RESUMO
OBJECTIVES: Muscarinic M3 (M3 ) receptors mediate cholinergic smooth muscle contraction of the bladder. Current drugs targeting bladder M3 receptors for micturition disorders have a risk of cholinergic side effects due to excessive receptor activation and insufficient selectivity. We investigated the effect of ASP8302, a novel positive allosteric modulator (PAM) of M3 receptors, on bladder function in rats. METHODS: Modulation of carbachol-induced increases in intracellular Ca2+ was assessed in cells expressing rat muscarinic receptors. Potentiation of bladder contractions was evaluated using isolated rat bladder strips and by measuring intravesical pressure in anesthetized rats. Conscious cystometry was performed to investigate the effects on residual urine volume and voiding efficiency in rat voiding dysfunction models induced by the α1 -adrenoceptor agonist midodrine and muscarinic receptor antagonist atropine, and bladder outlet obstruction. To assess potential side effects, the number of stools and tracheal insufflation pressure were measured in conscious and anesthetized rats, respectively. RESULTS: ASP8302 demonstrated PAM effects on the rat M3 receptor in cell assays, and augmented cholinergic bladder contractions both in vivo and in vitro. ASP8302 improved voiding efficiency and reduced residual urine volume in two voiding dysfunction models as effectively as distigmine bromide, but unlike distigmine bromide did not affect the number of stools or tracheal insufflation pressure. CONCLUSIONS: Our 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.
Assuntos
Agonistas Muscarínicos , Antagonistas Muscarínicos , Receptor Muscarínico M3 , Bexiga Urinária , Animais , Agonistas Muscarínicos/farmacologia , Antagonistas Muscarínicos/farmacologia , Contração Muscular , Ratos , Ratos Sprague-Dawley , Receptor Muscarínico M3/fisiologiaRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Plectranthus vettiveroides (Jacob) N.P. Singh & B.D. Sharma is a traditional medicinal plant used in Siddha System of Medicine and its aromatic root is used to reduce the elevated blood pressure. AIM: The aim of the present study was to study vasorelaxant property of the root essential oil nanoemulsion (EON) of P. vettiveroides. METHODS: The EON was formulated to enhance the solubility and bioavailability and characterized. The preliminary screening was performed by treating the EON with aortic rings pre-contracted with phenylephrine (1 µM) and potassium chloride (80 mM). The role of K⺠channels in EON induced vasorelaxation was investigated by pre-incubating the aortic rings with different K⺠channel inhibitors namely, glibenclamide (a non-specific ATP sensitive K⺠channel blocker, 10 µM), TEA (a Ca2⺠activated non-selective K⺠channel blocker, 10-2 M), 4-AP (a voltage-activated K⺠channel blocker, 10-3 M) and barium chloride (inward rectifier K⺠channel blocker, 1 mM). The involvement of extracellular Ca2+ was performed by adding cumulative dose of extracellular calcium in the presence and absence of EON and the concentration-response curve (CRC) obtained is compared. Similarly, the role of nitric oxide synthase, muscarinic and prostacyclin receptors on EON induced vasorelaxation were evaluated by pre-incubating the aortic rings with their inhibitors and the CRC obtained in the presence and absence of inhibitor were compared. RESULTS: The GC-MS and GC-FID analyses of the root essential oil revealed the presence of 62 volatile compounds. The EON exhibited significant vasorelaxant effect through nitric oxide-mediated pathway, G-protein coupled muscarinic (M3) receptor pathway, involvement of K+ channels (KATP, KIR, KCa), and blocking of the calcium influx by receptor-operated calcium channel. CONCLUSION: It is concluded that the root essential oil of P. vettiveroides is possessing marked vasorelaxant property. The multiple mechanisms of action of the essential oil of P. vettiveroides make it a potential source of antihypertensive drug.
Assuntos
Anti-Hipertensivos/farmacologia , Aorta Torácica/efeitos dos fármacos , Óleos Voláteis/farmacologia , Plectranthus , Vasodilatadores/farmacologia , Animais , Anti-Hipertensivos/química , Aorta Torácica/fisiologia , Cálcio/fisiologia , Canais de Cálcio/fisiologia , Emulsões , Receptores de Inositol 1,4,5-Trifosfato/fisiologia , Canais KATP/fisiologia , Masculino , Óxido Nítrico/fisiologia , Óleos Voláteis/química , Compostos Fitoquímicos/análise , Compostos Fitoquímicos/farmacologia , Raízes de Plantas , Canais de Potássio Corretores do Fluxo de Internalização/fisiologia , Ratos Wistar , Receptor Muscarínico M3/fisiologia , Vasodilatação/efeitos dos fármacos , Vasodilatadores/químicaRESUMO
Glial fibrillary acidic protein expressing (GFAP+) glia modulate nociceptive neuronal activity in both the peripheral nervous system (PNS) and the central nervous system (CNS). Resident GFAP+ glia in dorsal root ganglia (DRG) known as satellite glial cells (SGCs) potentiate neuronal activity by releasing pro-inflammatory cytokines and neuroactive compounds. In this study, we tested the hypothesis that SGC Gq-coupled receptor (Gq-GPCR) signaling modulates pain sensitivity in vivo using Gfap-hM3Dq mice. Complete Freund's adjuvant (CFA) was used to induce inflammatory pain, and mechanical sensitivity and thermal sensitivity were used to assess the neuromodulatory effect of glial Gq-GPCR activation in awake mice. Pharmacogenetic activation of Gq-GPCR signaling in sensory SGCs decreased heat-induced nociceptive responses and reversed inflammation-induced mechanical allodynia via peripheral adenosine A1 receptor activation. These data reveal a previously unexplored role of sensory SGCs in decreasing afferent excitability. The identified molecular mechanism underlying the analgesic role of SGCs offers new approaches for reversing peripheral nociceptive sensitization.
Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/fisiologia , Hiperalgesia/prevenção & controle , Inflamação/fisiopatologia , Neuroglia/enzimologia , Nociceptividade/fisiologia , Receptor A1 de Adenosina/fisiologia , Receptor Muscarínico M3/fisiologia , Animais , Benzilatos/farmacologia , Clozapina/análogos & derivados , Clozapina/farmacologia , Adjuvante de Freund/toxicidade , Genes Sintéticos , Temperatura Alta , Hiperalgesia/fisiopatologia , Inflamação/induzido quimicamente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Agonistas Muscarínicos/farmacologia , Neuroglia/fisiologia , Nortropanos/farmacologia , Regiões Promotoras Genéticas , Agonistas do Receptor Purinérgico P1/farmacologia , Antagonistas de Receptores Purinérgicos P1/farmacologia , Receptor A1 de Adenosina/efeitos dos fármacos , Receptor Muscarínico M3/efeitos dos fármacos , Receptor Muscarínico M3/genética , Receptores Acoplados a Proteínas G , Proteínas Recombinantes de Fusão/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Teofilina/análogos & derivados , Teofilina/farmacologia , Tato , Xantinas/farmacologiaRESUMO
Previously, we reported that hepatic muscarinic receptors modulate both acute and chronic liver injury, however, the role of muscarinic receptors in fatty liver disease is unclear. We observed in patients who underwent weight loss surgery, a decrease in hepatic expression of M3 muscarinic receptors (M3R). We also observed that fat loading of hepatocytes, increased M3R expression. Based on these observations, we tested the hypothesis that M3R regulate hepatocyte lipid accumulation. Incubation of AML12 hepatocytes with 1â¯mM oleic acid resulted in lipid accumulation that was significantly reduced by co-treatment with a muscarinic agonist (pilocarpine or carbachol), an effect blocked by atropine (a muscarinic antagonist). Similar treatment of Hepa 1-6 cells, a mouse hepatoblastoma cell line, showed comparable results. In both, control and fat-loaded AML12 cells, pilocarpine induced time-dependent AMPKα phosphorylation and significantly up-regulated lipolytic genes (ACOX1, CPT1, and PPARα). Compound C, a selective and reversible AMPK inhibitor, significantly blunted pilocarpine-mediated reduction of lipid accumulation and pilocarpine-mediated up-regulation of lipolytic genes. BAPTA-AM, a calcium chelator, and STO-609, a calcium/calmodulin-dependent protein kinase kinase inhibitor, attenuated agonist-induced AMPKα phosphorylation. Finally, M3R siRNA attenuated agonist-induced AMPKα phosphorylation as well as agonist-mediated reduction of hepatocyte steatosis. In conclusion, this proof-of-concept study demonstrates that M3R has protective effects against hepatocyte lipid accumulation by activating AMPK pathway and is a potential therapeutic target for non-alcoholic fatty liver disease.
Assuntos
Proteínas Quinases Ativadas por AMP/fisiologia , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/fisiologia , Hepatócitos/metabolismo , Metabolismo dos Lipídeos , Receptor Muscarínico M3/fisiologia , Animais , Células Cultivadas , Humanos , Camundongos , PPAR alfa/fisiologia , Fosforilação , Receptor Muscarínico M1/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Calcium influx via store-operated calcium entry (SOCE) has an important role for regulation of vast majority of cellular physiological events. MAPK signalling is also another pivotal modulator of many cellular functions. However, the relationship between SOCE and MAPK is not well understood. In this study, we elucidated the involvement of SOCE in Gαq/11 protein-mediated activation of p38 MAPK in an intestinal epithelial cell line HT-29/B6. In this cell line, we previously showed that the stimulation of M3 muscarinic acetylcholine receptor (M3-mAChR) but not histamine H1 receptor (H1R) led to phosphorylation of p38 MAPK which suppressed tumor necrosis factor-α (TNF-α)-induced NF-κB signalling through ADAM17 protease-mediated shedding of TNF receptor-1 (TNFR1). First, we found that stimulation of M3-mAChR and protease-activated receptor-2 (PAR-2) but not H1R induced persistent upregulation of cytosolic Ca2+ concentration through SOCE. Activation of M3-mAChR or PAR-2 also suppressed TNF-α-induced NF-κB phosphorylation, which was dependent on the p38 MAPK activity. Time course experiments revealed that M3-mAChR stimulation evoked intracellular Ca2+-dependent early phase p38 MAPK phosphorylation and extracellular Ca2+-dependent later phase p38 MAPK phosphorylation. This later phase p38 MAPK phosphorylation, evoked by M3-mAChRs or PAR-2, was abolished by inhibition of SOCE. Thapsigargin or ionomycin also phosphorylate p38 MAPK by Ca2+ influx through SOCE, leading to suppression of TNF-α-induced NF-κB phosphorylation. Finally, we showed that p38 MAPK was essential for thapsigargin-induced cleavage of TNFR1 and suppression of TNF-α-induced NF-κB phosphorylation. In conclusion, SOCE is important for p38 MAPK phosphorylation and is involved in TNF-α signalling suppression.
