Muscarinic Receptor Signaling Research Articles

Strain differences in muscarinic cholinergic receptor antagonism of fat intake and acquisition and expression of fat-conditioned flavor preferences in male BALB/c, C57BL/6 and SWR mice

Murine strain differences occur for both intakes of and preferences for sugars and fats. Previous studies demonstrated that the muscarinic cholinergic receptor antagonist, scopolamine (SCOP) more potently reduced sucrose and saccharin intakes in inbred C57BL/6 and BALB/c than SWR mice, sucrose-conditioned flavor preferences (CFP) expression in BALB/c, but not C57BL/6 or SWR mice, and sucrose-CFP acquisition in BALB/c relative to SWR and C57BL/6 mice. Although fat intake and fat-CFP are observed in all three strains, strain-specific effects were previously observed following dopamine D1, opiate and NMDA receptor antagonism of sweet and fat intake and CFP. The present study investigated whether muscarinic receptor antagonism differentially affected fat (Intralipid) intake and preferences in these strains by examining whether SCOP altered fat (Intralipid) intake and fat-CFP expression and acquisition in BALB/c, C57BL/6 and SWR mice. SCOP (0.1–10 mg/kg) significantly reduced Intralipid (5%) intake in all three strains across 2 h. In fat-CFP expression experiments, food-restricted mice consumed one flavored (conditioned stimulus (CS)+, 5 sessions) Intralipid (5%) solution and a differently-flavored (CS−, 5 sessions) Intralipid (0.5%) solution. Two-bottle CS choice tests with the two flavors mixed in 0.5% Intralipid occurred following vehicle and two SCOP doses (1, 5 mg/kg). SCOP elicited small, but significant reductions in fat-CFP expression in BALB/c and C57BL/6, but not SWR mice. In fat-CFP acquisition experiments, separate groups of BALB/c, C57BL/6 and SWR mice were treated prior to the ten acquisition training sessions with vehicle or two SCOP (2.5, 5 mg/kg) doses followed by six two-bottle choice tests without injections. SCOP eliminated fat-CFP acquisition in all three strains. Thus, muscarinic receptor signaling mediates learning, and to a lesser degree maintenance of fat-CFP while maximally inhibiting fat intake in the three strains.

Role of Muscarinic Acetylcholine Signaling in Gastrointestinal Cancers.

In the tumor microenvironment, various stromal and immune cells accumulate and interact with cancer cells to contribute to tumor progression. Among stromal players, nerves have recently been recognized as key regulators of tumor growth. More neurotransmitters, such as catecholamines and acetylcholine (ACh), are present in tumors, as the cells that secrete neurotransmitters accumulate by the release of neurotrophic factors from cancer cells. In this short review, we focus on the role of nerve signaling in gastrointestinal (GI) cancers. Given that muscarinic acetylcholine receptor signaling seems to be a dominant regulator of GI stem cells and cancers, we review the function and mechanism of the muscarinic ACh pathway as a regulator of GI cancer progression. Accumulating evidence suggests that ACh, which is secreted from nerves and tuft cells, stimulates GI epithelial stem cells and contributes to cancer progression via muscarinic receptors.

Effects of cholinergic compounds and TNF-alpha on human erythroleukemia K562 cell proliferation and caspase expression

Objective: The purpose of this study was to investigate ifstimulating auto-paracrine muscarinic receptor signalling pathwaycould change human erythroleukemia K562 cell proliferation andcaspase 3, 8 and 9 expression levels. To better understand the role ofmuscarinic receptors in cell signalling mechanism, we investigatedthe effects of several compounds on human erythroleukemiaK562 cell proliferation and caspase 3, 8 and 9 expression. Thesecompounds were M3 muscarinic receptor agonist, pilocarpine, proinflammatorycytokine, tumor necrosis factor (TNF)-alpha, andthe wortmannin which is a phosphoinositide 3-kinase inhibitor.Materials and Methods: Cell proliferation and cell viabilitywere evaluated by the trypan blue exclusion test and 5-Bromo-2-deoxy-uridine (BrdU) Labelling and Detection Kits. Caspase 3, 8and 9 expression levels were determined by immunoblot analysis.Results: Both pilocarpine and TNF-alpha caused a small increasein human erythroleukemia K562 cell proliferation. However, whenall the compounds were treated together, proliferation of humanerythroleukemia K562 cells increased significantly when compared tountreated control cells. TNF-alpha and wortmannin treatment increasedcaspase 3 and caspase 8 expression patterns significantly in humanerythroleukemia K562 cells. TNF-alpha and wortmannin treatmentincreased caspase 9 expression level (P>0.05) but it was not significant.Conclusion: These findings partly demonstrated that M3muscarinic receptor mediated an increase in K562 cell proliferation.Pilocarpine prevented TNF-alpha and wortmannin inducedcaspase 3 and 8 expression and indirectly showed apoptosis inhuman erythroleukemia K562 cells.

