RESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Different parts of Mimosa pigra (MPG) are used in traditional medicine in Madagascar, tropical Africa, South America and Indonesia for various troubles including cardiovascular disorders. AIM OF THE STUDY: To investigate the mechanisms underlying the vascular effects of MPG by assessing in vitro its antioxidant and anti-inflammatory properties, and its vascular relaxing effects, and in vivo, its action on hypoxic pulmonary hypertension (PAH) in rats. MATERIAL AND METHODS: The antioxidant activity of MPG leaf hydromethanolic extract was determined by using both the 1,1-diphenyl-2-picrylhydrazyl radical scavenging and the oxygen radical absorbance capacity in vitro assays. Anti-inflammatory properties were assayed on TNFα-induced VCAM-1 expression in endothelial cells. The vasorelaxant effect of MPG extract was studied on rat arterial rings pre-contracted with phenylephrine (1µM) in the presence or absence of the endothelium. In vivo MPG extract effects were analyzed in chronic hypoxic PAH, obtained by housing male Wistar rats, orally treated or not with MPG extract (400mg/kg/d), in a hypobaric chamber for 21 days. RESULTS: MPG leaf extract had antioxidant and anti-inflammatory properties. It induced endothelium-dependent, NO-mediated relaxation of rat aorta and pulmonary artery. In vivo, chronic MPG treatment reduced hypoxic PAH in rat by decreasing by 22.3% the pulmonary arterial pressure and by 20.0% and 23.9% the pulmonary artery and cardiac remodelling, respectively. This effect was associated with a restoration of endothelium function and a 2.3-fold increase in endothelial NO synthase phosphorylation. MPG leaf hydromethanolic extract contained tryptophan and flavonoids, including quercetin glycosides. Both compounds also efficiently limit hypoxia-induced PAH. CONCLUSIONS: Our results show endothelial protective action of MPG leaf hydromethanolic extract which is likely to be due to its antioxidant action. MPG successfully attenuated the development of PAH, thus demonstrating the protective effect of MPG on cardiovascular diseases.
Asunto(s)
Antioxidantes/uso terapéutico , Cardiotónicos/uso terapéutico , Hipertensión Pulmonar/tratamiento farmacológico , Mimosa , Extractos Vegetales/uso terapéutico , Vasodilatadores/uso terapéutico , Animales , Antioxidantes/farmacología , Aorta/efectos de los fármacos , Aorta/fisiología , Cardiotónicos/farmacología , Células Cultivadas , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/fisiología , Humanos , Hipertensión Pulmonar/patología , Hipertensión Pulmonar/fisiopatología , Hipertrofia Ventricular Derecha/tratamiento farmacológico , Hipertrofia Ventricular Derecha/patología , Hipertrofia Ventricular Derecha/fisiopatología , Hipoxia/complicaciones , Técnicas In Vitro , Masculino , Fitoterapia , Extractos Vegetales/farmacología , Hojas de la Planta , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/patología , Arteria Pulmonar/fisiología , Ratas , Ratas Wistar , Vasodilatadores/farmacologíaRESUMEN
Endothelial dysfunction is one of the main consequences of the toxic effects of cigarette smoke on the vascular system. Increasing evidence suggests that the small G-protein RhoA and its downstream effectors, the Rho-kinases (ROCKs), are involved in systemic endothelial dysfunction induced by cigarette smoke. This study aimed to evaluate the role of the RhoA/ROCKs pathway in pulmonary artery endothelial function in current smokers with normal lung function. Lung tissues were obtained from nonsmokers and smokers who underwent lobectomy for lung carcinoma. Arterial relaxation in response to acetylcholine (ACh) was assessed in isolated pulmonary arterial rings. Protein expressions and activities of endothelial nitric oxide synthase (eNOS), ROCKs and the myosin phosphatase subunit 1 (MYPT-1) were sought. Relaxation in response to ACh was significantly lower in smokers as compared with nonsmokers (n = 8 in each group), consistent with reduced eNOS activity in the former compared with the latter. eNOS protein expression remained, however, the same in both groups. Expression of ROCKs, guanosine triphosphate-RhoA and phosphorylated MYPT-1 were significantly increased in smokers compared with controls. Pulmonary endothelial dysfunction is present in smokers whose lung function has not yet been impaired. Reduced activity of eNOS accounts at least in part for this endothelial dysfunction. Increased expression and activity of ROCKs accounts for another part through direct or indirect inhibition of the Rho-A/ROCKs pathway on nitric oxide synthesis and sustained pulmonary vasoconstriction through inhibition of myosin phosphatase.
