Hepatitis B virus X(HBx) protein plays a pivotal role in the development of hepatitis B virus(HBV)-associated hepatocellular carcinoma.Although regulation of cytosolic calcium is essential for HBV replication and is m...Hepatitis B virus X(HBx) protein plays a pivotal role in the development of hepatitis B virus(HBV)-associated hepatocellular carcinoma.Although regulation of cytosolic calcium is essential for HBV replication and is mediated by HBx protein,the mechanism of HBx protein regulating intracellular calcium level remains poorly understood.The present study examined whether HBx protein elevated the intracellular calcium through interacting with storeoperated calcium entry(SOCE) components,Orai1 and stromal interaction molecule 1,and then identified the targets of HBx protein,with an attempt to understand the mechanism of HBx protein upsetting intracellular calcium homeostasis.By employing co-immunoprecipitation and GST-pull-down assay,we found that Orai1 protein interacted with HBx protein,and the C-terminus of Orai1 was implicated in the interaction.Confocal microscopy also revealed that HBx protein could co-localize with full-length Orai1 protein in HEK293 cells.Moreover,live cell calcium imaging exhibited that HBx protein elevated intracellular calcium,possibly by binding to SOCE components.Our results suggest that HBx protein binds to STIM1-Orai1 complexes to positively regulate the activity of plasma membrane store-operated calcium channels.展开更多
Using laser scanning confocal microscopy, we have found that the in cells loaded with fluo-3/AM, highest intracellular Ca(2+) in the perinuclear region is associated with the Golgi apparatus. The spatiotemporal subcel...Using laser scanning confocal microscopy, we have found that the in cells loaded with fluo-3/AM, highest intracellular Ca(2+) in the perinuclear region is associated with the Golgi apparatus. The spatiotemporal subcellu lar distribution of Ca(2+) in living human fibroblasts exposing to calcium-free medium in response to agonists has been investigated. PDGF, which releases Ca(2+) from intracellular stores by inositol(1, 4, 5)-trisphosphate pathway,produced a biphasic transient rise in intracellular calcium.The initial rise was resulted from a direct release of calcium from the Golgi apparatus. Calcium could be also released from and reaccumulated into the Golgi apparatus by the stimulation of thapsigargin, an inhibitor of the Ca(2+) transport ATPase of intracellular calcium store. Permeablizing the plasma membrane by 10 μM digitonin resulted in the calcium release from the Golgi apparatus and depletion of the internal calcium store. These results suggest that the Golgi apparatus plays a role in Ca(2+) regulation in signal transduction.展开更多
Objective: To explore the effects of total flavonoids of Hippophae rhamnoides L. (TFH), quercetin (Que) and isorhamnetin (Isor) on the intracellular free calcium (Ca~ 2+ _ i) in vascular smooth muscle cells (VSMC) of ...Objective: To explore the effects of total flavonoids of Hippophae rhamnoides L. (TFH), quercetin (Que) and isorhamnetin (Isor) on the intracellular free calcium (Ca~ 2+ _ i) in vascular smooth muscle cells (VSMC) of spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY).Methods: Fluo 3-acetoxymethylester(Fluo-3/AM) was used to observe the effects of TFH (100mg/L) and its essential monomers, namely Que (10~ -4 mol/L) and Isor (10~ -4 mol/L) on changes of Ca~ 2+ _ i in cultured SHR and WKY VSMC (abbr. to Ca-SHR & Ca-WKY) following exposure to high K^+, norepinephrine (NE) and angiotensin Ⅱ(AngⅡ), and to compare with the effects of verapamil (Ver). Results: (1) TFH, Que and Isor had inhibitory effects on resting Ca-SHR (P<0.05), but had no significant effects on Ca-WKY (P>0.05). (2) High K^+ could increase Ca-SHR more significantly than Ca-WKY (P<0.05); TFH, Que and Isor could inhibit the elevation of Ca~ 2+ _ i induced by high K^+-depolarization, with the effects similar to that of Ver, and the effect on Ca-SHR was more significant than that on Ca-WKY (P<0.05). (3) NE and AngⅡcould increase Ca-SHR more significantly than Ca-WKY (P<0.05), TFH, Que and Isor had remarkably inhibitory effect on the elevation of Ca-SHR and Ca-WKY induced by NE or AngⅡ. (4) In the absence of extracellular Ca~ 2+ , TFH, Que and Isor also had certain inhibitory effect on Ca-SHR and Ca-WKY induced by NE, and the effect on the former was more significant than that on the latter(P<0.05). Conclusion: TFH, Que and Isor might decrease the levels of Ca~ 2+ _ i in VSMCs by blocking both voltage-dependent calcium channels (VDC) and receptor-operated calcium channels (ROC) in physiological or pathological state, which may be one of the important mechanisms of their hypotensive and protective effects on target organs in patients with hypertension.展开更多
