Bioinformatic Analysis on the Prognostic Value of Neurotransmitter Receptor-Related Genes in Kidney Renal Clear Cell Carcinoma.

Context: Kidney renal clear-cell carcinoma (KIRC) is a malignant tumor. At an early stage, KIRC patients may experience only mild fever and fatigue or even no symptoms, and these early nonspecific indications can delay treatment. Neurotransmitters and their receptors may be very useful in determining tumorigenesis and predicting metastasis. Objective: The study intended to investigate the predictive value of neurotransmitter receptor-related genes (NRRGs) using public KIRC data, by determining the biological processes that implicate the prognostic NRRGs and establishing a predictive NR-related risk model, to provide an empirical basis for identifying and treating KIRC patients. Design: The research team performed a genetic case-control study. Setting: The study took place at Research Center of Health, Big Data Mining and Applications, Wannan Medical College, Wuhu, China. Methods: The research team: (1) obtained the transcriptome data related to KIRC from the Cancer Genome Atlas (TCGA) and ArrayExpress databases; (2) developed the differentially expressed NRRGs (DENRRGs) by identifying the NRRGs that intersected with DEGs in KIRC and normal samples; (3) carried out functional enrichment analyses of the DENRRGs; (4) screened the characteristic genes of the DENRRGs using machine learning; (5) created a predictive model using multivariate Cox analyses of the distinctive genes; (6) obtained independent prognostic factors for KIRC patients and established a nomograph model; (7) investigated the sensitivity of KIRC patients to therapeutic agents to examine the variations in immunological features between high-risk and low-risk individuals. Results: Differential analysis found that 115 NRRGs intersected with 5275 DEGs to provide 52 DENRRGs. Functional enrichment showed that DENRRGs were mainly involved in signal transduction in the nervous system. The machine learning on the 52 DENRRGs filtered out nine characteristic genes. Subsequently, the research team found eight prognostic biomarkers-histamine receptor H2 (HRH2), gamma-aminobutyric acid (GABA) receptor subunit epsilon (GABRE), cholinergic receptor nicotinic delta subunit (CHRND), glutamate receptor ionotropic subunit 2D (GRIN2D), glutamate metabotropic receptor 4 (GRM4), glycine receptor alpha 3 (GLRA3), cholinergic receptor nicotinic beta 4 subunit (CHRNB4), and cholinergic receptor muscarinic-1 (CHRM1)-and established a predictive model. Furthermore, the team precisely predicted the KIRC patients' prognoses using a nomogram that combined their ages, risk scores, and M stages. The infiltration levels of 21 immune cells also significantly differed between the high-risk and low-risk groups, with neutrophils having a significant positive correlation with GABRE and HRH2 and a significant negative correlation with CHRNB4 and GRM4. Finally, the 50% inhibitory concentration (IC50) values for various drugs, such as 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR), 8-hydroxy-7-(6-sulfonaphthalen-2-yl)diazenyl-quinoline-5-sulfonic acid (NSC-87877), Sunitinib, c-Jun N-terminal kinase (JNK) inhibitor VIII, and tanespimyci (X17.AAG) were significantly lower for high-risk group. Conclusions: By studying the relevance of biomarkers to the immunological microenvironment of KIRC, the current research team was able to propose a new predictive model for KIRC based on NRRGs, to offer a novel viewpoint for investigating KIRC. The study's results suggest new avenues for research into the pathophysiology and therapy of KIRC. Determining the precise molecular processes by which predictive biomarkers regulate KIRC requires further evidence and analysis.

The Mechanism of Crocetin Targeting Cardiovascular Disease Based on Network Pharmacology Constrained by Spectral Experiments.

