凝血酶对荧光标记的人单个血小板游离[Ca^2＋]和膜电位的动态影响

本文将ｆｌｕｏ－３和ｄ_ｉ－ＢＡ－Ｃ４（３）荧光标记的血小板固定于纤维蛋白原表面,以５７０型粘附式细胞仪（ＡＣＡＳ５７０）动态观察了凝血酶激活的人单个血小板细胞内游离［Ｃａ~（２＋）］（钙离子浓度）和膜电位的变化。静息状态时细胞游离［Ｃａ~（２＋）］和膜电位荧光较低,波动不明显。当加入０．１Ｕ／ｍｌ凝血酶激活时,［Ｃａ~（２＋）］（细胞内游离钙离子浓度）与膜电位迅速升高,随后［Ｃａ~（２＋）］出现反复振荡,幅度达约５００荧光单位,而膜电位基本上保持峰值水平。［Ｃａ~（２＋）］_ｉ升高与膜电位变化在时间和程度上不一致。本文结果提示,凝血酶引起血小板［Ｃａ~（２＋）］振荡和膜去极化,后者不是Ｃａ~（２＋）内流引起的。     

AG555对猪血小板胞浆钙离子浓度的影响

将荧光标记物Ｆｕｒａ２－ＡＭ参入到血小板中,利用荧光分光光度计检测胞浆钙离子浓度的变化来研究ＡＧ５５５（一种合成的酪氨酸蛋白激酶抑制剂）对猪血小板胞浆钙离子浓度的影响．结果发现ＡＧ５５５可降低血小板胞浆钙离子浓度,并对凝血酶诱导的胞浆钙离子浓度的升高有明显的抑制作用．ＡＧ５５５可能对血小板功能有一定的影响,这对于进一步阐明ＡＧ５５５的作用机理将有重要意义．     

凝血酶抑制神经细胞突起生长的研究

本实验研究了凝血酶抑制CHP-100原始神经外胚叶瘤细胞神经分化的信号传导机制。凝血酶能抑制CHP-100细胞在无血清培养中神经突起的生长,这种作用与凝血酶激活细胞内磷酸肌醇/钙离子信号传导途径有关。凝血酶明显刺激Ins(1,4,5)P3的产生及细胞内游离钙离子浓度的升高。凝血酶的抑制剂水蛭素能抑制凝血酶引起的钙离子反应,并能拮抗凝血酶抑制CHP-100细胞神经突起生长的作用。结果提示,凝血酶信号传导系统可能在神经系统生长发育中具有重要调节作用。     

荧光探针测定人血小板内游离钙浓度及抗钙药物的影响

Quin 2是一种对钙离子敏感的荧光素。它的乙酰化形式Quin2-AM具有亲脂性,可穿过细胞膜进入细胞内,水解后可特异性地与细胞内钙离子结合发出荧光。我们利用Quin 2对人血小板静息状态下及加凝血酶后激动状态下的血小板内钙离子浓度进行了     

竹红菌乙素光敏作用对于小鼠肝癌细胞内游离钙浓度的影响

本文用Quin 2/AM荧光探针作为细胞内部钙离子指示剂,研究了竹红菌乙素光敏损伤后引起小鼠腹水肝癌细胞的钙离子浓度变化。实验结果表明,细胞内的钙离子浓度随着乙素光敏作用增强而上升。并且钙离子浓度的升高与细胞的存活率下降呈正比关系;用数种单线态氧淬灭剂(L-His,NaN_3);羟自由基清除剂(PABGA)观察了乙素光敏过程中产生的活性氧与细胞内的钙离子浓度增加相关。用膜去极化方法研究了细胞在光敏损伤过程中钙离子浓度变化与去极化的关系。     

