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Diabetic bladder dysfunction (DBD) is the most common complication in diabetes mellitus (DM). Myogenic abnormalities are common in DBD, however, the underlying mechanisms leading to these remains unclear. To understand the importance of smooth muscle insulin receptor (IR)-mediated signaling in the pathogenesis of DBD, we conditionally deleted it to achieve either heterozygous (SMIR+/-) or homozygous (SMIR-/-) deletion in smooth muscle cells. Despite impaired glucose and insulin tolerance seen with SMIR-/- mice, both SMIR+/- and SMIR-/- mice exhibited normal blood glucose and plasma insulin levels. Interestingly, these mice had abnormal voiding phenotypes, that included urinary frequency and small voids, and bladder smooth muscle (BSM) had significantly diminished contraction force. Morphology revealed a dilated bladder with thinner BSM layer, and BSM bundles were disorganized with penetrating interstitial tissue. Deletion of IR elevated FoxO and decreased mTOR protein expression, which further decreased the expression of Chrm3, P2x1, Sm22, and Cav1.2, crucial functional proteins for BSM contraction. Furthermore, we determined the expression of adiponectin in BSM, and deletion of IR in BSM inhibited adiponectin-mediated signaling. In summary, disruption of IR-mediated signaling in BSM caused abnormalities in proliferation and differentiation, leading to diminished BSM contractility and a voiding dysfunction phenotype that recapitulates human DBD.

Hypoxia can lead to adult middle cerebral artery (MCA) dysfunction and increase the risk of cerebrovascular diseases. It is largely unknown whether intrauterine hypoxia affects fetal MCA vasodilatation. This study investigated the effects and mechanisms of intrauterine hypoxia on fetal MCA vasodilatation. Near-term fetal sheep were exposed to intrauterine hypoxia. Human umbilical vein endothelial cells (HUVECs) were exposed to hypoxia in cellular experiments. Vascular tone measurement, molecular analysis, and transmission electron microscope (TEM) were utilized to determine vascular functions, tissue anatomy, and molecular pathways in fetal MCA. In fetal MCA, acetylcholine (ACh) induced reliable relaxation, which was markedly attenuated by intrauterine hypoxia. Atropine, P-F-HHSiD, L-NAME, and u0126 blocked most ACh-mediated dilation, while AF-DX 116 and tropicamide partially inhibited the dilation. Indomethacin and SB203580 did not significantly change ACh-mediated dilation. Tempol and PS-341 could restore the attenuated ACh-mediated vasodilatation following intrauterine hypoxia. The mRNA expression levels of CHRM2 and CHRM3 and the protein levels of CHRM3, p-NOS3, SOD2, ERK1/2, p-ERK1/2, MAPK14, and p-MAPK14 were significantly reduced by intrauterine hypoxia. The dihydroethidium assay showed that the production of ROS was increased under intrauterine hypoxia. TEM analysis revealed endothelial cells damaged by intrauterine hypoxia. In HUVECs, hypoxia increased ROS formation and decreased the expression of CHRM3, p-NOS3, SOD1, SOD2, SOD3, ERK1/2, p-ERK1/2, and p-MAPK14, while tempol and PS-341 potentiated p-NOS3 protein expression. In conclusion, in utero hypoxia reduced ACh-mediated vasodilatation in ovine MCA predominantly via decreased CHRM3 and p-NOS3, and the decreased NOS3 bioactivities might be attributed to ROS and ERK1/2.

The aim of this study was to find differentially expressed long noncoding RNAs (lncRNAs), microRNAs (miRNAs) and mRNAs and related signaling pathways, contributing to understanding the molecular mechanism of acute recurrent pancreatitis (ARP).First, peripheral whole blood samples from five acute pancreatitis (AP) patients, five ARP patients and five healthy individuals ( N ) were collected for RNA sequencing. Second, differentially/specifically expressed lncRNAs, miRNAs and mRNAs were identified in AP vs. N , ARP vs. N and ARP. Third, the ceRNA (lncRNA-miRNA-mRNA) networks of common/specifical lncRNAs, miRNAs and mRNAs were constructed in AP vs. N , ARP vs. N and ARP. Finally, functional analysis of common mRNAs in AP vs. N and ARP vs. N was performed.A total of 315 common lncRNAs, 12 common miRNAs and 909 common mRNAs were identified between AP and ARP. Ninety-four specifically expressed lncRNAs, one specifically expressed miRNAs and 286 specifically expressed mRNAs were found in ARP. Some interaction pairs were identified in AP and ARP, such as LUCAT1/NEAT1-hsa-miR-16-2-3p-HK2, CHRM3-AS2-hsa-miR-122-5p/hsa-miR-145-3p-DBH/CACNA1C, CHRM3-AS2-hsa-miR-200a-3p-PDGFD, RBM26-AS1-hsa-miR-200b-3p-FHIT and LINC00891/KTN1-AS1-hsa-miR-143-3p-tyrosine kinase (TXK). ASAP1-IT2/DGCR9-hsa-miR-342-5p-ABCC5/MAP2K6 was the only one specific interaction pair identified in ARP. Four significantly enriched signaling pathways were identified in AP vs. N and ARP vs. N , including amino sugar and nucleotide sugar metabolism (involved NPL and HK2), MAPK signaling pathway (involved CACNA1C and PDGFD), metabolic pathways (involved DBH and FHIT) and leukocyte transendothelial migration (involved TXK).The identified altered lncRNAs, miRNAs, mRNAs and related signaling pathways may be involved in the AP development and recurrence.