CPLA2α-/- sympathetic neurons exhibit increased membrane excitability and loss of N-Type Ca2+ current inhibition by M1 muscarinic receptor signaling.

Group IVa cytosolic phospholipase A2 (cPLA2α) mediates GPCR-stimulated arachidonic acid (AA) release from phosphatidylinositol 4,5-bisphosphate (PIP2) located in plasma membranes. We previously found in superior cervical ganglion (SCG) neurons that PLA2 activity is required for voltage-independent N-type Ca2+ (N-) current inhibition by M1 muscarinic receptors (M1Rs). These findings are at odds with an alternative model, previously observed for M-current inhibition, where PIP2 dissociation from channels and subsequent metabolism by phospholipase C suffices for current inhibition. To resolve cPLA2α’s importance, we have investigated its role in mediating voltage-independent N-current inhibition (~40%) that follows application of the muscarinic agonist oxotremorine-M (Oxo-M). Preincubation with different cPLA2α antagonists or dialyzing cPLA2α antibodies into cells minimized N-current inhibition by Oxo-M, whereas antibodies to Ca2+-independent PLA2 had no effect. Taking a genetic approach, we found that SCG neurons from cPLA2α-/- mice exhibited little N-current inhibition by Oxo-M, confirming a role for cPLA2α. In contrast, cPLA2α antibodies or the absence of cPLA2α had no effect on voltage-dependent N-current inhibition by M2/M4Rs or on M-current inhibition by M1Rs. These findings document divergent M1R signaling mediating M-current and voltage-independent N-current inhibition. Moreover, these differences suggest that cPLA2α acts locally to metabolize PIP2 intimately associated with N- but not M-channels. To determine cPLA2α’s functional importance more globally, we examined action potential firing of cPLA2α+/+ and cPLA2α-/- SCG neurons, and found decreased latency to first firing and interspike interval resulting in a doubling of firing frequency in cPLA2α-/- neurons. These unanticipated findings identify cPLA2α as a tonic regulator of neuronal membrane excitability.

Decanoic acid attenuates the excitability of nociceptive trigeminal primary and secondary neurons associated with hypoalgesia.

BackgroundAcute application of decanoic acid (DA) in vivo suppresses the excitability of spinal trigeminal nucleus caudalis (SpVc) wide dynamic range (WDR) neurons associated with the short-term mechanical hypoalgesia via muscarinic M2 receptor signaling; however, the effect of DA on nociceptive trigeminal ganglion (TG) and SpVc nociceptive-specific (NS) neuronal excitability under in vivo conditions remains to be determined. The present study investigated whether this effect could be observed in naive rats.ResultsExtracellular single-unit recordings were made from TG and SpVc NS neurons of pentobarbital-anesthetized rats in response to orofacial noxious mechanical stimuli. DA inhibited the mean firing frequency of both TG and SpVc NS neurons, reaching a maximum inhibition of discharge frequency within 1–5 minutes and reversing after approximately 10-minutes; however, this DA-induced suppression of SpVc NS neuronal firing frequency did not occur in rats administered with methoctramine intravenously prior to stimulation.ConclusionThis in vivo study indicated that firing of TG and SpVc NS neurons induced by mechanical hypoalgesia through peripheral M2 receptors could be inhibited by acutely administered DA, implicating the potential of DA in the future treatment of trigeminal pain.PerspectiveThis article presents that the acute DA application suppresses the excitability of TG and SpVc NS neurons associated with mechanical hypoalgesia via peripheral M2 receptor signaling, supporting DA as a potential therapeutic agent in complementary and alternative medicine for the attenuation of nociception.

Spicatoside A in red Liriopeplatyphylla displays a laxative effect in a constipation rat model via regulating mAChRs and ER stress signaling.

Red Liriope platyphylla extract (EtRLP) has been used as an oriental medicine for treatment of several chronic conditions, such as neurodegenerative disorders, diabetes, and obesity. To investigate the laxative activity of EtRLP, the levels of key constipation markers and their molecular regulators were examined following administration of EtRLP in constipation Sprague Dawley (SD) rats treated with loperamide (Lop). Compared with the Lop+Vehicle-treated group, the excretion levels of urine and stool were significantly enhanced in the Lop+EtRLP-treated group, even though feeding levels were kept constant. There was a significant improvement in histological structure, cytological ultrastructure and mucin secretion in transverse colon sections from the Lop+EtRLP-treated group, compared with the Lop+Vehicle-treated group. The Lop+EtRLP-treated group exhibited a rapid recovery of the muscarinic acetylcholine receptor (mAChR) signaling pathway and of the endoplasmic reticulum (ER) stress response, compared with Lop+Vehicle-treated group. Spicatoside A, one of the key components detected in EtRLP, recovered the levels of inositol triphosphate (IP3) and Gα in primary rat intestinal smooth muscle cells (pRISMCs). Taken together, the present results indicated that spicatoside A-containing EtRLP had therapeutic effects against Lop-induced constipation in SD rats via improvement of the mAChR downstream signaling pathway and the ER stress response.

