RESUMEN
The modulation of K+ channels plays a crucial role in cell migration and proliferation, but the effect of K+ channels on human cutaneous wound healing (CWH) remains underexplored. This study aimed to determine the necessity of modulating K+ channel activity and expression for human CWH. The use of 25 mM KCl as a K+ channel blocker markedly improved wound healing in vitro (in keratinocytes and fibroblasts) and in vivo (in rat and porcine models). K+ channel blockers, such as quinine and tetraethylammonium, aided in vitro wound healing, while Ba2+ was the exception and did not show similar effects. Single-channel recordings revealed that the Ba2+-insensitive large conductance Ca2+-activated K+ (BKCa) channel was predominantly present in human keratinocytes. NS1619, an opener of the BKCa channel, hindered wound healing processes like proliferation, migration, and filopodia formation. Conversely, charybdotoxin and iberiotoxin, which are BKCa channel blockers, dramatically enhanced these processes. The downregulation of BKCa also improved CWH, whereas its overexpression impeded these healing processes. These findings underscore the facilitative effect of BKCa channel suppression on CWH, proposing BKCa channels as potential molecular targets for enhancing human cutaneous wound healing.
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Fibroblastos , Hidrolasas , Humanos , Animales , Ratas , Porcinos , Movimiento Celular , Regulación hacia Abajo , Canales de Potasio de Gran Conductancia Activados por el Calcio , Cicatrización de HeridasRESUMEN
Acute lung injury (ALI) is a condition associated with acute respiratory failure, resulting in significant morbidity and mortality. It involves cellular changes such as disruption of the alveolar-capillary membrane, excessive neutrophil migration, and release of inflammatory mediators. Broncho-Vaxom® (BV), a lyophilized product containing cell membrane components derived from eight bacteria commonly found in the respiratory tract, is known for its potential to reduce viral and bacterial lung infections. However, the specific effect of BV on ALI has not been clearly defined. This study explored the preventive effects of BV and its underlying mechanisms in a lipopolysaccharide (LPS)-induced ALI mouse model. Oral BV (1 mg/kg) gavage was administered one hour before the intratracheal injection of LPS to evaluate its preventive effect on the ALI model. The pre-administration of BV significantly mitigates inflammatory parameters, including the production of inflammatory mediators, macrophage infiltration, and NF-κB activation in lung tissue, and the increase in inflammatory cells in bronchoalveolar lavage fluid (BALF). Moreover, BV (3 µg/mL) pretreatment reduced the expression of M1 macrophage markers, interleukins (IL-1ß, IL-6), tumor necrosis factor α, and cyclooxygenase-2, which are activated by LPS, in both mouse alveolar macrophage MH-S cells and human macrophage THP-1 cells. These findings showed that BV exhibits anti-inflammatory effects by suppressing inflammatory mediators through the NF-κB pathway, suggesting its potential to attenuate bronchial and pulmonary inflammation.
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Lesión Pulmonar Aguda , Modelos Animales de Enfermedad , Lipopolisacáridos , Animales , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/patología , Lesión Pulmonar Aguda/metabolismo , Lesión Pulmonar Aguda/etiología , Lesión Pulmonar Aguda/tratamiento farmacológico , Ratones , Humanos , Inflamación/patología , Inflamación/metabolismo , Inflamación/tratamiento farmacológico , Masculino , Extractos Celulares/farmacología , Extractos Celulares/uso terapéutico , FN-kappa B/metabolismo , Líquido del Lavado Bronquioalveolar , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/efectos de los fármacos , Citocinas/metabolismo , Mediadores de Inflamación/metabolismo , Pulmón/patología , Pulmón/metabolismo , Pulmón/efectos de los fármacos , Lisados BacterianosRESUMEN
Two-pore domain K+ channels (K2P channels), active as dimers, produce inhibitory currents regulated by a variety of stimuli. Among them, TWIK1-related alkalinization-activated K+ channel 1 (TALK1), TWIK1-related alkalinization-activated K+ channel 2 (TALK2), and TWIK1-related acid-sensitive K+ channel 2 (TASK2) form a subfamily of structurally related K2P channels stimulated by extracellular alkalosis. The human genes encoding these proteins are clustered at chromosomal region 6p21 and coexpressed in multiple tissues, including the pancreas. The question whether these channels form functional heteromers remained open. By analyzing single-cell transcriptomic data, we show that these channels are coexpressed in insulin-secreting pancreatic ß-cells. Using in situ proximity ligation assay and electrophysiology, we show that they form functional heterodimers both upon heterologous expression and under native conditions in human pancreatic ß-cells. We demonstrate that heteromerization of TALK2 with TALK1 or with TASK2 endows TALK2 with sensitivity to extracellular alkalosis in the physiological range. We further show that the association of TASK2 with TALK1 and TALK2 increases their unitary conductance. These results provide a new example of heteromerization in the K2P channel family expanding the range of the potential physiological and pathophysiological roles of TALK1/TALK2/TASK2 channels, not only in insulin-secreting cells but also in the many other tissues in which they are coexpressed.