Assuntos
Cálcio/fisiologia , Receptor Muscarínico M3/fisiologia , Receptor PAR-2/fisiologia , Receptores Histamínicos H1/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Células HT29 , Humanos , NF-kappa B/metabolismoRESUMO
AIM: To examine putative interaction between adrenergic and muscarinic contractile activation in the human urinary outflow tract. METHODS: Tissue from the trigone and prostatic urethra was obtained from 12 cystectomy and 16 prostatectomy specimen. Contractions were elicited by exposure to exogenous agonists before and after inhibition of Rho kinase and protein kinase c (PKC). Immunofluorescence and Western-blot studies were performed using antibodies to muscarinic M3-receptors (M3-R) and alpha1A-adrenoreceptors (alpha1A-AR). The study is registered with ClinicalTrials.gov, number NCT01227447. RESULTS: There was strong co-localization of M3-R and alpha1A-AR on trigonal and urethral myocytes. Western blot analysis revealed a significantly higher expression of alpha1A-AR in the superficial than in the deep trigone. Phenylephrine (PE, 1 µm) augmented contractions induced by carbachol (CA, 3 µm) to more than threefold control in the male superficial trigone, and to about sevenfold control in the proximal urethra. No such potentiation could be detected in female bladder outlet. Both PKC inhibitor GF 109203X and Rho kinase inhibitor Y-27632 reduced responses to 1 µM PE as well as to 3 µM CA significantly. However, the synergistic effect of the combined intervention remained proportionally unaffected. CONCLUSIONS: Muscarinic and adrenergic receptor activation exerts a strong synergistic effect in the male human bladder trigone and proximal urethra.
Assuntos
Receptor Muscarínico M3/fisiologia , Receptores Adrenérgicos alfa 1/fisiologia , Sistema Urinário/inervação , Agonistas alfa-Adrenérgicos/farmacologia , Idoso , Idoso de 80 Anos ou mais , Carbacol/farmacologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Agonistas Muscarínicos/farmacologia , Contração Muscular/efeitos dos fármacos , Fenilefrina/farmacologia , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Receptor Muscarínico M3/efeitos dos fármacos , Receptores Adrenérgicos alfa 1/efeitos dos fármacos , Caracteres Sexuais , Uretra/efeitos dos fármacos , Uretra/fisiologia , Quinases Associadas a rho/antagonistas & inibidoresRESUMO
Within the gastrointestinal stem cell niche, nerves help to regulate both normal and neoplastic stem cell dynamics. Here, we reveal the mechanisms underlying the cancer-nerve partnership. We find that Dclk1+ tuft cells and nerves are the main sources of acetylcholine (ACh) within the gastric mucosa. Cholinergic stimulation of the gastric epithelium induced nerve growth factor (NGF) expression, and in turn NGF overexpression within gastric epithelium expanded enteric nerves and promoted carcinogenesis. Ablation of Dclk1+ cells or blockade of NGF/Trk signaling inhibited epithelial proliferation and tumorigenesis in an ACh muscarinic receptor-3 (M3R)-dependent manner, in part through suppression of yes-associated protein (YAP) function. This feedforward ACh-NGF axis activates the gastric cancer niche and offers a compelling target for tumor treatment and prevention.
Assuntos
Acetilcolina/fisiologia , Fator de Crescimento Neural/fisiologia , Transdução de Sinais/fisiologia , Neoplasias Gástricas/etiologia , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Animais , Proteínas de Ciclo Celular , Quinases Semelhantes a Duplacortina , Mucosa Gástrica/inervação , Camundongos , Camundongos Endogâmicos C57BL , Fosfoproteínas/fisiologia , Proteínas Serina-Treonina Quinases/análise , Receptor Muscarínico M3/fisiologia , Proteínas de Sinalização YAPRESUMO
The aim of this study was to compare the effect of antimuscarinic antagonists on carbachol-induced contraction of normal human bladder and detrusor overactivity associated with benign prostatic hyperplasia (DO/BPH). Samples of human bladder muscle were obtained from patients undergoing total cystectomy for bladder cancer (normal bladder), and those undergoing retropubic prostatectomy for BPH. All of the patients with DO/BPH had detrusor overactivity according to urodynamic studies. Detrusor muscle strips were mounted in 10-ml organ baths containing Krebs solution, and concentration-response curves for carbachol were obtained in the presence of antimuscarinic antagonists (4-DAMP, methoctramine, pirenzepine, tolterodine, solifenacin, trospium, propiverine, oxybutynin, and imidafenacin) or vehicle. All antagonists competitively antagonized concentration-response curves to carbachol with high affinities in normal bladder. The rank order of mean pA2 values was as follows: trospium (10.1) > 4-DAMP (9.87), imidafenacin (9.3) > solifenacin (8.8) > tolterodine (8.6) > oxybutynin (8.3) > propiverine (7.7) > pirenzepine (7.4) > methoctramine (6.6). The effects of these antimuscarinic antagonists did not change when tested with DO/BPH bladder, suggesting that each antimuscarinic antagonist has a similar effect in this condition. Schild plots showed a slope corresponding to unity, except for propiverine with DO/BPH detrusor. In conclusion, M3-receptors mainly mediate contractions in human bladder strips with normal state and DO/BPH.
