RESUMO
PURPOSE: Muscarinic acetylcholine receptors (mAChRs) regulate a number of important physiological functions. Alteration of mAChR expression or function has been associated in the etiology of several pathologies including functional bladder disorders (e.g bladder pain syndrome/interstitial cystitis - BPS/IC). In a previous study we found specific mAChR expression patterns associated with BPS/IC, while correlation between protein and gene expression was lacking. Posttranslational regulatory mechanisms, e.g. altered intracellular receptor trafficking, could explain those differences. In addition, alternative G protein (GP) coupling could add to the pathophysiology via modulation of muscarinic signaling. In our proof-of-principle study, we addressed these questions in situ. We established PLA in combination with confocal laserscanning microscopy (CLSM) and 3D object reconstruction for highly specific detection and analysis of muscarinic 3 receptors (M3), G protein (GP) coupling and intracellular trafficking in human detrusor samples. MATERIAL AND METHODS: Paraffin sections of formalin-fixed bladder tissue (FFPE) of BPS/IC patients receiving transurethral biopsy were examined by Cy3-PLA for M3 expression, coupling of M3 to GPs (Gαq/11, Gαs, Gαi) and interaction of M3 with endocytic regulator proteins. Membranes were labeled with wheat germ agglutinin-Alexa Fluor®488, nuclei were stained with DAPI. Object density and co-localization were analyzed in 3D-reconstruction of high resolution confocal z-stacks. RESULTS: Confocal image stack processing resulted in well demarcated objects. Calculated receptor densities correlated significantly with existing confocal expression data, while significantly improved specificity of M3 detection by PLA was verified using bladder tissue samples from transgenic mice. 50-60% of the M3 receptor complexes were plasma membrane associated in human bladder detrusor. Application of PLA for M3 and GPs allowed visualization of M3-GP interactions and revealed individual GP-subtype coupling patterns. Detection of M3 interactions with endocytic trafficking proteins by PLA resulted in object sizes correlating with well-documented vesicle sizes of the endocytosis pathway. CONCLUSION: PLA enabled highly specific detection of M3 receptor expression, demonstration of M3/GP differential coupling and intracellular M3 trafficking in human detrusor smooth muscle cells. This new approach minimized background fluorescence and antibody cross-reactions resulting from single antibody application, and enhanced specificity due to the use of two primary antibodies. Use of subcellular markers allowed visualization of subcellular receptor location. PLA/CLSM allows analyses of muscarinic "receptor - G protein - promiscuity" and intracellular trafficking even in bladder paraffin sections and may give new insights into the etiology and pathology of BPS/IC.
Assuntos
Receptor Muscarínico M3/análise , Bexiga Urinária Hiperativa , Bioensaio , Imunofluorescência , Humanos , Limite de Detecção , Microscopia Confocal , Receptor Muscarínico M3/metabolismo , Transdução de Sinais , Bexiga Urinária Hiperativa/metabolismoRESUMO
Endocytosis has numerous functions in cellular homeostasis. Defects in the endocytic pathway of receptors may lead to dysfunction of salivary gland secretion. Therefore, elucidating the complex mechanisms of endocytosis may facilitate solutions for disease treatment and prevention. The muscarinic type 3 receptor (M3R), a G-protein-coupled receptor (GPCR) located in the plasma membrane, is involved in numerous physiological activities such as smooth muscle contraction and saliva secretion. M3R enters cells through clathrin-mediated endocytosis (CME), while flotillins (flot-1 and -2), highly conserved proteins residing in lipid-raft microdomains, make use of clathrin-independent endocytosis (CIE) for their internalization. Since these two proteins use two discrete pathways for endocytic entry, the association of flotillins with CME is poorly understood. We examined whether flotillins play a role in CME of M3R using immunoblotting, immunocytochemistry, confocal immunofluorescence microscopy, co-immunoprecipitation, and RNA interference techniques in secretory epithelial cells. Upon stimulation with a cholinergic agonist, M3R, flot-1, and flot-2 each internalized from the plasma membrane into intracellular sites. The addition of chlorpromazine and cytochalasin D, well-known inhibitors of CME, inhibited internalization of M3R via CME. Filipin III and methyl-ß-cyclodextrin (mßCD) acting as lipid raft inhibitors disrupted internalization of flot-1/2 via CIE. Interestingly, filipin III and mßCD slightly reduced expression level of M3R whereas chlorpromazine and cytochalasin D did not affect internalization of the flotillin isoforms. M3R and flot-1/2 colocalized and interacted with each other as they entered the cytosol during limited periods of incubation. Moreover, knockdown of flot-1 or -2 by flotillin-specific siRNA prevented internalization and reduced the endocytic efficiency of M3R. Our results suggest that flot-1 and -2 are partially involved in CME of M3R by facilitating its internalization.
