M1 Acetylcholine Receptors Research Articles

Expression of muscarinic acetylcholine receptors M3 and M5 in osteoporosis.

BackgroundCholinergic signaling via muscarinic acetylcholine receptors (mAChR) is known to influence various physiological functions. In bone, M3 mAChR and M5 mAChR were identified on the membrane of osteoblast-like cells. M3 mAChR seems to be particularly relevant for bone physiology, as signaling via this receptor was reported to increase bone formation and decrease bone resorption. Thus, in the present study we investigated the relative mRNA expression of M3 and M5 mAChR in bones of a rat osteoporosis model.Material/MethodsOsteoporosis was induced in Sprague-Dawley rats by bilateral ovariectomy and additional feeding of a diet deficient in calcium, vitamins C, D2, D3, and phosphorus, and free of soy and phytoestrogen. After a period of 3, 12, and 14 months, relative mRNA expression of M3 mAChR and M5 mAChR was analyzed in the 11th thoracic vertebra by real-time RT-PCR.ResultsRelative mRNA expression of M3 mAChR was significantly reduced in bones of osteoporotic rats compared to sham operated animals that served as controls. Further, M3 mAChR mRNA expression was significantly down-regulated when comparing 14-month osteoporotic rats to 3-month osteoporotic rats. Relative M5 mAChR mRNA was expressed to a lesser extent than M3 mAChR and did not show significant differences in mRNA expression level between the experimental groups.ConclusionsM3 mAChR mRNA expression was reduced upon induction of osteoporosis and progression of disease was associated with further decrease of this receptor, indicating that M3 mAChR is involved in the development and regulation of osteoporosis.

May disordered protein cause serious drug side effect?

Insomnia is a self-reported disease where patients lose their ability to initiate and maintain sleep, leading to daytime performance impairment. Several drug targets to ameliorate insomnia symptoms have been discovered; however, these drug targets lead to serious side effects. Thus, we characterize the structural properties of these sleep-related receptors and the clock complex and discuss a possible drug design that will reduce side effects. Computational prediction shows that disordered property is shared. Over 30% of the structure of CLOCK, PER1/2/3, BMAL-1, muscarinic acetylcholine receptor-M1, melatonin receptor and casein kinase I are structurally disordered (the remaining proteins represent <30%). Investigations support the principle that the failures of insomnia drugs might be closely related to the protein architecture.

Early Stress Prevents the Potentiation of Muscarinic Excitation by Calcium Release in Adult Prefrontal Cortex

The experience of early stress contributes to the etiology of several psychiatric disorders and can lead to lasting deficits in working memory and attention. These executive functions require activation of the prefrontal cortex (PFC) by muscarinic M1 acetylcholine (ACh) receptors. Such Gαq-protein coupled receptors trigger the release of calcium (Ca(2+)) from internal stores and elicit prolonged neuronal excitation. In brain slices of rat PFC, we employed multiphoton imaging simultaneously with whole-cell electrophysiological recordings to examine potential interactions between ACh-induced Ca(2+) release and excitatory currents in adulthood, across postnatal development, and following the early stress of repeated maternal separation, a rodent model for depression. We also investigated developmental changes in related genes in these groups. Acetylcholine-induced Ca(2+) release potentiates ACh-elicited excitatory currents. In the healthy PFC, this potentiation of muscarinic excitation emerges in young adulthood, when executive function typically reaches maturity. However, the developmental consolidation of muscarinic ACh signaling is abolished in adults with a history of early stress, where ACh responses retain an adolescent phenotype. In prefrontal cortex, these rats show a disruption in the expression of multiple developmentally regulated genes associated with Gαq and Ca(2+) signaling. Pharmacologic and ionic manipulations reveal that the enhancement of muscarinic excitation in the healthy adult PFC arises via the electrogenic process of sodium/Ca(2+) exchange. This work illustrates a long-lasting disruption in ACh-mediated cortical excitation following early stress and raises the possibility that such cellular mechanisms may disrupt the maturation of executive function.

