From structure to clinic: Design of a muscarinic M1 receptor agonist with potential to treatment of Alzheimer's disease.

Current therapies for Alzheimer's disease seek to correct for defective cholinergic transmission by preventing the breakdown of acetylcholine through inhibition of acetylcholinesterase, these however have limited clinical efficacy. An alternative approach is to directly activate cholinergic receptors responsible for learning and memory. The M1-muscarinic acetylcholine (M1) receptor is the target of choice but has been hampered by adverse effects. Here we aimed to design the drug properties needed for a well-tolerated M1-agonist with the potential to alleviate cognitive loss by taking a stepwise translational approach from atomic structure, cell/tissue-based assays, evaluation in preclinical species, clinical safety testing, and finally establishing activity in memory centers in humans. Through this approach, we rationally designed the optimal properties, including selectivity and partial agonism, into HTL9936-a potential candidate for the treatment of memory loss in Alzheimer's disease. More broadly, this demonstrates a strategy for targeting difficult GPCR targets from structure to clinic.

Integration of Feedforward and Feedback Information Streams in the Modular Architecture of Mouse Visual Cortex.

Radial cell columns are a hallmark feature of cortical architecture in many mammalian species. It has long been held, based on the lack of orientation columns, that such functional units are absent in rodent primary visual cortex (V1). These observations led to the view that rodent visual cortex has a fundamentally different network architecture than that of carnivores and primates. While columns may be lacking in rodent V1, we describe in this review that modular clusters of inputs to layer 1 and projection neurons in the layers below are prominent features of the mouse visual cortex. We propose that modules organize thalamocortical inputs, intracortical processing streams, and transthalamic communications that underlie distinct sensory and sensorimotor functions.

IP3-mediated Ca2+ signals regulate larval to pupal transition under nutrient stress through the H3K36 methyltransferase Set2.

Persistent loss of dietary protein usually signals a shutdown of key metabolic pathways. In Drosophila larvae that have reached a 'critical weight' and can pupariate to form viable adults, such a metabolic shutdown would needlessly lead to death. Inositol 1,4,5-trisphosphate-mediated calcium (IP3/Ca2+) release in some interneurons (vGlutVGN6341) allows Drosophila larvae to pupariate on a protein-deficient diet by partially circumventing this shutdown through upregulation of neuropeptide signaling and the expression of ecdysone synthesis genes. Here, we show that IP3/Ca2+ signals in vGlutVGN6341 neurons drive expression of Set2, a gene encoding Drosophila Histone 3 Lysine 36 methyltransferase. Furthermore, Set2 expression is required for larvae to pupariate in the absence of dietary protein. IP3/Ca2+ signal-driven Set2 expression upregulates key Ca2+-signaling genes through a novel positive-feedback loop. Transcriptomic studies, coupled with analysis of existing ChIP-seq datasets, identified genes from larval and pupal stages that normally exhibit robust H3K36 trimethyl marks on their gene bodies and concomitantly undergo stronger downregulation by knockdown of either the intracellular Ca2+ release channel IP3R or Set2. IP3/Ca2+ signals thus regulate gene expression through Set2-mediated H3K36 marks on select neuronal genes for the larval to pupal transition.

Biased M1 muscarinic receptor mutant mice show accelerated progression of prion neurodegenerative disease.

There are currently no treatments that can slow the progression of neurodegenerative diseases, such as Alzheimer's disease (AD). There is, however, a growing body of evidence that activation of the M1 muscarinic acetylcholine receptor (M1-receptor) can not only restore memory loss in AD patients but in preclinical animal models can also slow neurodegenerative disease progression. The generation of an effective medicine targeting the M1-receptor has however been severely hampered by associated cholinergic adverse responses. By using genetically engineered mouse models that express a G protein-biased M1-receptor, we recently established that M1-receptor mediated adverse responses can be minimized by ensuring activating ligands maintain receptor phosphorylation/arrestin-dependent signaling. Here, we use these same genetic models in concert with murine prion disease, a terminal neurodegenerative disease showing key hallmarks of AD, to establish that phosphorylation/arrestin-dependent signaling delivers neuroprotection that both extends normal animal behavior and prolongs the life span of prion-diseased mice. Our data point to an important neuroprotective property inherent to the M1-receptor and indicate that next generation M1-receptor ligands designed to drive receptor phosphorylation/arrestin-dependent signaling would potentially show low adverse responses while delivering neuroprotection that will slow disease progression.

