共查询到20条相似文献，搜索用时 31 毫秒
1.
AIM: To investigate the interaction of Ca2+-sensing proteins, stromal interaction molecule 1 (STIM1) and calcium release-activated calcium channel protein 1 (Orai1), in Ca2+-sensing receptor (CaSR)-mediated extracellular Ca2+ influx and production of nitric oxide (NO). METHODS: Human umbilical vein endothlial cells (HUVECs) were incubated with CaSR agonist spermine [activating store-operated calcium channels (SOC) and receptor-operated calcium channels (ROC)] alone or combined with CaSR negative allosteric modulator Calhex 231+ROC analogue TPA (activating ROC, blocking SOC), protein kinase C (PKC) inhibitor Ro 31-8220, or PKCα/β1 selective inhibitor Go 6976 (activate SOC, blocking ROC). The protein expression of STIM1 and Orai1 was determined by the method of immunofluorescence. The interaction between STIM1 and Orai1 was examined by co-immunoprecipitation. The second to third passages of HUVECs were divided into STIM1 and Orai1 short hairpin RNA group (shSTIM1+shOrai1 group), vehicle-STIM1+vehicle-Orai1 group and control group, and then incubated with the 4 different treatments above. The intracellular Ca2+ concentration ([Ca2+]i) was detected using the fluorescent Ca2+ indicator Fura-2/AM. The production of NO was also determined by DAF-FM DA fluorescent probe. RESULTS: The protein expression of STIM1 and Orai1 was located in the cytoplasm. Compared with control group, the localization of STIM1 and Orai1 in the cytoplasm was reduced after the HUVECs were incubated with Calhex 231+TPA, Ro 31-8220 or Go 6976, and the interaction of STIM1 and Orai1 was decreased significantly. The [Ca2+]i and the net NO fluorescence intensity in shSTIM1+shOrai1 group were significantly reduced after the 4 different treatments (P<0.05). CONCLUSION: STIM1 and Orai1 are components of SOC and ROC in store-and receptor-operated Ca2+ entry and NO generation. 相似文献
2.
AIM: To investigate the role of reactive oxygen species (ROS) and calcium overload in the apoptosis of MC3T3-E1 cells induced by high glucose. METHODS: Cultured mouse skull bone-derived osteoblast cell line MC3T3-E1 was treated with high concentration of D-glucose to induce apoptosis. The proliferation of MC3T3-E1 cells was detected by MTT assay after treated with different concentrations of D-glucose for 24 h and 48 h. The apoptotic rate and the intracellular levels of calcium and ROS were also measured after the cells were treated with high glucose (35 mmol/L) for 24 h. RESULTS: After high glucose treatment, the cell proliferation was inhibited. The early apoptosis and total cell death increased to (24.16?3.53)% and (63.74?4.32)%,respectively. High glucose treatment significantly increased intracellular levels of ROS and Ca2+. The increased apoptotic rate was reduced by addition of antioxidant N-acetylcysteine and calcium chelator BAPTA-AM. Inhibition of store-operated Ca2+ channels by La3+ also decreased the intracellular level of Ca2+ and cell apoptosis induced by high glucose. CONCLUSION: High glucose increases intracellular ROS level and the release of Ca2+ through the store-operated Ca2+ channels, thus resulting in intracellular Ca2+ overload and leading to apoptosis of osteoblasts. 相似文献
3.
AIM: The changes of myocardial nuclear membrane Ca2+ -ATPase function was investigated in ischemia/reperfusion injury. METHODS: The model of myocardial ischemia/reperfusion injury was established in rats. Myocardial nuclei were purified with sucrose density centrifugation, the activity of Ca2+ -ATPase was measured and calcium uptake was assayed with [45 Ca2+ ] . RESULTS: Plasma levels of malondialdehyde (MDA) and free fatty acid (FFA) in myocardial ischemia/reperfusion injury increased significantly( P<0.01 vs control). Ca2+ -ATPase activity and [45 Ca2+ ] uptake was lower than normal at below 10 μmol/L, while higher at 50 μmol/L. CONCLUSION:These data indicate dysfunction of nuclear menbrane calcium pump and [45 Ca2+ ] uptake function in myocardial ischemia/reperfusion injury. 相似文献
4.