BackgroundCholinergic urticaria (CholU), a frequent form of chronic inducible urticaria, is characterized by itchy wheals and angioedema in response to sweating. As of now, the rate and pathophysiological relevance of impaired sweating in patients with CholU are ill-defined.AimTo assess in CholU patients the rate and extent of impaired sweating and its links to clinical and pathophysiological features of CholU.Patients and methodsWe assessed sweating in patients with CholU (n = 13) subjected to pulse-controlled ergometry (PCE) provocation testing. Pre- and post-PCE biopsies of lesional (L) and non-lesional (NL) skin were analyzed for the expression of acetylcholine receptor M3 (CHRM3) and acetylcholine esterase (ACh-E) by quantitative histomorphometry and compared to those of healthy control subjects (HCs). CholU patients were assessed for disease duration and severity as well as other clinical features.ResultsOf the 13 patients with CholU, 10 showed reduced sweating in response to PCE provocation, and 3 had severely reduced sweating. Reduced sweating was linked to long disease duration and high disease severity. CholU patients with impaired sweating responses showed reduced sweat gland epithelial expression of CHRM3 and ACh-E.ConclusionReduced sweating is common in CholU patients, especially in those with long-standing and severe disease, and it can be severe. Reduced expression of CHRM3 and ACh-E may be the cause or consequence of CholU in patients with impaired sweating, and this should be explored by further studies.

Gastric cancer represents a real public health problem as far as incidence, aggressiveness and unfavorable prognosis are concerned. The autonomous nervous system might be one of the major factors involved in the onset, progression, and metastasis, both sympathetically and parasympathetically. The increased activation of the M3 muscarinic acetylcholine receptors (mAChRs) triggers pro-oncogenic mechanisms, especially at a gastric level, through the activation of the Hippo signaling pathway and the increase of the nerve growth factor.In this study, biopsy or postoperative gastric resection pieces have been evaluated by histopathological (HP) and immunohistochemical (IHC) examination in a group of 77 gastric patients and 23 patients without an oncological diagnosis. To quantify the IHC signal, also considering the HP aspect, light microscopy images were obtained.The M3 mAChR expression analysis has been correlated with the different gastric adenocarcinoma differentiation degrees (G1-G3). M3 mAChR presence has been observed also in the non-malignant gastric tissue, but it was significantly increased in the tumor tissue. The highest receptor expression was recorded in patients with a poorly-differentiated (G3) adenocarcinoma, these expressions decreasing with the increase of the differentiation degree towards moderately-differentiated (G2) and well-differentiated (G1).Surgical or pharmacological parasympathetic activity inhibition could decrease the development and progression of gastric tumors and could improve the gastric cancer patient's prognosis.

Whether 7,8-dihydroxyflavone (7,8-DHF), a tyrosine kinase receptor B (TrkB) agonist, modulates colonic smooth muscle motility and/or alleviates constipation has not yet been studied. Here, we aimed to determine how 7,8-DHF influences carbachol (CCh)-stimulated contraction of colonic strips and the in vivo effect of 7,8-DHF on constipation. Muscle strips were isolated from rat colons for recording contractile tension and performing western blotting. Constipation was induced in rats with loperamide. Although it specifically activated TrkB, 7,8-DHF applied alone neither activated PLCγ1 in the colonic strips nor induced colonic strip contraction. However, 7,8-DHF enhanced CCh-stimulated PLCγ1 activation and strip contraction. The PLCγ1 antagonist U73122 suppressed both CCh-stimulated and 7,8-DHF-enhanced/CCh-stimulated contraction. While clarifying the underlying mechanism, we revealed that 7,8-DHF augmented muscarinic M3 receptor expression in the colonic strips. The M3-selective antagonist tarafenacin specifically inhibited the 7,8-DHF-enhanced/CCh-stimulated contraction of the colonic strips. Since 7,8-DHF increased Akt phosphorylation, and LY294002 (an antagonist of PI3K upstream of Akt) dramatically inhibited both 7,8-DHF-augmented M3 expression and 7,8-DHF-enhanced/CCh-stimulated contractions, we assumed that 7,8-DHF/TrkB/Akt was associated with the modulation of M3 expression in the colonic strips. ANA-12, a specific TrkB antagonist, not only inhibited TrkB activation by 7,8-DHF but also suppressed 7,8-DHF-enhanced cholinergic contraction, 7,8-DHF/CCh-mediated activation of PLCγ1/Akt, and M3 overexpression in colonic strips. In vivo 7,8-DHF, also by promoting intestinal motility and M3 expression, significantly alleviated loperamide-induced functional constipation in rats. Our results suggest that 7,8-DHF regulates colonic motility possibly via a TrkB/Akt/M3 pathway and may be applicable for alleviating constipation.

Objectives Pancreatic acinar necrosis is a typical feature in the early phase of severe acute pancreatitis (SAP). Muscarinic acetylcholine receptor M3 (CHRM3) has been reported to play important roles in promoting insulin secretion and tumor cell proliferation, but its effect on necrosis remains unknown. This study revealed the important role of CHRM3 in regulating L-arginine-induced SAP and the molecular mechanisms. Methods To verify the function of CHRM3, pancreatic tissues and primary acinar cells of CRISPR/Cas9-mediated Chrm3 knockout mice were used in CHRM3 knockdown experiments, and to ascertain the CHRM3 overexpression, PLV-EGFP-Chrm3 plasmids were transfected in acinar cells in vitro. Results In L-arginine-induced SAP, CHRM3 is activated and regulates SAP through the mitogen-activated protein kinase/p38 pathway. Moreover, the expression of miR-31-5p decreased in the SAP model both in vitro and in vivo. Mir-31-5p effects the necrosis of acinar cells in SAP by upregulating the target gene RIP3, and miR-31-5p is a downstream miRNA of CHRM3. Conclusions Necrosis in L-arginine-induced SAP is promoted by CHRM3 through the mitogen-activated protein kinase-p38/miR-31-5p/RIP3 axis.

Lower urinary tract symptoms (LUTS) induced by anticholinergic drug action impair the QOL of patients and are associated with a poor prognosis. Therefore, it is expedient to develop methods of predicting the anticholinergic side effects of drugs, which we aimed to achieve in this study using a quantitative structure-activity relationship (QSAR) and docking study with molecular operations environment (MOE; Molecular Simulation Informatics Systems [MOLSIS], Inc.) In the QSAR simulation, the QSAR model built using the partial least squares regression (PLS) and genetic algorithm-multiple linear regression (GA-MLR) methods showed remarkable coefficient of determination (R2) and XR2 values. In the docking study, a specific relationship was identified between the adjusted docking score (-S) and bioactivity (pKi) values. In conclusion, the methods developed could be useful for in silico risk assessment of LUTS, and plans are potentially applicable to numerous drugs with anticholinergic activity that induce serious side effects, limiting their use.

