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1.
Rio de Janeiro; s.n; 2020. xiv, 79 p. ilus.
Thesis in Portuguese | LILACS | ID: biblio-1128725

ABSTRACT

O timo é um órgão linfoide primário responsável pelo desenvolvimento e seleção de células T. Diversos fatores podem afetar o desenvolvimento de células T, como citocinas, quimiocinas e moléculas da matriz extracelular, mas também hormônios, neuropeptídeos e neurotransmissores. O timo recebe densa inervação simpática, liberando majoritariamente noradrenalina (NA), timócitos e células tímicas não linfoides expressam receptores adrenérgicos e podem sintetizar catecolaminas, sugerindo modulação de NA por diferentes vias. Por outro lado, poucas evidências anatômicas suportam a hipótese da inervação tímica parassimpática. Entretanto, acetilcolina (ACh) parece ser endogenamente produzida no órgão, uma vez que diferentes células no timo expressam a enzima sintetizadora de ACh (ChAT) e receptores colinérgicos. Sendo assim, o objetivo deste trabalho foi determinar o papel funcional de ACh e NA sobre os componentes linfoide e microambiental do timo, e sobre as interações entre células epiteliais e timócitos. Avaliamos a expressão de receptores adrenérgicos α1A, α1D, α2C, ß2 e colinérgicos M1, M3, M5 e α7 em timócitos e TECs, através do método de PCR quantitativa. Todas as populações celulares testadas expressavam os receptores selecionados.


Além disso, buscamos caracterizar o efeito de drogas análogas aos neurotransmissores sobre a morte e a proliferação de timócitos, através da marcação com anexina-V/iodeto de propídio (IP) e CFSE respectivamente. Timócitos obtidos do timo de camundongos C57BL/6 fêmeas, foram tratados com carbacol (análogo de Ach - em concentrações variando de 10 nM a 1000 nM) ou arterenol (análogo de NA - em concentrações variando de 10 nM a 1 mM). Verificamos que apenas arterenol na concentração de 1 mM induziu a apoptose nas células tratadas. Nenhum dos análogos modulou a proliferação celular. Avaliamos ainda o efeito das drogas sobre a migração de timócitos, agindo como quimioatrente ou modulando a migração induzida por fibronectina. Nenhuma das drogas demonstrou efeito quimioatraente ou alterou a migração induzida por fibronectina. Além disso, avaliamos o efeito das drogas sobre a interação TEC/timócitos atraves de ensaios de adesão e observamos que carbacol foi capaz de diminuir a adesão de timócitos a TECs em todas as concentrações testadas. Essa diminuição é refletida no número de timócitos que aderem às TECs e parece estar associada a modulação da expressão do receptor de laminina VLA-6. Nossos dados sugerem que timócitos e as linhagens de TECs testadas expressam receptores e que neurotransmissores, em especial a ACh, podem modular a interação entre TECs e timócitos. (AU)


Subject(s)
Animals , Mice , Thymus Gland , Carbachol , Acetylcholine
2.
Article in English | WPRIM | ID: wpr-772280

ABSTRACT

Corticosteroids are used in the treatment of many diseases; however, they also induce various side effects. Dexamethasone is one of the most potent corticosteroids, and it has been reported to induce the side effect of impaired salivary gland function. This study aimed to evaluate the effects of dexamethasone on mouse submandibular gland function to gain insight into the mechanism of dexamethasone-induced salivary hypofunction. The muscarinic agonist carbachol (CCh) induced salivary secretion and was not affected by short-term dexamethasone treatment but was decreased following long-term dexamethasone administration. The expression levels of the membrane proteins Na-K-2Cl cotransporter, transmembrane member 16A, and aquaporin 5 were comparable between the control and long-term dexamethasone treatment groups. The CCh-induced increase in calcium concentration was significantly lower in the presence of extracellular Ca in the long-term dexamethasone treatment group compared to that in the control group. Furthermore, CCh-induced salivation in the absence of extracellular Ca and Ca ionophore A23187-induced salivation was comparable between the control and long-term dexamethasone treatment groups. Moreover, salivation induced by the Ca-ATPase inhibitor thapsigargin was diminished in the long-term dexamethasone treatment group. In summary, these results demonstrate that short-term dexamethasone treatment did not impair salivary gland function, whereas long-term dexamethasone treatment diminished store-operated Ca entry, resulting in hyposalivation in mouse submandibular glands.


Subject(s)
Acinar Cells , Metabolism , Animals , Calcium , Metabolism , Calcium Signaling , Carbachol , Pharmacology , Dexamethasone , Therapeutic Uses , Mice , Muscarinic Agonists , Pharmacology , Saliva , Metabolism , Salivation , Submandibular Gland , Metabolism
3.
Article in English | WPRIM | ID: wpr-761784

ABSTRACT

The transient receptor potential canonical (TRPC) 5 channel, known as a nonselective cation channel, has a crucial role in calcium influx. TRPC5 has been reported to be activated by muscarinic receptor activation and extracellular pH change and inhibited by the protein kinase C pathway. Recent studies have also suggested that TRPC5 is extracellularly activated by englerin A (EA), but the mechanism remains unclear. The purpose of this study is to identify the EA-interaction sites in TRPC5 and thereby clarify the mechanism of TRPC5 activation. TRPC5 channels are over-expressed in human embryonic kidney (HEK293) cells. TRPC5 mutants were generated by site-directed mutagenesis. The whole-cell patch-clamp configuration was used to record TRPC5 currents. Western analysis was also performed to observe the expression of TRPC5 mutants. To identify the EA-interaction site in TRPC5, we first generated pore mutants. When screening the mutants with EA, we observed the EA-induced current increases of TRPC5 abolished in K554N, H594N, and E598Q mutants. The current increases of other mutants were reduced in different levels. We also examined the functional intactness of the mutants that had no effect by EA with TRPC5 agonists, such as carbachol or GTPγS. Our results suggest that the three residues, Lys-554, His-594, and Glu-598, in TRPC5 might be responsible for direct interaction with EA, inducing the channel activation. We also suggest that although other pore residues are not critical, they could partly contribute to the EA-induced channel activation.


