RESUMO
Neural stem cells (NSCs) in the subventricular zone (SVZ) located along the lateral ventricles (LVs) of the mammalian brain continue to self-renew to produce new neurons after birth and into adulthood. Quiescent LV cells, which are situated close to the ependymal cells lining the LVs, are activated by choline acetyltransferase-positive (ChAT+) neurons within the subependymal (subep) region of the SVZ when these neurons are stimulated by projections from the anterior cingulate cortex (ACC). Here, we uncovered a signaling pathway activated by the ACC-subep-ChAT+ circuit responsible for the activation and proliferation of quiescent LV NSCs specifically in the ventral area of the SVZ. This circuit activated muscarinic M3 receptors on quiescent LV NSCs, which subsequently induced signaling mediated by the inositol 1,4,5-trisphosphate receptor type 1 (IP3R1). Downstream of IP3R1 activation, which would be expected to increase intracellular Ca2+, Ca2+-/calmodulin-dependent protein kinase II δ and the MAPK10 signaling pathway were stimulated and required for the proliferation of quiescent LV NSCs in the SVZ. These findings reveal the mechanisms that regulate quiescent LV NSCs and underscore the critical role of projections from the ACC in promoting their proliferative activity within the ventral SVZ.
Assuntos
Receptores de Inositol 1,4,5-Trifosfato , Ventrículos Laterais , Células-Tronco Neurais , Transdução de Sinais , Animais , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/citologia , Ventrículos Laterais/metabolismo , Ventrículos Laterais/citologia , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Camundongos , Colina O-Acetiltransferase/metabolismo , Colina O-Acetiltransferase/genética , Proliferação de Células , Receptor Muscarínico M3/metabolismo , Receptor Muscarínico M3/genética , Giro do Cíngulo/metabolismo , Giro do Cíngulo/citologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismoRESUMO
EP4 prostanoid receptor (EP4R) contributes to the intestinal epithelial Cl- secretion, and inhibition of prostaglandin E (PGE) production by non-steroidal anti-inflammatory drugs (NSAIDs) plays a central role in NSAID-induced enteropathy. Although M3 muscarinic acetylcholine receptor (M3R) also contributes to the intestinal epithelial Cl- secretion, it remains unclear whether M3R is involved in NSAID-induced enteropathy due to a lack of selective agents. The present study explored how M3R is involved in the regulation of the intestinal epithelial Cl- secretion and its pathophysiological role in NSAID-induced enteropathy. Using the novel highly-selective M3 positive allosteric modulator PAM-369 that we recently developed, we evaluated the role of M3R in the intestinal epithelial secretion ex vivo by measuring the short circuit current (Isc) of intestinal epithelium with a Ussing chamber system and examined whether or not M3R protects against small intestinal injury in indomethacin-treated mice. Both the PGE1 derivative misoprostol and carbachol similarly increased the Isc in a concentration-dependent manner. The Isc increases were abolished either by receptor antagonists (an EP4R antagonist and a M3R antagonist, respectively) or by removal of extracellular Cl-. PAM-369 enhanced the carbachol-induced Isc by potentiating M3R, which could contribute to enhanced intestinal epithelial secretion. Treatment with PAM-369 ameliorated small intestinal injury in indomethacin-treated mice. Importantly, the M3R expression was significantly up-regulated, and PAM-369 potentiation of M3R was augmented in indomethacin-treated mice compared to untreated mice. These findings show that M3R plays a role in maintaining the intestinal epithelial secretion, which could contribute to protection against indomethacin-induced small intestinal injury. M3R is a promising target for treating or preventing NSAID-induced enteropathy. KEY MESSAGES: PAM-369, the M3 positive allosteric modulator, was used to potentiate M3R. PAM-369 enhanced carbachol-induced Isc in mouse ileum. PAM-369 ameliorated small intestinal injury in indomethacin-treated mice. M3R is a promising target for treating or preventing NSAID-induced enteropathy.
Assuntos
Indometacina , Intestino Delgado , Receptor Muscarínico M3 , Animais , Masculino , Camundongos , Anti-Inflamatórios não Esteroides/farmacologia , Anti-Inflamatórios não Esteroides/efeitos adversos , Carbacol/farmacologia , Indometacina/efeitos adversos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/metabolismo , Intestino Delgado/patologia , Intestino Delgado/lesões , Camundongos Endogâmicos C57BL , Misoprostol/farmacologia , Receptor Muscarínico M3/metabolismoRESUMO
The muscarinic acetylcholine receptor M3 (M3-mAChR) is involved in various physiological and pathological processes. Owing to specific cardioprotective effects, M3-mAChR is an ideal diagnostic and therapeutic biomarker for cardiovascular diseases (CVDs). Growing evidence has linked M3-mAChR to the development of multiple CVDs, in which it plays a role in cardiac protection such as anti-arrhythmia, anti-hypertrophy, and anti-fibrosis. This review summarizes M3-mAChR's expression patterns, functions, and underlying mechanisms of action in CVDs, especially in ischemia/reperfusion injury, cardiac hypertrophy, and heart failure, opening up a new research direction for the treatment of CVDs.