Pulsatile insulin from pancreatic islets is crucial for glucose homeostasis, but the mechanism behind coordinated pulsatility is still under investigation. One hypothesis suggests that cholinergic stimulation of islets by pancreatic ganglia resets these endocrine units, producing synchronization. Previously, it was shown that intracellular Ca2+ oscillations within islets can be entrained by pulses of a cholinergic agonist, carbachol (CCh). Although these proxy measurements of Ca2+ provided insight into the synchronization mechanism, measurement of insulin output would be more direct evidence. To this end, a fluorescence anisotropy competitive immunoassay for online insulin detection from single and grouped islets in a microfluidic system was developed using a piezoelectric pressure-driven fluid delivery system and a squaraine rotaxane fluorophore, SeTau-647, as the fluorescent label for insulin. Due to SeTau-647 having a longer lifetime and higher brightness compared to the previously used Cy5 fluorophore, a 45% increase in the anisotropy range was observed with enhanced signal-to-noise ratio (S/N) of the measurements. This new system was tested by measuring glucose-stimulated insulin secretion from single and groups of murine and human islets. Distinct islet entrainment of groups of murine islets by pulses of CCh was also observed, providing further evidence for the hypothesis that pulsatile output from the ganglia can synchronize islet behavior. We expect that this relatively straightforward, homogeneous assay can be widely used for examining not only insulin secretion but other secreted factors from different tissues.

Background Postoperative nausea and vomiting (PONV) is the most frequent side effect following anaesthesia. Predisposition to developing PONV is multifactorial with patient risk factors and anaesthetic techniques both being contributory. However, there is also a genetic susceptibility to PONV, and several studies have aimed to identify polymorphisms contributing to a genetic PONV risk. Objective We summarised previous published studies investigating genetic contribution to PONV risk. Design Systematic review without meta-analysis. Data source We searched MEDLINE until June 2019. Eligibility criteria Articles were chosen for review when PONV and polymorphisms were included. Exclusion criteria were reviews/meta-analysis/comments, articles not in the English language, nonappropriate content (e.g. PONV not as primary aim of the study, study investigated opioid-induced nausea) or if articles were pharmacogenetic studies addressing treatment of PONV. Results A total of 59 studies were screened and 14 articles were reviewed including one genome-wide association study (GWAS). Seven studies were performed in East Asians, and seven in Caucasians. Seventeen polymorphisms have been positively associated with PONV in at least one study. Allele frequency of the investigated polymorphisms differs widely between the ethnicities. Furthermore, the anaesthesia regimen and the postoperative time point at which the association with PONV was reported were quite different. Only two polymorphisms, the CHRM3 rs2165870 and the KCNB2 rs349358 (both first associated with PONV in a GWAS), have been significantly associated with PONV incidence in Caucasians in independent studies. Conclusion There is a genetic susceptibility to the development of PONV. Two single nucleotide polymorphisms (SNPs), the CHRM3 rs2165870 and the KCNB2 rs349358 SNP, seem to have a major influence on PONV incidence, at least in Caucasians. Both SNPs were primarily identified in a GWAS and this association may lead to a better understanding of the disease aetiology. Further high-quality studies are needed to reveal more insights in genetic PONV susceptibility, particularly so in non-Caucasian ethnicities.

A series of novel 1,4-dioxane analogues of the muscarinic acetylcholine receptor (mAChR) antagonist 2 was synthesized and studied for their affinity at M1–M5 mAChRs. The 6-cyclohexyl-6-phenyl derivative 3b, with a cis configuration between the CH2N+(CH3)3 chain in the 2-position and the cyclohexyl moiety in the 6-position, showed pKi values for mAChRs higher than those of 2 and a selectivity profile analogous to that of the clinically approved drug oxybutynin. The study of the enantiomers of 3b and the corresponding tertiary amine 33b revealed that the eutomers are (2S,6S)-(−)-3b and (2S,6S)-(−)-33b, respectively. Docking simulations on the M3 mAChR-resolved structure rationalized the experimental observations. The quaternary ammonium function, which should prevent the crossing of the blood–brain barrier, and the high M3/M2 selectivity, which might limit cardiovascular side effects, make 3b a valuable starting point for the design of novel antagonists potentially useful in peripheral diseases in which M3 receptors are involved.

Background Berardinelli-Seip congenital lipodystrophy (BSCL) is a heterogeneous autosomal recessive disorder characterized by an almost total lack of adipose tissue in the body. Mutations in the AGPAT2, BSCL2, CAV1 and PTRF genes define I-IV subtype of BSLC respectively and clinical data indicate that new causative genes remain to be discovered. Here, we retrieved 341 cases from 60 BSCL-related studies worldwide and aimed to explore genotype-phenotype correlations based on mutations of AGPAT2 and BSCL2 genes from 251 cases. We also inferred new candidate genes for BSCL through protein-protein interaction and phenotype-similarity. Results Analysis results show that BSCL type II with earlier age of onset of diabetes mellitus, higher risk to suffer from premature death and mental retardation, is a more severe disorder than BSCL type I, but BSCL type I patients are more likely to have bone cysts. In BSCL type I, females are at higher risk of developing diabetes mellitus and acanthosis nigricans than males, while in BSCL type II, males suffer from diabetes mellitus earlier than females. In addition, some significant correlations among BSCL-related phenotypes were identified. New candidate genes prediction through protein-protein interaction and phenotype-similarity was conducted and we found that CAV3, EBP, SNAP29, HK1, CHRM3, OBSL1 and DNAJC13 genes could be the pathogenic factors for BSCL. Particularly, CAV3 and EBP could be high-priority candidate genes contributing to pathogenesis of BSCL. Conclusions Our study largely enhances the current knowledge of phenotypic and genotypic heterogeneity of BSCL and promotes the more comprehensive understanding of pathogenic mechanisms for BSCL.