Subject(s)
Calcium , Carbachol , Humans , Hydrogen-Ion Concentration , Ion Channels , Kidney , Mass Screening , Mutagenesis, Site-Directed , Mutant Proteins , Protein Kinase C , Receptors, Muscarinic
4.
Lima; IETSI; 2018.
Non-conventional in Spanish | LILACS, BIGG | ID: biblio-911693

ABSTRACT

La catarata es definida por la Organización Mundial de la Salud (OMS) como la opacificación del lente cristalino que generalmente ocurre por el envejecimiento, trauma, o alguna enfermedad sistémica, afectando la capacidad visual de la persona. Esta disminución de la capacidad visual o incluso la ceguera, es un problema de salud pública en adultos y adultos mayores. En el Perú, aproximadamente el 0.6 % de la población tiene ceguera, cuya causa en el 47 % de los casos son por las cataratas. El tratamiento indicado para la catarata es la intervención quirúrgica, la cual consiste en reemplazar el cristalino opacificado o catarata por un lente intraocular. Hay dos formas de realizar esto, mediante la extracción extracapsular del cristalino opacificado, o mediante la facoemulsificación del cristalino, que consiste en un proceso de destrucción mediante ondas vibratorias ultrasónicas. Luego de ello, se realiza la implantación de un nuevo lente intraocular con soporte capsular, el cual reemplaza al cristalino permitiendo que el paciente vuelva tener una visión adecuada. Posterior a la implantación del lente intraocular se realiza la inducción de miosis pupilar para mantener el lente dentro de la bolsa capsular, evitar la captura del lente por el iris y el prolapso del iris por las heridas operatorias. La inducción de miosis luego de una intervención por catarata debe realizarse inmediatamente después de la implantación del lente, por lo que la vía de administración indicada es la inyección intraocular del agente miótico. De este modo, se espera que el efecto miótico se prolongue hasta por 24 horas, mientras que la aplicación tópica de un agente miótico se limita a un efecto que bordea las ocho horas, y sólo se puede realizar en el periodo postoperatorio, por lo que se genera un periodo entre la intervención quirúrgica y la aplicación del agente miótico que expone al paciente a las potenciales complicaciones anteriormente mencionadas. Aunque algunos estudios de serie de casos clínicos indican el uso de pilocarpina 2 %, el cual se encuentra disponible en el Petitorio Farmacológico de EsSalud, este medicamento no ha sido aprobado por la FDA para uso intraocular, sólo para uso tópico, dado que su uso intraocular puede ser tóxico o incrementar el riesgo de infecciones intraoculares. Así, en la actualidad EsSalud no cuenta con un medicamento miótico de uso intraocular autorizado, por lo cual, surge la necesidad de evaluar otras alternativas que pudieran ser de beneficio para dichos pacientes. OBJETIVO: objetivo del presente dictamen fue evaluar la eficacia y seguridad del uso intraocular de carbacol 0.01 % para la inducción de miosis intraoperatoria en las intervenciones quirúrgicas por catarata. Carbacol es um colinérgico o parasimpaticomimético potente, que actúa como agonista del receptor de acetilcolina, inhibiendo la acetilcolinesterasa y estimulando tanto los receptores muscarínicos como nicotínicos, produciendo miosis a través de la constricción del iris y del cuerpo ciliar, y reduciendo la presión intraocular. TECNOLOGÍA SANITARIA DE INTERÉS: CARBACOL. Carbacol, también conocido como carbamilcolina, es un colinérgico o parasimpaticomimético potente, que actúa como agonista del receptor de acetilcolina, inhibiendo la acetilcolinesterasa y estimulando tanto los receptores muscarínicos como nicotínicos, produciendo constricción del iris y del cuerpo ciliar y además reduciendo la presión intraocular. El agente colinérgico fue aprobado por la Administración de Drogas y Alimentos (FDA, por sus siglas en inglés) en el año 2002 (FDA, 2015). METODOLOGÍA: Se realizó una búsqueda sin restricción de idioma hasta mayo del 2018. La formulación de la estrategia de búsqueda incluyó los criterios de elegibilidad, los términos controlados propios de cada base y términos libres. Asimismo, se buscaron otros documentos potencialmente elegibles a través de la revisión del listado de referencias de los documentos seleccionados para lectura a texto completo. Por último, la selección de la evidencia siguió el flujograma mostrado en la subsección de resultados. RESULTADOS: Luego de la búsqueda sistemática realizada, se identificaron dos ensayos clínicos aleatorizados (ECA) Beasley, 1972 y Solomon et al., 1998; y el estudio de serie de casos de Pekel et al., 2014. Si bien estos estudios muestran algunas limitaciones que serán analizadas más adelante, es la evidencia de mayor relevancia en torno al uso de carbacol para la inducción de miosis intraoperatoria en las intervenciones quirúrgicas por catarata. Con respecto a la eficacia de carbacol en la inducción de miosis, el estudio de Beasley, 1972 muestra que el efecto miótico a los dos minutos de la inyección intraocular es significativamente mayor en carbacol, con respecto a placebo (p<0.01) y que el efecto miótico persiste por lo menos por 15 horas. Por otro lado, a la séptima semana postoperatoria, se observó una incidencia significativamente menor de sinequias anteriores periféricas (SAP) en el grupo que recibió carbacol (11 %), en comparación al grupo que recibió a placebo (35 %). Al analizar el impacto sobre la calidad de vida por parte de carbacol mediante el cuestionario modificado de SF-36, el cual mide la percepción del paciente sobre su estado de salud, el estudio de Solomon et al., 1998 muestra que carbacol incrementa la agudeza visual durante el primer día postoperatorio, con respecto a placebo, y muestra una diferencia estadísticamente significativa en el porcentaje de sujetos que pueden descender las escaleras sin ayuda durante la primera semana posterior a la intervención quirúrgica, tanto en un ambiente con luz brillante (p=0.007) o con luz tenue (p=0.037), siendo un potencial factor protector en pacientes con riesgo de presentar caídas acci Con respecto a los eventos adversos, los estudios evaluados no encontraron casos de inflamación intraocular ni de cefalea frontal. El estudio Pekel et al., 2015 muestra que sólo en el primer día postoperatorio hubo un menor volumen macular total (VMD y del grosor macular central (GMC) con respecto al volumen preoperatorio. Mientras que, durante el seguimiento (al primer día, a la primera semana y al primer mes) no se encontraron diferencias estadísticamente significativas en la presencia de edema macular, ni en el calibre de los vasos retinianos (CVR) al comparar los pacientes que recibieron carbacol con los que no lo recibieron. dentales y fractura de caderas. CONCLUSIONES: El presente dictamen preliminar muestra la evidencia disponible hasta mayo 2018 con respecto al uso intraocular de carbacol 0.01 % en comparación con placebo para la inducción de miosis intraoperatoria en pacientes operados de cataratas. No se encontraron guías de práctica clínica, revisiones sistemáticas ni evaluaciones de tecnologías sanitarias que respondan la pregunta PICO de la presente evaluación. Finalmente, se identificaron dos ECA y un estudio de serie de casos como sustento para la elaboración del presente dictamen preliminar. Al evaluar la eficacia de carbacol en la inducción de miosis, el estudio Beasley, 1972 muestra que carbacol genera miosis dentro de los dos minutos de ser aplicado y que su efecto perdura por más de 15 horas. Esto se traduce en una menor incidencia de SAP a la sétima semana postoperatorio, con respecto a placebo. Aunque no se aprecia diferencia en la conservación de la integridad de la cámara vítrea. Con respecto a los eventos adversos, los estudios evaluados no observaron casos de inflamación intraocular ni de cefalea frontal, mientras que el estudio Pekel et al., 2015 muestra que carbacol disminuye el volumen macular total (VMT) y el grosor macular central (GMT) en el primer día postoperatorio, mas no en la primera semana, ni primer mes. Por otro lado, no se ve afectado el calibre de los vasos retinianos (CVR). Estos resultados muestran una cierta protección ante el edema macular en el postoperatorio inmediato y ausencia de secuelas en la morfología macular, sin embargo, se debe tener en cuenta el posible sesgo de medición que conlleva. En resumen, carbacol es un agente miótico que inicia su acción dentro de los dos minutos de su inyección, manteniendo su efecto por más de 15 horas, generando un beneficio en el periodo postoperatorio inmediato en la agudeza visual y en el volumen macular total. Asimismo, los estudios no reportan eventos adversos como edema macular, cefalea, y, por el contrario, refieren una reducción en la incidencia de SAP, mas no hay una diferencia en la preservación de la integridad de la cámara vítrea, al ser comparado con placebo. Con respecto a la calidad de vida en el postoperatorio, se observa un inicio más temprano de la deambulación y autonomía en el uso de las escaleras, lo que podría tener un impacto positivo en el estilo de vida de los pacientes intervenidos. Por lo expuesto, el Instituto de Evaluaciones de Tecnologías en Salud e Investigación - IETSI aprueba el uso de carbacol, según lo establecido en el Anexo N.° 1. La vigencia del presente dictamen preliminar es de dos años a partir de la fecha de publicación.