Myths and realities of the cardiac vagus.

There is continuing belief that cardiac parasympathetic postganglionic fibres are sparse or absent from the ventricles. This review of the literature shows that the supposition is a myth. Early studies considered that fine silver-stained fibres coursing amongst ventricle myocardial cells were most likely cardiac parasympathetic postganglionic fibres. The conclusions were later supported by acetyl cholinesterase staining using a method that appeared not to be associated with noradrenaline nerve fibres. The conclusion is critically examined in the light of several recent histological studies using the acetyl cholinesterase method and also a more definitive technique (CHAT), that suggest a widespread location of parasympathetic ganglia and a relatively dense parasympathetic innervation of ventricular muscle in a range of mammals including man. The many studies demonstrating acetylcholine release in the ventricle on vagal nerve stimulation and a high density of acetylcholine M2 receptors is in accord with this as are tests of ventricular performance from many physiological studies. Selective control of cardiac functions by anatomically segregated parasympathetic ganglia is discussed. It is argued that the influence of vagal stimulation on ventricular myocardial action potential refractory period, duration, force and rhythm is evidence that vagal fibres have close apposition to myocardial fibres. This is supported by clear evidence of accentuated antagonism between sympathetic activity and vagal activity in the ventricle and also by direct effects of vagal activity independent of sympathetic activity. The idea of differential control of atrial and ventricular physiology by vagal C and vagal B preganglionic fibres is examined as well as differences in chemical phenotypes and their function. The latter is reflected in medullary and supramedullary control. Reference is made to the importance of this knowledge to understanding the normal physiology of cardiac autonomic control and significance to pathology.

Substituted Benzylspiroindolin-2-one Analogues as Positive Allosteric Modulators of the Muscarinic Acetylcholine Receptor M1

ADVERTISEMENT RETURN TO ISSUEPREVPatent HighlightNEXTSubstituted Benzylspiroindolin-2-one Analogues as Positive Allosteric Modulators of the Muscarinic Acetylcholine Receptor M1David P. Rotella*View Author Information Department of Chemistry and Biochemistry, Montclair State University, 1 Normal Avenue, Montclair, New Jersey 07043, United States*Tel: 973-655-7204. Fax: 973-655-7772. E-mail: [email protected]Cite this: ACS Med. Chem. Lett. 2013, 4, 9, 822Publication Date (Web):August 9, 2013Publication History Received16 July 2013Published online9 August 2013Published inissue 12 September 2013https://doi.org/10.1021/ml400279jCopyright © 2013 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views514Altmetric-Citations2LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (177 KB) Get e-AlertsSUBJECTS:Amines,Ligands,Modulators,Nervous system diseases,Receptors Get e-Alerts

Impaired β-cell function in the adult offspring of rats fed a protein-restricted diet during lactation is associated with changes in muscarinic acetylcholine receptor subtypes

Impaired pancreatic β-cell function, as observed in the cases of early nutrition disturbance, is a major hallmark of metabolic diseases arising in adulthood. In the present study, we aimed to investigate the function/composition of the muscarinic acetylcholine receptor (mAChR) subtypes, M2 and M3, in the pancreatic islets of adult offspring of rats that were protein malnourished during lactation. Neonates were nursed by mothers that were fed either a low-protein (4%, LP) or a normal-protein (23%, NP) diet. Adult rats were pre-treated with anti-muscarinic drugs and subjected to the glucose tolerance test; the function and protein expression levels of M2mAChR and M3mAChR were determined. The LP rats were lean and hypoinsulinaemic. The selective M2mAChR antagonist methoctramine increased insulinaemia by 31% in the NP rats and 155% in the LP rats, and insulin secretion was increased by 32% in the islets of the NP rats and 88% in those of the LP rats. The selective M3mAChR antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide decreased insulinaemia by 63% in the NP rats and 40% in the LP rats and reduced insulin release by 41% in the islets of the NP rats and 28% in those of the LP rats. The protein expression levels of M2mAChR and M3mAChR were 57% higher and 53% lower, respectively, in the islets of the LP rats than in those of the NP rats. The expression and functional compositions of M2mAChR and M3mAChR were altered in the islets of the LP rats, as a result of metabolic programming caused by the protein-restricted diet, which might be another possible effect involved in the weak insulin secretion ability of the islets of the programmed adult rats.

