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High-altitude pulmonary edema (HAPE) is a fatal threat for sojourners who ascend rapidly without sufficient acclimatization. Acclimatized sojourners and adapted natives are both insensitive to HAPE but have different physiological traits and molecular bases. In this study, based on GSE52209, the gene expression profiles of HAPE patients were compared with those of acclimatized sojourners and adapted natives, with the common and divergent differentially expressed genes (DEGs) and their hub genes identified, respectively. Bioinformatic methodologies for functional enrichment analysis, immune infiltration, diagnostic model construction, competing endogenous RNA (ceRNA) analysis and drug prediction were performed to detect potential biological functions and molecular mechanisms. Next, an array of in vivo experiments in a HAPE rat model and in vitro experiments in HUVECs were conducted to verify the results of the bioinformatic analysis. The enriched pathways of DEGs and immune landscapes for HAPE were significantly different between sojourners and natives, and the common DEGs were enriched mainly in the pathways of development and immunity. Nomograms revealed that the upregulation of TNF-α and downregulation of RPLP0 exhibited high diagnostic efficiency for HAPE in both sojourners and natives, which was further validated in the HAPE rat model. The addition of TNF-α and RPLP0 knockdown activated apoptosis signaling in endothelial cells (ECs) and enhanced endothelial permeability. In conclusion, TNF-α and RPLP0 are shared biomarkers and molecular bases for HAPE susceptibility during the acclimatization/adaptation/maladaptation processes in sojourners and natives, inspiring new ideas for predicting and treating HAPE.
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OBJECTIVE: Exposure to microgravity results in postflight cardiovascular deconditioning in astronauts. Vascular oxidative stress injury and mitochondrial dysfunction have been reported during this process. To elucidate the mechanism for this condition, we investigated whether mitochondrial oxidative stress regulates calcium homeostasis and vasoconstriction in hindlimb unweighted (HU) rat cerebral arteries. METHODS: Three-week HU was used to simulate microgravity in rats. The contractile responses to vasoconstrictors, mitochondrial fission/fusion, Ca 2+ distribution, inositol 1,4,5-trisphosphate receptor (IP 3R) abundance, and the activities of voltage-gated K + channels (K V) and Ca 2+-activated K + channels (BK Ca) were examined in rat cerebral vascular smooth muscle cells (VSMCs). RESULTS: An increase of cytoplasmic Ca 2+ and a decrease of mitochondrial/sarcoplasmic reticulum (SR) Ca 2+ were observed in HU rat cerebral VSMCs. The abundance of fusion proteins (mitofusin 1/2 [MFN1/2]) and fission proteins (dynamin-related protein 1 [DRP1] and fission-mitochondrial 1 [FIS1]) was significantly downregulated and upregulated, respectively in HU rat cerebral VSMCs. The cerebrovascular contractile responses to vasoconstrictors were enhanced in HU rats compared to control rats, and IP 3R protein/mRNA levels were significantly upregulated. The current densities and open probabilities of K V and BK Ca decreased and increased, respectively. Treatment with the mitochondrial-targeted antioxidant mitoTEMPO attenuated mitochondrial fission by upregulating MFN1/2 and downregulating DRP1/FIS1. It also decreased IP 3R expression levels and restored the activities of the K V and BK Ca channels. MitoTEMPO restored the Ca 2+ distribution in VSMCs and attenuated the enhanced vasoconstriction in HU rat cerebral arteries. CONCLUSION: The present results suggest that mitochondrial oxidative stress enhances cerebral vasoconstriction by regulating calcium homeostasis during simulated microgravity.
Assuntos
Cálcio/metabolismo , Homeostase , Mitocôndrias/fisiologia , Miócitos de Músculo Liso/fisiologia , Estresse Oxidativo , Vasoconstrição/fisiologia , Simulação de Ausência de Peso , Animais , Artérias Cerebrais , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Zaoren Anshen prescription preparations(ZRASs), which are prepared from three traditional Chinese herb medicines, namely fried Zizyphi Spinosae Semen, Salvia Miltiorrhizae Radix et Rhizoma and vinegar-processed Schisandrae Chinensis Fructus, are a series of proprietary Chinese medicines for the treatment of insomnia, amnesia and dizzy in clinic. In recent years, pharmacodynamic effect, chemical constituents and quality control of ZRASs had been extensively studied for the purpose of ensuring their safety, efficacy and stability, and a great progress had been made. However, there is no review of the research advance of ZRASs up to date. The present review summarized the research advance of ZRASs in quality control standards, chemical constituents, pharmacodynamic effects, and chemical analysis for the first time, with the aim to provide a reference for further studies on the effective constituents and quality control of ZRASs.
