound reepithelialization were facilitated by M4 and inhibited by M3. Additional studies showed that M4 increases expression of "migratory" integrins alpha5beta1, alphaVbeta5, and alphaVbeta6, whereas M3 up-regulates "sedentary" integrins alpha2beta1 and alpha3beta1. Inhibition of migration by M3 was mediated through Ca2+-dependent guanylyl cyclase-cyclic GMP-protein kinase G signaling pathway. The M4 effects resulted from inhibition of the inhibitory pathway involving the adenylyl cyclase-cyclic AMP-protein kinase A pathway. Both signaling pathways intersected at Rho, indicating that Rho kinase provides a common effector for M3 and M4 regulation of cell migration. These findings offer novel insights into the mechanisms of ACh-mediated modulation of KC migration and wound reepithelialization, and may aid the development of novel methods to promote wound healing.
ESTHER : Chernyavsky_2004_J.Cell.Biol_166_261
PubMedSearch : Chernyavsky_2004_J.Cell.Biol_166_261
PubMedID: 15263021

Title : Role of specific muscarinic receptor subtypes in cholinergic parasympathomimetic responses, in vivo phosphoinositide hydrolysis, and pilocarpine-induced seizure activity - Bymaster_2003_Eur.J.Neurosci_17_1403
Author(s) : Bymaster FP , Carter PA , Yamada M , Gomeza J , Wess J , Hamilton SE , Nathanson NM , McKinzie DL , Felder CC
Ref : European Journal of Neuroscience , 17 :1403 , 2003
Abstract : 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.
ESTHER : Bymaster_2003_Eur.J.Neurosci_17_1403
PubMedSearch : Bymaster_2003_Eur.J.Neurosci_17_1403
PubMedID: 12713643

Title : The M4 muscarinic receptor-selective effects on keratinocyte crawling locomotion - Chernyavsky_2003_Life.Sci_72_2069
Author(s) : Chernyavsky AI , Nguyen VT , Arredondo J , Ndoye A , Zia S , Wess J , Grando SA
Ref : Life Sciences , 72 :2069 , 2003
Abstract : 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.
ESTHER : Chernyavsky_2003_Life.Sci_72_2069
PubMedSearch : Chernyavsky_2003_Life.Sci_72_2069
PubMedID: 12628458

Title : Muscarinic receptor subtypes mediating central and peripheral antinociception studied with muscarinic receptor knockout mice: a review - Wess_2003_Life.Sci_72_2047
Author(s) : Wess J , Duttaroy A , Gomeza J , Zhang W , Yamada M , Felder CC , Bernardini N , Reeh PW
Ref : Life Sciences , 72 :2047 , 2003
Abstract : To gain new insight into the physiological and pathophysiological roles of the muscarinic cholinergic system, we generated mutant mouse strains deficient in each of the five muscarinic acetylcholine receptor subtypes (M(1)-M(5)). In this chapter, we review a set of recent studies dealing with the identification of the muscarinic receptor subtypes mediating muscarinic agonist-dependent analgesic effects by central and peripheral mechanisms. Most of these studies were carried out with mutant mouse strains lacking M(2) or/and M(4) muscarinic receptors. It is well known that administration of centrally active muscarinic agonists induces pronounced analgesic effects. To identify the muscarinic receptors mediating this activity, wild-type and muscarinic receptor mutant mice were injected with the non-subtype-selective muscarinic agonist, oxotremorine (s.c., i.t., and i.c.v.), and analgesic effects were assessed in the tail-flick and hot-plate tests. These studies showed that M(2) receptors play a key role in mediating the analgesic effects of oxotremorine, both at the spinal and supraspinal level. However, studies with M(2)/M(4) receptor double KO mice indicated that M(4) receptors also contribute to this activity. Recent evidence suggests that activation of muscarinic receptors located in the skin can reduce the sensitivity of peripheral nociceptors. Electrophysiological and neurochemical studies with skin preparations from muscarinic receptor mutant mice indicated that muscarine-induced peripheral antinociception is mediated by M(2) receptors. Since acetylcholine is synthesized and released by different cell types of the skin, it is possible that non-neuronally released acetylcholine plays a role in modulating peripheral nociception. Our results highlight the usefulness of muscarinic receptor mutant mice to shed light on the functional roles of acetylcholine released from both neuronal and non-neuronal cells.
ESTHER : Wess_2003_Life.Sci_72_2047
PubMedSearch : Wess_2003_Life.Sci_72_2047
PubMedID: 12628455

Title : Use of knockout mice reveals involvement of M2-muscarinic receptors in control of the kinetics of acetylcholine release - Slutsky_2003_J.Neurophysiol_89_1954
Author(s) : Slutsky I , Wess J , Gomeza J , Dudel J , Parnas I , Parnas H
Ref : Journal of Neurophysiology , 89 :1954 , 2003
Abstract : We have previously suggested that presynaptic M(2)-muscarinic receptors (M(2)R) are involved in the control of the time course of evoked acetylcholine release in the frog neuromuscular junction. The availability of knockout mice lacking functional M(2)R (M(2)-KO) enabled us to address this issue in a more direct way. Using the phrenic diaphragm preparation, we show that in wild-type (WT) mice experimental manipulations known to affect Ca(2+) entry and removal, greatly affected the amount of acetylcholine released (quantal content). However, the time course of release remained unaltered under all these experimental treatments. On the other hand, in the M(2)-KO mice, similar experimental treatments affected both the quantal content and the time course of release. In general, a larger quantal content was accompanied by a longer duration of release. Similarly, the rise time of the postsynaptic current produced by axon stimulation was sensitive to changes in [Ca(2+)](o) or [Mg(2+)](o) in M(2)-KO mice but not in WT mice. Measurements of Ca(2+) currents revealed that the shorter rise time of the postsynaptic current seen in high [Mg(2+)](o) in M(2)-KO mice was not produced by a shorter wave of the presynaptic Ca(2+) current. These results support our earlier findings and provide direct evidence for the major role that presynaptic M(2)-muscarinic receptors play in the control of the time course of evoked acetylcholine release under physiological conditions.
ESTHER : Slutsky_2003_J.Neurophysiol_89_1954
PubMedSearch : Slutsky_2003_J.Neurophysiol_89_1954
PubMedID: 12686574

Title : Heterogeneity of presynaptic muscarinic receptors mediating inhibition of sympathetic transmitter release: a study with M2- and M4-receptor-deficient mice - Trendelenburg_2003_Br.J.Pharmacol_138_469
Author(s) : Trendelenburg AU , Gomeza J , Klebroff W , Zhou H , Wess J
Ref : British Journal of Pharmacology , 138 :469 , 2003
Abstract : 1 Presynaptic muscarinic receptors modulate sympathetic transmitter release. The goal of the present study was to identify the muscarinic receptor subtype(s) mediating inhibition of sympathetic transmitter release in mouse atria, urinary bladder and vas deferens. To address this question, electrically evoked noradrenaline release was assessed using tissue preparations from NMRI, M(2)- and M(4)-knockout, and the corresponding M(2)- and M(4)-wildtype mice, after preincubation with (3)H-noradrenaline. 2 The muscarinic agonist carbachol decreased evoked tritium overflow (20 pulses/50 Hz) in each tissue and strain investigated. After deletion of the M(2)-receptor the maximal inhibition by carbachol was significantly reduced (by 41-72%), but not abolished, in all tissues. After deletion of the M(4)-receptor a moderate and significant reduction of the maximal inhibition by carbachol (by 28%) was observed only in the vas deferens. 3 Experiments with the muscarinic antagonists methoctramine and pirenzepine confirmed that the presynaptic muscarinic receptors were predominantly M(2) in atria and bladder and probably a mixture of M(2) and M(4) in the vas deferens. 4 Experiments in the urinary bladder with the cholinesterase inhibitor physostigmine and the muscarinic antagonist ipratropium demonstrated that endogenously released acetylcholine predominantly acted through M(2)-receptors to inhibit noradrenaline release. However, the results do not exclude a minor contribution of M(4)-receptors to this endogenous inhibition. 5 In conclusion, our results clearly indicate that the release-inhibiting muscarinic receptors on postganglionic sympathetic axons in mouse atria, bladder and vas deferens represent mixtures of M(2)- and non-M(2)-receptors. The non-M(2)-receptors remain unknown in atria and the bladder, and may represent primarily M(4)-receptors in the vas deferens. These results reveal an unexpected heterogeneity among the muscarinic receptors mediating inhibition of noradrenaline release.
ESTHER : Trendelenburg_2003_Br.J.Pharmacol_138_469
PubMedSearch : Trendelenburg_2003_Br.J.Pharmacol_138_469
PubMedID: 12569072

Title : Role for M5 muscarinic acetylcholine receptors in cocaine addiction - Fink-Jensen_2003_J.Neurosci.Res_74_91
Author(s) : Fink-Jensen A , Fedorova I , Wortwein G , Woldbye DP , Rasmussen T , Thomsen M , Bolwig TG , Knitowski KM , McKinzie DL , Yamada M , Wess J , Basile A
Ref : Journal of Neuroscience Research , 74 :91 , 2003
Abstract : Muscarinic cholinergic receptors of the M5 subtype are expressed by dopamine-containing neurons of the ventral tegmentum. These M5 receptors modulate the activity of midbrain dopaminergic neurons, which play an important role in mediating reinforcing properties of abused psychostimulants like cocaine. The potential role of M5 receptors in the reinforcing effects of cocaine was investigated using M5 receptor-deficient mice in a model of acute cocaine self-administration. The M5-deficient mice self-administered cocaine at a significantly lower rate than wild-type controls. In the conditioned place preference procedure, a classic test for evaluating the rewarding properties of drugs, M5-deficient mice spent significantly less time in the cocaine-paired compartment than control mice. Moreover, the severity of the cocaine withdrawal syndrome (withdrawal-associated anxiety measured in the elevated plus-maze) was significantly attenuated in mice lacking the M5 receptor. These results demonstrate that M5 receptors play an important role in mediating both cocaine-associated reinforcement and withdrawal.
ESTHER : Fink-Jensen_2003_J.Neurosci.Res_74_91
PubMedSearch : Fink-Jensen_2003_J.Neurosci.Res_74_91
PubMedID: 13130510

Title : Random mutagenesis of the M3 muscarinic acetylcholine receptor expressed in yeast. Identification of point mutations that silence a constitutively active mutant M3 receptor and greatly impair receptor\/G protein coupling - Schmidt_2003_J.Biol.Chem_278_30248
Author(s) : Schmidt C , Li B , Bloodworth L , Erlenbach I , Zeng FY , Wess J
Ref : Journal of Biological Chemistry , 278 :30248 , 2003
Abstract : The M3 muscarinic receptor is a prototypical member of the class I family of G protein-coupled receptors (GPCRs). To facilitate studies on the structural mechanisms governing M3 receptor activation, we generated an M3 receptor-expressing yeast strain (Saccharomyces cerevisiae) that requires agonist-dependent M3 receptor activation for cell growth. By using receptor random mutagenesis followed by a genetic screen in yeast, we initially identified a point mutation at the cytoplasmic end of transmembrane domain (TM) VI (Q490L) that led to robust agonist-independent M3 receptor signaling in both yeast and mammalian cells. To explore further the molecular mechanisms by which point mutations can render GPCRs constitutively active, we subjected a region of the Q490L mutant M3 receptor that included TM V-VII to random mutagenesis. We then applied a yeast genetic screen to identify second-site mutations that could suppress the activating effects of the Q490L mutation and restore wild-type receptor-like function to the Q490L mutant receptor. This analysis led to the identification of 12 point mutations that allowed the Q490L mutant receptor to function in a fashion similar to the wild-type receptor. These amino acid substitutions mapped to two distinct regions of the M3 receptor, the exofacial segments of TM V and VI and the cytoplasmic ends of TM V-VII. Strikingly, in the absence of the activating Q490L mutation, all recovered point mutations severely reduced the efficiency of receptor/G protein coupling, indicating that the targeted residues play important roles in receptor activation and/or receptor/G protein coupling. This strategy should be generally applicable to identify sites in GPCRs that are critically involved in receptor function.
ESTHER : Schmidt_2003_J.Biol.Chem_278_30248
PubMedSearch : Schmidt_2003_J.Biol.Chem_278_30248
PubMedID: 12750375