Objective To investigate the effect of oleanolic acid (OA) on apoptosis,correlation between apoptosis and intracellular calcium,and its mechanism in human lung adenocarcinoma cell line A549. Methods Human lung adenoca...Objective To investigate the effect of oleanolic acid (OA) on apoptosis,correlation between apoptosis and intracellular calcium,and its mechanism in human lung adenocarcinoma cell line A549. Methods Human lung adenocarcinoma A549 cells were incubated in vitro and assigned with OA concentrations of 0,10,20 and 40μg/mL. The apoptosis status of A549 cell line was detected with Annexin V-FITC/PI by flow cytometry (FCM); fluorescence intensity (FI) of A549 cells was assessed and the level of intracellular calcium was calculated at 24 hour of OA intervention. The relation between apoptosis and calcium FI was illustrated by curve fitting. Results FCM showed that 10,20 and 40μg/mL of OA could induce A549 cell apoptosis,which followed a concentration-effect pattern; 24-hour intervention with 20μg/mL and 40μg/mL OA showed increased A549 cell apoptosis,and was significantly different from that with 0μg/mL OA (P<0.01). The FI of intracellular calcium concentration in 10,20 and 40μg/mL OA groups was significantly higher than that in 0μg/mL group after 24 hours' intervention,and the FI showed a trend of increase with increased OA concentration (P<0.01). Curve fitting showed a significant correlation between apoptosis rate and intracellular calcium concentration in A549 cells (r=0.981,P<0.01). Regression equation was Y=0.508X-1.627. Conclusion OA plays a role in inducing apoptosis of human lung adenocarcinoma cells in a concentration-dependent manner. The OA-induced apoptosis is responsible for intracellular calcium overload of the tumor.展开更多
Objective:To investigate the involvement of Ca^(2+)in dengue virus(DENV)-infected human umbilical vein endothelial cells(HUVECs)and the disruption of endothelial integrity.Methods:HUVECs were infected with DENV-2 in t...Objective:To investigate the involvement of Ca^(2+)in dengue virus(DENV)-infected human umbilical vein endothelial cells(HUVECs)and the disruption of endothelial integrity.Methods:HUVECs were infected with DENV-2 in the presence of intracellular Ca^(2+)or endoplasmic reticulum Ca^(2+)chelators.Virus infectivity was measured by focus-forming assay and quantitative RT-PCR.Intracellular Ca^(2+)was measured using Fluo-4-AM dye.VE-cadherin and focal adhesion kinase(FAK)expressions were investigated by immunofluorescence and immunoblotting assays,respectively.Results:DENV infection increased intracellular cytosolic Ca^(2+)levels and caused disassembly of the adherens junction protein,VEcadherin as evidenced by decreased VE-cadherin expression at the periphery of DENV-2 infected HUVECs.Depletion of intracellular Ca^(2+)stores,particularly those of the endoplasmic reticulum Ca^(2+),significantly decreased DENV yield in HUVECs.Decreased virus yield following the depletion of intracellular Ca^(2+)was caused by the inhibition of viral entry into HUVECs and not the inhibition of viral binding or attachment.DENV-2 infection also resulted in Ca^(2+)-dependent activation of FAK.Conclusions:Intracellular Ca^(2+)is required for the early phases of DENV infection in endothelial cells.Increased cytosolic Ca^(2+)levels in endothelial cells during DENV infection activated FAK,disrupted adherens junctions and compromised barrier integrity.Thus,Ca^(2+)plays an important role in DENV infection in endothelial cells.展开更多
Objective To investigate the relationship between the alteration of intracellular calcium concentration and proliferation in cultured glomerular mesangial cells. Methods Rat mesangial cells were cultured. Intracellula...Objective To investigate the relationship between the alteration of intracellular calcium concentration and proliferation in cultured glomerular mesangial cells. Methods Rat mesangial cells were cultured. Intracellular calcium concentrations were measured by confocal Laser Scanning Microscopy and Fura-3 fluorescence dyeing techniques. Cell growth was measured by MTT assay. Results PDGF-BB increased intracellular calcium concentrations in a dose-dependent manner, and at the same time promote the proliferation of mesangial cells. After preincubation with calcium channel blocker nifedipine or angiotensin converting enzyme inhibitor captopril, both the increase of intracellular calcium concentrations and cell proliferations induced by PDGF-BB were inhibited. Tripterigium Wilfordii Glycosides (TMG) significantly inhibited the mesangial cell proliferations, but it had no significant effect on intracellular calcium concentrations. Conclusion There was a positive relationship between the elevation of intracellular calcium concentration and cell proliferation in glomerular mesangial cells, but the increase of intracellular calcium concentrations wasn’t the only way for proliferation.展开更多
