RESUMEN
Vascular calcification, the deposition of calcium in the arterial wall, is often linked to increased stiffness of the vascular wall. Vascular calcification is one of the important factors for high morbidity and mortality of cardiovascular and cerebrovascular diseases, as well as an important biomarker in atherosclerotic cardiovascular events, stroke and peripheral vascular diseases. The mechanism of vascular calcification has not been fully elucidated. Recently, non-coding RNAs have been found to play an important role in the process of vascular calcification. In this paper, the main types of non-coding RNAs and their roles involved in vascular smooth muscle cell calcification are reviewed, including the changes of osteoblast-related proteins, calcification signaling pathways and intracellular Ca2+.
Asunto(s)
Músculo Liso Vascular , Calcificación Vascular , Humanos , Músculo Liso Vascular/metabolismo , Calcificación Vascular/genética , Calcificación Vascular/metabolismo , Miocitos del Músculo Liso/metabolismoRESUMEN
Pulmonary arterial hypertension (PAH) is a rare disease with a complex aetiology characterized by elevated pulmonary artery resistance, which leads to progressive right ventricular failure and ultimately death. The aberrant metabolism of arachidonic acid in the pulmonary vasculature plays a central role in the pathogenesis of PAH. The levels of 15-lipoxygenase (15-LO) and 15-hydroxyeicosatetraenoic acid (15-HETE) are elevated in the pulmonary arterial endothelial cells (PAECs), pulmonary smooth muscle cells (PASMCs) and fibroblasts of PAH patients. Under hypoxia condition, 15-LO/15-HETE induces pulmonary artery contraction, promotes the proliferation of PAECs and PASMCs, inhibits apoptosis of PASMCs, promotes fibrosis of pulmonary vessels, and then leads to the occurrence of PAH. Here, we review the research progress on the relationship between 15-LO/15-HETE and hypoxic PAH, in order to clarify the significance of 15-LO/15-HETE in hypoxic PAH.
Asunto(s)
Araquidonato 15-Lipooxigenasa , Hipertensión Arterial Pulmonar , Proliferación Celular , Células Cultivadas , Células Endoteliales , Humanos , Ácidos Hidroxieicosatetraenoicos , Hipoxia , Miocitos del Músculo Liso , Arteria PulmonarRESUMEN
Abnormal airway smooth muscle cells (ASMCs) proliferation is an important pathological process in airway remodeling contributes to increased mortality in asthma. Mitochondrial dynamics and metabolism have a central role in the maintenance of the cell function. In this study, lipopolysaccharide (LPS)-induced ASMCs proliferative model was used to investigate the effect of mitochondria on the proliferation of ASMCs and the possible mechanism. We used cell and molecular biology to determine the effect of dynamin-related protein 1 (Drp1) on LPS-mediated ASMCs cell cycle progression and glycolysis. The major findings of the current study are as follows: LPS promoted an increased mitochondrial fission and phosphorylation of Drp1 at Ser616 (p-Drp1 Ser616). LPS-induced ASMCs proliferation and cell cycle progression, which was significantly inhibited application of Drp1 RNA interfering. Glycolysis inhibitor 2-deoxyglucose (2-DG) depressed ASMCs proliferative process induced by LPS stimulation. LPS caused mitochondrial metabolism disorders and aerobic glycolysis in a dependent on Drp1 activation. These results indicated that Drp1 may function as a key factor in asthma airway remodeling by mediating ASMC proliferation and cell cycle acceleration through an effect on mitochondrial metabolic disturbance.