The network pharmacology under conditions is a recent development trend. We use network pharmacology methods to analyze the mechanism of crocetin (CRO) that regulates cardiovascular diseases. In this work, the spectral experimental data of CRO-Protein interaction is first time combined with constraint conditions to solve the problems of targeting redundancy and lack of verification. CRO targets and cardiovascular disease targets were obtained by the target database. The STRING platform was used for PPI analysis. The GO and KEGG pathways of the target were analyzed using the Metascape platform; The core functional targets of CRO were screened by molecular docking techniques and the spectra of CRO and human serum albumin (HSA). Under the collaborative constraint conditions, the core targets of CRO that regulate cardiovascular diseases are ADRA1A, ADRA1B, CHRM1, CHRM2, GABRA1, and PTGS2; This study incorporates spectroscopy and molecular docking as constraints into the network pharmacological analysis, which significantly improves the credibility of network pharmacological analysis compared with unconstrained conditions. This method provides theoretical references for the in-depth study of the mechanism between active substances and protein targets for other medicines in network pharmacology.

Study on the mechanism of Qiju Dihuang pill in the treatment of ophthalmic diseases based on systems pharmacology.

Qiju Dihuang pill is one of the common Traditional Chinese Medicine to treat ophthalmic diseases. In vivo studies have suggested that Qiju Dihuang pill can be used for treating glaucoma, and it can also be used clinically to treat cataract patients. However, the bioactive ingredients and the therapeutic mechanism of Qiju Dihuang pill on treating these ophthalmic diseases remained unclear. Presently, a systems pharmacology approach which combines pharmacokinetic screening, targeted fishing, biological function enrichment, network pharmacology, and molecular docking analysis, was employed. A total of 134 active ingredients with 72 corresponding targets are identified from Qiju Dihuang pill. Additionally, 3 core targets including CHRM1, ESR1, and AR are obtained from the ingredients and drug targets network analysis. Besides, gen ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis reveal 3 important biological pathways, that is, calcium signaling pathway, insulin signaling pathway and Vascular endothelial growth factor signaling pathway. In final, a molecular docking model was constructed to study the interaction mechanism between active components and drug targets at the molecular level. All the findings show that Qiju Dihuang pill achieves therapeutic effects on treating ophthalmic diseases by regulating the crucial targets of the compounds in it. This work not only provides insight into the therapeutic mechanism of herbal medicine in the treatment of ophthalmic diseases from a multiscale perspective, but also offers an effective approach for drug discovery and development of Traditional Chinese Medicine.

Reimagining cholinergic therapy for Alzheimer's disease.

Currently, enhancement of cholinergic neurotransmission via cholinesterase inhibitors represents the main available approach to treat cognitive and behavioural symptoms of the early as well as late stages of Alzheimer's disease. Restoring the cholinergic system has been a primary means of improving cognition in Alzheimer's disease, as four of the six approved therapies are acetylcholinesterase inhibitors. Memantine is an N-methyl-d-aspartate antagonist with a well-documented clinical effect on behavioural symptoms, which is often added to cholinesterase inhibitors to potentiate their effect and aducanumab, targeting the amyloid pathology, has recently been approved. The early, progressive and selective degeneration of the cholinergic system together and its close relation to cognitive deficits supports the use of cholinergic therapy for Alzheimer's disease. This review provides an updated view of the basal forebrain cholinergic system, its relation to cognition and its relevance for therapy of Alzheimer's disease. It deals with the three main aspects that form the basis of the cholinergic-oriented therapy of Alzheimer's disease, its origin, its mechanism of action, its clinical effects, advantages and limits of a cholinergic therapeutic approach. It includes a new and updated overview of the involvement of muscarinic receptors in Alzheimer's disease as well as the recent development of new and highly selective M1 muscarinic receptor agonists with disease-modifying potential. It also addresses the discovery of a novel nerve growth factor metabolic pathway responsible for the trophic maintenance of the basal forebrain system and its deregulation in Alzheimer's disease. It discusses new clinical studies and provides evidence for the long-term efficacy of cholinesterase inhibitor therapy suggesting a disease-modifying effect of these drugs. The classical symptomatic cholinergic therapy based on cholinesterase inhibitors is judiciously discussed for its maximal efficacy and best clinical application. The review proposes new alternatives of cholinergic therapy that should be developed to amplify its clinical effect and supplement the disease-modifying effect of new treatments to slow down or arrest disease progression.