AIM: To investigate the changes of cytosolic free calcium concentration([Ca2+]i) and expression of Bcl-2 in HL-60 cells treated by 6F isolated from Pteris semipinnata L.(PSL), and to discuss the relations between calcium ion and cytotoxicity and DNA fragment induction effects of 6F. METHODS: HL-60 cells were used as in vitro model. [Ca2+]i was measured on fluorescent spectrophotometry using Fura-2/AM as Ca2+ indicator. Bcl-2 expressing level was measured by flow cytometry. Tetrazolium salt(MTT) and diphenylamine staining methods were applied for cytotoxicity assay and DNA fragmentation detection, respectively. RESULTS: [Ca2+]i increased obviously in a dose and time dependent manner after treated HL-60 cells with 6F. 6F decreased the expressing level of Bcl-2. Adding 2 mmol/L Ca2+ to the medium, or 1 mmol/L EDTA to chelate Ca2+, or 4 μmol/L calcium ionophore A 23187 to increase the concentration of cytosolic Ca2+, the DNA fragment induction by 6F was not affected, whereas the cytotoxicity of 6F was enhanced. 250 μmol/L Zn2+ attenuated the DNA fragment induction, and the cytotoxicity of 6F against HL-60 cells was enhanced significantly. CONCLUSION: It was speculated that the decreased expressing of Bcl-2 by compound 6F was related to increased [Ca2+]i in HL-60 cells, and DNA fragment induction was possibly catalyzed by Ca2+ - independent DNase. 相似文献
5.
AIM: To determine whether nuclear Ca2+ is independently regulated from the cytosolic Ca2+ and nuclear Ca2+ oscillation induced by many modulating factors in cultured rat neonatal myocytes and its mechanism. METHODS: Rat neonatal cardiac myocytes were cultured, and fluo-4/AM was loaded as calcium probe. The changes of cytosolic and nuclear Ca2+ were observed by confocal laser microscopy. RESULTS: Calcium fluorescent intensity oscillated slightly in myocardiocytes and the average intensity was much higher in the nucleus than that in the cytosole. Ca2+ oscillation in nucleus and cytosole induced by norepinephrine, isoproperenol, ATP were completely blocked by Ca2+-ATPase antagonist thapsigargin (10-6 mol/L),L-type Ca2+ channel blocker verapermil (500 μmol/L) and KCl (20 mmol/L). Ca2+-ATPase antagonist thapsigargin completely blocked the propagation of Ca2+ waves and simutaneouly induced a temporary Ca2+ increase followed by a magnificient drop and loss of response to norepinephrine. CONCLUSIONS: The results suggested that generation and maintenance of calcium oscillation both in cytosole and nucleus depended on extracellular Ca2+ influx, membrane potential, Ca2+ release and uptake of cytosolic and nuclear calcium stores. The difference between cytosolic calcium and nuclear calcium indicated that calcium regulating system relatively independent of cytosole may exist in nucleus. 相似文献
6.
AIM: To explore the mechanism of ET-1, NO and PGI2 release from coronary artery endothelial cells(CAEC) induced by acute hypoxia. METHODS: Bovine coronary artery endothelial cells were cultured and [45 Ca2+] was used to investigate the difference of calcium uptake between normoxia group and hypoxia group(3% O2). The contents of ET-1, NO and PGI2 in media of normoxia group, hypoxia group and hypoxia + verapamil group were measured 24 h after hypoxia. RESULTS: [ 45 Ca2+] uptake by CAEC in hypoxia group was 1.9 times more than normoxia group(P< 0.01). Hypoxia + verapamil group released more PGI2, ET-1 and less NO than hypoxia group(P< 0.05). CONCLUSION: Changes of ET-1, NO and PGI2 releases during hypoxia may be caused by the inflow of Ca2+ into coronary artery endothelial cells. 相似文献
7.