脉冲电场对鸡胚脑细胞Ca~(2+)和cAMP含量的影响

利用ｆｕｒａ－２／ＡＭ荧光方法和放射免疫方法分别研究了脉冲电场对１１天和１４天胚龄的悬浮鸡胚脑细胞内Ｃａ２＋和环腺苷酸（ｃＡＭＰ）含量的影响,并探讨了在脉冲电场作用下这两种信号物质变化的相关性。实验结果表明：当电激液中［Ｃａ２＋］为１ｍｍｏｌ／Ｌ时,在宽度为１００微秒．强度为０．５,１．０,２．０ｋＶ／ｃｍ的脉冲电场作用下,１１天胚龄的鸡胚脑细胞内钙离子浓度（以［Ｃａ２＋］ｉ表示）与对照相比显著上升,其升高值随着脉冲强度的增加而增加,而细胞内ｃＡＭＰ含量与对照相比显著降低;用ＥＧＴＡ络合电激液中游离Ｃａ２＋以后,虽然细胞内［Ｃａ２＋］ｉ显著上升,细胞内ｃＡＭＰ含量没有显著变化。１４天胚龄的鸡胚脑细胞内［Ｃａ２＋］ｉ在脉冲电场作用下显著上升,而细胞内ｃＡＭＰ含量没有显著变化。脉冲电场对１１天和１４天胚龄的鸡胚脑细胞膜和细胞内Ｃａ２＋库Ｃａ２＋转移系统的开放均有显著作用,此作用具有对脉冲强度的依赖性。为了解物理信号跨细胞膜传导的方式提供了初步的实验根据     

脉冲电场对鸡胚脑细胞Ca~(2 )和cAMP含量的影响

利用ｆｕｒａ－２／ＡＭ荧光方法和放射免疫方法分别研究了脉冲电场对１１天和１４天胚龄的悬浮鸡胚脑细胞内Ｃａ２＋和环腺苷酸（ｃＡＭＰ）含量的影响,并探讨了在脉冲电场作用下这两种信号物质变化的相关性。实验结果表明：当电激液中［Ｃａ２＋］为１ｍｍｏｌ／Ｌ时,在宽度为１００微秒．强度为０．５,１．０,２．０ｋＶ／ｃｍ的脉冲电场作用下,１１天胚龄的鸡胚脑细胞内钙离子浓度（以［Ｃａ２＋］ｉ表示）与对照相比显著上升,其升高值随着脉冲强度的增加而增加,而细胞内ｃＡＭＰ含量与对照相比显著降低;用ＥＧＴＡ络合电激液中游离Ｃａ２＋以后,虽然细胞内［Ｃａ２＋］ｉ显著上升,细胞内ｃＡＭＰ含量没有显著变化。１４天胚龄的鸡胚脑细胞内［Ｃａ２＋］ｉ在脉冲电场作用下显著上升,而细胞内ｃＡＭＰ含量没有显著变化。脉冲电场对１１天和１４天胚龄的鸡胚脑细胞膜和细胞内Ｃａ２＋库Ｃａ２＋转移系统的开放均有显著作用,此作用具有对脉冲强度的依赖性。为了解物理信号跨细胞膜传导的方式提供了初步的实验根据     

电刺激和低镁引起的大鼠内嗅皮层游离钙和钾离子浓度的动态变化

电刺激或降低细胞外的镁离子浓度,会引起Ｎ－甲基－Ｄ－天门冬氨酸受体通道的开放,造成胞内外离子浓度失平衡．使用离子选择性微电极结合脑片技术,对电刺激和低镁溶液引起的大鼠内嗅皮层游离钙和钾离子浓度及电位的动态变化的规律进行了研究。实验结果表明,电刺激和低镁溶液引起的内嗅皮层游离钙和钾离子浓度的改变,在细胞层Ⅳ－Ⅴ（皮层表面下９００－１１００μｍ）变化最大。低镁溶液引起游离钙离子浓度下降,同时钾离子浓度呈双相变化,即先增加后减少。低镁溶液灌流内嗅皮层脑片数小时后,胞外钙离子浓度持续地停留在低浓度水平,而钾离子浓度受影响较小．     

不同钙离子浓度对日本沼虾感光器细胞超微结构的影响

为了进一步研究细胞外钙离子浓度变化对甲壳动物感光细胞超微结构的影响,应用透射电子显微镜显示了日本沼虾感光细胞,在暗适应时高钙离子浓度中温育的感光器细胞的感杆束直径下降,微绒毛排列零乱;多囊体、板膜体数量增加;色素颗粒散布在细胞质中,呈现出光适应的结构特征。而温育在低钙离子溶液和生理溶液中的感光器细胞结构相同,呈现出暗适应的结构特征。另外,细胞器中储存的钙离子也受细胞外钙离子浓度的影响,在高钙离子溶液中温育后细胞器储存的钙离子量增加,膜下储泡囊、多囊体、线粒体、色素颗粒等细胞器中的焦锑酸钙结晶颗粒比温育在低钙溶液中的细胞明显增多。结果显示,细胞外钙离子浓度变化引起细胞内钙离子浓度变化,从而影响感光器细胞的结构而影响其生理功能。     