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Medicamentos de Ervas Chinesas , Salvia miltiorrhiza , Medicamentos de Ervas Chinesas/farmacologia , Medicina Tradicional Chinesa , Prescrições , RizomaRESUMO
OBJECTIVES: Postflight orthostatic intolerance has been regarded as a major adverse effect after microgravity exposure, in which cerebrovascular adaptation plays a critical role. Our previous finding suggested that dedifferentiation of vascular smooth muscle cells (VSMCs) might be one of the key contributors to cerebrovascular adaptation under simulated microgravity. This study was aimed to confirm this concept and elucidate the underlying mechanisms. MATERIALS AND METHODS: Sprague Dawley rats were subjected to 28-day hindlimb-unloading to simulate microgravity exposure. VSMC dedifferentiation was evaluated by ultrastructural analysis and contractile/synthetic maker detection. The role of T-type CaV 3.1 channel was revealed by assessing its blocking effects. MiR-137 was identified as the upstream of CaV 3.1 channel by luciferase assay and investigated by gain/loss-of-function approaches. Calcineurin/nuclear factor of activated T lymphocytes (NFAT) pathway, the downstream of CaV 3.1 channel, was investigated by detecting calcineurin activity and NFAT nuclear translocation. RESULTS: Simulated microgravity induced the dedifferentiation and proliferation in rat cerebral VSMCs. T-type CaV 3.1 channel promoted the dedifferentiation and proliferation of VSMC. MiR-137 and calcineurin/NFATc3 pathway were the upstream and downstream signalling of T-type CaV 3.1 channel in modulating the dedifferentiation and proliferation of VSMCs, respectively. CONCLUSIONS: The present work demonstrated that miR-137 and its target T-type CaV 3.1 channel modulate the dedifferentiation and proliferation of rat cerebral VSMCs under simulated microgravity by regulating calcineurin/NFATc3 pathway.
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Calcineurina/metabolismo , Canais de Cálcio Tipo T/metabolismo , Artérias Cerebrais/citologia , MicroRNAs/metabolismo , Miócitos de Músculo Liso/citologia , Fatores de Transcrição NFATC/metabolismo , Animais , Encéfalo/irrigação sanguínea , Canais de Cálcio Tipo T/genética , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Artérias Cerebrais/metabolismo , Regulação da Expressão Gênica , MicroRNAs/genética , Miócitos de Músculo Liso/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Simulação de Ausência de PesoRESUMO
The functional and structural adaptations in cerebral arteries could be one of the fundamental causes in the occurrence of orthostatic intolerance after space flight. In addition, emerging studies have found that many cardiovascular functions exhibit circadian rhythm. Several lines of evidence suggest that space flight might increase an astronaut's cardiovascular risks by disrupting circadian rhythm. However, it remains unknown whether microgravity disrupts the diurnal variation in vascular contractility and whether microgravity impacts on circadian clock system. Sprague-Dawley rats were subjected to 28-day hindlimb-unweighting to simulate the effects of microgravity on vasculature. Cerebrovascular contractility was estimated by investigating vasoconstrictor responsiveness and myogenic tone. The circadian regulation of CaV1.2 channel was determined by recording whole-cell currents, evaluating protein and mRNA expressions. Then the candidate miRNA in relation with Ca2+ signal was screened. Lastly, the underlying pathway involved in circadian regulation of cerebrovascular contractility was determined. The major findings of this study are: (1) The clock gene BMAL1 could induce the expression of miR-103, and in turn modulate the circadian regulation of CaV1.2 channel in rat cerebral arteries at post-transcriptional level; and (2) simulated microgravity disrupted intrinsic diurnal oscillation in rat cerebrovascular contractility by altering circadian regulation of BMAL1/miR-103/CaV1.2 signal pathway.
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Fatores de Transcrição ARNTL/genética , Canais de Cálcio Tipo L/metabolismo , Circulação Cerebrovascular/genética , Ritmo Circadiano , MicroRNAs/genética , Vasoconstrição/genética , Ausência de Peso , Fatores de Transcrição ARNTL/metabolismo , Animais , Linhagem Celular , Regulação da Expressão Gênica , Masculino , Modelos Biológicos , Ratos , Transdução de SinaisRESUMO
Vessel disease is a kind of severe complication in diabetic patients. However, few pharmacologic agents can directly recover diabetic vascular function. Salidroside (SAL), a major ingredient from Rhodiola rosea, has been found to have an obvious hypoglycemic effect and a beneficial protection on vascular function in diabetes. However, whether SAL is a suitable treatment for diabetes has not so far been evaluated and the underlying mechanisms remain unknown. The present work aims to (1) investigate the potential effects of SAL on cerebrovascular relaxation in streptozotocin-induced diabetic rats or when exposed to acute hyperglycemia condition and (2) examine whether function of the BKCa channel is involved in SAL treatment for diabetic vascular relaxation. Our results indicate that chronic administration of 100 mg/kg/day SAL not only improves cerebrovascular relaxation but also increases BKCa ß1-subunit expressions at both protein and mRNA levels and enhances BKCa whole-cell and single-channel activities in cerebral VSMCs of diabetic rats. Correspondingly, acute application of 100 µM SAL induces cerebrovascular relaxation by activation of the BKCa channel. Furthermore, SAL activated the BKCa channel mainly through acting on the ß1-subunit in HEK293 cells transfected with hSloα+ß1 constructs. We concluded that SAL improved vasodilation in diabetic rats through restoring the function of the BKCa-ß1 subunit in cerebrovascular smooth muscle cells, which may be the underlying mechanism responsible for the vascular protection of SAL in diabetes.
