An allosteric enhancer of M4 muscarinic acetylcholine receptor function inhibits behavioral and neurochemical effects of cocaine

Rationale

The mesostriatal dopamine system plays a key role in mediating the reinforcing effects of psychostimulant drugs like cocaine. The muscarinic M₄ acetylcholine receptor subtype is centrally involved in the regulation of dopamine release in striatal areas. Consequently, striatal M₄ 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 M₄ 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 M₄ receptors, its inhibitory effects on cocaine-induced increases in striatal dopamine were abolished in M₄ receptor knockout mice. Furthermore, selective deletion of the M₄ receptor gene in dopamine D₁ receptor-expressing neurons resulted in a partial reduction of the VU0152100 effect, indicating that VU0152100 partly regulates dopaminergic neurotransmission via M₄ receptors co-localized with D₁ receptors.

Conclusions

These results show that positive allosteric modulators of the M₄ receptor deserve attention as agents in the future treatment of cocaine abuse.     

Pharmacological magnetic resonance imaging of muscarinic acetylcholine receptor activation in rat brain

The central cholinergic system is involved in several cognitive functions such as attention, consciousness, learning and memory. Functional imaging of this neurotransmitter system may provide novel opportunities in the diagnosis and evaluation of cognitive disorders. The aim of this study was to investigate the spatial and temporal activation patterns of muscarinic acetylcholine receptor (mAChR) stimulation in rat brain with pharmacological magnetic resonance imaging (phMRI). We performed blood oxygenation level-dependent (BOLD) MRI and contrast-enhanced cerebral blood volume (CBV)-weighted MRI combined with injection of pilocarpine, a non-selective mAChR agonist. BOLD and CBV responses were assessed after pretreatment with methyl-scopolamine in order to block peripheral muscarinic effects. Region-of-interest analysis in individual animals and group-level independent component analysis failed to show significant BOLD signal changes following pilocarpine injection. However, with contrast-enhanced CBV-weighted MRI, positive CBV responses were detected in the cerebral cortex, thalamus, and hippocampus whereas a negative CBV response was observed in the striatum. Thus, pilocarpine-induced significant activation responses in brain regions that are known to have a high density of muscarinic receptors. Our study demonstrates that phMRI of mAChR stimulation in rats allows functional assessment of the cholinergic system in vivo.     

Modulation of arousal and sleep/wake architecture by M1 PAM VU0453595 across young and aged rodents and nonhuman primates

Degeneration of basal forebrain cholinergic circuitry represents an early event in the development of Alzheimer’s disease (AD). These alterations in central cholinergic function are associated with disruptions in arousal, sleep/wake architecture, and cognition. Changes in sleep/wake architecture are also present in normal aging and may represent a significant risk factor for AD. M₁ muscarinic acetylcholine receptor (mAChR) positive allosteric modulators (PAMs) have been reported to enhance cognition across preclinical species and may also provide beneficial effects for age- and/or neurodegenerative disease-related changes in arousal and sleep. In the present study, electroencephalography was conducted in young animals (mice, rats and nonhuman primates [NHPs]) and in aged mice to examine the effects of the selective M₁ PAM VU0453595 in comparison with the acetylcholinesterase inhibitor donepezil, M₁/M₄ agonist xanomeline (in NHPs), and M₁ PAM BQCA (in rats) on sleep/wake architecture and arousal. In young wildtype mice, rats, and NHPs, but not in M₁ mAChR KO mice, VU0453595 produced dose-related increases in high frequency gamma power, a correlate of arousal and cognition enhancement, without altering duration of time across all sleep/wake stages. Effects of VU0453595 in NHPs were observed within a dose range that did not induce cholinergic-mediated adverse effects. In contrast, donepezil and xanomeline increased time awake in rodents and engendered dose-limiting adverse effects in NHPs. Finally, VU0453595 attenuated age-related decreases in REM sleep duration in aged wildtype mice. Development of M₁ PAMs represents a viable strategy for attenuating age-related and dementia-related pathological disturbances of sleep and arousal.Subject terms: Neuroscience, Circadian rhythms and sleep     

Muscarinic acetylcholine receptors for psychotic disorders: bench-side to clinic

Modern interest in muscarinic acetylcholine receptor (mAChR) activators for schizophrenia began in the 1990s when xanomeline, an M₁/M₄-preferring mAChR agonist developed for cognitive symptoms of Alzheimer's disease (AD), had unexpected antipsychotic activity. However, strategies to address tolerability concerns associated with activation of peripheral mAChRs were not available at that time. The discovery of specific targeted ligands and combination treatments to reduce peripheral mAChR engagement have advanced the potential of mAChR activators as effective treatments for psychotic disorders. This review provides perspectives on the background of the identification of mAChRs as potential antipsychotics, advances in the preclinical understanding of mAChRs as targets, and the current state of mAChR activators under active clinical development for schizophrenia.     