Asunto(s)
Dinaminas/metabolismo , Glucólisis/efectos de los fármacos , Lipopolisacáridos/farmacología , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Aerobiosis , Animales , Proliferación Celular/efectos de los fármacos , Pulmón/citología , Masculino , Mitocondrias/metabolismo , Dinámicas Mitocondriales , Miocitos del Músculo Liso/efectos de los fármacos , Interferencia de ARN , Ratas WistarRESUMEN
To investigate the effect and regulatory mechanism of puerarin on pulmonary arterial hypertension due to hypoxia and the possible accompanying pulmonary fibrosis, The rat model of hypoxic pulmonary hypertension and the rat model of hypoxia were established. Totally 18 clean-grade SD rats were fed and randomly divided into normal control group, model group and hypoxia+medicine group. Each group received intraperitoneal injection 30 min before modeling every day; hypoxia+medicine group was injected with 20 mg·kg⻹ puerarin. Normal control group and model group were injected with the equal volume of 0.9% NaCl solution. Normal control group was cultured under normal conditions in the laboratory, while model group and hypoxia+medicine group were cultured in ahypoxia environment for 21 days to observe rat hypoxic characteristics and make the preliminary judgment about modeling. Afterwards, small animal echocardiography, right cardiac catheterization, HE dyeing and other experiments were used to verify the successful modeling, and puerarin has a therapeutic effect in pulmonary hypertension caused by hypoxia in SD rats. Fluorescence quantitative PCR, Western blot and immunofluorescence method were used to detect the changes caused by hypoxia pulmonary fibrosis-associated protein. It was found that puerarin could be given in anoxia to promote the expressions of CD31, VE-cadherin, inhibit the expressions of α-SMA, vimentin and fibronection, namely the inhibition of vascular wall thickening. Puerarin has the therapeutic effect on the pulmonary hypertension and accompanying pulmonary fibrosis in rats induced by hypoxia.
Asunto(s)
Hipertensión Pulmonar/tratamiento farmacológico , Hipoxia/tratamiento farmacológico , Isoflavonas/farmacología , Fibrosis Pulmonar/tratamiento farmacológico , Animales , Distribución Aleatoria , Ratas , Ratas Sprague-DawleyRESUMEN
To investigate the effect of taurine(Tau) on ICAM-1, VCAM-1 by p-p38 pathway in bovine pulmonary artery endothelial cells(PAECs) and explore its mechanism of action. Generation 4-12 cells in primary cultures of PAECs were used in experiments and divided into five groupsï¼ control group, hypoxia(hyp) group, inhibitor(SB203580) group, treatment(Tau) group, and treatment+inhibitor(SB+Tau) group. The concentration of Tauï¼100 mmolâ¢L⻹; p38 inhibitor SB203580ï¼ 20 µmolâ¢L⻹; and the treatment time was 12 h. MTT assay was used to detect the inhibitory effect of different concentrations of Tau on PAECs. Western blot and Real-time PCR method were used to detect the p38 pathway proteins and ICAM-1, VCAM-1 expression levels. Immunofluorescence was used to investigate p38 nuclear displacement situation. The results of MTT showed that the inhibitory effect was gradually increased with increasing concentrations of Tau. Western blot and RT-PCR revealed that the protein and mRNA expression levels of ICAM-1, VCAM-1 were reduced by Tau. Western blot and immunofluorescence showed Tau can inhibit p38 activation. Tau may decrease the expression levels of VCAM-1 and ICAM-1 in endothelial cells induced by hypoxia through MAPK p38 pathway.
Asunto(s)
Células Endoteliales/efectos de los fármacos , Molécula 1 de Adhesión Intercelular/metabolismo , Taurina/farmacología , Molécula 1 de Adhesión Celular Vascular/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Bovinos , Hipoxia de la Célula , Células CultivadasRESUMEN
To discuss the effect of puerarin (Pue) on the proliferation of hypoxia-induced pulmonary artery smooth muscle cells (PASMCs) and discuss whether its mechanism is achieved by regulating reactive oxygen. PASMCs of primarily cultured rats (2-5 generations) were selected in the experiment. MTT, Western blot, FCM and DCFH-DA were used to observe Pue's effect the proliferation of PASMCs. The Western blot was adopted to detect whether ROS participated in Pue's effect in inhibiting PASMC proliferation. The PASMCs were divided into five groups: the normoxia group, the hypoxia group, the hypoxia + Pue group, the hypoxia + Pue + Rotenone group and the hypoxia + Rotenone group, with Rotenone as the ROS blocker. According