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Alcalosis , Células Secretoras de Insulina , Canales de Potasio de Dominio Poro en Tándem , Humanos , Canales de Potasio de Dominio Poro en Tándem/genética , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Células Secretoras de Insulina/metabolismo , Concentración de Iones de Hidrógeno , Insulina/metabolismo , Potasio/metabolismoRESUMEN
Another affiliation: 2 Department of Anesthesiology and Pain Medicine, Gyeongsang National University College of Medicine, Jinju-si, Gyeongsangnam-do, Republic of Korea was added for the author Kyeong-Eon Park at his own request.
RESUMEN
This study examined the effect of chloroquine on vasodilation induced by levcromakalim in isolated endothelium-denuded rat aortas and clarified the underlying mechanisms. We examined the effects of chloroquine, hydroxychloroquine, lipid emulsion, reactive oxygen species (ROS) scavenger N-acetyl-Ê-cysteine (NAC), and KATP channel inhibitor glibenclamide on levcromakaliminduced vasodilation. The effects of chloroquine, hydroxychloroquine, NAC, and levcromakalim on membrane hyperpolarization and ROS production were examined in aortic vascular smooth muscle cells (VSMCs). Chloroquine inhibited levcromakalim-induced vasodilation more than hydroxychloroquine. NAC attenuated chloroquine-mediated inhibition of levcromakalim-induced vasodilation, while lipid emulsion had no effect. Glibenclamide eliminated levcromakalim-induced vasodilation in aortas pretreated with chloroquine. Chloroquine and hydroxychloroquine inhibited levcromakalim-induced membrane hyperpolarization in VSMCs. Chloroquine and hydroxychloroquine both produced ROS, but chloroquine produced more. NAC inhibited chloroquine-induced ROS production in VSMCs. Collectively, these results suggest that, partially through ROS production, chloroquine inhibits levcromakalim-induced vasodilation. In addition, chloroquine-induced KATP channel-induced vasodilation impairment was not restored by lipid emulsion.
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Vasodilatación , Vasodilatadores , Ratas , Animales , Cromakalim/farmacología , Vasodilatadores/farmacología , Canales KATP , Gliburida/farmacología , Especies Reactivas de Oxígeno , Hidroxicloroquina/farmacología , Cloroquina/farmacología , Emulsiones/farmacología , Canales de Potasio , Aorta , LípidosRESUMEN
The TRPM4 gene encodes a Ca2+-activated monovalent cation channel called transient receptor potential melastatin 4 (TRPM4) that is expressed in various tissues. Dysregulation or abnormal expression of TRPM4 has been linked to a range of diseases. We introduced the hemagglutinin (HA) tag into the extracellular S6 loop of TRPM4, resulting in an HA-tagged version called TRPM4-HA. This TRPM4-HA was developed to investigate the purification, localization, and function of TRPM4 in different physiological and pathological conditions. TRPM4-HA was successfully expressed in the intact cell membrane and exhibited similar electrophysiological properties, such as the current-voltage relationship, rapid desensitization, and current size, compared to the wild-type TRPM4. The presence of the TRPM4 inhibitor 9-phenanthrol did not affect these properties. Furthermore, a wound-healing assay showed that TRPM4-HA induced cell proliferation and migration, similar to the native TRPM4. Co-expression of protein tyrosine phosphatase, non-receptor type 6 (PTPN6 or SHP-1) with TRPM4-HA led to the translocation of TRPM4-HA to the cytosol. To investigate the interaction between PTPN6 and tyrosine residues of TRPM4 in enhancing channel activity, we generated four mutants in which tyrosine (Y) residues were substituted with phenylalanine (F) at the N-terminus of TRPM4. The YF mutants displayed properties and functions similar to TRPM4-HA, except for the Y256F mutant, which showed resistance to 9-phenanthrol, suggesting that Y256 may be involved in the binding site for 9-phenanthrol. Overall, the creation of HA-tagged TRPM4 provides researchers with a valuable tool to study the role of TRPM4 in different conditions and its potential interactions with other proteins, such as PTPN6.
