毛木耳多糖对小鼠腹腔巨噬细胞胞浆游离Ca^2＋浓度的影响

为探讨毛木耳多糖（APSP－A）对免疫细胞信号转导的影响，采用荧光分光光度法，测定毛木耳多糖（APSP－A）对小鼠腹腔巨噬细胞（Mφ）胞浆游离Ca2 浓度（[Ca2 ]i）的影响。结果表明：APSP-A能剂量依赖性地引起小鼠腹腔Mφ[Ca2 ]i明显升高，[Ca2 ]i的升高是由胞外钙内流和胞内钙释放共同作用的结果;与Ca2 通道有关，而与细胞膜电位无关。     

小檗胺对ROCC介导的血管平滑肌细胞内游离钙的影响

目的　研究小檗胺 (BA)对受体调控性Ca2 +通道介导的家兔胸主动脉血管平滑肌细胞内游离钙 ([Ca2 +]i)的影响。方法　家兔主动脉血管平滑肌以Fluo 3/AM负载 ,通过激光扫描共聚焦显微镜 (LSCM )测定 [Ca2 +]i。结果　在细胞外Ca2 +存在的条件下 ,BA 30 μmol·L-1不影响静息[Ca2 +]i;但对去甲肾上腺素 (NE) 30mmol·L-1、5 羟色胺 (5 HT) 1μmol·L-1诱导的 [Ca2 +]i 升高有明显的抑制作用。在无胞外钙时 ,对咖啡因 40mmol·L-1诱导的 [Ca2 +]i 升高没有作用。结论　BA对ROCC激活后的外钙内流有明显的抑制作用 ,对内钙释放没有影响。其作用与维拉帕米相似     

灯盏花素对人脐静脉内皮细胞胞内Ca~(2+)水平的调控作用

目的探讨灯盏花素对培养的人脐静脉内皮细胞(HUVECs)胞内Ca2+水平([Ca2+]i)的调控作用。方法采用新一代Ca2+荧光探针Fluo-3/AM标记培养的HUVECs,激光共聚焦显微镜检测细胞胞内钙荧光信号,观察灯盏花素对培养的HUVECs胞内Ca2+水平的调控作用。结果在胞外有Ca2+或无Ca2+的情况下,灯盏花素均可引起[Ca2+]i的短暂性升高;灯盏花素的Ca2+释放作用与钙泵抑制剂CPA存在着交迭;灯盏花素能够抑制由KCl所引起的[Ca2+]i的升高;灯盏花素对胞内Ca2+池耗竭后胞外复Ca2+所引起的钙内流无明显阻断作用。结论灯盏花素可引起胞内Ca2+池的Ca2+释放,其释放的Ca2+来自CPA敏感的Ca2+池。灯盏花素也可抑制经电压依赖性Ca2+通道的Ca2+内流,对Ca2+池耗竭后引起的Ca2+内流通道无明显阻断作用。     

羊角拗苷对豚鼠心室肌细胞内游离钙离子浓度的影响

目的观察羊角拗苷(Div)对豚鼠心室肌细胞内游离钙离子([Ca2+]i)浓度的影响,以探讨Div正性肌力作用的机制。方法Fura-2/AM荧光探针标记豚鼠心室肌细胞,应用荧光离子成像系统观察Div800nmol.L-1对心室肌细胞[Ca2+]i的影响。结果在正常台氏液中,Div使心室肌细胞[Ca2+]i显著升高(243±36)%,在无钙液中对[Ca2+]i无明显影响。在无Na+、无K+台氏液中,Div使[Ca2+]i升高(96±20)%。给予L型钙通道阻滞剂CdCl2100μmol.L-1预处理后,Div仍使[Ca2+]i升高(63±10)%。给予T型钙通道阻滞剂NiCl240μmol.L-1预处理后,Div引起的[Ca2+]i升高基本被阻断。结论体外应用Div800nmol.L-1时使豚鼠心室肌细胞[Ca2+]i升高,该升高作用依赖于细胞外Ca2+的存在,可能主要由T型钙通道介导,L型钙通道和Na+-Ca2+交换蛋白亦参与其中。其正性肌力作用与心室肌细胞[Ca2+]i升高有关。     