to the results, under the conditions of normoxia, Pue had no effect on the PASMC proliferation; But, under the conditions of hypoxia, it could inhibit the PASMC proliferation; Under the conditions of normoxia and hypoxia, Pue had no effect on the expression of the tumor necrosis factor-α (TNF-α) among PASMCs, could down-regulate the expression of hypoxia-induced cell cycle protein Cyclin A and proliferative nuclear antigen (PCNA). DCFH-DA proved Pue could reverse ROS rise caused by hypoxia. Both Rotenone and Pue could inhibit the up-regulated expressions of HIF-1α, Cyclin A, PCNA caused by anoxia, with a synergistic effect. The results suggested that Pue could inhibit the hypoxia-induced PASMC proliferation. Its mechanism may be achieved by regulating ROS.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Isoflavonas/farmacología , Miocitos del Músculo Liso/efectos de los fármacos , Arteria Pulmonar/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Animales , Ciclo Celular/efectos de los fármacos , Células Cultivadas , Hipoxia/patología , Masculino , Miocitos del Músculo Liso/fisiología , Antígeno Nuclear de Célula en Proliferación/análisis , Arteria Pulmonar/citología , Ratas , Ratas WistarRESUMEN
To discuss the effect of puerarin (Pue) on the proliferation of hypoxia-induced pulmonary artery smooth muscle cells (PASMCs) and discuss whether the extracellular signal PI3K/AKT pathway was involved in the Pue-induced PASMC apoptosis. With the serum starvation group (SD group) as the control group, the MTT colorimetry method, Annexin V-FITC apoptosis detection kit and Western blot were used to detect Pue's effect on apoptosis of rat PASMCs. The protein immunoblot assay was used to detect whether PI3K/AKT pathway was involved in the inhibition of hypoxia-induced PASMC apoptosis process. The results show that under normoxic conditions, Pue had no effect on PASMC apoptosis; Under hypoxia conditions, Pue can inhibit PASMC apoptosis; Under normoxic and hypoxic conditions, Pue had no effect on TNF-α expression. Pue can reverse hypoxia-induced Bcl-2 (P <0.01), up-regulate it and down-regulated Bax (P <0.01). Under normoxic conditions, Pue had no effect on P-AKT expression. Both LY294002 and Pue can inhibit hypoxia-induced Bcl-2, up-regulation of P-AKT expression and down-regulation of Bax expression. Compared with the hypoxia + Pue group or the hypoxia + LY294002 group, the hypoxia + Pue + LY294002 group showed more significantly changes in Bcl-2, Bax, P-AKT expressions. The results show that, Pue can inhibit the hypoxic-induced PASMC apoptosis, which may be regulated through PI3K/AKT pathway.
Asunto(s)
Apoptosis/efectos de los fármacos , Isoflavonas/farmacología , Miocitos del Músculo Liso/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Arteria Pulmonar/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Animales , Células Cultivadas , Cromonas/farmacología , Morfolinas/farmacología , Arteria Pulmonar/citología , Ratas , Ratas WistarRESUMEN
BACKGROUND: Acquisition of exogenous genetic material is a key event in bacterial speciation. It seems reasonable to assume that recombination of the incoming DNA into genome would be more efficient with higher levels of relatedness between the DNA donor and recipient. If so, bacterial speciation would be a smooth process, leading to a continuous spectrum of genomic divergence of bacteria, which, however, is not the case as shown by recent findings. The goal of this study was todetermine if DNA transfer efficiency is correlated with the levels of sequence identity. RESULTS: To compare the relative efficiency of exogenous DNA acquisition among closely related bacteria, we carried out phage-mediated transduction and plasmid-mediated transformation in representative Salmonella strains with different levels of relatedness. We found that the efficiency was remarkably variable even among genetically almost identical bacteria. Although there was a general tendency that more closely related DNA donor-recipient pairs had higher transduction efficiency, transformation efficiency exhibited over a thousand times difference among the closely related Salmonella strains. CONCLUSION: DNA acquisition efficiency is greatly variable among bacteria that have as high as over 99% identical genetic background, suggesting that bacterial speciation involves highly complex processes affected not only by whether beneficial exogenous DNA may exist in the environment but also the "readiness" of the bacteria to accept it.