Impaired object-location learning and recognition memory but enhanced sustained attention in M2 muscarinic receptor-deficient mice

RationaleMuscarinic acetylcholine receptors are known to play key roles in mediating cognitive processes, and impaired muscarinic cholinergic neurotransmission is associated with normal ageing processes and Alzheimer’s disease. However, the specific contributions of the individual muscarinic receptor subtypes (M1–M5) to cognition are presently not well understood.ObjectivesThe aim of this study was to investigate the contribution of M2-type muscarinic receptor signalling to sustained attention, executive control and learning and memory.MethodsM2 receptor-deficient (M2−/−) mice were tested on a touchscreen-operated task battery testing visual discrimination, behavioural flexibility, object-location associative learning, attention and response control. Spontaneous recognition memory for real-world objects was also assessed.ResultsWe found that M2−/− mice showed an enhancement of attentional performance, but significant deficits on some tests of learning and memory. Executive control and visual discrimination were unaffected by M2-depletion.ConclusionsThese findings suggest that M2 activation has heterogeneous effects across cognitive domains, and provide insights into how acetylcholine may support multiple specific cognitive processes through functionally distinct cholinergic receptor subtypes. They also suggest that therapeutics involving M2 receptor-active compounds should be assessed across a broad range of cognitive domains, as they may enhance some cognitive functions, but impair others.

Murine genetic variance in muscarinic cholinergic receptor antagonism of acquisition and expression of sucrose-conditioned flavor preferences in three inbred mouse strains

Conditioned flavor preferences (CFP) are elicited by sucrose relative to saccharin in inbred mice with both the robustness of the preferences and sensitivity to pharmacological receptor antagonists sensitive to genetic variance. Dopamine, opioid and N-methyl-d-aspartate receptor antagonists differentially interfere with the acquisition (learning) and expression (maintenance) of sucrose-CFP in BALB/c and SWR inbred mice. Further, the muscarinic cholinergic receptor antagonist, scopolamine (SCOP) more potently reduces both sucrose and saccharin intake in BALB/c and C57BL/6 relative to SWR inbred mice. The present study examined whether SCOP altered the expression and acquisition of sucrose-CFP in BALB/c, C57BL/6 and SWR mice. In expression experiments, food-restricted mice alternately consumed a flavored (CS+, e.g., cherry, 5 sessions) 16% sucrose solution and a differently-flavored (CS−, e.g., grape, 5 sessions) 0.05% saccharin solution. Two-bottle CS choice tests with the two flavors mixed in 0.2% saccharin solutions occurred following vehicle or SCOP at doses of 1 or 5 mg/kg. SCOP significantly reduced the magnitude of the expression of sucrose-CFP in BALB/c, but not either C57BL/6 or SWR mice. In acquisition experiments, separate groups of BALB/c, C57BL/6 and SWR mice were treated prior to acquisition training sessions with vehicle or 2.5 or 5 mg/kg SCOP doses that was followed by six two-bottle CS choice tests without injections. SCOP dose-dependently reduced (1 mg/kg) and eliminated (2.5 mg/kg) the acquisition of sucrose-CFP in BALB/c mice, and reduced the magnitude of acquisition of sucrose-CFP in SWR mice. In contrast, neither SCOP dose affected the acquisition of sucrose-CFP in C57BL/6 mice. Thus, muscarinic cholinergic receptor signaling is essential for the learning of sucrose-CFP in BALB/c mice, to a lesser degree in SWR mice, but not in C57BL/6 mice. Murine genetic variance differentially modulates muscarinic cholinergic receptor control of sweet intake per se relative to learned conditioned flavor preferences of sweets.