Assuntos
Doenças Cardiovasculares , Receptor Muscarínico M3 , Humanos , Doenças Cardiovasculares/metabolismo , Animais , Receptor Muscarínico M3/metabolismo , Receptor Muscarínico M3/genéticaRESUMO
Exposure to particulate matter (PM10) can induce respiratory diseases that are closely related to bronchial hyperresponsiveness. However, the involved mechanism remains to be fully elucidated. This study aimed to demonstrate the effects of PM10 on the acetylcholine muscarinic 3 receptor (CHRM3) expression and the role of the ERK1/2 pathway in rat bronchial smooth muscle. A whole-body PM10 exposure system was used to stimulate bronchial hyperresponsiveness in rats for 2 and 4 months, accompanied by MEK1/2 inhibitor U0126 injection. The whole-body plethysmography system and myography were used to detect the pulmonary and bronchoconstrictor function, respectively. The mRNA and protein levels were determined by Western blotting, qPCR, and immunofluorescence. Enzyme-linked immunosorbent assay was used to detect the inflammatory cytokines. Compared with the filtered air group, 4 months of PM10 exposure significantly increased CHRM3-mediated pulmonary function and bronchial constriction, elevated CHRM3 mRNA and protein expression levels on bronchial smooth muscle, then induced bronchial hyperreactivity. Additionally, 4 months of PM10 exposure caused an increase in ERK1/2 phosphorylation and increased the secretion of inflammatory factors in bronchoalveolar lavage fluid. Treatment with the MEK1/2 inhibitor, U0126 inhibited the PM10 exposure-induced phosphorylation of the ERK1/2 pathway, thereby reducing the PM10 exposure-induced upregulation of CHRM3 in bronchial smooth muscle and CHRM3-mediated bronchoconstriction. U0126 could rescue PM10 exposure-induced pathological changes in the bronchus. In conclusion, PM10 exposure can induce bronchial hyperresponsiveness in rats by upregulating CHRM3, and the ERK1/2 pathway may be involved in this process. These findings could reveal a potential therapeutic target for air pollution induced respiratory diseases.
Assuntos
Hiper-Reatividade Brônquica , Material Particulado , Receptor Muscarínico M3 , Animais , Hiper-Reatividade Brônquica/induzido quimicamente , Hiper-Reatividade Brônquica/fisiopatologia , Hiper-Reatividade Brônquica/metabolismo , Masculino , Material Particulado/toxicidade , Receptor Muscarínico M3/metabolismo , Receptor Muscarínico M3/genética , Ratos , Regulação para Cima/efeitos dos fármacos , Brônquios/efeitos dos fármacos , Brônquios/metabolismo , Brônquios/patologia , Ratos Sprague-Dawley , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Broncoconstrição/efeitos dos fármacos , Citocinas/metabolismo , Citocinas/genética , Butadienos , NitrilasRESUMO
Purpose: Through network pharmacology combined with molecular docking and in vivo validation, the study examines the unexplored molecular mechanisms of Tongxieyaofang (TXYF) in the treatment of irritable bowel syndrome (IBS). In particular, the potential pharmacological mechanism of TXYF alleviating IBS by regulating CHRM3 and intestinal barrier has not been studied. Patients and Methods: LC-MS technique and TCMSP database were used in combination to identify the potential effective components and target sites of TXYF. Potential targets for IBS were obtained from Genecards and OMIM databases. PPI and cytoHub analysis for targets. Molecular docking was used to validate the binding energy of effective components with related targets and for visualization. GO and KEGG analysis were employed to identify target functions and signaling pathways. In the in vivo validation, wrap restraint stress-induced IBS model was employed to verify the change for cytoHub genes and CHRM3 expression. Furthermore, inflammatory changes of colon were observed by HE staining. The changes of Ach were verified by ELISA. IHC and WB validated CHRM3 and GNAQ/PLC/MLCK channel variations. AB-PAS test and WB test confirmed the protection of TXYF on gut barrier. The NF-κB/MLCK pathway was also verified. Results: In TXYF decoction, LC-MS identified 559 chemical components, with 23 remaining effective components after screening in TCMSP. KEGG analysis indicated that calcium plays a crucial role in TXYF treated for IBS. Molecular docking validated the binding capacity of the effective components Naringenin and Nobiletin with cytoHub-gene and CHRM3. In vivo validation demonstrated that TXYF inhibits the activation of Ach and CHRM3 in IBS, and inhibits for the GNAQ/PLC/MLCK axis. Additionally, TXYF downregulates TNF-α, MMP9, and NF-κB/MLCK, while modulating goblet cell secretion to protect gut barrier. Conclusion: TXYF inhibits Ach and CHRM3 expression, regulating the relaxation of intestinal smooth muscle via GNAQ/PLC/MLCK. Additionally, TXYF inhibits NF-κB/MLCK activated and goblet cell secretion to protect gut barrier.