Assuntos
Carbacol/farmacologia , Agonistas Muscarínicos/farmacologia , Antagonistas Muscarínicos/farmacologia , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Hiperplasia Prostática/complicações , Hiperplasia Prostática/fisiopatologia , Receptor Muscarínico M3/fisiologia , Bexiga Urinária Hiperativa/etiologia , Bexiga Urinária Hiperativa/fisiopatologia , Bexiga Urinária/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Técnicas In Vitro , MasculinoRESUMO
BACKGROUND: The role of muscarinic receptors in mucosal homeostasis, response to enteric pathogens, and modulation of immune cell function is undefined. METHODS: The contribution of type 3 muscarinic receptors (M3R) to mucosal homeostasis within the colon and host defense against Citrobacter rodentium was determined in uninfected and C. rodentium-infected WT and M3R-deficient (Chrm3) mice. In addition, WT and Chrm3 bone marrow-derived macrophages were studied to determine the ability of M3R to modulate macrophage phenotype and function. RESULTS: In Chrm3 mice, clearance of C. rodentium was delayed despite an amplified TH1/TH17 response. Delayed clearance of C. rodentium from Chrm3 mice was associated with prolonged adherence of bacteria to colonic mucosa, decreased goblet cell number, and decreased mucin 2 gene expression. Treatment of bone marrow-derived macrophages with bethanechol, a muscarinic-selective agonist, induced a classically activated macrophage phenotype, which was dependent on M3R expression. Chrm3 bone marrow-derived macrophages retained their ability to attain a classically activated macrophage phenotype when treated with the TH1 cytokine IFN-γ. CONCLUSIONS: In Chrm3 mice, mucin production is attenuated and is associated with prolonged adherence of C. rodentium to colonic mucosa. The immune response, as characterized by production of TH1/TH17 cytokines, in C. rodentium-infected Chrm3 mice is intact. In addition, M3R activity promotes the development of classically activated macrophages. Our data establish a role for M3R in host defense against C. rodentium through effects on goblet cell mucus production and in the modulation of macrophage phenotype and function.
Assuntos
Citrobacter rodentium/imunologia , Infecções por Enterobacteriaceae/microbiologia , Infecções por Enterobacteriaceae/prevenção & controle , Células Caliciformes/imunologia , Macrófagos/imunologia , Receptor Muscarínico M3/fisiologia , Animais , Proliferação de Células , Citocinas/genética , Citocinas/metabolismo , Infecções por Enterobacteriaceae/imunologia , Feminino , Imunofluorescência , Células Caliciformes/microbiologia , Células Caliciformes/patologia , Macrófagos/microbiologia , Macrófagos/patologia , Masculino , Camundongos , Camundongos Knockout , Mucinas/genética , Mucinas/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Type 2 diabetes (T2D) has emerged as a major threat to human health in most parts of the world. Therapeutic strategies aimed at improving pancreatic ß cell function are predicted to prove beneficial for the treatment of T2D. In the present study, we demonstrate that drug-mediated, chronic, and selective activation of ß cell G(q) signaling greatly improve ß cell function and glucose homeostasis in mice. These beneficial metabolic effects were accompanied by the enhanced expression of many genes critical for ß cell function, maintenance, and differentiation. By employing a combination of in vivo and in vitro approaches, we identified a novel ß cell pathway through which receptor-activated G(q) leads to the sequential activation of ERK1/2 and IRS2 signaling, thus triggering a series of events that greatly improve ß cell function. Importantly, we found that chronic stimulation of a designer G(q)-coupled receptor selectively expressed in ß cells prevented both streptozotocin-induced diabetes and the metabolic deficits associated with the consumption of a high-fat diet in mice. Since ß cells are endowed with numerous receptors that mediate their cellular effects via activation of G(q)-type G proteins, our findings provide a rational basis for the development of novel antidiabetic drugs targeting this class of receptors.