Objective:To evaluate the effect of serum ionized calcium levels on the prognosis of severe sepsis patients.Methods:This retrospective cross-sectional study included sepsis patients who were hospitalized in an intensi...Objective:To evaluate the effect of serum ionized calcium levels on the prognosis of severe sepsis patients.Methods:This retrospective cross-sectional study included sepsis patients who were hospitalized in an intensive care unit between January 2011 and December 2014.The demographic and baseline data of the patients who died and survived were compared.The cutoff value of ionized calcium for in-hospital mortality was determined by the receiver operating characteristics curve(ROC).In-hospital mortalities and the survival rates were compared between patients with different ionized calcium levels.Besides,the risk factor of in-hospital mortality was determined.Results:This study included 145 patients with 113 patients who died in the hospital.The patients who died had significantly lower ionized calcium levels(U=2.25,P=0.034).A cut-off value of 0.93 mmol/L of ionized calcium was determined by the ROC curve.The patients with ionized calcium>0.93 mmol/L showed a significantly lower morality(χ2=9.90,P=0.002)and higher survival rate than with≤0.93 mmol/L(log rank=6.20,P=0.010).Multivariate Cox regression revealed that ionized calcium≤0.93 mmol/L was a risk factor of in-hospital mortality.Conclusions:Ionized calcium level≤0.93 mmol/L was an independent predictor of in-hospital mortality of severe sepsis.展开更多
Corticotropin-releasing factor(CRF), which activates the hypothalamic-pituitary-adrenal axis under stress, also has proinflammatory peripheral effects possibly through mast cells. The purpose of this study was to inve...Corticotropin-releasing factor(CRF), which activates the hypothalamic-pituitary-adrenal axis under stress, also has proinflammatory peripheral effects possibly through mast cells. The purpose of this study was to investigate the effect of urocortin (UCN), a 40-amino-acid CRF family peptide, on degranulation and intracellular calcium of rat lung mast cells. The activation and degranulation of mast cells were observed by Toluidine blue staining and transmission electron microscope. The intracellular calcium was investigated using confocal laser scanning microscopy and flow cytometry. The results indicated that all the three different concentrations of UCN(0.1, 1 and 10 mu M) significantly induced the activation and degranulation of rat lung mast cells in vitro. This effect was markedly blocked by selective CRF receptor 1(CRF-R1) antagonist antalarmin, but not by specific CRF receptor 2(CRF-R2) antagonist antisauvagine-30(anti-Svg-30). The results also showed that UCN caused a rapid peak increase inCa2+(i) at point of 300s after UCN treatment, followed by a decrease to a sustained plateau phase. The peak increase inCa2+(i) induced by UCN was significantly inhibited by antalarmin, but not by anti-Svg-30. This effect of UCN onCa2+(i) in rat lung mast cells was also found by flow cytometry. Regression analysis revealed a positive correlation between mast cells degranulation extent and the maximum value ofCa2+(i)(P < 0.01). Taken together, our present study suggested that UCN induced the increase of Ca2+(i) and degranulation of rat lung mast cells through CRF-R1. These findings may have implications for the pathophysiology of allergic and inflammatory lung disorders such as asthma, which is closely associated with mast cell activation and degranulation. Copyright (c) 2008 S. Karger AG, Basel.展开更多
BACKGROUND:Studies have shown that voltage-dependent calcium influx,and enhancement of certain calcium-dependent processes in neurons,is related to aging.OBJECTIVE:To observe changes in intracellular calcium([Ca2+]i) ...BACKGROUND:Studies have shown that voltage-dependent calcium influx,and enhancement of certain calcium-dependent processes in neurons,is related to aging.OBJECTIVE:To observe changes in intracellular calcium([Ca2+]i) in neurons of aged rats,and to compare with young rats.DESIGN,TIME AND SETTING:A randomized control experiment of neurophysiology was performed at the Central Laboratory of School of Pharmaceutical Science,China Medical University from June to August 2004.MATERIALS:Ten male,healthy,Wistar rats,19 months old,were selected for the aged group.Ten male,3-month-old,Wistar rats were selected for the young control group.Fura-2/AM was provided by the Institute of Pharmaceutical Research of Chinese Academy of Medical Sciences,and the F-2000 fluorospectrophotometer was a product of Hitachi,Japan.METHODS:Fluorescence Fura-2 spectrophotometer was used to measure [Ca2+]i in acutely dissociated brain cells of aged and young rats.The concentration of extracellular potassium was controlled by adding different volumes of chloridated potassium solution of high concentration.MAIN OUTCOME MEASURES:[Ca2+]i in neurons of young and aged rats in the presence of 1 mmol/L extracellular calcium concentration and 0 mmol/L(resting state),5,10,20,and 40 mmol/L extracellular potassium.Absolute increase of [Ca2+]i in neurons of young and aged rats when extracellular potassium was 5,10,20,40 mmol/L.RESULTS:In the presence of 1 mmol/L extracellular Ca2+ and 0 mmol/L(resting state),5,10,20,and 40 mmol/L extracellular potassium,[Ca2+]i in the neurons of aged rats was significantly less than that in young rats(P < 0.05).However,there was no significant difference in absolute [Ca2+]i increase induced by different concentrations of KCl between the aged and the young rats(P > 0.05).CONCLUSION:The overload of [Ca2+]i in neurons of aged rats is greater than that of young rats under the same circumstances.展开更多