Asunto(s)
ADN/genética , Recombinación Genética , Salmonella/genética , Transducción Genética , ADN/metabolismo , ADN Bacteriano/química , ADN Bacteriano/genética , Especiación Genética , Datos de Secuencia Molecular , Salmonella/metabolismo , Análisis de Secuencia de ADN , Transformación BacterianaRESUMEN
OBJECTIVE: To discuss the effect of taurine (Tau) on the proliferation of hypoxia-induced pulmonary artery smooth muscle cells (PASMCs), and study whether the extracellular signal-regulated kinase 1/2 (ERK1/2) signal pathway participated in the Tau-inhibited PASMC proliferation process and the possible molecular mechanism. METHOD: The primary culture was performed for PASMCs in rats. The second to fifth generations were adopted for the experiment. The Tau concentration was 80 mmol x L(-1). The concentration of ERK1/2 blocker (PD98059) was 50 micromol x L(-1). The drug administration time was 24 h. The effect of Tau on the PASMC proliferation was detected by MTT assay, immunofluorescence staining method and western blot under different conditions. The PASMCs were growing were divided into four groups: the normoxia group, the normoxia + Tau group, the hypoxia group and the hypoxia + Tau group. The Western blot was adopted to detect whether the ERK1/2 signal pathway participated in the Tau-inhibited PASMC proliferation process. Subsequently, the PASMCs were divided into five groups: the normoxia group, the hypoxia group, the hypoxia + Tau group, the hypoxia + Tau + PD98059 group and the hypoxia + PD98059 group. RESULT: Hypoxia could induce the PASMC proliferation. Under the conditions of normoxia, Tau had no effect on the PASMC proliferation. Under the conditions of normoxia and hypoxia, Tau had no effect on the expression of the tumor necrosis factor-alpha (TNF-alpha) among PASMCs. Tau could reverse the expression up-regulation of hypoxia-induced proliferative cell nuclear antigen (PCNA) (P < 0.01) and Cyclin A (Cyclin A) (P < 0. 05). Under the conditions of normoxia, Tau had no effect on the expression of phosphoryl extracellular signal-regulated kinase 1/2 (p-ERK1/2). Hypoxia could up-regulate the p-ERK1/2 expression (P < 0.01). Tau could reverse the up-regulation of the hypoxia-induced p-ERK1/2 expression(P < 0.01). Both PD98059 and Tau could inhibit the up-regulated expressions of PCNA, Cyclin A and p-ERK1/2. According to the comparison between the single addition of Tau and PD98059 under conditions of hypoxia, the hypoxia + Tau + PD98059 group showed more significant down-regulation in the expressions of PCNA, Cyclin A and p-ERK1/2. CONCLUSION: Tau could inhibit the hypoxia-induced PASMC proliferation, and may regulate it through ERK1/2 pathway.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Miocitos del Músculo Liso/citología , Oxígeno/metabolismo , Arteria Pulmonar/citología , Taurina/farmacología , Animales , Hipoxia de la Célula/efectos de los fármacos , Células Cultivadas , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/metabolismo , Arteria Pulmonar/fisiopatología , Ratas , Ratas Wistar , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
DT104 emerged as a new branch of Salmonella typhimurium with resistance to multiple antimicrobials. To reveal some general genomic features of DT104 for clues of evolutionary events possibly associated with the emergence of this relatively new type of this pathogen, we mapped 11 independent DT104 strains and compared them with non-DT104 S. typhimurium strains. We found that all 11 DT104 strains contained three insertions absent in non-DT104 strains, i.e., the previously reported ST104, ST104B and ST64B. However, SGI-1, a genomic island known to be responsible for DT104 multidrug resistance, was not present in all DT104 strains examined in this study: one DT104 strain did not contain SGI-1 but carried a 144 kb plasmid, suggesting possible evolutionary relationships between the two DNA elements in the development of antimicrobial resistance.
Asunto(s)
Genoma Bacteriano/genética , Genómica , Infecciones por Salmonella/microbiología , Salmonella typhimurium/genética , Evolución Biológica , Mapeo Cromosómico , ADN Bacteriano/química , ADN Bacteriano/genética , Desoxirribonucleasas de Localización Especificada Tipo II , Farmacorresistencia Bacteriana Múltiple/genética , Endodesoxirribonucleasas , Reordenamiento Génico , Islas Genómicas/fisiología , Plásmidos/genética , Especificidad de la EspecieRESUMEN
Salmonella enterica serovar Pullorum is a chicken-adapted pathogen, causing pullorum disease. Its strict host adaptation has been suspected to result in gene decay. To validate this hypothesis and identify the decayed genes, we sequenced the complete genome of S. Pullorum RKS5078. We found 263 pseudogenes in this strain and conducted functional analyses of the decayed genes.
Asunto(s)
Genoma Bacteriano , Enfermedades de las Aves de Corral/microbiología , Salmonelosis Animal/microbiología , Salmonella enterica/genética , Animales , Proteínas Bacterianas/genética , Secuencia de Bases , Pollos , Datos de Secuencia Molecular , Salmonella enterica/aislamiento & purificaciónRESUMEN
Chemical investigation of the ethanol extract of the aerial parts of Hydrangea macrophylla collected in the Sichuan Province of China resulted in the isolation of two new cyanogenic glucosides. Their structures were elucidated as [(2R)-2-ß-D-glucopyranosyloxy)-2-(3,4-dimethoxy-phenyl)] acetonitrile (1) and {(2R)-2-[α-D-glucopyranosyl(1-->6)ß-D-glucopyranosyloxy]-2-(3-hydroxy-4-methoxy-phenyl)}acetonitrile (2) on the basis of extensive spectroscopic analysis (1D, 2D NMR and HRESIMS) and chemical studies.