Monoclonal Antibody against α-Actinin 4 from Human Umbilical Vein Endothelial Cells Inhibits Endothelium-Dependent Vasorelaxation

Background: This study was attempted to identify new molecules expressed on the plasma membrane of human umbilical vein endothelial cells (HUVECs) using monoclonal antibody-based proteomics technology and to determine the effect of the identified antibody on vascular reactivity. Methods: Twenty-two antibodies were developed from rats inoculated with HUVECs, and their effects were determined by observing vascular reactivity. Results: Among the 22 antibodies, the C-7 antibody significantly inhibited endothelium-dependent vasorelaxation in response to acetylcholine (ACh) but not to histamine. Moreover, the C-7 antibody did not affect norepinephrine-induced contraction in either the endothelium-intact or -denuded aorta. A proteomics study involving immunoprecipitation of the C-7 antibody with biotinylated HUVECs showed that this antibody binds to plasma membrane proteins corresponding to immunoglobulin heavy chain (VHDJ region), chaperonin-containing T-complex polypeptide 1 and α-actinin 4. The muscarinic M3 ACh receptor and α-actinin 4 were colocalized on the plasma membrane of HUVECs, and the colocalization was found to increase in response to ACh and was inhibited by pretreatment with the C-7 antibody. Conclusions: These results demonstrate that monoclonal C-7 antibody exerts an inhibitory effect on endothelium-dependent vasorelaxation induced by ACh and that this response may at least partially result from the inhibition of α-actinin 4.

The muscarinic M3 acetylcholine receptor exists as two differently sized complexes at the plasma membrane

The literature on GPCR (G-protein-coupled receptor) homo-oligomerization encompasses conflicting views that range from interpretations that GPCRs must be monomeric, through comparatively newer proposals that they exist as dimers or higher-order oligomers, to suggestions that such quaternary structures are rather ephemeral or merely accidental and may serve no functional purpose. In the present study we use a novel method of FRET (Förster resonance energy transfer) spectrometry and controlled expression of energy donor-tagged species to show that M(3)Rs (muscarinic M(3) acetylcholine receptors) at the plasma membrane exist as stable dimeric complexes, a large fraction of which interact dynamically to form tetramers without the presence of trimers, pentamers, hexamers etc. That M(3)R dimeric units interact dynamically was also supported by co-immunoprecipitation of receptors synthesized at distinct times. On the basis of all these findings, we propose a conceptual framework that may reconcile the conflicting views on the quaternary structure of GPCRs.

Collagen-induced arthritis impairs osteoclastogenesis and reactivity to sympathetic neurotransmitter stimuli in bone marrow-derived macrophages from DA rats

Osteoclasts (OC) are key players in bone destruction in rheumatoid arthritis (RA). Recent work showed that catecholaminergic nerve fibers are reduced in RA synovial tissue. Studies on sweat gland innervation revealed that catecholaminergic fibers are capable of phenotypic transition to cholinergic nerves. The sympathetic neurotransmitters norepinephrine (NE) and acetylcholine (ACh) affect osteoclastogenesis oppositely and that is why we wanted to study osteoclastogenesis at different phases of collagen-induced arthritis (CIA) in an altered neurotransmitter microenvironment. The influence of NE and ACh on differentiation and activity of bone marrow macrophage (BMM)-derived osteoclasts from arthritic and control animals were compared at various time-points after arthritis induction. The expression profile for adrenergic and ACh receptors was analyzed on mRNA level. The number of OCs was tendentially lower in arthritic animals. Stimulation with ACh yielded significantly more OCs in controls. NE decreased osteoclastogenesis via β-adrenoceptors and enhanced via α-adrenoceptor stimulation. Cells from arthritic animals were less affected. In form of a trend, osteoclasts from arthritic animals showed decreased activity in cathepsin K activity and in a pit formation assay. The receptor gene expression profile changed in the time course of arthritis. After 20 days past immunization, muscarinic ACh receptors M3 and M5 were significantly upregulated whereas after 40 days adrenoceptors α1D and α2B were significantly downregulated. We conclude that CIA suppresses OC differentiation and activity as well as reactivity to neurotransmitter stimulation but the underlying processes remain to be demonstrated.