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Glucosídeos/farmacologia , Hipoglicemiantes/farmacologia , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Fenóis/farmacologia , Vasodilatação/efeitos dos fármacos , Animais , Linhagem Celular , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/patologia , Células HEK293 , Humanos , Masculino , Miócitos de Músculo Liso/metabolismo , Ratos , Ratos Wistar , EstreptozocinaRESUMO
Hyperglycemia and hypertension are considered to be the two leading risk factors for vascular disease in diabetic patients. However, few pharmacologic agents could provide a combinational therapy for controlling hyperglycemia and hypertension at the same time in diabetes. The objectives of this study are to investigate whether berberine treatment could directly reduce blood pressure and identify the molecular mechanism underlying the vascular protection of berberine in diabetic rats. Berberine was intragastrically administered with different dosages of 50, 100 and 200 mg/kg/day to diabetic rats for 8 weeks since the injection of streptozotocin. The endothelium-dependent/-independent relaxation in middle cerebral arteries was investigated. The activity of large-conductance Ca2+-activated K+ channel (BKCa) was investigated by recording whole-cell currents, analyzing single-channel activities and assessing the expressions of α- and ß1-subunit at protein or mRNA levels. Results of the study suggest that chronic administration of 100 mg/kg/day berberine not only lowered blood glucose but also reduced blood pressure and improved vasodilation in diabetic rats. Furthermore, berberine markedly increased the function and expression of BKCa ß1-subunit in cerebral vascular smooth muscle cells (VSMCs) isolated from diabetic rats or when exposed to hyperglycemia condition. The present study provided initial evidences that berberine reduced blood pressure and improved vasodilation in diabetic rats by activation of BKCa channel in VSMCs, which suggested that berberine might provide a combinational therapy for controlling hyperglycemia and blood pressure in diabetes. Furthermore, our work indicated that activation of BKCa channel might be the underlying mechanism responsible for the vascular protection of berberine in diabetes.
Assuntos
Berberina/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Animais , Berberina/administração & dosagem , Pressão Sanguínea/genética , Diabetes Mellitus Experimental , Relação Dose-Resposta a Droga , Expressão Gênica , Hiperglicemia/metabolismo , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Masculino , Artéria Cerebral Média/efeitos dos fármacos , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Ratos , Fatores de Tempo , Vasodilatação/genéticaRESUMO
BACKGROUND: Vascular disease is a common and often severe complication in diabetes mellitus. Hyperglycemia and hypertension are considered to be two of the leading risk factors for vascular complications in diabetic patients. However, few pharmacologic agents could provide a combinational therapy for controlling hyperglycemia and blood pressure in diabetic patients at the same time. Salidroside (SAL) is the major active ingredient derived from Rhodiola. Recently, it has been reported that SAL have an obvious hypoglycemic effect in diabetes and show a beneficial activity in diabetic vascular dysfunction. However, it remains unknown whether or not SAL treatment could directly reduce blood pressure in diabetes. Furthermore, it is not clear what is the molecular mechanism underlying the vascular protection of SAL treatment in diabetes. METHODS: Male diabetic Goto-Kakizaki (GK) and non-diabetic control Wistar-Kyoto (WKY) rats were administrated with different dosages of SAL (50, 100 and 200 mg/kg/day) for 4 weeks. Contractile responsiveness of cerebral artery to KCl or 5-HT was investigated by Pressure Myograph System. The activity of CaL channel was investigated by recording whole-cell currents, assessing the expressions of CaL channel α1C-subunit and its downstream kinase, MLCK, at protein or mRNA levels. RESULTS: We showed that administration of 100 mg/kg/day SAL for 4 weeks not only lowered blood glucose, but also reduced blood pressure and alleviated cerebrovascular contractile activity in diabetic GK rats, which suggested that SAL treatment may provide a combinational therapy for lowering blood glucose and reducing blood pressure in diabetes at the same time. Furthermore, SAL treatment markedly inhibited the function and expression of CaL channel in cerebral VSMCs isolated from diabetic GK rats or when exposed to hyperglycemia condition, which may be the underlying mechanism responsible for the vascular protection of SAL in diabetes. CONCLUSIONS: The present study provided evidences that SAL contributes to reducing blood pressure and alleviating cerebrovascular contractile activity in diabetic GK rats by inhibition of CaL channel in smooth muscle cells, which may provide a novel approach to treat vascular complications in diabetic patients.