Title : Novel insights into M5 muscarinic acetylcholine receptor function by the use of gene targeting technology - Yamada_2003_Life.Sci_74_345
Author(s) : Yamada M , Basile AS , Fedorova I , Zhang W , Duttaroy A , Cui Y , Lamping KG , Faraci FM , Deng CX , Wess J
Ref : Life Sciences , 74 :345 , 2003
Abstract : Until recently, little was known about the possible physiological functions of the M(5) muscarinic acetylcholine receptor subtype, the last member of the muscarinic receptor family (M(1)-M(5)) to be cloned. To learn more about the potential physiological roles of this receptor subtype, we generated and analyzed M(5) receptor-deficient mice (M5 -/- mice). Strikingly, acetylcholine, a potent dilator of most vascular beds, virtually lost the ability to dilate cerebral arteries and arterioles in M5 -/- mice, suggesting that endothelial M(5) receptors mediate this activity in wild-type mice. This effect was specific for cerebral blood vessels, since acetylcholine-mediated dilation of extra-cerebral arteries remained fully intact in M5 -/- mice. In addition, in vitro neurotransmitter release experiments indicated that M(5) receptors located on dopaminergic nerve terminals play a role in facilitating muscarinic agonist-induced dopamine release in the striatum, consistent with the observation that the dopaminergic neurons innervating the striatum almost exclusively express the M(5) receptor subtype. We also found that the rewarding effects of morphine, the prototypical opiate analgesic, were substantially reduced in M5 -/- mice, as measured in the conditioned place preference paradigm. Furthermore, both the somatic and affective components of naloxone-induced morphine withdrawal symptoms were significantly attenuated in M5 -/- mice. It is likely that these behavioral deficits are caused by the lack of mesolimbic M(5) receptors, activation of which is known to stimulate dopamine release in the nucleus accumbens. These results convincingly demonstrate that the M(5) muscarinic receptor is involved in modulating several important pharmacological and behavioral functions. These findings may lead to novel therapeutic strategies for the treatment of drug addiction and certain cerebrovascular disorders.
ESTHER : Yamada_2003_Life.Sci_74_345
PubMedSearch : Yamada_2003_Life.Sci_74_345
PubMedID: 14607263

Title : Deletion of the M5 muscarinic acetylcholine receptor attenuates morphine reinforcement and withdrawal but not morphine analgesia - Basile_2002_Proc.Natl.Acad.Sci.U.S.A_99_11452
Author(s) : Basile AS , Fedorova I , Zapata A , Liu X , Shippenberg T , Duttaroy A , Yamada M , Wess J
Ref : Proc Natl Acad Sci U S A , 99 :11452 , 2002
Abstract : Little is known about the physiological roles of the M5 muscarinic receptor, the last member of the muscarinic receptor family (M1-M5) to be cloned. In the brain, the M5 receptor subtype is preferentially expressed by dopaminergic neurons of the substantia nigra and the ventral tegmental area. Dopaminergic neurons located in the ventral tegmental area are known to play important roles in mediating both the rewarding effects of opiates and other drugs of abuse and the manifestations of opiate/drug withdrawal symptoms. We therefore speculated that acetylcholine-dependent activation of M5 receptors might modulate the manifestations of opiate reward and withdrawal. This hypothesis was tested in a series of behavioral, biochemical, and neurochemical studies using M5 receptor-deficient mice (M5-/- mice) as novel experimental tools. We found that the rewarding effects of morphine, as measured in the conditioned place preference paradigm, were substantially reduced in M5-/- mice. Furthermore, both the somatic and affective components of naloxone-induced morphine withdrawal symptoms were significantly attenuated in M5-/- mice. In contrast, the analgesic efficacy of morphine and the development of tolerance to the analgesic effects of morphine remained unaltered by the lack of M5 receptors. The finding that M5 receptor activity modulates both morphine reward and withdrawal processes suggests that M5 receptors may represent a novel target for the treatment of opiate addiction.
ESTHER : Basile_2002_Proc.Natl.Acad.Sci.U.S.A_99_11452
PubMedSearch : Basile_2002_Proc.Natl.Acad.Sci.U.S.A_99_11452
PubMedID: 12154229

Title : Heterogeneity of release-inhibiting muscarinic autoreceptors in heart atria and urinary bladder: a study with M(2)- and M(4)-receptor-deficient mice - Zhou_2002_Naunyn.Schmiedebergs.Arch.Pharmacol_365_112
Author(s) : Zhou H , Meyer A , Starke K , Gomeza J , Wess J , Trendelenburg AU
Ref : Naunyn Schmiedebergs Arch Pharmacol , 365 :112 , 2002
Abstract : Release-inhibiting muscarinic autoreceptors were studied in heart atria and the urinary bladder of NMRI mice, M(2)-receptor-deficient mice, M(4)-receptor-deficient mice, and wildtype mice sharing the genetic background of the knockout animals. Segments of the tissues were preincubated with (3)H-choline and then superfused and stimulated electrically. In atrial segments taken from adult mice and stimulated with 120 pulses at 1 Hz, the muscarinic receptor agonist oxotremorine-M reduced the evoked overflow of tritium. Its concentration-response curves in atria from NMRI, M(2)-wildtype, M(4)-wildtype and M(2)-knockout mice were similar, with maximal inhibition by about 75%. In atria from M(4)-knockout mice, the maximal inhibitory effect of oxotremorine-M was reduced to 57%. The concentration-response curves of oxotremorine-M were shifted to the right by ipratropium, methoctramine and pirenzepine. Methoctramine and pirenzepine were approximately equipotent antagonists in all strains except in M(4)-knockout atria in which methoctramine was more potent than pirenzepine. When atria from adult NMRI mice were stimulated by 360 pulses at 3 Hz, ipratropium increased the evoked overflow of tritium both in the absence and in the presence of physostigmine (0.1 microM). In atria taken from 1-day-old NMRI mice, oxotremorine-M failed to reduce the evoked overflow of tritium. In bladder segments taken from adult mice, superfused with medium containing oxotremorine-M (1 microM), and stimulated by 360 pulses at 3 Hz, ipratropium increased the evoked overflow of tritium. Its concentration-response curves in preparations from NMRI, M(2)-wildtype, M(4)-wildtype and M(2)-knockout mice were similar. There was one exception: ipratropium failed to cause an increase in bladder pieces from M(4)-knockout mice. Methoctramine and pirenzepine also increased the evoked overflow of tritium in all strains except the M(4)-knockout. The two antagonists were approximately equipotent in NMRI, M(4)-wildtype and M(2)-knockout preparations but methoctramine was less potent than pirenzepine in M(2)-wildtype preparations. When bladder pieces from adult NMRI mice were superfused with oxotremorine-M-free medium and stimulated by 360 pulses at 3 Hz, ipratropium increased the evoked overflow of tritium in the presence of physostigmine (0.1 microM) but not in its absence. In bladder segments taken from 1-day-old NMRI mice and superfused with medium containing oxotremorine-M (1 microM), ipratropium increased the evoked overflow of tritium in the same way as in adult tissue. It is concluded that NMRI mice and the two wildtype strains are similar in their muscarinic autoreceptors. In atria, the autoreceptors are heterogeneous. Some are M(4). The non-M(4)-autoreceptors probably are M(2). In the bladder, the autoreceptors are exclusively M(4). In both tissues, the autoreceptors are activated by previously released acetylcholine under appropriate conditions.
ESTHER : Zhou_2002_Naunyn.Schmiedebergs.Arch.Pharmacol_365_112
PubMedSearch : Zhou_2002_Naunyn.Schmiedebergs.Arch.Pharmacol_365_112
PubMedID: 11819029

Title : M1 muscarinic receptor signaling in mouse hippocampus and cortex - Porter_2002_Brain.Res_944_82
Author(s) : Porter AC , Bymaster FP , DeLapp NW , Yamada M , Wess J , Hamilton SE , Nathanson NM , Felder CC
Ref : Brain Research , 944 :82 , 2002
Abstract : The five subtypes (M1-M5) of muscarinic acetylcholine receptors signal through G(alpha)(q) or G(alpha)(i)/G(alpha)(o). M1, M3 and M5 receptors couple through G(alpha)(q) and function predominantly as postsynaptic receptors in the central nervous system. M1 and M3 receptors are localized to brain regions involved in cognition, such as hippocampus and cortex, but their relative contribution to function has been difficult to ascertain due to the lack of subtype specific ligands. A functional and genetic approach was used to identify the predominant muscarinic receptor subtype(s) mediating responses in mouse hippocampus and cortex, as well as the relative degree of spare muscarinic receptors in hippocampus. The nonselective muscarinic agonist oxotremorine-M stimulated G(alpha)(q)/11-specific GTP-gamma-35S binding in a concentration dependent manner with a Hill slope near unity in wild type mouse hippocampus and cortex. Muscarinic receptor stimulated G(alpha)(q)/11-specific GTP-gamma-35S binding was virtually abolished in both the hippocampus and cortex of M1 receptor knockout (KO) mice. In contrast, there was no loss of signaling in M3 receptor KO mice in either brain region. Muscarinic receptor reserve in wildtype mouse hippocampus was measured by Furchgott analysis after partial receptor alkylation with propylbenzylcholine mustard. Occupation of just 15% of the M1 receptors in mouse hippocampus was required for maximal efficacy of oxotremorine-M-stimulated GTP-gamma-35S binding indicating a substantial level of spare receptors. These findings support a role for the M1 receptor subtype as the primary G(alpha)(q)/11-coupled muscarinic receptor in mouse hippocampus and cortex.
ESTHER : Porter_2002_Brain.Res_944_82
PubMedSearch : Porter_2002_Brain.Res_944_82
PubMedID: 12106668

Title : Evaluation of muscarinic agonist-induced analgesia in muscarinic acetylcholine receptor knockout mice - Duttaroy_2002_Mol.Pharmacol_62_1084
Author(s) : Duttaroy A , Gomeza J , Gan JW , Siddiqui N , Basile AS , Harman WD , Smith PL , Felder CC , Levey AI , Wess J
Ref : Molecular Pharmacology , 62 :1084 , 2002
Abstract : Centrally active muscarinic agonists display pronounced analgesic effects. Identification of the specific muscarinic acetylcholine receptor (mAChR) subtype(s) mediating this activity is of considerable therapeutic interest. To examine the roles of the M(2) and M(4) receptor subtypes, the two G(i)/G(o)-coupled mAChRs, in mediating agonist-dependent antinociception, we generated a mutant mouse line deficient in both M(2) and M(4) mAChRs [M(2)/M(4) double-knockout (KO) mice]. In wild-type mice, systemic, intrathecal, or intracerebroventricular administration of centrally active muscarinic agonists resulted in robust analgesic effects, indicating that muscarinic analgesia can be mediated by both spinal and supraspinal mechanisms. Strikingly, muscarinic agonist-induced antinociception was totally abolished in M(2)/M(4) double-KO mice, independent of the route of application. The nonselective muscarinic agonist oxotremorine showed reduced analgesic potency in M(2) receptor single-KO mice, but retained full analgesic activity in M(4) receptor single-KO mice. In contrast, two novel muscarinic agonists chemically derived from epibatidine, CMI-936 and CMI-1145, displayed reduced analgesic activity in both M(2) and M(4) receptor single-KO mice, independent of the route of application. Radioligand binding studies indicated that the two CMI compounds, in contrast to oxotremorine, showed >6-fold higher affinity for M(4) than for M(2) receptors, providing a molecular basis for the observed differences in agonist activity profiles. These data provide unambiguous evidence that muscarinic analgesia is exclusively mediated by a combination of M(2) and M(4) mAChRs at both spinal and supraspinal sites. These findings should be of considerable relevance for the development of receptor subtype-selective muscarinic agonists as novel analgesic drugs.
ESTHER : Duttaroy_2002_Mol.Pharmacol_62_1084
PubMedSearch : Duttaroy_2002_Mol.Pharmacol_62_1084
PubMedID: 12391271

Title : M(3)-receptor knockout mice: muscarinic receptor function in atria, stomach fundus, urinary bladder, and trachea - Stengel_2002_Am.J.Physiol.Regul.Integr.Comp.Physiol_282_R1443
Author(s) : Stengel PW , Yamada M , Wess J , Cohen ML
Ref : American Journal of Physiology Regul Integr Comp Physiol , 282 :R1443 , 2002
Abstract : Negative chronotropic and smooth muscle contractile responses to the nonselective muscarinic agonist carbamylcholine were compared in isolated tissues from M(3)-muscarinic receptor knockout and wild-type mice. Carbamylcholine (10(-8)-3.0 x 10(-5) M) induced a concentration-dependent decrease in atrial rate that was similar in atria from M(3)-receptor knockout and wild-type mice, indicating that M(3) receptors were not involved in muscarinic receptor-mediated atrial rate decreases. In contrast, the M(3) receptor was a major muscarinic receptor involved in smooth muscle contraction of stomach fundus, urinary bladder, and trachea, although differences existed in the extent of M(3)-receptor involvement among the tissues. Contraction to carbamylcholine was virtually abolished in urinary bladder from M(3)-receptor knockout mice, suggesting that contraction was predominantly due to M(3)-receptor activation. However, approximately 50-60% maximal contraction to carbamylcholine occurred in stomach fundus and trachea from M(3)-receptor knockout mice, indicating that contraction in these tissues was also due to M(2)-receptor activation. High concentrations of carbamylcholine relaxed the stomach fundus from M(3)-receptor knockout mice by M(1)-receptor activation. Thus M(3)-receptor knockout mice provided unambiguous evidence that M(3) receptors 1) play no role in carbamylcholine-induced atrial rate reduction, 2) are the predominant receptor mediating carbamylcholine-induced urinary bladder contractility, and 3) share contractile responsibility with M(2) receptors in mouse stomach fundus and trachea.
ESTHER : Stengel_2002_Am.J.Physiol.Regul.Integr.Comp.Physiol_282_R1443
PubMedSearch : Stengel_2002_Am.J.Physiol.Regul.Integr.Comp.Physiol_282_R1443
PubMedID: 11959688

