RESUMEN
Mechanosensitive cation channels and Ca2+ influx through these channels play an important role in the regulation of endothelial cell functions. Transient receptor potential canonical channel 6 (TRPC6) is a diacylglycerol-sensitive nonselective cation channel that forms receptor-operated Ca2+ channels in a variety of cell types. Piezo1 is a mechanosensitive cation channel activated by membrane stretch and shear stress in lung endothelial cells. In this study, we report that TRPC6 and Piezo1 channels both contribute to membrane stretch-mediated cation currents and Ca2+ influx or increase in cytosolic-free Ca2+ concentration ([Ca2+]cyt) in human pulmonary arterial endothelial cells (PAECs). The membrane stretch-mediated cation currents and increase in [Ca2+]cyt in human PAECs were significantly decreased by GsMTX4, a blocker of Piezo1 channels, and by BI-749327, a selective blocker of TRPC6 channels. Extracellular application of 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane permeable analog of diacylglycerol, rapidly induced whole cell cation currents and increased [Ca2+]cyt in human PAECs and human embryonic kidney (HEK)-cells transiently transfected with the human TRPC6 gene. Furthermore, membrane stretch with hypo-osmotic or hypotonic solution enhances the cation currents in TRPC6-transfected HEK cells. In HEK cells transfected with the Piezo1 gene, however, OAG had little effect on the cation currents, but membrane stretch significantly enhanced the cation currents. These data indicate that, while both TRPC6 and Piezo1 are involved in generating mechanosensitive cation currents and increases in [Ca2+]cyt in human PAECs undergoing mechanical stimulation, only TRPC6 (but not Piezo1) is sensitive to the second messenger diacylglycerol. Selective blockers of these channels may help develop novel therapies for mechanotransduction-associated pulmonary vascular remodeling in patients with pulmonary arterial hypertension.
Asunto(s)
Células Endoteliales , Canales Iónicos , Mecanorreceptores , Canal Catiónico TRPC6 , Calcio/metabolismo , Cationes/metabolismo , Diglicéridos/metabolismo , Diglicéridos/farmacología , Células Endoteliales/metabolismo , Humanos , Soluciones Hipotónicas/metabolismo , Soluciones Hipotónicas/farmacología , Canales Iónicos/genética , Canales Iónicos/metabolismo , Mecanorreceptores/metabolismo , Mecanotransducción Celular/genética , Mecanotransducción Celular/fisiología , Arteria Pulmonar/citología , Arteria Pulmonar/metabolismo , Canal Catiónico TRPC6/genética , Canal Catiónico TRPC6/metabolismoRESUMEN
Concentric pulmonary vascular wall thickening due partially to increased pulmonary artery (PA) smooth muscle cell (PASMC) proliferation contributes to elevating pulmonary vascular resistance (PVR) in patients with pulmonary hypertension (PH). Although pulmonary vasoconstriction may be an early contributor to increasing PVR, the transition of contractile PASMCs to proliferative PASMCs may play an important role in the development and progression of pulmonary vascular remodeling in PH. A rise in cytosolic Ca2+ concentration ([Ca2+]cyt) is a trigger for PASMC contraction and proliferation. Here, we report that upregulation of Piezo1, a mechanosensitive cation channel, is involved in the contractile-to-proliferative phenotypic transition of PASMCs and potential development of pulmonary vascular remodeling. By comparing freshly isolated PA (contractile PASMCs) and primary cultured PASMCs (from the same rat) in a growth medium (proliferative PASMCs), we found that Piezo1, Notch2/3, and CaSR protein levels were significantly higher in proliferative PASMCs than in contractile PASMCs. Upregulated Piezo1 was associated with an increase in expression of PCNA, a marker for cell proliferation, whereas downregulation (with siRNA) or inhibition (with GsMTx4) of Piezo1 attenuated PASMC proliferation. Furthermore, Piezo1 in the remodeled PA from rats with experimental PH was upregulated compared with PA from control rats. These data indicate that PASMC contractile-to-proliferative phenotypic transition is associated with the transition or adaptation of membrane channels and receptors. Upregulated Piezo1 may play a critical role in PASMC phenotypic transition and PASMC proliferation. Upregulation of Piezo1 in proliferative PASMCs may likely be required to provide sufficient Ca2+ to assure nuclear/cell division and PASMC proliferation, contributing to the development and progression of pulmonary vascular remodeling in PH.