Assuntos
Endocitose , Células Epiteliais/metabolismo , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/metabolismo , Receptor Muscarínico M3/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Linhagem Celular , Células Epiteliais/citologia , Humanos , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Receptor Muscarínico M3/análiseRESUMO
OBJECTIVES: This study determined if muscarinic receptors could mediate the cold stress-induced detrusor overactivity induced in type 2 diabetes mellitus rats. METHODS: Ten-week-old female Goto-Kakizaki diabetic rats (n = 12) and Wister Kyoto non-diabetic rats (n = 12) were maintained on a high-fat diet for 4 weeks. Cystometric investigations of the unanesthetized rats were carried out at room temperature (27 ± 2°C) for 20 min. They were intravenously administered imidafenacin (0.3 mg/kg, n = 6) or vehicle (n = 6). After 5 min, the rats were transferred to a low temperature (4 ± 2°C) for 40 min where the cystometry was continued. The rats were then returned to room temperature for the final cystometric measurements. Afterwards, expressions of bladder muscarinic receptor M3 and M2 messenger ribonucleic acids and proteins were assessed by reverse transcription polymerase chain reaction and immunohistochemistry. RESULTS: In non-diabetic Wister Kyoto rats, imidafenacin did not reduce cold stress-induced detrusor overactivity. In diabetic Goto-Kakizaki rats, just after transfer to a low temperature, the cold stress-induced detrusor overactivity in imidafenacin-treated rats was reduced compared with vehicle-treated rats. Within the urinary bladders, the ratio of M3 to M2 receptor messenger ribonucleic acid in the diabetic Goto-Kakizaki rats was significantly higher than that of the non-diabetic Wister Kyoto rats. The proportion of muscarinic M3 receptor-positive area within the detrusor in diabetic Goto-Kakizaki rats was also significantly higher than that in non-diabetic Wister Kyoto rats. CONCLUSIONS: Imidafenacin partially inhibits cold stress-induced detrusor overactivity in diabetic Goto-Kakizaki rats. In this animal model, muscarinic M3 receptors partially mediate cold stress-induced detrusor overactivity.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Imidazóis/farmacologia , RNA Mensageiro/análise , Receptor Muscarínico M2/análise , Receptor Muscarínico M3/análise , Bexiga Urinária Hiperativa/fisiopatologia , Animais , Temperatura Baixa , Diabetes Mellitus Tipo 2/complicações , Feminino , Ratos , Ratos Endogâmicos WKY , Receptor Muscarínico M2/antagonistas & inibidores , Receptor Muscarínico M2/genética , Receptor Muscarínico M3/antagonistas & inibidores , Receptor Muscarínico M3/genética , Estresse Fisiológico/efeitos dos fármacos , Bexiga Urinária Hiperativa/complicações , Bexiga Urinária Hiperativa/metabolismo , Micção/efeitos dos fármacos , Urodinâmica/efeitos dos fármacosRESUMO
BACKGROUND: IGLEs represent the only low-threshold vagal mechanosensory terminals in the tunica muscularis of the esophagus. Previously, close relationships of vesicular glutamate transporter 2 (VGLUT2) immunopositive IGLEs and cholinergic varicosities suggestive for direct contacts were described in almost all mouse esophageal myenteric ganglia. Possible cholinergic influence on IGLEs requires specific acetylcholine receptors. In particular, the occurrence and location of neuronal muscarinic acetylcholine receptors (mAChR) in the esophagus were not yet characterized. METHODS: This study aimed at specifying relationships of VGLUT2 immunopositive IGLEs and vesicular acetylcholine transporter (VAChT)-immunopositive varicosities using pre-embedding electron microscopy and the location of mAChR1-3 (M1-3) within esophagus and nodose ganglia using multilabel immunofluorescence and retrograde tracing. KEY RESULTS: Electron microscopy confirmed synaptic contacts between cholinergic varicosities and IGLEs. M1- and M2-immunoreactivities (-iry; -iries) were colocalized with VGLUT2-iry in subpopulations of IGLEs. Retrograde Fast Blue tracing from the esophagus showed nodose ganglion neurons colocalizing tracer and M2-iry. M1-3-iries were detected in about 80% of myenteric ganglia and in about 67% of myenteric neurons. M1- and M2-iry were present in many fibers and varicosities within myenteric ganglia. Presynaptic M2-iry was detected in all, presynaptic M3-iry in one-fifth of motor endplates of striated esophageal muscles. M1-iry could not be detected in motor endplates of the esophagus, but in sternomastoid muscle. CONCLUSIONS & INFERENCES: Acetylcholine probably released from varicosities of both extrinsic and intrinsic origin may influence a subpopulation of esophageal IGLEs via M2 and M1-receptors.