F144. MUSCARINIC M1 RECEPTOR SIGNALLING UNDERLYING COGNITION IN PSYCHOTIC DISORDERS

BackgroundAntipsychotic treatment has failed to improve cognitive deficits associated with psychotic disorders. This has led to an increased interest to revisit earlier implications from post-mortem studies that lowered muscarinic M1 receptor signaling may underlie these symptoms. This receptor is highly expressed in important regions for cognition such as the dorsolateral prefrontal cortex (DLPFC) and hippocampus, and administration of anti-muscarinic agents gives induce cognitive deficits in healthy volunteers. Administration of xanomeline, a M1/4 agonist in patients with schizophrenia resulted in improved learning and memory scores and decreased psychotic symptom severity. Therefore, the current study sought to examine alterations in muscarinic M1 receptor signaling in relation to cognitive functioning in medication free subjects with psychotic disorders and matched controls.MethodsMuscarinic M1 binding potential (BPND) was measured using single photon emission computed tomography (SPECT) with the M1 selective radiopharmaceutical 123I-iododexetimide in the DLPFC and hippocampus in the psychotic group. Pharmacological functional magnetic resonance imaging (phMRI) with the M1 antagonist biperiden was used to assess differences in functional response on the paired associate learning task (PAL) and emotion recognition task (ERT) adapted for fMRI from The Cambridge Neuropsychological Test Automated Battery in all subjects. The PAL task assessed encoding phase (learning) and retrieval (memory) of figure-place associations and the ERT task social cognition, both highly predictive of functional outcome. Cluster significance was set at Z>2.3, with cluster threshold correction at p<0.05.ResultsThe current study included 26 (mean age: 27.68; 19male/7 female) subjects with a psychotic disorder and 29 (mean age 25.63; 20 male/9 female) matched controls. Subjects with psychotic disorders recalled less figure place associations than controls (t=2.9, p=0.005) and were worse in recognizing different intensities of disgust emotions (t=2.26, p=0.03). Psychotic subjects showed a blunted response in functional reactivity to biperiden in the bilateral superior and medial frontal gyri with decreasing intensity of disgust facial expressions compared to controls, this blunted response was greatest in those with lower M1 BPND in the DLPFC. During encoding processes, psychotic subjects also showed differential reactivity to biperiden in the left middle frontal gyrus, insula, and caudate nucleus showing hypoactivation compared to controls. Greater hypoactivation was significantly associated with lower hippocampal M1 BPND. For retrieval both groups showed lowered activation under biperiden in the inferior frontal gyrus, but psychotic subjects failed to show increased activation with increasing cognitive load in the placebo condition, like the controls. Lower hippocampal M1 BPND in psychotic subjects was associated with lower activation of this region.DiscussionResults show preliminary evidence for altered M1 signaling of prefrontal areas in psychotic disorders underlying social cognition and learning and memory processes. Additionally, results show an important role for the M1 receptor in the DLPFC and hippocampus in altered fronto-striatal activation underlying encoding processes. Lower hippocampal M1 BPND is related to more severe alterations in underlying functional activation in encoding and retrieval processes. Results further support the need for development of therapeutic strategies that focus on the M1 receptor to improve cognitive functioning and functional outcome in psychosis.

Murine genetic variance in muscarinic cholinergic receptor antagonism of sucrose and saccharin solution intakes in three inbred mouse strains

Nutritive (e.g., sucrose) and non-nutritive (e.g., saccharin) sweeteners stimulate intake in inbred mouse strains. BALB/c, SWR and C57BL/6 mice differ in the ability of dopamine (DA) D1 (SCH23390) and opioid (naltrexone) receptor antagonism to alter sucrose intake. Whereas SCH23390 comparably reduced cumulative sucrose intake in all three strains, naltrexone reduced cumulative sucrose intake maximally in C57/BL/6 mice, in intermediate fashion in BALB/c mice, but not in SWR mice. Whereas cumulative saccharin intake was reduced by DA D1 receptor antagonism in BALB/c and SWR mice, naltrexone was more potent in SWR relative to BALB/c mice. The present study first examined whether SCH23390 (50–1600nmol/kg) and naltrexone (0.01–5mg/kg) altered saccharin intake in C57BL/6 mice. Given that scopolamine (SCOP), a muscarinic cholinergic receptor antagonist, reduces sweet intake in outbred rats, a second experiment examined whether SCOP (0.1–10mg/kg) altered 0.2% saccharin and 10% sucrose intakes in BALB/c, SWR and C57BL/6 mice. Cumulative saccharin intake was significantly reduced by SCH23390 (200–1600nmol/kg; ID40=175nmol/kg) and naltrexone (0.1–5mg/kg; ID40>5mg/kg) in C57BL/6 mice. Cumulative sucrose intake was significantly reduced following SCOP in C57BL/6 (0.1–10mg/kg; ID40=2.32mg/kg) and BALB/c (2.5–10mg/kg; ID40=0.52mg/kg) mice. In contrast, SWR mice (ID40=41.61mg/kg) only displayed transient (15min) reductions in sucrose intake following SCOP (2.5–10mg/kg). Cumulative saccharin intake was significantly reduced following SCOP in C57BL/6 and BALB/c mice (0.1–10mg/kg; ID40<0.1mg/kg). In contrast, SWR mice (ID40=2.28mg/kg) displayed smaller significant reductions in saccharin intake following SCOP (0.1–10mg/kg). These data indicate that although both nutritive and non-nutritive sweet intakes are governed by muscarinic cholinergic receptor signaling, this process is subject to murine genetic variance with greater sensitivity observed in C57BL/6 and BALB/c relative to SWR inbred mouse strains.