Asunto(s)
Glucósidos/aislamiento & purificación , Glicósidos/aislamiento & purificación , Hydrangea/química , Componentes Aéreos de las Plantas/química , Hojas de la Planta/química , Espectroscopía de Resonancia Magnética , Estructura MolecularRESUMEN
OBJECTIVE: The present study investigates the influence of Qingkailing injection on rat liver CYP1A2 and CYP2D6 activity in vivo and in vitro, respectively. METHOD: We employed HPLC to measure the metabolites of caffeine in the whole blood and calculated the ratio be between the metabolite and caffeine, which was used as index to evaluate the effect of Qingkailing injection on rat CYP1A2 activity in vivo; We also detected the CYP1A2 and CYP2D6 activity in microsomal reconstituted system by analysis of phenacetin metabolism and dextromethorphan metabolism with HPLC. RESULT: The metabolism of caffeine in treated groups was (15.9 +/- 3.8)%, (14.5 +/- 1.8)%, (12.3 +/- 1.2)%, with different concentration of Qingkailing injection (0.15, 0.3, 0.6 mL x kg(-1)) compared with (16.8 +/- 5.9)% in the control group, which was no significant difference among groups. In rat liver microsomal reconstituted system, Qingkailing injection has no inhibitory effect on CYP2D6 activity while the group with high dose has inhibitory effect on rat CYP1A2. CONCLUSION: Qingkailing injection has no inhibitory effect on rat CYP1A2 and CYP2D6 in vivo and in vitro.
Asunto(s)
Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2D6/metabolismo , Medicamentos Herbarios Chinos/farmacología , Microsomas Hepáticos/efectos de los fármacos , Animales , Cafeína/sangre , Cafeína/metabolismo , Cromatografía Líquida de Alta Presión/métodos , Relación Dosis-Respuesta a Droga , Combinación de Medicamentos , Medicamentos Herbarios Chinos/administración & dosificación , Medicamentos Herbarios Chinos/aislamiento & purificación , Inyecciones , Masculino , Microsomas Hepáticos/enzimología , Plantas Medicinales/química , Distribución Aleatoria , Ratas , Ratas WistarRESUMEN
OBJECTIVES: Angiogenesis promotes neurobehavioral recovery after cerebral ischemic stroke. 15(S)-hydroxyeicosatetraenoic acid (15-HETE) is one of the major metabolites of arachidonic acid by 15-lipoxygenase (15-LO) and stimulates the production of vascular endothelial growth factor (VEGF), thus, inducing autocrine-mediated angiogenesis. The present study aimed to investigate the role of 15-LO/15-HETE system on VEGF expression and angiogenesis in brain ischemia. METHODS: Rat cerebral arterial vascular endothelial cells were used to set up a cell injury model of oxygen-glucose deprivation and reoxygenation (OGD/R), mimicking a condition of brain ischemia. A mouse model of middle cerebral artery occlusion (MCAO) was established. RESULTS: Oxygen-glucose deprivation increased cellular expression of 15-LO-1 and VEGF. Transfection of 15-LO-1 siRNA depleted cells of 15-LO-1, and sequentially induced downregulation of VEGF expression; while, incubation of 15-HETE increased the expression of VEGF. Incubation of 15-HETE attenuated the reduction in cell viability induced by oxygen-glucose deprivation, and promoted cell migration, while transfection of 15-LO-1 siRNA showed an opposite effect. In animal experiments, the density of microvessels in hypoxic regions of brains was significantly increased after MCAO, while intracerebroventricular delivery of 15-LO-1 siRNA significantly reduced the density of microvessels, and downregulates VEGF expression. DISCUSSION: The results indicate that the 15-LO-1/15-HETE system promotes angiogenesis in ischemic brains by upregulation of VEGF, representing a potential target for improving neurobehavioral recovery after cerebral ischemic stroke.