Assuntos
Medicamentos de Ervas Chinesas , Síndrome do Intestino Irritável , Simulação de Acoplamento Molecular , Receptor Muscarínico M3 , Síndrome do Intestino Irritável/tratamento farmacológico , Síndrome do Intestino Irritável/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/química , Animais , Receptor Muscarínico M3/metabolismo , Receptor Muscarínico M3/antagonistas & inibidores , Humanos , Masculino , Ratos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Ratos Sprague-Dawley , Farmacologia em RedeRESUMO
In cholinergic urticaria (CholU), small, itchy wheals are induced by exercise or passive warming and reduced sweating has been reported. Despite the described reduced muscarinic receptor expression, sweat duct obstruction, or sweat allergy, the underlying pathomechanisms are not well understood. To gain further insights, we collected skin biopsies before and after pulse-controlled ergometry and sweat after sauna provocation from CholU patients as well as healthy controls. CholU patients displayed partially severely reduced local sweating, yet total sweat volume was unaltered. However, sweat electrolyte composition was altered, with increased K+ concentration in CholU patients. Formalin-fixed, paraffin-embedded biopsies were stained to explore sweat leakage and tight junction protein expression. Dermcidin staining was not found outside the sweat glands. In the secretory coils of sweat glands, the distribution of claudin-3 and -10b as well as occludin was altered, but the zonula occludens-1 location was unchanged. In all, dermcidin and tight junction protein staining suggests an intact barrier with reduced sweat production capability in CholU patients. For future studies, an ex vivo skin model for quantification of sweat secretion was established, in which sweat secretion could be pharmacologically stimulated or blocked. This ex vivo model will be used to further investigate sweat gland function in CholU patients and decipher the underlying pathomechanism(s).
Assuntos
Urticária Crônica Induzida , Glândulas Sudoríparas , Suor , Junções Íntimas , Suor/química , Junções Íntimas/metabolismo , Glândulas Sudoríparas/metabolismo , Ergometria , Proteínas de Junções Íntimas/metabolismo , Urticária Crônica Induzida/metabolismo , Urticária Crônica Induzida/patologia , Humanos , Masculino , Feminino , Adulto , Receptor Muscarínico M3/metabolismo , Biópsia por AgulhaRESUMO
The smooth muscle-walled blood vessels control blood pressure. The vessel lumen is lined by an endothelial cell (ECs) layer, interconnected to the surrounding smooth muscle cells (SMCs) by myoendothelial gap junctions. Gap junctions also maintain homo-cellular ECs-ECs and SMCs-SMCs connections. This gap junction network nearly equalises both cells' membrane potential and cytosolic ionic composition, whether in resting or stimulated conditions. When acetylcholine (ACh) activates ECs M3 receptors, a complex signalling cascade involving second messengers and ion channels is triggered to induce vasodilation.