Asunto(s)
Endotelio Vascular/enzimología , Arteria Pulmonar/enzimología , Fumar/metabolismo , Quinasas Asociadas a rho/biosíntesis , Acetilcolina/farmacología , Anciano , Carcinoma Broncogénico/enzimología , Carcinoma Broncogénico/cirugía , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/patología , Humanos , Pulmón/efectos de los fármacos , Pulmón/enzimología , Pulmón/patología , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/cirugía , Masculino , Persona de Mediana Edad , Fosfatasa de Miosina de Cadena Ligera/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/patología , Pruebas de Función Respiratoria , Fumar/patología , Vasoconstricción/efectos de los fármacos , Proteína de Unión al GTP rhoA/biosíntesisRESUMEN
BACKGROUND AND PURPOSE: Pulmonary arterial hypertension (PAH) is associated with increased contraction and proliferation of pulmonary vascular smooth muscle cells. The anti-diabetic drug metformin has been shown to have relaxant and anti-proliferation properties. We thus examined the effect of metformin in PAH. EXPERIMENTAL APPROACH: Metformin effects were analysed in hypoxia- and monocrotaline-induced PAH in rats. Ex vivo and in vitro analyses were performed in lungs, pulmonary artery rings and cells. KEY RESULTS: In hypoxia- and monocrotaline-induced PAH, the changes in mean pulmonary arterial pressure and right heart hypertrophy were nearly normalized by metformin treatment (100 mg.kg(-1).day(-1)). Pulmonary arterial remodelling occurring in both experimental models of PAH was also inhibited by metformin treatment. In rats with monocrotaline-induced PAH, treatment with metformin significantly increased survival. Metformin increased endothelial nitric oxide synthase phosphorylation and decreased Rho kinase activity in pulmonary artery from rats with PAH. These effects are associated with an improvement of carbachol-induced relaxation and reduction of phenylephrine-induced contraction of pulmonary artery. In addition, metformin inhibited mitogen-activated protein kinase activation and strongly reduced pulmonary arterial cell proliferation during PAH. In vitro, metformin directly inhibited pulmonary artery smooth muscle cell growth. CONCLUSIONS AND IMPLICATIONS: Metformin protected against PAH, regardless of the initiating stimulus. This protective effect may be related to its anti-remodelling property involving improvement of endothelial function, vasodilatory and anti-proliferative actions. As metformin is currently prescribed to treat diabetic patients, assessment of its use as a therapy against PAH in humans should be easier.
Asunto(s)
Hipertensión Pulmonar/prevención & control , Hipoglucemiantes/farmacología , Metformina/farmacología , Animales , Proliferación Celular/efectos de los fármacos , Enfermedad Crónica , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiopatología , Activación Enzimática , Hemodinámica/efectos de los fármacos , Hipertensión Pulmonar/patología , Hipertensión Pulmonar/fisiopatología , Hipertrofia Ventricular Derecha/prevención & control , Hipoglucemiantes/uso terapéutico , Técnicas In Vitro , Masculino , Metformina/uso terapéutico , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Contracción Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/patología , Músculo Liso Vascular/fisiopatología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Fosforilación , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/patología , Arteria Pulmonar/fisiopatología , Ratas , Ratas Wistar , Quinasas Asociadas a rho/metabolismoRESUMEN
As the cellular and molecular mechanisms of major arterial diseases such as atherosclerosis and hypertension are being more clearly defined, it is becoming apparent that these pathological processes share a number of functional and biochemical features in the vessel wall. Typically, arterial diseases are associated with functional and structural wall alterations including modified contractile properties, smooth muscle cell hypertrophy and proliferation, endothelial dysfunction, excessive extracellular matrix accumulation and inflammation. Small G proteins of the Rho family are defined as major regulators of cell functions including migration, proliferation, differentiation and gene transcription. Recent studies have demonstrated that activation of Rho proteins appears to be a common component for the pathogenesis of hypertension and vascular proliferative disorders. Functional analyses have further revealed that RhoA-dependent pathways are involved in excessive contraction, migration and proliferation associated with arterial diseases. This review focuses on the role of Rho proteins, in particular RhoA, in vascular smooth muscle cells and the involvement of Rho-dependent signaling pathways in vascular diseases.
Asunto(s)
Arteriosclerosis/metabolismo , Reestenosis Coronaria/metabolismo , Hipertensión Pulmonar/metabolismo , Hipertensión/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Animales , Arteriosclerosis/prevención & control , Movimiento Celular , Reestenosis Coronaria/prevención & control , Humanos , Hipertensión/prevención & control , Hipertensión Pulmonar/prevención & control , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
The P2Y12 ADP receptor is one of the major regulators of platelet activation and the target of antithrombotic thienopyridines (ticlopidine and clopidogrel). It has been recently cloned but the signaling pathways triggered by this receptor are still poorly documented. Here, we show that stimulation of the human P2Y12 receptor stably expressed in Chinese hamster ovary cells activates two major intracellular signaling mechanisms leading either to cell proliferation or to actin cytoskeleton reorganization. Both effects were blocked by the active metabolite of clopidogrel, a specific antagonist of P2Y12. The P2Y12-mediated stimulation of proliferation required the pertussis toxin-sensitive activation of PI3-kinase/Akt upstream of MAP-kinases. A partial contribution of a transactivation mechanism, through the tyrosine kinase receptor platelet-derived growth factor (PDGF)-R-beta, was also observed. Conversely, the P2Y12-mediated reorganization of the actin cytoskeleton was Gi-independent, requiring activation of RhoA and Rho-kinase. Our results provide new insights into the molecular basis of P2Y12-mediated intracellular signaling. These data may prove to be useful for a better understanding of the physiological role of P2Y12, particularly in platelets and glial cells which express this important therapeutic target.