Title : Characterization of central inhibitory muscarinic autoreceptors by the use of muscarinic acetylcholine receptor knock-out mice - Zhang_2002_J.Neurosci_22_1709
Author(s) : Zhang W , Basile AS , Gomeza J , Volpicelli LA , Levey AI , Wess J
Ref : Journal of Neuroscience , 22 :1709 , 2002
Abstract : Forebrain muscarinic acetylcholine (ACh) receptors (mAChRs; M1-M5) are predicted to play important roles in many fundamental central functions, including higher cognitive processes and modulation of extrapyramidal motor activity. Synaptic ACh levels are known to be regulated by the activity of presynaptic muscarinic autoreceptors mediating inhibition of ACh release. Primarily because of the use of ligands with limited receptor subtype selectivity, classical pharmacological studies have led to conflicting results regarding the identity of the mAChR subtypes mediating this activity in different areas of the brain. To investigate the molecular identity of hippocampal, cortical, and striatal inhibitory muscarinic autoreceptors in a more direct manner, we used genetically altered mice lacking functional M2 and/or M4 mAChRs [knock-out (KO) mice]. After labeling of cellular ACh pools with [3H]choline, potassium-stimulated [3H]ACh release was measured in superfused brain slices, either in the absence or the presence of muscarinic drugs. The nonsubtype-selective muscarinic agonist, oxotremorine (0.1-10 microm), inhibited potassium-stimulated [3H]ACh release in hippocampal, cortical, and striatal slices prepared from wild-type mice by up to 80%. This activity was totally abolished in tissues prepared from M2-M4 receptor double KO mice. Strikingly, release studies with brain slices from M2 and M4 receptor single KO mice indicated that autoinhibition of ACh release is mediated primarily by the M2 receptor in hippocampus and cerebral cortex, but predominantly by the M4 receptor in the striatum. These results, together with additional receptor localization studies, support the novel concept that autoinhibition of ACh release involves different mAChRs in different regions of the brain.
ESTHER : Zhang_2002_J.Neurosci_22_1709
PubMedSearch : Zhang_2002_J.Neurosci_22_1709
PubMedID: 11880500

Title : Multiple muscarinic acetylcholine receptor subtypes modulate striatal dopamine release, as studied with M1-M5 muscarinic receptor knock-out mice - Zhang_2002_J.Neurosci_22_6347
Author(s) : Zhang W , Yamada M , Gomeza J , Basile AS , Wess J
Ref : Journal of Neuroscience , 22 :6347 , 2002
Abstract : A proper balance between striatal muscarinic cholinergic and dopaminergic neurotransmission is required for coordinated locomotor control. Activation of striatal muscarinic acetylcholine receptors (mAChRs) is known to modulate striatal dopamine release. To identify the mAChR subtype(s) involved in this activity, we used genetically altered mice that lacked functional M1-M5 mAChRs [knock-out (KO) mice]. In superfused striatal slices from wild-type mice, the non-subtype-selective muscarinic agonist oxotremorine led to concentration-dependent increases in potassium-stimulated [3H]dopamine release (by up to 60%). The lack of M1 or M2 receptors had no significant effect on the magnitude of these responses. Strikingly, oxotremorine-mediated potentiation of stimulated striatal [3H]dopamine release was abolished in M4 receptor KO mice, significantly increased in M3 receptor-deficient mice, and significantly reduced (but not abolished) in M5 receptor KO mice. Additional release studies performed in the presence of tetrodotoxin suggested that the dopamine release-stimulating M4 receptors are probably located on neuronal cell bodies, but that the release-facilitating M5 and the release-inhibiting M3 receptors are likely to be located on nerve terminals. Studies with the GABA(A) receptor blocker bicuculline methochloride suggested that M3 and M4 receptors mediate their dopamine release-modulatory effects via facilitation or inhibition, respectively, of striatal GABA release. These results provide unambiguous evidence that multiple mAChR subtypes are involved in the regulation of striatal dopamine release. These findings should contribute to a better understanding of the important functional roles that the muscarinic cholinergic system plays in striatal function.
ESTHER : Zhang_2002_J.Neurosci_22_6347
PubMedSearch : Zhang_2002_J.Neurosci_22_6347
PubMedID: 12151512

Title : Poster: Functional expression of M1 M3, and M5 muscarinic acetylcholine receptors in yeast -
Author(s) : Erlenbach I , Kostenis E , Schmidt C , Hamdan F , Pausch MH , Wess J
Ref : Life Sciences , 68 :2640 , 2001

Title : Investigations into the physiological role of muscarinic M2 and M4 muscarinic and M4 receptor subtypes using receptor knockout mice - Bymaster_2001_Life.Sci_68(22-23)_2473
Author(s) : Bymaster FP , Carter PA , Zhang L , Falcone JF , Stengel PW , Cohen ML , Shannon HE , Gomeza J , Wess J , Felder CC
Ref : Life Sciences , 68 :2473 , 2001
Abstract : Determination of muscarinic agonist-induced parasympathomimetic effects in wild type and M2 and M4 muscarinic receptor knockout mice revealed that M2 receptors mediated tremor and hypothermia, but not salivation. The M4 receptors seem to play a modest role in salivation, but did not alter hypothermia and tremor. In the M2 knockout mice, agonist-induced bradycardia in isolated spontaneously beating atria was completely absent compared to their wild type litter mates, whereas agonist-induced bradycardia was similar in the M4 knockout and wild type mice. The potency of carbachol to stimulate contraction of isolated stomach fundus, urinary bladder and trachea was reduced by a factor of about 2 in the M2 knockout mice, but was unaltered in the M4 knockout mice. The binding of the muscarinic agonist, [3H]-oxotremorine-M, was reduced in cortical tissue from the M2 knockout mice and to a lesser extent from the M4 knockout mice, and was reduced over 90% in the brain stem of M2 knockout mice. The data demonstrate the usefulness of knockout mice in determining the physiological function of peripheral and central muscarinic receptors.
ESTHER : Bymaster_2001_Life.Sci_68(22-23)_2473
PubMedSearch : Bymaster_2001_Life.Sci_68(22-23)_2473
PubMedID: 11392615

Title : Identification of subtypes of muscarinic receptors that regulate Ca2+ and K+ channel activity in sympathetic neurons - Shapiro_2001_Life.Sci_68(22-23)_2481
Author(s) : Shapiro MS , Gomeza J , Hamilton SE , Hille B , Loose MD , Nathanson NM , Roche JP , Wess J
Ref : Life Sciences , 68 :2481 , 2001
Abstract : Many different G protein-coupled receptors modulate the activity of Ca2+ and K+ channels in a variety of neuronal types. There are five known subtypes (M1-M5) of muscarinic acetylcholine receptors. Knockout mice lacking the M1, M2, or M4 subtypes are studied to determine which receptors mediate modulation of voltage-gated Ca2+ channels in mouse sympathetic neurons. In these cells, muscarinic agonists modulate N- and L-type Ca2+ channels and the M-type K+ channel through two distinct, G-protein mediated pathways. The fast and voltage-dependent pathway is lacking in the M2 receptor knockout mice. The slow and voltage-independent pathway is absent in the M1 receptor knockout mice. Neither pathway is affected in the M4 receptor knockout mice. Muscarinic modulation of the M current is absent in the M1 receptor knockout mice, and can be reconstituted in a heterologous expression system using cloned channels and M1 receptors. Our results using knockout mice are compared with pharmacological data in the rat.
ESTHER : Shapiro_2001_Life.Sci_68(22-23)_2481
PubMedSearch : Shapiro_2001_Life.Sci_68(22-23)_2481
PubMedID: 11392616

Title : Elucidating the role of muscarinic receptors in psychosis - Felder_2001_Life.Sci_68(22-23)_2605
Author(s) : Felder CC , Porter AC , Skillman TL , Zhang L , Bymaster FP , Nathanson NM , Hamilton SE , Gomeza J , Wess J , McKinzie DL
Ref : Life Sciences , 68 :2605 , 2001
Abstract : Muscarinic receptors have been implicated in the regulation of cognition and psychosis based on pharmacological evidence from pre-clinical and clinical studies. Muscarinic agonists have shown promise in the clinic in improving cognition and reducing psychotic episodes in Alzheimer's patients. However, lack of selective muscarinic ligands has limited their use due to troublesome side effects observed at higher doses. Without selective ligands, it has been difficult to assign a specific muscarinic receptor subtype to these high order mental processes. Recent development of muscarinic receptor knockout mice has provided additional tools to investigate cognition and psychosis in behavioral assays and to determine the receptor subtypes associated with parasympathomimetic physiology. Biochemical studies indicate that the M1 receptor plays a significant role in regulating G alpha q-mediated signal transduction in the hippocampus and cortex. Behavioral studies suggest that the M4 receptor is involved in movement regulation and prepulse inhibition of the startle reflex, a measure of attention. These findings support a role for the development of M1 and M4 receptor agonists for diseases in which symptoms include cognitive impairment and psychotic behaviors.
ESTHER : Felder_2001_Life.Sci_68(22-23)_2605
PubMedSearch : Felder_2001_Life.Sci_68(22-23)_2605
PubMedID: 11392633

Title : Functional expression of M(1), M(3) and M(5) muscarinic acetylcholine receptors in yeast - Erlenbach_2001_J.Neurochem_77_1327
Author(s) : Erlenbach I , Kostenis E , Schmidt C , Hamdan FF , Pausch MH , Wess J
Ref : Journal of Neurochemistry , 77 :1327 , 2001
Abstract : The goal of this study was to functionally express the three G(q)-coupled muscarinic receptor subtypes, M(1), M(3) and M(5), in yeast (Saccharomyces cerevisiae). Transformation of yeast with expression constructs coding for the full-length receptors resulted in very low numbers of detectable muscarinic binding sites (B(max) < 5 fmol/mg). Strikingly, deletion of the central portion of the third intracellular loops of the M(1), M(3) and M(5) muscarinic receptors resulted in dramatic increases in B(max) values (53-214 fmol/mg). To monitor productive receptor/G-protein coupling, we used specifically engineered yeast strains that required agonist-stimulated receptor/G-protein coupling for cell growth. These studies showed that the shortened versions of the M(1), M(3) and M(5) receptors were unable to productively interact with the endogenous yeast G protein alpha-subunit, Gpa1p, or a Gpa1 mutant subunit that contained C-terminal mammalian Galpha(s) sequence. In contrast, all three receptors gained the ability to efficiently couple to a Gpa1/Galpha(q) hybrid subunit containing C-terminal mammalian Galpha(q) sequence, indicating that the M(1), M(3) and M(5) muscarinic receptors retained proper G-protein coupling selectivity in yeast. This is the first study to report the expression of muscarinic receptors in a coupling-competent form in yeast. The strategy described here, which involves structural modification of both receptors and co-expressed G proteins, should facilitate the functional expression of other classes of G protein-coupled receptors in yeast.
ESTHER : Erlenbach_2001_J.Neurochem_77_1327
PubMedSearch : Erlenbach_2001_J.Neurochem_77_1327
PubMedID: 11389184

Title : M1 muscarinic acetylcholine receptors activate extracellular signal-regulated kinase in CA1 pyramidal neurons in mouse hippocampal slices - Berkeley_2001_Mol.Cell.Neurosci_18_512
Author(s) : Berkeley JL , Gomeza J , Wess J , Hamilton SE , Nathanson NM , Levey AI
Ref : Molecular & Cellular Neurosciences , 18 :512 , 2001
Abstract : Activation of extracellular signal-regulated kinases (ERK) is crucial for many neural functions, including learning, memory, and synaptic plasticity. As muscarinic acetylcholine receptors (mAChR) modulate many of the same higher brain functions as ERK, we examined mAChR-mediated ERK activation in mouse hippocampal slices. The cholinergic agonist carbachol caused an atropine-sensitive ERK activation in the dendrites and somata CA1 pyramidal neurons. To determine the responsible mAChR subtype, we combined pharmacologic and genetic approaches. Pretreatment with M1 antagonists inhibited ERK activation. Furthermore, mAChR-induced ERK activation was absent in slices from M1 knockout mice. ERK activation was normal in slices derived from other mAChR subtype knockouts (M2, M3, and M4), although these other subtypes are expressed in many of the same neurons. Thus, we demonstrate divergent functions for the different mAChR subtypes. We conclude that M1 is responsible for mAChR-mediated ERK activation, providing a mechanism by which M1 may modulate learning and memory.
ESTHER : Berkeley_2001_Mol.Cell.Neurosci_18_512
PubMedSearch : Berkeley_2001_Mol.Cell.Neurosci_18_512
PubMedID: 11922142