Magnolol upregulates CHRM1 to attenuate Amyloid-ß-triggered neuronal injury through regulating the cAMP/PKA/CREB pathway.

Alzheimer's disease (AD) is a common neurodegenerative disease characterized by neuronal degeneration and hyperphosphorylated Tau. Magnolol is an active component isolated from Magnolia officinalis with potential neuroprotection activity. However, the function and mechanism of magnolol in AD progression is largely uncertain. In present study, the biomarkers related to AD and magnolol were predicted by bioinformatics analyses. The key biomarker levels were predicted by GSE5281 and GSE36980 using AlzData. Cell viability was detected by CCK-8 assay. mRNA and protein levels were examined by qRT-PCR and western blotting assays. Cell apoptosis was investigated by caspase-3 activity and flow cytometry analyses. The cAMP/PKA/CREB signaling was evaluated by ELISA and western blotting analyses. The results showed that CHRM1 was a key biomarker for magnolol against AD progression. Magnolol attenuated Aß-induced viability inhibition, Tau hyperphosphorylation and apoptosis in SH-SY5Y cells by upregulating CHRM1. In addition, the cAMP signaling might be a potential pathway of CHRM1 in AD. Magnolol contributed to activation of the cAMP/PKA/CREB pathway through enhancing CHRM1 level. Inactivation of the cAMP/PKA/CREB signaling reversed the suppressive effect of magnolol on Tau hyperphosphorylation and apoptosis in Aß-treated SH-SY5Y cells. As a conclusion, magnolol mitigated Aß-induced Tau hyperphosphorylation and neuron apoptosis by upregulating CHRM1 and activating the cAMP/PKA/CREB pathway.

[Mechanism of Magnoliae Officinalis Cortex in treatment of peptic ulcer based on network pharmacology and molecular docking].

Magnoliae Officinalis Cortex(Houpo) can treat peptic ulcer disease(PUD), the mechanism of which remains unclear. In this study, network pharmacology and molecular docking were employed to predict the mechanism of Houpo in the treatment of PUD. Through literature review and TCMSP screening, 15 main active ingredients were obtained. The SwissTargetPrediction database was used to predict the potential targets of the ingredients, and Therapeutic Target Database(TTD), DrugBank, and Human Phenotype Ontology(HPO) to screen the disease-related targets. A total of 49 potential targets were obtained by the intersection of active ingre-dients-related targets and disease-related targets. Cytoscape 3.6.1 was employed to construct the protein-protein interaction network for the targets with high confidence(score>0.700) screened out by STRING. The DAVID database was used for GO and KEGG pathway enrichment of potential targets. GO enrichment analysis showed that the treatment mechanism was mostly related to nuclear receptor activity, ligand-activated transcription factor activity, and G protein-coupled acetylcholine receptor activity. KEGG enrichment analysis found that Houpo could regulate material metabolism, endocrine system, p53 signaling pathway, and PPAR signaling pathway. Molecu-lar docking verified that all 15 ingredients had good binding activities with key targets(CHRM1, CHRM2, FABP1, mTOR, and STAT3). The results mean that Houpo can treat PUD by participating in cell metabolism, inhibiting inflammatory cytokines, and regulating cell proliferation and apoptosis.

Effects of prenatal ethanol exposure on choline-induced long-term depression in the hippocampus.