GABA-gated chloride channel mutation (Rdl) induces cholinergic physiological compensation resulting in cross resistance in Drosophila melanogaster.

The Drosophila melanogaster MD-RR strain contains an Rdl mutation (A301S) resulting in resistance to several insecticide classes viz. phenyl pyrazoles (e.g., fipronil), cyclodienes (e.g., dieldrin), and chlorinated aliphatic hydrocarbons (e.g., lindane). Fitness costs are commonly observed with resistant insect populations as side effects of the genetic change conferring the resistant phenotype. Because of fitness costs, reversion from the resistant to susceptible genotype and phenotype is common. However, the Rdl genotype in D. melanogaster appears to allow the flies to maintain the resistant genotype/phenotype without selective pressure and with minimal fitness costs. We provide evidence that compensation for the Rdl mutation influences the cholinergic system, where an increase in acetylcholinesterase gene expression and enzyme activity results in neurophysiological changes and cross resistance to a carbamate insecticide (propoxur oral resistance ratio (RR) of 63) and an organophosphate insecticide (dichlorvos oral RR of 7). Such cross resistance was not previously reported with the initial collection and testing of this strain. In addition to acetylcholinesterase, the Rdl mutation influences the expression of the muscarinic acetylcholine receptor subtype-B, resulting in resistance to non-selective muscarinic compounds (pilocarpine and atropine). Collectively, these results indicate that the Rdl mutation (A301S) at GABA-gated ionophore complex influences the physiology of the cholinergic system, leading to resistance to established insecticide classes. Additionally, this mutation may impact the effectiveness of insecticides targeting novel sites, like muscarinic receptors.

The Role of Muscarinic Acetylcholine Receptor M3 in Cardiovascular Diseases.

The muscarinic acetylcholine receptor M3 (M3-mAChR) is involved in various physiological and pathological processes. Owing to specific cardioprotective effects, M3-mAChR is an ideal diagnostic and therapeutic biomarker for cardiovascular diseases (CVDs). Growing evidence has linked M3-mAChR to the development of multiple CVDs, in which it plays a role in cardiac protection such as anti-arrhythmia, anti-hypertrophy, and anti-fibrosis. This review summarizes M3-mAChR's expression patterns, functions, and underlying mechanisms of action in CVDs, especially in ischemia/reperfusion injury, cardiac hypertrophy, and heart failure, opening up a new research direction for the treatment of CVDs.

Chronic exposure to the anti-M3 muscarinic acetylcholine receptor autoantibody in pemphigus vulgaris contributes to disease pathophysiology.

Pemphigus vulgaris (PV) is a potentially lethal autoimmune mucocutaneous blistering disease characterized by binding of IgG autoantibodies (AuAbs) to keratinocytes (KCs). In addition to AuAbs against adhesion molecules desmogleins 1 and 3, PV patients also produce an AuAb against the M3 muscarinic acetylcholine (ACh) receptor (M3AR) that plays an important role in regulation of vital functions of KCs upon binding endogenous ACh. This anti-M3AR AuAb is pathogenic because its adsorption eliminates the acantholytic activity of PV IgG; however, the molecular mechanism of its action is unclear. In the present study, we sought to elucidate the mode of immunopharmacologic action of the anti-M3AR AuAb in PV. Short-term exposures of cultured KCs to PV IgG or the muscarinic agonist muscarine both induced changes in the expression of keratins 5 and 10, consistent with the inhibition of proliferation and upregulated differentiation and in keeping with the biological function of M3AR. In contrast, long-term incubations induced a keratin expression pattern consistent with upregulated proliferation and decreased differentiation, in keeping with the hyperproliferative state of KCs in PV. This change could result from desensitization of the M3AR, representing the net antagonist-like effect of the AuAb. Therefore, chronic exposure of KCs to the anti-M3AR AuAb interrupts the physiological regulation of KCs by endogenous ACh, contributing to the onset of acantholysis. Since cholinergic agents have already demonstrated antiacantholytic activity in a mouse model of PV and in PV patients, our results have translational significance and can guide future development of therapies for PV patients employing cholinergic drugs.

Neuroproteomic mapping of kinases and their substrates downstream of acetylcholine: finding and implications.