Title : Generation and pharmacological analysis of M2 and M4 muscarinic receptor knockout mice - Gomeza_2001_Life.Sci_68(22-23)_2457
Author(s) : Gomeza J , Zhang L , Kostenis E , Felder CC , Bymaster FP , Brodkin J , Shannon HE , Xia B , Duttaroy A , Deng CX , Wess J
Ref : Life Sciences , 68 :2457 , 2001
Abstract : Muscarinic acetylcholine receptors (M1-M5) play important roles in the modulation of many key functions of the central and peripheral nervous system. To explore the physiological roles of the two Gi-coupled muscarinic receptors, we disrupted the M2 and M4 receptor genes in mice by using a gene targeting strategy. Pharmacological and behavioral analysis of the resulting mutant mice showed that the M2 receptor subtype is critically involved in mediating three of the most striking central muscarinic effects, tremor, hypothermia, and analgesia. These studies also indicated that M4 receptors are not critically involved in these central muscarinic responses. However, M4 receptor-deficient mice showed an increase in basal locomotor activity and greatly enhanced locomotor responses following drug-induced activation of D1 dopamine receptors. This observation is consistent with the concept that M4 receptors exert inhibitory control over D1 receptor-mediated locomotor stimulation, probably at the level of striatal projection neurons where the two receptors are known to be coexpressed. These findings emphasize the usefulness of gene targeting approaches to shed light on the physiological and pathophysiological roles of the individual muscarinic receptor subtypes.
ESTHER : Gomeza_2001_Life.Sci_68(22-23)_2457
PubMedSearch : Gomeza_2001_Life.Sci_68(22-23)_2457
PubMedID: 11392613

Title : Poster: Ml muscarinic acetylcholine receptors activate MAPK in brain -
Author(s) : Berkeley JL , Hamilton SE , Nathanson NM , Wess J , Levey AI
Ref : Life Sciences , 68 :2624 , 2001

Title : Poster: Evaluation of muscarinic agonist-induced analgesia in muscarinic receptor knockout mice -
Author(s) : Duttaroy A , Gomeza J , Gan JW , Basile AS , Harman WD , Smith PL , Felder CC , Levey AI , Wess J
Ref : Life Sciences , 68 :2631 , 2001

Title : Poster: Regional brain uptake of the muscarinic ligand, [18F]FP-TZTP, is decreased in M2 knockout but not in M4 knockout mice. -
Author(s) : Jagoda EM , Kiesewetter DO , Shimoji K , Gomeza J , Wess J , Eckelman WC
Ref : Life Sciences , 68 :2634 , 2001

Title : Poster: Enhanced sensitivity to the locomotor-activating and disrupting effects of phencyclidine in M4 muscarinic receptor knock-out mice -
Author(s) : Felder CC , Bymaster FP , Wess J , Zhang L , Shaw DB , McKinzie DL
Ref : Life Sciences , 68 :2636 , 2001

Title : Assignment of muscarinic receptor subtypes mediating G-protein modulation of Ca(2+) channels by using knockout mice - Shapiro_1999_Proc.Natl.Acad.Sci.U.S.A_96_10899
Author(s) : Shapiro MS , Loose MD , Hamilton SE , Nathanson NM , Gomeza J , Wess J , Hille B
Ref : Proc Natl Acad Sci U S A , 96 :10899 , 1999
Abstract : There are five known subtypes of muscarinic receptors (M(1)-M(5)). We have used knockout mice lacking the M(1), M(2), or M(4) receptors to determine which subtypes mediate modulation of voltage-gated Ca(2+) channels in mouse sympathetic neurons. Muscarinic agonists modulate N- and L-type Ca(2+) channels in these neurons through two distinct G-protein-mediated mechanisms. One pathway is fast and membrane-delimited and inhibits N- and P/Q-type channels by shifting their activation to more depolarized potentials. The other is slow and voltage-independent and uses a diffusible cytoplasmic messenger to inhibit both Ca(2+) channel types. Using patch-clamp methods on acutely dissociated sympathetic neurons, we isolated each pathway by pharmacological and kinetic means and found that each one is nearly absent in a particular knockout mouse. The fast and voltage-dependent pathway is lacking in the M(2) receptor knockout mice; the slow and voltage-independent pathway is absent from the M(1) receptor knockout mice; and neither pathway is affected in the M(4) receptor knockout mice. The knockout effects are clean and are apparently not accompanied by compensatory changes in other muscarinic receptors.
ESTHER : Shapiro_1999_Proc.Natl.Acad.Sci.U.S.A_96_10899
PubMedSearch : Shapiro_1999_Proc.Natl.Acad.Sci.U.S.A_96_10899
PubMedID: 10485923

Title : Poster: Development of a disulfide cross-linking strategy to study muscarinic receptor structure -
Author(s) : Zeng FY , Wess J
Ref : Life Sciences , 64 :565 , 1999

Title : Structure-function analysis of muscarinic receptors and their associated G proteins - Kostenis_1999_Life.Sci_64(6-7)_355
Author(s) : Kostenis E , Zeng FY , Wess J
Ref : Life Sciences , 64 :355 , 1999
Abstract : Each member of the muscarinic receptor family (M1-M5) can interact only with a limited subset of the many structurally closely related heterotrimeric G proteins expressed within a cell. To understand how this selectivity is achieved at a molecular level, we have used the G(i/0)-coupled M2 and the Gq/11-coupled M3 muscarinic receptors as model systems. We developed a genetic strategy involving the coexpression of wild type or mutant muscarinic receptors with hybrid or mutant G protein alpha subunits to identify specific, functionally relevant receptor/G protein contact sites. This approach led to the identification of N- and C-terminal amino acids on alpha(q) and alpha(i) that are critical for maintaining proper receptor/G protein coupling. Moreover, several receptor sites were identified that are likely to be contacted by these functionally critical G alpha residues. To gain deeper insight into muscarinic receptor structure, we recently developed a cysteine disulfide cross-linking strategy, using the M3 muscarinic receptor as a model system. Among other structural modifications, this approach involves the removal of most native cysteine residues by site-directed mutagenesis, the insertion of three factor Xa cleavage sites into the third intracellular loop, and systematic 'reintroduction' of pairs of cysteine residues. Following treatment of receptor-containing membrane preparations with factor Xa and oxidizing agents, disulfide cross-linked products can be identified by immunoprecipitation and immunoblotting studies. This approach should greatly advance our knowledge of the molecular architecture of muscarinic and other G protein-coupled receptors.
ESTHER : Kostenis_1999_Life.Sci_64(6-7)_355
PubMedSearch : Kostenis_1999_Life.Sci_64(6-7)_355
PubMedID: 10069496

Title : Poster: Development of a sandwich ELISA strategy to study mechanisms involved in muscarinic receptor assembly -
Author(s) : Jakubik J , Wess J
Ref : Life Sciences , 64 :565 , 1999

Title : Molecular basis of receptor\/G protein coupling selectivity studied by coexpression of wild type and mutant m2 muscarinic receptors with mutant G alpha(q) subunits - Kostenis_1997_Biochemistry_36_1487
Author(s) : Kostenis E , Conklin BR , Wess J
Ref : Biochemistry , 36 :1487 , 1997
Abstract : The molecular basis of receptor/G protein coupling selectivity was studied by using the m2 muscarinic receptor, a prototypical G(i/o)-coupled receptor as a model system. We could recently show that the m2 receptor can efficiently interact with mutant G protein alpha(q) subunits in which the last five amino acids were replaced with alpha(i2) or alpha(o) sequence [Liu, J., Conklin, B. R., Blin, N., Yun, J., & Wess, J. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 11642-11646]. Additional mutagenesis studies led to the identification of a four-amino-acid motif on the m2 receptor (Val385, Thr386, Ile389, and Leu390) that is predicted to functionally interact with the C-terminal portion of alpha(i/o) subunits. To further investigate the structural requirements for this interaction to occur, these four m2 receptor residues were replaced, either individually or in combination, with the corresponding residues present in the G(q/11)-coupled muscarinic receptors (m1, m3, and m5). The ability of the resulting mutant m2 receptors to interact with a mutant alpha(q) subunit (qo5) in which the last five amino acids were replaced with alpha(o) sequence was investigated in co-transfected COS-7 cells [studied biochemical response: stimulation of phosphatidyl inositol (PI) hydrolysis]. Our data suggest that the presence of three of the four targeted m2 receptor residues (Val385, Thr386, and Ile389) is essential for efficient recognition of C-terminal alpha(i/o) sequences. To study which specific amino acids within the C-terminal segment of alpha(i/o) subunits are critical for this interaction to occur, the wild type m2 receptor was co-expressed with a series of mutant alpha(q) subunits containing single or multiple alpha(q) --> alpha(i1,2) point mutations at their C-terminus. Remarkably, the wild type m2 receptor, while unable to efficiently stimulate wild type alpha(q), gained the ability to productively interact with three alpha(q) single-point mutants, providing the first example that the receptor coupling selectivity of G protein alpha subunits can be switched by single amino acid substitutions. Given the high degree of structural homology among different G protein-coupled receptors and among different classes of G protein alpha subunits, our results should be of broad general relevance.
ESTHER : Kostenis_1997_Biochemistry_36_1487
PubMedSearch : Kostenis_1997_Biochemistry_36_1487
PubMedID: 9063897

Title : Structural basis of receptor\/G protein coupling selectivity studied with muscarinic receptors as model systems - Wess_1997_Life.Sci_60(13-14)_1007
Author(s) : Wess J , Liu J , Blin N , Yun J , Lerche C , Kostenis E
Ref : Life Sciences , 60 :1007 , 1997
Abstract : Different muscarinic acetylcholine receptor subtypes were used as model systems to study the structural basis of receptor/G protein coupling selectivity. Extensive mutagenesis studies have previously led to the identification of single amino acids on the m3 muscarinic receptor protein (located in the second intracellular loop (i2) and at the N- and C-terminus of the third intracellular loop (i3)) that dictate selective recognition of Gq/11 proteins by this receptor subtype. Based on these results, we proposed a model of the intracellular m3 receptor surface in which the functionally critical residues project into the interior of the transmembrane receptor core. To identify specific regions on the G protein(s) that are contacted by these different, functionally critical receptor sites, we recently employed a novel experimental strategy involving the coexpression of hybrid m2/m3 muscarinic receptors with hybrid G alpha-subunits. Using this approach, we could demonstrate that the C-terminus of G protein alpha i/o-subunits is recognized by a short sequence element in the m2 muscarinic receptor ("VTIL") that is located at the junction between the sixth transmembrane domain (TM VI) and the i3 loop. We could show that this interaction is critically involved in determining coupling selectivity and triggering G protein activation. By using a similar strategy (coexpression of mutant muscarinic receptors with hybrid G alpha-subunits), other major receptor/G protein contact sites are currently being identified. These studies, complemented by biochemical and biophysical approaches, should eventually lead to a detailed structural model of the ligand-receptor-G protein complex.
ESTHER : Wess_1997_Life.Sci_60(13-14)_1007
PubMedSearch : Wess_1997_Life.Sci_60(13-14)_1007
PubMedID: 9121341