Probing the Stoichiometry and Geometry of M3 Acetylcholine Receptors at the Plasma Membrane

G-protein-coupled receptors (GPCRs) are the largest family of transmembrane proteins in nature. GPCRs cascade signals from outer environment into cells and hence, are the target of more than 60% of modern clinical drugs. Determination of oligomerization of GPCRs is subject to significant controversy in the literature. We have investigated the quaternary structure of human muscarinic acetylcholine receptor type 3 (hM3) in living cells using Forster Resonance Energy Transfer (FRET) and two-photon excitation in an optical micro-spectroscopic set-up [Raicu et al, Nature Photonics, 2009]. The wild-type form of the hM3 receptor was fused to Cerulean while its mutated form, activated solely by a synthetic ligand (RASSL), was fused to Citrine and expressed constitutively in Flp-InTM T-RExTM 293 cells [Alvarez-Curto et al, Journal of Biological Chemistry, 2010]. When WT-hM3-Citrine and RASSL-hM3-Cerulean were co-expressed in same cells, excited donor (Cerulean), transferred its energy to nearby acceptors (Citrine) through FRET. Apparent FRET efficiency (Eapp) distribution maps were obtained for the imaged section of the cells by unmixing acquired spectrally resolved images using elementary donor and acceptor spectra. By selecting pixels in the Eapp image corresponding to the plasma membrane, a FRET efficiency histogram was obtained displaying the number of pixels in a particular range of Eapp values versus the corresponding Eapp value. The Eapp histograms were analyzed using a FRET theory [V. Raicu, Journal of Biological Physics, 2007], predicting various numbers and positions of peaks in the histograms for various sizes and geometry of oligomers. Eapp histograms of all cells were best fitted by a rhombus tetramer model. By also simulating the amplitudes of the peaks, we determined that hM3 receptors form both dimers and rhombus tetramers at the plasma membrane, and that their proportion remained largely unaffected by CNO binding.

Acetylcholine Attenuates Hypoxia/ Reoxygenation-Induced Mitochondrial and Cytosolic ROS Formation in H9c2 Cells via M2 Acetylcholine Receptor

Background: The anti-infammatory and cardioprotective effect of acetylcholine (ACh) has been reported; nevertheless, whether and how ACh exhibits an antioxidant property against ischemia/reperfusion (I/R)-induced oxidative stress remains obscure. Methods: In the present study, H9c2 rat cardiomyocytes were exposed to hypoxia/reoxygenation (H/R) to mimic I/R injury. We estimated intracellular different sources of reactive oxygen species (ROS) by measuring mitochondrial ROS (mtROS), mitochondrial DNA (mtDNA) copy number, xanthine oxidase (XO) and NADPH oxidase (NOX) activity and expression of rac 1. Cell injury was determined by lactate dehydrogenase (LDH) release and cleaved caspase-3 expression. The siRNA transfection was performed to knockdown of M2 acetylcholine receptor (M2 AChR) expression. Results: 12-h hypoxia followed by 2-h reoxygenation resulted in an abrupt burst of ROS in H9c2 cells. Administration of ACh reduced the levels of ROS in a concentration-dependent manner. Compared to the H/R group, ACh decreased mtROS, recovered mtDNA copy number, diminished XO and NOX activity, rac 1 expression as well as cell injury. Co- treatment with atropine rather than hexamethonium abolished the antioxidant and cardioprotective effect of ACh. Moreover, knockdown of M2 AChR by siRNA showed the similar trends as atropine co-treatment group. Conclusions: ACh inhibits mitochondria-, XO- and NOX-derived ROS production thus protecting H9c2 cells against H/R-induced oxidative stress, and these benefcial effects are mainly mediated by M2 AChR. Our findings suggested that increasing ACh release could be a potential therapeutic strategy for treatment and prevention of I/R injury.