By using Fura-2/AM, the effects of magnesium (Mg 2+) on the glutamate-induced increase of intracellular free calcium ([Ca 2+]i) in the cultured hippocampal neurons and the features were investigated by integrated phot...By using Fura-2/AM, the effects of magnesium (Mg 2+) on the glutamate-induced increase of intracellular free calcium ([Ca 2+]i) in the cultured hippocampal neurons and the features were investigated by integrated photoelectric detecting system. The experiments were designed to three groups (The drug was spit to the cells for 20 s): Group A receiving 1×10 —5 mol/L glutamate; Group B receiving 1×10 —5 mol/L glutamate and1×10 —5 mol/L Mg 2+ simultaneously; Group C receiving 1×10 —5 mol/L glutamate again after [Ca 2+]i in group B back to the baseline. The results showed that in group A, [Ca 2+]i was obviously increased. In group B, the changes in [Ca 2+]i and the peak value were significantly decreased. Moreover, the elevation of Phase 1 was slowed down and Phase 2 was shortened to some extent, and the plateau phase between them was relatively prolonged. In group C, calcium oscillation similar to that in group A occurred, but both the Phase 1 and Phase 2 were shortened and the △[Ca 2+]i was slightly decreased. It was suggested that Mg 2+ could quickly inhibit the rise of [Ca 2+]i induced by glutamate in the cultured hippocampal neurons in rats.展开更多
The isolated cardiac myocytes of rats were immediately infected by cosackievirus B3 (CVB3) to investigate the effects of such procedure on the cell cycle, apoptosis and intracellular ionized calcium (Ca 2+ i) of cardi...The isolated cardiac myocytes of rats were immediately infected by cosackievirus B3 (CVB3) to investigate the effects of such procedure on the cell cycle, apoptosis and intracellular ionized calcium (Ca 2+ i) of cardiac myocytes. Newborn Balb/c murine cardiac myocytes were cultivated, then infected by CVB3. Intracellular Ca 2+ i was measured by flow cytometer. The calcium in the medium for culturing cardiac myocytes was detected by using atom absorb spectrum test. It was found that CVB3 could markedly inhibit the differentiation and proliferation of the infected cardiac myocytes and induce the apoptosis. The intracellular Ca 2+ i level in the infected group was significantly higher than in the control group (P<0.01). The calcium concentration in the medium for culturing cardiac myocytes in the infected group was significantly lower than in the control group (P<0.05). It was suggested that the apoptosis and intracellular calcium overload of the CVB3-affected cardiac myocytes are likely to play an important role in the pathogenesis of viral myocarditis.展开更多
The interphase NIH3T3 cells were vitally fluorescentstained with calcium indicator fluo-3 and Glogi probe C6NBD-ceramide, and then the single cells were examined by laser scanning confocal microscopy (LSCFM) for subce...The interphase NIH3T3 cells were vitally fluorescentstained with calcium indicator fluo-3 and Glogi probe C6NBD-ceramide, and then the single cells were examined by laser scanning confocal microscopy (LSCFM) for subcellular distributions of Ca2+ and the location of Golgi apparatus. In these cells, the intracellular Ca2+ were found to be highly concentrated in the Golgi apparatus. The changes of distribution of cytosolic high Ca2+ region and the Golgi apparatus coincided with the cell cycle phase.In calcium free medium, when the plasma membrane of the cells which had been loaded with fluo-3/AM were permeated by digitonin, the fluorescence of the Golgi region decreased far less than that of the cytosol. Our results indicated that the Glogi lumen retained significantly high concentration of free calcium.展开更多
Both calcium ionophore A23187 and endoplasmic reticulum Ca2+- ATPase inhibitor thapsigargin (Tg) could increase intracellular free calcium concentration and induce apoptosis in some cell lines. In the present study, w...Both calcium ionophore A23187 and endoplasmic reticulum Ca2+- ATPase inhibitor thapsigargin (Tg) could increase intracellular free calcium concentration and induce apoptosis in some cell lines. In the present study, we found that HL-60 cells treated with A23187 (1μg/ml) for 4 h or with Tg (0.5μg/ml) for 2 h showed typical characteristics of apoptosis. Pretreatment with nontoxic concentration of cyclosporin A (CsA) (1μg/ml) Could block these effects. Flow cytometric analysis of intracellular Ca2+ after staining with fluo-3 AM showed that CsA did not prevent the increase of intracellular calcium induced by A23187 or Tg, but it could maintain the high level of intracellular Ca2+ for a long time. These results suggest that CsA may prevent calcium- induced apoptosis by blocking the transportation of Ca2+ in HL-60cells.展开更多