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Canais de Cálcio Tipo L/efeitos dos fármacos , Artérias Cerebrais/efeitos dos fármacos , Cardiomiopatias Diabéticas/tratamento farmacológico , Glucosídeos/uso terapêutico , Hipoglicemiantes/uso terapêutico , Músculo Liso Vascular/efeitos dos fármacos , Fenóis/uso terapêutico , Animais , Glicemia/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Canais de Cálcio Tipo L/genética , Células Cultivadas , Diabetes Mellitus Experimental , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , RNA Mensageiro/metabolismo , Ratos Endogâmicos WKY , Vasodilatação/efeitos dos fármacosRESUMO
DEAD (Asp-Glu-Ala-Asp) box helicase 5 (DDX5) is an ATP-dependent RNA helicase that is overexpressed in various malignancies. Increasing evidence suggests that DDX5 participates in carcinogenesis and cancer progression via promoting cell proliferation and metastasis. However, the functional role of DDX5 in gastric cancer is largely unknown. In this study, we observed that DDX5 was significantly up-regulated in gastric cancer tissues compared with the paired adjacent normal tissues. The expression of DDX5 correlated strongly with Ki67 index and pathological stage of gastric cancer. In vitro and in vivo studies suggested that knockdown of DDX5 inhibited gastric cancer cell proliferation, colony formation and xenografts growth, whereas ectopic expression of DDX5 promoted these cellular functions. Mechanically, DDX5 induced gastric cancer cell growth by activating mTOR/S6K1. Treatment of everolimus, the specific mTOR inhibitor, significantly attenuated DDX5-mediated cell proliferation. Interestingly, the expression of DDX5 and p-mTOR in gastric cancer tissues demonstrated a positive correlation. Taken together, these results revealed a novel role of DDX5 in gastric cancer cell proliferation via the mTOR pathway. Therefore, DDX5 may serve as a therapeutic target in gastric cancer.
Assuntos
RNA Helicases DEAD-box/genética , Transdução de Sinais , Neoplasias Gástricas/patologia , Regulação para Cima , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , RNA Helicases DEAD-box/metabolismo , Everolimo/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Estadiamento de Neoplasias , Transplante de Neoplasias , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
BACKGROUND: Vascular dysfunction is a distinctive phenotype in diabetes mellitus. Current treatments mostly focus on the tight glycemic control and few of these treatments have been designed to directly recover the vascular dysfunction in diabetes. As a classical natural medicine, berberine has been explored as a possible therapy for DM. In addition, it is reported that berberine has an extra-protective effect in diabetic vascular dysfunction. However, little is known whether the berberine treatment could ameliorate the smooth muscle contractility independent of a functional endothelium under hyperglycemia. Furthermore, it remains unknown whether berberine affects the arterial contractility by regulating the intracellular Ca(2+) handling in vascular smooth cells (VSMCs) under hyperglycemia. METHODS: Sprague-Dawley rats were used to establish the diabetic model with a high-fat diet plus injections of streptozotocin (STZ). Berberine (50, 100, and 200 mg/kg/day) were intragastrically administered to control and diabetic rats for 8 weeks since the injection of STZ. The intracellular Ca(2+) handling of isolated cerebral VSMCs was investigated by recording the whole-cell L-type Ca(2+) channel (CaL) currents, assessing the protein expressions of CaL channel, and measuring the intracellular Ca(2+) in response to caffeine. Our results showed that chronic administration of 100 mg/kg/day berberine not only reduced glucose levels, but also inhibited the augmented contractile function of cerebral artery to KCl and 5-hydroxytryptamine (5-HT) in diabetic rats. Furthermore, chronic administration of 100 mg/kg/day berberine significantly inhibited the CaL channel current densities, reduced the α1C-subunit expressions of CaL channel, decreased the resting intracellular Ca(2+) ([Ca(2+)]i) level, and suppressed the Ca(2+) releases from RyRs in cerebral VSMCs isolated from diabetic rats. Correspondingly, acute application of 10 µM berberine could directly inhibit the hyperglycemia-induced CaL currents and suppress the hyperglycemia-induced Ca(2+) releases from RyRs in cerebral VSMCs isolated from normal control rats. CONCLUSIONS: Our study indicated that berberine alleviated the cerebral arterial contractility in the rat model of streptozotocin-induced diabetes via regulating the intracellular Ca(2+) handling of smooth muscle cells.
Assuntos
Berberina/farmacologia , Cálcio/metabolismo , Diabetes Mellitus Experimental/tratamento farmacológico , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Animais , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Canais de Cálcio Tipo L/efeitos dos fármacos , Canais de Cálcio Tipo L/metabolismo , Diabetes Mellitus Experimental/metabolismo , Dieta Hiperlipídica , Hiperglicemia/tratamento farmacológico , Hiperglicemia/metabolismo , Masculino , Miócitos de Músculo Liso/metabolismo , Ratos Sprague-DawleyRESUMO
The aim of this study was to investigate the effects of nitric oxide (NO) and reactive oxygen species (ROS) on L-type calcium channel (LTCC) gating properties of cardiomyocytes during long-term isoproterenol (ISO) stimulation. Expression and activity of nNOS as well as S-nitrosylation of LTCC α1C subunit significantly decreased in the myocardium of SUS rats. Long-term ISO stimulation increased ROS in cardiomyocytes of SUS rats. ISO-enhanced calcium current (I Ca,L) in the SUS group was less than that in the CON group. The maximal I Ca,L decreased to about 80% or 60% of initial value at the 50th minute of ISO treatment in CON or SUS group, respectively. Specific inhibitor NAAN of nNOS reduced maximal I Ca,L to 50% of initial value in the CON group; in contrast, NO donor SNAP maintained maximal I Ca,L in SUS group to similar extent of CON group after 50 min of ISO treatment. Long-term ISO stimulation also changed steady-state activation (P < 0.01), inactivation (P < 0.01), and recovery (P < 0.05) characteristics of LTCC in SUS group. In conclusion, NO-induced S-nitrosylation of LTCC α1C subunit may competitively prevent oxidation from ROS at the same sites. Furthermore, LTCC can be protected by NO during long-term ISO stimulation.