AC-260584, an orally bioavailable M1 muscarinic receptor allosteric agonist, improves cognitive performance in an animal model

The recent discovery of allosteric potentiators and agonists of the muscarinic M₁ receptor represents a significant advance in the muscarinic receptor pharmacology. In the current study we describe the receptor pharmacology and pro-cognitive action of the allosteric agonist AC-260584. Using in vitro cell-based assays with cell proliferation, phosphatidylinositol hydrolysis or calcium mobilization as endpoints, AC-260584 was found to be a potent (pEC₅₀ 7.6-7.7) and efficacious (90-98% of carbachol) muscarinic M₁ receptor agonist. Furthermore, as compared to orthosteric binding agonists, AC-260584 showed functional selectivity for the M₁ receptor over the M₂, M₃, M₄ and M₅ muscarinic receptor subtypes. Using GTPγS binding assays, its selectivity was found to be similar in native tissues expressing mAChRs to its profile in recombinant systems. In rodents, AC-260584 activated extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation in the hippocampus, prefrontal cortex and perirhinal cortex. The ERK1/2 activation was dependent upon muscarinic M₁ receptor activation since it was not observed in M₁ knockout mice. AC-260584 also improved the cognitive performance of mice in the novel object recognition assay and its action is blocked by the muscarinic receptor antagonist pirenzepine. Taken together these results indicate for the first time that a M₁ receptor agonist selective over the other mAChR subtypes can have a symptomatically pro-cognitive action. In addition, AC-260584 was found to be orally bioavailable in rodents. Therefore, AC-260584 may serve as a lead compound in the development of M₁ selective drugs for the treatment of cognitive impairment associated with schizophrenia and Alzheimer's disease.     

Muscarinic and Nicotinic Acetylcholine Receptor Agonists and Allosteric Modulators for the Treatment of Schizophrenia

Muscarinic and nicotinic acetylcholine (ACh) receptors (mAChRs and nAChRs) are emerging as important targets for the development of novel treatments for the symptoms associated with schizophrenia. Preclinical and early proof-of-concept clinical studies have provided strong evidence that activators of specific mAChR (M₁ and M₄) and nAChR (α₇ and α₂β₄) subtypes are effective in animal models of antipsychotic-like activity and/or cognitive enhancement, and in the treatment of positive and cognitive symptoms in patients with schizophrenia. While early attempts to develop selective mAChR and nAChR agonists provided important preliminary findings, these compounds have ultimately failed in clinical development due to a lack of true subtype selectivity and subsequent dose-limiting adverse effects. In recent years, there have been major advances in the discovery of highly selective activators for the different mAChR and nAChR subtypes with suitable properties for optimization as potential candidates for clinical trials. One novel strategy has been to identify ligands that activate a specific receptor subtype through actions at sites that are distinct from the highly conserved ACh-binding site, termed allosteric sites. These allosteric activators, both allosteric agonists and positive allosteric modulators, of mAChR and nAChR subtypes demonstrate unique mechanisms of action and high selectivity in vivo, and may provide innovative treatment strategies for schizophrenia.     

Impact of species variability and 'probe-dependence' on the detection and in vivo validation of allosteric modulation at the M4 muscarinic acetylcholine receptor

BACKGROUND AND PURPOSE

We recently characterized LY2033298 as a novel allosteric modulator and agonist at M₄ muscarinic acetylcholine receptors (mAChRs). Evidence also suggested a difference in the potency of LY2033298 at rodent relative to human M₄ mAChRs. The current study investigated the basis for the species difference of this modulator and used this knowledge to rationalize its in vivo actions.