基金supported by grants from the National Natural Science Foundation of China(No.81001063)the Fundamental Research Funds for the Central Universities(No.2015QN150)
文摘Hepatitis B virus X(HBx) protein plays a pivotal role in the development of hepatitis B virus(HBV)-associated hepatocellular carcinoma.Although regulation of cytosolic calcium is essential for HBV replication and is mediated by HBx protein,the mechanism of HBx protein regulating intracellular calcium level remains poorly understood.The present study examined whether HBx protein elevated the intracellular calcium through interacting with storeoperated calcium entry(SOCE) components,Orai1 and stromal interaction molecule 1,and then identified the targets of HBx protein,with an attempt to understand the mechanism of HBx protein upsetting intracellular calcium homeostasis.By employing co-immunoprecipitation and GST-pull-down assay,we found that Orai1 protein interacted with HBx protein,and the C-terminus of Orai1 was implicated in the interaction.Confocal microscopy also revealed that HBx protein could co-localize with full-length Orai1 protein in HEK293 cells.Moreover,live cell calcium imaging exhibited that HBx protein elevated intracellular calcium,possibly by binding to SOCE components.Our results suggest that HBx protein binds to STIM1-Orai1 complexes to positively regulate the activity of plasma membrane store-operated calcium channels.
文摘Using laser scanning confocal microscopy, we have found that the in cells loaded with fluo-3/AM, highest intracellular Ca(2+) in the perinuclear region is associated with the Golgi apparatus. The spatiotemporal subcellu lar distribution of Ca(2+) in living human fibroblasts exposing to calcium-free medium in response to agonists has been investigated. PDGF, which releases Ca(2+) from intracellular stores by inositol(1, 4, 5)-trisphosphate pathway,produced a biphasic transient rise in intracellular calcium.The initial rise was resulted from a direct release of calcium from the Golgi apparatus. Calcium could be also released from and reaccumulated into the Golgi apparatus by the stimulation of thapsigargin, an inhibitor of the Ca(2+) transport ATPase of intracellular calcium store. Permeablizing the plasma membrane by 10 μM digitonin resulted in the calcium release from the Golgi apparatus and depletion of the internal calcium store. These results suggest that the Golgi apparatus plays a role in Ca(2+) regulation in signal transduction.
基金Supported by One-hundred-people Plan of Hygiene Systemin Shanghai (No .990122)
文摘Objective: To explore the effects of total flavonoids of Hippophae rhamnoides L. (TFH), quercetin (Que) and isorhamnetin (Isor) on the intracellular free calcium (Ca~ 2+ _ i) in vascular smooth muscle cells (VSMC) of spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY).Methods: Fluo 3-acetoxymethylester(Fluo-3/AM) was used to observe the effects of TFH (100mg/L) and its essential monomers, namely Que (10~ -4 mol/L) and Isor (10~ -4 mol/L) on changes of Ca~ 2+ _ i in cultured SHR and WKY VSMC (abbr. to Ca-SHR & Ca-WKY) following exposure to high K^+, norepinephrine (NE) and angiotensin Ⅱ(AngⅡ), and to compare with the effects of verapamil (Ver). Results: (1) TFH, Que and Isor had inhibitory effects on resting Ca-SHR (P<0.05), but had no significant effects on Ca-WKY (P>0.05). (2) High K^+ could increase Ca-SHR more significantly than Ca-WKY (P<0.05); TFH, Que and Isor could inhibit the elevation of Ca~ 2+ _ i induced by high K^+-depolarization, with the effects similar to that of Ver, and the effect on Ca-SHR was more significant than that on Ca-WKY (P<0.05). (3) NE and AngⅡcould increase Ca-SHR more significantly than Ca-WKY (P<0.05), TFH, Que and Isor had remarkably inhibitory effect on the elevation of Ca-SHR and Ca-WKY induced by NE or AngⅡ. (4) In the absence of extracellular Ca~ 2+ , TFH, Que and Isor also had certain inhibitory effect on Ca-SHR and Ca-WKY induced by NE, and the effect on the former was more significant than that on the latter(P<0.05). Conclusion: TFH, Que and Isor might decrease the levels of Ca~ 2+ _ i in VSMCs by blocking both voltage-dependent calcium channels (VDC) and receptor-operated calcium channels (ROC) in physiological or pathological state, which may be one of the important mechanisms of their hypotensive and protective effects on target organs in patients with hypertension.