血小板激活时胞质游离钙浓度与两种颗粒数变化的相关性

用电镜形态计量法检测血小板α颗粒（αＧ）和致密颗粒（ｄＧ）的数密度,用钙荧光指示剂Ｆｕｒａ２检测血小板胞质游离Ｃａ＾２＋浓度（「Ｃａ＾２＋」ｉ）,观察到在钙离子导体Ａ２３１８７作用下,血小板「Ｃａ＾２＋」ｉ明显升高。凝血酶与ＡＤＰ也都分别引起「Ｃａ＾２＋」ｉ升高,且有浓度依赖性,选用三种激动剂的不同量以反映血小板不同程度激活时,测定「Ｃａ＾２＋」与颗粒数密度,分析两者间的相关性,发现αＧ和ｄＧ的数     

Flow cytometric measurements of cytoplasmic calcium changes in human platelets

Thrombin-induced stimulation of human platelets is accompanied by a dramatic increase in cytoplasmic calcium concentrations followed by a slow decrease. These changes are very rapid, are maximal by 10-15 s, and can be detected with probes such as Indo-1. Suspension studies using spectrofluorometry, which reflect a value which is the average of 3 x 10(7) cells per ml, indicate a thrombin dose-dependent increase in cytoplasmic calcium at doses up to 0.025 units per ml. We show here, using flow cytometry, that at less than half-saturating thrombin doses only subpopulations of platelets rather than the entire sample are responding. The extent of these responses, however, still depends on thrombin concentration. When the thrombin doses are between half and fully saturating, one subpopulation responds fully (i.e., its extent of increase in cytoplasmic calcium concentration, [Ca++]in is 100% of that seen at saturating thrombin concentrations) while the remaining platelets respond partially or not at all. There is thus evidence of positive cooperativity leading to disproportionate thrombin receptor occupancy on different subpopulations when platelets are subjected to subsaturating doses of thrombin. The existence of responding subpopulations may explain how the reported multiple stimulations of the same suspension of platelets at low thrombin doses occur.     

Erythropoietin increases cytosolic free calcium concentration and thrombin induced changes in cytosolic free calcium in platelets from spontaneously hypertensive rats

Using fura-2 cytosolic free calcium concentrations were measured in intact washed platelets from 9 spontaneously hypertensive rats (SHR) and from 9 age-matched normotensive Wistar-Kyoto rats (WKY). In resting platelets cytosolic free calcium concentration was significantly higher in SHR than in WKY (171.8 +/- 64.4 nM vs 93.1 +/- 59.0 nM, p less than 0.05). After preincubation with erythropoietin cytosolic free calcium concentration was significantly higher in SHR than in WKY (197.5 +/- 83.2 vs 93.0 +/- 60.1, p less than 0.01). Using platelets from SHR erythropoietin increased mean resting cytosolic free calcium concentration by 14.9% (p less than 0.05) and mean thrombin induced changes of cytosolic free calcium by 58.3% (p less than 0.01). In contrast, erythropoietin caused no significant increase in the resting calcium concentration or in thrombin induced changes of cytosolic free calcium in platelets from WKY. It is concluded that erythropoietin is involved in the pathogenesis of hypertension by elevating cytosolic free calcium concentration.     