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Canais de Cálcio Tipo L/metabolismo , Cardiotônicos/farmacologia , Isoproterenol/farmacologia , Miócitos Cardíacos/metabolismo , Óxido Nítrico/metabolismo , Animais , Decúbito Inclinado com Rebaixamento da Cabeça/fisiologia , Masculino , Miócitos Cardíacos/fisiologia , Ratos , Ratos Sprague-Dawley , Superóxidos/metabolismoRESUMO
Microgravity-induced vascular remodelling may play an important role in post-spaceflight orthostatic intolerance. In this study, we aimed to investigate the effects of simulated microgravity on monocyte adhesion to aortic endothelium in hindlimb unweighted rats and to elucidate the underlying mechanisms associated with this event. Sprague-Dawley rats were subjected to 4-week hindlimb unweighting to simulate microgravity. The recruitment of monocytes to the abdominal aorta was investigated by en face immunofluorescence staining and monocyte binding assays. The expression of the adhesion molecules E-selectin and vascular cell adhesion molecule-1 as well as the cytokine monocyte chemoattractant protein (MCP)-1 was evaluated by immunohistochemical staining, western blot, and quantitative reverse transcription polymerase chain reaction analyses. Additionally, nuclear factor-κB (NF-κB) activation and the messenger RNA expression levels of E-selectin, vascular cell adhesion molecule-1, and MCP-1 were assessed with the administration of an NF-κB inhibitor, pyrrolidine dithiocarbamate. Results showed that simulated microgravity significantly increased monocyte recruitment to the aortic endothelium, protein expression of E-selectin and MCP-1, and NF-κB activation in the abdominal aorta of rats. Pyrrolidine dithiocarbamate treatment not only significantly inhibited NF-κB activity but also reduced the messenger RNA levels of E-selectin, vascular cell adhesion molecule-1, and MCP-1 as well as monocyte recruitment in the abdominal aorta of hindlimb unweighted rats. These results suggest that simulated microgravity increases monocyte adhesion to rat aortic endothelium via the NF-κB-mediated expression of the adhesion molecule E-selectin and the cytokine MCP-1. Therefore, an NF-κB-mediated inflammatory response may be one of the cellular mechanisms responsible for arterial remodelling during exposure to microgravity.
Assuntos
Aorta Abdominal/citologia , Endotélio Vascular/citologia , Monócitos/citologia , NF-kappa B/metabolismo , Simulação de Ausência de Peso , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Adesão Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Quimiocina CCL2/genética , Selectina E/genética , Endotélio Vascular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Masculino , Monócitos/efeitos dos fármacos , NF-kappa B/antagonistas & inibidores , Pirrolidinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Tiocarbamatos/farmacologia , Molécula 1 de Adesão de Célula Vascular/genéticaRESUMO
Post-spaceflight orthostatic intolerance is one of the most important adverse effects after exposure to space microgravity, and there are still no effective countermeasures. It has been considered that arterial remodeling may play an important role in the occurrence of post-spaceflight orthostatic intolerance, but the cellular mechanisms remain unknown. In this study, we investigated whether an inflammatory response exists in the common carotid artery of rats exposed to simulated microgravity. For this, Sprague-Dawley rats were subjected to 4 weeks of hindlimb unweighting to simulate microgravity. The expression levels of the adhesion molecules E-selectin and vascular cell adhesion molecule-1 (VCAM-1), and the cytokine monocyte chemoattractant protein-1 (MCP-1) in the common carotid artery of simulated microgravity rats were evaluated by immunohistochemical staining, quantitative RT-PCR, and Western blot analyses. The recruitment of monocytes in the common carotid artery of rats exposed to simulated microgravity was investigated by en face immunofluorescence staining and monocyte binding assays. Our results provided convincing evidence that there is an inflammatory response in the common carotid artery of rats exposed to simulated microgravity. Our work suggests that the inflammatory response may be a novel cellular mechanism that is responsible for the arterial remodeling that occurs during exposure to microgravity.