Asunto(s)
Adenosina Difosfato/análogos & derivados , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Proteínas de la Membrana/metabolismo , Receptores Purinérgicos P2/metabolismo , Actinas/metabolismo , Adenosina Difosfato/farmacología , Animales , Células CHO , División Celular/efectos de los fármacos , Cricetinae , Humanos , Sistema de Señalización de MAP Quinasas , Modelos Biológicos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptores Purinérgicos P2Y12 , Proteínas Recombinantes/metabolismo , Transducción de Señal , Tionucleótidos/farmacología , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Although the use of stents has limited the incidence of restenosis, in-stent restenosis remains an important problem. In-stent restenosis is the result of a healing process that induced neointimal hyperplasia through mechanisms that are still not understood. The aim of this study was to analyze the histological consequences of the healing process following stent implantation. Internal mammary arteries from atheroslerotic patients undergoing coronary artery bypass surgery were stented and maintained in culture for 0-28 days. Stent implantation after predilatation induced an extensive loss of endothelial cells whereas direct stenting preserved endothelium between the struts. Morphometric analysis shows that stent placement induced neointimal thickening. Smooth muscle alpha-actin labeling indicates that neo-intimal formation was mainly due to proliferation and migration of smooth muscle cells. Smooth muscle cell proliferation, assessed by MIB-1 staining, was maximal at day 14 after stent insertion. Human mammary artery organ culture thus provides valuable information on histological consequences of stent implantation with or without predilatation regarding endothelial cell disappearance and neointimal hyperplasia. These data also demonstrate that neointimal thickening induced by stent implantation comprises an intrinsic component resulting from the vessel wall response to stent insertion and suggest that blood factors could play an amplifying but not necessary role.
Asunto(s)
Arterias Mamarias/patología , Stents , División Celular , Reestenosis Coronaria , Endotelio Vascular/patología , Humanos , Hiperplasia , Técnicas de Cultivo de Órganos , Túnica Íntima/patologíaRESUMEN
During pregnancy, the uterus undergoes major functional and structural remodelling. It is well known that during the major part of pregnancy, the myometrium normally remains relatively quiescent but is able to generate powerful contractions at the time of parturition. However, the intracellular molecular events regulating myometrial contractility during pregnancy still remain poorly understood. We applied differential gene expression screening using cDNA array technology to probe myometrium samples from non-pregnant and mid-pregnant (15 days) rabbits. Among the differentially expressed genes, the farnesylated small G-protein of the Rho family, Rnd3, was found to be upregulated (3.6-fold) at mid-pregnancy. Upregulation of Rnd3 was confirmed at the protein level by a 3.4-fold increase in Rnd3 expression in mid-pregnant myometrium. Measurements of contractile properties of beta-escin permeabilized smooth muscle strips revealed that the upregulation of Rnd3 correlated with an inhibition of RhoA-Rho kinase-mediated Ca2+ sensitization at mid-pregnancy. Treatment of muscle strips from mid-pregnant myometrium with the farnesyl-transferase inhibitor manumycin A (10 muM) led to the recovery of RhoA-Rho kinase-dependent Ca2+ sensitization. At late pregnancy (31 days), upregulation of RhoA and Rho kinase expression was associated with an increase in Ca2+ sensitivity of contractile proteins that was inhibited by the Rho kinase inhibitor Y-27632 (10 muM). These data thus demonstrate the time-dependent regulation of the RhoA-Rho kinase-mediated Ca2+ sensitization during the course of pregnancy. The depression of this mechanism at mid-pregnancy followed by its constitutive activation near term is associated with a co-ordinated modulation of Rnd3, RhoA and Rho kinase expression. The RhoA-Rho kinase signalling pathway and its regulators might thus represent potential targets for the development of new treatments for pre-term labour.