EXPERIMENTAL APPROACH

LY2033298 was investigated in vitro in CHO cells stably expressing human or mouse M₄ mAChRs, using assays of agonist-induced ERK1/2 or GSK-3α phosphorylation, [³⁵S]-GTPγS binding, or effects on equilibrium binding of [³H]-NMS and ACh. The in vivo actions of LY2033298 were investigated in a mouse model of amphetamine-induced locomotor activity. The function of LY2033298 was examined in combination with ACh, oxotremorine or xanomeline.

KEY RESULTS

LY2033298 had similar affinities for the human and mouse M₄ mAChRs. However, LY2033298 had a lower positive co-operativity with ACh at the mouse relative to the human M₄ mAChR. At the mouse M₄ mAChR, LY2033298 showed higher co-operativity with oxotremorine than with ACh or xanomeline. The different degrees of co-operativity between LY2033298 and each agonist at the mouse relative to the human M₄ mAChR necessitated the co-administration of LY2033298 with oxotremorine in order to show in vivo efficacy of LY2033298.

CONCLUSIONS AND IMPLICATIONS

These results provide evidence for species variability when comparing the allosteric interaction between LY2033298 and ACh at the M₄ mAChR, and also highlight how the interaction between LY2033298 and different orthosteric ligands is subject to ‘probe dependence’. This has implications for the validation of allosteric modulator actions in vivo.     

Pharmacological characterization of M1 muscarinic acetylcholine receptor-mediated Gq activation in rat cerebral cortical and hippocampal membranes

This study aimed to pharmacologically characterize the response derived from functional activation of Gq proteins coupled with native muscarinic acetylcholine receptors in rat cerebral cortex and hippocampus. Rat cerebral cortical and hippocampal membranes were prepared, and the effects of a range of mAChR agonists and antagonists, allosteric modulators, and muscarinic toxins were determined by an antibody-capture scintillation proximity assay combined with [³⁵S]GTPγS binding, using the anti-Gα_q antibody sc-393. Increased specific [³⁵S]GTPγS binding, elicited by carbachol (CCh), was selectively inhibited by the muscarinic toxin MT7, and was resistant to membrane pretreatment with N-ethylmaleimide, indicating that the response derived exclusively from Gα_q, selectively coupled with the M₁ mAChR. In addition to CCh, many mAChR agonists, including oxotremorine, arecholine, and methacholine, stimulated binding in a concentration-dependent manner with varied potencies and efficacies. The intrinsic activities of partial M₁ mAChR agonists in the present study were generally lower than previously reported in M₁-expressing cells. Xanomeline and N-desmethylclozapine had negligible or minimal agonist properties. CCh-stimulated [³⁵S]GTPγS binding to Gα_q was inhibited by mAChR antagonists, including scopolamine, ipratropium, atropine, 4-DAMP, pirenzepine, and AF-DX 116, with a rank order of potency consistent with previous studies of M₁-expressing cells. There was a highly significant correlation between the potencies of 13 agonists and 19 antagonists in the cerebral cortex and hippocampus. The effects of several allosteric mAChR modulators were also investigated. These data provide a comprehensive pharmacological profile of the G_q-coupled M₁ mAChR subtype natively expressed at physiological levels in rat cerebral cortex and hippocampus.     

Muscarinic receptor subtypes controlling the cationic current in guinea-pig ileal smooth muscle

1. The effects of muscarinic antagonists on cationic current evoked by activating muscarinic receptors with the stable agonist carbachol were studied by use of patch-clamp recording techniques in guinea-pig single ileal smooth muscle cells.
2. Ascending concentrations of carbachol (3–300 μM) activated the cationic conductance in a concentration-dependent manner with conductance at a maximally effective carbachol concentration (G_max) of 27.4±1.4 nS and a mean −log EC₅₀ of 5.12±0.03 (mean±s.e.mean) (n=114).
3. Muscarinic antagonists with higher affinity for the M₂ receptor, methoctramine, himbacine and tripitramine, produced a parallel shift of the carbachol concentration-effect curve to the right in a concentration-dependent manner with pA₂ values of 8.1, 8.0 and 9.1, respectively.
4. All M₃ selective muscarinic antagonists tested, 4-DAMP, p-F-HHSiD and zamifenacin, reduced the maximal response in a concentration-dependent and non-competitive manner. This effect could be observed even at concentrations which did not produce any increase in the EC₅₀ for carbachol. At higher concentrations M₃ antagonists shifted the agonist curve to the right, increasing the EC₅₀, and depressed the maximum conductance response. Atropine, a non-selective antagonist, produced both reduction in G_max (M₃ effect) and significant increase in the EC₅₀ (M₂ effect) in the same concentration range.
5. The depression of the conductance by 4-DAMP, zamifenacin and atropine could not be explained by channel block as cationic current evoked by adding GTPγS to the pipette (without application of carbachol) was unaffected.
6. The results support the hypothesis that carbachol activates M₂ muscarinic receptors so initiating the opening of cationic channels which cause depolarization; this effect is potentiated by an unknown mechanism when carbachol activates M₃ receptors. As an increasing fraction of M₃ receptors are blocked by an antagonist, the effects on cationic current of an increasing proportion of activated M₂ receptors are disabled.