INTRODUCTION: Since the emergence of the cholinergic hypothesis of Alzheimer's disease (AD), acetylcholine has been viewed as a mediator of learning and memory. Donepezil improves AD-associated learning deficits and memory loss by recovering brain acetylcholine levels. However, it is associated with side effects due to global activation of acetylcholine receptors. Muscarinic acetylcholine receptor M1 (M1R), a key mediator of learning and memory, has been an alternative target. The importance of targeting a specific pathway downstream of M1R has recently been recognized. Elucidating signaling pathways beyond M1R that lead to learning and memory holds important clues for AD therapeutic strategies. AREAS COVERED: This review first summarizes the role of acetylcholine in aversive learning, one of the outputs used for preliminary AD drug screening. It then describes the phosphoproteomic approach focused on identifying acetylcholine intracellular signaling pathways leading to aversive learning. Finally, the intracellular mechanism of donepezil and its effect on learning and memory is discussed. EXPERT OPINION: The elucidation of signaling pathways beyond M1R by phosphoproteomic approach offers a platform for understanding the intracellular mechanism of AD drugs and for developing AD therapeutic strategies. Clarifying the molecular mechanism that links the identified acetylcholine signaling to AD pathophysiology will advance the development of AD therapeutic strategies.

Design and synthesis of 2-(2,2-diarylethyl)-cyclamine derivatives as M3 receptor antagonists and functional evaluation on COPD.

Muscarine acetylcholine receptors (mAChRs) regulate a variety of central and peripheral physiological functions and emerge as important therapeutic targets for a number of diseases including chronic obstructive pulmonary disease (COPD). Inspired by two active natural products, we designed and synthesized a series of 2-(2,2-diarylethyl)-cyclamine derivatives for screening M3 mAChR antagonists. On this skeleton, the structural units including N heterocycle, aryl groups and its substituents on aryl were examined and resulted in a clear structure-activity relationships on the M3 mAChR. In general, these 2-(2,2-diarylethyl)-cyclamine derivatives exhibited good to excellent M3 antagonistic potency and receptor selectivity. The most active 5b-C1 had an IC50 value of 3 nM and the most of compound 6 displayed inactivity against histamine H1 receptor closely related to M3. In in vitro and in vivo evaluations of tracheo-relaxation function, some compounds even showed comparable activity to tiotropium bromide, a known blockbuster drug for COPD. Such excellent properties made these novel compounds potential candidates for COPD drug development.

Determination of Region-Specific Roles of the M3 Muscarinic Acetylcholine Receptor in Gastrointestinal Motility.

BACKGROUND: The specific role of the M3 muscarinic acetylcholine receptor in gastrointestinal motility under physiological conditions is unclear, due to a lack of subtype-selective compounds. AIMS: The objective of this study was to determine the region-specific role of the M3 receptor in gastrointestinal motility. METHODS: We developed a novel positive allosteric modulator (PAM) for the M3 receptor, PAM-369. The effects of PAM-369 on the carbachol-induced contractile response of porcine esophageal smooth muscle and mouse colonic smooth muscle (ex vivo) and on the transit in mouse small intestine and rat colon (in vivo) were examined. RESULTS: PAM-369 selectively potentiated the M3 receptor under the stimulation of its orthosteric ligands without agonistic or antagonistic activity. Half-maximal effective concentrations of PAM activity for human, mouse, and rat M3 receptors were 0.253, 0.345, and 0.127 µM, respectively. PAM-369 enhanced carbachol-induced contraction in porcine esophageal smooth muscle and mouse colonic smooth muscle without causing any contractile responses by itself. The oral administration of 30 mg/kg PAM-369 increased the small intestinal transit in both normal motility and loperamide-induced intestinal dysmotility mice but had no effects on the colonic transit, although the M3 receptor mRNA expression is higher in the colon than in the small intestine. CONCLUSIONS: This study provided the first direct evidence that the M3 receptor has different region-specific roles in the motility function between the small intestine and colon in physiological and pathophysiological contexts. Selective PAMs designed for targeted subtypes of muscarinic receptors are useful for elucidating the subtype-specific function.

Structural Insights into M1 Muscarinic Acetylcholine Receptor Signaling Bias between Gαq and ß-Arrestin through BRET Assays and Molecular Docking.