Genetic variation in the parasympathetic signaling pathway in patients with reflex syncope

Reflex syncope is defined by a self-terminating transient loss of consciousness associated with an exaggerated response of the vagal reflexes upon orthostatic challenges. A hereditary component has previously been suggested. We hypothesized that variations in genes encoding proteins mediating the vagal signaling in the heart may be involved in reflex syncope pathogenesis. We systematically resequenced the entire coding regions and flanking intron sequences in 5 genes in the cardiac post-synaptic parasympathetic signaling pathway [muscarinic acetylcholine receptor M2 (CHRM2); G-protein beta-1 subunit (GNB1); G-protein gamma-2 subunit (GNG2); potassium inwardly rectifying channel, subfamily J, member 3 (KCNJ3); and potassium inwardly rectifying channel, subfamily J, member 5 (KCNJ5)] in 74 patients with well-characterized reflex syncope of either cardioinhibitory [Vasovagal Syncope International Study (VASIS-IIB), N = 38] or vasodepressor (VASIS-III, N = 36) type. We identified 2 novel genetic variants (CHRM2 c.1114C>G and GNG2 c.87+34G>A) and several known variants (GNB1: c.267+14G>A, c.267+19C>T, and c.738C>T; KCNJ3: c.119A>G, c.591C>T, c.1038T>C, and c.1494T>C; KCNJ5: c. 171T>C, c.810T>G, c.834T>C, c.844C>G, c.938+7C>T, and c.938-10G>A). The minor allele frequency of the KCNJ5 c.938+7C>T variant was significantly lower in patients than in the control group (0.014 versus 0.089, P = 0.001), and the frequency of heterozygosity and homozygosity was lower in cardioinhibitory patients compared to controls. Genetic variations in genes responsible for the vagal signaling in the heart, including CHRM2, GNB1, GNG2, KCNJ3, and KCNJ5, are not major contributors to the pathogenesis of reflex syncope of vasodepressor or cardioinhibitory types.

Anti-M3 muscarinic acetylcholine receptor antibodies in patients with Sjögren’s syndrome

Sjögren's syndrome (SS) is an autoimmune disease that affects exocrine glands including salivary and lacrimal glands. Recently, autoantibodies against muscarinic acetylcholine receptor M3 (M3R) have been detected in serum from 9 to 100% of patients with SS in addition to anti-SS-A and anti-SS-B antibodies. These observations suggest the possibility that anti-M3R antibodies could serve as a new diagnostic test in patients with SS. Some anti-M3R antibodies are directly responsible for salivary underproduction in patients with SS. Thus, strategies designed to eliminate such pathogenic antibodies could help cure SS sufferers. In this review, we summarize the current state of knowledge of anti-M3R autoantibodies in patients with SS and the correlation between B cell epitopes and the function of anti-M3R antibodies.