Muscarinic receptor signaling contributes to atypical antipsychotic drug reversal of the phencyclidine-induced deficit in novel object recognition in rats.

Enhancement of cholinergic function via muscarinic acetylcholine receptor M1 agonism improves cognition in some schizophrenia patients. Most atypical antipsychotic drugs, including clozapine and its active metabolite, N-desmethylclozapine, and lurasidone, enhance the release of acetylcholine in key brain regions involved in cognition (e.g. hippocampus). We determined the effect of muscarinic acetylcholine receptor M1 stimulation on novel object recognition and its contribution to the ability of atypical antipsychotic drugs to reverse the novel object recognition deficit in rats withdrawn from subchronic phencyclidine, a rodent model of cognitive impairment in schizophrenia. In control rats, the non-specific muscarinic acetylcholine receptor antagonist, scopolamine, and the M1 selective antagonist, VU0255035, induced a novel object recognition deficit, which was reversed by the M1 agonist, AC260584. Scopolamine fully blocked the effect of clozapine and N-desmethylclozapine, but not lurasidone, to restore novel object recognition in subchronic phencyclidine-treated rats. VU0255035 also blocked these effects of clozapine and N-desmethylclozapine, but not lurasidone; however, the blockade was not as complete as that achieved with scopolamine. Furthermore, subchronic phencyclidine increased hippocampal M1 mRNA expression. These data suggest that M1 agonism is required for clozapine and N-desmethylclozapine to ameliorate the phencyclidine-induced deficit in novel object recognition, additional evidence that M1 agonism is a potential target for treating cognitive impairment in schizophrenia.

MP94-15 MUSCARINIC M3- AND M2- RECEPTOR-ACTIVATED SIGNALING IN THE BLADDER IS INVERSELY REGULATED BY CAVEOLAE.

You have accessJournal of UrologyBladder & Urethra: Anatomy, Physiology & Pharmacology II1 Apr 2017MP94-15 MUSCARINIC M3- AND M2- RECEPTOR-ACTIVATED SIGNALING IN THE BLADDER IS INVERSELY REGULATED BY CAVEOLAE. Vivian Cristofaro and Maryrose P. Sullivan Vivian CristofaroVivian Cristofaro More articles by this author and Maryrose P. SullivanMaryrose P. Sullivan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.2918AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Bladder smooth muscle (BSM) caveolae are cholesterol-enriched membrane microdomains that augment or attenuate detrusor functional responses by differentially regulating specific receptor-activated signaling pathways. However, BSM contractions induced by cholinergic activation in the rat are not altered by depletion of caveolae, unlike other smooth muscle systems in which muscarinic receptor signaling is evidently mediated by caveolae. Although this discrepancy may reflect the highly specific regulation imparted by caveolae among different tissues and species, a differential regulation of muscarinic M3 and M2 receptor subtypes in the bladder cannot be excluded. This study examined the functional and molecular relationship between caveolae and muscarinic acetylcholine receptor (mAChR) subtypes M3 and M2. METHODS BSM tissue strips were prepared from Sprague Dawley rat bladders after removing the mucosa. Tissue was suspended in organ baths for isometric tension studies. Dose response curves to carbachol (CCh, 1nM-10μM) were generated at baseline, as well as in the presence of 4-DAMP (10nM) or AFDX (0.1μM) to inactive M3 or M2 receptors respectively. Responses to CCh were repeated after incubation with methyl-β-cyclodextrin (mβCD, 15mM), an agent that disrupts caveolae by depleting membrane cholesterol. Interaction between caveolin-1 (Cav-1, a protein required for caveolae biogenesis) and M3 or M2 mAChR subtype were investigated by co-immunoprecipitation. RESULTS Compared to baseline responses, 4-DAMP decreased CCh-induced contractions at each dose. After mβCD treatment and in the presence of 4-DAMP, contractile responses to CCh were significantly enhanced. AFDX had little effect on CCh dose-response curves. However, the subsequent disruption of caveolae in the presence of AFDX attenuated significantly contractions induced by CCh. Immunoreactive bands corresponding to M3 and M2 mAChR subtypes were detected in Cav-1 immunoprecipitates. CONCLUSIONS The opposite effect of mβCD on CCh responses in the presence of M2 or M3 antagonists suggests that caveolae negatively regulate M2- and positively regulate M3-mediated signaling respectively, but this interaction is masked when only the aggregate effect of CCh is examined. Molecular interaction of Cav-1 and mAChRs is consistent with their localization within caveolae. Changes in the balance among caveolin-mAChR interactions, due to loss of caveolae or changes in mAChR subtype expression, may alter responses to cholinergic activation or the efficacy of anti-muscarinic agents in the bladder. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e1252 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Vivian Cristofaro More articles by this author Maryrose P. Sullivan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