     

The M1/M4 preferring muscarinic agonist xanomeline modulates functional connectivity and NMDAR antagonist-induced changes in the mouse brain

Cholinergic drugs acting at M1/M4 muscarinic receptors hold promise for the treatment of symptoms associated with brain disorders characterized by cognitive impairment, mood disturbances, or psychosis, such as Alzheimer’s disease or schizophrenia. However, the brain-wide functional substrates engaged by muscarinic agonists remain poorly understood. Here we used a combination of pharmacological fMRI (phMRI), resting-state fMRI (rsfMRI), and resting-state quantitative EEG (qEEG) to investigate the effects of a behaviorally active dose of the M1/M4-preferring muscarinic agonist xanomeline on brain functional activity in the rodent brain. We investigated both the effects of xanomeline per se and its modulatory effects on signals elicited by the NMDA-receptor antagonists phencyclidine (PCP) and ketamine. We found that xanomeline induces robust and widespread BOLD signal phMRI amplitude increases and decreased high-frequency qEEG spectral activity. rsfMRI mapping in the mouse revealed that xanomeline robustly decreased neocortical and striatal connectivity but induces focal increases in functional connectivity within the nucleus accumbens and basal forebrain. Notably, xanomeline pre-administration robustly attenuated both the cortico-limbic phMRI response and the fronto-hippocampal hyper-connectivity induced by PCP, enhanced PCP-modulated functional connectivity locally within the nucleus accumbens and basal forebrain, and reversed the gamma and high-frequency qEEG power increases induced by ketamine. Collectively, these results show that xanomeline robustly induces both cholinergic-like neocortical activation and desynchronization of functional networks in the mammalian brain. These effects could serve as a translatable biomarker for future clinical investigations of muscarinic agents, and bear mechanistic relevance for the putative therapeutic effect of these class of compounds in brain disorders.Subject terms: Schizophrenia, Translational research, Preclinical research     

Allosteric modulation of the group III mGlu(4) receptor provides functional neuroprotection in the 6-hydroxydopamine rat model of Parkinson's disease

BACKGROUND AND PURPOSE

We recently reported that broad spectrum agonist-induced activation of presynaptic group III metabotropic glutamate (mGlu) receptors within the substantia nigra pars compacta using L-2-amino-4-phosphonobutyrate provided functional neuroprotection in the 6-hydroxydopamine lesion rat model of Parkinson''s disease. The aim of this study was to establish whether selective activation of the mGlu₄ receptor alone could afford similar functional neuroprotection.

EXPERIMENTAL APPROACH

The neuroprotective effects of 8 days of supranigral treatment with a positive allosteric modulator of mGlu₄ receptors, (+/−)-cis-2-(3,5-dichlorphenylcarbamoyl)cyclohexanecarboxylic acid (VU0155041), were investigated in rats with unilateral 6-hydroxydopamine lesions. The effects of VU0155041 treatment on motor function were assessed using both habitual (cylinder test) and forced (adjusted stepping, amphetamine-induced rotations) behavioural tests. Nigrostriatal tract integrity was examined by analysis of tyrosine hydroxylase, dopa decarboxylase or dopamine levels in the striatum and tyrosine hydroxylase-positive cell counts in the substantia nigra pars compacta.

KEY RESULTS

VU0155041 provided around 40% histological protection against a unilateral 6-hydroxydopamine lesion as well as significant preservation of motor function. These effects were inhibited by pre-treatment with (RS)-α-cyclopropyl-4-phosphonophenylglycine, confirming a receptor-mediated response. Reduced levels of inflammatory markers were also evident in the brains of VU0155041-treated animals.