This study analyzed the effect of inflammation on acetylcholine (ACh)-induced muscarinic receptors (MR)2 and MR3 conducted contractility of the porcine uterus. On Day 3 of the estrous cycle, either E.coli suspension (E.coli group) or saline (SAL group) was injected into uterine horns or laparotomy was performed (CON group). Eight days later, infected gilts developed severe acute endometritis. Compared to the period before ACh treatment, ACh (10−5 M) increased the tension in myometrium (MYO) and endometrium/myometrium (ENDO/MYO) of the CON group (P 0.05) the tension. ACh (10−5 M) in ENDO/MYO of the E.coli group, reduced the tension compared to the CON group (P 0.05) the tension, but it was lower (P

OBJECTIVES Blood pressure (BP) is comparable among different mammalian orders, despite their evolution divergence. Because of it, fundamental mechanisms should connect humans and rodents by their shared BP physiology. We hypothesized that similar quantitative trait loci (QTLs) function in both humans and rodents in controlling BP. METHODS We utilized inbred hypertensive Dahl salt-sensitive rats (DSS) as a functional proxy to evaluate the relevance of human genome-wide association studies (GWAS) genes in BP regulation. RESULTS First, three DSS BP QTLs functionally captured three specific human GWAS genes. Each QTL has a major biological impact, not a miniscule effect, on BP, in causation by function. Second, noncoding single-nucleotide polymorphisms (SNPs) found in GWAS are by products of primate evolution, instead of mechanistic drivers in regulating BP, because their absence did not impact on BP of mammals. Third, a missense mutation, rather than a noncoding GWAS SNP marking it nearby, is the priority functional basis for a given QTL. Depleting such a noncoding GWAS SNP had no impact, whereas eliminating the muscarinic cholinergic receptor 3 (M3R) signaling decreased BP. Finally, epistatic modularity biologically organizes multiple QTLs with redundant functions, and is the genetic mechanism that modulates the BP homeostasis when QTLs function collectively. CONCLUSIONS Two pathogenic pathways of hypertension biologically unify mechanisms of BP regulations for humans and their functional surrogates. The mechanism-based biology for the M3R-mediated pathway in raising BP has established M3R as a novel pathogenesis-driven target for antihypertension therapies.

Eosinophils mediate airway hyperresponsiveness by increasing vagally mediated reflex bronchoconstriction. Here, we tested whether circulating or airway eosinophils change nerve function. Airway resistance in response to aerosolized 5-hydroxytryptamine (5-HT, 10–300 mM) was measured in wild-type mice or transgenic mice that overexpress IL5 in T cells (+IL5T), overexpress IL5 in airway epithelium (+IL5AE), or overexpress IL5 but are devoid of eosinophils (+IL5AE/−Eos). Inflammatory cells in bronchoalveolar lavage (BAL), blood, and bone marrow were quantified. Blood eosinophils were increased in +IL5T and +IL5AE mice compared with wild-type mice. +IL5T mice had increased eosinophils in bone marrow while +IL5AE mice had increased eosinophils in BAL. Eosinophils surrounding large airways were significantly increased only in +IL5AE mice. With intact vagal innervation, aerosolized 5-HT significantly increased airway resistance in +IL5AE mice. 5-HT-induced bronchoconstriction was blocked by vagotomy or atropine, demonstrating that it was mediated via a vagal reflex. Airway resistance was not increased in +IL5AE/−Eos mice, demonstrating that it required lung eosinophils, but was not affected by increased bone marrow or blood eosinophils or by increased IL5 in the absence of eosinophils. Eosinophils did not change M3 function on airway smooth muscle, since airway responses to methacholine in vagotomized mice were not different among strains. Eosinophils surrounding large airways were sufficient, even in the absence of increased IL5 or external insult, to increase vagally mediated reflex bronchoconstriction. Specifically blocking or reducing eosinophils surrounding large airways may effectively inhibit reflex hyperresponsiveness mediated by vagus nerves in eosinophilic asthma.

The objective of this study was to explore the effect of Alpiniae oxyphyllae Fructus (AOF) on a rat model of chronic intermittent hypoxia (CIH)–induced enuresis. Findings of this study may help identify therapeutic targets in children with nocturnal enuresis (NE). Female rats were randomly divided into a control group (saline gavage, 4 weeks of normal air), CIH group (saline gavage, 4 weeks of CIH), and AOF group (AOF gavage, 4 weeks of CIH). The variables measured in this study included water intake, urine output, bladder leak point pressure (BLPP), malondialdehyde (MDA) levels, and superoxide dismutase (SOD) activity. The expression levels of the purinergic P2X3 receptor, muscarinic M3 receptor, and s3-adrenergic receptor (s3-AR) in the bladder were also measured. The bladder was subjected to haematoxylin and eosin (HE) and Weigert staining, and histological changes were observed under a light microscope to evaluate the morphological changes in the bladder in each group. Compared with the control group, urine output was increased, and the BLPP was decreased in the CIH group, but AOF administration decreased urine output and increased BLPP. In addition, the serum MDA level increased and the SOD activity decreased in the CIH group compared with the control group. Administration of AOF decreased the MDA level and increased the SOD activity. Additionally, compared with the control group, HE and Weigert staining in the CIH group showed that the bladder detrusor muscle bundles were disordered and loose, some muscle bundles were broken, the content of collagen fibres in the gap was reduced, and the gap was significantly widened. However, following the administration of AOF, the bladder detrusor muscle bundles were neatly arranged, and the content of collagen fibres in the gap was increased. Furthermore, compared with the control group, the purinergic P2X3 receptor and muscarinic M3 receptor were expressed at higher levels, and s3-AR was expressed at lower levels in the CIH group, but AOF administration decreased the expression of the purinergic P2X3 receptor and muscarinic M3 receptor and increased the expression of the s3-AR. AOF improves enuresis by inhibiting oxidative stress and regulating the expression of the purinergic P2X3 receptor, muscarinic M3 receptor, and s3 adrenergic receptor.