Assuntos
Clozapina/análogos & derivados , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Hipoglicemiantes/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Receptores Acoplados a Proteínas G/agonistas , Animais , Linhagem Celular Tumoral , Proliferação de Células , Clozapina/farmacologia , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/prevenção & controle , Avaliação Pré-Clínica de Medicamentos , Feminino , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Expressão Gênica/efeitos dos fármacos , Proteínas Substratos do Receptor de Insulina/genética , Proteínas Substratos do Receptor de Insulina/metabolismo , Proteínas Substratos do Receptor de Insulina/fisiologia , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/fisiologia , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Terapia de Alvo Molecular , Agonistas Muscarínicos/farmacologia , Engenharia de Proteínas , Receptor Muscarínico M3/agonistas , Receptor Muscarínico M3/fisiologia , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Recombinantes/agonistas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
We and others have shown that epithelial Na(+) channels (ENaC) in alveolar type 2 (AT2) cells are activated by ß2 agonists, steroid hormones, elevated oxygen tension, and by dopamine. Although acetylcholine receptors (AChRs) have been previously described in the lung, there are few reports of whether cholinergic agonists alter sodium transport in the alveolar epithelium. Therefore, we investigated how cholinergic receptors regulate ENaC activity in primary cultures of rat AT2 cells using cell-attached patch-clamp recordings to assess ENaC activity. We found that the muscarinic agonists, carbachol (CCh) and oxotremorine, activated ENaC in a dose-dependent manner but that nicotine did not. CCh-induced activation of ENaC was blocked by atropine. Western blotting and immunohistochemistry suggested that muscarinic M2 and M3 receptors (mAChRs) but not nicotinic receptors were present in AT2 cells. Endogenous RhoA and GTP-RhoA increased in response to CCh and the increase was reduced by pretreatment with atropine. We showed that Y-27632, an inhibitor of Rho-associated protein kinase (ROCK), abolished endogenous ENaC activity and inhibited the activation of ENaC by CCh. We also showed that ROCK signaling was necessary for ENaC stability in 2F3 cells, a model for AT2 cells. Our results showed that muscarinic agonists activated ENaC in rat AT2 cells through M2 and/or M3 mAChRs probably via a RhoA/ROCK signaling pathway.
Assuntos
Células Epiteliais Alveolares/metabolismo , Canais Epiteliais de Sódio/metabolismo , Células Epiteliais Alveolares/efeitos dos fármacos , Células Epiteliais Alveolares/fisiologia , Amidas/farmacologia , Animais , Atropina/farmacologia , Carbacol/farmacologia , Células Cultivadas , Ativação Enzimática , Agonistas do Canal de Sódio Epitelial/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Agonistas Muscarínicos/farmacologia , Antagonistas Muscarínicos/farmacologia , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Oxotremorina/farmacologia , Técnicas de Patch-Clamp , Piridinas/farmacologia , Ratos , Ratos Sprague-Dawley , Receptor Muscarínico M2/metabolismo , Receptor Muscarínico M2/fisiologia , Receptor Muscarínico M3/metabolismo , Receptor Muscarínico M3/fisiologia , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
M3 subtype muscarinic receptors (CHRM3) are over-expressed in colon cancer. In this study, we used Apc(min/+) mice to identify the role of Chrm3 expression in a genetic model of intestinal neoplasia, explored the role of Chrm3 in intestinal mucosal development and determined the translational potential of inhibiting muscarinic receptor activation. We generated Chrm3-deficient Apc(min/+) mice and compared intestinal morphology and tumor number in 12-week-old Apc(min/+)Chrm3(-/-) and Apc(min/+)Chrm3(+/+) control mice. Compared with Apc(min/+)Chrm3(+/+) mice, Apc(min/+)Chrm3(-/-) mice showed a 70 and 81% reduction in tumor number and volume, respectively (P < 0.01). In adenomas, ß-catenin nuclear staining was reduced in Apc(min/+)Chrm3(-/-) compared with Apc(min/+)Chrm3(+/+) mice (P < 0.02). Whereas Apc gene mutation increased the number of crypt and Paneth cells and decreased villus goblet cells, these changes were absent in Apc(min/+)Chrm3(-/-) mice. To determine whether pharmacological inhibition of muscarinic receptor activation attenuates intestinal neoplasia, we treated 6-week-old Apc(min/+) mice with scopolamine butylbromide, a non-subtype-selective muscarinic receptor antagonist. After 8 weeks of continuous treatment, scopolamine butylbromide-treated mice showed a 22% reduction in tumor number (P = 0.027) and a 36% reduction in tumor volume (P = 0.004) as compared with control mice. Compared with Chrm3 gene ablation, the muscarinic antagonist was less efficacious, most probably due to shorter duration of treatment and incomplete blockade of muscarinic receptors. Overall, these findings indicate that interplay of Chrm3 and ß-catenin signaling is important for intestinal mucosal differentiation and neoplasia and provide a proof-of-concept that pharmacological inhibition of muscarinic receptor activation can attenuate intestinal neoplasia in vivo.