Cytoplasmic free calcium concentration in porcine platelets. Regulation by an intracellular nonmitochondrial calcium pump and increase after thrombin stimulation

Mechanisms are assumed to exist in the resting platelet which maintain the concentration of cytoplasmic free calcium below that level required to activate cellular responses. To assess such processes the porcine platelet plasma membrane was selectively lysed with digitonin and the uptake (or flux) of free calcium monitored by an extracellular calcium electrode. Lysis resulted in an immediate lowering of the extracellular free calcium, due to the action of intracellular organelle(s) acting on the extracellular space through the permeabilized plasma membrane. In resting platelets, the rate of calcium uptake was first order with respect to the extracellular prelytic calcium concentration, and hence the cytoplasmic free concentration was found to be 1 X 10(-7) M by extrapolation to a point of zero flux (i.e., the null point). This approach could not be used with thrombin-stimulated platelets, as external calcium was required for both secretion of ATP + ADP and aggregation. Nevertheless, evidence for an increase in cytoplasmic free calcium after thrombin stimulation was obtained. Metabolic inhibitors and agents known to inhibit calcium uptake by mitochondria had no effect on the calcium flux following lysis, indicating different mechanisms for calcium homeostasis in the platelet when compared with other cell types (e.g., liver). Levels of ionophore A23187, which caused platelet aggregation, gave a massive release of the nonmitochondrial pool of calcium into the cytoplasmic space. Thus, in porcine platelets an intracellular energy-requiring calcium pump, which sequesters calcium in a nonmitochondrial membranous compartment, is crucial for intracellular calcium homeostasis.     

氨对脑细胞胞浆游离钙含量的影响

目的与方法：采用Ｆｕｒａ－２／ＡＭ探针技术观察ＮＨ４Ｃｌ对离体急性分离之Ｗｉｓｔａｒ乳鼠大脑细胞胞头游离钙「Ｃａ＾２＋」ｉ含量的影响。结果：ＮＨ４＾＋浓度为２．５ｍｍｏｌ／Ｌ时脑细胞内「Ｃａ＾２＋」ｉ含量升高。在一定范围内,随着ＮＨ４＾＋浓度的加大,细胞内Ｃａ＾２＋持续升高。ＮＨ４＾＋的升钙作用主要被Ｎｉｃａｒｄｉｐｉｎｅ所阴断,其变化特征类以ＫＣｌ。结论：ＮＨ４＾＋主要通过影响电压依赖性钙离子通     

Intracellular calcium mobilization is triggered by clustering of membrane glycoproteins in concanavalin A-stimulated platelets

Stimulation of human platelets with concanavalin A resulted in a significant increase in the concentration of cytoplasmic free Ca²⁺. This effect was due to two different processes: Ca²⁺ mobilization from internal stores and Ca²⁺ influx from the extracellular medium. Kinetic analysis revealed that the release of Ca²⁺ from internal storage sites occurred sooner than the opening of plasma membrane Ca²⁺ channels. The ability of concanavalin A to induce a sustained increase in cytoplasmic Ca²⁺ concentration was antagonized and reversed by methyl ∝-D -mannopyranoside, demonstrating that it was promoted by the interaction of the lectin with cell surface glycoproteins. Succinyl–concanavalin A, a dimeric derivative of the lectin, that does not promote patching/capping of the receptor, was able to bind to the platelet surface, and antagonized the effects of native concanavalin A. In addition, succinyl–concanavalin A, per se, was unable to induce Ca²⁺ mobilization in human platelets. Therefore, the action of the native concanavalin A was mediated by receptor clustering events. Concanavalin A mobilized Ca²⁺ from the same internal stores from which Ca²⁺ was mobilized in response to strong platelet agonists, such as thrombin and arachidonic acid. However, while thrombin was ineffective in inducing Ca²⁺ release after stimulation of platelets with Con A, Con A was able to cause a full discharge of Ca²⁺ from internal stores even in platelets previously stimulated with thrombin. These results demonstrate for the first time that the clustering of specific membrane glycoproteins can trigger platelet activation. The physiological implications during platelet aggregation are discussed.     

Cytoplasmic Ca2+ in platelets is controlled by cyclic AMP: antagonism between stimulators and inhibitors of adenylate cyclase

Activation of platelets by thrombin rapidly increases cytoplasmic free calcium, [Ca2+]i, measured by Quin -2, and induces secretion. Stimulators of adenylate cyclase (i.e. PGI2, PGD2, forskolin) suppressed or reversed the increase of [Ca2+]i. Inhibitors of adenylate cyclase (i.e. epinephrine, ADP), added before or after thrombin, counteracted PGI2, PGD2 and forskolin and thereby increased [Ca2+]i and restored secretion. Responses to epinephrine (via alpha-2 adrenoreceptors) and ADP were independent of extracellular Ca2+, but required maintained occupancy of thrombin receptors and intact cAMP-phosphodiesterase activity. These results indicate that cAMP serves as an inhibitory second-messenger that antagonizes the mobilization of Ca2+, an activator second-messenger.     