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This study investigated changes in nutrients (fatty acids, amino acids, and minerals), ginsenosides, and volatile flavors, and antioxidant activities during food processing of mountain-cultivated ginseng (MCG) with the cocktail lactic acid bacteria. Fatty acid content increased, but the free amino acid content decreased, and minerals were practically unaffected during processing. Total phenolic and flavonoid contents and maillard reaction products increased markedly according to processing stage. The total ginsenosides levels increased from 31.25 mg/g (DMCG) to 32.36 mg/g (red MCG, RMCG) and then decreased (27.27 mg/g, at fermented RMCG) during processing. Particularly, the contents of F2 (0.31 â 1.02 â 2.27 mg/g), Rg3 (0.36 â 0.77 â 1.93 mg/g), and compound K (0.5 â 1.68 â 4.13 mg/g) of ginsenosides and ß-panasinsene (17.28 â 22.69 â 31.61%), biocycloelemene (0.11 â 0.84 â 0.92%), δ-cadinene (0.39 â 0.5 â 0.94%), and alloaromadendrene (1.64 â 1.39 â 2.6%) of volatile flavor compounds increased during processing, along with to the antioxidant effects (such as DPPH, ABTS, and hydroxyl radical scavenging activities, and FRAP). This study may provide several choices for the use of ginseng in functional foods and functional cosmetics.
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Ginsenósidos , Panax , Antioxidantes/química , Ginsenósidos/química , Radical Hidroxilo/química , Panax/química , Fenoles/químicaRESUMEN
Models created by the intraperitoneal injection of lipopolysaccharide (LPS) and D-galactosamine (D-GalN) have been widely used to study the pathogenesis of human acute liver failure (ALF) and drug development. Our previous study reported that oyster (Crassostrea gigas) hydrolysate (OH) had a hepatoprotective effect in LPS/D-GalN-injected mice. This study was performed to identify the hepatoprotective effect of the tyrosine-alanine (YA) peptide, the main component of OH, in a LPS/D-GalN-injected ALF mice model. We analyzed the effect of YA on previously known mechanisms of hepatocellular injury in the model. LPS/D-GalN-injected mice showed inflammatory, apoptotic, ferroptotic, and pyroptotic liver injury. The pre-administration of YA (10 mg/kg or 50 mg/kg) significantly reduced the liver damage factors. The hepatoprotective effect of YA was higher in the 50 mg/kg YA pre-administered group than in the 10 mg/kg YA pre-administered group. These results showed that YA had a hepatoprotective effect by reducing inflammation, apoptosis, ferroptosis, and pyroptosis in the LPS/D-GalN-injected ALF mouse model. We suggest that YA can be used as a functional peptide for the prevention of acute liver injury.