内皮细胞乙酰胆碱作用靶标对胞内钙信号的调节

目的研究激活血管内皮细胞乙酰胆碱作用靶标(ETA)对不同类型血管内皮细胞[Ca2+]i水平的影响;以[Ca2+]i水平作为鉴定ETA功能的指标之一,观察其在传代过程中的改变,为研究ETA的分子结构及其信号转导途径提供实验依据.方法采用激光共聚焦技术,以荧光指示剂Fluo-3为探针,观察乙酰胆碱(ACh)和氨甲酰胆碱(carbachol,Car)对内皮细胞[Ca2+]i水平的影响.结果ACh和Car在10-5～10-2 mol*L-1可使3～10代牛主动脉内皮细胞标本的[Ca2+]i水平显著升高,ACh在 10-8～10-2 mol*L-1可使8代人冠脉内皮细胞标本的[Ca2+]i水平显著升高.结论内皮细胞在10代培养过程中外源性激动剂ACh和Car均可诱导胞内[Ca2+]i水平升高,ETA的功能与[Ca2+]i水平升高有关.     

ATP对大鼠三叉神经节小直径神经元胞内钙浓度的调制作用及其机制

目的探讨神经递质ATP通过何种途径引起大鼠三叉神经节(trigeminal ganglion,TG)小直径神经元胞内钙离子浓度升高。方法在急性分离的TG神经元上,应用钙离子成像技术检测胞内游离Ca2+浓度([Ca2+]i)的变化。结果在大鼠TG小直径神经元中,ATP(100μmol·L-1),thap-sigargin(1μmol·L-1,内质网钙泵抑制剂)和咖啡因(20mmol·L-1,内质网钙离子通道开放剂)在正常细胞外液和去除细胞外Ca2+的情况下,均能够引起细胞[Ca2+]i升高。在细胞外无Ca2+条件下,thapsigargin能够可逆地抑制ATP引起细胞内[Ca2+]i升高(n=8,P<0.01),而咖啡因对ATP引起的细胞内[Ca2+]i升高无影响(n=6,P>0.05)。然而在正常外液中,thapsigargin不能完全抑制ATP引起的细胞内[Ca2+]i升高,不过ATP引起的细胞内[Ca2+]i升高的幅度明显地低于thapsigargin处理前(n=7,P<0.05)。结论在大鼠TG小直径神经元中,存在有IP3敏感钙库和Ryanod-ine敏感钙库。ATP可通过激动P2Y受体引起IP3敏感钙库的Ca2+释放,也可通过激动P2X受体引起细胞外钙内流。     

青蒿琥酯对人前列腺癌PC-3细胞内游离Ca~(2+)的影响

目的:研究青蒿琥酯(ART)对人前列腺癌PC-3细胞内游离Ca2+浓度([Ca2+]i)的影响。方法:ART处理PC-3细胞,用Ca2+荧光探针Fluo-3/AM标记PC-3细胞,激光共聚焦扫描显微镜检测PC-3细胞内[Ca2+]i变化。结果:ART诱导PC-3细胞内[Ca2+]i在0～0.5h显著升高;在0.5～1h维持高水平;在4～24h降到初始水平。结论:ART能够诱导PC-3细胞内[Ca2+]i显著持续升高,[Ca2+]i升高可能在ART诱导PC-3细胞凋亡中起重要作用。     

铅对小鼠海马[Ca2+]的影响及L型钙通道的作用

目的　探讨铅对分离的小鼠海马细胞内游离钙的影响及其及L型钙通道的作用。方法　采用低浓度胰蛋白酶消化法分离小鼠海马细胞 ,并以莹光指标剂 (Fura 2 )作Ca2 荧光探针 ,用双波长荧光法测定海马细胞 [Ca2 ] i。结果　铅可致分离的小鼠海马细胞 [Ca2 ] i 升高 [由静息时的 (2 0 3 4± 10 86)nmol L升至 (4 2 3 3± 19 2 6)nmol L] ,而不论胞外是否有钙 ;铅可抑制钾诱发海马细胞 [Ca2 ] i 升高 ,尼莫地平可加强这种抑制作用 ,而BayK864 4则可部分消除这种抑制作用。结论　铅可致分离的小鼠海马细胞 [Ca2 ] i 升高作用与胞内钙库释放有关 ;铅的抑制钾诱发海马细胞 [Ca2 ] i 升高作用与其抑制L型钙通道有关。     