Discovery of an anticancer medicine using a single target protein has often been unsuccessful due to the complexity of pathogenic mechanisms as well as the presence of redundant signaling pathways. In this work, we attempted to find promising anticancer drug candidates by simultaneously targeting casein kinase 1 delta (CK1δ) and muscarinic acetylcholine receptor M3 (M3R). Through the structure-based virtual screening and de novo design with the modified potential function for protein-ligand binding, a series of benzo[4,5]imidazo[1,2-a][1,3,5]triazine-2-amine (BITA) derivatives were identified as CK1δ inhibitors and also as M3R antagonists. The biochemical potencies of these bifunctional molecules reached the nanomolar and low-micromolar levels with respect to CK1δ and M3R, respectively. A common interaction feature in the calculated CK1δ-inhibitor and M3R-antagonist complexes is that the BITA moiety is well-stabilized in the orthosteric site of M3R and the hinge region of CK1δ through the establishment of the three hydrogen bonds and the hydrophobic contacts in the vicinity. The computational and experimental results found in this work exemplify the efficiency of kinase and GPCR polypharmacology in developing anticancer medicines.

Muscarinic MModulation of carbachol-induced increases in intracellular CaASP8302 demonstrated PAM effects on the rat MOur results in rats indicate that ASP8302 improves voiding dysfunction by potentiating bladder contraction with fewer effects on cholinergic responses in other organs, and suggest a potential advantage over current cholinomimetic drugs for treating micturition disorders caused by insufficient bladder contraction.

Overexpression of M3 subtype muscarinic receptors (M3R) hastens colon cancer progression. As microRNA (miRNA) expression is commonly dysregulated in cancer, we used microarrays to examine miRNA profiles in muscarinic receptor agonist-treated human colon cancer cells. We used quantitative RT-PCR (qPCR) to validate microarray results and examine miRNA expression in colon cancers and adjacent normal colon. These assays revealed that acetylcholine (ACh) treatment robustly induced miR-222 expression; miR-222 levels were three-fold higher in cancer compared to normal colon. In kinetic studies, ACh induced a 4.6-fold increase in pri-miR-222 levels within 1 h, while mature miR-222 increased gradually to 1.8-fold within 4 h. To identify post-M3R signaling mediating these actions, we used chemical inhibitors and agonists. ACh-induced increases in pri-miR-222 were attenuated by pre-incubating cells with atropine and inhibitors of protein kinase C (PKC) and p38 MAPK. Treatment with a PKC agonist, phorbol 12-myristate 13-acetate, increased pri-miR-222 levels, an effect blocked by PKC and p38 MAPK inhibitors, but not by atropine. Notably, treatment with ACh or transfection with miR-222 mimics increased cell proliferation; atropine blocked the effects of ACh but not miR-222. These findings identify a novel mechanism whereby post-M3R PKC/p38 MAPK signaling stimulates miR-222 expression and colon cancer cell proliferation.

The mineralocorticoid aldosterone is produced in the adrenal zona glomerulosa (ZG) under the control of the renin–angiotensin II (AngII) system. Primary aldosteronism (PA) results from renin-independent production of aldosterone and is a common cause of hypertension. PA is caused by dysregulated localization of the enzyme aldosterone synthase (Cyp11b2), which is normally restricted to the ZG. Cyp11b2 transcription and aldosterone production are predominantly regulated by AngII activation of the Gq signaling pathway. Here, we report the generation of transgenic mice with Gq-coupled designer receptors exclusively activated by designer drugs (DREADDs) specifically in the adrenal cortex. We show that adrenal-wide ligand activation of Gq DREADD receptors triggered disorganization of adrenal functional zonation, with induction of Cyp11b2 in glucocorticoid-producing zona fasciculata cells. This result was consistent with increased renin-independent aldosterone production and hypertension. All parameters were reversible following termination of DREADD-mediated Gq signaling. These findings demonstrate that Gq signaling is sufficient for adrenocortical aldosterone production and implicate this pathway in the determination of zone-specific steroid production within the adrenal cortex. This transgenic mouse also provides an inducible and reversible model of hyperaldosteronism to investigate PA therapeutics and the mechanisms leading to the damaging effects of aldosterone on the cardiovascular system.

Here, we investigate the effects of exercise training on glucose- and cholinergic-induced insulin secretion in pancreatic islets from obese and lean rats. Male Wistar rats were treated with monosodium glutamate (MSG) for the first 5 days of life, while control (CON) rats received saline. At 21 days, the rats were divided into exercised (EXE) and sedentary (SED) groups. The EXE rats swam for 30 minutes, three times/week, for 10 weeks. After this, MSG-SED rats showed hyperglycaemia, hypertriglyceridaemia and hyperinsulinaemia. Besides, islets from MSG-SED rats exhibited increased glucose-stimulated insulin secretion (GSIS), followed by impaired glucose sensitivity, absence of glucose-amplifying pathway and weak cholinergic response. In contrast, adiposity, hyperinsulinaemia and hypertriglyceridaemia were reduced in MSG-EXE rats. Moreover, islets from MSG-EXE rats exhibited lower GSIS and improved islet glucose sensitivity, without restoration of the glucose-amplifying pathway or alteration in the weak cholinergic effect of these islets. In islets from CON-EXE rats we also observed reduced GSIS and absence of glucose-amplifying effects and an accentuated reduction in cholinergic insulinotropic responses, without effect on glucose sensitivity in pancreatic islets from this group. Neither obesity nor exercise modified Muscarinic Receptor 3 (M3R) immunocontent or its downstream pathways (PKC and PKA). Moreover, only CON-EXE showed increased GSIS in the presence of calcium blocker, Thapsigargin. In conclusion, swimming training reduces GSIS and cholinergic responsiveness in isolated pancreatic islets from lean and hypothalamic obese rats, which could be due to the inhibition of glucose-amplifying pathways.