CONCLUSIONS AND IMPLICATIONS

Allosteric potentiation of mGlu₄ receptors in the substantia nigra pars compacta provided neuroprotective effects in the 6-hydroxydopamine rat model A reduced inflammatory response may contribute, in part, to this action. In addition to the reported symptomatic effects, activation of mGlu₄ receptors may also offer a novel approach for slowing the progressive degeneration observed in Parkinson''s disease.     

Mapping dopamine D2/D3 receptor function using pharmacological magnetic resonance imaging

Rationale Regulation of dopamine release and synthesis occurs via pre-synaptic dopamine (DA) D2/D3 autoreceptors (DARs). Mapping of DAR function in vivo is difficult and is usually best assessed using invasive measures of DA release, such as microdialysis at discrete sites. We wished to show that pharmacological magnetic resonance imaging (phMRI) may prove useful for this purpose. Objective To demonstrate that the relative cerebral blood volume (rCBV) changes induced by amphetamine can be modulated by DA D2 receptor antagonists and agonists in a manner consistent with modulation of DAR function and to compare these effects with microdialysis. Methods We used phMRI with iron oxide contrast agents to map changes in rCBV in response to an amphetamine challenge, pre-treatment and post-treatment with varying doses of the D2 antagonist eticlopride and the D2 agonist quinpirole. We also compared the effects of D2 antagonism using microdialysis measurements of DA release. Results Antagonism of D2 receptors with eticlopride potentiated rCBV changes induced by amphetamine in the nucleus accumbens and caudate putamen in a dose-dependent manner. The amphetamine-induced increase in rCBV in the accumbens in animals pre-treated with eticlopride was paralleled by a similar percentage increase in DA release measured by means of microdialysis. Conversely, agonism of D2 receptors using quinpirole reduced amphetamine-induced rCBV changes in the caudate putamen and nucleus accumbens. The effects of both quinpirole and eticlopride on amphetamine-induced rCBV changes were largest in the nucleus accumbens. Conclusions These results suggest that phMRI may potentially prove useful to map DAR function non-invasively in multiple brain regions simultaneously.     

Brain M3 muscarinic receptors are involved in the ACTH-induced reversal of hemorrhagic shock

Summary In an experimental model of bleeding-induced hemorrhagic shock causing the death of all saline-treated rats within 30 min, the intravenous injection of ACTH-(1–24) at the dose of 160 g/kg induced a sustained reversal of the shock condition, with almost complete recovery of blood pressure, pulse amplitude, respiratory rate, heart rate, and 100% survival, at least for the 2 h of observation. This effect of ACTH-(1–24) was prevented by the intracerebroventricular injection of 4-DAMP (a highly selective antagonist for M₁ and M3 muscarinic receptors), but unaffected by the intracerebroventricular injection of pirenzepine (a highly selective antagonist for M₁ muscarinic receptors). These data indicate that an essential step in the complex mechanism of the ACTH-induced shock reversal may be the activation of brain M₃ muscarinic receptors. Send offprint requests to S. Guarini at the above address     

Novel N-Substituted Benzimidazolones as Potent,Selective, CNS-Penetrant,and Orally Active M1 mAChR Agonists

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Increased amphetamine-induced locomotor activity, sensitization, and accumbal dopamine release in M5 muscarinic receptor knockout mice

Introduction

Muscarinic M₅ receptors are the only muscarinic receptor subtype expressed by dopamine-containing neurons of the ventral tegmental area. These cells play an important role for the reinforcing properties of psychostimulants and M₅ receptors modulate their activity. Previous studies showed that M₅ receptor knockout (M ₅ ^?/? ) mice are less sensitive to the reinforcing properties of addictive drugs.

Materials and methods

Here, we investigate the role of M₅ receptors in the effects of amphetamine and cocaine on locomotor activity, locomotor sensitization, and dopamine release using M ₅ ^?/? mice backcrossed to the C57BL/6NTac strain.

Statistical analyses

Sensitization of the locomotor response is considered a model for chronic adaptations to repeated substance exposure, which might be related to drug craving and relapse. The effects of amphetamine on locomotor activity and locomotor sensitization were enhanced in M ₅ ^?/? mice, while the effects of cocaine were similar in M ₅ ^?/? and wild-type mice.