Asunto(s)
Calcio/fisiología , Proteínas Activadoras de GTPasa/fisiología , Miometrio/fisiología , Preñez/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Proteína de Unión al GTP rhoA/fisiología , Animales , Western Blotting , ADN Complementario/biosíntesis , ADN Complementario/genética , Femenino , Proteínas Activadoras de GTPasa/genética , Perfilación de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular , Contracción Isométrica/fisiología , Fibras Musculares Esqueléticas/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos , Embarazo , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/biosíntesis , Conejos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia Arriba/genética , Regulación hacia Arriba/fisiología , Quinasas Asociadas a rho , Proteína de Unión al GTP rhoA/genéticaRESUMEN
Migration and proliferation of arterial smooth muscle cells (SMCs) play a prominent role in the development of atherosclerotic plaques and restenosis lesions. Most of the growth-regulatory molecules potentially involved in these pathological conditions also demonstrate chemotactic properties. Extracellular purine and pyrimidine nucleotides have been shown to induce cell cycle progression and to elicit growth of cultured vascular SMCs. Moreover, the P2Y(2) ATP/UTP receptor was overexpressed in intimal thickening, suggesting a role of these nucleotides in vascular remodeling. Using the Transwell system migration assay, we demonstrate that extracellular ATP, UTP, and UDP exhibit a concentration-dependent chemotactic effect on cultured rat aortic SMCs. UTP, the most powerful nucleotide inducer of migration, elicited significant responses from 10 nmol/L. In parallel, UTP increased osteopontin expression dose-dependently. The blockade of osteopontin or its integrin receptors alpha(v)beta(3)/beta(5) by specific antibodies or antagonists inhibited UTP-induced migration. Moreover, the blockade of ERK-1/ERK-2 MAP kinase or rho protein pathways led to the inhibition of both UTP-induced osteopontin increase and migration, demonstrating the central role of osteopontin in this process. Taken together, these results suggest that extracellular nucleotides, and particularly UTP, can induce arterial SMC migration via the action of osteopontin.
Asunto(s)
Movimiento Celular/fisiología , Espacio Extracelular/metabolismo , Músculo Liso Vascular/metabolismo , Nucleótidos/farmacología , Sialoglicoproteínas/metabolismo , Adenosina Difosfato/farmacología , Adenosina Trifosfato/farmacología , Animales , Aorta , Calcio/metabolismo , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Quimiotaxis/efectos de los fármacos , Quimiotaxis/fisiología , Cámaras de Difusión de Cultivos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Péptidos y Proteínas de Señalización Intracelular , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Oligopéptidos/farmacología , Osteopontina , Fosforilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Ratas Wistar , Uridina Difosfato/farmacología , Uridina Trifosfato/farmacología , Quinasas Asociadas a rho , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
The aim of this work was to investigate the coupling of human urotensin II (hU-II) to RhoA activation and regulation of RhoA-dependent functions. The use of the Rho-kinase inhibitor Y-27632 and the development of a membrane-permeant RhoA inhibitor (TAT-C3) allowed us to demonstrate that hU-II induced arterial smooth muscle contraction, actin stress fiber formation, and proliferation through the activation of the small GTPase RhoA and its downstream effector Rho-kinase.
Asunto(s)
Toxinas Botulínicas , Músculo Liso Vascular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Urotensinas/farmacología , Vasoconstricción/fisiología , Proteína de Unión al GTP rhoA/metabolismo , ADP Ribosa Transferasas/genética , ADP Ribosa Transferasas/metabolismo , ADP Ribosa Transferasas/farmacología , Amidas/farmacología , Animales , Aorta Torácica/citología , Aorta Torácica/metabolismo , División Celular/efectos de los fármacos , Células Cultivadas , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Relación Dosis-Respuesta a Droga , Activación Enzimática/fisiología , Inhibidores Enzimáticos/farmacología , Humanos , Técnicas In Vitro , Péptidos y Proteínas de Señalización Intracelular , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Norepinefrina/antagonistas & inhibidores , Norepinefrina/farmacología , Cloruro de Potasio/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Piridinas/farmacología , Ratas , Ratas Wistar , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Urotensinas/metabolismo , Vasoconstricción/efectos de los fármacos , Vasoconstrictores/antagonistas & inhibidores , Vasoconstrictores/farmacología , Quinasas Asociadas a rho , Proteína de Unión al GTP rhoA/antagonistas & inhibidoresRESUMEN
We have addressed the role of Rho-kinase in the different steps of thrombin receptor agonist peptide (TRAP)-induced platelet activation. Interestingly, under physiological conditions, incubation of platelets with increasing concentrations of the specific Rho-kinase inhibitor Y-27632 resulted in a dose-dependent reversion of the aggregation induced by 10 microM TRAP, without affecting serotonin secretion. Addition of Y-27632 after three minutes of TRAP stimulation, when the maximal aggregation was reached, resulted in a rapid disaggregation of platelets. Accordingly, the early peak of myosin light chain (MLC) phosphorylation induced by TRAP was not affected by Y-27632 but its sustained phosphorylation, observed during the irreversible phase of aggregation, was dependent of Rho-kinase activity. The rapid decrease in MLC phosphorylation upon Y-27632 treatment correlated well with the specific disappearance of myosin heavy chain from the cytoskeleton and preceded platelet disaggregation. Finally, we provide evidence that secreted ADP, known to play a key role in TRAP-induced irreversible phase of aggregation, was involved in the sustained MLC phosphorylation through Rho-kinase and could be replaced by epinephrine.