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Antiinflamatorios/farmacología , Ostreidae , Péptidos/farmacología , Animales , Antiinflamatorios/química , Antiinflamatorios/uso terapéutico , Organismos Acuáticos , Modelos Animales de Enfermedad , Galactosamina , Lipopolisacáridos , Fallo Hepático Agudo/tratamiento farmacológico , Masculino , Ratones , Ratones Endogámicos C57BL , Péptidos/química , Péptidos/uso terapéutico , Piroptosis/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
We examined the effect of endothelium and lipid emulsion on vasodilation induced by minoxidil at a toxic dose and determined the underlying mechanism. The effects of endothelial denudation, NW-nitro-L-arginine methyl ester (L-NAME), methylene blue, 1H-[1,2,4]oxadiazolo[4,3-a] quinoxalin-1-one (ODQ), and glibenclamide, alone or in combination, on minoxidil-induced vasodilation in endothelium-intact rat aorta were examined. Additionally, the effects of lipid emulsion on minoxidil-induced membrane hyperpolarization and minoxidil concentration were examined. The vasodilatory effects of minoxidil at the toxic dose were higher in endothelium-intact aorta than in endothelium-denuded aorta. L-NAME, methylene blue, ODQ, and glibenclamide attenuated minoxidil-induced vasodilation of endothelium-intact rat aorta. Combined treatment with L-NAME and glibenclamide almost eliminated minoxidil-induced vasodilation. However, lipid emulsion pretreatment did not significantly alter minoxidil-induced vasodilation. Lipid emulsion did not significantly alter minoxidil-induced membrane hyperpolarization and minoxidil concentration. Overall, minoxidil-induced vasodilation is mediated by ATP-sensitive potassium channels and pathways involving nitric oxide and guanylate cyclase.
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Óxido Nítrico , Vasodilatación , Animales , Aorta , Endotelio Vascular , Minoxidil , NG-Nitroarginina Metil Éster , Óxido Nítrico Sintasa de Tipo III , RatasRESUMEN
The two-pore domain K+ (K2P) channel, which is involved in setting the resting membrane potential in neurons, is an essential target for receptor agonists. Activation of the γ-aminobutyric acid (GABA) receptors (GABAAR and GABABR) reduces cellular excitability through Cl- influx and K+ efflux in neurons. Relatively little is known about the link between GABAAR and the K+ channel. The present study was performed to identify the effect of GABAR agonists on K2P channel expression and activity in the neuroblastic B35 cells that maintain glutamic acid decarboxylase (GAD) activity and express GABA. TASK and TREK/TRAAK mRNA were expressed in B35 cells with a high level of TREK-2 and TRAAK. In addition, TREK/TRAAK proteins were detected in the GABAergic neurons obtained from GABA transgenic mice. Furthermore, TREK-2 mRNA and protein expression levels were markedly upregulated in B35 cells by GABAAR and GABABR agonists. In particular, muscimol, a GABAAR agonist, significantly increased TREK-2 expression and activity, but the effect was reduced in the presence of the GABAAR antagonist bicuculine or TREK-2 inhibitor norfluoxetine. In the whole-cell and single-channel patch configurations, muscimol increased TREK-2 activity, but the muscimol effect disappeared in the N-terminal deletion mutant. These results indicate that muscimol directly induces TREK-2 activation through the N-terminus and suggest that muscimol can reduce cellular excitability by activating the TREK-2 channel and by inducing Cl- influx in GABAergic neurons.
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Agonistas de Receptores de GABA-A/farmacología , Neuronas GABAérgicas/metabolismo , Potenciales de la Membrana , Muscimol/farmacología , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Receptores de GABA/química , Animales , Células Cultivadas , Neuronas GABAérgicas/efectos de los fármacos , Células HEK293 , Humanos , Masculino , Ratones , Canales de Potasio de Dominio Poro en Tándem/genética , RatasRESUMEN
The aim of this study is to explore anti-inflammatory phytochemicals from B. chinensis based on the inhibition of pro-inflammatory enzyme, human neutrophil elastase (HNE) and anti-inflammatory activities in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage. Three stereoisomers of iridal-type triterpenoids (1-3) were isolated from the roots of B. chinensis and their stereochemistries were completely identified by NOESY spectra. These compounds were confirmed as reversible noncompetitive inhibitors against HNE with IC50 values of 6.8-27.0 µM. The binding affinity experiment proved that iridal-type triterpenoids had only a single binding site to the HNE enzyme. Among them, isoiridogermanal (1) and iridobelamal A (2) displayed significant anti-inflammatory effects by suppressing the expressions of pro-inflammatory cytokines, such as iNOS, IL-1ß, and TNF-α through the NF-κB pathway in LPS-stimulated RAW264.7 cells. This is the first report that iridal-type triterpenoids are considered responsible phytochemicals for anti-inflammatory effects of B. chinensis.