Assuntos
Acetilcolina , Ácido Araquidônico , Endotélio Vascular , Junções Comunicantes , Vasodilatação , Vasodilatação/efeitos dos fármacos , Ácido Araquidônico/metabolismo , Humanos , Junções Comunicantes/metabolismo , Animais , Acetilcolina/metabolismo , Acetilcolina/farmacologia , Endotélio Vascular/metabolismo , Canais Iônicos/metabolismo , Células Endoteliais/metabolismo , Miócitos de Músculo Liso/metabolismo , Receptor Muscarínico M3/metabolismo , Músculo Liso Vascular/metabolismo , Transdução de SinaisRESUMO
Berberine, a natural isoquinoline alkaloid, exhibits a variety of pharmacological effects, but the pharmacological targets and mechanisms remain elusive. Here, we report a novel finding that berberine inhibits acetylcholine (ACh)-induced intracellular Ca2+ oscillations, mediated through an inhibition of the muscarinic subtype 3 (M3) receptor. Patch-clamp recordings and confocal Ca2+ imaging were applied to acute dissociated pancreatic acinar cells prepared from CD1 mice to examine the effects of berberine on ACh-induced Ca2+ oscillations. Whole-cell patch-clamp recordings showed that berberine (from 0.1 to 10 µM) reduced ACh-induced Ca2+ oscillations in a concentration-dependent manner, and this inhibition also depended on ACh concentrations. The inhibitory effect of berberine neither occurred in intracellular targets nor extracellular cholecystokinin (CCK) receptors, chloride (Cl-) channels, and store-operated Ca2+ channels. Together, the results demonstrate that berberine directly inhibits the muscarinic M3 receptors, further confirmed by evidence of the interaction between berberine and M3 receptors in pancreatic acinar cells.
Assuntos
Células Acinares , Berberina , Sinalização do Cálcio , Receptor Muscarínico M3 , Animais , Berberina/farmacologia , Receptor Muscarínico M3/metabolismo , Receptor Muscarínico M3/antagonistas & inibidores , Camundongos , Células Acinares/efeitos dos fármacos , Células Acinares/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Masculino , Acetilcolina/metabolismo , Cálcio/metabolismo , Relação Dose-Resposta a DrogaRESUMO
INTRODUCTION: The muscarinic M3 receptor antagonist, tiotropium, has a bronchodilatory effect on asthma patients. Additionally, tiotropium inhibits allergic airway inflammation and remodeling in a murine asthma model. However, the underlying mechanisms of this M3 receptor antagonist remain unclear. Therefore, we investigated the effect of muscarinic M3 receptor blockage on M2 macrophage development during allergic airway inflammation. METHODS: BALB/c mice were sensitized and challenged with ovalbumin to develop a murine model of allergic airway inflammation mimicking human atopic asthma. During the challenge phase, mice were treated with or without tiotropium. Lung cells were isolated 24 h after the last treatment and gated using CD68-positive cells. Relm-α and Arginase-1 (Arg1) (M2 macrophage markers) expression was determined by flow cytometry. Mouse bone marrow mononuclear cell-derived macrophages (mBMMacs) and human peripheral blood mononuclear cells (PBMCs)-derived macrophages were stimulated with IL-4 and treated with a muscarinic M3 receptor antagonist in vitro. RESULTS: The total cells, eosinophils, and IL-5 and IL-13 levels in BAL fluids were markedly decreased in the asthma group treated with tiotropium compared to that in the untreated asthma group. The Relm-α and Arg1 expression in macrophages was reduced considerably in the asthma group treated with tiotropium compared to that in the untreated asthma group, suggesting that the development of M2 macrophages was inhibited by muscarinic M3 receptor blockage. Additionally, muscarinic M3 receptor blockage in vitro significantly inhibited M2 macrophage development in both mBMMacs- and PBMCs-derived macrophages. CONCLUSIONS: Muscarinic M3 receptor blockage inhibits M2 macrophage development and prevents allergic airway inflammation. Moreover, muscarinic M3 receptors might be involved in the differentiation of immature macrophages into M2 macrophages.
Assuntos
Asma , Macrófagos , Camundongos Endogâmicos BALB C , Receptor Muscarínico M3 , Animais , Receptor Muscarínico M3/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Asma/imunologia , Asma/metabolismo , Asma/tratamento farmacológico , Humanos , Modelos Animais de Doenças , Brometo de Tiotrópio/farmacologia , Ovalbumina/imunologia , Feminino , Arginase/metabolismo , Citocinas/metabolismo , Antagonistas Muscarínicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Inflamação/imunologia , Inflamação/metabolismoRESUMO
The M3 muscarinic acetylcholine receptor (M3R) is a G protein-coupled receptor (GPCR) that regulates important physiologic processes, including vascular tone, bronchoconstriction, and insulin secretion. It is expressed on a wide variety of cell types, including pancreatic beta, smooth muscle, neuronal, and immune cells. Agonist binding to the M3R is thought to initiate intracellular signaling events primarily through the heterotrimeric G protein Gq. However, reports differ on the ability of M3R to couple to other G proteins beyond Gq. Using members from the four primary G protein families (Gq, Gi, Gs, and G13) in radioligand binding, GTP turnover experiments, and cellular signaling assays, including live cell G protein dissociation and second messenger assessment of cAMP and inositol trisphosphate, we show that other G protein families, particularly Gi and Gs, can also interact with the human M3R. We further show that these interactions are productive as assessed by amplification of classic second messenger signaling events. Our findings demonstrate that the M3R is more promiscuous with respect to G protein interactions than previously appreciated. SIGNIFICANCE STATEMENT: The study reveals that the human M3 muscarinic acetylcholine receptor (M3R), known for its pivotal roles in diverse physiological processes, not only activates intracellular signaling via Gq as previously known but also functionally interacts with other G protein families such as Gi and Gs, expanding our understanding of its versatility in mediating cellular responses. These findings signify a broader and more complex regulatory network governed by M3R and have implications for therapeutic targeting.