Monitoring targeted drug delivery: a key cornerstone of precision medicine

The application of Personalized Medicine strategies of matching specific disease genotypes and phenotypes with specific pharmaceutical drug treatment has shown tremendous efficacy in several important diseases. The most successful example is the targeting mutations of the epithelial growth factor receptor by small molecule antagonists of the tyrosine kinase in non small cell lung cancer. The expansion of this treatment paradigm into other diseases has led to new thinking considering the diagnosis and treatment of disease, including “Precision Medicine”, which seeks to provide a molecular taxonomy of disease that can be used for guiding targeted therapy in individual patients. Today we have only a cursory understanding of the patterns of actual uptake of drugs or their active metabolites at such targeted sites in the body. In the past such studies required extrinsic labeling of the parent drug (ie radioactivity in PET) that potentially changed the chemical and biological properties of the drug. New technologies such as MALDI-MSI mass spectrometry have revolutionized the label-less tracking of drugs within targeted disease tissue sites [1]. A spectral image of drug distribution in biopsy tissue compartments is created by monitoring the signal intensity of signature ion mass fingerprints unique to each compound and its metabolites at contiguous sampling windows separated by < thirty microns. In a Proof of Principle study, we have applied MALDI-MSI to track the uptake and distribution of an inhaled muscarinic receptor antagonist [2], ipratropium in the bronchial airways of COPD patients shortly after administration. Direct measurement of the unlabeled drug in brochial biopsies showed that the ipratropium (parent drug ion mass, m/z 332.332, daughter ion masses m/z 166.2, and m/z 290.2) was transported and localized to areas of airway smooth muscle that expressed the targeted acetylcholine receptor M3, as shown by immunohistochemistry performed following mass spectrometry analysis. This result is the first reported co-localization of the unlabeled drug and its targeted receptor in man. In addition to the drug position and intensity signatures it is possible to simultaneously map the patterns of thousands of ion masses representing peptides, proteins, phospholipids, and metabolites that characterize healthy and diseased states. Further, the effect of drug on the relative abundances and histological positions of these ion signatures may also provide important indices of response to therapy. Together, these catalogs will likely become an important part of the molecular taxonomy of disease being developed with Precision Medicine frameworks using genomic, genetic and proteomic approaches defining the micro-environment of disease.

Synthesis and biological characterization of a series of novel diaryl amide M1 antagonists

Utilizing a combination of high-throughput and multi-step synthesis, SAR in a novel series of M1 acetylcholine receptor antagonists was rapidly established. The efforts led to the discovery the highly potent M1 antagonists 6 (VU0431263), and 8f (VU0433670). Functional Schild analysis and radioligand displacement experiments demonstrated the competitive, orthosteric binding of these compounds; human selectivity data are presented.

Muscarinic acetylcholine receptor M3 in proliferation and perineural invasion of cholangiocarcinoma cells

Cholangiocarcinoma, a type of malignant tumor, originates from epithelial cells of the bile duct. Perineural invasion is common path for cholangiocarcinoma metastasis, and it is highly correlated with postoperative recurrence and poor prognosis. It has been reported that muscarinic acetylcholine receptor M3 (mAChR M3) is widely expressed in digestive tract cancer, and may play an important role in the proliferation, differentiation, transformation and carcinogenesis of tumors. This study was to explore the effect of mAChR M3 on the growth of cholangiocarcinoma cells in vitro and provide a new approach to the pathogenesis and treatment of cholangiocarcinoma. Streptavidin-biotin complex immunohistochemistry was carried out to assess the expression of mAChR M3 in surgical specimens of cholangiocarcinomas (40 cases) and normal bile duct tissues (9), as well as to investigate nerve infiltration. The cholangiocarcinoma cells were treated with different concentrations of selective M-receptor agonist pilocarpine and M-receptor blocker atropine sulfate to induce changes in cell proliferation. The experimental data were analyzed by the Chi-square test. The strongly-positive expression rate of mAChR M3 was much higher in poorly-differentiated (69%, 9/13) than in well- and moderately-differentiated cholangiocarcinomas (30%, 8/27) (X2=5.631, P<0.05). The strongly-positive mAChR M3 expression rate in hilar cholangiocarcinoma (50%, 14/28) was higher than that in cholangiocarcinomas from the middle and lower common bile duct (25%, 3/12) (X2=2.148, P<0.05). Cholangiocarcinomas with distant metastasis had a strongly-positive expression rate (75%, 9/12), which was much higher than those without distant metastasis (29%, 8/28) (X2=7.410, P<0.01). The absorbance value in the pilocarpine+atropine group was significantly higher than the corresponding value in the pilocarpine group. The expression of mAChR M3 is influenced by the extent of differentiation, distant metastasis and the site of cholangiocarcinoma. It also plays a key role in the proliferation and metastasis of cholangiocarcinoma.