Subject(s)
Humans , Carbachol/administration & dosage , Cataract Extraction/methods , Miosis/chemically induced , Cost Efficiency Analysis , Randomized Controlled Trials as Topic , Treatment Outcome , Intraoperative Care/methods
5.
Article in English | WPRIM | ID: wpr-740725

ABSTRACT

BACKGROUND/AIMS: We investigated the role of representative endoplasmic reticulum proteins, stromal interaction molecule 1 (STIM1), and store-operated calcium entry-associated regulatory factor (SARAF) in pacemaker activity in cultured interstitial cells of Cajal (ICCs) isolated from mouse small intestine. METHODS: The whole-cell patch clamp technique applied for intracellular calcium ions ([Ca²+]i) analysis with STIM1 or SARAF overexpressed cultured ICCs from mouse small intestine. RESULTS: In the current-clamping mode, cultured ICCs displayed spontaneous pacemaker potentials. External carbachol exposure produced tonic membrane depolarization in the current-clamp mode, which recovered within a few seconds into normal pacemaker potentials. In STIM1-overexpressing cultured ICCs pacemaker potential frequency was increased, and in SARAF-overexpressing ICCs pacemaker potential frequency was strongly inhibited. The application of gadolinium (a non-selective cation channel inhibitor) or a Ca2+-free solution to understand Orai channel involvement abolished the generation of pacemaker potentials. When recording intracellular Ca²+ concentration with Fluo 3-AM, STIM1-overexpressing ICCs showed an increased number of spontaneous intracellular Ca²+ oscillations. However, SARAF-overexpressing ICCs showed fewer spontaneous intracellular Ca2+ oscillations. CONCLUSION: Endoplasmic reticulum proteins modulated the frequency of pacemaker activity in ICCs, and levels of STIM1 and SARAF may determine slow wave patterns in the gastrointestinal tract.


Subject(s)
Animals , Calcium , Carbachol , Endoplasmic Reticulum , Gadolinium , Gastrointestinal Motility , Gastrointestinal Tract , Interstitial Cells of Cajal , Intestine, Small , Ions , Membranes , Mice
6.
Article in English | WPRIM | ID: wpr-728587

ABSTRACT

Conflicting evidence has been obtained regarding whether transient receptor potential cation channels (TRPC) are store-operated channels (SOCs) or receptor-operated channels (ROCs). Moreover, the Ca/Na permeability ratio differs depending on whether the current-voltage (I-V) curve has a doubly rectifying shape or inward rectifying shape. To investigate the calcium permeability of TRPC4 channels, we attached GCaMP6s to TRPC4 and simultaneously measured the current and calcium signals. A TRPC4 specific activator, (–)-englerin A, induced both current and calcium fluorescence with the similar time course. Muscarinic receptor stimulator, carbachol, also induced both current and calcium fluorescence with the similar time course. By forming heteromers with TRPC4, TRPC1 significantly reduced the inward current with outward rectifying I-V curve, which also caused the decrease of calcium fluorescence intensity. These results suggest that GCaMP6s attached to TRPC4 can detect slight calcium changes near TRPC4 channels. Consequently, TRPC4-GCaMP6s can be a useful tool for testing the calcium permeability of TRPC4 channels.