文摘Objective To investigate the effect of oleanolic acid (OA) on apoptosis,correlation between apoptosis and intracellular calcium,and its mechanism in human lung adenocarcinoma cell line A549. Methods Human lung adenocarcinoma A549 cells were incubated in vitro and assigned with OA concentrations of 0,10,20 and 40μg/mL. The apoptosis status of A549 cell line was detected with Annexin V-FITC/PI by flow cytometry (FCM); fluorescence intensity (FI) of A549 cells was assessed and the level of intracellular calcium was calculated at 24 hour of OA intervention. The relation between apoptosis and calcium FI was illustrated by curve fitting. Results FCM showed that 10,20 and 40μg/mL of OA could induce A549 cell apoptosis,which followed a concentration-effect pattern; 24-hour intervention with 20μg/mL and 40μg/mL OA showed increased A549 cell apoptosis,and was significantly different from that with 0μg/mL OA (P<0.01). The FI of intracellular calcium concentration in 10,20 and 40μg/mL OA groups was significantly higher than that in 0μg/mL group after 24 hours' intervention,and the FI showed a trend of increase with increased OA concentration (P<0.01). Curve fitting showed a significant correlation between apoptosis rate and intracellular calcium concentration in A549 cells (r=0.981,P<0.01). Regression equation was Y=0.508X-1.627. Conclusion OA plays a role in inducing apoptosis of human lung adenocarcinoma cells in a concentration-dependent manner. The OA-induced apoptosis is responsible for intracellular calcium overload of the tumor.
基金funding from the Ministry of Higher Education Malaysia via the Higher Institution Centre of Excellence(HICo E)program(MO002-2019)Development of Research Institute for Excellent Enterprises(ATC+)Project,Republic of Korea(IF001-2021)
文摘Objective:To investigate the involvement of Ca^(2+)in dengue virus(DENV)-infected human umbilical vein endothelial cells(HUVECs)and the disruption of endothelial integrity.Methods:HUVECs were infected with DENV-2 in the presence of intracellular Ca^(2+)or endoplasmic reticulum Ca^(2+)chelators.Virus infectivity was measured by focus-forming assay and quantitative RT-PCR.Intracellular Ca^(2+)was measured using Fluo-4-AM dye.VE-cadherin and focal adhesion kinase(FAK)expressions were investigated by immunofluorescence and immunoblotting assays,respectively.Results:DENV infection increased intracellular cytosolic Ca^(2+)levels and caused disassembly of the adherens junction protein,VEcadherin as evidenced by decreased VE-cadherin expression at the periphery of DENV-2 infected HUVECs.Depletion of intracellular Ca^(2+)stores,particularly those of the endoplasmic reticulum Ca^(2+),significantly decreased DENV yield in HUVECs.Decreased virus yield following the depletion of intracellular Ca^(2+)was caused by the inhibition of viral entry into HUVECs and not the inhibition of viral binding or attachment.DENV-2 infection also resulted in Ca^(2+)-dependent activation of FAK.Conclusions:Intracellular Ca^(2+)is required for the early phases of DENV infection in endothelial cells.Increased cytosolic Ca^(2+)levels in endothelial cells during DENV infection activated FAK,disrupted adherens junctions and compromised barrier integrity.Thus,Ca^(2+)plays an important role in DENV infection in endothelial cells.