Assuntos
Doenças das Artérias Carótidas/metabolismo , Artéria Carótida Primitiva/metabolismo , Elevação dos Membros Posteriores/efeitos adversos , Animais , Peso Corporal , Doenças das Artérias Carótidas/etiologia , Doenças das Artérias Carótidas/patologia , Artéria Carótida Primitiva/patologia , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Selectina E/genética , Selectina E/metabolismo , Endotélio Vascular/metabolismo , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Masculino , Monócitos/metabolismo , Ratos Sprague-Dawley , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismo , Remodelação VascularRESUMO
The variability of peak current of L-type calcium channel (I(Ca,L)) shows an increase in cardiomyocytes after 6 h of preservation when the acutely isolated cardiomyocytes are preserved in a small volume buffer solution. The mechanism of the increased variability of I(Ca,L) is not clear. In order to obtain more accurately and stably experimental data of I(Ca,L), the aim of this study was to observe the pH changes of preservation buffer solution with acutely isolated rat cardiomyocytes, and the effects of pH changes on the shape of cardiomyocytes, the function of mitochondria and the gating property of L-type calcium channel. The results indicated that the pH was kept stable in 100 mL buffer solution, but was decreased from 7.20 to 6.95 in 20 mL buffer solution during 10 h of cardiomyocyte preservation. Therefore, 100 mL or 20 mL preservation solution was used as a normal control or acidotic group, respectively. The ratio of abnormal to normal rod-shaped cardiomyocytes increased in the acidotic group after 6 h of preservation. The acidosis induced a reduction in mitochondrial membrane potential indicated by JC-1 fluorescent probe after 8 h of cardiomyocyte preservation. The acidosis also shifted the autofluorescence of NADPH from blue to green after 8 h of cardiomyocyte preservation. The above changes in mitochondrial function induced a significant decrease in the peak I(Ca,L) and a shift in the clamped voltage at peak I(Ca,L) from +10 mV to 0 mV, after 10 h of cardiomyocyte preservation. These results suggest that the best way to preserve acutely isolated cardiomyocytes is to use a larger volume buffer system. In order to get stable peak I(Ca,L), we need to not only select a normal shape of cardiomyocyte at a bright field but also a blue fluorescent myocyte at an ultraviolet excitation.
Assuntos
Canais de Cálcio Tipo L/fisiologia , Miócitos Cardíacos/fisiologia , Animais , Soluções Tampão , Células Cultivadas , Potencial da Membrana Mitocondrial , Preservação Biológica , RatosRESUMO
BACKGROUND: Zoledronic acid, one of the most potent nitrogen-containing biphosphonates, has been demonstrated to have direct anti-tumor and anti-metastatic properties in breast cancer in vitro and in vivo. In particular, tumor-cell apoptosis has been recognized to play an important role in the treatment of metastatic breast cancer with zoledronic acid. However, the precise mechanisms remain less clear. In the present study, we investigated the specific role of large conductance Ca(2+)-activated potassium (BK(Ca)) channel in zoledronic acid-induced apoptosis of estrogen receptor (ER)-negative MDA-MB-231 breast cancer cells. METHODOLOGY/PRINCIPAL FINDINGS: The action of zoledronic acid on BK(Ca) channel was investigated by whole-cell and cell-attached patch clamp techniques. Cell apoptosis was assessed with immunocytochemistry, analysis of fragmented DNA by agarose gel electrophoresis, and flow cytometry assays. Cell proliferation was investigated by MTT test and immunocytochemistry. In addition, such findings were further confirmed with human embryonic kidney 293 (HEK293) cells which were transfected with functional BK(Ca) α-subunit (hSloα). Our results clearly indicated that zoledronic acid directly increased the activities of BK(Ca) channels, and then activation of BK(Ca) channel by zoledronic acid contributed to induce apoptosis in MDA-MB-231 cells. The possible mechanisms were associated with the elevated level of intracellular Ca(2+) and a concomitant depolarization of mitochondrial membrane potential (Δψm) in MDA-MB-231 cells. CONCLUSIONS: Activation of BK(Ca) channel was here shown to be a novel molecular pathway involved in zoledronic acid-induced apoptosis of MDA-MB-231 cells in vitro.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Conservadores da Densidade Óssea/farmacologia , Neoplasias da Mama/tratamento farmacológico , Difosfonatos/farmacologia , Imidazóis/farmacologia , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Mama/efeitos dos fármacos , Mama/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/agonistas , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Ácido ZoledrônicoRESUMO
BACKGROUND: The differential adaptations of cerebrovasculature and small mesenteric arteries could be one of critical factors in postspaceflight orthostatic intolerance, but the cellular mechanisms remain unknown. We hypothesize that there is a differential regulation of intracellular Ca(2+) determined by the alterations in the functions of plasma membrane Ca(L) channels and ryanodine-sensitive Ca(2+) releases from sarcoplasmic reticulum (SR) in cerebral and small mesenteric vascular smooth muscle cells (VSMCs) of simulated microgravity rats, respectively. METHODOLOGY/PRINCIPAL FINDINGS: Sprague-Dawley rats were subjected to 28-day hindlimb unweighting to simulate microgravity. In addition, tail-suspended rats were submitted to a recovery period of 3 or 7 days after removal of suspension. The function of Ca(L) channels was evaluated by patch clamp and Western blotting. The function of ryanodine-sensitive Ca(2+) releases in response to caffeine were assessed by a laser confocal microscope. Our results indicated that simulated microgravity increased the functions of Ca(L) channels and ryanodine-sensitive Ca(2+) releases in cerebral VSMCs, whereas, simulated microgravity decreased the functions of Ca(L) channels and ryanodine-sensitive Ca(2+) releases in small mesenteric VSMCs. In addition, 3- or 7-day recovery after removal of suspension could restore the functions of Ca(L) channels and ryanodine-sensitive Ca(2+) releases to their control levels in cerebral and small mesenteric VSMCs, respectively. CONCLUSIONS: The differential regulation of Ca(L) channels and ryanodine-sensitive Ca(2+) releases in cerebral and small mesenteric VSMCs may be responsible for the differential regulation of intracellular Ca(2+), which leads to the altered autoregulation of cerebral vasculature and the inability to adequately elevate peripheral vascular resistance in postspaceflight orthostatic intolerance.