Assuntos
Proteínas de Ligação ao GTP , Receptor Muscarínico M3 , Transdução de Sinais , Animais , Humanos , Células CHO , Cricetulus , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Receptor Muscarínico M3/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Acetylcholine (ACh) is released from basal forebrain cholinergic neurons in response to salient stimuli and engages brain states supporting attention and memory. These high ACh states are associated with theta oscillations, which synchronize neuronal ensembles. Theta oscillations in the basolateral amygdala (BLA) in both humans and rodents have been shown to underlie emotional memory, yet their mechanism remains unclear. Here, using brain slice electrophysiology in male and female mice, we show large ACh stimuli evoke prolonged theta oscillations in BLA local field potentials that depend upon M3 muscarinic receptor activation of cholecystokinin (CCK) interneurons (INs) without the need for external glutamate signaling. Somatostatin (SOM) INs inhibit CCK INs and are themselves inhibited by ACh, providing a functional SOMâCCK IN circuit connection gating BLA theta. Parvalbumin (PV) INs, which can drive BLA oscillations in baseline states, are not involved in the generation of ACh-induced theta, highlighting that ACh induces a cellular switch in the control of BLA oscillatory activity and establishes an internally BLA-driven theta oscillation through CCK INs. Theta activity is more readily evoked in BLA over the cortex or hippocampus, suggesting preferential activation of the BLA during high ACh states. These data reveal a SOMâCCK IN circuit in the BLA that gates internal theta oscillations and suggest a mechanism by which salient stimuli acting through ACh switch the BLA into a network state enabling emotional memory.
Assuntos
Acetilcolina , Colecistocinina , Camundongos Endogâmicos C57BL , Ritmo Teta , Ritmo Teta/efeitos dos fármacos , Ritmo Teta/fisiologia , Animais , Masculino , Camundongos , Feminino , Acetilcolina/farmacologia , Acetilcolina/metabolismo , Colecistocinina/farmacologia , Colecistocinina/metabolismo , Interneurônios/fisiologia , Interneurônios/efeitos dos fármacos , Somatostatina/metabolismo , Somatostatina/farmacologia , Tonsila do Cerebelo/fisiologia , Tonsila do Cerebelo/efeitos dos fármacos , Complexo Nuclear Basolateral da Amígdala/fisiologia , Complexo Nuclear Basolateral da Amígdala/efeitos dos fármacos , Rede Nervosa/fisiologia , Rede Nervosa/efeitos dos fármacos , Receptor Muscarínico M3/fisiologia , Receptor Muscarínico M3/metabolismo , Parvalbuminas/metabolismoRESUMO
Muscarinic acetylcholine receptor M3 (M3R) has repeatedly been shown to be prominently expressed in human colorectal cancer (CRC), playing roles in proliferation and cell invasion. Its therapeutic targetability has been suggested in vitro and in animal models. We aimed to investigate the clinical role of MR3 expression in CRC for human survival. Surgical tissue samples from 754 CRC patients were analyzed for high or low immunohistochemical M3R expression on a clinically annotated tissue microarray (TMA). Immunohistochemical analysis was performed for established immune cell markers (CD8, TIA-1, FOXP3, IL 17, CD16 and OX 40). We used Kaplan-Meier curves to evaluate patients' survival and multivariate Cox regression analysis to evaluate prognostic significance. High M3R expression was associated with increased survival in multivariate (hazard ratio (HR) = 0.52; 95% CI = 0.35-0.78; p = 0.001) analysis, as was TIA-1 expression (HR = 0.99; 95% CI = 0.94-0.99; p = 0.014). Tumors with high M3R expression were significantly more likely to be grade 2 compared to tumors with low M3R expression (85.7% vs. 67.1%, p = 0.002). The 5-year survival analysis showed a trend of a higher survival rate in patients with high M3R expression (46%) than patients with low M3R expression CRC (42%) (p = 0.073). In contrast to previous in vitro and animal model findings, this study demonstrates an increased survival for CRC patients with high M3R expression. This evidence is highly relevant for translation of basic research findings into clinically efficient treatments.