文摘Objective To investigate the relationship between the alteration of intracellular calcium concentration and proliferation in cultured glomerular mesangial cells. Methods Rat mesangial cells were cultured. Intracellular calcium concentrations were measured by confocal Laser Scanning Microscopy and Fura-3 fluorescence dyeing techniques. Cell growth was measured by MTT assay. Results PDGF-BB increased intracellular calcium concentrations in a dose-dependent manner, and at the same time promote the proliferation of mesangial cells. After preincubation with calcium channel blocker nifedipine or angiotensin converting enzyme inhibitor captopril, both the increase of intracellular calcium concentrations and cell proliferations induced by PDGF-BB were inhibited. Tripterigium Wilfordii Glycosides (TMG) significantly inhibited the mesangial cell proliferations, but it had no significant effect on intracellular calcium concentrations. Conclusion There was a positive relationship between the elevation of intracellular calcium concentration and cell proliferation in glomerular mesangial cells, but the increase of intracellular calcium concentrations wasn’t the only way for proliferation.
文摘Objective:To evaluate the effect of serum ionized calcium levels on the prognosis of severe sepsis patients.Methods:This retrospective cross-sectional study included sepsis patients who were hospitalized in an intensive care unit between January 2011 and December 2014.The demographic and baseline data of the patients who died and survived were compared.The cutoff value of ionized calcium for in-hospital mortality was determined by the receiver operating characteristics curve(ROC).In-hospital mortalities and the survival rates were compared between patients with different ionized calcium levels.Besides,the risk factor of in-hospital mortality was determined.Results:This study included 145 patients with 113 patients who died in the hospital.The patients who died had significantly lower ionized calcium levels(U=2.25,P=0.034).A cut-off value of 0.93 mmol/L of ionized calcium was determined by the ROC curve.The patients with ionized calcium>0.93 mmol/L showed a significantly lower morality(χ2=9.90,P=0.002)and higher survival rate than with≤0.93 mmol/L(log rank=6.20,P=0.010).Multivariate Cox regression revealed that ionized calcium≤0.93 mmol/L was a risk factor of in-hospital mortality.Conclusions:Ionized calcium level≤0.93 mmol/L was an independent predictor of in-hospital mortality of severe sepsis.
文摘Corticotropin-releasing factor(CRF), which activates the hypothalamic-pituitary-adrenal axis under stress, also has proinflammatory peripheral effects possibly through mast cells. The purpose of this study was to investigate the effect of urocortin (UCN), a 40-amino-acid CRF family peptide, on degranulation and intracellular calcium of rat lung mast cells. The activation and degranulation of mast cells were observed by Toluidine blue staining and transmission electron microscope. The intracellular calcium was investigated using confocal laser scanning microscopy and flow cytometry. The results indicated that all the three different concentrations of UCN(0.1, 1 and 10 mu M) significantly induced the activation and degranulation of rat lung mast cells in vitro. This effect was markedly blocked by selective CRF receptor 1(CRF-R1) antagonist antalarmin, but not by specific CRF receptor 2(CRF-R2) antagonist antisauvagine-30(anti-Svg-30). The results also showed that UCN caused a rapid peak increase inCa2+(i) at point of 300s after UCN treatment, followed by a decrease to a sustained plateau phase. The peak increase inCa2+(i) induced by UCN was significantly inhibited by antalarmin, but not by anti-Svg-30. This effect of UCN onCa2+(i) in rat lung mast cells was also found by flow cytometry. Regression analysis revealed a positive correlation between mast cells degranulation extent and the maximum value ofCa2+(i)(P < 0.01). Taken together, our present study suggested that UCN induced the increase of Ca2+(i) and degranulation of rat lung mast cells through CRF-R1. These findings may have implications for the pathophysiology of allergic and inflammatory lung disorders such as asthma, which is closely associated with mast cell activation and degranulation. Copyright (c) 2008 S. Karger AG, Basel.
文摘BACKGROUND:Studies have shown that voltage-dependent calcium influx,and enhancement of certain calcium-dependent processes in neurons,is related to aging.OBJECTIVE:To observe changes in intracellular calcium([Ca2+]i) in neurons of aged rats,and to compare with young rats.DESIGN,TIME AND SETTING:A randomized control experiment of neurophysiology was performed at the Central Laboratory of School of Pharmaceutical Science,China Medical University from June to August 2004.MATERIALS:Ten male,healthy,Wistar rats,19 months old,were selected for the aged group.Ten male,3-month-old,Wistar rats were selected for the young control group.Fura-2/AM was provided by the Institute of Pharmaceutical Research of Chinese Academy of Medical Sciences,and the F-2000 fluorospectrophotometer was a product of Hitachi,Japan.METHODS:Fluorescence Fura-2 spectrophotometer was used to measure [Ca2+]i in acutely dissociated brain cells of aged and young rats.The concentration of extracellular potassium was controlled by adding different volumes of chloridated potassium solution of high concentration.MAIN OUTCOME MEASURES:[Ca2+]i in neurons of young and aged rats in the presence of 1 mmol/L extracellular calcium concentration and 0 mmol/L(resting state),5,10,20,and 40 mmol/L extracellular potassium.Absolute increase of [Ca2+]i in neurons of young and aged rats when extracellular potassium was 5,10,20,40 mmol/L.RESULTS:In the presence of 1 mmol/L extracellular Ca2+ and 0 mmol/L(resting state),5,10,20,and 40 mmol/L extracellular potassium,[Ca2+]i in the neurons of aged rats was significantly less than that in young rats(P < 0.05).However,there was no significant difference in absolute [Ca2+]i increase induced by different concentrations of KCl between the aged and the young rats(P > 0.05).CONCLUSION:The overload of [Ca2+]i in neurons of aged rats is greater than that of young rats under the same circumstances.