Assuntos
Brometo de Butilescopolamônio/farmacologia , Genes APC , Neoplasias Intestinais/prevenção & controle , Intestino Delgado/patologia , Antagonistas Muscarínicos/farmacologia , Receptor Muscarínico M3/fisiologia , Animais , Feminino , Neoplasias Intestinais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptor Muscarínico M3/genética , beta Catenina/análiseRESUMO
BACKGROUND: Acetylcholine, the primary parasympathetic neurotransmitter in the airways, plays an important role in bronchoconstriction and mucus production. Recently, it has been shown that acetylcholine, by acting on muscarinic receptors, is also involved in airway inflammation and remodelling. The mechanism(s) by which muscarinic receptors regulate inflammatory responses are, however, still unknown. METHODS: The present study was aimed at characterizing the effect of muscarinic receptor stimulation on cytokine secretion by human airway smooth muscle cells (hASMc) and to dissect the intracellular signalling mechanisms involved. hASMc expressing functional muscarinic M2 and M3 receptors were stimulated with the muscarinic receptor agonist methacholine, alone, and in combination with cigarette smoke extract (CSE), TNF-α, PDGF-AB or IL-1ß. RESULTS: Muscarinic receptor stimulation induced modest IL-8 secretion by itself, yet augmented IL-8 secretion in combination with CSE, TNF-α or PDGF-AB, but not with IL-1ß. Pretreatment with GF109203X, a protein kinase C (PKC) inhibitor, completely normalized the effect of methacholine on CSE-induced IL-8 secretion, whereas PMA, a PKC activator, mimicked the effects of methacholine, inducing IL-8 secretion and augmenting the effects of CSE. Similar inhibition was observed using inhibitors of IκB-kinase-2 (SC514) and MEK1/2 (U0126), both downstream effectors of PKC. Accordingly, western blot analysis revealed that methacholine augmented the degradation of IκBα and the phosphorylation of ERK1/2 in combination with CSE, but not with IL-1ß in hASMc. CONCLUSIONS: We conclude that muscarinic receptors facilitate CSE-induced IL-8 secretion by hASMc via PKC dependent activation of IκBα and ERK1/2. This mechanism could be of importance for COPD patients using anticholinergics.
Assuntos
Acetilcolina/farmacologia , Brônquios/metabolismo , Mediadores da Inflamação/metabolismo , Músculo Liso/metabolismo , Receptor Muscarínico M2/metabolismo , Receptor Muscarínico M3/metabolismo , Receptores Muscarínicos/metabolismo , Células Cultivadas , Humanos , Mediadores da Inflamação/fisiologia , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Miócitos de Músculo Liso/metabolismo , Receptor Muscarínico M2/fisiologia , Receptor Muscarínico M3/fisiologia , Receptores Muscarínicos/fisiologia , Transdução de Sinais/imunologia , Fumar/metabolismoRESUMO
We investigated pharmacological characteristics of the itch-associated response to chronic dermatitis induced by 4-ethoxymethylene-2-phenyl-2-oxazolin-5-one (oxazolone) repeated application in mice. Application of an oxazolone challenge to mice with oxazolone-induced chronic dermatitis evoked severe and transient scratching behavior for up to 1h. Thereafter, mild and continuous scratching behavior was observed for at least 8 h. Both severe and continuous scratching behaviors were suppressed by the opioid-receptor antagonist naltrexone, but not by the H(1) histamine-receptor antagonist fexofenadine, 5-hydroxytryptamine-2 (5-HT(2))-receptor antagonist methysergide, NK(1)-receptor antagonist LY303870, cyclooxygenase inhibitor indomethacin, or the platelet-activating factor-receptor antagonist YM264. The severe scratching behavior was suppressed by the 5-lipoxygenase inhibitor zileuton and leukotriene B(4)-receptor antagonist ONO-4057, but not by the cysteinyl leukotriene-receptor antagonist montelukast. The continuous scratching behavior was suppressed by pretreatment with the non-selective muscarinic acetylcholine-receptor antagonist atropine and M(3) muscarinic acetylcholine-receptor antagonist darifenacin. These results suggest that leukotriene B(4) receptor and M(3) muscarinic acetylcholine receptor are involved in the itch-associated response induced by repeated application of oxazolone in mice.