Assuntos
Neoplasias Colorretais , Receptores Muscarínicos , Animais , Humanos , Neoplasias Colorretais/genética , Receptor Muscarínico M3/metabolismoRESUMO
Despite its increasing application in pursing potential ligands, the capacity of receptor affinity chromatography is greatly challenged as most current research studies lack a comprehensive characterization of the ligand-receptor interaction, particularly when simultaneously determining their binding thermodynamics and kinetics. This work developed an immobilized M3 muscarinic receptor (M3R) affinity column by fixing M3R on amino polystyrene microspheres via the interaction of a 6-chlorohexanoic acid linker with haloalkane dehalogenase. The efficiency of the immobilized M3R was tested by characterizing the binding thermodynamics and kinetics of three known drugs to immobilized M3R using a frontal analysis and the peak profiling method, as well as by analyzing the bioactive compounds in Daturae Flos (DF) extract. The data showed that the immobilized M3R demonstrated good specificity, stability, and competence for analyzing drug-protein interactions. The association constants of (-)-scopolamine hydrochloride, atropine sulfate, and pilocarpine to M3R were determined to be (2.39 ± 0.03) × 104, (3.71 ± 0.03) × 104, and (2.73 ± 0.04) × 104 M-1, respectively, with dissociation rate constants of 27.47 ± 0.65, 14.28 ± 0.17, and 10.70 ± 0.35 min-1, respectively. Hyoscyamine and scopolamine were verified as the bioactive compounds that bind to M3R in the DF extract. Our results suggest that the immobilized M3R method was capable of determining drug-protein binding parameters and probing specific ligands in a natural plant, thus enhancing the effectiveness of receptor affinity chromatography in diverse stages of drug discovery.
Assuntos
Pilocarpina , Receptor Muscarínico M3 , Receptor Muscarínico M3/metabolismo , Derivados da Escopolamina , Extratos Vegetais/farmacologiaRESUMO
Cholinergic receptor muscarinic 3 (CHRM3)-mediated focal adhesion kinase/YES-associated protein (YAP) signalling is essential for the growth of castration-resistant prostate cancer (CRPC) cells. Here, we evaluated the molecular mechanisms through which CHRM3 overexpression facilitates castration-resistant growth. Small RNA sequencing combined with in silico analyses revealed that CHRM3 was a putative target of miR-15b-5p. Notably, androgen deprivation suppressed miR-15b-5p expression and increased CHRM3 expression. Moreover, miR-15b-5p bound directly to CHRM3 and inhibited YAP activation induced by CHRM3 stimulation. Furthermore, miR-15b-5p abolished the growth of CRPC cells induced by CHRM3 stimulation. We conclude that the miR-15b-5p/CHRM3/YAP signalling axis promotes the castration-resistant growth of prostate cancer.
Assuntos
MicroRNAs , Neoplasias de Próstata Resistentes à Castração , Masculino , Humanos , MicroRNAs/genética , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/metabolismo , Antagonistas de Androgênios , Proliferação de Células/fisiologia , Castração , Linhagem Celular Tumoral , Receptores Colinérgicos/metabolismo , Colinérgicos , Regulação Neoplásica da Expressão Gênica , Receptor Muscarínico M3/genética , Receptor Muscarínico M3/metabolismoRESUMO
Chronic obstructive pulmonary disease (COPD) is a common respiratory disease with high disability and mortality. Clinical studies have shown that the Traditional Chinese Medicine Bufei Granule (BFG) has conspicuous effects on relieving cough and improving lung function in patients with COPD and has a reliable effect on the treatment of COPD, whereas the therapeutic mechanism is vague. In the present study, the latent bronchodilators and mechanism of BFG in the treatment of COPD were discussed through the method of network pharmacology. Then, the molecular docking and molecular dynamics simulation were performed to calculate the binding efficacy of corresponding compounds in BFG to muscarinic receptor. Finally, the effects of BFG on bronchial smooth muscle were validated by in vitro experiments. The network pharmacology results manifested the anti-COPD effect of BFG was mainly realized via restraining airway smooth muscle contraction, activating cAMP pathways and relieving oxidative stress. The results of molecular docking and molecular dynamics simulation showed alpinetin could bind to cholinergic receptor muscarinic 3. The in vitro experiment verified both BFG and alpinetin could inhibit the levels of CHRM3 and acetylcholine and could be potential bronchodilators for treating COPD. This study provides an integrating network pharmacology method for understanding the therapeutic mechanisms of traditional Chinese medicine, as well as a new strategy for developing natural medicines for treating COPD.