Subject(s)
Calcium , Carbachol , Fluorescence , Permeability , Receptors, Muscarinic
7.
Article in English | WPRIM | ID: wpr-728575

ABSTRACT

Intracellular calcium (Ca²⁺) oscillation is an initial event in digestive enzyme secretion of pancreatic acinar cells. Reactive oxygen species are known to be associated with a variety of oxidative stress-induced cellular disorders including pancreatitis. In this study, we investigated the effect of hydrogen peroxide (H₂O₂) on intracellular Ca²⁺ accumulation in mouse pancreatic acinar cells. Perfusion of H₂O₂ at 300 µM resulted in additional elevation of intracellular Ca²⁺ levels and termination of oscillatory Ca²⁺ signals induced by carbamylcholine (CCh) in the presence of normal extracellular Ca²⁺. Antioxidants, catalase or DTT, completely prevented H₂O₂-induced additional Ca²⁺ increase and termination of Ca²⁺ oscillation. In Ca²⁺-free medium, H₂O₂ still enhanced CCh-induced intracellular Ca²⁺ levels and thapsigargin (TG) mimicked H₂O₂-induced cytosolic Ca²⁺ increase. Furthermore, H₂O₂-induced elevation of intracellular Ca²⁺ levels was abolished under sarco/endoplasmic reticulum Ca²⁺ ATPase-inactivated condition by TG pretreatment with CCh. H₂O₂ at 300 µM failed to affect store-operated Ca²⁺ entry or Ca²⁺ extrusion through plasma membrane. Additionally, ruthenium red, a mitochondrial Ca²⁺ uniporter blocker, failed to attenuate H₂O₂-induced intracellular Ca²⁺ elevation. These results provide evidence that excessive generation of H₂O₂ in pathological conditions could accumulate intracellular Ca²⁺ by attenuating refilling of internal Ca²⁺ stores rather than by inhibiting Ca²⁺ extrusion to extracellular fluid or enhancing Ca²⁺ mobilization from extracellular medium in mouse pancreatic acinar cells.


Subject(s)
Acinar Cells , Animals , Antioxidants , Calcium , Carbachol , Catalase , Cell Membrane , Cytosol , Extracellular Fluid , Hydrogen Peroxide , Hydrogen , Ion Transport , Mice , Pancreatitis , Perfusion , Reactive Oxygen Species , Reticulum , Ruthenium Red , Thapsigargin
8.
Article in English | WPRIM | ID: wpr-212091

ABSTRACT

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are confirmed to be expressed in bladder interstitial Cajal-like cells (ICC-LCs), but little is known about their possible role in cystitis-associated bladder dysfunction. The present study aimed to determine the functional role of HCN channels in regulating bladder function under inflammatory conditions. Sixty female wild-type C57BL/6J mice and sixty female HCN1-knockout mice were randomly assigned to experimental and control groups, respectively. Cyclophosphamide (CYP)-induced cystitis models were successfully established in these mice. CYP treatment significantly enhanced HCN channel protein expression and I(h) density and significantly altered bladder HCN1 channel regulatory proteins. Carbachol (CCH) and forskolin (FSK) exerted significant effects on bladder ICC-LC [Ca²⁺]i in CYP-treated wild-type (WT) mice, and HCN1 channel ablation significantly decreased the effects of CCH and FSK on bladder ICC-LC [Ca²⁺]i in both naive and CYP-treated mice. CYP treatment significantly potentiated the spontaneous contractions and CCH (0.001-10 µM)-induced phasic contractions of detrusor strips, and HCN1 channel deletion significantly abated such effects. Finally, we demonstrated that the development of CYP-induced bladder overactivity was reversed in HCN1 -/- mice. Taken together, our results suggest that CYP-induced enhancements of HCN1 channel expression and function in bladder ICC-LCs are essential for cystitis-associated bladder hyperactivity development, indicating that the HCN1 channel may be a novel therapeutic target for managing bladder hyperactivity.


Subject(s)
Animals , Carbachol , Colforsin , Cyclophosphamide , Cystitis , Female , Humans , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels , Mice , Telocytes , Up-Regulation , Urinary Bladder
9.
Arq. bras. oftalmol ; 79(2): 105-110, Mar.-Apr. 2016. tab, graf
Article in English | LILACS | ID: lil-782803

ABSTRACT

ABSTRACT Purpose: The goal of the present study was to establish a protocol for primary culture of lacrimal gland acinar cells (LGACs) and to assess the effect of adding insulin to the culture media. Methods: LGACs were isolated and cultured from lacrimal glands of Wistar male rats. The study outcomes included cell number, viability, and peroxidase release over time and in response to three concentrations of insulin (0.5, 5.0, and 50.0 μg/mL). Results: In LGAC primary culture, cells started to form clusters by day 3. There was a time-response pattern of peroxidase release, which rose by day 6, in response to carbachol. Culture viability lasted for 12 days. An insulin concentration of 5.0 μg/mL in the culture medium resulted in higher viability and secretory capacity. Conclusions: The present method simplifies the isolation and culture of LGACs. The data confirmed the relevance of adding insulin to maintain LGACs in culture.