马尾松花粉硫酸酯化多糖调控脾细胞[Ca~(2+)]_i的研究

目的探讨马尾松花粉多糖硫酸酯化物(SPPM60-A)对小鼠脾细胞内游离钙离子浓度([Ca2+]i)调控的机制。方法热水浸提醇沉淀法提取马尾松花粉粗多糖,SephacrylS-400HR分离纯化,氯磺酸-吡啶法进行硫酸酯化,荧光分光光度计测定脾淋巴细胞[Ca2+]i。结果 LPS与SPPM60-A都能促进脾淋巴细胞[Ca2+]i升高,与对照相比升高率分别为11.7%和15.4%(P<0.01)。TAK-242、LY294002、U73122、低分子肝素与维拉帕米均可以抑制[Ca2+]i的升高。结论推测硫酸酯化马尾松花粉多糖可通过TLR4-PI3K-PLC-IP3R信号通路促进脾淋巴细胞内[Ca2+]i升高。     

异钩藤碱体外对家兔血小板聚集和胞浆游离钙离子浓度的影响

目的研究异钩藤碱对血小板内游离钙离子浓度([Ca2+]i)的影响,以探讨其抗血小板聚集作用的可能机制。方法比浊法测定家兔血小板聚集功能;双波长Fura-2荧光法测定血小板胞浆[Ca2+]i。结果异钩藤碱0.33~1.30mmol.L-1体外给药对ADP和凝血酶引起的血小板聚集有浓度依赖性的抑制作用。存在细胞外钙时,异钩藤碱对基础状态血小板的[Ca2+]i和ADP及凝血酶诱导的[Ca2+]i水平有浓度依赖性的降低作用,而无细胞外钙存在时,则均无明显影响,表明其可抑制血小板的外钙内流,对内钙释放无明显抑制作用。结论异钩藤碱可抑制血小板聚集,其作用机制可能与其抑制血小板胞浆[Ca2+]升高有关。     

银杏内酯B对血小板活化因子引起的巨噬细胞趋化及细胞骨架改变的影响

目的考察银杏内酯B(ginkgolide B，BN52021)对血小板活化因子(PAF)引起的小鼠腹腔巨噬细胞趋化反应和丝状肌动蛋白(F-actin)聚合作用的影响。方法Boyden小室法检测化合物对巨噬细胞趋化的影响；流式细胞术检测特异性标记的巨噬细胞中F-actin的变化。结果PAF可显著刺激小鼠腹腔巨噬细胞产生趋化效应，PAF受体拮抗剂BN52021(0.01 nmol·L^-1～0.1 μmol·L^-1)可明显抑制该作用。此外，在含钙缓冲液中，BN52021可显著抑制PAF引起的丝状肌动蛋白聚合。结论BN52021可能通过抑制丝状肌动蛋白的聚合作用，从而抑制PAF引起的巨噬细胞趋化，并且这种作用是钙依赖性的。表明BN52021的抗炎作用途径之一是抑制PAF诱导的趋化作用。     

Extract of motorcycle exhaust particles induced macrophages apoptosis by calcium-dependent manner

Large survey and experiments have reported that environment pollutants from fossil fuel combustion would cause immune system deleterious by enhancement of allergic reaction and damage to respiratory tract. In this study, we reported that the extract of motorcycle exhaust particles (MEP) might affect the immune system by inducing cell apoptosis on macrophages. The motorcycle exhaust particles were collected from a two-stoke engine and their cytotoxic effect on macrophages was investigated. We found MEP is cytotoxic and induced apoptosis in RAW 264.7 cells, murine peritoneal macrophage, and rat alveolar macrophage. Pretreatment with mitochondria permeability transition inhibitor (cyclosporin A), intracellular (BAPTA-AM) and extracellular (EGTA) Ca(2+) chelator, and antioxidants (NAC, GSH, catalase, SOD) attenuated the MEP-induced cell apoptosis, and BAPTA-AM was the most effective one. Utilized Fura-2/AM loaded RAW 264.7 cells to directly detect the change of intracellular Ca(2+) concentration ([Ca(2+)](i)), we found that MEP could induce a sustained increase of [Ca(2+)](i). The raise of [Ca(2+)](i) induced by MEP could be completely blocked by the intracellular Ca(2+) chelator, BAPTA-AM, however, only partially inhibited by the extracellular Ca(2+) chelator, EGTA. These results suggested that both influx of extracellular Ca(2+) and release of Ca(2+) from the internal storage were involved. We also found that MEP caused a decrease of mitochondria membrane potential and an increase of oxidative stress in RAW 264.7 cells. In conclusion, we found that the particles, collected from the motorcycle exhaust, contain chemicals that will induce apoptosis of macrophage in calcium-dependent manner.     

Phagocytic activity of ethyl alcohol fraction of deer antler in murine peritoneal macrophage.