Acinar cells in acute pancreatitis (AP) die through apoptosis and necrosis, the impacts of which are quite different. Early clinical interference strategies on preventing the progress of AP to severe acute pancreatitis (SAP) are the elimination of inflammation response and inhibition of necrosis. Muscarinic acetylcholine receptor M3 was encoded by Chrm3 gene. It is one of the best-characterized receptors of pancreatic β cells and regulates insulin secretion, but its function in AP remains unclear. In this study, we explored the function of Chrm3 gene in the regulation of cell death in l-arginine-induced SAP animal models. We found that Chrm3 was upregulated in pancreatitis, and we further confirmed the localization of Chrm3 resided in both pancreatic islets and acinar cell membranes. The reduction of Chrm3 decreased the pathological lesion of SAP and reduced amylase activities in serum. Consistently, Chrm3 can suppress acinar cells necrosis markedly, but has no effect on regulating apoptosis after l-arginine treatment. It was shown that Chrm3 attenuated acinar cells necrosis at least in part by stabilizing caspase-8. Thus, this study indicates that Chrm3 is critical participants in SAP, and regulation of Chrm3 expression might be a useful therapeutic strategy for preventing pathologic necrosis.

Background Alzheimer’s disease (AD) as a neurodegenerative disease occupies 3/5–4/5 cases among patients with dementia, yet its pathogenetic mechanism remains unclear. Geraniol, on the other hand, is a well-known extract from essential oils of aromatic plants and has been proven that it has outstanding neuroprotective effects as well as ameliorating influence in memory impairment. Therefore, the present study aims to elucidate the potential of geraniol against AD by network pharmacology-based approach combined with molecular modeling study. Materials and methods Firstly, we evaluated the druggability of geraniol by ADME method. Then, we obtained the geraniol targets and AD-related targets from multiple open data sources. Afterward, we calculated the intersection through a Venn diagram to find common targets, and via Panther classification system to categorize them. In order to gain a macroscopic understanding of these common targets, we carried out GO terms and KEGG pathways enrichment analyses, according to which we constructed a compound–target–pathway–disease network. In addition, we built a preliminary PPI network which was further analyzed both functionally and topologically. Consequently, five hub targets were sorted out. Finally, we conducted molecular docking and molecular dynamic simulation to validate our findings. Results In the present study, the pharmacological properties of geraniol were assessed according to ADME and Lipinski’s rule, which demonstrate promising druggability. Then, from 10,972 AD-related targets and 33 geraniol targets, 29 common targets were identified, among which 38.1% of them are metabolite interconversion enzymes, 23.8% are protein modifying enzymes, 33.3% are transmembrane receptors, and the rest are transporters. Enrichment analyses hint that geraniol is involved in cholinergic synapse, serotonergic synapse, and neuroactive ligand–receptor interaction. We also built a preliminary PPI network to investigate the interplay between these targets and their extensive interactions. Then, by functionally clustering the preliminary PPI network, we gained a cluster of proteins which formed a subnetwork with score of 8.476, and 22 nodes. Its results of GO terms and KEGG pathways enrichment analyses once again suggests that geraniol actively participates in cholinergic synapse, serotonergic synapse, and neuroactive ligand–receptor interaction, which are believed to be strongly associated with AD pathogenesis. Besides, topological analyses of the preliminary PPI network helped find 5 hub targets (i.e., CHRM3, PRKCA, PRKCD, JAK1, JAK2). To verify their interaction with geraniol molecule, we conducted molecular docking, and found that CHRM3 possesses the highest affinity in binding, indicating that geraniol molecules are closely bound to each hub target, and CHRM3 may serve as a key target of geraniol against AD. It was then further confirmed by molecular dynamic simulation, the result of which supports our hypothesis. Conclusion The present study shares a mechanistic insight of the potential of geraniol against AD, giving a reference to future experimental studies.

The M3 muscarinic acetylcholine receptor is predominantly expressed in the basal epidermal layer where it mediates the effects of the autocrine/paracrine cytotransmitter acetylcholine. Patients with the autoimmune blistering disease pemphigus develop autoantibodies to M3 muscarinic acetylcholine receptor and show alterations in keratinocyte adhesion, proliferation, and differentiation, suggesting that M3 muscarinic acetylcholine receptor controls these cellular functions. Chmr3-/- mice display altered epidermal morphology resembling that seen in patients with pemphigus vulgaris. In this study, we characterized the cellular and molecular mechanisms through which M3 muscarinic acetylcholine receptor controls epidermal structure and function. We used single-cell RNA sequencing to evaluate keratinocyte heterogeneity and identify differentially expressed genes in specific subpopulations of epidermal cells in Chmr3-/- neonatal mice. We found that Chmr3-/- mice feature abnormal epidermal morphology characterized by accumulation of nucleated basal cells, shrinkage of basal keratinocytes, and enlargement of intercellular spaces. These morphologic changes were associated with upregulation of cell proliferation genes and downregulation of genes contributing to epidermal differentiation, extracellular matrix formation, intercellular adhesion, and cell arrangement. These findings provide, to our knowledge, previously unreported insights into how acetylcholine controls epidermal differentiation and lay a groundwork for future translational studies evaluating the therapeutic potential of cholinergic drugs in dermatology.

Background: Olanzapine is one of the most commonly used antipsychotic drugs; however, its metabolic disorders are the main obstacle in the clinic. Olanzapine is a potent antagonist of the M3 acetylcholine muscarinic receptor (M3R), while the downregulated hepatic M3R-AMPKα signalling pathway is involved in metabolic disorders. Aim: This study investigated the effects of chronic co-treatment with cevimeline (an agonist of M3Rs) in attenuating olanzapine-induced metabolic disorders and the underlying mechanisms. Methods: Forty-eight adult female Sprague-Dawley rats were treated orally with olanzapine (2 mg/kg, 3 times/day (t.i.d.)) and/or cevimeline (9 mg/kg, t.i.d.), or control (vehicle) for 9 weeks. Results: Cevimeline co-treatment significantly attenuated olanzapine-induced body weight gain and glucolipid metabolic disorders. Importantly, cevimeline co-treatment attenuated olanzapine-induced upregulation of M3Rs, while the co-treatment improved olanzapine-induced downregulation of AMPKα in the liver. Cevimeline co-treatment attenuated olanzapine-induced dyslipidaemia by modulating the hepatic M3R-AMPKα downstream pathways. Cevimeline co-treatment also improved lower activated AKT-GSK3β signalling to reverse impairment of glucose metabolism and insulin resistance caused by chronic olanzapine treatment. Conclusion: These results not only support the important role of M3R antagonism and its related AMPKα and downstream pathways in antipsychotic-induced metabolic disorders but also indicate that these pathways might be promising targets for pharmacological intervention to control these side effects caused by antipsychotic therapy.