Asunto(s)
Miosinas/metabolismo , Fragmentos de Péptidos/farmacología , Agregación Plaquetaria/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/fisiología , Adenosina Difosfato/metabolismo , Adenosina Difosfato/farmacología , Amidas/farmacología , Plaquetas/citología , Plaquetas/efectos de los fármacos , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Péptidos y Proteínas de Señalización Intracelular , Cinética , Miosinas/efectos de los fármacos , Fosforilación , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Piridinas/farmacología , Quinasas Asociadas a rhoRESUMEN
1. Vasospasm of arterial conduits used for coronary artery surgery is an important cause of graft failure and is likely to result partly from raised levels of vasoconstrictor substances such as thromboxane A(2) and endothelin-1. Our aim was to find pharmacological agents that could prevent agonist-induced vasospasm. 2. Isometric tension was recorded from discarded segments of human left internal mammary artery (LIMA). Submaximal contraction evoked by the thromboxane A(2) mimetic U46619 (10 nM) was not inhibited by a blocker of store- and receptor-operated Ca(2+) channels (30 microM SKF96365) in the presence of diltiazem. Furthermore, contractions to < or =1 nM U46619 were preserved when extracellular Ca(2+) was reduced from 2.5 mM to 60 nM. Thus, sustained U46619-evoked contraction occurred without Ca(2+) influx. 3., We hypothesized that contraction might occur via Rho-kinase-mediated Ca(2+)-sensitization of myofilaments. Inhibitors of Rho-kinase (Y27632 and HA1077) were profound relaxants. If contraction was pre-evoked by 10 nM U46619, Y27632 and HA1077 caused full relaxation with EC(50)s of 1.67+/-0.22 microM and 3.58+/-0.35 microM respectively. Y27632 was also effective if applied before U46619, but was less potent. 4. Y27632 abolished contraction evoked by endothelin-1 and significantly reduced resting tone in the absence of a vasoconstrictor. 5. Rho-kinase-mediated Ca(2+)-sensitization appears to be a major mechanism of vasoconstriction in human LIMA. Rho-kinase inhibitors may have an important role in preventing vasospasm in arterial grafts used for coronary artery surgery.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Arterias Mamarias/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Vasoconstricción/efectos de los fármacos , Calcio/metabolismo , Canales de Calcio Tipo L/efectos de los fármacos , Señalización del Calcio/efectos de los fármacos , Endotelio Vascular/fisiología , Femenino , Humanos , Técnicas In Vitro , Péptidos y Proteínas de Señalización Intracelular , Contracción Isométrica/efectos de los fármacos , Arterias Mamarias/fisiología , Contracción Muscular/efectos de los fármacos , Relajación Muscular/efectos de los fármacos , Músculo Liso Vascular/fisiología , Quinasas Asociadas a rhoRESUMEN
In the cardiovascular system, activation of ionotropic (P2X receptors) and metabotropic (P2Y receptors) P2 nucleotide receptors exerts potent and various responses including vasodilation, vasoconstriction, and vascular smooth muscle cell proliferation. Here we examined the involvement of the small GTPase RhoA in P2Y receptor-mediated effects in vascular myocytes. Stimulation of cultured aortic myocytes with P2Y receptor agonists induced an increase in the amount of membrane-bound RhoA and stimulated actin cytoskeleton organization. P2Y receptor agonist-induced actin stress fiber formation was inhibited by C3 exoenzyme and the Rho kinase inhibitor Y-27632. Stimulation of actin cytoskeleton organization by extracellular nucleotides was also abolished in aortic myocytes expressing a dominant negative form of RhoA. Extracellular nucleotides induced contraction and Y-27632-sensitive Ca(2+) sensitization in aortic rings. Transfection of Swiss 3T3 cells with P2Y receptors showed that Rho kinase-dependent actin stress fiber organization was induced in cells expressing P2Y(1), P2Y(2), P2Y(4), or P2Y(6) receptor subtypes. Our data demonstrate that P2Y(1), P2Y(2), P2Y(4), and P2Y(6) receptor subtypes are coupled to activation of RhoA and subsequently to Rho-dependent signaling pathways.
