Hepatitis B Virus X Protein Upregulates Intracellular Calcium Signaling by Binding C-terminal of Orai1 Protein

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.

Effect of Low Dose Radiation on Intracellular Calcium and Protein Kinase C in Lymphocytes

It is first reported in the present paper that whole-body irradiation (WBI) with low dose X-rays could increase intracellular calcium ions ([Ca2+]i) and stimulate protein kinase C (PKC) activity of mouse lymphocytes. Following WBI of male Kunming micc With 75 mGy X-rays at a dose rate of 12.5 mGy/min the mobilization of [Ca2+]i with Con A in CD4+ and CD8+ Cells in the thymus and spleen was potentiated and the amplitude of [Ca2+], mobilization in thymocytes in response to anti-CD3 monoclonal antibody increased with time from 4 to 24 h following low dose radiation. The PKC activity in the homogenate of spleen was markedly stimulated 12 h after WBl with 75 mGy, reaching its peak value at 24-48 h and coming down to lower than normal on day 7. However, the PKC activity in the separated T lymphocytes reached its peak value at 12 h and that in the B lymphocytes reached its peak value on day 4, both coming down to below control on day 7. The implications of this facilitation of signal transduction in T lymphocytes in the mechanism of immunoenhancement after low dose radiation were discussed

Oleanolic acid-induced apoptosis and its relation with intracellular calcium in human lung adenocarcinoma A549 cells

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.

Changes in intracellular calcium in brain cells of aged rats

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 （[Ca^2＋]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 [Ca^2＋]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： [Ca^2＋]i in neurons of young and aged rats in the presence of I mmol/L extracellular calcium concentration and 0 mmol/L （resting state）, 5, 10, 20, and 40 mmol/L extracellular potassium. Absolute increase of [Ca^2＋]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 Ca〉 and 0 mmol/L （resting state）, 5, 10, 20, and 40 mmol/L extracellular potassium, [Ca^2＋]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 [Ca^2＋]i increase induced by different concentrations of KCI between the aged and the young rats （P 〉 0.05）. CONCLUSION： The overload of [Ca^2＋]i in neurons of aged rats is greater than that of young rats under the same circumstances.

Intracellular calcium ions facilitate dengue virus entry into endothelial cells and compromise endothelial barrier integrity

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.

Visualization of Golgia apparatus as an intracellular calcium store by laser scanning confocal microscope

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.

Enhanced intracellular calcium induced by urocortin is involved in degranulation of rat lung mast cells

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.

EFFECTS OF PDGF-BB ON INTRACELLULAR CALCIUM CONCENTRATION AND PROLIFERATION IN CULTURED GLOMERULAR MESANGIAL CELLS

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.

Formaldehyde increases intracellular calcium concentration in primary cultured hippocampal neurons partly through NMDA receptors and T-type calcium channels

Objective Formaldehyde at high concentrations is a contributor to air pollution. It is also an endogenous metabolic product in cells, and when beyond physiological concentrations, has pathological effects on neurons. Formaldehyde induces mis-folding and aggregation of neuronal tau protein, hippocampal neuronal apoptosis, cognitive impairment and loss of memory functions, as well as excitation of peripheral nociceptive neurons in cancer pain models. Intracellular calcium （[Ca2＋]i） is an important intracellular messenger, and plays a key role in many pathological processes. The present study aimed to investigate the effect of formaldehyde on [Ca2＋]i and the possible involvement of N-methyl-D-aspartate receptors （NMDARs） and T-type Ca2＋ channels on the cell membrane. Methods Using primary cultured hippocampal neurons as a model, changes of [Ca2＋]i in the presence of formaldehyde at a low concentration were detected by confocal laser scanning microscopy. Results Formaldehyde at 1 mmol/L approximately doubled [Ca2＋]i. （2R）-amino-5-phosphonopentanoate （AP5, 25 μmol/L, an NMDAR antagonist） and mibefradil （MIB, 1 μmol/L, a T-type Ca2＋ channel blocker）, given 5 min after formaldehyde perfusion, each partly inhibited the formaldehyde-induced increase of [Ca：＋]i, and this inhibitory effect was reinforced by combined application of AP5 and MIB. When applied 3 min before formaldehyde perfusion, AP5 （even at 50μmol/L） did not inhibit the formaldehyde-induced increase of [Ca2＋]i, but MIB （1 μmol/L） significantly inhibited this increase by 70%. Conclusion These results suggest that formaldehyde at a low concentration increases [Ca2＋]i in cultured hippocampal neurons; NMDARs and T-type Ca2＋ channels may be involved in this process.

