Activation of focal adhesion kinase via M1 muscarinic acetylcholine receptor is required in restitution of intestinal barrier function after epithelial injury.

Impairment of epithelial barrier is observed in various intestinal disorders including inflammatory bowel diseases (IBD). Numerous factors may cause temporary damage of the intestinal epithelium. A complex network of highly divergent factors regulates healing of the epithelium to prevent inflammatory response. However, the exact repair mechanisms involved in maintaining homeostatic intestinal barrier integrity remain to be clarified. In this study, we demonstrate that activation of M1 muscarinic acetylcholine receptor (mAChR) augments the restitution of epithelial barrier function in T84 cell monolayers after ethanol-induced epithelial injury, via ERK-dependent phosphorylation of focal adhesion kinase (FAK). We have shown that ethanol injury decreased the transepithelial electrical resistance (TER) along with the reduction of ERK and FAK phosphorylation. Carbachol (CCh) increased ERK and FAK phosphorylation with enhanced TER recovery, which was completely blocked by either MT-7 (M1 antagonist) or atropine. The CCh-induced enhancement of TER recovery was also blocked by either U0126 (ERK pathway inhibitor) or PF-228 (FAK inhibitor). Treatment of T84 cell monolayers with interferon-γ (IFN-γ) impaired the barrier function with the reduction of FAK phosphorylation. The CCh-induced ERK and FAK phosphorylation were also attenuated by the IFN-γ treatment. Immunological and binding experiments exhibited a significant reduction of M1 mAChR after IFN-γ treatment. The reduction of M1 mAChR in inflammatory area was also observed in surgical specimens from IBD patients, using immunohistochemical analysis. These findings provide important clues regarding mechanisms by which M1 mAChR participates in the maintenance of intestinal barrier function under not only physiological but also pathological conditions.

Diverse glutamatergic inputs target spines expressing M1 muscarinic receptors in the basolateral amygdala: An ultrastructural analysis.

Although it is known that acetylcholine acting through M1 muscarinic receptors (M1Rs) is essential for memory consolidation in the anterior basolateral nucleus of the amygdala (BLa), virtually nothing is known about the circuits involved. In the hippocampus M1R activation facilitates long-term potentiation (LTP) by potentiating NMDA glutamate receptor (NMDAR) currents. The majority of NMDAR+ profiles in the BLa are spines. Since about half of dendritic spines of BLa pyramidal neurons (PNs) receiving glutamatergic inputs are M1R-immunoreactive (M1R+) it is possible that the role of M1Rs in BLa mnemonic functions also involves potentiation of NMDAR currents in spines. However, the finding that only about half of BLa spines are M1R+ suggests that this proposed mechanism may only apply to a subset of glutamatergic inputs. As a first step in the identification of differential glutamatergic inputs to M1R+ spines in the BLa, the present electron microscopic study used antibodies to two different vesicular glutamate transporter proteins (VGluTs) to label two different subsets of glutamatergic inputs to M1R+ spines. These inputs are largely complimentary with VGluT1+ inputs arising mainly from cortical structures and the basolateral nucleus, and VGluT2+ inputs arising mainly from the thalamus. It was found that about one-half of the spines that were postsynaptic to VGluT1+ or VGluT2+ terminals were M1R+. In addition, a subset of the VGluT1+ or VGluT2+ axon terminals were M1R+, including those that synapsed with M1R+ spines. These results suggest that acetylcholine can modulate glutamatergic inputs to BLa spines by presynaptic as well as postsynaptic M1R-mediated mechanisms.

Evaluation of 11C-LSN3172176 as a Novel PET Tracer for Imaging M1 Muscarinic Acetylcholine Receptors in Nonhuman Primates.