Results

Consistent with the behavioral results, amphetamine-, but not cocaine, -elicited dopamine release in nucleus accumbens was enhanced in M ₅ ^?/? mice.

Discussion

The different effects of amphetamine and cocaine in M ₅ ^?/? mice may be due to the divergent pharmacological profile of the two drugs, where amphetamine, but not cocaine, is able to release intracellular stores of dopamine. In conclusion, we show here for the first time that amphetamine-induced hyperactivity and dopamine release as well as amphetamine sensitization are enhanced in mice lacking the M₅ receptor. These results support the concept that the M₅ receptor modulates effects of addictive drugs.     

Quantitative analysis of the loss of muscarinic receptors in various peripheral tissues in M1�CM5 receptor single knockout mice

Background and purpose:

To compare loss in binding to muscarinic receptor (mAChR) subtypes with their known functions, the total density of muscarinic receptors was measured in peripheral tissues from wild type (WT) and mAChR knockout (KO) mice.

Experimental approach:

Binding parameters of [N-methyl-³H]scopolamine methyl chloride ([³H]NMS) were determined in 10 peripheral tissues of WT and M₁–M₅ receptor KO mice. Competition between [³H]NMS and darifenacin (selective M₃ receptor antagonist) was also measured

Key results:

There was an extensive loss of [³H]NMS-binding sites (maximal number of binding sites, B_max) in heart and smooth muscle from M₂KO mice, compared with WT mice. Smooth muscle from M₃KO mice also showed a moderate loss of B_max. B_max fell in pancreas and bladder of M₄KO mice and in prostate in M₁KO and M₃KO mice. There was a large loss of B_max in exocrine and endocrine glands of M₃KO mice with a moderate decrease in M₂KO mice. Darifenacin inhibited specific [³H]NMS binding in submandibular gland and bladder of WT, M₂KO and M₃KO mice. K_i (inhibition constant) values for darifenacin in the submandibular gland were the same in WT and M₂KO mice but increased in M₃KO mice. However, K_i values in bladder were decreased in M₂KO mice and increased in M₃KO mice.

Conclusions and implications:

Single mAChR KO mice exhibit a loss of mAChR in peripheral tissues that generally paralleled the reported loss of function. Quantitative analysis of data, however, also suggested that, in some instances, normal expression of a receptor subtype depended on expression of other subtypes.     

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

Background and Purpose

Activation of muscarinic receptors results in catecholamine secretion in adrenal chromaffin cells in many mammals, and muscarinic receptors partly mediate synaptic transmission from the splanchnic nerve, at least in guinea pigs. To elucidate the physiological functions of muscarinic receptors in chromaffin cells, it is necessary to identify the muscarinic receptor subtypes involved in excitation.

Experimental Approach

To identify muscarinic receptors, pharmacological tools and strains of mice where one or several muscarinic receptor subtypes were genetically deleted were used. Cellular responses to muscarinic stimulation in isolated chromaffin cells were studied with the patch clamp technique and amperometry.

Key Results

Muscarinic M₁, M₄ and M₅ receptors were immunologically detected in mouse chromaffin cells, and these receptors disappeared after the appropriate gene deletion. Mouse cells secreted catecholamines in response to muscarinic agonists, angiotensin II and a decrease in external pH. Genetic deletion of M₁, but not M₃, M₄ or M₅, receptors in mice abolished secretion in response to muscarine, but not to other stimuli. The muscarine-induced secretion was suppressed by MT7, a snake peptide toxin specific for M₁ receptors. Similarly, muscarine failed to induce an inward current in the presence of MT7 in mouse and rat chromaffin cells. The binding affinity of VU0255035 for the inhibition of muscarine-induced currents agreed with that for the M₁ receptor.

Conclusions and Implications

Based upon the effects of genetic deletion of muscarinic receptors and MT7, it is concluded that the M₁ receptor alone is responsible for muscarine-induced catecholamine secretion.     

The GABA B receptor agonist CGP44532 and the positive modulator GS39783 reverse some behavioural changes related to positive syndromes of psychosis in mice

BACKGROUND AND PURPOSE

An important role of GABAergic neurotransmission in schizophrenia was proposed a long time ago, but there is limited data to support this hypothesis. In the present study we decided to investigate GABA_B receptor ligands in animal models predictive for the antipsychotic activity of drugs. The GABA_B receptor antagonists {"type":"entrez-protein","attrs":{"text":"CGP51176","term_id":"875489595","term_text":"CGP51176"}}CGP51176 and {"type":"entrez-protein","attrs":{"text":"CGP36742","term_id":"877561962","term_text":"CGP36742"}}CGP36742, agonist {"type":"entrez-protein","attrs":{"text":"CGP44532","term_id":"875097404","term_text":"CGP44532"}}CGP44532 and positive allosteric modulator GS39783 were studied.