The mechanism of phagocytic activity of the ethyl alcohol fraction of Cervus nippon (CN-E) was investigated in vivo. The administration of CN-E (100 mg/kg, p.o.) enhanced lucigenin chemiluminescence and the engulfment of fluorescein-conjugated E. coli particles in murine peritoneal macrophages. Phagocytic activity was suppressed by the treatment of S-nitrosoglutathione (GSNO) which is an exogenous nitric oxide donor depending on the concentration of dose. CN-E suppressed the production of nitric oxide and enhanced the concentration in [Ca2+]i. The enhancement in [Ca2+]i was diminished by the treatment of EGTA. These results indicate that CN-E enhances the phagocytic activity of murine peritoneal macrophage via a suppression of nitric oxide production and an increase in [Ca2+]i.     

Effect of azelastine on the intracellular Ca2+ mobilization in guinea pig peritoneal macrophages

Azelastine, an orally effective anti-allergic agent, has been demonstrated to inhibit the release of histamine and leukotrienes. This suggests that azelastine might alter the mobilization of intracellular Ca2+. We have examined the effect of azelastine on the change in intracellular free calcium concentration ([Ca2+])i) in guinea pig peritoneal macrophages induced by platelet activating factor (PAF-acether) or N-formylmethionyl-leucyl-phenylalanine (FMLP) using a fluorescent Ca2+ indicator, fura2. PAF-acether raised [Ca2+]i from 89 +/- 4 to 243 +/- 26 nM (n = 15) within 20 s after addition of PAF-acether in the presence of 2 mM EGTA. This indicates that the stimulation of macrophages by PAF-acether induced intracellular mobilization of Ca2+, and pretreatment with azelastine reduced the PAF-acether-induced increase in [Ca2+]i in a dose-dependent manner (IC50 = 16 microM). Azelastine also inhibited the FMLP-induced increase in [Ca2+]i. Furthermore, PAF-acether and FMLP both caused the release of prostaglandin E2 from macrophages, and pretreatment with azelastine reduced the PGE2 release dose dependently (IC50 = 10 microM). These results suggest that azelastine inhibits the release of Ca2+ from intracellular storage sites induced by PAF-acether or FMLP, and that this effect possibly causes reduction in the release of PGE2 from the cells.     

螺旋藻多糖对小鼠脾细胞中环腺苷酸浓度的影响

目的：对螺旋藻免疫调节作用的机理进行研究。方法：采用竞争性蛋白结合分析法，研究螺旋藻多糖（ Ｓｐｉｒｕｌｉｎａ ｐｌａｔｅｎｓｉｓ ｐｏｌｙｓａｃｃｈａｒｉｄｅｓ，ＳＰＰ） 对小鼠脾细胞中第二信使环腺苷酸（ｃＡＭＰ） 浓度的影响。结果：ＳＰＰ可剂量依赖性引起小鼠脾细胞中ｃＡＭＰ浓度的升高。结论：ＳＰＰ免疫调节作用的重要机制之一是对脾细胞中第二信使ｃＡＭＰ浓度的影响。关键词　螺旋藻多糖，环磷酸腺苷，脾细胞     

灵芝多糖体外对小鼠腹腔巨噬细胞pH的影响

目的　探讨灵芝多糖（ＧＬＰ）对免疫细胞信号转导过程的影响。方法　采用激光扫描共聚焦显微镜技术,动态监测ＧＬＰ均一体组分ＧＬＢ７对小鼠腹腔巨噬细胞（ＭΦ）胞内ｐＨ（［ｐＨ］ｉ）的影响。结果　ＧＬＢ７引起小鼠腹腔ＭΦ［ｐＨ］ｉ明显升高,［ｐＨ］ｉ的升高与Ｎａ＋／Ｈ＋交换系统及细胞［Ｃａ２＋］ｉ有关。结论　ＭΦ是灵芝多糖作用的靶细胞,ＧＬＢ７引起ＭΦ［ｐＨ］ｉ快速升高的现象,是灵芝多糖产生作用的重要途径。     

The inhibitory effect of ambroxol on respiratory burst, degranulation and cytosolic Ca2+ change in degraded immunoglobulin G-activated neutrophils