Effects of Total Flavonoids ofHippophae RhamnoidesL.on Intracellular Free Calciumin Cultured Vascular Smooth Muscle Cells of Spontaneously Hypertensive Rats and Wistar-Kyoto Rats

To explore the effects of total flavonoids of Hippophae rhamnoides L. （TFH） quercetin （Que） and isorhamnetin （Isor） on the intracellular free calcium （[Ca^2＋]） in vascular smooth muscle cells （VSMC） of spontaneously hypertensive rats （SHR） and Wistar-Kyoto rats （WKY）. Metheds： Fluo 3-acetoxymethylester（Fluo-3/AM） was used to observe the effects of TFH （100mg/L） and its essential monomers, namely Que （10^-4mol/L） and Isor （10^-4mol/L） on changes of [Ca^2＋]1 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＋]1 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 Ang Ⅱ could 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 Ang Ⅱ. （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）. Ceaclusiea： TFH, Que and Isor might decrease the levels of [Ca^2＋], in VSMCs by blocking both voltage-dependent calcium channels （VDC） and receptoroperated 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.

Role of intracellular calcium in contraction of internal anal sphincter

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Induction of human β-defensin-2 in airway epithelial cells by Streptococcus pneumoniae:involvement of IP3-dependent intracellular calcium release and NF-jB

Human β-definsin-2(hBD-2)is mainly induced by bacterial factors and pro-inflammation mediators in epithelial cells.As the major cause of community-acquired pneumonia,whether Streptococcus pneumoniae(S.pneumoniae)stimulation induces hBD-2 expression in airway epithelial cells is elusive.In this study,we found that S.pneumoniae stimulation induced hBD-2 expression in a time-and concentration-dependent manner in primary human airway epithelial cells.To further reveal the mechanism of S.pneumoniae inducing hBD-2,we found that S.pneumoniae stimulation activated NF-jB signaling pathway.Specific NF-jB inhibitor,PDTC,could reverse the induction of hBD-2 by S.pneumoniae.We also found that cellular inner Ca2+ signaling is involved in the S.pneumoniae-induced hBD-2.Taken together,our findings indicated that S.pneumoniae can stimulate the expression of hBD-2 in airway epithelial cells and NF-jB and inositol triphosphate-dependent intracellular calcium release is involved in this induction.

Increased intracellular calcium concentration causes electrical turbulence in guinea pig ventricular myocytes

Dysregulation of intracellular Ca2+ homeostasis is associated with various pathological conditions and arrhythmogenesis of the heart.The objective of this study was to investigate the effects of an acute increase in intracellular Ca2+ concentration ([Ca2+] i) on the electrophysiology of ventricular myocytes by mimicking intracellular Ca 2+ overload.The [Ca2+] i was clamped to either a controlled (65-100 nmol L-1) or increased (1 μmol L-1) level.The transmembrane action potentials and ionic currents were recorded using whole-cell patch clamp techniques.We found that the acute increase in [Ca2+] i shortened the action potential duration,reduced the action potential amplitude,maximum depolarization velocity and resting membrane potential,caused delayed after-depolarizations (DADs),and triggered activity--compared with these parameters in the control.The increased [Ca2+] i augmented late I Na in a time-dependent manner,reduced ICaL and IK1,and increased IKr but not IKs.The results of this study can be used to explain calcium overload-induced ventricular arrhythmias.

Separation and analysis of the soluble trimer of Aβ_(1-40) and its effects on the rise in intracellular calcium

The oligomers of Aβ1-40 peptide in PBS buffer solution were analyzed by SEC and native PAGE, and the trimer of Aβ1-40 was also isolated by SEC. In addition, the effects of the soluble Aβ1-40 trimer on intracellular free calcium (Ca2+) balance of hippocampal neurons of postnatal rats were investi- gated by fluorescence microscopy. The experimental results indicated that Aβ1-40 peptide existed in the form of low molecular weight oligomers in 0.231 mmol/L fresh Aβ1-40 solution (20 mmol/L sodium phosphate buffer, pH 7.4, 0.02% sodium azide) within 24 h and the soluble trimer was the most abundant species. Both the trimeric and the fibrillar Aβ1-40 were able to increase the intracellular Ca2+ concentration, but the Aβ1-40 trimer caused a gradual rise and the potential was also stronger than that of the fibrils at the same concentration. In addition there were dif- ferent response modes for trimeric and fibrillar Aβ1-40, meaning that there are different mechanisms of in- crease in intracellular Ca2+ caused by Aβ1-40.

Effects of simulated microgravity on the alkaline phosphatase activity and intracellular calcium concentration of cultured chondrocytes

The effects of simulated microgravity on matrix mineralization of chondrocytes were examined using cultured chicken embryonic chondrocytes as the model. In four days, there was a time course decrease in alkaline phosphatase activity of chondrocytes, a marker of matrix mineralization.Meanwhile, in two days, there was a significant drop in intracellular calcium concentration in contrast to the control. These results indicate that simulated microgravity can suppress matrix calcification of cultured chondrocytes, and intracellular calcium may be involved in the regulation of matrix calcification as the second messenger.

Rapid Inhibition of the Glutamate-induced Increase of Intracellular Free Calcium by Magnesium in Rat Hippocampal Neurons

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.