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Antiinflamatorios/farmacología , Iridaceae/química , Elastasa de Leucocito/antagonistas & inhibidores , Extractos Vegetales/farmacología , Triterpenos/farmacología , Animales , Antiinflamatorios/química , Antiinflamatorios/aislamiento & purificación , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Humanos , Elastasa de Leucocito/metabolismo , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/farmacología , Ratones , Conformación Molecular , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Células RAW 264.7 , Triterpenos/química , Triterpenos/aislamiento & purificaciónRESUMEN
Accumulative alcohol hangovers cause liver damage through oxidative and inflammatory stress. Numerous antioxidant and anti-inflammatory reagents have been developed to reduce alcohol hangovers, but these reagents are still insignificant and have limitations in that they can cause liver toxicity. Oyster hydrolysate (OH), another reagent that has antioxidant and anti-inflammatory activity, is a product extracted through an enzymatic hydrolysis process from oysters (Crassostrea gigas), which can be easily eaten in meals. This study was aimed at determining the effects of OH on alcohol metabolism, using a single high dose of ethanol (EtOH) administered to rodents, by monitoring alcohol metabolic enzymes, oxidative stress signals, and inflammatory mediators. The effect of tyrosine-alanine (YA) peptide, a main component of OH, on EtOH metabolism was also identified. In vitro experiments showed that OH pretreatment inhibited EtOH-induced cell death, oxidative stress, and inflammation in liver cells and macrophages. In vivo experiments showed that OH and YA pre-administration increased alcohol dehydrogenase, aldehyde dehydrogenase, and catalase activity in EtOH binge treatment. In addition, OH pre-administration alleviated CYP2E1 activity, ROS production, apoptotic signals, and inflammatory mediators in liver tissues. These results showed that OH and YA enhanced EtOH metabolism and had a protective effect against acute alcohol liver damage. Our findings offer new insights into a single high dose of EtOH drinking and suggest that OH and YA could be used as potential marine functional foods to prevent acute alcohol-induced liver damage.
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Crassostrea/química , Dipéptidos/farmacología , Etanol/metabolismo , Aldehído Deshidrogenasa/genética , Aldehído Deshidrogenasa/metabolismo , Animales , Dipéptidos/química , Etanol/administración & dosificación , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Hidrólisis , Masculino , Ratones , Ratones Endogámicos C57BL , Estrés Oxidativo/efectos de los fármacos , Ratas Sprague-DawleyRESUMEN
TWIK (tandem-pore domain weak inward rectifying K+)-related spinal cord K+ channel (TRESK), a member of the two-pore domain K+ channel family, is abundantly expressed in dorsal root ganglion (DRG) neurons. It is well documented that TRESK expression is changed in several models of peripheral nerve injury, resulting in a shift in sensory neuron excitability. However, the role of TRESK in the model of spinal cord injury (SCI) has not been fully understood. This study investigates the role of TRESK in a thoracic spinal cord contusion model, and in transgenic mice overexpressed with the TRESK gene (TGTRESK). Immunostaining analysis showed that TRESK was expressed in the dorsal and ventral neurons of the spinal cord. The TRESK expression was increased by SCI in both dorsal and ventral neurons. TRESK mRNA expression was upregulated in the spinal cord and DRG isolated from the ninth thoracic (T9) spinal cord contusion rats. The expression was significantly upregulated in the spinal cord below the injury site at acute time points (6, 24, and 48 h) after SCI (p < 0.05). In addition, TRESK expression was markedly increased in DRGs below and adjacent to the injury site. TRESK was expressed in inflammatory cells. In addition, the number and fluorescence intensity of TRESK-positive neurons increased in the dorsal and ventral horns of the spinal cord after SCI. TGTRESK SCI mice showed faster paralysis recovery and higher mechanical threshold compared to wild-type (WT)-SCI mice. TGTRESK mice showed lower TNF-α concentrations in the blood than WT mice. In addition, IL-1ß concentration and apoptotic signals in the caudal spinal cord and DRG were significantly decreased in TGTRESK SCI mice compared to WT-SCI mice (p < 0.05). These results indicate that TRESK upregulated following SCI contributes to the recovery of paralysis and mechanical pain threshold by suppressing the excitability of motor and sensory neurons and inflammatory and apoptotic processes.