Assuntos
Medicamentos de Ervas Chinesas , Doença Pulmonar Obstrutiva Crônica , Humanos , Pulmão/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/uso terapêutico , Broncodilatadores/farmacologia , Broncodilatadores/metabolismo , Broncodilatadores/uso terapêutico , Simulação de Acoplamento Molecular , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Receptor Muscarínico M3/metabolismo , Receptor Muscarínico M3/uso terapêuticoRESUMO
Emerging evidence suggested that long non-coding RNAs (lncRNAs) were involved in Parkinson's disease (PD) pathogenesis. Herein, we used gene expression profiles from GEO database to construct a PD-specific ceRNA network. Functional enrichment analysis suggested that ceRNA network might participate in the development of PD. PPI networks were constructed, and the ceRNA subnetwork based on five hub genes was set up. In a cohort of 32 PD patients and 31 healthy controls, the expression of 10 DElncRNAs (TTC3-AS1, LINC01259, ZMYND10-AS1, CHRM3-AS1, MYO16-AS1, AGBL5-IT1, HOTAIRM1, RABGAP1L-IT1, HLCS-IT1, and LINC00393) were further verified. Consistent with the microarray data, LINC01259 expression was significantly lower in PD patients compared with controls (P = 0.008). Intriguingly, such a difference was only observed among male patients and male controls when dividing study participants based on their gender (P = 0.016). However, the expression of other lncRNAs did not differ significantly between the two groups. Receiver operating characteristic (ROC) curve analysis revealed that the diagnostic power of LINC01259 was 0.694 for PD and 0.677 for early-stage PD. GSEA enrichment analysis revealed that LINC01259 was mainly enriched in biological processes associated with immune function and inflammatory response. Moreover, LINC01259 expression was not correlated with age of patients, disease duration, disease stage, MDS-UPDRS score, MDS-UPDRS III score, MMSE score, and MOCA score. The current study provides further evidence for the dysregulation of lncRNAs in circulating leukocytes of PD patients, revealing that LINC01259 has clinical potential as a novel immune and inflammatory biomarker for PD and early-stage PD diagnosis.
Assuntos
Doença de Parkinson , RNA Longo não Codificante , Humanos , Masculino , Biomarcadores Tumorais/genética , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Doença de Parkinson/genética , Receptor Muscarínico M3/genética , Receptor Muscarínico M3/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , FemininoRESUMO
Muscarinic acetylcholine receptor subtype 3 (M3 receptor) is a G Protein-Coupled Receptor (GPCR) that mediates many important physiological functions. Currently, most M3 receptor drugs also have high affinity for other subtypes of muscarinic acetylcholine receptors (mAChRs) and produce the risk of side effects. Therefore, in order to find M3 receptor drugs with high specificity, high activity and low side effects, we established a cell model and method for efficient and sensitive screening of M3 receptor based on calcium-activated chloride channels (CaCCs), and this method is also suitable for the screening of other GPCR drugs. This screening model consists of Fischer rat thyroid follicular epithelial (FRT) cells that endogenously express M3 receptors, CaCCs, and the indicator YFP-H148Q/I152L. We verified that the model can sensitively detect changes in intracellular Ca2+ concentration using fluorescence quenching kinetics experiments, confirmed the screening function of the model by applying available M3 receptor drugs, and also evaluated the good performance of the model in high-throughput screening.
Assuntos
Canais de Cloreto , Ensaios de Triagem em Larga Escala , Ratos , Animais , Receptor Muscarínico M3/metabolismo , Glândula Tireoide/metabolismoRESUMO
BACKGROUND: The specific role of the M3 muscarinic acetylcholine receptor in gastrointestinal motility under physiological conditions is unclear, due to a lack of subtype-selective compounds. AIMS: The objective of this study was to determine the region-specific role of the M3 receptor in gastrointestinal motility. METHODS: We developed a novel positive allosteric modulator (PAM) for the M3 receptor, PAM-369. The effects of PAM-369 on the carbachol-induced contractile response of porcine esophageal smooth muscle and mouse colonic smooth muscle (ex vivo) and on the transit in mouse small intestine and rat colon (in vivo) were examined. RESULTS: PAM-369 selectively potentiated the M3 receptor under the stimulation of its orthosteric ligands without agonistic or antagonistic activity. Half-maximal effective concentrations of PAM activity for human, mouse, and rat M3 receptors were 0.253, 0.345, and 0.127 µM, respectively. PAM-369 enhanced carbachol-induced contraction in porcine esophageal smooth muscle and mouse colonic smooth muscle without causing any contractile responses by itself. The oral administration of 30 mg/kg PAM-369 increased the small intestinal transit in both normal motility and loperamide-induced intestinal dysmotility mice but had no effects on the colonic transit, although the M3 receptor mRNA expression is higher in the colon than in the small intestine. CONCLUSIONS: This study provided the first direct evidence that the M3 receptor has different region-specific roles in the motility function between the small intestine and colon in physiological and pathophysiological contexts. Selective PAMs designed for targeted subtypes of muscarinic receptors are useful for elucidating the subtype-specific function.