Relationship between cytoplasmic free calcium and myosin light chain phosphorylation in intact platelets.

Human platelets were prepared and loaded with the fluorescent Ca2+ indicator quin2. The relation between cytoplasmic free calcium concentration, [Ca2+]i, and the extent of the phosphorylation of myosin light chains of Mr 20 000 could then be examined. When the calcium ionophore ionomycin is used to stimulate platelets, little phosphorylation is seen until [Ca2+]i exceeds 400 nM; half-maximal response occurs at 600 nM with a full response at about 1 microM-[Ca2+]i. Under optimal conditions, physiological stimuli such as platelet-activating factor and thrombin can increase [Ca2+]i to sufficiently high levels [Rink, Smith & Tsien (1982) FEBS Lett. 148, 21-26; Hallam, Sanchez & Rink (1984) Biochem. J. 218, 819-827] that Ca2+ ions could be the trigger for the myosin phosphorylation evoked by these agonists. However, in this paper we show that, in the absence of external calcium, platelet-activating factor and thrombin can stimulate myosin phosphorylation while [Ca2+]i remains at levels which are well below those needed when the calcium ionophore is the stimulus. This observation suggests that myosin light chain phosphorylation may be controlled by an additional pathway.     

Inhibitory effects of phenol derivatives on bovine platelet aggregation and their effects on Ca2+ mobilization

The effects of phenol derivatives on aggregation of bovine platelets induced by ADP, thrombin, platelet activating factor, collagen and A23187 were investigated. The phenol derivatives inhibited all these induced aggregations except that by the calcium ionophore. The derivatives each inhibited the aggregations induced by ADP, thrombin, platelet activating factor and collagen, respectively, within a similar concentration range. A linear relation was found between the inhibitory potencies of the phenol derivatives and their partition coefficients between n-octanol and water (Poct values), suggesting that their interaction with hydrophobic regions of the cell was important for inhibition. Fluorescence analyses with fura-2-loaded platelets showed that in the concentration ranges in which the phenol derivatives inhibited aggregation, they also inhibited agonist-induced increases in Ca2+ both in the presence and absence of extracellular Ca2+. Moreover, a high correlation was found between the inhibitory effects of the derivatives on aggregation and their effects on Ca2+ mobilization. These results suggest that inhibition of platelet aggregation by phenol derivatives is mainly due to inhibition of the increase in cytoplasmic Ca2+ by inhibition of both intracellular Ca2+ mobilization and Ca2+ uptake.     

Cytoplasmic free calcium concentration in porcine platelets: Regulation by an intracellular nonmitochondrial calcium pump and increase after thrombin stimulation

Mechanisms are assumed to exist in the resting platelet which maintain the concentration of cytoplasmic free calcium below that level required to activate cellular responses. To assess such processes the porcine platelet plasma membrane was selectively lysed with digitonin and the uptake (or flux) of free calcium monitored by an extracellular calcium electrode. Lysis resulted in an immediate lowering of the extracellular free calcium, due to the action of intracellular organelle(s) acting on the extracellular space through the permeabilized plasma membrane. In resting platelets, the rate of calcium uptake was first order with respect to the extracellular prelytic calcium concentration, and hence the cytoplasmic free concentration was found to be 1·10^?7 M by extrapolation to a point of zero flux (i.e., the null point). This approach could not be used with thrombin-stimulated platelets, as external calcium was required for both secretion of ATP + ADP and aggregation. Nevertheless, evidence for an increase in cytoplasmic free calcium after thromin stimulation was obtained. Metabolic inhibitors and agents known to inhibit calcium uptake by mitochondria had no effect on the calcium flux following lysis, indicating different mechanisms for calcium homeostasis in the platelet when compared with other cell types (e.g., liver). Levels of ionophore A23187, which caused platelet aggregation, gave a massive release of the nonmitochondrial pool of calcium into the cytoplasmic space. Thus, in porcine platelets an intracellular energy-requiring calcium pump, which sequesters calcium in a nonmitochondrial membranous compartment, is crucial for intracellular calcium homeostasis.