The M1 muscarinic acetylcholine receptor (mAChR) plays an important role in learning and memory, and therefore is a target for development of drugs for treatment of cognitive impairments in Alzheimer disease and schizophrenia. The availability of M1-selective radiotracers for PET will help in developing therapeutic agents by providing an imaging tool for assessment of drug dose-receptor occupancy relationship. Here we report the synthesis and evaluation of 11C-LSN3172176 (ethyl 4-(6-(methyl-11C)-2-oxoindolin-1-yl)-[1,4'-bipiperidine]-1'-carboxylate) in nonhuman primates. Methods:11C-LSN3172176 was radiolabeled via the Suzuki-Miyaura cross-coupling method. PET scans in rhesus macaques were acquired for 2 h with arterial blood sampling and metabolite analysis to measure the input function. Blocking scans with scopolamine (50 µg/kg) and the M1-selective agent AZD6088 (0.67 and 2 mg/kg) were obtained to assess tracer binding specificity and selectivity. Regional brain time-activity curves were analyzed with the 1-tissue-compartment model and the multilinear analysis method (MA1) to calculate regional distribution volume. Nondisplaceable binding potential values were calculated using the cerebellum as a reference region. Results:11C-LSN3172176 was synthesized with greater than 99% radiochemical purity and high molar activity. In rhesus monkeys, 11C-LSN3172176 metabolized rapidly (29% ± 6% parent remaining at 15 min) and displayed fast kinetics and extremely high uptake in the brain. Imaging data were modeled well with the 1-tissue-compartment model and MA1 methods. MA1-derived distribution volume values were high (range, 10-81 mL/cm3) in all known M1 mAChR-rich brain regions. Pretreatment with scopolamine and AZD6088 significantly reduced the brain uptake of 11C-LSN3172176, thus demonstrating its binding specificity and selectivity in vivo. The cerebellum appeared to be a suitable reference region for derivation of nondisplaceable binding potential, which ranged from 2.42 in the globus pallidus to 8.48 in the nucleus accumbens. Conclusion:11C-LSN3172176 exhibits excellent in vivo binding and imaging characteristics in nonhuman primates and appears to be the first appropriate radiotracer for PET imaging of human M1 AChR.

Expression of muscarinic receptor subtypes in salivary glands of rats, sheep and man.

In rat parotid, submandibular and sublingual glands and in ovine parotid and in human labial glands, the expression of muscarinic receptor subtypes was examined by immunoblotting and immunohistochemistry. Functional correlates were searched for in rat salivary glands. In the rat submandibular and sublingual glandular tissues clear signals of muscarinic M1 and M5 receptors could be detected in the immunoblotting and vague bands for muscarinic M3 and, in particular for, M4 receptors. The rat parotid gland differed. In this gland, the signal was less obvious for the muscarinic M1 receptor, and further, muscarinic M4 receptors appeared more strongly marked than in the submandibular glands. The results from the immunohistochemistry could be interpreted as the muscarinic M4 receptors are located on nerve fibres, since the outer layer of lobuli were densely stained. Intraglandular vessels in the rat submandibular and parotid glands showed expression of M3 receptors. In contrast to the parotid gland, the submandibular vessels also expressed M1 and M2 receptors. Occasionally M5 receptors appeared in the arteries and veins also. The functional studies in the rat confirmed muscarinic M1 receptor mediated secretion in the submandibular gland. Since the M1 receptor blockade did not affect submandibular blood flow, indirect vascular effects could not in total explain the secretory inhibition. Also in the human labial glands, muscarinic M1, M3 and M5 receptors occurred. No or low amounts of muscarinic M2 and M4 receptors could be detected. In patients with Sjögren-like symptoms an up-regulation of M3, M4 and M5 receptors was apparent in the labial glands. In ovine parotid glands all receptors could be detected, but constantly with vague bands for muscarinic M2 receptors. In conclusion, muscarinic M1 receptors seem to be expressed in seromucous/mucous glands. A secretory effect by muscarinic M5 receptors is not to be excluded, since they were expressed in all the glands examined. However, other functions, such as promotion of inflammation, cell growth and proliferation are possible as well.

Decrease of muscarinic cholinergic receptors expression in placenta from rats exposed to methyl parathion.

Placental transfer of methyl parathion (MP), an organophosphate pesticide, could involve effects on cholinergic system. To analyze whether placental cholinergic system is altered by prenatal exposure to MP, expression of muscarinic cholinergic receptors (M1 and M2 subtypes; mAChR) was determined in pregnant rats exposed to MP at 0.0, 1.0, 1.5, and 2.0 mg/kg. An immunohistochemical analysis for M1 and M2 mAChR was performed, and the density of the mAChR signal was measured by image analysis. M1 and M2 mAChR were found in the trophoblast present in the labyrinth, with an 18% predominance of M2 over M1 in the non-exposed group. The expression of M1 and M2 mAChR in placentas exposed to MP showed a decrease when compared with the non-exposed group (P < 0.05); a dose-response effect was not detected. These results demonstrate that prenatal exposure to MP causes changes in the placental expression of mAChR M1 and M2, suggesting that related placental cholinergic functions could be affected.

Distribution and function of muscarinic receptor subtypes in the ovine submandibular gland.