Choline is an essential nutrient under evaluation as a cognitive enhancing treatment for fetal alcohol spectrum disorders (FASD) in clinical trials. As a result, there is increased pressure to identify therapeutic mechanism(s) of action. Choline is not only a precursor for several essential cell membrane components and signaling molecules but also has the potential to directly affect synaptic mechanisms that are believed important for cognitive processes. In the current work, we study how the direct application of choline can affect synaptic transmission in the dentate gyrus (DG) of hippocampal slices obtained from adolescent (postnatal days 21-28) Sprague-Dawley rats (Rattus norvegicus). The acute administration of choline chloride (2 mM) reliably induced a long-term depression (LTD) of field excitatory postsynaptic potentials (fEPSPs) in the DG in vitro. The depression required the involvement of M1 receptors, and the magnitude of the effect was similar in slices obtained from male and female animals. To further study the impact of choline in an animal model of FASD, we examined offspring from dams fed an ethanol-containing diet (35.5% ethanol-derived calories) throughout gestation. In slices from the adolescent animals that experienced prenatal ethanol exposure (PNEE), we found that the choline induced an LTD that uniquely involved the activation of N-methyl-d-aspartate (NMDA) and M1 receptors. This study provides a novel insight into how choline can modulate hippocampal transmission at the level of the synapse and that it can have unique effects following PNEE.NEW & NOTEWORTHY Choline supplementation is a nutraceutical therapy with significant potential for a variety of developmental disorders; however, the mechanisms involved in its therapeutic effects remain poorly understood. Our research shows that choline directly impacts synaptic communication in the brain, inducing a long-term depression of synaptic efficacy in brain slices. The depression is equivalent in male and female animals, involves M1 receptors in control animals, but uniquely involves NMDA receptors in a model of FASD.

Glutathione peroxidase-1 and neuromodulation: Novel potentials of an old enzyme.

Glutathione peroxidase (GPx) acts in co-ordination with other signaling molecules to exert its own antioxidant role. We have demonstrated the protective effects of GPx,/GPx-1, a selenium-dependent enzyme, on various neurodegenerative disorders (i.e., Parkinson's disease, Alzheimer's disease, cerebral ischemia, and convulsive disorders). In addition, we summarized the recent findings indicating that GPx-1 might play a role as a neuromodulator in neuropsychiatric conditions, such as, stress, bipolar disorder, schizophrenia, and drug intoxication. In this review, we attempted to highlight the mechanistic scenarios mediated by the GPx/GPx-1 gene in impacting these neurodegenerative and neuropsychiatric disorders, and hope to provide new insights on the therapeutic interventions against these disorders.

Mechanism of Magnoliae Officinalis Cortex in treatment of peptic ulcer based on network pharmacology and molecular docking / 中国中药杂志

Magnoliae Officinalis Cortex(Houpo) can treat peptic ulcer disease(PUD), the mechanism of which remains unclear. In this study, network pharmacology and molecular docking were employed to predict the mechanism of Houpo in the treatment of PUD. Through literature review and TCMSP screening, 15 main active ingredients were obtained. The SwissTargetPrediction database was used to predict the potential targets of the ingredients, and Therapeutic Target Database(TTD), DrugBank, and Human Phenotype Ontology(HPO) to screen the disease-related targets. A total of 49 potential targets were obtained by the intersection of active ingre-dients-related targets and disease-related targets. Cytoscape 3.6.1 was employed to construct the protein-protein interaction network for the targets with high confidence(score>0.700) screened out by STRING. The DAVID database was used for GO and KEGG pathway enrichment of potential targets. GO enrichment analysis showed that the treatment mechanism was mostly related to nuclear receptor activity, ligand-activated transcription factor activity, and G protein-coupled acetylcholine receptor activity. KEGG enrichment analysis found that Houpo could regulate material metabolism, endocrine system, p53 signaling pathway, and PPAR signaling pathway. Molecu-lar docking verified that all 15 ingredients had good binding activities with key targets(CHRM1, CHRM2, FABP1, mTOR, and STAT3). The results mean that Houpo can treat PUD by participating in cell metabolism, inhibiting inflammatory cytokines, and regulating cell proliferation and apoptosis.

Muscarinic and Nicotinic Modulation of Neocortical Layer 6A Synaptic Microcircuits Is Cooperative and Cell-Specific.