Asunto(s)
Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/metabolismo , Ácidos Hidroxieicosatetraenoicos/metabolismo , Regulación hacia Arriba/fisiología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Inductores de la Angiogénesis/uso terapéutico , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Isquemia Encefálica/patología , Movimiento Celular , Células Cultivadas , Arterias Cerebrales/citología , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Glucosa/deficiencia , Ácidos Hidroxieicosatetraenoicos/genética , Ácidos Hidroxieicosatetraenoicos/uso terapéutico , Hipoxia/tratamiento farmacológico , Hipoxia/metabolismo , Ratones , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , ARN Interferente Pequeño/uso terapéutico , Ratas , Transducción de Señal , Factores de Tiempo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
We have reported that hypoxia increases the activation of 15-lipoxygenase (15-LO), which converts arachidonic acid (AA) into 15-hydroxyeicosatetraenoic acid (15-HETE) in small pulmonary arteries (PAs). Through inhibition of Kv channels, 15-HETE causes more robust concentration-dependent contraction of PA rings from the hypoxic compared to the normoxic controls. However, the subtypes of Kv channels inhibited by 15-HETE are incompletely understood. The aim of the present study was to identify the contribution of Kv3.4 channel in the process of pulmonary vasoconstriction induced by 15-HETE using the tension studies of PA rings from rat with Kv3.4 channel blocker in tissue bath; to explore the role of vascular endothelium in15-HETE-induced pulmonary vasoconstriction through denuded endothelia of PA rings; and to define the downregulation of 15-HETE on the expression of Kv3.4 channel in cultured pulmonary artery smooth muscle cells (PASMCs) with RT-PCR and Western blot. In the present study, healthy Wistar rats were divided randomly into two groups: Group A with normal oxygen supply and group B with hypoxia. Six days later, the rats were killed. Pulmonary artery rings were prepared for organ bath experiments. Firstly, different concentrations of 15-HETE (10~1 000 nmol/L) were added to the Krebs solution. The isometric tension was recorded using a four-channel force-displacement transducer. Then Kv3.4 channel blocker, 100 nmol/L BDS-I, was added, followed by adding 1 mumol/L 15-HETE, and the isometric tension was recorded. Furthermore, RT-PCR and Western blot were employed to identify the influence of 15-HETE on the expression of Kv3.4 channel in cultured rat PASMCs.The results showed the PA tension was significantly increased both in groups A and B by 15-HETE in a concentration-dependent manner (P<0.05), especially in group B (P<0.05 compared to control); denuded endothelia enhanced 15-HETE concentration-related constrictions in rat PA rings; Kv3.4 channel blocker, BDS-I, significantly decreased the PA ring constriction induced by 15-HETE (P<0.05); the expressions of Kv3.4 mRNA and protein in rat PASMCs were significantly downregulated by 15-HETE (P<0.05). Based on all the information above, we conclude that Kv3.4 channel is involved in vasoconstriction induced by 15-HETE in rat PAs.
Asunto(s)
Ácidos Hidroxieicosatetraenoicos/farmacología , Músculo Liso Vascular/citología , Arteria Pulmonar/fisiopatología , Canales de Potasio Shaw/metabolismo , Vasoconstricción/efectos de los fármacos , Animales , Células Cultivadas , Femenino , Hipertensión Pulmonar/fisiopatología , Hipoxia/fisiopatología , Masculino , Músculo Liso Vascular/patología , Arteria Pulmonar/citología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Canales de Potasio Shaw/antagonistas & inhibidores , Canales de Potasio Shaw/genéticaRESUMEN
AIM: To observe the effect of subtypes of Kv channels in rat pulmonary artery smooth muscle cells (PASMCs) on the process of pulmonary vasoconstriction induced by 15-HETE. METHODS: In the present study, ring of rabbit PA with specific Kv channel blockers were employed to functionally identify certain channel subtypes that took part in the process of 15-HETE induced pulmonary vasoconstriction; RT-PCR and Western blotting analysis were also used to measure the expression of subtypes of Kv in PASMCs exposed to 15-HETE,chronic hypoxia. RESULTS: Blocking of Kv1. 1, Kv1. 2, Kv1. 3 and Kv1. 6 channels did not affect 15-HETE induced vasoconstriction in normoxic rats; 15-HETE did not affect expression of Kv1. 1 and Kv1. 2 channels; 15-HETE significantly downregulated the expression of mRNA and protein of Kv1. 5 and Kv2. 1 in rat PASMCs. CONCLUSION: The results suggested that hypoxia may block Kv1. 5 and Kv2. 1 channels via 15-HETE mediated mechanism, leading to decrease numbers of functional Kv1. 5 and Kv2. 1 channels in PASMCs, leading to PA vasoconstriction.