文摘By using Fura-2/AM, the effects of magnesium (Mg 2+) on the glutamate-induced increase of intracellular free calcium ([Ca 2+]i) in the cultured hippocampal neurons and the features were investigated by integrated photoelectric detecting system. The experiments were designed to three groups (The drug was spit to the cells for 20 s): Group A receiving 1×10 —5 mol/L glutamate; Group B receiving 1×10 —5 mol/L glutamate and1×10 —5 mol/L Mg 2+ simultaneously; Group C receiving 1×10 —5 mol/L glutamate again after [Ca 2+]i in group B back to the baseline. The results showed that in group A, [Ca 2+]i was obviously increased. In group B, the changes in [Ca 2+]i and the peak value were significantly decreased. Moreover, the elevation of Phase 1 was slowed down and Phase 2 was shortened to some extent, and the plateau phase between them was relatively prolonged. In group C, calcium oscillation similar to that in group A occurred, but both the Phase 1 and Phase 2 were shortened and the △[Ca 2+]i was slightly decreased. It was suggested that Mg 2+ could quickly inhibit the rise of [Ca 2+]i induced by glutamate in the cultured hippocampal neurons in rats.
文摘The isolated cardiac myocytes of rats were immediately infected by cosackievirus B3 (CVB3) to investigate the effects of such procedure on the cell cycle, apoptosis and intracellular ionized calcium (Ca 2+ i) of cardiac myocytes. Newborn Balb/c murine cardiac myocytes were cultivated, then infected by CVB3. Intracellular Ca 2+ i was measured by flow cytometer. The calcium in the medium for culturing cardiac myocytes was detected by using atom absorb spectrum test. It was found that CVB3 could markedly inhibit the differentiation and proliferation of the infected cardiac myocytes and induce the apoptosis. The intracellular Ca 2+ i level in the infected group was significantly higher than in the control group (P<0.01). The calcium concentration in the medium for culturing cardiac myocytes in the infected group was significantly lower than in the control group (P<0.05). It was suggested that the apoptosis and intracellular calcium overload of the CVB3-affected cardiac myocytes are likely to play an important role in the pathogenesis of viral myocarditis.
文摘The interphase NIH3T3 cells were vitally fluorescentstained with calcium indicator fluo-3 and Glogi probe C6NBD-ceramide, and then the single cells were examined by laser scanning confocal microscopy (LSCFM) for subcellular distributions of Ca2+ and the location of Golgi apparatus. In these cells, the intracellular Ca2+ were found to be highly concentrated in the Golgi apparatus. The changes of distribution of cytosolic high Ca2+ region and the Golgi apparatus coincided with the cell cycle phase.In calcium free medium, when the plasma membrane of the cells which had been loaded with fluo-3/AM were permeated by digitonin, the fluorescence of the Golgi region decreased far less than that of the cytosol. Our results indicated that the Glogi lumen retained significantly high concentration of free calcium.
文摘Both calcium ionophore A23187 and endoplasmic reticulum Ca2+- ATPase inhibitor thapsigargin (Tg) could increase intracellular free calcium concentration and induce apoptosis in some cell lines. In the present study, we found that HL-60 cells treated with A23187 (1μg/ml) for 4 h or with Tg (0.5μg/ml) for 2 h showed typical characteristics of apoptosis. Pretreatment with nontoxic concentration of cyclosporin A (CsA) (1μg/ml) Could block these effects. Flow cytometric analysis of intracellular Ca2+ after staining with fluo-3 AM showed that CsA did not prevent the increase of intracellular calcium induced by A23187 or Tg, but it could maintain the high level of intracellular Ca2+ for a long time. These results suggest that CsA may prevent calcium- induced apoptosis by blocking the transportation of Ca2+ in HL-60cells.