Acetylcholine (ACh) is known to regulate cortical activity during different behavioral states, for example, wakefulness and attention. Here we show a differential expression of muscarinic ACh receptors (mAChRs) and nicotinic ACh receptors (nAChRs) in different layer 6A (L6A) pyramidal cell (PC) types of somatosensory cortex. At low concentrations, ACh induced a persistent hyperpolarization in corticocortical (CC) but a depolarization in corticothalamic (CT) L6A PCs via M 4 and M1 mAChRs, respectively. At ~ 1 mM, ACh depolarized exclusively CT PCs via α4ß2 subunit-containing nAChRs without affecting CC PCs. Miniature EPSC frequency in CC PCs was decreased by ACh but increased in CT PCs. In synaptic connections with a presynaptic CC PC, glutamate release was suppressed via M4 mAChR activation but enhanced by nAChRs via α4ß2 nAChRs when the presynaptic neuron was a CT PC. Thus, in L6A, the interaction of mAChRs and nAChRs results in an altered excitability and synaptic release, effectively strengthening CT output while weakening CC synaptic signaling.

Small-Scale Panel Comprising Diverse Gene Family Targets To Evaluate Compound Promiscuity.

Despite the recent advances in the life sciences and the remarkable investment in drug discovery research, the success rate of small-molecule drug development remains low. Safety is the second most influential factor of drug attrition in clinical studies; thus, the selection of compounds with fewer toxicity concerns is crucial to increase the success rate of drug discovery. Compounds that promiscuously bind to multiple targets are likely to cause unexpected pharmacological activity that may lead to adverse effects. Therefore, avoiding such compounds during early research stages would contribute to identifying compounds with a higher chance of success in the clinic. To evaluate the interaction profile against a wide variety of targets, we constructed a small-scale promiscuity panel (PP) consisting of eight targets (ROCK1, PDE4D2, GR, PPARγ, 5-HT2B, adenosine A3, M1, and GABAA) that were selected from diverse gene families. The validity of this panel was confirmed by comparison with the promiscuity index evaluated from larger-scale panels. Analysis of data from the PP revealed that both lipophilicity and basicity are likely to increase promiscuity, while the molecular weight does not significantly contribute. Additionally, the promiscuity assessed using our PP correlated with the occurrence of both in vitro cytotoxicity and in vivo toxicity, suggesting that the PP is useful to identify compounds with fewer toxicity concerns. In summary, this small-scale and cost-effective PP can contribute to the identification of safer compounds that would lead to a reduction in drug attrition due to safety issues.

M1 muscarinic receptor is a key target of neuroprotection, neuroregeneration and memory recovery by i-Extract from Withania somnifera.

Memory loss is one of the most tragic symptoms of Alzheimer's disease. Our laboratory has recently demonstrated that 'i-Extract' of Ashwagandha (Withania somnifera) restores memory loss in scopolamine (SC)-induced mice. The prime target of i-Extract is obscure. We hypothesize that i-Extract may primarily target muscarinic subtype acetylcholine receptors that regulate memory processes. The present study elucidates key target(s) of i-Extract via cellular, biochemical, and molecular techniques in a relevant amnesia mouse model and primary hippocampal neuronal cultures. Wild type Swiss albino mice were fed i-Extract, and hippocampal cells from naïve mice were treated with i-Extract, followed by muscarinic antagonist (dicyclomine) and agonist (pilocarpine) treatments. We measured dendritic formation and growth by immunocytochemistry, kallikrein 8 (KLK8) mRNA by reverse transcription polymerase chain reaction (RT-PCR), and levels of KLK8 and microtubule-associated protein 2, c isoform (MAP2c) proteins by western blotting. We performed muscarinic receptor radioligand binding. i-Extract stimulated an increase in dendrite growth markers, KLK8 and MAP2. Scopolamine-mediated reduction was significantly reversed by i-Extract in mouse cerebral cortex and hippocampus. Our study identified muscarinic receptor as a key target of i-Extract, providing mechanistic evidence for its clinical application in neurodegenerative cognitive disorders.

Contribution of M1 and M2 muscarinic receptor subtypes to convulsions in fasted mice treated with scopolamine and given food.