Both Hypoxic Endothelial Cell Conditioned Medium and Hypoxia Elevate Intracellular Free Calcium in Pulmonary Artery Smooth Mu

By using Ca2+ -sensitive fluorescent probe, Fura-2 , the effects of endothelial cell-conditioned medium and hypoxia on intracellular free calcium ( [Ca2+]i) in cultured pulmonary artery smooth muscle cell (PASMC) were studied. Normoxic porcine pulmonary artery endothelial cell-conditioned medium (NPAECCM) obviously elevated [Ca2+]i in PASMC,whereas the hypoxic porcine pulmonary artery endothelial cell conditioned medium (HPAECCM)significantly elevated [Ca2+]i in PASMC much more than NPAECCM. Both the effects of NPAECCM and HPAECCM were dependent on the cultured endothelial cell extracellular calcium concentrations, ranged from 1.8 mmol/L to 2. 4 mmol/L.Meanwhile, hypoxia directly increased, which was partially inhibited by verapamil,[Ca2+]i in PASMC through Ca2+ influx pathway.The data suggest that the augmented regulation of endothelial cell on PASMC via Ca2+ second messenger system and the hypoxia-induced Ca2+ influx into PASMC,particularly the former, may be components of mechanisms underlying hypoxic pulmonary vasoconstriction and chronic pulmonary hypertension.

Na^(+)/Ca^(2+)交换体抑制剂SN-6和维拉帕米降低肾上腺皮质癌细胞系NCI-H295R醛固酮合成酶表达

目的研究Na^(+)/Ca^(2+)交换体(NCX)抑制剂SN-6和钙通道阻滞剂(CCB)维拉帕米对钾离子(K^(+))刺激的肾上腺皮质癌细胞系NCI-H295R(H295R)醛固酮合成酶表达的影响。方法H295R细胞分为对照组、K^(+)(15 mmol/L)处理组、钙通道阻滞剂维拉帕米(verapamil)(10μmol/L)处理组、SN-6(10μmol/L)处理组、K^(+)+维拉帕米处理组、K^(+)+SN-6处理组、维拉帕米+SN-6处理组和K^(+)+维拉帕米+SN-6处理组,用实时荧光定量PCR检测醛固酮合成酶(CYP11B2)的mRNA表达,用FLIPR Calcium6检测细胞内钙离子水平。结果与对照组相比,K^(+)刺激醛固酮合成酶CYP11B2的mRNA表达(P<0.001);SN-6和维拉帕米均抑制K^(+)刺激的CYP11B2的mRNA表达(P<0.01);与K^(+)+SN-6组相比,K^(+)+SN-6+维拉帕米组更能显著抑制CYP11B2的mRNA表达(P<0.001)。SN-6和维拉帕米显著降低K^(+)刺激的细胞内钙离子水平(P<0.0001)。结论SN-6和维拉帕米均抑制K^(+)诱导的H295R细胞的醛固酮合成酶的表达;SN-6联合维拉帕米处理,抑制作用更显著。

载脂蛋白A1抗体通过抑制Ca^(2+)载体A23187诱导的Ca^(2+)内流降低精子活力

目的 分析载脂蛋白A1(apo A1)抗体对Ca^(2+)载体A23187诱导的精子Ca^(2+)内流的影响,探讨apo A1抗体与精子活力的关系。方法 收集健康男性精液样本38份。采用免疫荧光法观察apo A1在人精子中的定位和apo A1抗体对精子顶体反应的影响。采用免疫印迹法检测精子获能相关蛋白酪氨酸磷酸化情况。分别采用正常兔Ig G(正常兔Ig G组)和40μg·m L^(-1) apo A1抗体(apo A1抗体组)处理样本,以正常精液样本作为空白对照组,比较各组精子总活力和前向运动精子百分率差异。分别采用正常兔Ig G和10、20、40μg·m L^(-1) apo A1抗体处理Fluo-4AM负载的精子样本,以未作处理的Fluo-4AM负载的精子样本作为空白对照,采用10μmol·L^(-1) A23187诱导30 min,采用流式细胞术检测精子内Ca^(2+)水平。结果 apo A1蛋白定位于人精子头部。apo A1抗体组精子总活力和前向运动精子百分率均显著低于空白对照组和正常兔Ig G组(P<0.001),空白对照组与正常兔Ig G组之间2项指标差异无统计学意义(P>0.05)。流式细胞术结果显示,A23187诱导后,精子内Ca^(2+)荧光强度升高;采用apo A1抗体处理后,Ca^(2+)浓度显著降低(P<0.01),且呈剂量依赖性。40μg·m L^(-1) apo A1抗体+A23187组顶体反应率显著低于A23187组和正常兔Ig G+A23187组(P<0.001),且apo A1抗体对精子自发顶体反应无影响。免疫印迹法结果显示,apo A1抗体处理前后精子的蛋白酪氨酸磷酸化无显著变化。结论 apo A1抗体可以通过降低Ca^(2+)内流使精子活力下降,并抑制Ca^(2+)载体A23187诱导的顶体反应。