RESUMO Objetivo: O objetivo do estudo foi estabelecer um protocolo de cultura primária para o isolamento de células acinares da glândula lacrimal (CAGL) e avaliar a relevância de insulina no meio de cultura. Métodos: CAGL foram isoladas e cultivadas a partir das glândulas lacrimais de ratos Wistar machos. Os parâmetros analisados foram: o número de células, viabilidade e secreção da peroxidase ao longo do tempo e em resposta a três concentrações de insulina (0,5; 5,0 e 50,0 μg/ml). Resultados: Na cultura primária de CAGL as células passaram a se agrupar por volta do dia 3. A secreção de peroxidase em resposta ao carbacol aumentou no dia 6. O período de cultura viável foi limitado à 12 dias. Insulina à 5,0 μg/ml no meio de cultura resultou em viabilidade e capacidade secretora maior. Conclusão: o estudo descreveu um método para simplificar o isolamento e cultivo de CAGL. Os dados apresentados confirmam a importância da insulina na manutenção da cultura de CAGL.


Subject(s)
Animals , Male , Acinar Cells/cytology , Primary Cell Culture/standards , Insulin/pharmacology , Lacrimal Apparatus/cytology , Carbachol/metabolism , Cell Count/methods , Cell Separation/methods , Rats, Wistar , Peroxidase/metabolism , Acinar Cells/drug effects , Acinar Cells/metabolism , Insulin/metabolism , Lacrimal Apparatus/metabolism
10.
Article in Korean | WPRIM | ID: wpr-124491

ABSTRACT

Acetylcholine receptors (AChR) including muscarinic and nicotinic AChR are widely expressed and mediate a variety of physiological cellular responses in neuronal and non-neuronal cells. Notably, a functional cholinergic system exists in oral epithelial cells, and nicotinic AChR (nAChR) mediates cholinergic anti-inflammatory responses. However, the pathophysiological roles of AChR in periodontitis are unclear. Here, we show that activation of AChR elicits increased cytosolic Ca²⁺ ([Ca²⁺]ᵢ), transient cytotoxicity, and induction of receptor activator of nuclear factor kappa-B ligand (RANKL) expression. Intracellular Ca²⁺ mobilization in human gingival fibroblast-1 (hGF-1) cells was measured using the fluorescent Ca²⁺ indicator, fura-2/AM. Cytotoxicity and induction of gene expression were evaluated by measuring the release of glucose-6-phosphate dehydrogenase and RT-PCR. Activation of AChR in hGF-1 cells by carbachol (Cch) induced [Ca²⁺]ᵢ increase in a dose-dependent manner. Treatment with a high concentration of Cch on hGF-1 cells caused transient cytotoxicity. Notably, treatment of hGF-1 cells with Cch resulted in upregulated RANKL expression. The findings may indicate potential roles of AChR in gingival fibroblast cells in bone remodeling.


Subject(s)
Acetylcholine , Bone Remodeling , Carbachol , Cytosol , Epithelial Cells , Fibroblasts , Gene Expression , Glucosephosphate Dehydrogenase , Humans , Neurons , Osteoprotegerin , Periodontitis , Receptors, Cholinergic
11.
Biol. Res ; 48: 1-6, 2015. graf, tab
Article in English | LILACS | ID: biblio-950792

ABSTRACT

BACKGROUND: Ruta graveolens L. (R. graveolens) is a medicinal plant employed in non-traditional medicines that has various therapeutic properties, including anthelmintic, and vasodilatory actions, among others. We evaluated the trachea-relaxant effects of hydroalcoholic extract of R. graveolens against potassium chloride (KCl)- and carbachol-induced contraction of rat tracheal rings in an isolated organ bath. RESULTS: The results showed that the airway smooth muscle contraction induced by the depolarizing agent (KCl) and cholinergic agonist (carbachol) was markedly reduced by R. graveolens in a concentration-dependent manner, with maximum values of 109 ± 7.9 % and 118 ± 2.6 %, respectively (changes in tension expressed as positive percentages of change in proportion to maximum contraction), at the concentration of 45 µg/mL (half-maximal inhibitory concentration IC50: 35.5 µg/mL and 27.8 µg/mL for KCl- and carbachol-induced contraction, respectively). Additionally, the presence of R. graveolens produced rightward parallel displacement of carbachol dose-response curves and reduced over 35 % of the maximum smooth muscle contraction. CONCLUSIONS: The hydroalcoholic extract of R. graveolens exhibited relaxant activity on rat tracheal rings. The results suggest that the trachea-relaxant effect is mediated by a non-competitive antagonistic mechanism. More detailed studies are needed to identify the target of the inhibition, and to determine more precisely the pharmacological mechanisms involved in the observed biological effects.


Subject(s)
Animals , Rats , Parasympatholytics/pharmacology , Trachea/drug effects , Plant Extracts/pharmacology , Ruta/chemistry , Muscle, Smooth/drug effects , Neuromuscular Depolarizing Agents/pharmacology , Potassium Chloride/pharmacology , Furocoumarins/analysis , Quercetin/analysis , Rutin/analysis , Trachea/surgery , In Vitro Techniques , Carbachol/pharmacology , Plant Extracts/chemistry , Chromatography, Liquid , Rats, Sprague-Dawley , Cholinergic Agents/pharmacology , Inhibitory Concentration 50 , Plant Components, Aerial/chemistry , Muscle Contraction/drug effects , Muscle Tonus/drug effects
12.
Article in English | WPRIM | ID: wpr-176182

ABSTRACT

BACKGROUND/AIMS: It has been reported that proton pump inhibitors induce relaxation in different types of smooth muscles. The aim of this study is to investigate in vitro effects of proton pump inhibitors on human pylorus muscle. METHODS: Pyloric sphincters were studied in 10 patients who were operated for stomach cancer. In isolated organ bath, control and response to rabeprazole were recorded following contraction with carbachol. During the treatment experiment, while distilled water was applied during the control experiment in every 5 minutes, rabeprazole was administered in every 5 minutes at doses of 10-6, 10-5, 10-4, and 10-3 M respectively. Contraction frequencies, maximum contraction values and muscle tones were measured. RESULTS: The contraction frequencies in the control group were greater than the rabeprazole group in the second, third and fourth intervals while the maximum contraction values in the rabeprazole group were lower in the fourth interval. Even though muscles tones were not different in both groups during all intervals, it was remarkable that the muscle tone was significantly decreased in the rabeprazole group during the fourth interval compared to the first and second intervals. CONCLUSIONS: In the present study, high doses of rabeprazole reduced contraction frequencies, maximum contraction values, and muscle tone of human pylorus.