Assuntos
Esôfago/química , Cistos Glanglionares/química , Receptor Muscarínico M1/ultraestrutura , Receptor Muscarínico M2/ultraestrutura , Receptor Muscarínico M3/ultraestrutura , Proteína Vesicular 2 de Transporte de Glutamato/ultraestrutura , Animais , Esôfago/ultraestrutura , Cistos Glanglionares/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Receptor Muscarínico M1/análise , Receptor Muscarínico M2/análise , Receptor Muscarínico M3/análise , Proteína Vesicular 2 de Transporte de Glutamato/análiseRESUMO
BACKGROUND: This study was attempted to identify new molecules expressed on the plasma membrane of human umbilical vein endothelial cells (HUVECs) using monoclonal antibody-based proteomics technology and to determine the effect of the identified antibody on vascular reactivity. METHODS: Twenty-two antibodies were developed from rats inoculated with HUVECs, and their effects were determined by observing vascular reactivity. RESULTS: Among the 22 antibodies, the C-7 antibody significantly inhibited endothelium-dependent vasorelaxation in response to acetylcholine (ACh) but not to histamine. Moreover, the C-7 antibody did not affect norepinephrine-induced contraction in either the endothelium-intact or -denuded aorta. A proteomics study involving immunoprecipitation of the C-7 antibody with biotinylated HUVECs showed that this antibody binds to plasma membrane proteins corresponding to immunoglobulin heavy chain (VHDJ region), chaperonin-containing T-complex polypeptide 1 and α-actinin 4. The muscarinic M3 ACh receptor and α-actinin 4 were colocalized on the plasma membrane of HUVECs, and the colocalization was found to increase in response to ACh and was inhibited by pretreatment with the C-7 antibody. CONCLUSIONS: These results demonstrate that monoclonal C-7 antibody exerts an inhibitory effect on endothelium-dependent vasorelaxation induced by ACh and that this response may at least partially result from the inhibition of α-actinin 4.
Assuntos
Actinina/imunologia , Anticorpos Monoclonais/farmacologia , Endotélio Vascular/fisiologia , Células Endoteliais da Veia Umbilical Humana/imunologia , Vasodilatação/fisiologia , Acetilcolina/farmacologia , Actinina/análise , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Membrana Celular/química , Membrana Celular/metabolismo , Chaperonina com TCP-1/análise , Chaperonina com TCP-1/imunologia , Humanos , Hibridomas/imunologia , Masculino , Proteínas de Membrana/análise , Dados de Sequência Molecular , Norepinefrina/farmacologia , Proteômica/métodos , Ratos , Ratos Sprague-Dawley , Receptor Muscarínico M3/análise , Vasodilatação/efeitos dos fármacosRESUMO
AIMS: A non-neuronal cholinergic system has been described in epithelial cells including that of the urinary bladder (urothelium) and the upper gastrointestinal tract (esophagus). Epithelial dysfunction has been implicated in the pathophysiology of persistent pain conditions such as painful bladder syndrome as well as functional heartburn. For example, alterations in the ability to synthesize and release acetylcholine may contribute to changes in epithelial sensory and barrier function associated with a number of functional genitourinary and intestinal disorders. MAIN METHODS: We examined using immunoblot, acetylcholine (ACh)-synthesis and release components in cat esophageal mucosa and whether elements of these components are altered in a naturally occurring model of chronic idiopathic cystitis termed feline interstitial cystitis (FIC). KEY FINDINGS: We identified proteins involved in ACh synthesis and release (high affinity choline transporter, CHT1; ACh synthesizing enzyme choline acetyltransferase ChAT and carnitine acetyltransferase CarAT; vesicular ACh transporter VAChT and the organic cation transporter isoforms 1-3 or OCT-1-3) in cat esophageal mucosa. Significant alterations in CHT, ChAT, VAChT and OCT-1 were detected in the esophageal mucosa from FIC cats. Changes in the vesicular nucleotide transporter (VNUT) and the junctional protein pan-cadherin were also noted. SIGNIFICANCE: Taken together, these findings suggest that changes in the non-neuronal cholinergic system may contribute to alterations in cell-cell contacts and possibly communication with underlying cells that may contribute to changes in sensory function and visceral hyperalgesia in functional esophageal pain.