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Neuronas Motoras/patología , Canales de Potasio/genética , Recuperación de la Función , Células Receptoras Sensoriales/patología , Traumatismos de la Médula Espinal/genética , Traumatismos de la Médula Espinal/fisiopatología , Regulación hacia Arriba/genética , Animales , Ganglios Espinales/metabolismo , Ganglios Espinales/patología , Ratones Endogámicos C57BL , Neuronas Motoras/metabolismo , Canales de Potasio/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Sprague-Dawley , Células Receptoras Sensoriales/metabolismoRESUMEN
The goal of this study was to examine the effect of lipid emulsion on the vasodilation induced by ATP-sensitive potassium (KATP) channels in isolated rat aortae and the underlying mechanism. The effects of Intralipid, containing 100% long-chain fatty acids, and Lipofundin MCT/LCT, containing 50% long-chain fatty acids plus 50% medium-chain fatty acids, on the vasodilation induced by levcromakalim in endothelium-intact aorta with or without NW-nitro-L-arginine methyl ester (L-NAME) and in endothelium-denuded aorta were examined. The effects of L-arginine, L-NAME, glibenclamide, and Lipofundin MCT/LCT, alone or combined, on the levcromakalim-induced vasodilation were examined. Lipofundin MCT/LCT inhibited the levcromakalim-induced vasodilation of isolated endothelium-intact aortae, whereas Intralipid did not. In addition, Lipofundin MCT/LCT had no effect on the levcromakalim-induced vasodilation of endothelium-denuded rat aortae and endothelium-intact aortae with L-NAME. L-arginine and Lipofundin MCT/LCT produced more levcromakalim-induced vasodilation than Lipofundin MCT/LCT alone. Glibenclamide inhibited levcromakalim-induced vasodilation. Levcromakalim did not significantly alter endothelial nitric oxide synthase phosphorylation, whereas Lipofundin MCT/LCT decreased cyclic guanosine monophosphate. Lipofundin MCT/LCT did not significantly alter levcromakalim-induced membrane hyperpolarization. Taken together, these results suggest that Lipofundin MCT/LCT inhibits the vasodilation induced by levcromakalim by inhibiting basally released endothelial nitric oxide, which seems to occur through medium-chain fatty acids.
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Ácidos Grasos/química , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Fosfolípidos/farmacología , Sorbitol/farmacología , Vasodilatación/efectos de los fármacos , Animales , Aorta/metabolismo , Cromakalim , GMP Cíclico/metabolismo , Combinación de Medicamentos , Emulsiones , Células Endoteliales/metabolismo , Masculino , Potenciales de la Membrana , Fosforilación , Canales de Potasio/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
The present study evaluates the transdifferentiation potential of different region-derived same donor Wharton's jelly MSCs (WJMSCs) into functional smooth muscle-like cells (SMLCs). All regions showed baseline expression for early smooth muscle cell (SMC) markers (αSMA and SM22-α) whereas mid marker CALPONIN gradually reduced during in vitro culture expansion and late marker myosin heavy chain type-11 (MHY-11) was completely absent. Furthermore, WJMSCs were induced to SMLCs using DMEM containing 10% FBS supplemented with different concentrations/combinations of TGF-ß1 and PDGF-BB under normoxia (20% O2) condition. Three treatment groups namely group A: 2.5 ng/ml TGF-ß1, group B: 5 ng/ml PDGF-BB and group C: 2.5 ng/ml TGF-ß1 + 5 ng/ml PDGF-BB were used for the induction of WJMSCs into SMLCs. Cells were evaluated for SMC-specific marker expression at different time intervals. Finally, selection of the SMC-specific highly potent region along with the most suitable treatment group was done on the basis of highest outcome in terms of SMC-specific marker expression and functional competence of transdifferentiated cells. Among all regions, baby region-derived WJMSCs (B-WJMSCs) exhibited highest SMC marker expression and functional ability. To mimic the in vivo physiological conditions, hypoxic conditions (3% O2) were used to evaluate the effect of low oxygen on the SMLC differentiation potential of selected WJMSCs using previously used same parameters. Annexin-V assay was performed to check the effect of cytokines and different oxygen concentrations, which revealed no significant differences. It was concluded that different induction conditions have different but positive effects on the functional SMLC differentiation ability of WJMSCs.