Subject(s)
Baths , Carbachol , Humans , Muscle Tonus , Muscle, Smooth , Muscles , Proton Pump Inhibitors , Pylorus , Rabeprazole , Relaxation , Stomach Neoplasms , Water
13.
Article in English | WPRIM | ID: wpr-14536

ABSTRACT

BACKGROUND/AIMS: Inflammatory bowel disease is commonly accompanied by colonic dysmotility and causes changes in intestinal smooth muscle contractility. In this study, colonic smooth muscle contractility in a chronic inflammatory condition was investigated using smooth muscle tissues prepared from interleukin-10 knockout (IL-10(-/-)) mice. METHODS: Prepared smooth muscle sections were placed in an organ bath system. Cholinergic and nitrergic neuronal responses were observed using carbachol and electrical field stimulation with L-NG-nitroarginine methyl ester (L-NAME). The expression of interstitial cells of Cajal (ICC) networks, muscarinic receptors, neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) was observed via immunofluorescent staining. RESULTS: The spontaneous contractility and expression of ICC networks in the proximal and distal colon was significantly decreased in IL-10(-/-) mice compared to IL-10(+/+) mice. The contractility in response to carbachol was significantly decreased in the proximal colon of IL-10(-/-) mice compared to IL-10(+/+) mice, but no significant difference was found in the distal colon. In addition, the expression of muscarinic receptor type 2 was reduced in the proximal colon of IL-10(-/-) mice. The nictric oxide-mediated relaxation after electrical field stimulation was significantly decreased in the proximal and distal colon of IL-10(-/-) mice. In inflamed colon, the expression of nNOS decreased, whereas the expression of iNOS increased. CONCLUSIONS: These results suggest that damage to the ICC network and NOS system in the proximal and distal colon, as well as damage to the smooth muscle cholinergic receptor in the proximal colon may play an important role in the dysmotility of the inflamed colon.


Subject(s)
Animals , Baths , Carbachol , Colon , Inflammatory Bowel Diseases , Interleukin-10 , Interstitial Cells of Cajal , Mice , Mice, Knockout , Muscle, Smooth , Nitrergic Neurons , Nitric Oxide Synthase Type I , Nitric Oxide Synthase Type II , Receptors, Muscarinic , Relaxation
14.
Article in English | WPRIM | ID: wpr-727690

ABSTRACT

Transient receptor potential vanilloid subtype 1 (TRPV1) was originally found in sensory neurons. Recently, it has been reported that TRPV1 is expressed in salivary gland epithelial cells (SGEC). However, the physiological role of TRPV1 in salivary secretion remains to be elucidated. We found that TRPV1 is expressed in mouse and human submandibular glands (SMG) and HSG cells, originated from human submandibular gland ducts at both mRNA and protein levels. However, capsaicin (CAP), TRPV1 agonist, had little effect on intracellular free calcium concentration ([Ca2+]i) in these cells, although carbachol consistently increased [Ca2+]i. Exposure of cells to high temperature (>43degrees C) or acidic bath solution (pH5.4) did not increase [Ca2+]i, either. We further examined the role of TRPV1 in salivary secretion using TRPV1 knock-out mice. There was no significant difference in the pilocarpine (PILO)-induced salivary flow rate between wild-type and TRPV1 knock-out mice. Saliva flow rate also showed insignificant change in the mice treated with PILO plus CAP compared with that in mice treated with PILO alone. Taken together, our results suggest that although TRPV1 is expressed in SGEC, it appears not to play any direct roles in saliva secretion via transcellular pathway.


Subject(s)
Animals , Baths , Calcium , Capsaicin , Carbachol , Epithelial Cells , Humans , Mice , Mice, Knockout , Pilocarpine , RNA, Messenger , Saliva , Salivary Glands , Sensory Receptor Cells , Submandibular Gland , Transcytosis
15.
Article in English | WPRIM | ID: wpr-347180

ABSTRACT

<p><b>OBJECTIVE</b>To observe the relaxant effect of Aike Mixture (AKM) on isolated bladder and prostatic urethral smooth muscle of rabbits.</p><p><b>METHODS</b>The isolated bladder and prostatic urethral smooth muscle from male rabbits were placed in a Magnus bath and smooth muscle contraction was measured using a biological signal acquisition and analysis system. The effects of AKM in combination with methoxyamine, carbachol and CaCl2 on the contractile tension of muscle strips were determined by cumulative dosing.</p><p><b>RESULTS</b>AKM dose-dependently reduced contractile tension of bladder trigone smooth muscle (r=0.831, P<0.05), reduced contractile wave amplitude (r=0.837, P<0.05) and decreased contractile frequency (r=-0.917, P<0.01). AKM significantly inhibited the increases in smooth muscle contraction induced by methoxyamine, carbachol and CaCl2.</p><p><b>CONCLUSION</b>AKM dose-dependently inhibited the contraction of rabbit isolated bladder and prostatic urethral smooth muscle by antagonizing α1-adrenergic receptors and M-cholinergic receptors.</p>


Subject(s)
Animals , Calcium Chloride , Pharmacology , Carbachol , Pharmacology , Drugs, Chinese Herbal , Pharmacology , Hydroxylamines , Pharmacology , In Vitro Techniques , Male , Muscle Contraction , Muscle, Smooth , Physiology , Neuromuscular Agents , Pharmacology , Prostate , Physiology , Rabbits , Receptors, Adrenergic, alpha-1 , Metabolism , Receptors, Muscarinic , Metabolism , Urethra , Physiology , Urinary Bladder , Physiology
16.
Article in English | WPRIM | ID: wpr-727605

ABSTRACT

Interstitial cells of Cajal (ICCs) from the urinary bladder regulate detrusor smooth muscle activities. We cultured ICCs from the urinary bladder of mice and performed patch clamp and intracellular Ca2+ ([Ca2+]i) imaging to investigate whether cultured ICCs can be a valuable tool for cellular functional studies. The cultured ICCs displayed two types of spontaneous electrical activities which are similar to those recorded in intact bladder tissues. Spontaneous electrical activities of cultured ICCs were nifedipine-sensitive. Carbachol and ATP, both excitatory neurotransmitters in the urinary bladder, depolarized the membrane and increased the frequency of spike potentials. Carbachol increased [Ca2+]i oscillations and basal Ca2+ levels, which were blocked by atropine. These results suggest that cultured ICCs from the urinary bladder retain rhythmic phenotypes similar to the spontaneous electrical activities recorded from the intact urinary bladder. Therefore, we suggest that cultured ICCs from the urinary bladder may be useful for cellular and molecular studies of ICCs.