The M2 Muscarinic Receptor Signaling Complex Resolved by Single Molecule Tracking in Live Cells

Many aspects of the cellular signaling pathways via G protein-coupled receptors (GPCRs) are not completely understood. In particular, three questions have been the focus of much attention and debate: the oligomeric status of the receptor, the coupling strength between the receptor and the G protein, and the regulation of the receptors in response to external stimuli. Here we examine those questions by 2D tracking the M2 muscarinic receptors with Total Internal Reflection Fluorescence Microscopy (TIRFM) in the membrane of live cells.M2 Receptors and G proteins were genetically fused with fluorescent proteins (GFP and mCherry) and expressed in Chinese Hamster Ovary (CHO) cells. The distributions of oligomeric sizes and diffusion coefficients for both the receptors and the G proteins were extracted from single-particle tracking data obtained with TIRFM. Both of the receptors and G proteins were found to be localized in membrane micro-domains, showing multi-step photobleaching and exhibited a wide range of diffusion behaviors on a slower time scale than single protein controls. Corroborated with dual-color fluorescence correlation spectroscopy on the same samples, we propose that multiple oligomeric receptors and oligomeric G proteins sizes co-exist in close proximity inside a spatially confined signaling scaffold in the membrane of living cells. In addition, two-color simultaneous tracking of co-expressed receptors and G proteins shows preliminary evidence for receptor-G protein decoupling and receptor internalization upon induction with excessive agonist.

A unifying hypothesis for M1 muscarinic receptor signalling in pyramidal neurons.

Phasic release of acetylcholine (ACh) in the neocortex facilitates attentional processes. Acting at a single metabotropic receptor subtype, ACh exerts two opposing actions in cortical pyramidal neurons: transient inhibition and longer-lasting excitation. Cholinergic inhibitory responses depend on calcium release from intracellular calcium stores, and run down rapidly at resting membrane potentials when calcium stores become depleted. We demonstrate that cholinergic excitation promotes calcium entry at subthreshold membrane potentials to rapidly refill calcium stores, thereby maintaining the fidelity of inhibitory cholinergic signalling. We propose a 'unifying hypothesis' for M1 receptor signalling whereby inhibitory and excitatory responses to ACh in pyramidal neurons represent complementary mechanisms governing rapid calcium cycling between the endoplasmic reticulum, the cytosol and the extracellular space. Gq -coupled M1-type muscarinic acetylcholine (ACh) receptors (mAChRs) mediate two distinct electrophysiological responses in cortical pyramidal neurons: transient inhibition driven by calcium-dependent small conductance potassium ('SK') channels, and longer-lasting and voltage-dependent excitation involving non-specific cation channels. Here we examine the interaction of these two cholinergic responses with respect to their contributions to intracellular calcium dynamics, testing the 'unifying hypothesis' that rundown of inhibitory SK responses at resting membrane potentials (RMPs) reflects depletion of intracellular calcium stores, while mAChR-driven excitation acts to refill those stores by promoting voltage-dependent entry of extracellular calcium. We report that fidelity of cholinergic SK responses requires the continued presence of extracellular calcium. Inhibitory responses that diminished after repetitive ACh application at RMPs were immediately rescued by pairing mAChR stimulation with subthreshold depolarization (∼10mV from RMPs) initiated with variable delay (up to 500ms) after ACh application, but not by subthreshold depolarization preceding mAChR stimulation. Further, rescued SK responses were time-locked to ACh application, rather than to the timing of subsequent depolarizing steps, suggesting that cholinergic signal transduction itself is not voltage-sensitive, but that depolarization facilitates rapid cycling of extracellular calcium through the endoplasmic reticulum to activate SK channels. Consistent with this prediction, rescue of SK responses by subthreshold depolarization required the presence of extracellular calcium. Our results demonstrate that, in addition to gating calcium release from intracellular stores, mAChR activation facilitates voltage-dependent refilling of calcium stores, thereby maintaining the ongoing fidelity of SK-mediated inhibition in response to phasic release of ACh.