EXPERIMENTAL APPROACH

The effects of all ligands were investigated in MK-801- and amphetamine-induced hyperactivity tests. The anti-hallucinogenic-like effect of the compounds was screened in the model of head twitches induced by (±)1-(2.5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI). Furthermore, the effect of GS39783 and {"type":"entrez-protein","attrs":{"text":"CGP44532","term_id":"875097404","term_text":"CGP44532"}}CGP44532 on DOI-induced frequency of spontaneous excitatory postsynaptic currents (EPSCs) in slices from mouse brain frontal cortices was investigated. The anti-cataleptic properties of the compounds were also assessed.

KEY RESULTS

The GABA_B receptor activators {"type":"entrez-protein","attrs":{"text":"CGP44532","term_id":"875097404","term_text":"CGP44532"}}CGP44532 and GS39783 exhibited antipsychotic-like effects both in the MK-801- and amphetamine-induced hyperactivity tests, as well as in the head-twitch model in mice. Such effects were not observed for the GABA_B receptor antagonists. DOI-induced increased frequency of spontaneous EPSCs was also decreased by the compounds. Moreover, {"type":"entrez-protein","attrs":{"text":"CGP44532","term_id":"875097404","term_text":"CGP44532"}}CGP44532 and GS39783 inhibited haloperidol-induced catalepsy and EPSCs.

CONCLUSION AND IMPLICATIONS

These data suggest that selective GABA_B receptor activators may be useful in the treatment of psychosis.     

Discovery of VU6028418: A Highly Selective and Orally Bioavailable M4 Muscarinic Acetylcholine Receptor Antagonist

Herein, we report the SAR leading to the discovery of VU6028418, a potent M₄ mAChR antagonist with high subtype-selectivity and attractive DMPK properties in vitro and in vivo across multiple species. VU6028418 was subsequently evaluated as a preclinical candidate for the treatment of dystonia and other movement disorders. During the characterization of VU6028418, a novel use of deuterium incorporation as a means to modulate CYP inhibition was also discovered.     

M3 muscarinic acetylcholine receptor is associated with β-catenin in ventricular myocytes during myocardial infarction in the rat

1. The present study was designed to investigate whether the M₃ muscarinic acetylcholine receptors (mAChR) is associated with β‐catenin in the ventricular myocardium during ischaemic myocardial injury and to determine the possible mechanism/s involved. 2. Rat hearts were subjected to coronary artery ligation for 1 and 6 h or 1 month to establish a myocardial ischaemia (MI) model. In the acute MI model, 16 rats were randomized into four groups: (i) control; (ii) ischaemia (rats were subjected to 20 min coronary occlusion); (iii) choline (10 mg/kg, i.v., choline chloride, an M₃ receptor agonist, was administered 15 min before occlusion); and (iv) 4‐diphenylacetoxy‐N‐methylpiperidine methiodide (4‐DAMP; 0.12 mg/kg 4‐DAMP, an M₃ receptor antagonist, was administered 20 min before occlusion, followed 5 min later by 10 mg/kg, i.v., choline chloride). Immunochemistry, western blot analysis and immunoprecipitation were used to determine the expression and localization of β‐catenin and the M₃ mAChR. 3. Myocardial ischaemia caused a time‐dependent increase in the expression of β‐catenin. Moreover, a physical association was found between β‐catenin and the M₃ mAChR in intercalated discs. This association was enhanced by prolonged ischaemia. Administration of choline before ischaemia not only increased β‐catenin expression, but also strengthened the association between β‐catenin and the M₃ mAChR. However, blockade of M₃ mAChR by 4‐DAMP completely inhibited the effect of choline on the expression of β‐catenin. In addition, MI increased phosphorylation of the M₃ mAChR. 4. The results indicate that increased β‐catenin activity is associated with M₃ mAChR during MI. This association is likely to play a role in heart signal transduction during ischaemia between neighbouring ventricular myocardiocum.