Treatment of fasted mice and rats with the nonselective muscarinic antagonist, scopolamine or atropine, causes convulsions after food intake. This study evaluated the effect of fasting on the expression of M1 and M2 muscarinic receptors in the brain regions, the relationship between receptor expression and seizure stages, and the muscarinic receptor subtype which plays a role in the occurrence of convulsions. Mice were grouped as allowed to eat ad lib (fed) and deprived of food for 24h (fasted). Fasted animals developed convulsions after being treated with scopolamine (60%) or the selective M1 receptor antagonist pirenzepine (10mg/kg; 20% and 60mg/kg; 70%) and given food. Fasting increased expression of M1 receptors in the frontal cortex and M2 receptors in the hippocampus, but produced no change in the expression of both receptors in the amygdaloid complex. Food intake after fasting decreased M1 receptor expression in the frontal cortex and M1 and M2 receptor expression in the hippocampus. Seizure severity was uncorrelated with muscarinic receptor expression in the brain regions. Taken together, these findings provide evidence for the role of M1 muscarinic receptor antagonism and fasting-induced increases in M1 and M2 expression possible underlying mechanism in the occurrence of convulsions in fasted animals.

Effects of standardized isopropanolic black cohosh and estrogen on salivary function in ovariectomized rats.

Oral dryness is a common feature in menopausal women. Estrogen therapy can relieve this symptom; however, the underlying mechanism was not clear. Standardized isopropanolic black cohosh (Actaea racemosa; Remifemin) can also relieve menopausal symptoms, such as hot flashes and sweating. Our previous study showed that standardized isopropanolic black cohosh could protect the submandibular gland structure. To investigate the effects and possible mechanisms of action of estrogen and standardized isopropanolic black cohosh on submandibular gland function in ovariectomized (OVX) rats, we measured body weight, daily water consumption, and blood flow in the submandibular glands. Immunohistochemistry and western blotting were used to detect the expression of muscarinic acetylcholine receptors 1 (M1) and 3 (M3), and aquaporin 5 (AQP5) in the submandibular gland. OVX increased daily water consumption and reduced vasodilation in the submandibular gland. It suggested that ovariectomy could damage the salviary function. Moreover, the expression of M1 and M3 receptors decreased, whereas that of AQP5 increased. These changes may explain the dysfunction of saliva secretion in menopause. Estrogen and standardized isopropanolic black cohosh treatment had the same effect on daily water consumption and vasodilation in the submandibular gland. It indicated that estrogen and standardized isopropanolic black cohosh could relieve oral dryness in menopause. However, the mechanism of the two treatments may differ because standardized isopropanolic black cohosh only protected against changes in M1 expression, whereas estrogen protected against variations in M1, M3, and AQP5 expression.

Discovery of the first low-shift positive allosteric modulators for the muscarinic M1 receptor.

Positive modulation of the muscarinic M1-receptor has for a long time attracted scientists and drug developers for the potential treatment of Alzheimer's disease or Schizophrenia. The precognitive potential of M1 activation has however not been clinically demonstrated as a result of side effects associated both with agonists and positive allosteric modulators (PAM's) of the M1-receptor. To avoid excessive activation of the M1-receptor we have designed a new screening format and developed the first low-shift positive allosteric modulators for the M1 receptor. Low-shift PAM's offer the potential of "use-dependent" attenuation of transmitter-signaling while avoiding pseudo-agonistic behavior in vivo as a common limitation of the so far described high-shift PAM's. With these novel M1-PAM's, the M1 receptor is potentially the first GPCR for which both, high- and low shift PAM's have become available.

Recovery of hippocampal functions and modulation of muscarinic response by electroacupuncture in young diabetic rats.