Asunto(s)
Músculo Liso Vascular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores Purinérgicos P2/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Células 3T3 , Actinas/fisiología , Animales , Aorta/citología , Aorta/efectos de los fármacos , Aorta/metabolismo , Aorta/fisiología , Membrana Celular/metabolismo , Células Cultivadas , Citoesqueleto/fisiología , Activación Enzimática/fisiología , Fibroblastos/metabolismo , Membranas Intracelulares/fisiología , Péptidos y Proteínas de Señalización Intracelular , Ratones , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/fisiología , Nucleótidos/farmacología , Proteínas Serina-Treonina Quinasas/fisiología , Agonistas del Receptor Purinérgico P2 , Ratas , Ratas Wistar , Transducción de Señal/fisiología , Vasoconstricción/fisiología , Quinasas Asociadas a rhoRESUMEN
The potent vasodilator action of cyclic GMP-dependent protein kinase (cGK) involves decreasing the Ca(2+) sensitivity of contraction of smooth muscle via stimulation of myosin light chain phosphatase through unknown mechanisms (Wu, X., Somlyo, A. V., and Somlyo, A. P. (1996) Biochem. Biophys. Res. Commun. 220, 658-663). Myosin light chain phosphatase activity is controlled by the small GTPase RhoA and its target Rho kinase. Here we demonstrate cGMP effects mediated by cGK that inhibit RhoA-dependent Ca(2+) sensitization of contraction of blood vessels and actin cytoskeleton organization in cultured vascular myocytes. Ca(2+) sensitization and actin organization were inhibited by both 8-bromo-cGMP and sodium nitroprusside (SNP). SNP also caused translocation of activated RhoA from the membrane to the cytosol. SNP-induced actin disassembly was lost in vascular myocytes in culture after successive passages but was restored by transfection of cells with cGK I. Furthermore, cGK phosphorylated RhoA in vitro, and addition of cGK I inhibited RhoA-induced Ca(2+) sensitization in permeabilized smooth muscle. 8-Bromo-cGMP-induced actin disassembly was inhibited in vascular myocytes expressing RhoA(Ala-188), a mutant that could not be phosphorylated. Collectively, these results indicate that cGK phosphorylates and inhibits RhoA and suggest that the consequent inhibition of RhoA-induced Ca(2+) sensitization and actin cytoskeleton organization contributes to the vasodilator action of nitric oxide.
Asunto(s)
Señalización del Calcio/fisiología , Calcio/fisiología , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Contracción Isométrica/fisiología , Músculo Liso Vascular/fisiología , Proteína de Unión al GTP rhoA/metabolismo , Actinas/efectos de los fármacos , Actinas/metabolismo , Animales , Aorta/fisiología , Células Cultivadas , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacología , Citoesqueleto/efectos de los fármacos , Citoesqueleto/fisiología , Galopamilo/farmacología , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Cobayas , Técnicas In Vitro , Músculo Liso Vascular/efectos de los fármacos , Nitroprusiato/farmacología , Fenilefrina/farmacología , Fosforilación , Vena Porta/fisiología , Arteria Pulmonar/fisiología , Conejos , Ratas , Ratas Wistar , Transducción de Señal , Tapsigargina/farmacologíaRESUMEN
BACKGROUND: Peptide YY (PYY) is involved in the regulation of several gut functions, including secretion and motility. It exerts its effects through a family of six receptors, commonly named the Y receptor family. AIMS: To characterise the effects of PYY on strips of rat proximal colon in vitro, and to determine the pathways and receptors involved. METHODS: Contractions of strips removed from the muscle layer of rat proximal colon were recorded under isometric conditions, using PYY, Y receptor agonists and antagonists, and nerve blockers. Reverse transcription-polymerase chain reaction was also performed to detect the presence of mRNA coding for Y receptors. Finally, smooth muscle cells were isolated to estimate the cell length and intracellular Ca(2+) concentration in the presence and absence of PYY. RESULTS: PYY, neuropeptide Y (NPY), pancreatic polypeptide (PP) and [Leu31,Pro34]NPY induced a dose dependent contraction of strips from proximal colon. Tetrodotoxin partially inhibited the PYY and NPY induced contractions, and strongly inhibited the PP induced contraction. Specific antagonists showed the involvement of cholinergic nicotinic receptors and NK1 receptor. BIBP 3226, a specific Y1 antagonist, did not modify the colonic smooth muscle response to PYY, whereas blocking L-type Ca(2+) channels with D-600 abolished its effects. Moreover, PYY induced an increase in intracellular Ca(2+) concentration, associated with a reduction in cell length. mRNA encoding Y1 and Y4 receptors were detected in the muscle strips. CONCLUSIONS: These findings suggest that PYY stimulates colonic contractile activity in vitro through (a) a nervous Y4 dependent pathway and (b) a pathway involving a potential new receptor on myocytes.