Title : Molecular mechanisms involved in muscarinic acetylcholine receptor-mediated G protein activation studied by insertion mutagenesis - Liu_1996_J.Biol.Chem_271_6172
Author(s) : Liu J , Blin N , Conklin BR , Wess J
Ref : Journal of Biological Chemistry , 271 :6172 , 1996
Abstract : We have recently shown that a four-amino acid epitope (VTIL) on the m2 muscarinic receptor (corresponding to Val385, Thr386, Ile389, and Leu390) is essential for Gi/o coupling specificity and Gi/o activation (Liu, J., Conklin, B. R., Blin, N., Yun, J., and Wess, J. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 11642-11646). Because this sequence element is thought to be located at the junction between the third intracellular loop and the sixth transmembrane helix (TM VI), we speculated that agonist binding to the m2 receptor protein results in conformational changes that enable the VTIL motif to interact with Gi/o proteins. To test the hypothesis that such structural changes might involve a relative movement of TM VI toward the cytoplasm, we created a series of mutant m2 muscarinic receptors in which one to four extra Ala residues were inserted into TM VI immediately after Leu390. Based on the geometry of an alpha-helix, such mutations are predicted to "push" the VTIL sequence away from the lipid bilayer. Consistent with our working hypothesis, second messenger assays with transfected COS-7 cells showed that all mutant m2 receptors containing extra Ala residues C-terminal of Leu390 could activate the proper G proteins even in the absence of agonist. However, replacement of the VTIL motif in such constitutively active m2 receptors with the corresponding m3 muscarinic receptor sequence (AALS) or deletion of Ala391 from the wild type m2 receptor completely abolished G protein coupling. Interestingly, introduction of extra Ala residues C-terminal of the AALS motif in the m3 muscarinic receptor completely abolished functional activity. Mutant m2 and m3 receptors that contained extra Ala residues immediately N-terminal of the VTIL and AALS motif, respectively, displayed wild type-like coupling properties. Our data are consistent with a model in which agonist binding to the m2 muscarinic receptor leads to a relative movement of TM VI toward the cytoplasm, thus enabling the adjacent VTIL sequence to interact with the C terminus of Galpha(i/o) subunits.
ESTHER : Liu_1996_J.Biol.Chem_271_6172
PubMedSearch : Liu_1996_J.Biol.Chem_271_6172
PubMedID: 8626406

Title : Molecular aspects of muscarinic receptor assembly and function -
Author(s) : Wess J , Blin N , Yun J , Schoneberg T , Liu J
Ref : Prog Brain Res , 109 :153 , 1996
PubMedID: 9009702

Title : Influence of monovalent cations on the binding of a charged and an uncharged ('carbo'-)muscarinic antagonist to muscarinic receptors - Hou_1996_Br.J.Pharmacol_117_955
Author(s) : Hou X , Wehrle J , Menge W , Ciccarelli E , Wess J , Mutschler E , Lambrecht G , Timmerman H , Waelbroeck M
Ref : British Journal of Pharmacology , 117 :955 , 1996
Abstract : 1. The effect of the buffer concentration on binding of [3H]-N-methylscopolamine to muscarinic receptors M2 was tested in rat heart. Tracer binding was of low affinity in a 20 mM imidazole buffer (pKD 8.3), inhibited by an increase from 10 to 100 mM of the sodium phosphate buffer concentration (pKD 9.92 to 9.22), slightly inhibited by an increase of the Tris/HC1 buffer concentration from 20 to 100 mM (pKD 9.70 to 9.47) and unaffected by an increase of the histidine/HC1 buffer concentration from 20 to 100 mM (pKD 9.90 to 9.82). We chose the last buffer to analyse the effect of ions on antagonists binding to cardiac M2 receptors and to transiently expressed wild-type and (Y533-->F) mutant m3 muscarinic receptors in COS-7 cells. 2. Equilibrium [3H]-N-methylscopolamine binding to cardiac M2 receptors was inhibited, apparently competitively, by monovalent salts (LiCl > or = NaCl > or = KCl). In contrast, binding of the uncharged 3,3-dimethylbutan-1-ol ester of diphenylglycolic acid (BS-6181) was facilitated by addition of monovalent salts (LiCl > or = NaCl > or = KCl) to the binding buffer. This cation binding pattern is consistent with interaction with a large, negative field strength binding site, such as, for instance, a carboxylic acid. 3. In the presence of 100 mM NaCl, [3H]-N-methylscopolamine had a similar affinity for the wild-type m3 receptor (pKD 9.85) and for a (Y533-->F) mutant m3 receptor (pKD 9.68). However, in the absence of added salts, the tracer had a significantly lower affinity for the mutated (pKD 10.19) as compared to the wild-type (pKD 10.70) m3 receptor. BS-6181 had a significantly lower affinity for the (Y533-->F) mutant m3 muscarinic receptor, as compared to the wild-type m3 receptor, both in the absence (pKD 6.19-6.72) in the presence (pKD 6.48-7.40) of 100 mM NaCl. The effects of NaCl on binding of the uncharged ester and of [3H]-N-methylscopolamine to the m3 receptor were decreased by the mutation. 4. Taken together, these results support the hypothesis that monovalent cations from the buffer may interact with the cation binding site of the receptors (an aspartate residue in the third transmembrane helix of muscarinic receptors). Buffer cations may inhibit competitively the binding of (charged) muscarinic ligands having a tertiary amine or ammonium group, while facilitating the receptor recognition by uncharged, isosteric 'carbo-analogues'. Mutation of the (Y533-->F) of the m3 receptor decreased the affinity of the receptor for positive charges, including the sodium ion.
ESTHER : Hou_1996_Br.J.Pharmacol_117_955
PubMedSearch : Hou_1996_Br.J.Pharmacol_117_955
PubMedID: 8851517

Title : Muscarinic acetylcholine receptors: structural basis of ligand binding and G protein coupling - Wess_1995_Life.Sci_56(11-12)_915
Author(s) : Wess J , Blin N , Mutschler E , Bluml K
Ref : Life Sciences , 56(11-12) :915 , 1995
Abstract : Muscarinic acetylcholine receptors (m1-m5) were studied by a combined molecular genetic/pharmacologic approach to elucidate the molecular characteristics of the ligand binding site and of the receptor domains involved in G protein coupling. Site-directed mutagenesis studies of the rat m3 muscarinic receptor suggest that the acetylcholine binding domain is formed by a series of hydrophilic amino acids located in the "upper" half of transmembrane domains (TM) III, V, VI, and VII. Moreover, we showed that mutational modification of a TM VI Asn residue (Asn507 in the rat m3 receptor sequence) which is characteristic for the muscarinic receptor family has little effect on high-affinity acetylcholine binding and receptor activation, but results in dramatic reductions in binding affinities for certain subclasses of muscarinic antagonists. The N-terminal portion of the third intracellular loop (i3) of muscarinic and other G protein-coupled receptors has been shown to play a central role in determining the G protein coupling profile of a given receptor subtype. Insertion mutagenesis studies with the rat m3 muscarinic receptor suggest that this region forms an amphiphilic alpha-helix and that the hydrophobic side of this helix represents an important G protein recognition surface. Further mutational analysis of this receptor segment showed that Tyr254 located at the N-terminus of the i3 loop of the m3 muscarinic receptor plays a key role in muscarinic receptor-induced Gq activation. The studies described here, complemented by biochemical and biophysical approaches, should eventually lead to a detailed structural model of the ligand-receptor-G protein complex.
ESTHER : Wess_1995_Life.Sci_56(11-12)_915
PubMedSearch : Wess_1995_Life.Sci_56(11-12)_915
PubMedID: 10188793

Title : Identification of a receptor\/G-protein contact site critical for signaling specificity and G-protein activation - Liu_1995_Proc.Natl.Acad.Sci.U.S.A_92_11642
Author(s) : Liu J , Conklin BR , Blin N , Yun J , Wess J
Ref : Proc Natl Acad Sci U S A , 92 :11642 , 1995
Abstract : Each G protein-coupled receptor recognizes only a distinct subset of the many structurally closely related G proteins expressed within a cell. How this selectively is achieved at a molecular level is not well understood, particularly since no specific point-to-point contact sites between a receptor and its cognate G protein(s) have been identified. In this study, we demonstrate that a 4-aa epitope on the m2 muscarinic acetylcholine receptor, a prototypical Gi/o-coupled receptor, can specifically recognize the C-terminal 5 aa of alpha subunits of the Gi/o protein family. The m2 receptor residues involved in this interaction are predicted to be located on one side of an alpha-helical receptor region present at the junction between the third intracellular loop and the sixth transmembrane domain. Coexpression studies with hybrid m2/m3 muscarinic receptors and mutant G-protein alpha q subunits showed that the receptor/G-protein contact site identified in this study is essential for coupling specificity and G-protein activation.
ESTHER : Liu_1995_Proc.Natl.Acad.Sci.U.S.A_92_11642
PubMedSearch : Liu_1995_Proc.Natl.Acad.Sci.U.S.A_92_11642
PubMedID: 8524820

Title : Muscarinic receptor subtypes--pharmacological, molecular biological and therapeutical aspects -
Author(s) : Mutschler E , Moser U , Wess J , Lambrecht G
Ref : Pharm Acta Helv , 69 :243 , 1995
PubMedID: 7651968

Title : Poster: Mapping of muscarinic receptor domains conferring specificity of coupling to Gq -
Author(s) : Blin N , Wess J
Ref : Life Sciences , 56(11-12) :1032 , 1995

Title : Functional role in ligand binding and receptor activation of an asparagine residue present in the sixth transmembrane domain of all muscarinic acetylcholine receptors - Bluml_1994_J.Biol.Chem_269_18870
Author(s) : Bluml K , Mutschler E , Wess J
Ref : Journal of Biological Chemistry , 269 :18870 , 1994
Abstract : The molecular mechanisms through which muscarinic receptors are activated upon binding of the neurotransmitter acetylcholine (ACh) are still poorly understood. Classical structure-function relationship studies have previously established that the ACh ester moiety plays a key role in muscarinic receptor recognition and activation. Consistent with this notion, all recently proposed three-dimensional muscarinic receptor models predict that an asparagine residue present in transmembrane domain VI of all muscarinic receptors is critically involved in the binding of the ACh ester moiety by means of hydrogen bonding. To test the correctness of this hypothesis, we created several mutant m3 muscarinic receptors in which this residue (Asn507) was replaced with alanine, serine, or aspartic acid. Radioligand binding studies with transfected COS-7 cells showed that, in contrast to the predictions made based on molecular modeling studies, all three mutant receptors were able to bind ACh and the structurally related muscarinic agonist, carbachol, with high affinities which differed from the corresponding wild type values by less than 5-fold. However, all three mutations led to dramatic reductions (235-28,300-fold) in binding affinities for certain subclasses of muscarinic antagonists including atropine-like agents and pirenzepine. The m3(Asn507-->Ala) and m3(Asn507-->Asp) mutant receptors were able to mediate carbachol-induced phosphatidylinositol hydrolysis in a fashion similar to that of the wild type receptor. Interestingly, the m3(Asn507-->Ser) mutant receptor displayed about 2-fold increased basal inositol phosphate levels, raising the possibility that it is constitutively active. In conclusion, our data suggest that the asparagine residue present in transmembrane domain VI of all muscarinic receptors is not critical for ACh binding and agonist-induced receptor activation, but plays a key role in the binding of certain subclasses of muscarinic antagonists.
ESTHER : Bluml_1994_J.Biol.Chem_269_18870
PubMedSearch : Bluml_1994_J.Biol.Chem_269_18870
PubMedID: 8034642

Title : Functional role of a cytoplasmic aromatic amino acid in muscarinic receptor-mediated activation of phospholipase C - Bluml_1994_J.Biol.Chem_269_11537
Author(s) : Bluml K , Mutschler E , Wess J
Ref : Journal of Biological Chemistry , 269 :11537 , 1994
Abstract : The N-terminal portion of the third intracellular loop (i3) of muscarinic acetylcholine and other G protein-coupled receptors has been shown to largely determine the G protein coupling profile of a given receptor subtype. Using the rat m3 muscarinic receptor as a model system, we have recently demonstrated that a tyrosine residue (Tyr-254), located at the beginning of the i3 domain, is critically involved in muscarinic receptor-mediated stimulation of phosphatidylinositol (PI) hydrolysis (Bluml, K., Mutschler, E., and Wess, J. (1994) J. Biol. Chem. 269, 402-405). This study was designed to investigate the functional role of this amino acid in further molecular detail. Replacement of Tyr-254 (rat m3 receptor) with alanine or exchange of its position with Ile-253 virtually abolished receptor-mediated stimulation of PI hydrolysis studied in transfected COS-7 cells. In contrast, substitution of Tyr-254 by other aromatic residues such as phenylalanine or tryptophan resulted in mutant receptors that behaved functionally similar to the wild type m3 receptor. Introduction of Tyr-254 into the corresponding position (Ser-210) of the m2 muscarinic receptor (which is only poorly coupled to PI turnover) did not result in an enhanced PI response. However, "reinsertion" of Tyr-254 into a functionally inactive chimeric m3/m2 muscarinic receptor (containing m2 receptor sequence at the N terminus of the i3 loop) yielded a mutant receptor that was able to stimulate PI hydrolysis to a similar maximum extent as the wild type m3 receptor. Taken together, our data provide strong evidence that muscarinic receptor-mediated stimulation of PI metabolism is critically dependent on the presence and proper positioning of an aromatic residue at the beginning of the i3 loop.
ESTHER : Bluml_1994_J.Biol.Chem_269_11537
PubMedSearch : Bluml_1994_J.Biol.Chem_269_11537
PubMedID: 8157684