The selectivity of drugs for G protein-coupled receptor (GPCR) signaling pathways is crucial for their therapeutic efficacy. Different agonists can cause receptors to recruit effector proteins at varying levels, thus inducing different signaling responses, called signaling bias. Although several GPCR-biased drugs are currently being developed, only a limited number of biased ligands have been identified regarding their signaling bias for the M1 muscarinic acetylcholine receptor (M1mAChR), and the mechanism is not yet well understood. In this study, we utilized bioluminescence resonance energy transfer (BRET) assays to compare the efficacy of six agonists in inducing Gαq and ß-arrestin2 binding to M1mAChR. Our findings reveal notable variations in agonist efficacy in the recruitment of Gαq and ß-arrestin2. Pilocarpine preferentially promoted the recruitment of ß-arrestin2 (∆∆RAi = -0.5), while McN-A-343 (∆∆RAi = 1.5), Xanomeline (∆∆RAi = 0.6), and Iperoxo (∆∆RAi = 0.3) exhibited a preference for the recruitment of Gαq. We also used commercial methods to verify the agonists and obtained consistent results. Molecular docking revealed that certain residues (e.g., Y404, located in TM7 of M1mAChR) could play crucial roles in Gαq signaling bias by interacting with McN-A-343, Xanomeline, and Iperoxo, whereas other residues (e.g., W378 and Y381, located in TM6) contributed to ß-arrestin recruitment by interacting with Pilocarpine. The preference of activated M1mAChR for different effectors may be due to significant conformational changes induced by biased agonists. By characterizing bias towards Gαq and ß-arrestin2 recruitment, our study provides insights into M1mAChR signaling bias.

Muscarinic Acetylcholine Receptor M3 Expression and Survival in Human Colorectal Carcinoma-An Unexpected Correlation to Guide Future Treatment?

Muscarinic acetylcholine receptor M3 (M3R) has repeatedly been shown to be prominently expressed in human colorectal cancer (CRC), playing roles in proliferation and cell invasion. Its therapeutic targetability has been suggested in vitro and in animal models. We aimed to investigate the clinical role of MR3 expression in CRC for human survival. Surgical tissue samples from 754 CRC patients were analyzed for high or low immunohistochemical M3R expression on a clinically annotated tissue microarray (TMA). Immunohistochemical analysis was performed for established immune cell markers (CD8, TIA-1, FOXP3, IL 17, CD16 and OX 40). We used Kaplan-Meier curves to evaluate patients' survival and multivariate Cox regression analysis to evaluate prognostic significance. High M3R expression was associated with increased survival in multivariate (hazard ratio (HR) = 0.52; 95% CI = 0.35-0.78; p = 0.001) analysis, as was TIA-1 expression (HR = 0.99; 95% CI = 0.94-0.99; p = 0.014). Tumors with high M3R expression were significantly more likely to be grade 2 compared to tumors with low M3R expression (85.7% vs. 67.1%, p = 0.002). The 5-year survival analysis showed a trend of a higher survival rate in patients with high M3R expression (46%) than patients with low M3R expression CRC (42%) (p = 0.073). In contrast to previous in vitro and animal model findings, this study demonstrates an increased survival for CRC patients with high M3R expression. This evidence is highly relevant for translation of basic research findings into clinically efficient treatments.

Rab43 GTPase directs postsynaptic trafficking and neuron-specific sorting of G protein-coupled receptors.

G protein-coupled receptors (GPCRs) are important modulators of synaptic functions. A fundamental but poorly addressed question in neurobiology is how targeted GPCR trafficking is achieved. Rab GTPases are the master regulators of vesicle-mediated membrane trafficking, but their functions in the synaptic presentation of newly synthesized GPCRs are virtually unknown. Here, we investigate the role of Rab43, via dominant-negative inhibition and CRISPR-Cas9-mediated KO, in the export trafficking of α2-adrenergic receptor (α2-AR) and muscarinic acetylcholine receptor (mAChR) in primary neurons and cells. We demonstrate that Rab43 differentially regulates the overall surface expression of endogenous α2-AR and mAChR, as well as their signaling, in primary neurons. In parallel, Rab43 exerts distinct effects on the dendritic and postsynaptic transport of specific α2B-AR and M3 mAChR subtypes. More interestingly, the selective actions of Rab43 toward α2B-AR and M3 mAChR are neuronal cell specific and dictated by direct interaction. These data reveal novel, neuron-specific functions for Rab43 in the dendritic and postsynaptic targeting and sorting of GPCRs and imply multiple forward delivery routes for different GPCRs in neurons. Overall, this study provides important insights into regulatory mechanisms of GPCR anterograde traffic to the functional destination in neurons.