Superoxide and H2O2 production by neutrophils stimulated by 0.5 mg/ml degraded immunoglobulin G (IgG) and 1 microM N-formyl-methionyl-leucyl-phenylalanine (fMLP) was inhibited by ambroxol in a dose-dependent fashion, and at the concentration of 100 microM, 43.3% to 64.3% of inhibitions were detected. The inhibitory effect of ambroxol on H2O2 production by neutrophils was greater than that on superoxide production. The production of nitrite by lipopolysaccharide-activated murine peritoneal macrophages was significantly attenuated by ambroxol in a dose-dependent fashion and NG-monomethyl-L-arginine (NMMA). Ambroxol decreased the release of myeloperoxidase and lysozyme evoked by 0.5 mg/ml degraded immunoglobulin G and 1 microM fMLP in a dose-dependent fashion, and at the concentration of 100 microM, 37.1% to 64.2% of inhibitions were observed. The stimulatory effect of phorbol 12-myristate 13-acetate (PMA) (0.1 microg/ml) on superoxide production and myeloperoxidase, which is inhibited by 100 nM staurosporine, was not affected by 100 microM ambroxol. Degraded immunoglobulin G (0.5 mg/ml) caused an immediate elevation of [Ca2+]i in fura-2 load neutrophils in 1.23 mM Ca2+-containing medium. Preincubation of neutrophils with 10 microM to 100 microM ambroxol, 5 mM EGTA and 100 microM verapamil depressed the elevation of [Ca2+]i elicited by 0.5 mg/ml degraded immunoglobulin G. In conclusion, the inhibitory action of ambroxol on stimulated neutrophil responses, including respiratory burst and lysosomal enzyme release, appears to be attributed to its depressant action on the activation process, including the change in intracellular Ca2+ level. in which the role of protein kinase C is uncertain.     

毛木耳多糖对小鼠腹腔巨噬细胞蛋白激酶A活性的影响

目的 :观察毛木耳多糖 (APSP)体外对小鼠腹腔巨噬细胞 (MΦ)蛋白激酶A (PKA)活性的影响。方法 :采用反相离子对高效液相色谱法测定MΦ中PKA活力。结果 :毛木耳多糖 (80mg/L)能引起小鼠腹腔MΦ中PKA活性明显升高 ,8min达峰值 ,20min恢复到基础水平 ;8min毛木耳多糖与PKA活性的量效关系具有一定的浓度依赖性。结论 :毛木耳多糖的免疫增强作用与其增强MΦ中PKA活性有关。     

丁基苯酞对低糖低氧引起神经细胞内钙升高的作用

目的:探讨抗脑缺血新药丁基苯酞(NBP) 对脑缺血过程中细胞内游离钙升高的影响及其作用机制。方法:采用低糖低氧损伤胎鼠或新生鼠神经细胞以模拟脑缺血,用Fura-2/AM 作荧光指示剂,观察手性丁基苯酞对神经细胞内游离钙升高的抑制作用,并用内钙释放剂thapsigargin,外源性谷氨酸,高K+ 及钙离子载体A23187 作工具药,对其作用环节进行分析。结果:d-,l-,dl-丁基苯酞能完全抑制低糖低氧造成的神经细胞内钙升高。手性NBP能降低thapsigargin 引起的内质网钙库释放,d-NBP 还对谷氨酸引起的内钙升高表现出抑制作用。结论:丁基苯酞抑制低糖低氧条件下神经细胞内钙升高的作用环节主要是抑制细胞内钙库的释放。     

Differential sensitivity of macrophages to bradykinin

In this report, we investigated the responsiveness of subpopulations of elicited peritoneal macrophages between each other compared to resident tissue macrophages of alveoli of guinea pig to the action of bradykinin. Bradykinin stimulated the secretion of superoxide radical, arachidonic acid and prostaglandin E₂ (PGE₂) via the bradykinin B₂ receptor subtype in peritoneal macrophages, indicated by complete inhibitory effect of the bradykinin B₂ receptor antagonist HOE 140. The extent of the secretion, however, varied substantially between macrophages of different size. The highest level of the secretion was observed in the fraction containing the intermediate-size macrophages, while progressively lower level of the secretion was observed with decreasing size. In contrast, large macrophages obviously lost their secretory ability. Additionally, the bradykinin-stimulated release of cyclooxygenase products exerted an inhibitory action on NADPH-oxidase activity depending on size and stage of maturation/activation of macrophages, as judged by an increase in superoxide radical generation by indomethacin (100 μM) preincubation of cells. 2 receptor subtype. However, the receptor activity measured by bradykinin-induced increase in intracellular free calcium [Ca²⁺]_i was very low compared to elicited peritoneal macrophages. These findings indicate that the stage of differentiation/maturation and activation of macrophages may be important for the ability of bradykinin to stimulate these cells to inflammatory responses in vivo. Received: 27 June / Accepted: 17 October 1997