AIM:To investigate different intracellular concentration of Ca2+ in uterine myometrial cells at term and non-term.METHODS:The living cells suspensions were made to measure intracellular Ca2+ concentrations after stainned by calcium fluorescent indicator Fluo-3 AM, then examined by laser scanning confocal microscope (LSCM).RESULTS:Intracelluar Ca2+ showed very stronger red positive signal in myometrial cells at term than that in non-term cells. [Ca2+]i were (35±8.1) nmol/L at non term and (75±7.3) nmol/L at term, which had significant difference compared with each other (P＜0.05).CONCLUSION:Increase in [Ca2+]i in myometrial cells might play a very important role labor. 相似文献
8.
AIM: To investigate intracellular free calcium ( [Ca2+]i ) alterations in hypothalamus of febrile rabbits induced by endotoxin (ET), and compare with the effect of ET and IL-1β on i in hypothalamic neurocytes from normothermia rabbits. METHOD: The concentration of [Ca2+]i was determined by using spectrofluorometer and fluorescent Ca2+ probe fura-2 /Am. RESULTS: 1. A minute dose of ET (2 ng/mL) induced a significant rise in [Ca2+]i in hypothalamic neurocytes from normothermia rabbits. The rise in [Ca2+]i in hypothalamic neurocytes from febrile rabbits induced by intravenous injection of ET was also observed. 2. In hypothalamic neurocytes from normotheria rabbits, IL-1β failed to affect [Ca2+]i at concentrations of 100, 500, 1 000ng/mL, respectively. CONCLUSION:The action site of low concentration of calcium that plays a regulatory role during fever seems unlikely to be in cytosolic compartment of hypothalamic neurons. The change of [Ca2+]i in hypothalamic neurocytes by ET can not be considered the direct effect of IL-1β. 相似文献
9.
AIM: To study the roles and mechanisms of ERKs and intracellular free calcium in cardiomyocyte hypertrophic response induced by endothelin-1(ET-1). METHODS: (1) Neonatal rat cardiomyocyte hypertrophic response was assayed by measuring cell surface area and protein content; (2) ERKs activity was determined by Whatman Paper Filter method; (3) Intracellular free calcium concentration ([Ca2+]i) was measured using Fura-2/AM as a fluorescent indicator. RESULTS:(1)ET-1 could increase total protein production,surface area,ERKs activity and[Ca2+]i in cultured cardiomyocyte in dose-dependent manner at concentrations ranging from 10-9to 10-7mol/L.And this effect could be abolished by BQ123,an antagonist of ETA receptor,partly inhibited by PTX,but not by BQ788,an antagonist of ETB receptor.(2)The activation of ERKs and the increase of[Ca2+]i induced by ET-1 were obviously in hibited by PD98059,a selective ERKs kinase inhibitor,and nifedipine,a calcium channel blocker,respectively.Both antagonists partialy inhibited ET-1-stimulated cardiomyocyte hypertrophic response.(3)Staurosporine,a selective PKC inhibitor,could inhibit ET-1-stimulated cardiomyocyte hypertrophic response and increase of[Ca2+]i,but not af ect the activation of ERKs.CONCLUSION: Cardiomyocyte hypertrophic response induced by ET-1 is mediated by ETA receptor coupled to PTX-sensitive G-protein, which involves at least two signalling pathways: PKC-mediated increase of [Ca2+]i , and PKC-independent activation of ERKs. 相似文献
10.