Assuntos
Acetilcolina/metabolismo , Cistite Intersticial/veterinária , Células Epiteliais/metabolismo , Esôfago/metabolismo , Acetilcolinesterase/análise , Acetilcolinesterase/metabolismo , Animais , Caderinas/análise , Caderinas/metabolismo , Carnitina O-Acetiltransferase/análise , Carnitina O-Acetiltransferase/metabolismo , Gatos , Colina O-Acetiltransferase/análise , Colina O-Acetiltransferase/metabolismo , Cistite Intersticial/metabolismo , Células Epiteliais/citologia , Esôfago/citologia , Proteínas de Membrana Transportadoras/análise , Proteínas de Membrana Transportadoras/metabolismo , Mucosa/citologia , Mucosa/metabolismo , Proteínas de Transporte de Cátions Orgânicos/análise , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor Muscarínico M2/análise , Receptor Muscarínico M2/metabolismo , Receptor Muscarínico M3/análise , Receptor Muscarínico M3/metabolismo , Proteínas Vesiculares de Transporte de Acetilcolina/análise , Proteínas Vesiculares de Transporte de Acetilcolina/metabolismoRESUMO
OBJECTIVE: Chronic obstructive pulmonary disease (COPD) is characterized by irreversible progressive airflow limitation related to tobacco smoking. This limitation is caused by chronic inflammation of the airways and lung parenchyma and is associated with increased activity of parasympathetic system. The most effective bronchodilators in COPD are muscarinic receptor antagonists (MRA), which reverse, at least in part, compromised respiratory function. MRA also contribute to control inflammatory processes via interactions with inflammatory signaling molecules. The use of the long-acting cholinolytic bronchodilatator - tiotropium, with high affinity to M3 receptors, is suggested as a first line maintenance treatment in COPD patients. MATERIAL AND METHODS: In this study we assessed M3 receptor protein expression in induced sputum of 27 stable COPD patients before and after therapy consisting of 18 µg once daily tiotropium for 12 weeks. Lung function tests including spirometry, lung volumes, and DLCO were performed before and after therapy in all COPD patients. The patients were subjected to the sputum induction procedures before and after therapy. Sputum cells were isolated, sample-specific cell profiles were characterized, and the cells were processed to isolate pure cytosolic fractions. Cytosolic M3 protein and HDAC2 levels and nuclear acetylated histone H3 (AcH3) expression was quantified using specific antibodies against human proteins and Western blot with enhanced luminescence detection. RESULTS: Therapy significantly increased the mean forced expiratory volume in one second (FEV1) and forced vital capacity (FVC) volume (P<0.05). The mean expression of M3 protein was higher by 37% after therapy (P<0.05), HDAC2 expression was not altered, while AcH3 level was increased by about 90% (P<0.01), compared with the corresponding data before therapy. HDAC2 expression before therapy was positively correlated with AcH3 expression (r = 0.74), while after therapy no correlation was detected. FEV1, FCV, and cytosolic M3 protein expression did not correlate with other biochemical parameters tested. CONCLUSIONS: Twelve weeks of tiotropium therapy in COPD patients improves clinical indices of lung function and involves alterations in sputum cell chromatin acetylation and also increased cholinergic M3 receptor internalization.