The effects of muscarinic receptor antagonists on responses to electrical stimulation of the chorda-lingual nerve were determined in pentobarbitone-anesthetized sheep and correlated to the morphology of tissue specimens. Stimulation at 2 Hz continuously, or in bursts of 1 s at 20 Hz every 10 s, for 10 min induced similar submandibular fluid responses (19 +/- 3 vs. 21 +/- 3 microl x min(-1) x g gland(-1)), whereas vasodilatation was greater during stimulation in bursts (-52 +/- 4 vs. -43 +/- 5%; P < 0.01). Continuous stimulation at 8 Hz induced substantially greater responses (66 +/- 9 microl x min(-1) x g gland(-1) and -77 +/- 3%). While atropine (0.5 mg/kg iv) abolished the secretory response at 2 and 20 Hz (1:10 s), a small response persisted at 8 Hz (<5%). The "M1-selective" antagonist pirenzepine (40 microg/kg iv) reduced the fluid response at all frequencies tested (P < 0.05-0.01), most conspicuously at 2 Hz (reduced by 69%). Methoctramine ("M2/M4-selective"; 100 microg/kg iv; n = 5) had no effect on fluid or the vascular responses but increased the protein output at 2 (+90%, P < 0.05) and 8 Hz (+45%, P < 0.05). The immunoblotting showed distinct bands for muscarinic M1, M3, M4, and M5 receptors, and immunohistochemistry showed muscarinic M1 and M3 receptors to occur in the parenchyma. Thus muscarinic M1 receptors contribute to the secretory response to parasympathetic stimulation but have little effect on the vasodilatation in the ovine submandibular gland. Increased transmitter release caused by blockade of neuronal inhibitory receptors of the M4 subtype would explain the increase in protein output.

Muscarinic cholinoceptor activation modulates DNA synthesis and CD40 expression in fibroblast cells.

1 The aim of the present work was to examine the role of muscarinic acetylcholine receptors (mAChR) on DNA synthesis and CD40 expression in human fibroblast cells. Neonatal human skin fibroblast cultures were stimulated with carbachol in presence or absence of specific antagonists and the following parameters were measured: identification of mAChR subtypes, DNA synthesis, inositol phosphates (InsP) production and CD40 expression. 2 Human fibroblasts express mAChR with Kd 0.47 +/- 0.11 nm and Bmax 236 +/- 22 fmol mg protein(-1). Carbachol stimulates DNA synthesis, InsP and the expression of CD40. All these effects were inhibited by atropine, mustard hydrochloride (4-DAMP) and pirenzepine but not by AF-DX 116 and tropicamide, indicating that M3 and M1 mAChR are implicated in carbachol action. The relative Ki of the antagonists obtained by competition binding assay was in parallel to the relative potency for blocking both carbachol-stimulated InsP accumulation and DNA synthesis. 3 The intracellular pathway leading to carbachol-induced biological effects involved phospholipase C and calcium/calmodulin, as U-73122 and trifluoroperazine blocked carbachol effects, respectively. Calphostin C, a protein kinase C inhibitor, had no effect, indicating that this enzyme does not participate in the system. 4 These results may contribute to a better understanding of the modulatory role of the parasympathetic muscarinic system on normal human fibroblast function.

[The acute effects of dimethoate on the muscarinic-receptors of rat brains and the relationship between muscarinic-receptors and cholinesterase].

OBJECTIVE: To study the acute effects of dimethoate on the muscarinic-receptors(M1, M2) in the brain of rats. METHODS: 24 Sprague-Dawley rats were divided into 4 groups randomly. They were administered subcutaneously with 0, 25, 50, 100 mg/kg dimethoate, respectively. Brains were removed after 48 hours of administration. Radioligand binding assay was used to determine the density and affinity of M1 and M2 receptors. RESULTS: Rats in the treated group showed low density of M1 and M2 receptors compared with the control rats. The brain M1 receptor density of the rats in the highest dosage group was significantly lower than that in the control group while brain M2 receptors density had a decrease trend with increasing dosage, but the difference showed no significance. However, there were no differences of the affinity of both M1 and M2 among different treated groups. Correlation analysis showed there is positive relationship between cholinesterase activity and density of M1 receptors(r = 0.583, P < 0.01). CONCLUSION: M1 and M2 receptors density decreased with the increasing dosage of dimethoate. It is suggested that the alleviating of cholinergic symptoms may be due to the decrease of M1 and M2 receptors in rat brain.

Hypersensitive mAChRs are involved in the epiphora of transplanted glands.