Title : Identification of an intracellular tyrosine residue critical for muscarinic receptor-mediated stimulation of phosphatidylinositol hydrolysis - Bluml_1994_J.Biol.Chem_269_402
Author(s) : Bluml K , Mutschler E , Wess J
Ref : Journal of Biological Chemistry , 269 :402 , 1994
Abstract : Several lines of evidence suggest that the N-terminal portion of the third cytoplasmic loop (i3) of muscarinic and other G protein-coupled receptors is of pivotal importance for G protein recognition and activation. The present study was designed to identify specific amino acids within this domain required for muscarinic receptor-induced activation of G proteins mediating stimulation of phosphatidylinositol (PI) hydrolysis. Among the five mammalian muscarinic receptors (m1-m5), only the m1, m3, and m5 receptors are efficiently coupled to this second messenger pathway. Initially, we created a series of rat m3 receptor mutants in which short segments in the N terminus of the i3 loop were replaced with the corresponding m2 receptor sequences. The effect of these substitutions on m3 receptor-mediated stimulation of PI hydrolysis was studied in transiently transfected COS-7 cells. We found that a stretch of 4 amino acids (Arg252-Ile253-Tyr254-Lys255) located at the beginning of the i3 domain of the m3 muscarinic receptor is critically involved in receptor-mediated stimulation of PI hydrolysis. Further mutational analysis of this 4-amino acid segment by single amino acid substitutions demonstrated that only Tyr254 is essential for efficient activation of the PI pathway. This tyrosine residue is conserved among all PI-coupled muscarinic receptors as well as in many other biogenic amine and glycoprotein hormone receptors, suggesting that it may also play an important functional role in other G protein-coupled receptors.
ESTHER : Bluml_1994_J.Biol.Chem_269_402
PubMedSearch : Bluml_1994_J.Biol.Chem_269_402
PubMedID: 8276826

Title : Insertion mutagenesis as a tool to predict the secondary structure of a muscarinic receptor domain determining specificity of G-protein coupling - Bluml_1994_Proc.Natl.Acad.Sci.U.S.A_91_7980
Author(s) : Bluml K , Mutschler E , Wess J
Ref : Proc Natl Acad Sci U S A , 91 :7980 , 1994
Abstract : The N-terminal segment of the third intracellular loop (i3) of muscarinic acetylcholine receptors and other G protein-coupled receptors has been shown to largely determine the G-protein coupling selectivity displayed by a given receptor subtype. Based on secondary-structure prediction algorithms, we have tested the hypothesis that this region adopts an alpha-helical secondary structure. Using the rat m3 muscarinic receptor as a model system, a series of five mutant receptors, m3(+1A) to m3(+5A) were created in which one to five additional alanine residues were inserted between the end of the fifth transmembrane domain and the beginning of i3. We speculated that this manipulation should lead to a rotation of the N-terminal segment of the i3 domain (if it is in fact alpha-helically arranged), thus producing pronounced effects on receptor/G protein coupling. Pharmacological analysis of the various mutant receptors expressed in COS-7 cells showed that m3(+1A), m3(+3A), and m3(+4A) retained strong functional activity, whereas m3(+2A) and m3(+5A) proved to be virtually inactive. Helical wheel models show that this pattern is fully consistent with the notion that the N-terminal portion of i3 forms an amphiphilic alpha-helix and that the hydrophobic side of this helix represents the G-protein recognition surface.
ESTHER : Bluml_1994_Proc.Natl.Acad.Sci.U.S.A_91_7980
PubMedSearch : Bluml_1994_Proc.Natl.Acad.Sci.U.S.A_91_7980
PubMedID: 8058746

Title : Poster: Conserved threonine and tyrosine residues play important roles in acetylcholine binding and muscarinic receptor activation -
Author(s) : Vogel Z , Maggio R , Palmer JR , Wess J
Ref : Life Sciences , 52(5-6) :558 , 1993

Title : Coexpression studies with mutant muscarinic\/adrenergic receptors provide evidence for intermolecular cross-talk between G-protein-linked receptors - Maggio_1993_Proc.Natl.Acad.Sci.U.S.A_90_3103
Author(s) : Maggio R , Vogel Z , Wess J
Ref : Proc Natl Acad Sci U S A , 90 :3103 , 1993
Abstract : We have tested the hypothesis that guanine-nucleotide-binding-protein-coupled receptors may be able to interact with each other at a molecular level. To address this question, we have initially created two chimeric receptors, alpha 2/m3 and m3/alpha 2, in which the C-terminal receptor portions (containing transmembrane domains VI and VII) were exchanged between the alpha 2C-adrenergic and the m3 muscarinic receptor. Transfection of COS-7 cells with either of the two chimeric constructs alone did not result in any detectable binding activity for the muscarinic ligand N-[3H]methylscopolamine or the adrenergic ligand [3H]rauwolscine. However, cotransfection with alpha 2/m3 and m3/alpha 2 resulted in the appearance of specific binding sites (30-35 fmol/mg of membrane protein) for both radioligands. These sites displayed ligand binding properties similar to those of the two wild-type receptors. Furthermore, COS-7 cells cotransfected with alpha 2/m3 and m3/alpha 2 were able to mediate a pronounced stimulation of phosphatidylinositol hydrolysis upon stimulation with the muscarinic agonist carbachol (Emax approximately 40-50% of wild-type m3). A mutant m3 receptor (containing 16 amino acids of m2 receptor sequence at the N terminus of the third cytoplasmic loop) that was capable of binding muscarinic ligands but was virtually unable to stimulate phosphatidylinositol hydrolysis was also used in various cotransfection experiments. Coexpression of this chimeric receptor with other functionally impaired mutant muscarinic receptors (e.g., with an m3 receptor containing a Pro-->Ala point mutation in transmembrane region VII) resulted in a considerable stimulation of phosphatidylinositol breakdown after carbachol treatment (Emax approximately 40-50% of wild-type m3). Thus, these data suggest that guanine-nucleotide-binding-protein-coupled receptors can interact with each other at a molecular level. One may speculate that the formation of receptor dimers involving the intermolecular exchange of N- and C-terminal receptor domains (containing transmembrane domains I-V and VI and VII, respectively) may underlie this phenomenon.
ESTHER : Maggio_1993_Proc.Natl.Acad.Sci.U.S.A_90_3103
PubMedSearch : Maggio_1993_Proc.Natl.Acad.Sci.U.S.A_90_3103
PubMedID: 8385357

Title : Poster: Mutational analysis of the m3 muscarinic receptor: Functional role of proline residues that are highly conserved among all G protein-coupled receptors -
Author(s) : Wess J , Nanavati S , Vogel Z , Maggio R
Ref : Life Sciences , 52(5-6) :557 , 1993

Title : Functional role of proline and tryptophan residues highly conserved among G protein-coupled receptors studied by mutational analysis of the m3 muscarinic receptor - Wess_1993_EMBO.J_12_331
Author(s) : Wess J , Nanavati S , Vogel Z , Maggio R
Ref : EMBO Journal , 12 :331 , 1993
Abstract : Most G protein-coupled receptors contain a series of highly conserved proline and tryptophan residues within their hydrophobic transmembrane domains (TMD I-VII). To study their potential role in ligand binding and receptor function, the rat m3 muscarinic acetylcholine receptor was used as a model system. A series of mutant receptors in which the conserved proline and tryptophan residues were individually replaced with alanine and phenylalanine, respectively, was created and transiently expressed in COS-7 cells. [3H]N-methylscopolamine ([3H]NMS) saturation binding studies showed that three of the seven mutant receptors studied (Pro242-->Ala, TMD V; Pro505-->Ala, TMD VI; Pro540-->Ala, TMD VII) were expressed at 35-100 times lower levels than the wild-type receptor while displaying 'm3-like' antagonist binding affinities. Pro201-->Ala (TMD IV) showed drastically reduced binding affinities (up to 450-fold) for both muscarinic agonists and antagonists. Whereas most mutant receptors retained strong functional activity, Pro540-->Ala (TMD VII) was found to be severely impaired in its ability to stimulate carbachol-induced phosphatidyl inositol hydrolysis (Emax approximately 25% of wild type m3). Interestingly, this mutant receptor bound muscarinic agonists with 7- to 19-fold higher affinities than the wild type receptor. The Trp-->Phe substitutions (Trp192-->Phe, TMD IV; Trp503-->Phe, TMD VI; Trp530-->Phe, TMD VII) resulted in less pronounced changes (compared with the Pro-->Ala mutant receptors) in both ligand binding and receptor function. Our data indicate that the proline residues that are highly conserved across the entire superfamily of G protein-coupled receptors play key roles in receptor expression, ligand binding and receptor activation.
ESTHER : Wess_1993_EMBO.J_12_331
PubMedSearch : Wess_1993_EMBO.J_12_331
PubMedID: 7679072

Title : Poster: Coexpression of N- and C- terminal muscarinic receptor domains results in functional receptors -
Author(s) : Maggio R , Vogel Z , Nanavati S , Wess J
Ref : Life Sciences , 52(5-6) :558 , 1993

Title : Role of conserved threonine and tyrosine residues in acetylcholine binding and muscarinic receptor activation. A study with m3 muscarinic receptor point mutants - Wess_1992_J.Biol.Chem_267_19313
Author(s) : Wess J , Maggio R , Palmer JR , Vogel Z
Ref : Journal of Biological Chemistry , 267 :19313 , 1992
Abstract : Structure-function relationship studies of the m3 muscarinic acetylcholine receptor have recently identified a series of threonine and tyrosine residues (all located within the hydrophobic receptor core) that are critically involved in acetylcholine binding (Wess, J., Gdula, D., and Brann, M.R. (1991) EMBO J. 10, 3729-3734). To gain further insight into the functional roles of these amino acids, the agonist binding properties of six rat m3 muscarinic receptor point mutants, in which the critical threonine and tyrosine residues had been individually replaced by alanine and phenylalanine, respectively, were studied in greater detail following their transient expression in COS-7 cells. The binding profiles of a series of acetylcholine derivatives suggest that the altered threonine and tyrosine residues are primarily involved in the interaction of the acetylcholine ester moiety with the receptor protein. The two m3 receptor point mutants, Thr234----Ala and Tyr506----Phe, which showed the most pronounced decreases in acetylcholine binding affinities (approximately 40-60-fold as compared with the wild-type receptor), were stably expressed in CHO cells for further functional analysis. Both mutant receptors were found to be severely impaired in their ability to stimulate agonist-dependent phosphatidylinositol hydrolysis. Consistent with this observation, acetylcholine binding to the two mutant receptors was not significantly affected by addition of the GTP analog Gpp(NH)p (5'-guanylyl imidodiphosphate). Our data suggest that Thr234 and Tyr506 (located within transmembrane domains V and VI, respectively), which are conserved among all muscarinic receptors (m1-m5), may play an important role in agonist-induced muscarinic receptor activation.
ESTHER : Wess_1992_J.Biol.Chem_267_19313
PubMedSearch : Wess_1992_J.Biol.Chem_267_19313
PubMedID: 1527051

Title : Selectivity profile of the novel muscarinic antagonist UH-AH 37 determined by the use of cloned receptors and isolated tissue preparations - Wess_1991_Br.J.Pharmacol_102_246
Author(s) : Wess J , Lambrecht G , Mutschler E , Brann MR , Dorje F
Ref : British Journal of Pharmacology , 102 :246 , 1991
Abstract : 1. Functional in vitro experiments were carried out to determine the antimuscarinic potencies of the pirenzepine derivative UH-AH 37 (6-chloro-5,10-dihydro-5-[(1-methyl-4-piperidinyl)acetyl]-11H-dibenzo- [b,e] [1,4] diazepine-11-one hydrochloride) at M1 muscarinic receptors of rabbit vas deferens, M2 receptors of rat left atria and M3 receptors of rat ileum. Furthermore, N-[3H]-methylscopolamine competition binding experiments were performed to obtain its affinities for the five cloned human muscarinic receptors (m1-m5) stably expressed in CHO-K1 cells. Pirenzepine served as a reference drug throughout all experiments. 2. In all preparations used, UH-AH 37 interacted with muscarinic receptors in a fashion characteristic of a simple competitive antagonist. 3. In the functional studies, UH-AH 37, like pirenzepine, showed high affinity for M1 (pA2 8.49) and low affinity for M2 muscarinic receptors (pA2 6.63). In contrast to pirenzepine, UH-AH 37 also displayed high affinity for M3 receptors (pA2 8.04). 4. In agreement with its functional profile, UH-AH 37 bound with highest affinity to m1 (pKi 8.74) and with lowest affinity to m2 receptors (pKi 7.35). Moreover, it showed a 7 fold higher affinity for m3 (pKi 8.19) than for m2 receptors, whereas pirenzepine bound to both receptors with low affinities. 5. The binding affinity of UH-AH 37 for m4 and m5 receptors (pKi 8.32 for both receptors) was only ca. 2.5 fold lower than that for m1 receptors, while the corresponding affinity differences were 6 and 13 fold in case of pirenzepine. 6. In conclusion, the receptor selectivity profile of UH-AH 37 differs clearly from that of its parent compound, pirenzepine, in both functional and radioligand binding studies, the major characteristics being its pronounced M2 (m2)/M3 (m3) selectivity. UH-AH 37 thus represents a useful tool for the further pharmacological characterization of muscarinic receptor subtypes.
ESTHER : Wess_1991_Br.J.Pharmacol_102_246
PubMedSearch : Wess_1991_Br.J.Pharmacol_102_246
PubMedID: 2043926