The muscarinic receptor response to acetylcholine regulates the hippocampal-related learning, memory, neural plasticity and the production and processing of the pro-nerve growth factor (proNGF) by hippocampal cells. The development and progression of diabetes generate a mild cognitive impairment reducing the functions of the septo-hippocampal cholinergic circuitry, depressing neural plasticity and inducing proNGF accumulation in the brain. Here we demonstrate, in a rat model of early type-1 diabetes, that a physical therapy, the electroacupuncture, counteracts the diabetes-induced deleterious effects on hippocampal physiology by ameliorating hippocampal-related memory functions; recovering the impaired long-term potentiation at the dentate gyrus (DG-LTP) and the lowered expression of the vesicular glutamate transporter 1; normalizing the activity-dependent release of proNGF in diabetic rat hippocampus. Electroacupuncture exerted its therapeutic effects by regulating the expression and activity of M1- and M2-acetylcholine muscarinic receptors subtypes in the dentate gyrus of hippocampus. Our results suggest that a physical therapy based on repetitive sensory stimulation could promote hippocampal neural activity, neuronal metabolism and functions, and conceivably improve the diabetes-induced cognitive impairment. Our data can support the setup of therapeutic protocols based on a better integration between physical therapies and pharmacology for the cure of diabetes-associated neurodegeneration and possibly for Alzheimer's disease.

Contribution of muscarinic receptors to in vitro and in vivo effects of Ruscus extract.

The objectives of this study were to evaluate, in vitro and in vivo, the contribution of muscarinic receptors to the effects of Ruscus extract. Ruscus extract was tested in competition binding experiments at recombinant human muscarinic receptors, heterologous expressed in Chinese Hamster Ovary (CHO) cells and in cellular assays measuring Ca2+ liberation and activator protein-1 (AP-1) reporter gene activation. The impact of muscarinic blockade on prolonged treatment outcome was evaluated using the hamster cheek pouch (HCP) microcirculation examining macromolecular permeability increase induced by histamine or ischemia/reperfusion (I/R), mean arteriolar and venular diameters, functional capillary density and I/R-induced leukocyte rolling and sticking. Ruscus extract exhibited affinities for muscarinic receptor subtypes at a range of 50-100µg/ml and behaved as partial agonist at human recombinant M1 and M3 receptors for Ca2+ liberation, confirmed in an AP-1 reporter gene assay. In the HCP model, topical application of atropine completely or partially blocked Ruscus extract-induced reductions of histamine- and I/R-induced increases of macromolecular permeability and leukocyte-endothelium interaction. Our results showed that Ruscus extract in vitro binds and activates different subtypes of muscarinic receptors and in vivo its anti-inflammatory effects are, at least partially, mediated via muscarinic receptors.

A unifying hypothesis for M1 muscarinic receptor signalling in pyramidal neurons.

KEY POINTS: Phasic release of acetylcholine (ACh) in the neocortex facilitates attentional processes. Acting at a single metabotropic receptor subtype, ACh exerts two opposing actions in cortical pyramidal neurons: transient inhibition and longer-lasting excitation. Cholinergic inhibitory responses depend on calcium release from intracellular calcium stores, and run down rapidly at resting membrane potentials when calcium stores become depleted. We demonstrate that cholinergic excitation promotes calcium entry at subthreshold membrane potentials to rapidly refill calcium stores, thereby maintaining the fidelity of inhibitory cholinergic signalling. We propose a 'unifying hypothesis' for M1 receptor signalling whereby inhibitory and excitatory responses to ACh in pyramidal neurons represent complementary mechanisms governing rapid calcium cycling between the endoplasmic reticulum, the cytosol and the extracellular space. ABSTRACT: Gq -coupled M1-type muscarinic acetylcholine (ACh) receptors (mAChRs) mediate two distinct electrophysiological responses in cortical pyramidal neurons: transient inhibition driven by calcium-dependent small conductance potassium ('SK') channels, and longer-lasting and voltage-dependent excitation involving non-specific cation channels. Here we examine the interaction of these two cholinergic responses with respect to their contributions to intracellular calcium dynamics, testing the 'unifying hypothesis' that rundown of inhibitory SK responses at resting membrane potentials (RMPs) reflects depletion of intracellular calcium stores, while mAChR-driven excitation acts to refill those stores by promoting voltage-dependent entry of extracellular calcium. We report that fidelity of cholinergic SK responses requires the continued presence of extracellular calcium. Inhibitory responses that diminished after repetitive ACh application at RMPs were immediately rescued by pairing mAChR stimulation with subthreshold depolarization (∼10 mV from RMPs) initiated with variable delay (up to 500 ms) after ACh application, but not by subthreshold depolarization preceding mAChR stimulation. Further, rescued SK responses were time-locked to ACh application, rather than to the timing of subsequent depolarizing steps, suggesting that cholinergic signal transduction itself is not voltage-sensitive, but that depolarization facilitates rapid cycling of extracellular calcium through the endoplasmic reticulum to activate SK channels. Consistent with this prediction, rescue of SK responses by subthreshold depolarization required the presence of extracellular calcium. Our results demonstrate that, in addition to gating calcium release from intracellular stores, mAChR activation facilitates voltage-dependent refilling of calcium stores, thereby maintaining the ongoing fidelity of SK-mediated inhibition in response to phasic release of ACh.