Asunto(s)
Hipertensión Pulmonar , Proteínas de la Membrana/metabolismo , Arteria Pulmonar , Animales , Señalización del Calcio/fisiología , Proliferación Celular , Células Cultivadas , Humanos , Hipertensión Pulmonar/metabolismo , Canales Iónicos/genética , Canales Iónicos/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Arteria Pulmonar/metabolismo , Ratas , Remodelación VascularRESUMEN
Idiopathic pulmonary arterial hypertension (PAH) is a fatal and progressive disease. Sustained vasoconstriction due to pulmonary arterial smooth muscle cell (PASMC) contraction and concentric arterial remodeling due partially to PASMC proliferation are the major causes for increased pulmonary vascular resistance and increased pulmonary arterial pressure in patients with precapillary pulmonary hypertension (PH) including PAH and PH due to respiratory diseases or hypoxemia. We and others observed upregulation of TRPC6 channels in PASMCs from patients with PAH. A rise in cytosolic Ca2+ concentration ([Ca2+]cyt) in PASMC triggers PASMC contraction and vasoconstriction, while Ca2+-dependent activation of PI3K/AKT/mTOR pathway is a pivotal signaling cascade for cell proliferation and gene expression. Despite evidence supporting a pathological role of TRPC6, no selective and orally bioavailable TRPC6 antagonist has yet been developed and tested for treatment of PAH or PH. In this study, we sought to investigate whether block of receptor-operated Ca2+ channels using a nonselective blocker of cation channels, 2-aminoethyl diphenylborinate (2-APB, administered intraperitoneally) and a selective blocker of TRPC6, BI-749327 (administered orally) can reverse established PH in mice. The results from the study show that intrapulmonary application of 2-APB (40 µM) or BI-749327 (3-10 µM) significantly and reversibly inhibited acute alveolar hypoxia-induced pulmonary vasoconstriction. Intraperitoneal injection of 2-APB (1 mg/kg per day) significantly attenuated the development of PH and partially reversed established PH in mice. Oral gavage of BI-749327 (30 mg/kg, every day, for 2 wk) reversed established PH by â¼50% via regression of pulmonary vascular remodeling. Furthermore, 2-APB and BI-749327 both significantly inhibited PDGF- and serum-mediated phosphorylation of AKT and mTOR in PASMC. In summary, the receptor-operated and mechanosensitive TRPC6 channel is a good target for developing novel treatment for PAH/PH. BI-749327, a selective TRPC6 blocker, is potentially a novel and effective drug for treating PAH and PH due to respiratory diseases or hypoxemia.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Hipertensión Pulmonar/patología , Músculo Liso Vascular/patología , Arteria Pulmonar/patología , Canal Catiónico TRPC6/metabolismo , Vasoconstricción , Animales , Compuestos de Boro/farmacología , Señalización del Calcio , Células Cultivadas , Humanos , Hipertensión Pulmonar/tratamiento farmacológico , Hipertensión Pulmonar/metabolismo , Ratones , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/metabolismo , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Canal Catiónico TRPC6/antagonistas & inhibidores , Canal Catiónico TRPC6/genéticaRESUMEN
Astragali Radix is commonly used as bulk medicinal materials. Chinese Pharmacopoeia contains about 150 compound preparations of Astragali Radix, but the sample preparation method under the determination of Astragali Radix content in Chinese Pharmacopoeia is tedious and time-consuming, not convenient for the test of a large number of samples. Therefore, it is of great significance to simplify the sample preparation method and improve the practicability of the method for the quality control of Astragali Radix and its preparations. In this study, ultrasonic extraction method was used instead of heated reflux extraction, and solid phase extraction method was used to enrich and prepare the samples. A set of practical quality evaluation method was established for Astragali Radix slices and standard decoction, greatly shortening the sample preparation time and improving the accuracy of the method. The results of Astragali Radix standard decoction analysis showed that the transfer rate of calycosin 7-O-ß-D-glucospyranoside,(96.5±28.7)%, had great variation, which was found to be related to the conversion of mulberry isoflavone glucoside into calycosin 7-O-ß-D-glucospyranoside during the preparation of standard decoction. The transfer rates were(59.4±14.4)% and(101.3±12.3)% for calycosin and astragaloside â £ respectively, which were relatively stable. Therefore, it is suggested that Astragali Radix slices and water decoction preparations should be evaluated by using calycosin and astragaloside â £ as the quality evaluation index. The results provide a scientific and practical method for quality control of Astragali Radix slices and its standard decoction, and also provide scientific evidence for quality evaluation of the preparations.
Asunto(s)
Planta del Astrágalo/química , Medicamentos Herbarios Chinos/análisis , Astragalus propinquus , Medicamentos Herbarios Chinos/normas , Glucósidos/análisis , Raíces de Plantas/química , Control de Calidad , Extracción en Fase SólidaRESUMEN
Decoction of single medicinal herb is a reference for the standardization of different dosage forms of Chinese medicine and it provides a new direction for solving the problems existing in the quality of Chinese medicinal granules such as no uniform dosage form and no clear quality standard. In this paper, the quality evaluation method of standard decoction of rhubarb was established to provide reference for the quality control of common dosage forms such as clinical decoction and formula granule. 10 batches of representative Rhei Radix et Rhizoma were collected to establish UPLC fingerprints were established. The chemical structures of main peaks were identified with ultra-performance liquid chromatography with quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS) and the main components in the decoction were Anthraquinones. The extraction ratio of the standard decoction was (28.1±3.8)% and the transfer rate was (19.9±6.3)%. The method for the quality evaluation of standard decoction of Rhei Radix et Rhizoma was established in this study, providing reference for the quality control method of terminal products from decoction of Rhei Radix et Rhizoma.