Funder: Biotechnology and Biological Sciences Research Council, Acetylcholine release in the hippocampus plays a central role in the formation of new memory representations. An influential but largely untested theory proposes that memory formation requires acetylcholine to enhance responses in CA1 to new sensory information from entorhinal cortex whilst depressing inputs from previously encoded representations in CA3. Here, we show that excitatory inputs from entorhinal cortex and CA3 are depressed equally by synaptic release of acetylcholine in CA1. However, feedforward inhibition from entorhinal cortex exhibits greater depression than CA3 resulting in a selective enhancement of excitatory-inhibitory balance and CA1 activation by entorhinal inputs. Entorhinal and CA3 pathways engage different feedforward interneuron subpopulations and cholinergic modulation of presynaptic function is mediated differentially by muscarinic M3 and M4 receptors, respectively. Thus, our data support a role and mechanisms for acetylcholine to prioritise novel information inputs to CA1 during memory formation.

Assisted reproductive technology (ART) has been used globally among infertile couples. However, many epidemiological investigations have indicated that ART is associated with a range of long-term adverse health outcomes in offspring, including cardiovascular disease, obesity, and increased plasma lipid levels. Until now, direct evidence has been limited regarding the pathological changes in vascular function in fetuses with ART. In this study, human umbilical cords were collected from healthy normal pregnancies and in vitro fertilization and embryo transfer (IVF-ET) pregnancies. Vascular functional studies involving acetylcholine (ACh), antagonists of its specific receptors, and L-type calcium channel/PKC-MLC20 phosphorylation pathway specific inhibitors were conducted. Quantitative real-time PCR, Western blotting, and methylation analyses were performed on umbilical vein samples. We found that the umbilical vein constriction induced by ACh in the IVF-ET group was significantly attenuated compared with that in the healthy normal pregnancy group, which was not only associated with the hypermethylation of ACh muscarinic receptor subtype 3 (CHRM3) and decreased expression of CHRM3, PKCβ, and CaV1.2, but was also related to the reduced phosphorylation of MLC20. This study revealed that the hypermethylation of CHRM3, leading to a reduction in CHRM3 expression and downregulation of the CaV1.2/PKC-MLC20 phosphorylation pathway, was responsible for the decreased sensitivity to ACh observed in the umbilical vein under IVF-ET conditions. The hypermethylation of CHRM3 caused by IVF-ET might play an important role in altered vasoconstriction and impact cardiovascular systems in the long run.

A signaling cascade composing M3, EphB/ephrin-B, and MEK synergizes to guide the homeostasis of intestinal epithelial cell growth and differentiation following modifications of the cholinergic intestinal niche., Acetylcholine (ACh) signaling through activation of nicotinic and muscarinic ACh receptors regulates expression of specific genes that mediate and sustain proliferation, differentiation, and homeostasis in the intestinal crypts. This signaling plays a pivotal role in the regulation of intestinal stem cell function, but the details have not been clarified. Here, we performed experiments using type 3 muscarinic acetylcholine receptor (M3) knockout mice and their intestinal organoids and report that endogenous ACh affects the size of the intestinal stem niche via M3 signaling. RNA sequencing of crypts identified up-regulation of the EphB/ephrin-B signaling pathway. Furthermore, using an MEK inhibitor (U0126), we found that mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling, which is downstream of EphB/ephrin-B signaling, is activated in M3-deficient crypts. Collectively, M3, EphB/ephrin-B, and the MAPK/ERK signaling cascade work together to maintain the homeostasis of intestinal epithelial cell growth and differentiation following modifications of the cholinergic intestinal niche.

Acquired idiopathic generalized anhidrosis (AIGA) is a rare disorder in which systemic anhidrosis/hypohidrosis occurs without causative dermatological, metabolic or neurological disorder. Most cases of AIGA have been reported in Asia, especially in Japan, but there have been only a few reports in Europe and the United States. Severe AIGA may result in heatstroke and can reduce quality of life due to restriction of exercise and outdoor works. AIGA is often accompanied by cholinergic urticaria (CholU), and it is thought that AIGA and CholU with anhidrosis/hypohidrosis belong to the same spectrum of the disease. However, the pathophysiology of AIGA has not yet been clarified. Decreased expression of cholinergic receptor M3 on the epithelial cells of eccrine sweat glands is often accompanied by T cell infiltration around eccrine apparatus, suggesting an immunological mechanism of disordered perspiration. AIGA is occasionally associated with various complications indicative of autoimmune disorders. The association of autoimmune complications further suggests that AIGA is an autoimmune disorder. Studies on complications may lead to a better understanding of the pathophysiology of AIGA.

In this paper, we report the discovery of dual M

The parasympathetic nervous system is critically involved in the regulation of tear secretion by activating muscarinic acetylcholine receptors. Hence, various animal models targeting parasympathetic signaling have been developed to induce dry eye disease (DED). However, the muscarinic receptor subtype (M1–M5) mediating tear secretion remains to be determined. This study was conducted to test the hypothesis that the M3 receptor subtype regulates tear secretion and to evaluate the ocular surface phenotype of mice with targeted disruption of the M3 receptor (M3R−/−). The experimental techniques included quantification of tear production, fluorescein staining of the ocular surface, environmental scanning electron microscopy, assessment of proliferating cells in the corneal epithelium and of goblet cells in the conjunctiva, quantification of mRNA for inflammatory cytokines and prooxidant redox enzymes and quantification of reactive oxygen species. Tear volume was reduced in M3R−/− mice compared to age-matched controls at the age of 3 months and 15 months, respectively. This was associated with mild corneal epitheliopathy in the 15-month-old but not in the 3-month-old M3R−/− mice. M3R−/− mice at the age of 15 months also displayed changes in corneal epithelial cell texture, reduced conjunctival goblet cell density, oxidative stress and elevated mRNA expression levels for inflammatory cytokines and prooxidant redox enzymes. The findings suggest that the M3 receptor plays a pivotal role in tear production and its absence leads to ocular surface changes typical for DED at advanced age.