Asunto(s)
Ácidos Hidroxieicosatetraenoicos/farmacología , Miocitos del Músculo Liso/metabolismo , Arteria Pulmonar/fisiopatología , Vasoconstricción/efectos de los fármacos , Animales , Hipoxia de la Célula , Células Cultivadas , Hipoxia/fisiopatología , Canal de Potasio Kv1.5/biosíntesis , Canal de Potasio Kv1.5/genética , Masculino , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/citología , Canales de Potasio con Entrada de Voltaje/antagonistas & inhibidores , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas Wistar , Canales de Potasio Shab/biosíntesis , Canales de Potasio Shab/genéticaRESUMEN
Hypoxia-induced 15-hydroxyeicosatetraenoic acid (15-HETE) is an essential mediator to constrict pulmonary arteries (PA). The signaling pathway involved in 15-HETE-induced PA vasoconstriction remains obscure. The aim of the present study was to test the hypothesis that hypoxic PA constriction induced by 15-HETE was possibly regulated by the extracellular signal-regulated kinase-1/2 (ERK1/2) pathway. PA ring tension measurement, Western blot and immunocytochemistry were used in the study to determine the possible role of ERK1/2 in 15-HETE-induced PA vasoconstriction. The organ bath for PA rings tension study was employed. Adult male Wistar rats were raised in hypoxic environment with fractional inspired oxygen (FIO2, 0.12) for 9 d. PA 1~1.5 mm in diameter were dissected and cut into 3 mm long rings for tension study. ERK1/2 up-stream kinase (MEK) inhibitor PD98059, which blocks the activation of ERK1/2, was used. The results showed that pretreatment of PD98059 significantly blunted 15-HETE-induced PA vasoconstrictions in the rings from hypoxic rat. Moreover, in endothelium-denuded rings, PD98059 also significantly attenuated 15-HETE-induced vasoconstriction. Phosphorylation of ERK1/2 in pulmonary arterial smooth muscle cells (PASMCs) of rat was enhanced evidently when stimulated by 15-HETE. Thus, the data suggest that ERK1/2 signaling pathway is involved in 15-HETE-induced hypoxic pulmonary vasoconstriction.
Asunto(s)
Ácidos Hidroxieicosatetraenoicos/farmacología , Hipoxia/fisiopatología , Sistema de Señalización de MAP Quinasas/fisiología , Arteria Pulmonar/fisiopatología , Vasoconstricción , Animales , Flavonoides/farmacología , Ácidos Hidroxieicosatetraenoicos/antagonistas & inhibidores , Masculino , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/efectos de los fármacos , Arteria Pulmonar/citología , Arteria Pulmonar/efectos de los fármacos , Ratas , Ratas Wistar , Vasoconstricción/efectos de los fármacosRESUMEN
15-hydroxyeicosatetraenoic acid (15-HETE) plays an important role in hypoxia-induced pulmonary vasoconstriction. Release of nitric oxide (NO) is apparently decreased and activity of endothelial nitric oxide synthase (eNOS) is impaired in chronic hypoxia. However, little is known whether 15-HETE contributes to eNOS/NO pathway in the constriction induced by 15-HETE. We examined the response of rat pulmonary artery (PA) rings to 15-HETE, the production of NO, total eNOS expression and the phosphorylation of eNOS in bovine pulmonary artery endothelial cells (BPAECs) stimulated by 15-HETE. Rat PA rings were divided into three groups: endothelium intact group, endothelium denuded group, and nitro-L-arginine methyl ester (L-NAME, 0.1 mmol/L, an inhibitor of eNOS) group. Constrictions to 15-HETE were significantly enhanced in endothelium denuded group and L-NAME group (both P< 0.05 vs endothelium intact group, n= 9); BPAECs were incubated in different conditions to test nitrite production by Greiss method. Nitrite production was significantly reduced by 1 mumol/L 15-HETE (P<0.05), and increased by the lipoxygenase inhibitors, 10 mumol/L cinnamyl 3,4- dihydroxy-[alpha] -cyanocinnamate (CDC, P< 0.05) and 0.1 mmol/L nordihydroguiairetic acid (NDGA, P< 0.01 ); Western blot analysis of extracts from BPAECs incubated with 15-HETE in different time was carried out to test total eNOS expression, and the expression was changed unobviously. Immunoprecipitation (IP) and Western blot analysis of cell extracts from BPAECs treated with 2 mumol/L 15-HETE in different length of time were accomplished, using phospo-eNOS-threonine 495 (Thr495, an inhibitory site) antibody for IP, and eNOS or 15-lipoxygenase (15-LO) antibodies for Western blot. 15-HETE depressed eNOS activity by increasing the levels of phospho-eNOS-Thr 495. The data suggest that eNOS/NO pathway is involved in PA constrictions induced by 15-HETE and that 15-HETE depresses eNOS activity by phosphorylation in Thr495 site. The protein interaction between phospho-eNOS (Thr495) and 15-LO is discovered for the first time.