Assuntos
Antialérgicos/farmacologia , Antipruriginosos/farmacologia , Dermatite Atópica/fisiopatologia , Prurido/fisiopatologia , Receptor Muscarínico M3/fisiologia , Receptores do Leucotrieno B4/fisiologia , Animais , Antialérgicos/uso terapêutico , Antipruriginosos/uso terapêutico , Comportamento Animal/efeitos dos fármacos , Dermatite Atópica/tratamento farmacológico , Dermatite Atópica/imunologia , Relação Dose-Resposta a Droga , Haptenos/administração & dosagem , Haptenos/imunologia , Antagonistas de Leucotrienos/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Antagonistas Muscarínicos/uso terapêutico , Oxazolona/administração & dosagem , Oxazolona/imunologia , Prurido/induzido quimicamente , Prurido/tratamento farmacológico , Prurido/imunologia , Receptor Muscarínico M3/antagonistas & inibidores , Receptores do Leucotrieno B4/antagonistas & inibidores , Fatores de TempoRESUMO
OBJECTIVES: To investigate the effects of various muscarinic acetylcholine receptor (mAChR) antagonists, including selective M2 and M3 mAChR antagonists, on bladder overactivity. It has been proposed that the urothelium modulates the activity of bladder afferent pathways. However, the differential roles of mAChR subtypes in local bladder afferent activation remain unclear. METHODS: Cystometry was performed in urethane-anesthetized female rats. We examined the effects of intravesical administration of antimuscarinic agents (nonselective mAChR antagonists: atropine sulfate, tolterodine tartrate, and propiverine hydrochloride; M2-selective antagonists: dimethindene maleate and methoctramine hemihydrate; M3-selective antagonists: darifenacin hydrobromide and 4-DAMP) on bladder overactivity induced by oxotremorine-M (oxo-M; nonselective mAChR agonist). RESULTS: Intravesical administration of oxo-M (200 microM) elicited bladder overactivity as evidenced by decreased intercontraction interval, bladder capacity, and pressure threshold. These effects were blocked by intravesical administration of nonselective or M2-selective antagonists (30-60 microM), whereas M3-selective antagonists (150 microM) did not suppress the overactivity. When instilled intravesically by itself, none of the antimuscarinic agents (nonselective, M2-selective or M3-selective antagonists) affected any cystometric parameters. CONCLUSIONS: The M2 mAChR subtype plays an important role in the local cholinergic modulation of bladder afferent activity that contributes to bladder overactivity in normal rats. Therefore, it is expected that antimuscarinic agents that have antagonistic activity against M2 mAChR can be more beneficial for the treatment of patients with overactive bladder if enhanced acetylcholine mechanisms are involved in pathogenesis of overactive bladder.
Assuntos
Receptor Muscarínico M2/fisiologia , Receptor Muscarínico M3/fisiologia , Bexiga Urinária Hiperativa/fisiopatologia , Bexiga Urinária/inervação , Bexiga Urinária/fisiologia , Vias Aferentes , Animais , Feminino , Ratos , Ratos Sprague-Dawley , Receptor Muscarínico M2/antagonistas & inibidores , Receptor Muscarínico M3/antagonistas & inibidoresRESUMO
BACKGROUND: Cholinergic receptors are expressed in small cell lung cancer (SCLC); however, the distinct functions of muscarinic cholinergic receptor 3 (mAChR3) and the nicotinic cholinergic receptor (nAChR) in SCLC have not yet been completely elucidated. MATERIALS AND METHODS: RT-PCR and Western blotting were used to investigate the expression of cholinergic receptors. Flow cytometry was used to detect the integrin expression. Cell proliferation, adhesion and migration assays were carried out in vitro to determine the roles of the cholinergic receptors in SBC3 human SCLC cells. RESULTS: Both mAChR3 and nAChR were expressed in the SBC3 cells. Acetylcholine iodide (Ach) stimulated SBC3 cell proliferation, adhesion and migration toward fibronectin (Fn). The mAChR3 antagonist, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), or the nAChR antagonist, mecamylamine hydrochloride (Meca), inhibited SBC3 cell proliferation in the presence or the absence of exogenous Ach. 4-DAMP abrogated cell adhesion and migration toward Fn induced by Ach, while Meca had no effect. Interestingly, Ach did not alter Fn receptor (alphavbeta1 or alpha5beta1 integrin) expression, while anti-beta1 integrin antibody or anti-alphav and anti-alpha5 integrin antibody completely abrogated cell adhesion to Fn induced by Ach. CONCLUSION: Both mAChR3 and nAChR are expressed in SCLC. SBC3 cell proliferation is regulated in vitro through both cholinergic receptors. In contrast, SBC3 cell migration and adhesion toward Fn are modulated only by mAChR. Moreover, the stimulatory effects of Ach on cell adhesion and migration through mAChR3 are presumably modulated by functional alteration of alphavbeta1 and alpha5beta1 integrin, but not by any variation in their expression. The mAChR3 antagonist may therefore be a beneficial therapeutic modality for SCLC patients, especially those with chronic obstructive pulmonary disease (COPD) as a comorbidity.
Assuntos
Carcinoma de Células Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Receptor Muscarínico M3/fisiologia , Receptores Nicotínicos/fisiologia , Western Blotting , Carcinoma de Células Pequenas/genética , Carcinoma de Células Pequenas/patologia , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Agonistas Colinérgicos/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Antagonistas Muscarínicos/farmacologia , Piperidinas/farmacologia , Polissacarídeos/farmacologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptor Muscarínico M3/biossíntese , Receptor Muscarínico M3/genética , Receptores Nicotínicos/biossíntese , Receptores Nicotínicos/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
OBJECTIVES: The aim was to characterize the effect of penehyclidine hydrochloride, which mediates the relaxation of guinea-pig isolated gastrointestinal smooth muscle, on muscarinic receptor subtypes. METHODS: Radioimmune assay was used to determine cAMP levels in isolated guinea-pig gastrointestinal smooth muscle to compare the selective effects of penehyclidine hydrochloride on muscarinic receptor subtypes. KEY FINDINGS: The results indicated that the relaxing effect of penehyclidine hydrochloride on isolated gastrointestinal smooth muscle contraction induced by acetylcholine was stronger than that of atropine (based on PA2 values). In the radioimmune assay, penehyclidine hydrochloride increased the cAMP content in isolated guinea-pig stomach smooth muscle and decreased the cAMP content in isolated guinea-pig intestinal smooth muscle, but the difference was not statistically significant at a dose of 10 mumol/l. CONCLUSIONS: The results suggest that penehyclidine hydrochloride has little or no effect on M2 receptor subtypes in guinea-pig gastrointestinal smooth muscle.