Acetylcholine is released from taste cells, enhancing taste signalling

Acetylcholine (ACh), a candidate neurotransmitter that has been implicated in taste buds, elicits calcium mobilization in Receptor (Type II) taste cells. Using RT-PCR analysis and pharmacological interventions, we demonstrate that the muscarinic acetylcholine receptor M3 mediates these actions. Applying ACh enhanced both taste-evoked Ca2+ responses and taste-evoked afferent neurotransmitter (ATP) secretion from taste Receptor cells. Blocking muscarinic receptors depressed taste-evoked responses in Receptor cells, suggesting that ACh is normally released from taste cells during taste stimulation. ACh biosensors confirmed that, indeed, taste Receptor cells secrete acetylcholine during gustatory stimulation. Genetic deletion of muscarinic receptors resulted in significantly diminished ATP secretion from taste buds. The data demonstrate a new role for acetylcholine as a taste bud transmitter. Our results imply specifically that ACh is an autocrine transmitter secreted by taste Receptor cells during gustatory stimulation, enhancing taste-evoked responses and afferent transmitter secretion.

Real-time detection of acetylcholine release from the human endocrine pancreas

Neurons, sensory cells and endocrine cells secrete neurotransmitters and hormones to communicate with other cells and to coordinate organ and system function. Validation that a substance is used as an extracellular signaling molecule by a given cell requires a direct demonstration of its secretion. In this protocol we describe the use of biosensor cells to detect neurotransmitter release from endocrine cells in real-time. Chinese hamster ovary cells expressing the muscarinic acetylcholine (ACh) receptor M3 were used as ACh biosensors to record ACh release from human pancreatic islets. We show how ACh biosensors loaded with the Ca(2+) indicator Fura-2 and pressed against isolated human pancreatic islets allow the detection of ACh release. The biosensor approach is simple; the Ca(2+) signal generated in the biosensor cell reflects the presence (release) of a neurotransmitter. The technique is versatile because biosensor cells expressing a variety of receptors can be used in many applications. The protocol takes ∼3 h.

Smooth muscle adaptation and recovery of contractility after massive small bowel resection in rats

Previous studies have suggested that massive small bowel resection (mSBR) compromises the normal intestinal processes of digestion and absorption, and requires an adaptive response to regain full function and reinstate coordinated contractile activity of the circular smooth muscle. This study was designed to investigate spontaneous contractile activity of circular smooth muscle using the mSBR rat model and to determine the functional role of M(2) and M(3) muscarinic acetylcholine receptors (mAChR) in this process. Male Sprague-Dawley rats underwent an 80% proximal SBR or sham operation. Markers of adaptation, including villus and microvillus height, were analyzed by hematoxylin and eosin staining and transmission electron microscopy. Contractility was measured by attaching the distal ileum strips to strain gauge transducers and exposing the tissue to varying doses of the cholinergic agonist carbachol. Protein expressions of M(2)- and M(3)-mAChR in intestinal smooth muscle (ISM) were detected by Western blot. Following mSBR, the ISM showed perturbed spontaneous rhythmic contraction, irregular amplitude and slow frequency by muscle strip test. However, by two weeks after mSBR, the contractile function of circular smooth muscle was found to have returned to normal levels. Protein expression of M(2)-mAChR was down-regulated following mSBR but up-regulated during the adaptive process when contractile activity of circular smooth muscle was regained. These results indicate that smooth muscle contractility was spontaneously restored in rats following mSBR, and involved the acetylcholine receptors M(2) and M(3). Thus, the disrupted contractile response of smooth muscle in short bowel syndrome may be corrected by therapeutic intervention to restore the expressions of M(2)- and M(3)-mAChR to pre-mSBR levels.

Muscarinic acetylcholine receptor M2 (CHRM2; HM2)

Muscarinic acetylcholine receptor M2 (CHRM2; HM2)