Asunto(s)
Medicamentos Herbarios Chinos/normas , Control de Calidad , Rheum/química , Antraquinonas/química , Cromatografía Líquida de Alta Presión , Rizoma/químicaRESUMEN
INTRODUCTION: Piezo1 is an important mechanosensitive channel implicated in vascular remodeling. However, the role of Piezo1 in different types of vascular cells during the development of pulmonary hypertension (PH) induced by high shear stress is largely unknown. MATERIALS AND METHODS: We used a rat PH model established by left pulmonary artery ligation (LPAL, for 2-5 weeks), which mimics the high flow and hemodynamic stress, to study Piezo1 contribution to pulmonary vascular remodeling. RESULTS: Right ventricular systolic pressure (RVSP), a surrogate measure for pulmonary arterial systolic pressure, and right ventricular wall thickness, a measure for right ventricular hypertrophy, were significantly increased in LPAL rats compared with Sham-control (SHAM) rats. Rats in LPAL-5w groups developed remarkable pulmonary vascular remodeling, while phenylephrine-induced contraction and acetylcholine-induced relaxation were both significantly inhibited in these rats. Upregulation of Piezo1, in association with increase in cytosolic Ca2+ concentration ([Ca2+]cyt), was observed in pulmonary arterial smooth muscle cells (PASMCs) from LPAL-2w and LPAL-5w rats in comparison to the SHAM controls. Piezo1 upregulation in PASMCs from LPAL rats was directly related to Yes-associated protein (YAP)/ TEA domain transcription factor 4 (TEAD4). Piezo1 expression was also upregulated in the whole-lung tissue of LPAL rats. The endothelial upregulation of Piezo1 was related to transcriptional regulation by RELA (p65) and lung inflammation. CONCLUSION: The upregulation of Piezo1 in both PASMCs and ECs coordinates with each other via different cell signaling pathways to cause pulmonary vascular remodeling in LPAL-PH rats, providing novel insights into the cell-type specific pathogenic roles of Piezo1 in shear stress-associated experimental PH.
Asunto(s)
Hipertensión Pulmonar , Proteínas de la Membrana , Animales , Ratas , Acetilcolina/metabolismo , Proliferación Celular , Hipertensión Pulmonar/etiología , Proteínas de la Membrana/metabolismo , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Fenilefrina/metabolismo , Arteria Pulmonar/patología , Factor de Transcripción 4/metabolismo , Regulación hacia Arriba , Remodelación Vascular , Proteínas Señalizadoras YAPRESUMEN
Due to the complex nature of traditional medicines, quality control methods need to cover two aspects: compliance of raw materials with quality standards and process control. Astragali radix (AR), the roots of Astragalus mongholicus Bunge, was selected in this study as an example of a widely used traditional medicine in various formulations. Astragaloside IV (AG IV) and calycosin 7-O-ß-D-glucoside (CG) are used as the markers for the quality control of AR and its products in the Chinese Pharmacopoeia. However, in the raw materials, malic acid esters of the CG and acetate esters of the astragaloside are easily decomposed into CG and AG IV during storage and processing of AR to make extracts for various preparations. The thermal stability of the isoflavonoids and astragalosides in decoction was studied. The level of CG and astragalosides (AG I/AG II/AG IV) was strongly affected by prolonged heat during processing, while calycosin was stable in the conditions. Also the major astragalosides in AR could fully converted into AG IV which eventually reaches a stable level under certain conditions. With calycosin and AG IV as marker components, practical, reproducible, and precise methods were established and applied to the quality analysis of AR from its raw materials to its intermediates and products. This study demonstrates that a full chemical profiles analysis of the whole manufacturing process (from "raw materials-intermediates/extracts-final product") is important to identify quality markers (Q-markers) and even to establish proper analysis methods for traditional Chinese medicine products.
RESUMEN
Saponins are a class of important active ingredients. Analysis of saponin-containing herbal medicines is a major challenge for the quality control of medicinal herbs in companies. Taking the medicine Astragali radix (AR) as an example, it has been shown that the existing evaporative light scattering detection (ELSD) methods of astragaloside IV (AG IV) has the disadvantages of time-consuming sample preparation and low sensitivity. The universality of ELSD results in an inapplicable fingerprint with huge signals from primary compounds and smaller signals from saponins. The purpose of this study was to provide a practical and comprehensive method for the quality control of the astragalosides in AR. A simple sample preparation method with sonication extraction and ammonia hydrolyzation was established, which shortens the preparation time from around 2 days to less than 2 h. A UPLC-QDA method with the SIM mode was established for the quantification of AG IV in AR. Methanol extract was subjected to UPLC-QDA for fingerprinting analysis, and the common peaks were assigned simultaneously with the QDA. The results showed that with the newly established method, the preparation time for a set of samples was less than 90 min. The fingerprints can simultaneously detect both saponins and flavonoids in AR. This simple, rapid, and comprehensive UPLC-QDA method is suitable for quality assessment of RA and its products in companies, and also provides references for the quality control of other saponin ingredients without UV absorption.