Autologous transplantation of the submandibular gland is an effective treatment for severe dry eye syndrome. However, more than 40% of patients experience epiphora 3 to 6 months after transplantation. The underlying mechanism of epiphora remains to be elucidated. To investigate the potential roles of muscarinic acetylcholine receptors (mAChRs) in the induction of epiphora in transplanted glands, we assessed and found elevated mRNA and protein expression of M1- and M3-mAChR in transplanted glands from epiphora patients. The content of inositol 1, 4, 5-trisphosphate was also elevated. Moreover, carbachol (5 and 10 µM) induced greater increase of [Ca(2+)]i in isolated epiphora submandibular cells than in controls. Although aquaporin-5 (AQP5) content and distribution in the apical and lateral plasma of epiphora glands did not change, AQP5 content was reduced in lipid microdomains (lipid rafts and caveolae) but increased in non-lipid microdomains compared with controls. Carbachol (10 µM) increased the ratio of non-lipid microdomain to total AQP5 in the cultured control submandibular gland tissue. Taken together, these results indicated that hypersensitive mAChRs might be involved in the epiphora of transplanted submandibular glands by modulating AQP5 trafficking.

Muscarinic acetylcholine receptors in the mouse esophagus: focus on intraganglionic laminar endings (IGLEs).

BACKGROUND: IGLEs represent the only low-threshold vagal mechanosensory terminals in the tunica muscularis of the esophagus. Previously, close relationships of vesicular glutamate transporter 2 (VGLUT2) immunopositive IGLEs and cholinergic varicosities suggestive for direct contacts were described in almost all mouse esophageal myenteric ganglia. Possible cholinergic influence on IGLEs requires specific acetylcholine receptors. In particular, the occurrence and location of neuronal muscarinic acetylcholine receptors (mAChR) in the esophagus were not yet characterized. METHODS: This study aimed at specifying relationships of VGLUT2 immunopositive IGLEs and vesicular acetylcholine transporter (VAChT)-immunopositive varicosities using pre-embedding electron microscopy and the location of mAChR1-3 (M1-3) within esophagus and nodose ganglia using multilabel immunofluorescence and retrograde tracing. KEY RESULTS: Electron microscopy confirmed synaptic contacts between cholinergic varicosities and IGLEs. M1- and M2-immunoreactivities (-iry; -iries) were colocalized with VGLUT2-iry in subpopulations of IGLEs. Retrograde Fast Blue tracing from the esophagus showed nodose ganglion neurons colocalizing tracer and M2-iry. M1-3-iries were detected in about 80% of myenteric ganglia and in about 67% of myenteric neurons. M1- and M2-iry were present in many fibers and varicosities within myenteric ganglia. Presynaptic M2-iry was detected in all, presynaptic M3-iry in one-fifth of motor endplates of striated esophageal muscles. M1-iry could not be detected in motor endplates of the esophagus, but in sternomastoid muscle. CONCLUSIONS & INFERENCES: Acetylcholine probably released from varicosities of both extrinsic and intrinsic origin may influence a subpopulation of esophageal IGLEs via M2 and M1-receptors.

A fluorescence anisotropy assay for the muscarinic M1 G-protein-coupled receptor.

In the search for new chemical entities that interact with G-proteincoupled receptors (GPCRs), assays that quantify efficacy and affinity are employed. Traditional methods for measuring affinity involve radiolabeled ligands. To address the need for homogeneous biochemical fluorescent assays to characterize orthosteric ligand affinity and dissociation rates, we have developed a fluorescence anisotropy (FA) assay for the muscarinic M1 receptor that can be conducted in a 384-well plate. We used membranes from a muscarinic M1 cell line optimized for high-throughput functional assays and the previously characterized fluorescent antagonist BODIPY FL pirenzepine. The affinities of reference compounds were determined in the competitive FA assay and compared with those obtained with a competitive filter-based radioligand-binding assay using [(3)H] N-methylscopolamine. The IC(50) values produced from the FA assay were well-correlated with the radioligand-binding K(i) values (R(2) = 0.98). The dissociation of the BODIPY FL pirenzepine was readily monitored in real time using the FA assay and was sensitive to the presence of the allosteric modulator gallamine. This M1 FA assay offers advantages over traditional radioligandbinding assays as it eliminates radioactivity while allowing investigation of orthosteric or allosteric muscarinic M1 ligands in a homogeneous format.

Cortical M1 receptor concentration increases without a concomitant change in function in Alzheimer's disease.