Title : Antagonist binding profiles of five cloned human muscarinic receptor subtypes - Dorje_1991_J.Pharmacol.Exp.Ther_256_727
Author(s) : Dorje F , Wess J , Lambrecht G , Tacke R , Mutschler E , Brann MR
Ref : Journal of Pharmacology & Experimental Therapeutics , 256 :727 , 1991
Abstract : A variety of muscarinic antagonists are currently used as tools to pharmacologically subclassify muscarinic receptors into M1, M2 and M3 subtypes. In the present study, we have determined the affinity profiles of several of these antagonists at five cloned human muscarinic receptors (m1-m5) stably expressed in Chinese hamster ovary cells (CHO-K1). At all five receptors, the (R)-enantiomers of trihexyphenidyl and hexbutinol displayed considerably higher affinities (up to 525-fold) than their corresponding (S)-isomers. The stereoselectivity ratios [inhibition constant(S)/inhibition constant(R)] for both pairs of enantiomers were lowest at m2 receptors, suggesting that less stringent configurational demands are made by this receptor subtype. The "M1-selective" antagonist (R)-trihexyphenidyl displayed high affinities for m1 and m4 receptors. The "M2-selective" antagonists himbacine, (+-)-5,11-dihydro-11- ([(2-[(dipropylamino)methyl]-1- piperidinyl)ethyl)amino]carbonyl)-6H-pyrido(2,3-b)(1,4)benzodiazepine-6- one (AF-DX 384), 11-[4-[4-(diethylamino)butyl]-1-piperidinyl)acetyl)-5,11- dihydro-6H-pyrido(2,3-b) (1,4)benzodiazepine-6-one (AQ-RA 741) and (+)-(11-[2-[(diethylamino) methyl]-1-piperidinyl)acetyl)-5,11-di-hydro-6H-pyrido(2,3-b)(1,4) benzodiazepine-6-one [AF-DX 250; the (+)-enantiomer of AF-DX 116] exhibited high affinities for m2 and m4, intermediate affinities for m1 and m3 and low affinities for m5 receptors. This selectivity profile was most prominent for AQ-RA 741, which displayed 195- and 129-fold higher affinities for m2 and m4 receptors than for m5 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)
ESTHER : Dorje_1991_J.Pharmacol.Exp.Ther_256_727
PubMedSearch : Dorje_1991_J.Pharmacol.Exp.Ther_256_727
PubMedID: 1994002

Title : Chimeric m2\/m3 muscarinic receptors: role of carboxyl terminal receptor domains in selectivity of ligand binding and coupling to phosphoinositide hydrolysis - Wess_1990_Mol.Pharmacol_38_872
Author(s) : Wess J , Bonner TI , Brann MR
Ref : Molecular Pharmacology , 38 :872 , 1990
Abstract : The cloning and expression of five mammalian muscarinic receptor genes (m1-m5) have shown that the individual receptor subtypes differ in their functional and ligand-binding properties. To study the role of the carboxyl terminal receptor domains in this pharmacological diversity, we constructed chimeric m2/m3 receptors in which a region comprising part of transmembrane domain VI, the third extracellular loop, transmembrane region VII, and the cytoplasmic tail (collectively referred to as C-terminal domains) was exchanged between the human m2 and the rat m3 receptor. The ability of the cloned receptors to mediate stimulation of phosphoinositide hydrolysis and to bind subtype-selective muscarinic ligands was studied after their transient expression in COS-7 cells. Whereas wild-type m3 strongly stimulated phosphoinositide breakdown, wild-type m2 gave only a poor response. Exchange of the C-terminal domains between m2 and m3 had no significant effect on the magnitude of these responses. In N-[3H]methylscopolamine competition binding studies, the muscarinic antagonists AF-DX 116 and methoctramine showed 11- and 23-fold higher affinities, respectively, for m2 than for m3, whereas hexahydro-silad-ifenidol (HHSiD) and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) displayed the reverse selectivity profile, having approximately 10-fold higher affinities for m3. In comparison with wild-type m3, the mutant m3 receptor containing the C-terminal domains of m2 displayed 2.5- and 8-fold higher affinities for AF-DX 116 and methoctramine but 7- and 3-fold lower affinities for HHSiD and 4-DAMP, respectively. The mutant m2 receptor with the C-terminal domains of m3 showed 2-3-fold lower affinities for AF-DX 116 and methoctramine but 2-3-fold higher affinities for HHSiD and 4-DAMP, as compared with wild-type m2. These data suggest that the C-terminal domains of the muscarinic receptors are not involved in conferring selectivity of coupling to phosphoinositide hydrolysis but contain major structural determinants of antagonist binding selectivity.
ESTHER : Wess_1990_Mol.Pharmacol_38_872
PubMedSearch : Wess_1990_Mol.Pharmacol_38_872
PubMedID: 2174507

Title : Delineation of muscarinic receptor domains conferring selectivity of coupling to guanine nucleotide-binding proteins and second messengers - Wess_1990_Mol.Pharmacol_38_517
Author(s) : Wess J , Bonner TI , Dorje F , Brann MR
Ref : Molecular Pharmacology , 38 :517 , 1990
Abstract : The cloning and functional expression of five mammalian muscarinic acetylcholine receptor genes (m1-m5) has revealed that m1, m3, and m5 primarily couple to stimulation of phosphoinositide (PI) turnover, whereas m2 and m4 are strongly linked to inhibition of adenylate cyclase, albeit not exclusively. To identify the structural domains responsible for this functional specificity, cDNAs encoding chimeric m2/m3 receptors were constructed. The abilities of these receptors to mediate stimulation of PI hydrolysis and inhibition of prostaglandin E2-stimulated cAMP accumulation, as well as the pertussis toxin (PTX) sensitivity of these responses, were examined after stable expression in mouse A9 L cells. Substitution of the putative third cytoplasmic loop (i3) of m2 with the corresponding m3 sequence resulted in a chimeric receptor that, similar to m3, stimulated PI breakdown by a PTX-insensitive mechanism but did not inhibit adenylate cyclase. Conversely, a chimeric m3 receptor containing the i3 domain of m2 showed the same functional profile as m2 (i.e., inhibition of adenylate cyclase and weak stimulation of PI turnover by a PTX-sensitive mechanism), indicating that the i3 loop is sufficient to determine coupling selectivity. Similarly, exchange of a short N-terminal segment of i3 (16 or 17 amino acids) between m2 and m3 resulted in chimeric receptors that gained the ability to mediate the functional responses of the wild-type receptor from which the segment was derived, although with substantially reduced efficiency. However, the chimeric m2 receptor containing the 17-amino acid m3 sequence in the N-terminal portion of i3 retained its ability to inhibit adenylate cyclase. Carbachol binding studies involving the use of the GTP analog 5'-guanylyl imidodiphosphate and PTX-pretreated cells generally correlated well with the functional findings. Our data indicate that the N-terminal portion of i3 is a sufficient but not the exclusive determinant of coupling selectivity displayed by the various muscarinic receptors.
ESTHER : Wess_1990_Mol.Pharmacol_38_517
PubMedSearch : Wess_1990_Mol.Pharmacol_38_517
PubMedID: 2172767

Title : Molecular genetics of signal transduction by muscarinic acetylcholine receptors -
Author(s) : Brann MR , Wess J , Jones SV
Ref : Soc Gen Physiol Ser , 45 :105 , 1990
PubMedID: 2198665

Title : Synthesis and muscarinic activity of a series of tertiary and quaternary N-substituted guvacine esters structurally related to arecoline and arecaidine propargyl ester - Wolf-Pflugmann_1989_Arzneimittelforschung_39_539
Author(s) : Wolf-Pflugmann M , Lambrecht G , Wess J , Mutschler E
Ref : Arzneimittelforschung , 39 :539 , 1989
Abstract : A series of tertiary and quaternary N-substituted guvacine (1,2,5,6-tetrahydro-3-carboxy-pyridine) methyl and propargyl esters have been synthesized and tested for muscarinic/antimuscarinic activity on rat ileum and electrically paced left atria. Arecoline and arecaidine propargyl ester (APE) as well as their corresponding N-demethyl derivatives, guvacoline (norarecoline) and guvacine propargyl ester, acted as full agonists at both atrial and ileal muscarinic receptors (range of pD2-values 6.09-8.07). However, in both preparations arecoline and APE were clearly more potent (up to 15-fold) than their N-demethyl analogues. Replacement of the N-methyl group in arecoline and APE by larger substituents (ethyl, n-propyl, n-butyl, benzyl, phenylethyl) as well as N-methylation resulted in a decrease or even a complete loss of agonistic activity. In both organs, the propargyl esters usually showed higher potency than the corresponding methyl ester analogues. N-Ethylguvacine propargyl ester and APE methiodide displayed pronounced agonistic activity in the atria (pD2 approximately 6.5; intrinsic activity = 0.79 and 0.67, respectively) but behaved as competitive antagonists in the ileum (pA2 = 6.06 and 5.62, respectively). Beside the lower sensitivity to muscarinic agonists of the rat ileum as compared to rat atria, the cardioselective stimulant action of both agents may also be due to their ability to recognize structural differences between atrial M2 alpha and ileal M2 beta muscarinic receptor subtypes.
ESTHER : Wolf-Pflugmann_1989_Arzneimittelforschung_39_539
PubMedSearch : Wolf-Pflugmann_1989_Arzneimittelforschung_39_539
PubMedID: 2757669

Title : Identification of a small intracellular region of the muscarinic m3 receptor as a determinant of selective coupling to PI turnover - Wess_1989_FEBS.Lett_258_133
Author(s) : Wess J , Brann MR , Bonner TI
Ref : FEBS Letters , 258 :133 , 1989
Abstract : Molecular cloning studies have demonstrated the existence of five different muscarinic receptors (m1-m5). While m1, m3 and m5 strongly couple to stimulation of phosphoinositide (PI) hydrolysis, m2 and m4 are more efficiently linked to inhibition of adenylate cyclase. The sequences of m1-m5 have a short segment at the N-terminal portion of the putative third cytoplasmic loop (i3) which is highly conserved among m1, m3 and m5, but different from the sequence which is well conserved among m2 and m4. To study the role of this region in conferring coupling selectivity, we constructed cDNAs encoding chimeric m2/m3 receptors. Transient expression of these receptor hybrids in COS-7 cells showed that a 17 amino acid segment at the N-terminal portion of i3 is a major determinant of how efficiently the different muscarinic receptors are coupled to PI hydrolysis.
ESTHER : Wess_1989_FEBS.Lett_258_133
PubMedSearch : Wess_1989_FEBS.Lett_258_133
PubMedID: 2556294

Title : Structure-activity relationships of new analogues of arecaidine propargyl ester at muscarinic M1 and M2 receptor subtypes - Moser_1989_Br.J.Pharmacol_96_319
Author(s) : Moser U , Lambrecht G , Wagner M , Wess J , Mutschler E
Ref : British Journal of Pharmacology , 96 :319 , 1989
Abstract : 1. The potency of arecaidine propargyl ester (APE) and of several analogues containing a modified ester side chain has been assessed at M1 and M2 muscarinic receptor subtypes. APE was shown to act as a potent agonist at ganglionic M1 receptors in the pithed rat, at M2 receptors in guinea-pig isolated atria (-log EC50 = 8.22) and ileum (-log EC50 = 7.77). 2. The arecaidine 2-butynyl and 2-pentynyl esters were approximately equipotent with APE at M1 and M2 receptors, whereas the 2-hexynyl derivative was found to be less potent than APE in atria (-log EC50 = 6.80) and ileum (-log EC50 = 6.70) by about one order of magnitude. The 2-heptynyl and 3-phenyl propargyl esters exhibited no agonist actions in atria and ileum. 3. Shifting the triple bond from the 2 to the 3 position and introducing a bulky group at position 1 of the ester side chain of APE and analogues resulted in competitive antagonists (pA2 ranging from 4.9 to 7.3). 4. APE and its 2-butynyl analogue showed some agonistic selectivity for cardiac M2 receptors (potency ratio, ileum/atria = 2.8 and 4.6 respectively). All antagonists in this series of compounds were not selective in terms of affinity since their pA2 values at cardiac and ileal M2 receptors were similar (potency ratios, ileum/atria = 0.4 to 1.2).
ESTHER : Moser_1989_Br.J.Pharmacol_96_319
PubMedSearch : Moser_1989_Br.J.Pharmacol_96_319
PubMedID: 2924082