Asunto(s)
Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Péptido YY/farmacología , Animales , Colon/efectos de los fármacos , Colon/fisiología , Relación Dosis-Respuesta a Droga , Masculino , Contracción Muscular/fisiología , Músculo Liso/fisiología , Péptido YY/antagonistas & inhibidores , Ratas , Ratas Wistar , Receptores Colinérgicos/efectos de los fármacos , Receptores Colinérgicos/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Mamba intestinal toxin (MIT(1)) isolated from Dendroaspis polylepis venom is a 81 amino acid polypeptide cross-linked by five disulphide bridges. MIT(1) has a very potent action on guinea-pig intestinal contractility. MIT(1) (1 nM) potently contracts longitudinal ileal muscle and distal colon, and this contraction is equivalent to that of 40 mM K(+). Conversely MIT(1) relaxes proximal colon again as potently as 40 mM K(+). The MIT(1)-induced effects are antagonised by tetrodotoxin (1 microM) in proximal and distal colon but not in longitudinal ileum. The MIT(1)-induced relaxation of the proximal colon is reversibly inhibited by the NO synthase inhibitor L-NAME (200 microM). (125)I-labelled MIT(1) binds with a very high affinity to both ileum and brain membranes (K(d)=1.3 pM and 0.9 pM, and B(max)=30 fmol/mg and 26 fmol/mg, respectively). MIT(1) is a very highly selective toxin for a receptor present both in the CNS and in the smooth muscle and which might be an as yet unidentified K(+) channel.
Asunto(s)
Venenos Elapídicos/química , Motilidad Gastrointestinal/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Péptidos/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Sitios de Unión , Química Encefálica , Colon/efectos de los fármacos , Cistina/química , Cobayas , Íleon/efectos de los fármacos , Péptidos y Proteínas de Señalización Intercelular , Datos de Secuencia Molecular , Contracción Muscular/efectos de los fármacos , Especificidad de Órganos , Péptidos/química , Péptidos/farmacología , Ratas , Tetrodotoxina/farmacologíaRESUMEN
1. The small GTP-binding Rho proteins are involved in the agonist-induced Ca2+ sensitization of smooth muscle. The action and the expression of Rnd1, a new member of the Rho protein family constitutively bound to GTP, has been studied in rat smooth muscle. 2. Recombinant prenylated Rnd1 (0.01-0.1 mg ml-1) dose dependently inhibited carbachol- and GTPgammaS-induced Ca2+ sensitization in beta-escin-permeabilized ileal smooth muscle strips but had no effect on the tension at submaximal [Ca2+] (pCa 6.3). Rnd1 inhibited GTPgammaS-induced tension without shifting the dose-response curves to GTPgammaS. 3. pCa-tension relationships were not modified by Rnd1 and the rise in tension induced through the inhibition of myosin light chain phosphatase by calyculin A was not affected by Rnd1. 4. The Ca2+ sensitization induced by recombinant RhoA was completely abolished when RhoA and Rnd1 were applied together. 5. Rnd1 was expressed at a low level in membrane fractions prepared from intestinal or arterial smooth muscles. The expression of Rnd1 was strongly increased in ileal and aortic smooth muscle from rats treated with progesterone or oestrogen. Progesterone-treated ileal muscle strips showed a decrease in agonist-induced Ca2+ sensitization. 6. The present study shows that (i) Rnd1 inhibits agonist- and GTPgammaS-induced Ca2+ sensitization of smooth muscle by specifically interfering with a RhoA-dependent mechanism and (ii) an increase in Rnd1 expression may account, at least in part, for the steroid-induced decrease in agonist-induced Ca2+ sensitization.
Asunto(s)
Señalización del Calcio/efectos de los fármacos , Proteínas de Unión al GTP/farmacología , Músculo Liso/fisiología , Proteínas de Unión al GTP rho , Animales , Western Blotting , Carbacol/antagonistas & inhibidores , Carbacol/farmacología , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Inhibidores Enzimáticos/farmacología , Proteínas de Unión al GTP/biosíntesis , Hormonas Esteroides Gonadales/farmacología , Guanosina 5'-O-(3-Tiotrifosfato)/antagonistas & inhibidores , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Guanosina Trifosfato/metabolismo , Técnicas In Vitro , Contracción Isométrica/efectos de los fármacos , Toxinas Marinas , Agonistas Muscarínicos/farmacología , Músculo Liso/efectos de los fármacos , Músculo Liso/metabolismo , Oxazoles/farmacología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Ratas , Ratas Wistar , Proteínas Recombinantes/farmacologíaRESUMEN
In cutaneous veins where purinergic neurotransmission is more prominent compared with in deep vessels, physiological and pathological roles of nerve-released ATP have been described. Neuronally released ATP has been reported to act through activation of unidentified ionotropic P2X receptor(s). This study analyzed P2X receptor subtypes expressed in human saphenous vein smooth muscle and their physiological functions. Transcripts for both hP2X1 receptors, already identified in other smooth muscles, and, surprisingly, hP2X7 receptors known to be responsible for the cytotoxic effect of ATP in macrophages were detected by Northern blot analysis in total RNA from saphenous vein smooth muscle. ATP and other P2X receptor agonists [alphabeta-methylene-ATP, 2-methylthio-ATP, and 2',3'-(4-benzoyl)benzoyl-ATP] dose-dependently contracted venous rings, but the contraction induced by 2-methylthio-ATP was more transient than that evoked by the other P2X agonists. The effect of hP2X1 agonists involved the activation of a rapidly desensitizing cation current recorded in freshly isolated myocytes. The action of hP2X7 receptor agonists was related to a maintained nondesensitizing cation current. In addition, hP2X7 receptor activation formed membrane pores that were permeable to large molecules. hP2X1 and hP2X7 receptors coexpressed in COS cells did not associate to form heteromultimers. Our data indicate that both hP2X1 and hP2X7 receptors are expressed as 2 separated populations of channels in human saphenous vein myocytes and are involved in ATP-induced tension. We suggest that cell lysis consequent to hP2X7 receptor-induced pore formation contributes to the disorganization and decrease in the amount of contractile myocytes in the media of varicose veins.