Assuntos
Citosol/química , Histonas/metabolismo , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Receptor Muscarínico M3/efeitos dos fármacos , Derivados da Escopolamina/farmacologia , Escarro/metabolismo , Acetilação , Volume Expiratório Forçado , Humanos , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Receptor Muscarínico M3/análise , Escarro/citologia , Brometo de Tiotrópio , Capacidade VitalRESUMO
INTRODUCTION: The parasympathetic autonomous nervous system exerts control over thyroid function by activation of the muscarinic receptors in follicular cells. Various pharmacological and molecular subtypes of muscarinic receptors (M(1), M(2), M(3), M(4), M(5)) have been identified in central nervous system and peripheral tissues. Controversy surrounds receptor characterization in thyroid cells. MATERIALS AND METHODS: Undifferentiated Fisher rat thyroid epithelial cells (FRT) were cultured. Association and dissociation kinetics assays and antagonist competition studies of the binding of (3)H-N-methylscopolamine ((3)H-NMS) to muscarinic receptors were performed to demonstrate the presence of muscarinic receptors. RESULTS: Specific muscarinic receptors in the plasma membrane of FRT cells were observed with an equilibrium dissociation constant (K(d)) of 0.44 nmol. The order of affinities obtained fitting the data to one binding site model in competition experiments with the muscarinic receptor antagonist was: dicyclomine > hexahydrosiladifenidol (HHSD) = 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) > pirenzepine > himbacine = 11-[[2-[(diethylamino)methyl]- 1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido (414)benzodiazepine (AF-DX 116). CONCLUSIONS: The results obtained indicate the existence of specific (3)H-NMS muscarinic binding sites located in the plasma membrane of FRT cells. The results obtained in competition experiments suggest that the receptors present in FRT cells belong to the M(3) subtype.
Assuntos
Células Epiteliais/química , Receptor Muscarínico M3/análise , Glândula Tireoide/citologia , Animais , Ligação Competitiva , Diferenciação Celular , Membrana Celular/química , Células Cultivadas/química , Células Cultivadas/efeitos dos fármacos , Antagonistas Muscarínicos/metabolismo , Antagonistas Muscarínicos/farmacologia , Ensaio Radioligante , Ratos , Ratos Endogâmicos F344 , Receptor Muscarínico M3/efeitos dos fármacos , Receptor Muscarínico M3/metabolismo , Glândula Tireoide/metabolismoAssuntos
Extrofia Vesical/patologia , Receptor Muscarínico M1/análise , Receptor Muscarínico M2/análise , Receptor Muscarínico M3/análise , Bexiga Urinária/patologia , Biópsia , Extrofia Vesical/cirurgia , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Microscopia de Fluorescência , Bexiga Urinária/inervação , Bexiga Urinária/cirurgia , Urodinâmica/fisiologiaRESUMO
Studies on salivary secretion are usually focused on parotid and submandibular glands. However, the film of mucin, that protects the oral structures and is responsible for the feeling of oral comfort, is produced by the submucosal glands. The submucosal zygomatic and molar glands are particularly large in carnivores such as the ferret. Comparisons between the mucous sublingual, zygomatic and molar glands, serous parotid and sero-mucous submandibular glands showed the acetylcholine synthesis, in terms of concentration, to be three to four times higher in the mucous glands than in the parotid and submandibular glands. Bromoacetylcholine inhibited 95-99% of the synthesis of acetylcholine in the incubates of the five types of glands, showing the acetylcholine synthesis to depend on the activity of choline acetyltransferase. The high acetylcholine synthesis in the zygomatic gland was of nervous origin, since cutting the buccal nerve, aiming at parasympathetic denervation, and allowing time for nerve degeneration, reduced the acetylcholine synthesising capacity of the gland by 95%. A similar reduction (96%) in the parotid gland followed upon the avulsion of the parasympathetic auriculo-temporal nerve. Zygomatic saliva was very viscous. The salivary flow rate in response to electrical stimulation (20 Hz) of the buccal nerve (zygomatic gland), expressed per gland weight, was one-third of that to stimulation of the auriculo-temporal nerve (parotid gland) or the chorda-lingual nerve (submandibular gland). As previously shown for the parotid and submandibular gland, a certain fraction (25%) of the parasympathetic secretory response of the zygomatic gland depended on non-adrenergic, non-cholinergic transmission mechanisms, probably involving substance P and vasoactive intestinal peptide and possibly calcitonin gene-related peptide. Particularly, high concentrations of vasoactive intestinal peptide were found in the sublingual and molar glands, and of substance P in the submandibular, zygomatic and molar glands; notably, the concentration of calcitonin gene-related peptide of the sublingual gland was not detectable. All five muscarinic receptor subtypes were detected in the five glands. The receptor protein profile, as judged by immunoblotting and semi-quantitative estimations, was about the same in the glands: high level of M3, low level of M2 and levels roughly in the same range of M1, M4 and M5. Compared to the parotid and submandibular glands, the M5 receptor level was particularly low in the mucin-secreting glands. The present study points out both similarities and dissimilarities between the five types of glands investigated. The zygomatic gland, in particular, appears to be a suitable model for future studies aiming at causing relief of dry mouth by local pharmacological treatment.