Assuntos
Cálcio/metabolismo , Cérebro/citologia , Espaço Intracelular/metabolismo , Artérias Mesentéricas/citologia , Miócitos de Músculo Liso/metabolismo , Simulação de Ausência de Peso , Animais , Western Blotting , Peso Corporal/efeitos dos fármacos , Cafeína/farmacologia , Canais de Cálcio Tipo L/metabolismo , Separação Celular , Fêmur/anatomia & histologia , Fêmur/efeitos dos fármacos , Fluorescência , Espaço Intracelular/efeitos dos fármacos , Ativação do Canal Iônico/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Subunidades Proteicas/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
It has been reported that diabetic vascular dysfunction is associated with impaired function of large conductance Ca(2+) -activated K(+) (BK(Ca) ) channels. However, it is unclear whether impaired BK(Ca) channel directly participates in regulating diabetic vascular remodeling by altering cell growth in response to hyperglycemia. In the present study, we investigated the specific role of BK(Ca) channel in controlling apoptosis and proliferation under high glucose concentration (25 mM). The cDNA encoding the α+ß1 subunit of BK(Ca) channel, hSloα+ß1, was transiently transfected into human embryonic kidney 293 (HEK293) cells. Cloned BK(Ca) currents were recorded by both whole-cell and cell-attached patch clamp techniques. Cell apoptosis was assessed with immunocytochemistry and analysis of fragmented DNA by agarose gel electrophoresis. Cell proliferation was investigated by flow cytometry assays, MTT test, and immunocytochemistry. In addition, the expression of anti-apoptotic protein Bcl-2, intracellular Ca(2+) , and mitochondrial membrane potential (Δψm) were also examined to investigate the possible mechanisms. Our results indicate that inhibition of cloned BK(Ca) channels might be responsible for hyperglycemia-altered apoptosis and proliferation in HEK-hSloα+ß1 cells. However, activation of BK(Ca) channel by NS1619 or Tamoxifen significantly induced apoptosis and suppressed proliferation in HEK-hSloα+ß1 cells under hyperglycemia condition. When rat cerebral smooth muscle cells were cultured in hyperglycemia, similar findings were observed. Moreover, the possible mechanisms underlying the activation of BK(Ca) channel were associated with decreased expression of Bcl-2, elevation of intracellular Ca(2+) , and a concomitant depolarization of Δψm in HEK-hSloα+ß1 cells. In conclusion, cloned BK(Ca) channel directly regulated apoptosis and proliferation of HEK293 cell under hyperglycemia condition.
Assuntos
Apoptose/efeitos dos fármacos , Glucose/farmacologia , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/antagonistas & inibidores , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/antagonistas & inibidores , Animais , Benzimidazóis/farmacologia , Cálcio/metabolismo , Proliferação de Células/efeitos dos fármacos , Clonagem Molecular , Regulação para Baixo/efeitos dos fármacos , Vetores Genéticos/genética , Células HEK293 , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Peptídeos/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Sprague-Dawley , Tamoxifeno/farmacologia , TransfecçãoRESUMO
1. Cardiomyocyte apoptosis plays an important role in the transition from cardiac hypertrophy to heart failure. Hyper-trophic cardiomyocytes show an increased susceptibility to apoptotic stimuli, but the mechanisms remain unclear. 2. We hypothesized that activated protein kinase Cδ (PKCδ) associated with cardiomyocyte hypertrophy could move from the cytoplasm to mitochondria, and subsequently trigger the apoptotic signalling pathway. 3. Hypertrophy was induced in cultured neonatal rat cardiomyocytes using endothelin-1 (ET-1), insulin-like growth factor-1 (IGF-1), thyroid hormone (T(3) ) or angiotensin-II (AngII). AngII at high concentrations (1 and 10 nmol/L) also induced apoptosis. Hypertrophic cells were then treated with AngII with or without specific inhibitors of the angiotensin receptors AT(1) and AT(2) (losartan and PD123319, respectively), endothelin receptor A (BQ-123) and PKCδ (rottlerin). ET-1 plus AngII had a threefold and significant increase in apoptosis in the hypertrophic cultures compared with AngII alone. In association with the increase in apoptosis, this treatment also promoted mitochondrial translocation of PKCδ, and increased expression of cleaved caspase 9 and activity of caspase 3. All of these increases were modulated by concurrent use of the PKCδ inhibitor, rottlerin. 4. The results suggest that apoptotic signalling in hypertrophic cardiomyocytes is determined by mitochondrial pathways involving PKCδ.