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Diferenciación Celular , Transdiferenciación Celular , Células Madre Mesenquimatosas , Miocitos del Músculo Liso , Biomarcadores/metabolismo , Células Cultivadas , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Músculo Liso/citología , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Cordón Umbilical/citología , Gelatina de Wharton/citologíaRESUMEN
In recent years, noise has become a serious hazard and can have permanent biological and psychological effects on humans and other organisms in nature. Textile materials are commonly used as absorbent acoustic materials for noise reduction. This work examines the use of electrospun nylon-6 and polyurethane nanofibres (PU NFs) to improve the sound absorption and sound insulation properties of polyurethane foam. In this work, nylon-6 and polyurethane nanofibres were prepared by an electrospinning technique and were glued to a polyurethane foam. The sound absorption coefficient of the materials was measured by the impedance tube method. An impedance tube was used to measure the sound absorption and airborne sound insulation. The results showed decreased sound absorption properties, whereas the sound insulation was highly enhanced when polyurethane/nanofibre hybrids were used, as compared to the pristine polyurethane foam. Furthermore, the sound insulation properties of polyurethane foam were highly enhanced when the foam was combined with nylon-6 NFs, compared with the polyurethane foam with PU NFs. Therefore, by investigating the acoustic characteristics of electrospun nylon-6 and PU nanofibres, we believe that this study can broaden the application of electrospun nanofibres for sound pollution control.
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Caprolactama , Nanofibras , Caprolactama/análogos & derivados , Humanos , Polímeros , PoliuretanosRESUMEN
The tandem of pore domain in a weak inwardly rectifying K(+) channel (Twik)-related acid-arachidonic activated K(+) channel (TRAAK) and Twik-related K(+) channels (TREK) 1 and TREK2 are active as homodimers gated by stretch, fatty acids, pH, and G protein-coupled receptors. These two-pore domain potassium (K2P) channels are broadly expressed in the nervous system where they control excitability. TREK/TRAAK KO mice display altered phenotypes related to nociception, neuroprotection afforded by polyunsaturated fatty acids, learning and memory, mood control, and sensitivity to general anesthetics. These channels have emerged as promising targets for the development of new classes of anesthetics, analgesics, antidepressants, neuroprotective agents, and drugs against addiction. Here, we show that the TREK1, TREK2, and TRAAK subunits assemble and form active heterodimeric channels with electrophysiological, regulatory, and pharmacological properties different from those of homodimeric channels. Heteromerization occurs between all TREK variants produced by alternative splicing and alternative translation initiation. These results unveil a previously unexpected diversity of K2P channels that will be challenging to analyze in vivo, but which opens new perspectives for the development of clinically relevant drugs.
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Canales de Potasio de Dominio Poro en Tándem/química , Animales , Dimerización , Perros , Humanos , Células de Riñón Canino Madin Darby , RatonesRESUMEN
Transient receptor-potential, cation channel, subfamily M, member 4 (TRPM4) channels regulate a variety of physiological and pathological processes; however, their roles as functional channels under diverse conditions remain unclear. In this study, cytosolic protein tyrosine phosphatase non-receptor type 6 (PTPN6) interacted with TRPM4 channels. We confirmed their interaction by performing co-immunoprecipitation (Co-IP) assays following heterologous PTPN6 and TRPM4 channel expression in HEK293 cells. Furthermore, biomolecular fluorescence complementation (BiFC) image analysis confirmed TRPM4-PTPN6 binding. In addition, immunoblotting and Co-IP analyses revealed that TRPM4 expression significantly decreased in the membrane fraction of cells after PTPN6 was silenced with a specific short-hairpin RNA (shRNA-PTPN6). In agreement, TRPM4-induced changes in whole-cell currents were not detected in PTPN6-silenced HEK cells, in contrast to cells transfected with a scrambled RNA (scRNA) or in naïve HEK cells. These data suggest that PTPN6 inhibits TRPM4 channel activity by disrupting TRPM4 expression. Furthermore, TRPM4 channels were expressed in the membrane of naïve cells and scRNA transfectants, but not in those of PTPN6-silenced cells. These results indicated that PTPN6 is critically associated with TRPM4 trafficking. This role of PTPN6 in TRPM4 membrane localization was also demonstrated in HeLa cells. TRPM4 overexpression significantly enhanced cell proliferation in untreated HeLa cells, but not in HeLa cells with silenced PTPN6 expression. These findings indicate that PTPN6-dependent TRPM4 expression and trafficking to the plasma membrane is critical for cell proliferation in both HEK293 and HeLa cells. Therefore, PTPN6 is a novel therapeutic target for treating pathologic diseases involving TRPM4.