Assuntos
Acetilcolina/biossíntese , Neuropeptídeos/biossíntese , Receptores Muscarínicos/análise , Glândulas Salivares Menores/metabolismo , Glândulas Salivares/metabolismo , Animais , Peptídeo Relacionado com Gene de Calcitonina/fisiologia , Nervo da Corda do Tímpano/fisiologia , Estimulação Elétrica , Feminino , Furões , Nervo Lingual/fisiologia , Mucinas/metabolismo , Sistema Nervoso Parassimpático/fisiologia , Glândula Parótida/inervação , Glândula Parótida/metabolismo , Receptor Muscarínico M3/análise , Receptor Muscarínico M5/análise , Receptores Muscarínicos/classificação , Saliva/metabolismo , Glândulas Salivares Menores/inervação , Taxa Secretória/fisiologia , Glândula Sublingual/metabolismo , Glândula Submandibular/inervação , Glândula Submandibular/metabolismo , Substância P/fisiologia , Peptídeo Intestinal Vasoativo/fisiologiaRESUMO
1 The aim of the present work was to examine the role of muscarinic acetylcholine receptors (mAChR) on DNA synthesis and CD40 expression in human fibroblast cells. Neonatal human skin fibroblast cultures were stimulated with carbachol in presence or absence of specific antagonists and the following parameters were measured: identification of mAChR subtypes, DNA synthesis, inositol phosphates (InsP) production and CD40 expression. 2 Human fibroblasts express mAChR with Kd 0.47 +/- 0.11 nm and Bmax 236 +/- 22 fmol mg protein(-1). Carbachol stimulates DNA synthesis, InsP and the expression of CD40. All these effects were inhibited by atropine, mustard hydrochloride (4-DAMP) and pirenzepine but not by AF-DX 116 and tropicamide, indicating that M3 and M1 mAChR are implicated in carbachol action. The relative Ki of the antagonists obtained by competition binding assay was in parallel to the relative potency for blocking both carbachol-stimulated InsP accumulation and DNA synthesis. 3 The intracellular pathway leading to carbachol-induced biological effects involved phospholipase C and calcium/calmodulin, as U-73122 and trifluoroperazine blocked carbachol effects, respectively. Calphostin C, a protein kinase C inhibitor, had no effect, indicating that this enzyme does not participate in the system. 4 These results may contribute to a better understanding of the modulatory role of the parasympathetic muscarinic system on normal human fibroblast function.
Assuntos
Antígenos CD40/biossíntese , DNA/biossíntese , Fibroblastos/efeitos dos fármacos , Agonistas Muscarínicos/farmacologia , Receptores Muscarínicos/efeitos dos fármacos , Atropina/farmacologia , Calmodulina/antagonistas & inibidores , Calmodulina/metabolismo , Carbacol/farmacologia , Células Cultivadas , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Estrenos/farmacologia , Fibroblastos/imunologia , Fibroblastos/metabolismo , Citometria de Fluxo , Humanos , Fosfatos de Inositol/metabolismo , Antagonistas Muscarínicos/farmacologia , Pirenzepina/farmacologia , Pirrolidinonas/farmacologia , Quinuclidinil Benzilato , Ensaio Radioligante , Receptor Muscarínico M1/análise , Receptor Muscarínico M1/efeitos dos fármacos , Receptor Muscarínico M1/metabolismo , Receptor Muscarínico M3/análise , Receptor Muscarínico M3/efeitos dos fármacos , Receptor Muscarínico M3/metabolismo , Receptores Muscarínicos/análise , Receptores Muscarínicos/metabolismo , Trifluoperazina/farmacologia , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/metabolismoRESUMO
In guinea-pig isolated vasa deferentia, purinergic neurogenic contractions and responses to applied adenosine 5'-triphosphate (ATP) were potentiated by carbachol; responses to adrenergic transmission and applied noradrenaline were not. Following blockade of P2 receptors and alpha-adrenoceptors, the residual neurogenic response was massively potentiated by carbachol, suggesting the presence of a non-purinergic, non-adrenergic component. In the presence of guanethidine, carbachol had no significant effect, indicating that sympathetic transmission was the only element involved. Use of oxotremorine and selective muscarinic receptor antagonists suggested that the potentiating effect of carbachol and oxotremorine was mediated via M3 muscarinic receptors without involvement of nicotinic receptors.