Protective effect of quercetin on ER stress-related apoptosis induced by thapsigargin in macrophages
AIM: To investigate the effects and possible mechanisms of quercetin (Que) on endoplasmic reti-culum stress (ERS)-related apoptosis induced by thapsigargin (TG) in RAW264.7 cells. METHODS: ER stress of RAW264.7 cells were induced by TG at concentration of 1 μmol/L for 24 h. After treated with different concentrations of Que (80, 120 and 160 μmol/L), the cell viability was determined by MTT assay.The apoptotic rate and the changes of intracellular Ca2+ concentration ([Ca2+]i) were determined by flow cytometry, and the cell apoptotic morphology was observed under laser scanning confocal microscope.The protein levels of glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP) were detected by Western blotting. The effect of Que on GRP78 and CHOP induced by TG with phosphatidylinositol 3-kinase (PI3K) inihibitor LY294002 at concentration of 15 nmol/L was measured by Western blotting. RESULTS: Que suppressed ER stress-related injury induced by TG in RAW264.7 cells. Compared with TG group, the cell viability increased (P<0.05), apoptotic rate and [Ca2+]i decreased (P<0.05) and the changes of apoptotic morphology were alleviated. The increase in GRP78 and CHOP induced by TG as an ER stress marker was suppressed by Que (P<0.05). The suppressive effect of Que on GRP78 and CHOP was reproduced by LY294002 (P<0.05), but they failed to exhibit additive suppression. CONCLUSION: Que suppresses the ER stress induced by TG in RAW264.7 cells. The protective effect may be related to its suppression on PI3K signaling pathway. 相似文献
11.
AIM:To investigate the effect of lipopolysaccharide (LPS) priming on macrophage(MΦ).METHODS:Macrophage cell line RAW264.7 were pretreated with or without LPS for 1 h, then challenged with PMA, or LPS, muramyl dipeptide(MDP), Zymosan, formyl-methionyl-leucyl-phenylalanine(FMLP) for 1 h. O2- production in supernatants and intracellular free calcium([Ca2+]i) were measured, and changes in [Ca2+]i and LPS induced O2- production were compared.RESULTS:LPS pretreatment significantly increased O2- production in RAW264.7 cells challenged with the stimuli, and in a certain extent, both O2- production and increase of resting intracellular [Ca2+]i were dose- and time-dependent on LPS pretreatment.Furthermore, the peak [Ca2+]i was significantly higher in LPS pretreated groups than that of LPS unpretreated groups when challenged with PMA. Pretreatment with Ca2+ inophore A23187 mimicked the LPS priming effects on O2- production, but pretreatment with Ca2+ chelator BAPTA and EGTA blocked this priming effect.CONCLUSION:LPS-induced MΦ priming effect on O2- production is dependent on elevation of resting intracellular [Ca2+]i. 相似文献
12.
AIM:We examined the effect of interleukin-2 (IL-2) on calcium handling of rat cardiomyocytes. METHODS:The effects of steady state and transient changes in stimulus frequency on the intracellular calcium transient were investigated in the isolated ventricular myocytes with spectrofluorometry technique. RESULTS: Under the steady state (0.2 Hz), IL-2 at 2×105U/L decreased the peak [Ca2+] i and amplitude of the [Ca2+]i transient, increased the diastolic calcium level, and prolonged the decay of the calcium transient. At 1.25 mmol/L of extracellular [Ca2+], when increasing the stimulus frequency from 0.2 to 1.0 Hz, diastolic calcium level and peak [Ca2+] i as well as the amplitude of the transient were increased. The positive frequency relationship was blunted in the IL-2-treated myocytes and this was not normalized by increasing extracellular [Ca2+] to 2.5 mmol/L. The caffeine induced Ca2+ release was increased with increase in stimulus frequency. IL-2 inhibited the frequency relationship of caffeine induced Ca2+ release. The restitution was not different between control and IL-2 groups at the 1.25 mmol/L of extracellular [Ca2+], which was slowed in IL-2-treated myocytes when the extracellular [Ca2+] was increased to 2.5 mmol/L. CONCLUSIONS:It is concluded that the blunted frequency response of IL-2-treated myocytes was resulted from the decrease in SR Ca2+ release, which was related to depression of SR function. Despite the evidence of depressed SR Ca2+ uptake, the restitution of calcium transient at 1.25 mmol/L of extracellular remains unchanged, which maybe due to the increase in the Na+/Ca2+ exchanger activity. 相似文献
13.