Title : Poster: Identification of a small intracellular region of the rat m3 receptor responsible for selective coupling to PI turnover -
Author(s) : Wess J , Brann MR , Bonner TI
Ref : Trends in Pharmacological Sciences , Suppl :115 , 1989

Title : Presynaptic muscarinic receptors mediating inhibition of neurogenic contractions in rabbit vas deferens are of the ganglionic M1-type - Eltz_1988_Eur.J.Pharmacol_158_233
Author(s) : Eltz M , Gmelin G , Wess J , Strohmann C , Tacke R , Mutschler E , Lambrecht G
Ref : European Journal of Pharmacology , 158 :233 , 1988
Abstract : The present study was designed to further characterize the presynaptic muscarinic M1-receptor responsible for the inhibition of neurogenic contractions in the isolated rabbit vas deferens. Electrically induced twitch contractions of this preparation were inhibited by the M1-agonist, McN-A-343, and by some of its analogs: 4-chloro-phenyl derivative greater than McN-A-343 greater than trans-olefinic analog greater than cis-olefinic analog. The same rank order of potency was observed for these agonists to raise the blood pressure of pithed rats by stimulation of M1-receptors in sympathetic ganglia. A highly significant correlation was found between the antimuscarinic potencies of atropine, pirenzepine and a series of 9 antagonists structurally related to the ganglionic M1 beta-receptor selective compounds, hexocyclium and hexahydro-difenidol, to antagonize the McN-A-343-induced inhibition of twitch contractions in rabbit vas deferens or the muscarine-induced depolarization in rat isolated superior cervical ganglia. It is suggested that the presynaptic muscarinic receptor that mediates inhibition of neurogenic contractions in rabbit vas deferens is of the ganglionic M1 beta-type.
ESTHER : Eltz_1988_Eur.J.Pharmacol_158_233
PubMedSearch : Eltz_1988_Eur.J.Pharmacol_158_233
PubMedID: 2472963

Title : Methoctramine selectively blocks cardiac muscarinic M2 receptors in vivo - Wess_1988_Naunyn.Schmiedebergs.Arch.Pharmacol_338_246
Author(s) : Wess J , Angeli P , Melchiorre C , Moser U , Mutschler E , Lambrecht G
Ref : Naunyn Schmiedebergs Arch Pharmacol , 338 :246 , 1988
Abstract : The antimuscarinic effects of methoctramine (N, N'-bis[6-[(2-methoxybenzyl)amino]hexyl]-1, 8-octanediamine tetrahydrochloride), a polymethylene tetraamine endowed with high cardioselectivity in vitro, were assessed in two in vivo preparations. Methoctramine (300 micrograms/kg i.v.) strongly inhibited the methacholine- and muscarine-induced bradycardia in the anaesthetized an pithed rat, respectively. The same dose of methoctramine did not significantly affect the depressor action of methacholine in the anaesthetized rat mediated by vascular M2 receptors. Furthermore, even high doses of methoctramine (up to 1 mg/kg i.v.) did not reduce the ganglionic M1 receptor-mediated tachycardia and pressor response to muscarine or McN-A-343 in the pithed rat. These data suggest that methoctramine while showing high affinity for cardiac M2 alpha receptors has rather low affinity for ganglionic M1 and vascular M2 receptors. This in vivo study thus provides further evidence to support the view that methoctramine is a potent and highly selective antagonist of cardiac M2 alpha receptors.
ESTHER : Wess_1988_Naunyn.Schmiedebergs.Arch.Pharmacol_338_246
PubMedSearch : Wess_1988_Naunyn.Schmiedebergs.Arch.Pharmacol_338_246
PubMedID: 3057387

Title : Stimulation of ganglionic muscarinic M1 receptors by a series of tertiary arecaidine and isoarecaidine esters in the pithed rat - Wess_1987_Eur.J.Pharmacol_134_61
Author(s) : Wess J , Lambrecht G , Moser U , Mutschler E
Ref : European Journal of Pharmacology , 134 :61 , 1987
Abstract : The cardiovascular effects of a series of tertiary esters of arecaidine (1-methyl-1,2,5,6-tetrahydro-3-carboxy-pyridine) and isoarecaidine (1-methyl-1,2,5,6-tetrahydro-4-carboxy-pyridine) were investigated in the pithed rat. For some esters (e.g. arecoline, arecaidine propargyl ester, isoarecoline) a prominent elevation in mean arterial pressure and heart rate was observed following an initial short-lasting and atropine-sensitive depressor response and bradycardia (dose range: 0.1-10 mumol/kg i.v.). The increase in blood pressure and heart rate was not affected by pretreatment with mecamylamine (0.5 and 5 mg/kg i.v.), but could be totally blocked by N-methylatropine (500 micrograms/kg i.v.). Furthermore, the M1 receptor antagonist pirenzepine (300 micrograms/kg i.v.) selectively antagonized these stimulatory cardiovascular responses, indicating that these effects are due to an activation of muscarinic M1 receptors in sympathetic ganglia. As tertiary arecaidine and isoarecaidine esters easily penetrate the blood-brain barrier, they might also stimulate central M1 receptors and thus become lead compounds in the search for an effective drug treatment of Alzheimer's disease.
ESTHER : Wess_1987_Eur.J.Pharmacol_134_61
PubMedSearch : Wess_1987_Eur.J.Pharmacol_134_61
PubMedID: 3556399

Title : Selective blockade in vivo of cardiac muscarinic M2 receptors by a polymethylene tetramine, BHC-9C - Wess_1987_Eur.J.Pharmacol_142_475
Author(s) : Wess J , Lambrecht G , Mutschler E , Melchiorre C , Angeli P
Ref : European Journal of Pharmacology , 142 :475 , 1987
Abstract : The antimuscarinic effects of BHC-9C (N,N'-bis[6-[(2-methoxybenzyl)amino]hexyl]-1,9-nonanediamine tetrahydrochloride), a member of a series of polymethylene tetraamines with unprecedented in vitro selectivity for cardiac muscarinic M2 receptors, were assessed in several in vivo test systems. BHC-9C (300 micrograms/kg i.v.) proved to be a potent antagonist at cardiac M2 receptors that mediate the decrease in heart rate in the pithed rat. In contrast, it displayed no considerable blocking activity at vascular M2 receptors subserving vasodepression and at ganglionic M1 receptors that mediate cardiovascular stimulation in the anaesthetized and pithed rat, respectively. These in vivo data are consistent with the suggestion based on in vitro experiments that BHC-9C is a highly selective antagonist of cardiac muscarinic M2 receptors.
ESTHER : Wess_1987_Eur.J.Pharmacol_142_475
PubMedSearch : Wess_1987_Eur.J.Pharmacol_142_475
PubMedID: 3428357

Title : Antimuscarinic action of methoctramine, a new cardioselective M-2 muscarinic receptor antagonist, alone and in combination with atropine and gallamine - Melchiorre_1987_Eur.J.Pharmacol_144_117
Author(s) : Melchiorre C , Angeli P , Lambrecht G , Mutschler E , Picchio MT , Wess J
Ref : European Journal of Pharmacology , 144 :117 , 1987
Abstract : The antimuscarinic effects of methoctramine (N,N'-bis[6- [(2-methoxybenzyl)amino]hexyl]-1,8-octanediamine tetrahydrochloride) were investigated in vitro in isolated paced left (force) and spontaneously beating right (force and rate) atria of guinea pigs as well as ileum of guinea pig and rat. Methoctramine was a potent competitive antagonist of M-2 muscarinic receptors in myocardium and pacemaker cells over a wide range of concentrations. The pA2 values ranged from 7.74 to 7.93. They were not significantly different in the two cardiac preparations and were independent of the agonist used (muscarine and carbachol). A combination of methoctramine with atropine resulted in addition of the dose ratios for left atria, which is expected for two antagonists interacting competitively with the same receptor site. In contrast, a combination of methoctramine with gallamine produced a less than additive shift of the dose-response curve for carbachol, confirming that gallamine acts as an allosteric antagonist at cardiac muscarinic receptors. Methoctramine was 54 to 132-fold less potent in ileal than in atrial preparations (pA2 values ranging from 5.81 to 6.20) which makes it the most cardioselective antimuscarinic agent now available. A combination of methoctramine with atropine gave a slight supra-additive antagonism on guinea pig ileum, which suggests that methoctramine interacts to some extent with a second independent site. These results strongly reinforce the view that M-2 muscarinic receptors are not a homogeneous population.
ESTHER : Melchiorre_1987_Eur.J.Pharmacol_144_117
PubMedSearch : Melchiorre_1987_Eur.J.Pharmacol_144_117
PubMedID: 3436364

Title : Muscarinic ganglionic stimulants: conformationally restrained analogues related to [4-[[N-(3-chlorophenyl)carbamoyl]oxy]-2-butynyl]trimethylammonium chloride - Lambrecht_1986_J.Med.Chem_29_1309
Author(s) : Lambrecht G , Moser U , Mutschler E , Walther G , Wess J
Ref : Journal of Medicinal Chemistry , 29 :1309 , 1986
Abstract : The synthesis of a series of tertiary and quaternary cyclic analogues (isoarecolinol, dihydroisoarecolinol, arecolinol, and 3-pyrroline-3-carbinol derivatives) of [4-[[N-(3-chlorophenyl)carbamoyl]oxy]-2-butynyl]trimethylammonium chloride (McN-A-343) (1), a selective stimulant of muscarinic receptors in sympathetic ganglia (so-called M1 receptors), is reported. The compounds 3-10 were tested for muscarinic ganglion-stimulating activity by recording blood pressure responses in pithed rats. All tertiary compounds tested had no ganglion-stimulating activity. Among the series of quaternary derivatives, only the isoarecolinol analogues 4a and 4b showed considerable ganglion-stimulating effects, whereas the dihydroisoarecolinol (8), the arecolinol (6a, 6b), and the 3-pyrroline-3-carbinol derivatives (10) were much less potent. Our experiments therefore demonstrate that in this series a quaternary nitrogen atom, unsaturation at C2 of the ammonium side chain, and a certain spatial arrangement of the ammonium and the phenylcarbamate groups are essential for potent M1-receptor stimulating activity.
ESTHER : Lambrecht_1986_J.Med.Chem_29_1309
PubMedSearch : Lambrecht_1986_J.Med.Chem_29_1309
PubMedID: 3806582

Title : Poster: Hexahydrosiladifenidol: a selective antagonist at muscarinic M2 receptor subtypes -
Author(s) : Lambrecht G , Moser U , Wess J , Riotte J , Fuder H , Kilbinger H , Milller H , Linoh H , Tacke R , Zilch H , Mutschler E
Ref : Trends in Pharmacological Sciences , Suppl :91 , 1986

Title : A comparison of the antimuscarinic effects of pirenzepine and N-methylatropine on ganglionic and vascular muscarinic receptors in the rat - Wess_1984_Life.Sci_35_553
Author(s) : Wess J , Lambrecht G , Moser U , Mutschler E
Ref : Life Sciences , 35 :553 , 1984
Abstract : The antimuscarinic properties of pirenzepine and N-methylatropine were evaluated in two intact preparations by measuring A) the inhibition of increase in mean arterial pressure evoked by McN-A-343 in pithed rats through activation of ganglionic muscarinic receptors and B) the inhibition of fall in arterial pressure evoked by methacholine in anaesthetized rats through activation of vascular muscarinic receptors. To characterize the antimuscarinic potencies of pirenzepine and N-methylatropine, for both antagonists doses were calculated that produce a 10-fold shift to the right of the dose-response curves for A) the pressor response to McN-A-343 (i.v. administration) in pithed rats (D10-p.r.) and B) for the depressor effect to methacholine (i.v. administration) in anaesthetized rats (D10-an.r.), respectively. Whereas N-methylatropine was virtually equieffective in blocking both muscarinic responses (D10-an.r./D10-p.r. approximately equal to 1), pirenzepine, however, was considerably more potent at ganglionic than at vascular muscarinic receptors (D10-an.r./D10-p.r. approximately equal to 16). These data confirm the existence of excitatory ganglionic muscarinic receptors with high affinity for pirenzepine (M1) and provide evidence for the presence of M2 receptors - receptors which show a low sensitivity to pirenzepine - on vascular smooth muscle cells. To further characterize the anticholinergic properties of pirenzepine, its effect on the pressor response to DMPP, a nicotinic ganglionic stimulant, was investigated in pithed rats. A high dose of pirenzepine (1.13 mumol/kg), given i.v., did not affect nicotinic ganglionic transmission.
ESTHER : Wess_1984_Life.Sci_35_553
PubMedSearch : Wess_1984_Life.Sci_35_553
PubMedID: 6379356