Assuntos
Trato Gastrointestinal/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Quinuclidinas/farmacologia , Receptor Muscarínico M2/fisiologia , Receptor Muscarínico M3/fisiologia , Acetilcolina/farmacologia , Animais , Atropina/farmacologia , AMP Cíclico/biossíntese , Relação Dose-Resposta a Droga , Feminino , Trato Gastrointestinal/fisiologia , Cobaias , Técnicas In Vitro , Masculino , Contração Muscular/efeitos dos fármacos , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso/fisiologia , Ensaio Radioligante , Receptor Muscarínico M2/antagonistas & inibidores , Receptor Muscarínico M3/antagonistas & inibidoresRESUMO
Acetylcholine (ACh) is a major excitatory neurotransmitter in the myenteric plexus, and it regulates its own release acting via muscarinic autoreceptors. Adenosine released from stimulated myenteric neurons modulates ACh release preferentially via facilitatory A(2A) receptors. In this study, we investigated how muscarinic and adenosine receptors interplay to regulate ACh from the longitudinal muscle-myenteric plexus of the rat ileum. Blockade of the muscarinic M(2) receptor with 11-[[2-1[(diethylamino) methyl-1-piperidinyl]- acetyl]]-5,11-dihydro-6H-pyrido [2,3-b][1,4] benzodiazepine-6-one (AF-DX 116), 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and atropine facilitated [3H]ACh release evoked by short stimulation trains (5 Hz, 200 pulses). Prolonging stimulus train length (>750 pulses) shifted muscarinic autoinhibition towards facilitatory M(3) receptors activation, as predicted by blockade with J104129 (a selective M(3) antagonist), 4-DAMP and atropine, whereas the selective M(2) antagonist, AF-DX 116, was without of effect. Blockade of A(2A) receptors with ZM 241385, inhibition of adenosine transport with dipyridamole, and inhibition of ecto-5'-nucleotidase with concanavalin A, all attenuated release inhibition caused by 4-DAMP. J104129 and 4-DAMP, but not AF-DX 116, decreased ( approximately 60%) evoked adenosine outflow (5 Hz, 3000 pulses). Oxotremorine (300 micromol L(-1)) facilitated the release of [3H]ACh (34 +/- 4%, n = 5) and adenosine (57 +/- 3%, n = 6) from stimulated myenteric neurons. 4-DAMP, dipyridamole and concanavalin A prevented oxotremorine-induced facilitation. ZM 241385 blocked oxotremorine facilitation of [3H]ACh release, but kept adenosine outflow unchanged. Thus, ACh modulates its own release from myenteric neurons by activating inhibitory M(2) and facilitatory M(3) autoreceptors. While the M(2) inhibition is prevalent during brief stimulation periods, muscarinic M(3) facilitation is highlighted during sustained nerve activity as it depends on extracellular adenosine accumulation leading to activation of facilitatory A(2A) receptors.
Assuntos
Acetilcolina/metabolismo , Agonistas do Receptor A2 de Adenosina , Adenosina/metabolismo , Plexo Mientérico/metabolismo , Neurônios/metabolismo , Receptor Muscarínico M3/fisiologia , Nucleotídeos de Adenina/farmacologia , Adenosina/fisiologia , Animais , AMP Cíclico/fisiologia , Relação Dose-Resposta a Droga , Estimulação Elétrica , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Feminino , Íleo/efeitos dos fármacos , Técnicas In Vitro , Masculino , Agonistas Muscarínicos/farmacologia , Antagonistas Muscarínicos/farmacologia , Contração Muscular/efeitos dos fármacos , Músculo Liso/inervação , Músculo Liso/fisiologia , Neurônios/fisiologia , Ratos , Ratos Wistar , Receptor Muscarínico M3/efeitos dos fármacos , Sinapses/efeitos dos fármacosRESUMO
Extracellular tau promotes an increase in the level of intracellular calcium in cultured neuronal cells. We have found that such increase is impaired in the presence of antagonists of muscarinic receptors. In order to identify the nature of those receptors, we have tested the effect of different specific muscarinic receptor antagonists on tau promoted calcium increase. Our results indicate that the increase does not take place in the presence of antagonists of muscarinic (mainly M1 and M3) receptors. A similar increase in intracellular calcium was found in non-neuronal cells transfected with cDNA of M1 and M3 muscarinic receptors when tau was added. These results suggest that observed effect of tau protein on neuronal (neuroblastoma and primary cultures of hippocampal and cortical neurons) cells is through M1 and M3 muscarinic receptors. Therefore blocking M1 and for M3 receptors, by using specific receptor antagonists, can prevent that tau toxic effect that could take place in tauopathies.