AIM:To study the effects of oxidized high-density lipoprotein (oxHDL) on the expression of monocyte chemoattractant protein-1(MCP-1) and intercellular adhesion molecule-1(ICAM-1) and intracellular free calcium concentration ([Ca2+]i) level in cultured human umbilical venous endothelial cells(HUVECs). METHODS:The MCP-1 protein content in the medium of conditioned HUVEC was measured by ELISA, and the ICAM-1 on HUVECs was detected by indirect immunofluorescence, and [Ca2+]i was determined by Fluo-3/AM, the injury of cells was observed by scanning electron microscopy (SEM).RESULTS:oxHDL could induce the expression of MCP-1 and ICAM-1 in HUVECs. In oxHDL group (HUVECs were incubated with 100 mg protein/L oxHDL for 24 h), the levels of MCP-1, ICAM-1 and [Ca2+]i increased by 160%, 60% and 70% respectively compared with the control group (P＜0.01). When HUVECs were incubated with 300 mg protein/L oxHDL for 24 h, cells were injured obviously. CONCLUSION:By inducing the expression of ICAM-1 and MCP-1 in endothelial cells, oxHDL may promote monocyte-endothelium adhesion and monocyte migration to intima, it may promote atherosclerosis as oxidized low-density lipoprotein (oxLDL). 相似文献
14.
AIM: To investigate the role of potassium channels in the regulation of intracellular free calcium concentration ( [Ca2+]i) of pulmonary artery smooth muscle cells (PASMCs) in rats. METHODS: The fluorescence Ca2+ indicator Fura-2/AM was used to observe [Ca2+]i of rat PASMCs in normal and chronic hypoxic condition. The influences of potassium channels on PASMCs proliferation were assessed by MTT assay. RESULTS: 1. In normoxic condition, [Ca2+]i was (156.91±8.60) nmol/L, and in hypoxic condition, [Ca2+]i was (294.01±16.81) nmol/L. 2. In normoxic condition, the voltage-dependent K+-channel antagonist 4-aminopyridine (4AP), but not the Ca2+-activated K+-channel antagonist tetraethylammonium (TEA) and the ATP-sensitive K+-channel antagonist glibenclamide (Glib) increased [Ca2+]i. 3. In hypoxic condition, 4AP and TEA caused the rise in [Ca2+]i , but Glib had no effect on [Ca2+]i. 4. MTT assay showed that 4AP increased the value of absorbing light degree (A value) in normoxic and hypoxic condition (0.582±0.062,0.873±0.043,respectively, P＜0.01), TEA increased A value only in hypoxic condition, and Glib had no effect on the proliferation of PASMCs. CONCLUSIONS: KV plays an important role in the regulation of [Ca2+]i and proliferation of PASMCs. KCa serves as distinct responsive roles in the regulation of proliferation of PASMCs in hypoxic condition. KATP has no effect on [Ca2+]i and proliferation of PASMCs in normoxic and hypoxic conditions. 相似文献
15.
AIM:To investigate the change in myocardial mitochondrial Ca2＋ concentration ( [Ca2+]m) and its mechanism in the early stage of severe burn. METHODS:An experimental model of 30%TBSA full-thickness skin scalding was reproduced in rats. [Ca2+]m, cytosolic Ca2＋ concentration ( c) and mitochondrial Ca2＋ transport velocity were determined. RESULTS: ① [Ca2+]m increased evidently at 1st hour postburn, and continuously at 3rd hour, reached the peak at 6th hour postburn, then, it decreased at 12th and 24th hour, but remained in higher level than that of the control. ② There was no significant difference in c between 1st hour postburn and the control, but c increased evidently at 3rd, 6th, 12th, 24th hour postburn. ③ mitochondrial Ca2＋ uptake velocity at 1st hour postburn was higher than that of control, and Ca2＋ release velocity didn't change obviously, but both of them were decreased at 3rd, 6th, 12th, 24th hour postburn. ④ [Ca2+]m was positive correlated with c after burn, and negative correlated with mitochondrial Ca2＋ release velocity at 3rd, 6th, 12th, 24th hour postburn, respectively. CONCLUSION: There was obvious Ca2＋ overload in myocardial mitochondria after severe burn, the mechanism of which might include ascent of c and disorder of Ca2＋ transport in mitochondria. 相似文献
16.