Pharmacological and molecular characterization of the A-type muscarinic acetylcholine receptor from Anopheles gambiae.

Muscarinic acetylcholine receptors (mAChRs) which are G protein-coupled receptors play key roles in insect physiology. Whereas vertebrate mAChRs are important targets for pharmaceutical drugs, insect mAChRs are under-exploited by the agro-chemical industry. Moreover, insect mAChRs have been less well studied than their vertebrate counterparts. Their critical functions mean that a better knowledge of the insect mAChRs is crucial for the effort to develop a new molecular-level strategy for insect pest management. Almost all insects possess three mAChRs named A, B and C which differ according to their coupling effector systems and their pharmacological profile. The aim of this study was to characterize the A-type mAChR (mAChR-A) from Anopheles gambiae which is the major vector of malaria in order to develop new strategies in pest management. In this paper, we reported that mAChR-A is more expressed in adult mosquitoes than in larvae. Furthermore, using calcium imaging recordings, we found that the An. gambiae mAChR-A expressed in Sf9 cells is activated by specific muscarinic agonists acetylcholine, muscarine and oxotremorine M and blocked by several mAChR antagonists. Moreover, using inhibitors of phosphoinositide pathway such as Gαq/11 protein blocker, we have shown that an increased intracellular calcium concentration elicited by the acetylcholine application was mediated by PLC/IP3R pathway. As a rise in intracellular calcium concentration could lead to an increase in the insecticide target sensitivity, these results suggest that An. gambiae mAChR-A should not be only considered as a potential target for new molecules but also as a key element to optimize the efficacy of insecticide in vector control.

Discovery of a potent M5 antagonist with improved clearance profile. Part 2: Pyrrolidine amide-based antagonists.

This Letter describes our ongoing effort to improve the clearance of selective M5 antagonists. Herein, we report the replacement of the previously disclosed piperidine amide (4, disclosed in Part 1) with a pyrrolidine amide core. Several compounds within this series provided good potency, subtype selectivity, and low to moderate clearance profiles. Interestingly, the left-hand side SAR for this series diverged from our earlier efforts.

Discovery of a potent M5 antagonist with improved clearance profile. Part 1: Piperidine amide-based antagonists.

The lack of potent and selective tool compounds with pharmaceutically favorable properties limits the in vivo understanding of muscarinic acetylcholine receptor subtype 5 (M5) biology. Previously, we presented a highly potent and selective M5 antagonist VU6019650 with a suboptimal clearance profile as our second-generation tool compound. Herein, we disclose our ongoing efforts to generate next-generation M5 antagonists with improved clearance profiles. A mix and match approach between VU6019650 (lead) and VU0500325 (HTS hit) generated a piperidine amide-based novel M5 antagonist series. Several analogs within this series, including 29f, provided good on-target potency with improved clearance profiles, though room for improvement remains.

Synthesis and characterization of chiral 6-azaspiro[2.5]octanes as potent and selective antagonists of the M4 muscarinic acetylcholine receptor.

In this manuscript, we report a series of chiral 6-azaspiro[2.5]octanes and related spirocycles as highly potent and selective antagonists of the muscarinic acetylcholine receptor subtype 4 (mAChR4). Chiral separation and subsequent X-ray crystallographic analysis of early generation analogs revealed the R enantiomer to possess excellent human and rat M4 potency, and further structure-activity relationship (SAR) studies on this chiral scaffold led to the discovery of VU6015241 (compound 19). Compound 19 is characterized by high M4 potency and selectivity across multiple species, excellent aqueous solubility, and moderate brain exposure in rodents after intraperitoneal administration.

Non-neuronal Cholinergic Muscarinic Acetylcholine Receptors in the Regulation of Immune Function.