Asunto(s)
Endotelio Vascular/efectos de los fármacos , Ácidos Hidroxieicosatetraenoicos/farmacología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Arteria Pulmonar/enzimología , Arteria Pulmonar/fisiología , Animales , Bovinos , Regulación hacia Abajo/efectos de los fármacos , Endotelio Vascular/citología , Endotelio Vascular/enzimología , Técnicas In Vitro , Masculino , Arteria Pulmonar/citología , Ratas , Ratas WistarRESUMEN
This study investigated the role of 15-hydroxyeicosatetraenoic acid (15-HETE) in rabbit pulmonary arterial smooth muscle cells (PASMCs) under hypoxia by using organ bath and whole cell patch-clamp techniques. Neonatal rabbits born into normoxic environment were transferred after first feeding into normal and hypoxic environments with respectively 0.21 and 0.12 fractional inspired oxygen (FiO2). Pulmonary arteries were extracted after 9 d and cut into rings 1.0 approximately 1.5 mm in length for organ bath experiments. Whole cell patch-clamp technique was used to measure the potassium current in the freshly dispersed rabbit PASMCs. The results showed that 15-HETE-induced vasoconstriction was blocked by 4-aminopyridine (5 mmol/L), a Kv channel blocker. The K(ATP) channel blocker glyburide (1 micromol/L) and the BKCa channel blocker tetraethylammonium (10 mmol/L) did not abolish this vasoconstriction. 15-HETE decreased the whole-cell voltage-gated K+ current in the PASMCs. These findings demonstrate that hypoxia blocks Kv channels through a 15-HETE mediated mechanism, leading to PA vasoconstriction.
Asunto(s)
Ácidos Hidroxieicosatetraenoicos/farmacología , Músculo Liso Vascular/metabolismo , Bloqueadores de los Canales de Potasio/farmacología , Vasoconstrictores/farmacología , Animales , Animales Recién Nacidos , Hipoxia de la Célula , Células Cultivadas , Femenino , Contracción Muscular/efectos de los fármacos , Músculo Liso Vascular/citología , Músculo Liso Vascular/fisiología , Canales de Potasio con Entrada de Voltaje/efectos de los fármacos , Embarazo , Arteria Pulmonar/citología , Arteria Pulmonar/metabolismo , Arteria Pulmonar/fisiología , ConejosRESUMEN
BACKGROUND: The bacterial genus Salmonella contains thousands of serotypes that infect humans or other hosts, causing mild gastroenteritis to potentially fatal systemic infections in humans. Pathogenically distinct Salmonella serotypes have been classified as individual species or as serological variants of merely one or two species, causing considerable confusion in both research and clinical settings. This situation reflects a long unanswered question regarding whether the Salmonella serotypes exist as discrete genetic clusters (natural species) of organisms or as phenotypic (e.g. pathogenic) variants of a single (or two) natural species with a continuous spectrum of genetic divergence among them. Our recent work, based on genomic sequence divergence analysis, has demonstrated that genetic boundaries exist among Salmonella serotypes, circumscribing them into clear-cut genetic clusters of bacteria. METHODOLOGIES/PRINCIPAL FINDINGS: To further test the genetic boundary concept for delineating Salmonella into clearly defined natural lineages (e.g., species), we sampled a small subset of conserved genomic DNA sequences, i.e., the endonuclease cleavage sites that contain the highly conserved CTAG sequence such as TCTAGA for XbaI. We found that the CTAG-containing cleavage sequence profiles could be used to resolve the genetic boundaries as reliably and efficiently as whole genome sequence comparisons but with enormously reduced requirements for time and resources. CONCLUSIONS: Profiling of CTAG sequence subsets reflects genetic boundaries among Salmonella lineages and can delineate these bacteria into discrete natural clusters.