AIM: To investigate the effects of intracellular free calcium ([Ca2+]i) from different resources on the proliferation mediated by mitogen activated protein kinase (MAPK) in vascular smooth muscle cells (VSMCs). METHODS: Cultured VSMCs were used in all experiments. Calcium influx was stimulated by angiotension Ⅱ(Ang Ⅱ). The release of intracellular calcium stores was induced by inositol trisphosphate (IP3) and ryanodine (RY). MAPK activity was measured by [γ-32P]-ATP incorporation MAPK protein expression by western blot, VSMCs proliferation by [3H]-Leucine ([3H]-Leu) and [3H]-Thymidine ([3H]-TdR) incorporation. RESULTS: Compared to the control VSMCs, Ang Ⅱ, IP3 and RY significantly increased [Ca2+]i concentration activity of MAPK and its protein content in VSMCs. The promotion of [3H]-Leu and [3H]-TdR incorporation in VSMCs was also observed (P<0.01). CONCLUSION: The study indicated that calcium activator-induced increase in the activity and protein content of MAPK was involved in the proliferation of VSMCs, which was closely related to the [Ca2+]i concentration but independent to its origin. 相似文献
17.
AIM: To determine the effects of catestatin (CST) on calcium handling abnormalities and ventri-cular arrhythmia (VA) after myocardial infarction (MI) in rats. METHODS: The adult male SD rats (n=85) were randomly divided into sham group (n=20) and operation group (n=65). MI was induced by ligation of the left anterior descending coronary artery in operation group. The rats in sham group underwent pericardiotomy but without ligating the artery. The rats survived for 1 week after operation were randomly assigned to MI group and CST group. The rats in CST group was treated with CST (30 mg·kg-1·d-1, intraperitoneal administration) for 4 weeks, while saline was applied to the rats in sham group and MI group. The calcium imaging study was performed by loading isolated ventricular cardiomyocytes with Fura-2 AM. In the whole Langendorff-perfused hearts, the programmed electrical stimulation was used to induce action potential duration (APD) alternans and VA. The protein levels of ryanodine receptor 2 (RyR2), phosphorylated RyR2 (p-RyR2), calcium/calmodulin-dependent protein kinase II (CaMKII) and phosphorylated CaMKII (p-CAMKII) were determined by Western blot. RESULTS: Compared with sham group, the protein levels of p-RyR2 and p-CaMKII, the diastolic intracellular Ca2+ concentrations and the inducibility of VA were significantly increased, whereas the thresholds of Ca2+ transient (CaT) and APD alternans and the CaT amplitude were markedly decreased in MI group (P<0.01). Compared with MI group, the protein levels of p-RyR2 and p-CaMKII, the diastolic intracellular Ca2+ concentration and the inducibility of VA were significantly decreased, while the thresholds of CaT and APD alternans and the CaT amplitude were markedly increased in CST group (P<0.01). No significant difference of the protein expression of RyR2 and CaMKII among the 3 groups was observed (P>0.05). CONCLUSION: CST reduces the susceptibility to VA after MI via preventing calcium handling abnormalities. 相似文献
18.
WANG Wan-tie XU Tao XU Zheng-jie JIN Ke-ke PAN Xue-rong LI Dong FANG Zhou-xi 《园艺学报》2004,20(11):2090-2094
AIM: To study the effect of L-arginine (L-Arg) on function and structure of mitochondria in ischemia-reperfusion (MRI) myocardial cells. METHODS: Thirty rabbits were randomly divided into three groups (n=10 in each), control group, MIR group and MIR+L-Arg group. The mitochondrial respiratory function, Ca2+ concentration ([Ca2+]m), malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were determined. Meanwhile, the contents of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), total adenylic acid number (TAN) and energy charge (EC) in the myocardial tissue were respectively measured. Moreover, the ultrastructure changes in myocardial mitochondria were observed during MIR. RESULTS: The mitochondrial respiratory control rate (RCR), velocity 3 (V3), SOD, surface density (Sv) and specific surface (δ) in MIR+L-Arg group were higher than those in MIR group, velocity 4 (V4), [Ca2+]m, MDA, volume density (Vv), horizental diameter (Hd) were lower than those in MIR group. ATP, ADP, TAN and EC levels of myocardial tissue were higher than those in MIR group. There was no significant difference between MIR+L-Arg and control group in V3, V4, SOD, MDA, Vv, Sv, δ, Nv, Vd, AMP and TNA. CONCLUSION: It is suggested that L-Arg improves the function and structure of mitochondria in myocardial cells in the reperfusion injury after myocardial ischemia by decreasing oxygen free radical level and Ca2+ overload in the mitochondria. 相似文献
19.