Metformin is an antidiabetic drug used for the treatment of diabetes and metabolic diseases. Imbalance in the autonomic nervous system (ANS) is associated with metabolic diseases. This study aimed to test whether metformin could improve ANS function in obese rats. Obesity was induced by neonatal treatment with monosodium L-glutamate (MSG). During 21–100 days of age, MSG-rats were treated with metformin 250 mg/kg body weight/day or saline solution. Rats were euthanized to evaluate biometric and biochemical parameters. ANS electrical activity was recorded and analyzed. Metformin normalized the hypervagal response in MSG-rats. Glucose-stimulated insulin secretion in isolated pancreatic islets increased in MSG-rats, while the cholinergic response decreased. Metformin treatment normalized the cholinergic response, which involved mostly the M3 muscarinic acetylcholine receptor (M3 mAChR) in pancreatic beta-cells. Protein expression of M3 mAChRs increased in MSG-obesity rats, while metformin treatment decreased the protein expression by 25%. In conclusion, chronic metformin treatment was effective in normalizing ANS activity and alleviating obesity in MSG-rats.

Long‑term hypertension leads to alterations in the structure and function of blood vessels, and abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are important factors for these changes. Linalool is a natural compound extracted from plants. The present study aimed to explore the role and underlying mechanism of linalool in the physiological behavior of VSMCs. Angiotensin II (Ang II) was utilized to treat VSMCs, and MTT and western blotting assays were then employed to detect the effect of linalool on the induced proliferation and migration of VSMCs. The target gene of linalool was predicted by the SwissTargetPrediction website, and its expression level in VSMCs was determined using reverse transcription‑quantitative PCR and western blotting. Next, the role of the target gene in the physiological behavior of VSMCs treated with linalool was examined, and the signaling pathway was explored. The results revealed that the proliferation and migration of VSMCs treated with Ang II were significantly promoted, and linalool could alleviate these effects in a dose‑dependent manner. Cholinergic receptor muscarinic 3 (CHRM3), as a predicted target, was found to be highly expressed in Ang II‑induced VSMCs, and CHRM3 overexpression could prevent the inhibitory effect of linalool on cell proliferation and migration. In addition, its overexpression caused an increase in the expression of proteins related to the MAPK signaling pathway. In conclusion, linalool inhibited the proliferation and migration of Ang II‑induced VSMCs and blocked the MAPK signaling pathway by downregulating CHRM3.

Numerous human clinical studies have suggested that decreased locomotor activity is a common symptom of major depressive disorder (MDD), as well as other psychiatric diseases. In MDD, the midbrain ventral tegmental area (VTA) dopamine (DA) neurons are closely related to regulate the information processing of reward, motivation, cognition, and aversion. However, the neural circuit mechanism that underlie the relationship between VTA-DA neurons and MDD-related motor impairments, especially hypolocomotion, is still largely unknown. Herein, we investigate how the VTA-DA neurons contribute to the hypolocomotion performance in chronic social defeat stress (CSDS), a mouse model of depression-relevant neurobehavioral states. The results show that CSDS could affect the spontaneous locomotor activity of mice, but not the grip strength and forced locomotor ability. Chemogenetic activation of VTA-DA neurons alleviated CSDS-induced hypolocomotion. Subsequently, quantitative whole-brain mapping revealed decreased projections from VTA-DA neurons to substantia nigra pars reticulata (SNr) after CSDS treatment. Optogenetic activation of dopaminergic projection from VTA to SNr with the stimulation of phasic firing, but not tonic firing, could significantly increase the locomotor activity of mice. Moreover, chemogenetic activation of VTA-SNr dopaminergic circuit in CSDS mice could also rescued the decline of locomotor activity. Taken together, our data suggest that the VTA-SNr dopaminergic projection mediates CSDS-induced hypolocomotion, which provides a theoretical basis and potential therapeutic target for MDD.

Muscarinic M

Aim: Food intake regulated by a complex of physiologic mechanisms in the nervous system. Muscarinergic system has an important role in the central regulation of appetite in mammals, but the...

Rationale. We previously demonstrated increased expression of programmed cell death 5 (PDCD5) in asthmatic patients and ovalbumin-induced allergic asthma. International guidelines (GINA 2019) have included the use of tiotropium bromide for chronic treatment of the most severe and frequently exacerbated asthma in patients ≥6 years old, who do not have good response to inhaled corticosteroids. Objective. To explore the role of tiotropium and its effect on PDCD5 level in a mouse model of chronic asthma. Methods. We divided 12 female mice into 2 groups: untreated asthma (n = 6) and tiotropium-treated asthma (n = 6). The impact of tiotropium was assessed by histology of lung tissue and morphometry. Pulmonary function was tested by using pressure sensors. The number of cells in bronchoalveolar lavage fluid (BALF) was detected. Levels of PDCD5, active caspase-3, and muscarinic acetylcholine receptors M2 (ChRM2) and M3 (ChRM3) were examined. Results. Tiotropium treatment significantly reduced airway inflammation and remodeling in asthmatic mice and intensified the lung function. PDCD5 level was reduced with tiotropium (p<0.05). Moreover, active caspase-3 level was decreased with tiotropium (p<0.001), and ChRM3 level was increased. Conclusions. Tiotropium treatment may alleviate the pathological changes with asthma by regulating apoptosis.

CHRM3 codes for the M3 muscarinic acetylcholine receptor that is located on the surface of smooth muscle cells of the detrusor, the muscle that effects urinary voiding. Previously, we reported brothers in a family affected by a congenital prune belly‐like syndrome with mydriasis due to homozygous CHRM3 frameshift variants. In this study, we describe two sisters with bladders that failed to empty completely and pupils that failed to constrict fully in response to light, who are homozygous for the missense CHRM3 variant c.352G > A; p.(Gly118Arg). Samples were not available for genotyping from their brother, who had a history of multiple urinary tract infections and underwent surgical bladder draining in the first year of life. He died at the age of 6 years. This is the first independent report of biallelic variants in CHRM3 in a family with a rare serious bladder disorder associated with mydriasis and provides important evidence of this association.