Immune cells such as T and B cells, monocytes and macrophages all express most of the cholinergic components of the nervous system, including acetylcholine (ACh), choline acetyltransferase (ChAT), high affinity choline transporter, muscarinic and nicotinic ACh receptors (mAChRs and nAChRs, respectively), and acetylcholinesterase (AChE). Because of its efficient cleavage by AChE, ACh synthesized and released from immune cells acts only locally in an autocrine and/or paracrine fashion at mAChRs and nAChRs on themselves and other immune cells located in close proximity, leading to modification of immune function. Immune cells generally express all five mAChR subtypes (M1-M5) and neuron type nAChR subunits α2-α7, α9, α10, ß2-ß4. The expression pattern and levels of mAChR subtypes and nAChR subunits vary depending on the tissue involved and its immunological status. Immunological activation of T cells via T-cell receptor-mediated pathways and cell adhesion molecules upregulates ChAT expression, which facilitates the synthesis and release of ACh. At present, α7 nAChRs expressed in macrophages are receiving much attention because they play a central role in anti-inflammatory cholinergic pathways. However, it now appears that through modification of cytokine synthesis, Gq/11-coupled mAChRs play a prominent role in regulation of T cell proliferation and differentiation and B cell immunoglobulin class switching. It is anticipated that greater understanding of Gq/11-coupled mAChRs on immune cells will provide an opportunity to develop new and effective treatments for immunological disorders.

Molecular cloning of cholecystokinin (CCK) and CCK-A receptor and mechanism of CCK-induced gastrointestinal motility in Suncus murinus.

Cholecystokinin (CCK) is a peptide hormone mainly secreted by small intestinal endocrine I-cells and functions as a regulator of gallbladder contraction, gastric emptying, gastrointestinal (GI) motility, and satiety. The cellular effects of CCK in these peripheral tissues are predominantly mediated via CCK-A receptors which are found in smooth muscles, enteric neurons, and vagal afferent neurons in humans and animal models. Although various functions of CCK have been reported to be neurally mediated, it can also stimulate contraction via the CCK receptor on the smooth muscle. However, the entire underlying neural and cellular mechanisms involved in CCK-induced GI contractions are not clearly understood. Here, we first determined the cDNA and amino acid sequences of CCK and CCK-A receptor along with the distributions of cck mRNA and CCK-producing cells in house musk shrew (Suncus murinus, the laboratory strain named as suncus) and examined the mechanism of CCK-induced contraction in the GI tract. Mature suncus CCK-8 was identical to other mammalian species tested here, and suncus CCK-A receptor presented high nucleotide and amino acid homology with that of human, dog, mouse, and rat, respectively. Suncus CCK mRNA and CCK-producing cells were found mainly in small intestine and colon. In the organ bath study, CCK-8 induced dose-dependent contractions in the suncus stomach, duodenum, and jejunum, and these contractions were inhibited by atropine and CCK-A receptor antagonist. These results suggest that CCK-8-induced contraction is mediated in the myenteric cholinergic neural network and that CCK-A receptor is partly responsible for CCK-8-induced contractions. This study indicates that suncus is a useful animal model to study the functions of CCK involved in GI motility.

Histamine via histamine H1 receptor enhances the muscarinic receptor-induced calcium response to acetylcholine in an enterochromaffin cell model.

As a prerequisite for serotonin secretion, the P-STS ileal enterochromaffin cell line responds to acetylcholine (ACh) stimulation with an increase in intracellular calcium mediated by the muscarinic ACh receptor M3 (M3R). Histamine increases intracellular calcium via histamine H1 receptor (H1R) in P-STS cells and pre-incubation with histamine specifically augments the response to ACh but not to epinephrine or nicotine. We aimed to elucidate whether histamine receptors are involved in this synergism. Astonishingly, HEK-293 T cells-known to express M3R, but only a very low amount of histamine receptor messenger RNA-showed a similar enhancement of the calcium response to ACh by pre-incubation with histamine. Despite the much lower level of H1R protein detected in HEK-293 T cells as compared to P-STS cells, in both cell lines pre-treatment with H1R antagonists inhibited the synergism between histamine and ACh. No indication for an involvement of histamine H2 or H4 receptors in the synergism was found. Furthermore, pre-incubation with the cAMP-inducing compound forskolin had no influence on the intracellular calcium response to ACh. Serotonin secretion from P-STS cells was increased after challenge with ACh and histamine added simultaneously compared to ACh alone, suggesting that histamine increases ACh-induced serotonin secretion from enterochromaffin cells. In conclusion, our data suggest that histamine enhances the M3R-mediated intracellular calcium response to ACh via activation of H1R. This probably increases serotonin secretion from enterochromaffin cells and thereby affects intestinal motility in histamine intolerance, food allergies and irritable bowel syndrome.