Asunto(s)
Proteínas Musculares/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Receptores Purinérgicos P2/efectos de los fármacos , Vasoconstricción/efectos de los fármacos , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Animales , Células COS , Femenino , Humanos , Activación del Canal Iónico/efectos de los fármacos , Canales Iónicos/efectos de los fármacos , Canales Iónicos/fisiología , Transporte Iónico/efectos de los fármacos , Masculino , Persona de Mediana Edad , Proteínas Musculares/genética , Proteínas Musculares/fisiología , Músculo Liso Vascular/fisiología , Técnicas de Placa-Clamp , Reacción en Cadena de la Polimerasa , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/fisiología , Receptores Purinérgicos P2X7 , Proteínas Recombinantes de Fusión/metabolismo , Vena Safena , Tionucleótidos/farmacología , Vasoconstricción/fisiologíaRESUMEN
1. Effects of oestrogen on the current evoked by ATP and benzoylbenzoyl ATP (BzATP) in CV-1 monkey kidney cells transformed by SV 40 (COS cells) expressing the human P2X7 (hP2X7) purinoceptor were studied using standard patch-clamp techniques. 2. 17beta-Oestradiol rapidly and reversibly inhibited the whole-cell hP2X7 receptor cation current. This inhibitory action resulted in a rightward shift of the dose-response curve to ATP and BzATP in the presence of physiological as well as low divalent cation concentrations. 3. The inhibitory effect of 17beta-oestradiol on the BzATP- or ATP-induced cation current was concentration dependent. The half-maximal inhibition was obtained with 3 microM 17beta-oestradiol. Progesterone and 17alpha-oestradiol had almost no effect on the hP2X7 receptor cation current. 4. The inhibition of the hP2X7 receptor cation current by 17beta-oestradiol did not depend on the membrane potential. 17beta-Oestradiol added to the extracellular side of outside-out patches inhibited BzATP-activated single-channel currents. 5. Activation of the hP2X7 receptor in both COS and U937 (human macrophage) cells did not induce the formation of large non-specific pores. 6. Since COS cells do not express endogenous nuclear oestrogen receptor, this study shows that, at pharmacological concentrations, 17beta-oestradiol inhibited the hP2X7 receptor cation channel in a non-genomic manner.
Asunto(s)
Estradiol/farmacología , Antagonistas del Receptor Purinérgico P2 , Receptores Purinérgicos P2/fisiología , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Marcadores de Afinidad/farmacología , Animales , Células COS/efectos de los fármacos , Células COS/fisiología , Calcio/farmacología , Cationes/metabolismo , Inhibidores Enzimáticos/farmacocinética , Terapia de Reemplazo de Estrógeno , Etidio/farmacocinética , Humanos , Magnesio/farmacología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Menopausia , Técnicas de Placa-ClampRESUMEN
The effect of the centrally acting antihypertensive agent, rilmenidine, was examined on the contractile properties of isolated rat portal vein strips and on the free cytosolic [Ca2+] ([Ca2+]i) in isolated myocytes. Rilmenidine (1-30 microM) relaxed strips precontracted with noradrenaline. This effect was not inhibited by the alpha2-adrenoceptor antagonist, yohimbine, and was not mimicked by the alpha2-adrenoceptor agonist, 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK 14,304). Rilmenidine dose dependently shifted to the right the concentration-response curves to noradrenaline and to phenylephrine but not that to carbachol. Rilmenidine alone (0.1-30 microM) caused a contraction which maximally corresponded to 18% of the maximal noradrenaline-induced contraction. This effect was not produced by UK 14,304, was not affected by yohimbine, but was inhibited by the alpha1-adrenoceptor antagonist, prazosin. In isolated myocytes, rilmenidine reduced the noradrenaline-induced [Ca2+]i increase but alone, it produced a rise in [Ca2+]i, the peak amplitude of which averaged 15% of the noradrenaline-induced transient [Ca2+]i rise. It is concluded that rilmenidine acts as a partial agonist of alpha1-adrenoceptors of vascular smooth muscle, causing relaxation of vessels precontracted by full agonists of alpha1-adrenoceptors.