Inositol Phosphate Accumulation in Vivo Provides a Measure of Muscarinic M1 Receptor Activation.

The rationale for using M1 selective muscarinic acetylcholine receptor activators for the treatment of cognitive impairment associated with psychiatric and neurodegenerative disease is well-established in the literature. Here, we investigate measurement of inositol phosphate accumulation, an end point immediately downstream of the M1 muscarinic acetylcholine receptor signaling cascade, as an in vivo biochemical readout for M1 muscarinic acetylcholine receptor activation. Five brain penetrant M1-subtype selective activators from three structurally distinct chemical series were pharmacologically profiled for functional activity in vitro using recombinant cell calcium mobilization and inositol phosphate assays, and a native tissue hippocampal slice electrophysiology assay, to show that all five compounds presented a positive allosteric modulator agonist profile, within a narrow range of potencies. In vivo characterization using an amphetamine-stimulated locomotor activity behavioral assay and the inositol phosphate accumulation biochemical assay demonstrated that the latter has utility for assessing functional potency of M1 activators. Efficacy measured by inositol phosphate accumulation in mouse striatum compared favorably to efficacy in reversing amphetamine-induced locomotor activity, suggesting that the inositol phosphate accumulation assay has utility for the evaluation of M1 muscarinic acetylcholine receptor activators in vivo. The benefits of this in vivo biochemical approach include a wide response window, interrogation of specific brain circuit activation, an ability to model responses in the context of brain exposure, an ability to rank order compounds based on in vivo efficacy, and minimization of animal use.

Muscarinic, nicotinic and GABAergic receptor signaling differentially mediate fat-conditioned flavor preferences in rats

Rats display conditioned flavor preferences (CFP) for fats. Previous studies demonstrated that whereas expression of an already-acquired corn oil (CO)-CFP was mildly reduced by dopamine (DA) D1, DA D2, NMDA or opioid receptor antagonists, the acquisition or learning of CO-CFP was eliminated by NMDA antagonists, and significantly reduced by DA D1 and D2, but not opioid antagonists. Previous studies of fructose-CFP demonstrated that muscarinic (scopolamine) and nicotinic (mecamylamine) cholinergic receptor antagonists and GABAB (baclofen) receptor agonism reduced the expression of this acquired response, and that scopolamine, but not mecamylamine or baclofen eliminated the acquisition or learning of this response. The present study examined scopolamine, mecamylamine or baclofen effects upon expression or acquisition of CO-CFP. For expression, rats were trained over 10 sessions with CS+ (3.5% CO) and CS− (0.9% CO) flavored solutions without drugs. Two-bottle choice tests with CS+ and CS− flavors in 0.9% CO examined preferences following vehicle, scopolamine (1–10mg/kg), mecamylamine (1–8mg/kg) and baclofen (1.5–5mg/kg). In acquisition, eight groups of rats received vehicle, scopolamine (1, 2.5mg/kg), mecamylamine (4, 6mg/kg), baclofen (3, 5mg/kg) or a limited intake vehicle control 0.5h prior to all 10 CS+ and CS− training sessions followed by six 2-bottle CS+ and CS− choice tests in 0.9% CO. CO-CFP expression (percent CS+ intake) was significantly but marginally reduced by scopolamine (70%), mecamylamine (85%) and baclofen (74%) relative to vehicle (98%). CO-CFP acquisition was eliminated (41%) by scopolamine relative to vehicle (88%) and limited control (98%) conditions. Neither mecamylamine nor baclofen altered CO-CFP acquisition. Thus, the muscarinic cholinergic receptor system is essential for acquisition (learning) of both fat-induced and sugar (fructose)-induced preferences. In contrast, muscarinic, nicotinic and GABAB receptors were minimally involved in the expression (maintenance) of fat- and fructose-CFP.

Abstract 1865: Areca nut-induced JNK/ATF2/Jun axis regulates TGF-beta signaling contributing to pre-cancerous oral submucous fibrosis