Although the M(1) muscarinic receptor is a potential therapeutic target for Alzheimer's disease (AD) based on its wide spread distribution in brain and its association with learning and memory processes, whether its receptor response is altered during the onset of AD remains unclear. A novel [(35)S]GTPgammaS binding/immunocapture assay was employed to evaluated changes in M(1) receptor function in cortical tissue samples harvested from people who had no cognitive impairment (NCI), mild cognitive impairment (MCI), or AD. M(1) function was stable across clinical groups. However, [(3)H]-oxotremorine-M radioligand binding studies revealed that the concentration of M(1) cortical receptors increased significantly between the NCI and AD groups. Although M(1) receptor function did not correlate with cognitive function based upon mini-mental status examination (MMSE) or global cognitive score (GCS), functional activity was negatively correlated with the severity of neuropathology determined by Braak staging and NIA-Reagan criteria for AD. Since M(1) agonists have the potential to modify the pathologic hallmarks of AD, as well as deficits in cognitive function in animal models of this disease, the present findings provide additional support for targeting the M(1) receptor as a potential therapeutic for AD.

Suitability of muscarinic acetylcholine receptor antibodies for immunohistochemistry evaluated on tissue sections of receptor gene-deficient mice.

Acetylcholine (ACh) is a major regulator of visceral function exerting pharmacologically relevant effects upon smooth muscle tone and epithelial function via five types of muscarinic receptors (M1R-M5R). In this paper, we assessed the specificity of muscarinic receptor (MR) antibodies in immunohistochemical labelling on tissue sections by analysing specimens from wild-type and respective gene-deficient mice. Of 24 antibodies evaluated in this study, 16 were tested at 18 different conditions each, and eight of them in 21 different protocols, resulting in a total number of 456 antibody/protocol combinations. Each of them was tested at four antibody dilutions at minimum, so that finally, at least 1,824 conditions were evaluated. For each of them, dorsal root ganglia, urinary bladder and cross-sections through all thoracic viscera were investigated. In all cases where the antigen was available, at least one incubation condition was identified in which only select cell types were immunolabelled in the positive control but remained unlabelled in the pre-absorption control. With two exceptions (M2R antibodies), however, all antibodies produced identical immunohistochemical labelling patterns in tissues taken from corresponding gene-deficient mice even when the pre-absorption control in wild-type mice suggested specificity. Hence, the present data demonstrate the unpleasant fact that reliable immunohistochemical localisation of MR subtypes with antibodies is the exception rather than the rule. Immunohistochemical detection of MR subtype localisation in tissue sections of peripheral organs is limited to the M2R subtype utilising the most commonly used methodological approaches.

Cholinergic stimulation of amylase secretion from pancreatic acinar cells studied with muscarinic acetylcholine receptor mutant mice.

Muscarinic acetylcholine receptors (mAChRs) expressed by pancreatic acinar cells play an important role in mediating acetylcholine-dependent stimulation of digestive enzyme secretion. To examine the potential roles of M(1) and M(3) mAChRs in this activity, we used M(1) and M(3) receptor single knockout (KO) and M(1)/M(3) receptor double KO mice as novel experimental tools. Specifically, we examined the ability of the muscarinic agonist carbachol to stimulate amylase secretion in vitro, using dispersed pancreatic acini prepared from wild-type and mAChR mutant mice. Quantitative reverse transcription-polymerase chain reaction studies using RNA prepared from mouse pancreatic acini showed that deletion of the M(1) or M(3) mAChR genes did not lead to significantly altered mRNA levels of the remaining mAChR subtypes. Moreover, immunoprecipitation studies with M(1) and M(3) mAChR-selective antisera demonstrated that both mAChR subtypes are expressed by mouse pancreatic acini. Strikingly, carbachol-induced stimulation of amylase secretion was significantly impaired in acinar preparations from both M(1) and M(3) receptor single KO mice and abolished in acinar preparations from M(1)/M(3) receptor double KO mice. However, another pancreatic secretagogue, bombesin, retained its ability to fully stimulate amylase secretion in acinar preparations from M(1)/M(3) receptor double KO mice. Together, these studies support the concept that cholinergic stimulation of pancreatic amylase secretion is mediated by a mixture of M(1) and M(3) mAChRs and that other mAChR subtypes do not make a significant contribution to this activity. These findings clarify the long-standing question regarding the molecular nature of the mAChR subtypes mediating the secretion of digestive enzymes from the exocrine pancreas.