Subject(s)
Action Potentials , Adenosine Triphosphate , Animals , Atropine , Carbachol , Interstitial Cells of Cajal , Membranes , Mice , Muscle, Smooth , Neurotransmitter Agents , Phenotype , Urinary Bladder
17.
Article in English | WPRIM | ID: wpr-235313

ABSTRACT

<p><b>OBJECTIVE</b>Endothelial apoptosis plays an important role in the initiation of atherosclerosis. It would be useful to clarify whether activation of non-neuronal muscarinic receptor (NNMR) could prevent endothelial apoptosis and atherosclerosis. We investigated the effects of NNMR activation on regulating rat aortic endothelial cells (RAECs) apoptosis induced by homocysteine, an independent risk factor of atherosclerosis, and further studied its molecular mechanism.</p><p><b>METHODS</b>RAECs were incubated using homocysteine at the concentration of 2.7 mmol/L for 36 h. RAECs were also pre-treated with carbachol or arecoline to examine their effects. RT-PCR was used to assess changes in the gene expression related to cell apoptosis.</p><p><b>RESULTS</b>Incubation of RAECs with homocysteine at the concentration of 2.7 mmol/L resulted in morphologic changes, such as cellular shrinkage, membrane blebbing, chromatin condensation and margination. These could be attenuated by pretreatment with carbachol and arecoline at the concentration of 10 micromol/L for 12 h. Homocysteine induced apoptosis in RAECs and the molecular mechanisms were associated with the regulation of fas, fas-L and caspase-8 in the death receptor pathway, bcl-2, bcl-xL and bax in the mitochondrial pathway, caspase-12 in the endoplasmic reticulum pathway and caspase-3, caspase-6 and p53 as downstream effectors. Carbachol and arecoline attenuated the effects of homocysteine on genes in the death receptor pathway, in the mitochondrial pathway and in the downstream pathway. Atropine could reverse all of the effects of arecoline.</p><p><b>CONCLUSION</b>Activation of NNMR by carbacol and arecoline inhibits homocysteine-induced endothelial cell apoptosis mainly through regulation of death receptor pathway, mitochondrial pathway and downstream effectors.</p>


Subject(s)
Animals , Aorta , Cell Biology , Apoptosis , Apoptosis Regulatory Proteins , Metabolism , Arecoline , Carbachol , Cell Cycle , Endoplasmic Reticulum , Metabolism , Endothelial Cells , Cell Biology , Homocysteine , Mitochondria , Metabolism , Rats , Receptors, Muscarinic , Metabolism
18.
Arch. venez. farmacol. ter ; 31(4): 72-79, 2012. graf
Article in Spanish | LILACS | ID: lil-699601

ABSTRACT

Muscarinic activation of bovine tracheal smooth muscle (BTSM) leading to smooth muscle contraction involves the generation of two cGMP signals (20 and 60 s), being 20s peak associated with soluble (sGC) and the second (60s) to membrane-bound Natriuretic Peptide- receptor-Guanylylcy clases (NPR-GC). In this study, we showed that pre-incubation of isolated BTSM strips with mastoparan and superactive mastoparan (mastoparan 7) decreased significantly the muscarinic dependent contractile smooth muscle responses in dose-dependent and non-competitive manner. Moreover, mastoparan (50 nM) inhibited completely the BTSM-muscarinic contractile responses and affected dramatically the carbachol-dependent cGMP signals being the first cGMP signal inhibited in a 63 ± 5%, whereas the second signal disappeared. Mastoparan inhibition of muscarinic activation is specific since other spasmogens as serotonin and histamine fully contracted these BTSM strips under mastoparan treatment. Cyclic GMP levels were evaluated by exposing BTSM strips to activators of NO-sensitive sGC as Sodium Nitroprussiate (SNP) and Natriuretic Peptides as CNP-53 for membrane-bound NPR-GC. Thus, SNP and CNP increased in a binary way, in more than 20 fold cGMP levels at 30-40 s being both increments inhibited by mastoparan. Furthermore, the Gi/o-protein involvement on mastoparan inhibition of cGMP elevations induced by CNP and SNP is suggested by Pertussis toxin pre-treatment, which reversed mastoparan effects. These results indicate that muscarinic signal transduction cascades leading to airway smooth muscle contractions involved two different guanylyl cyclases being both regulated by mastoparan-sensitive G-proteins. ANP, Natriuretic Peptide type A; ASM, Airway Smooth Muscle; BTSM, Bovine Tracheal Smooth Muscle; CNP-53, Natriuretic Peptide type C-53; GPCR, G-Protein Coupled Receptor; Gq16, Heterotrimeric G protein subtype 16; Gi/o, Heterotrimeric G protein subtype...