Assuntos
Motilidade Gastrointestinal , Receptor Muscarínico M3 , Animais , Humanos , Camundongos , Ratos , Carbacol/farmacologia , Motilidade Gastrointestinal/genética , Motilidade Gastrointestinal/fisiologia , Contração Muscular , Receptor Muscarínico M2/genética , Receptor Muscarínico M2/metabolismo , Receptor Muscarínico M3/genética , Receptor Muscarínico M3/metabolismo , Receptores Muscarínicos/fisiologia , SuínosRESUMO
Background: Cholinergic 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. Aim: To assess in CholU patients the rate and extent of impaired sweating and its links to clinical and pathophysiological features of CholU. Patients and methods: We 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. Results: Of 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. Conclusion: Reduced 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.
Assuntos
Acetilcolinesterase , Receptor Muscarínico M3 , Glândulas Sudoríparas , Sudorese , Urticária , Acetilcolina/metabolismo , Acetilcolinesterase/biossíntese , Acetilcolinesterase/metabolismo , Colinérgicos , Humanos , Receptor Muscarínico M3/metabolismo , Receptores Colinérgicos , Glândulas Sudoríparas/metabolismo , Glândulas Sudoríparas/patologia , Sudorese/fisiologia , Urticária/complicações , Urticária/metabolismoRESUMO
Endothelial cells derived from human induced pluripotent stem cells (hiPSC-ECs) provide a new opportunity for mechanistic research on vascular regeneration and drug screening. However, functions of hiPSC-ECs still need to be characterized. The objective of this study was to investigate electrophysiological and functional properties of hiPSC-ECs compared with primary human cardiac microvascular endothelial cells (HCMECs), mainly focusing on ion channels and membrane receptor signaling, as well as specific cell functions. HiPSC-ECs were derived from hiPS cells that were generated from human skin fibroblasts of three independent healthy donors. Phenotypic and functional comparison to HCMECs was performed by flow cytometry, immunofluorescence staining, quantitative reverse-transcription polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA), tube formation, LDL uptake, exosome release assays and, importantly, patch clamp techniques. HiPSC-ECs were successfully generated from hiPS cells and were identified by endothelial markers. The mRNA levels of KCNN2, KCNN4, KCNMA1, TRPV2, and SLC8A1 in hiPSC-ECs were significantly higher than HCMECs. AT1 receptor mRNA level in hiPSC-ECs was higher than in HCMECs. AT2 receptor mRNA level was the highest among all receptors. Adrenoceptor ADRA2 expression in hiPSC-ECs was lower than in HCMECs, while ADRA1, ADRB1, ADRB2, and G-protein GNA11 and Gai expression were similar in both cell types. The expression level of muscarinic and dopamine receptors CHRM3, DRD2, DRD3, and DRD4 in hiPSC-ECs were significantly lower than in HCMECs. The functional characteristics of endothelial cells, such as tube formation and LDL uptake assay, were not statistically different between hiPSC-ECs and HCMECs. Phenylephrine similarly increased the release of the vasoconstrictor endothelin-1 (ET-1) in hiPSC-ECs and HCMECs. Acetylcholine also similarly increased nitric oxide generation in hiPSC-ECs and HCMECs. The resting potentials (RPs), ISK1-3, ISK4 and IK1 were similar in hiPSC-ECs and HCMECs. IBK was larger and IKATP was smaller in hiPSC-ECs. In addition, we also noted a higher expression level of exosomes marker CD81 in hiPSC-ECs and a higher expression of CD9 and CD63 in HCMECs. However, the numbers of exosomes extracted from both types of cells did not differ significantly. The study demonstrates that hiPSC-ECs are similar to native endothelial cells in ion channel function and membrane receptor-coupled signaling and physiological cell functions, although some differences exist. This information may be helpful for research using hiPSC-ECs.