Asunto(s)
Membrana Celular/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Canales Catiónicos TRPM/metabolismo , Células HEK293 , Células HeLa , Humanos , Unión Proteica , Transporte de ProteínasRESUMEN
Glomus cells isolated from rabbit and rat/mouse carotid bodies have been used for many years to study the role of ion channels in hypoxia sensing. Studies show that hypoxia inhibits the inactivating K+ channels (Kv4) in rabbits, but inhibits TASK in rats/mice to elicit the hypoxic response. Because the role of TASK in rabbit glomus cells is not known, we isolated glomus cells from rabbits and studied the expression of TASK mRNA in the whole carotid body (CB), changes in [Ca2+]i and TASK activity. RT-PCR showed that rabbit CB expressed mRNA for TASK-3 and several Kv (Kv2.1, Kv3.1 and Kv3.3). In rabbit glomus cells in which 20 mM KClo elevated [Ca2+], anoxia also elicited a strong rise in [Ca2+]. In cell-attached patches with 140 mM KCl in the pipette, basal openings of ion channels with single-channel conductance levels of 16-pS, 34-pS, and 42-pS were present. TREK-like channels were also observed. In inside-out patches with high [Ca2+]i, BK was activated. The 42-pS channel opened spontaneously and briefly. The 16-pS and 34-pS channels showed properties similar to those of TASK-1 and TASK-3, respectively. TASK activity in cell-attached patches was lower than that in rat glomus cells under identical recording conditions. Hypoxia (~0.5%O2) reduced TASK activity by ~52% and depolarized the cells by ~30 mV. Our results show that the O2-sensitive TASK contributes to the hypoxic response in rabbit glomus cells.
Asunto(s)
Cuerpo Carotídeo/citología , Canales de Potasio de Dominio Poro en Tándem/fisiología , Animales , Calcio/fisiología , Hipoxia de la Célula , Potenciales de la Membrana , Ratones , Proteínas del Tejido Nervioso , Técnicas de Placa-Clamp , Conejos , RatasRESUMEN
Tandem pore domain weak inward rectifier potassium channel (TWIK)-related spinal cord K⺠(TRESK; K2P18.1) channel is the only member of the two-pore domain K⺠(K2P) channel family that is activated by an increase in intracellular Ca2+ concentration ([Ca2+]i) and linked to migraines. This study was performed to identify the effect of verapamil, which is an L-type Ca2+ channel blocker and a prophylaxis for migraines, on the TRESK channel in trigeminal ganglion (TG) neurons, as well as in a heterologous system. Single-channel and whole-cell currents were recorded in TG neurons and HEK-293 cells transfected with mTRESK using patch-clamping techniques. In TG neurons, changes in [Ca2+]i were measured using the fluo-3-AM Ca2+ indicator. Verapamil, nifedipine, and NiCl2 inhibited the whole-cell currents in HEK-293 cells overexpressing mTRESK with IC50 values of 5.2, 54.3, and >100 µM, respectively. The inhibitory effect of verapamil on TRESK channel was also observed in excised patches. In TG neurons, verapamil (10 µM) inhibited TRESK channel activity by approximately 76%. The TRESK channel activity was not dependent on the presence of extracellular Ca2+. In addition, the inhibitory effect of verapamil on the TRESK channel remained despite the absence of extracellular Ca2+. These findings show that verapamil inhibits the TRESK current independently of the blockade of Ca2+ influx in TG neurons. Verapamil will be able to exert its pharmacological effects by modulating TRESK, as well as Ca2+ influx, in TG neurons in vitro. We suggest that verapamil could be used as an inhibitor for identifying TRESK channel in TG neurons.