La activación muscarínica del músculo liso de las vías aéreasrelacionada a la contracción de dicho músculo liso esta asociada a la generación de dos señales de GMPc (20 y 60 s), siendo la señal de los 20s relacionado a la activación de la guanililciclasa soluble mientras que el pico de los 60s a la guanililciclasa unida membranas y sensible a péptidos natriuréticos (NPR-GC). En este trabajo, nosotros mostramos que la pre-incubación de fragmentos del músculo liso traqueal de bovino (BTSM) con mastoparan y su análogo superactivo (mastoparan 7), en una forma dosis dependiente, son capaces de disminuir de manera significativa la actividad contráctil dependiente de agentes muscarinicos. Adicionalmente, mastoparan (50 nM) inhibió completamente la respuesta contráctil muscarinica del BTSM y afectó dramáticamente los picos de GMPc asociados a la activación muscarinica siendola primera señal inhibida en un 63 ± 5%, mientras que la segunda señal desapareció completamente. Esta inhibición del mastoparan de la activación muscarínica es especifica ya que otros espamogenos como la serotonina y la histamina fueron capaces de inducir respuestas máximas en presencia del mastoparan y su análogos. Este efecto del mastoparan sobre los niveles del GMPc fue evaluado en presencia de otros agentes generadores de este segundo mensajero como son el nitroprusiato de sodio (SNP) que activa la guanililciclasa soluble sensible a NO y los péptidos natriureticos como el CNP-53 (CNP) activador de la NPR-GC asociada a membranas plasmáticas. Tanto, el SNP como el CNP aumentaronen mas de 50 veces los niveles de GMPc a los 30-40 s en forma bifasica, siendo estos incrementos inhibidos de manera significativa por el mastoparan. Ademas, se sugiere la participación de proteínas Gi/o en los efectos inhibitoriosdel mastoparan, porque la Toxina pertussis revertió los efectos inhibitorios. Estos resultados indican que la cascada de activación muscarinica que conduce...


Subject(s)
Animals , Carbachol/therapeutic use , Guanylate Cyclase/therapeutic use , Muscle, Smooth , Natriuretic Peptides/therapeutic use
19.
Acta Physiologica Sinica ; (6): 303-307, 2012.
Article in Chinese | WPRIM | ID: wpr-335909

ABSTRACT

To investigate the mechanisms underlying the cholinergic agonist carbachol-induced cardiovascular responses, changes of renin-angiotensin system were examined in fetal hormonal systems. In the ovine fetal model under stressless condition, the cardiovascular function was recorded. Blood samples were collected before (during baseline period) and after the intravenous administration of carbachol. Simultaneously, the levels of angiotensin I (Ang I), angiotensin II (Ang II) and vasopressin in the fetal plasma were detected by immunoradiological method. Also, blood gas, plasma osmolality and electrolyte concentrations were analyzed in blood samples. Results showed that in chronically prepared ovine fetus, intravenous infusion of carbachol led to a significant decrease of heart rate (P < 0.05), and a transient decrease followed by an increase of blood pressure (P < 0.05) within 30 min. After the intravenous infusion of carbachol, blood concentrations of Ang I and Ang II in near-term ovine fetus were both significantly increased (P < 0.05); however, blood concentration of vasopressin, values of blood gas, electrolytes and plasma osmolality in near-term ovine fetus were not significantly changed (P > 0.05). Blood levels of Ang I and Ang II in the atropine (M receptor antagonist) + carbachol intravenous administration group was lower than those in the carbachol group without atropine administration (P < 0.05). In conclusion, this study indicates that the near-term changes of cardiovascular system induced by intravenous administration of carbachol in ovine fetus, such as blood pressure and heart rate, are associated with the changes of hormones of circulatory renin-angiotensin system.


Subject(s)
Angiotensin I , Blood , Angiotensin II , Blood , Animals , Blood Pressure , Carbachol , Pharmacology , Cholinergic Agonists , Pharmacology , Fetus , Heart Rate , Renin-Angiotensin System , Sheep , Vasopressins , Blood
20.
Bol. latinoam. Caribe plantas med. aromát ; 10(5): 414-422, sept. 2011. tab, graf
Article in English | LILACS | ID: lil-618822

ABSTRACT

Ruta chalepensis, is used, in traditional medicine, as emmenagogue, abortive, and analgesic. We analyzed, in male Wistar rats, the effects of the chronic intake of an infusion of Ruta chalepensis (20 g/L) on the vasomotor responses of, either intact or endothelium-denuded aortic rings, to phenylephrine or carbachol. Only in rings with endothelium significant effects were observed. The infusion induced a leftward shift of the concentration-response curve to phenylephrine and an increase in maximal tension development. These effects were abolished by indomethacin. In these rings, inhibiting the synthesis of nitric oxide, in the presence of indomethacin, induced a leftward shift of the concentration response curve to phenylephrine, as well as an increase in maximal tension. These results suggest that the chronic ingestion of a Ruta chalepensis infusion induces an endothelium dependent increase in the synthesis/release of cyclooxygenase-dependent vasoconstrictor prostanoids, and an increase in the basal synthesis/release of nitric oxide.


Ruta chalepensis se utiliza en la medicina tradicional como emenagogo, abortivo y analgésico. Se analizaron, en ratas Wistar macho, los efectos de la ingesta crónica de una infusión de Ruta chalepensis (20 g /L), sobre las respuestas vasomotoras de anillos de aorta con y sin endotelio, a la fenilefrina o al carbacol Se observaron efectos significativos sólo en anillos con endotelio. La infusión indujo un desplazamiento a la izquierda de la curva de concentración-respuesta a fenilefrina y un incremento en la tensión máxima desarrollada. Estos efectos fueron abolidos por la indometacina. La inhibición de la síntesis de óxido nítrico, en presencia de indometacina, produjo un desplazamiento a la izquierda de la curva de concentración-respuesta a la fenilefrina, así como un incremento en la tensión máxima. Estos resultados sugieren que la ingesta crónica de una infusión de Ruta chalepensis induce un incremento en la síntesis/liberación de prostanoides vasoconstrictores dependientes de la ciclooxigenasa y un aumento en la síntesis /liberación basal de óxido nítrico.


Subject(s)
Male , Animals , Rats , Aorta , Endothelium, Vascular , Plant Extracts/administration & dosage , Ruta/chemistry , Carbachol/pharmacology , Dose-Response Relationship, Drug , Drug Interactions , Plant Extracts/pharmacology , Phenylephrine/pharmacology , Nitric Oxide , Rats, Wistar
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