Assuntos
Apoptose/fisiologia , Cardiomegalia/enzimologia , Mitocôndrias/enzimologia , Miócitos Cardíacos/enzimologia , Proteína Quinase C-delta/metabolismo , Acetofenonas/farmacologia , Angiotensina II/farmacologia , Animais , Apoptose/efeitos dos fármacos , Benzopiranos/farmacologia , Cardiomegalia/induzido quimicamente , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Caspase 3/biossíntese , Caspase 3/metabolismo , Caspase 9/biossíntese , Células Cultivadas , Endotelina-1/farmacologia , Imidazóis/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Mitocôndrias/fisiologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Peptídeos Cíclicos/farmacologia , Proteína Quinase C-delta/antagonistas & inibidores , Transporte Proteico/efeitos dos fármacos , Piridinas/farmacologia , Ratos , Ratos Sprague-Dawley , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/metabolismo , Receptores de Endotelina/metabolismo , Transdução de Sinais , Tri-Iodotironina/farmacologiaRESUMO
Cerebral arterial remodeling is one of the critical factors in the occurrence of postspaceflight orthostatic intolerance. We hypothesize that large-conductance calcium-activated K(+) (BK(Ca)) channels in vascular smooth muscle cells (VSMCs) may play an important role in regulating cerebrovascular adaptation during microgravity exposure. The aim of this work was to investigate whether activation of BK(Ca) channels is involved in regulation of apoptotic remodeling of cerebral arteries in simulated microgravity rats. In animal studies, Sprague-Dawley rats were subjected to 1-wk hindlimb unweighting to simulate microgravity. Alterations of BK(Ca) channels in cerebral VSMCs were investigated by patch clamp and Western blotting; apoptosis was assessed by electron microscopy and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labeling (TUNEL). To evaluate the correlation of BK(Ca) channel and apoptosis, channel protein and cell nucleus were double-stained. In cell studies, hSloalpha+beta1 channel was coexpressed into human embryonic kidney 293 (HEK293) cells to observe the effects of BK(Ca) channels on apoptosis. In rats, enhanced activities and expression of BK(Ca) channels were found to be correlated with increased apoptosis in cerebral VSMCs after simulated microgravity. In transfected HEK293 cells, activation of cloned BK(Ca) channel induced apoptosis, whereas inhibition of cloned BK(Ca) channel decreased apoptosis. In conclusion, activation of BK(Ca) channels is associated with increased apoptosis in cerebral VSMCs of simulated microgravity rats.
Assuntos
Apoptose , Cálcio/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Simulação de Ausência de Peso , Animais , Western Blotting , Linhagem Celular , Artérias Cerebrais/metabolismo , Artérias Cerebrais/patologia , Elevação dos Membros Posteriores , Humanos , Marcação In Situ das Extremidades Cortadas , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/genética , Masculino , Potenciais da Membrana , Microscopia Eletrônica , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , TransfecçãoRESUMO
The large conductance Ca(2+)-activated K(+) (BK(Ca)) channels are highly expressed in vascular smooth muscle cells (VSMCs) and play an essential role in the regulation of various physiological functions. Besides its electrophysiological function in vascular relaxation, BK(Ca) has also been reported to be implicated in nitric oxide (NO)-induced apoptosis of VSMCs. However, the molecular mechanism is not clear and has not been determined on cloned channels. The present study was designed to clarify whether activation of cloned BK(Ca) channel was involved in NO-induced apoptosis in human embryonic kidney 293 (HEK293) cell. The cDNA encoding the alpha-subunit of BK(Ca) channel, hSloalpha, was transiently transfected into HEK293 cells. The apoptotic death in HEK-hSloalpha cells was detected using immunocytochemistry, analysis of fragmented DNA by agarose gel electrophoresis, MTT test, and flow cytometry assays. Whole-cell and single-channel characteristics of HEK-hSloalpha cells exhibited functional features similar to native BK(Ca) channel in VSMCs. Exposuring of HEK- hSloalpha cells to S-nitroso-N-acetyl-penicillamine increased the hSloalpha channel activities of whole-cell and single-channel, and then increased percentage of cells undergoing apoptosis. However, blocking hSloalpha channels with 1 mM tetraethylammonia or 100 nM iberiotoxin significantly decreased the NO-induced apoptosis, whereas 30 microM NS1619, the specific agonist of BK(Ca), independently increased hSloalpha currents and induced apoptosis. These results indicated that activation of cloned BK(Ca) channel was involved in NO-induced apoptosis of HEK293 cells.