Abstract Oral Submucous Fibrosis (OSF) is an inflammatory, pre-cancerous condition of the oral submucosa. It affects individuals with prolonged areca nut chewing habit and therefore areca nut is regarded as the etiological agent for OSF manifestation. Transforming Growth Factor beta is an established promoter of cancer and fibrosis. Up-regulation of TGF-β and its target genes has also been reported in OSF. However, the underlying mechanism for the regulation of TGF-β by areca nut has not been elucidated. The present study demonstrates that TGF-β pathway is activated in epithelial cells after two hour exposure to areca nut extract. Areca nut increases TGF-β transcript as well as protein and thereby the downstream canonical Smad2/3 signaling. Pre-treatment with various pathway inhibitors showed JNK pathway activation as a pre-requisite to TGF-β pathway activation by areca nut. Areca nut activated JNK pathway within half an hour of treatment and this was reversed by atropine treatment suggesting involvement of muscarinic acid receptor signaling. Second messengers upstream to JNK activation and regulated by areca nut were calcium/CAMKII and reactive oxygen species. Further, areca nut treatment resulted in JNK dependent phosphorylation of ATF2 and c-Jun transcription factors. These were found to bind TGF-β promoter, as revealed by chromatin immunoprecipitation experiments. Expectedly, knockdown of these transcription factors abrogated areca nut induced TGF-β pathway activation. Based on the data obtained a model is proposed to elucidate activation of TGF-β pathway by areca nut extract involving activation of JNK and ATF2, c-Jun resulting in the over expression of TGF-β in epithelial cells which may contribute to the manifestation of OSF. Thus, this study highlights ATF2/c-Jun as the critical nodes for therapeutic intervention to prevent TGF-β over production in OSF. Funding by DST and CSIR is acknowledged. Citation Format: Ila Pant, Paturu Kondaiah. Areca nut-induced JNK/ATF2/Jun axis regulates TGF-beta signaling contributing to pre-cancerous oral submucous fibrosis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1865.

Muscarinic receptor signaling and colon cancer progression.

Due to the lack of effective treatments, advanced colorectal cancer (CRC) remains a leading cause of cancer death in the United States. Emerging evidence supports the observation that muscarinic receptor (MR) signaling plays a critical role in growth and progression of CRC. MR activation by acetylcholine and bile acids results in transactivation of epidermal growth factor receptors (EGFR) and post-EGFR signal transduction that enhances cell proliferation, migration, and invasion. Here, the authors review recent progress in understanding the molecular mechanisms underlying MR-mediated CRC progression and its therapeutic implications.

Optogenetic stimulation of brain cholinergic networks suppresses inflammation

Abstract Brain cholinergic muscarinic acetylcholine receptor (mAChR) signaling regulates peripheral inflammation via the inflammatory reflex (Nat Rev Endocrinol, 2012, 8:743). Here we provide further insight into this regulation by utilizing optogenetic stimulation, a method allowing precise spatiotemporal neuronal activation. Laser light (473 nm) and transgenic mice expressing light activated channelrhodopsin-2 coupled to a fluorescent protein (ChR2-YFP) under the control of the choline acetyl transferase (ChAT) promoter were used. Optogenetic stimulation of the medial septum, a major source of brain cholinergic output to areas with abundant M1 mAChR localization significantly suppressed serum TNF levels, as compared to sham stimulation (P&amp;lt;0.0398) in ChAT-ChR2-YFP mice during endotoxemia. In contrast to ChAT-ChR2-YFP mice, using the same approach in control (non-carrier) endotoxemic mice did not alter serum TNF. In addition, intracerebroventricular administration of the selective M1 mAChR agonist benzyl quinolone carboxylic acid (5 μg/kg) significantly decreased serum TNF, as compared to vehicle administration (P&amp;lt;0.0180) in C57Bl/6 mice during endotoxemia. These results from ongoing studies reveal a role for forebrain cholinergic M1 mAChR-mediated signaling in controlling peripheral inflammation. These findings may be of interest for development of new approaches for selective brain modulation for the treatment of inflammatory conditions.

The Sensorless Pore Module of Voltage-gated K+ Channel Family 7 Embodies the Target Site for the Anticonvulsant Retigabine

KCNQ (voltage-gated K(+) channel family 7 (Kv7)) channels control cellular excitability and underlie the K(+) current sensitive to muscarinic receptor signaling (the M current) in sympathetic neurons. Here we show that the novel anti-epileptic drug retigabine (RTG) modulates channel function of pore-only modules (PMs) of the human Kv7.2 and Kv7.3 homomeric channels and of Kv7.2/3 heteromeric channels by prolonging the residence time in the open state. In addition, the Kv7 channel PMs are shown to recapitulate the single-channel permeation and pharmacological specificity characteristics of the corresponding full-length proteins in their native cellular context. A mutation (W265L) in the reconstituted Kv7.3 PM renders the channel insensitive to RTG and favors the conductive conformation of the PM, in agreement to what is observed when the Kv7.3 mutant is heterologously expressed. On the basis of the new findings and homology models of the closed and open conformations of the Kv7.3 PM, we propose a structural mechanism for the gating of the Kv7.3 PM and for the site of action of RTG as a Kv7.2/Kv7.3 K(+) current activator. The results validate the modular design of human Kv channels and highlight the PM as a high-fidelity target for drug screening of Kv channels.