Hydroxycinnamic acid amides from Scopolia tangutica inhibit the activity of M1 muscarinic acetylcholine receptor in vitro.

Scopolia tangutica Maxim (S. tangutica) extracts have been traditionally used as antispasmodic, sedative, and analgesic agents in Tibet and in the Qinghai province of China. Their active compositions are however poorly understood. We have recently isolated five new hydroxycinnamic acid (HCA) amides along with two known HCA amides, one cinnamic acid amide from these extracts. In this study, we evaluate their abilities to inhibit carbacol-induced activity of M1 muscarinic acetylcholine receptor along with the crude extracts. Chinese hamster ovary cells stably expressing the recombinant human M1 receptor (CHO-M1 cells) were employed to evaluate the anticholinergic potentials. Intracellular Ca(2+) changes were monitored using the FLIPR system. Five HCA amides as well as the crude S. tangutica extract displayed dose-dependent inhibitory effects against M1 receptor. These findings demonstrate that HCA amides are part of the M1 receptor-inhibiting principles of S. tangutica. Since blockade of parasympathetic nerve impulse transmission through the inhibition of the M1 receptor lessens smooth muscle spasms, our findings provided a molecular explanation for the traditional use of S. tangutica against spasm.

AF710B, a Novel M1/σ1 Agonist with Therapeutic Efficacy in Animal Models of Alzheimer's Disease.

We previously developed orthosteric M1 muscarinic agonists (e.g. AF102B, AF267B and AF292), which act as cognitive enhancers and potential disease modifiers. We now report on a novel compound, AF710B, a highly potent and selective allosteric M1 muscarinic and σ1 receptor agonist. AF710B exhibits an allosteric agonistic profile on the M1 muscarinic receptor; very low concentrations of AF710B significantly potentiated the binding and efficacy of carbachol on M1 receptors and their downstream effects (p-ERK1/2, p-CREB). AF710B (1-30 µg/kg, p.o.) was a potent and safe cognitive enhancer in rats treated with the M1 antagonist trihexyphenidyl (passive avoidance impairment). These effects of AF710B involve σ1 receptor activation. In agreement with its antiamnesic properties, AF710B (at 30 nM), via activation of M1 and a possible involvement of σ1 receptors, rescued mushroom synapse loss in PS1-KI and APP-KI neuronal cultures, while AF267B (1 µM) was less potent in PS1-KI and ineffective in APP-KI models, respectively. In female 3xTg-AD mice, AF710B (10 µg/kg, i.p./daily/2 months) (i) mitigated cognitive impairments in the Morris water maze; (ii) decreased BACE1, GSK3ß activity, p25/CDK5, neuroinflammation, soluble and insoluble Aß40, Aß42, plaques and tau pathologies. AF710B differs from conventional σ1 and M1 muscarinic (orthosteric, allosteric or bitopic) agonists. These results highlight AF710B as a potential treatment for Alzheimer's disease (e.g. improving cognitive deficits, synaptic loss, amyloid and tau pathologies, and neuroinflammation) with a superior profile over a plethora of other therapeutic strategies.