Assuntos
Agonistas alfa-Adrenérgicos/farmacologia , Agonistas Colinérgicos/farmacologia , Junção Neuromuscular/efeitos dos fármacos , Norepinefrina/farmacologia , Receptor Muscarínico M3/agonistas , Ducto Deferente/efeitos dos fármacos , Ducto Deferente/inervação , Acetilcolina/farmacologia , Trifosfato de Adenosina/farmacologia , Antagonistas Adrenérgicos alfa/farmacologia , Animais , Estimulação Elétrica , Potenciais Evocados/efeitos dos fármacos , Cobaias , Técnicas In Vitro , Masculino , Antagonistas Muscarínicos/farmacologia , Junção Neuromuscular/metabolismo , Antagonistas do Receptor Purinérgico P2 , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/farmacologia , Receptor Muscarínico M3/análise , Receptores Purinérgicos P2XRESUMO
BACKGROUND: Using an original model, the Donor Rat Model, we showed that bile-pancreatic juice (BPJ) exclusion from gut exacerbates ligation-induced acute pancreatitis in rats. We also showed that muscarinic cholinergic M3 and CCK-A receptor expression is induced following duct ligation. Increased receptor number potentially could exacerbate cytokine production. We hypothesize that BPJ exclusion is responsible for M3 and CCK-A receptor induction and increased interleukin-6 (IL-6) production. METHODS: M3 and CCK-A receptor expression and IL-6 production were compared in rat pancreata 1 to 3 hours after duct ligation with or without BPJ replacement. RESULTS: Our studies showed that BPJ replacement attenuates duct ligation-induced increases in M3 and CCK-A receptor expression and IL-6 production. CONCLUSIONS: In this model, BPJ exclusion from gut induces M3 and CCK-A receptor expression and increases IL-6 production. In this experimental corollary of gallstone pancreatitis, BPJ exclusion from gut may play a key role in the mechanism of disease pathogenesis.
Assuntos
Interleucina-6/biossíntese , Suco Pancreático/metabolismo , Pancreatite/metabolismo , Receptor de Colecistocinina A/metabolismo , Receptor Muscarínico M3/metabolismo , Análise de Variância , Animais , Biomarcadores/metabolismo , Modelos Animais de Doenças , Immunoblotting , Ligadura/métodos , Masculino , Probabilidade , Ratos , Ratos Sprague-Dawley , Receptor de Colecistocinina A/análise , Receptor Muscarínico M3/análise , Receptores Colinérgicos/análise , Receptores Colinérgicos/metabolismo , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Cholera toxin (CT) acts on intestinal epithelial cells both directly and indirectly via activation of a secretory neural reflex. The reflex may release acetylcholine as one of its final neurotransmitters. This opens up the possibility of a third mechanism of action for CT, namely a synergistic interaction between two secretagogues acting on different second messenger systems within the epithelial cell. AIMS: To establish evidence for cholinergic innervation to human ileal epithelial cells and to investigate whether CT potentiates the action of acetylcholine on human intestinal epithelial cells. METHODS: Transverse sections of human ileum were examined for mucosal cholinergic nerves and M3 muscarinic receptors using antibodies raised to choline acetyltransferase and M3 receptors. Short circuit current (Isc) responses and ion flux movements were elicited from T84 epithelial cell monolayers set up in Ussing chambers. RESULTS: Immunohistochemistry of native human ileal mucosa revealed the presence of both cholinergic nerves and muscarinic M3 receptors located to the basolateral domain of epithelial cells. Secretory responses of T84 cell monolayers to acetylcholine were greatly potentiated in the presence of CT. This effect, substituting forskolin for CT, was mirrored by increases in basolateral 86Rb and apical 125I efflux. Charybdotoxin plus apamin reduced both Isc and 86Rb efflux evoked by acetylcholine, in the presence of forskolin. CONCLUSIONS: Human ileal mucosa receives a direct cholinergic innervation to its epithelial cells. Secretory effects of acetylcholine on epithelial cells are augmented in the presence of CT. Such a synergistic response is dependent on optimum opening of basolateral potassium channels by acetylcholine and apical chloride channels by CT. The interaction may contribute to the mechanism of action of cholera toxin induced secretory diarrhoea.