AIM:To investigate the effects of lentivirus-mediated transfection of shRNA targeting α1D-adrenergic receptor (Adra1d) gene on calcium ion (Ca2+) and calmodulin (CaM) in vascular smooth muscle cells (VSMCs) of rat aorta. METHODS:Single oligonucleotide sequences of shRNA targeting rat Adra1d gene were design and synthesized, and then the shRNA was constructed and cloned into GV248 vector. The U6-shRNA carrier and expression vector were transfected into 293T cells together and packed with lentivirus, and the supernatant was collected and concentrated by overspeed centrifugation. The VSMCs of rat aorta were transfected with recombinant lentivirus vector. The interference effects were identified by RT-qPCR and Western blot. The concentration of Ca2+ in VSMCs was detected by laser confocal inspection, and the expression of CaM at mRNA and protein levels in the VSMCs was determined by RT-qPCR and Western blot. RESULTS:The lentiviral shRNA expression vector was successfully constructed. The titer of the concentrated virus was 3×1011 TU/L. The mRNA and protein expression levels of Adra1d in the rat aortic VSMCs were significantly reduced after transfection. The interference efficiency of Lv-shRNA4-Adr to Adrald gene was greater than 85%. After target silencing of Adra1d gene, compared with scrambled group, the Ca2+ fluorescence intensity of rat aortic VSMCs was significantly increased. Moreover, the mRNA and protein expression levels of CaM were also increased significantly. CONCLUSION:A lentiviral shRNA expression vector targeting rat Adra1d gene was successfully constructed, which significantly increased Ca2+ concentration and CaM expression in rat aortic VSMCs. 相似文献
20.
MO Xian-gang ZHANG Li ZHANG Luo-chao WANG Long XIANG Ning YANG Juan SONG Xiang 《园艺学报》2015,31(11):1992-1997
AIM: To examine the effects of hypoxia on sodium-hydrogen exchange 1(NHE1) expression, intracellular Ca2+ concentration ([Ca2+]i) and calpain activity, and to explore the effect of amiloride on adenosine triphosphate-binding cassette transporter A1(ABCA1) degradation and its calpain-related mechanism. METHODS: RAW264.7 cells were exposed to hypoxia for 0 h, 12 h, 24 h and 48 h. The cell viability was measured by MTT assay and the expression of NHE1 at mRNA and protein levels was detected by real-time PCR and Western blot. [Ca2+]i was analyzed by flow cytometry. Calpain activity was assessed by the method of Suc-LLVY-aminoluciferin. Furthermore, the protein levels of ABCA1 in the RAW264.7 cells exposed to hypoxia for 24 h were determined after 6 h or 12 h treatment with NHE1 inhibitor amiloride in the presence of cycloheximide. ABCA1 protein levels and calpain activity were detected after 12 h incubation with calpain inhibitor ALLN or intracellular calcium-chelating agent BAPTA. RESULTS: Hypoxia inhibited the cell viability in a time-dependent manner. Hypoxia up-regulated the mRNA and protein expression of NHE1, and increased [Ca2+]i and calpain activity. Hypoxia increased the degradation of ABCA1 and amiloride slowed down the ABCA1 degradation. ALLN or BAPTA increased ABCA1 protein level and decreased calpain activity. CONCLUSION: NHE1 inhibitor amiloride attenuates the calpain-mediated degradation of ABCA1, indicating that hypoxia-induced NHE1 might, at least in part, participate in the ABCA1 degradation. 相似文献