白细胞介素-2对缺氧/复氧心肌细胞[Ca2+]i的作用

目的:观察白细胞介素-2(IL-2)对心肌细胞在缺氧/复氧过程中电刺激诱导的[Ca²⁺]i的作用。方法:采用酶解分离成年大鼠心室肌细胞化学缺氧模型, 以Fura-2/AM为钙探针, 用细胞内双波长钙荧光系统检测心肌[Ca²⁺]i的变化。结果:①缺氧/复氧过程中, 缺氧5min时, 心肌[Ca²⁺]i幅度降低、舒张末期[Ca²⁺]i升高, [Ca²⁺]i达峰时间(TTP)延长, 恢复时间(RT)延长。复氧10min后, 心肌[Ca²⁺]i幅度、舒张末期[Ca²⁺]i、TTP及RT逐渐回复, 但不能完全恢复到对照水平;②在缺氧期间加入IL-2(2×10⁵U/L), 复氧期间[Ca²⁺]i各参数回复减慢;③用κ-阿片受体拮抗剂nor-BNI(10^-8mol/L)预处理后, 缺氧+IL-2对复氧时[Ca²⁺]i作用的影响被减弱, 而δ-阿片受体拮抗剂纳曲吲哚(10^-6mol/L)预处理则无此作用。结论:缺氧时同时存在IL-2, 可加剧复氧时心肌[Ca²⁺]i的变化, 其机制可能是IL-2通过心肌κ-阿片受体而发挥作用。     

噻庚啶和山莨菪碱拮抗TNFα诱导的内皮细胞[Ca2+]i增高

目的：研究噻庚啶（Cyp）和山莨菪碱（Ani）对肿瘤坏死因子（TNFα）诱导单个内皮细胞内Ca²⁺浓度（[Ca²⁺]_i）变化的影响，以探TNFα介导休克和Cyp、Ani的抗休克的机制。方法：人脐静脉内皮细胞株（ECV304）接种于35 mm含2 mL DMEM培养基的组织培养盘中培养。Fluo-3/AM负载细胞，激光扫描共聚焦显微技术（LSCM）测定单个内皮细胞[Ca²⁺]_i。结果：TNFα使单个内皮细胞[Ca²⁺]_i呈剂量依赖性升高，在60 s内达到峰值，然后下降并保持在基础水平之上。共聚焦扫描图像显示细胞核区[Ca²⁺]_i升高比胞浆区明显，下降比胞浆区慢。Cyp（3×10^-5 mol/L或6×10^-5 mol/L）、Ani（2×10^-5 mol/L或4×10^-5 mol/L）均能显著抑制由TNFα（1.2×10^-9 mol/L）诱导的单个内皮细胞[Ca²⁺]_i升高。结论：TNFα诱导内皮细胞[Ca²⁺]_i升高可能是TNFα介导休克的重要机制；Cyp和Ani抑制TNFα诱导的[Ca²⁺]_i升高可能是其抗休克作用的机制之一。     

红景天苷促进培养乳鼠心肌细胞肌质网钙离子的释放

目的: 观察红景天苷对乳鼠心肌细胞胞浆Ca²⁺浓度的影响并分析其可能的作用机制。方法: 应用荧光指示剂Fluo-3/AM负载培养大鼠乳鼠的心肌细胞,用激光共聚焦显微镜动态观察胞内游离钙荧光信号强度的变化,检测不同浓度红景天苷对培养心肌细胞胞内游离钙离子浓度([Ca²⁺]_i)的影响。结果: 红景天苷浓度为15 mg/L、30 mg/L和60 mg/L时,细胞内的平均[Ca²⁺]_i升高,峰值分别为574.08±4.65、591.86±3.64和618.66±4.27(均P<0.01);有剂量依赖性而无时间依赖性。用维拉帕米阻断细胞膜外钙内流时,红景天苷同样引起细胞内[Ca²⁺]_i升高,峰值由357.74±3.13、387.17±2.37和391.43±1.34分别上升到480.86±3.98、496.70±3.08和522.18±3.19(均P<0.01)。结论: 红景天苷能升高乳鼠心肌细胞中[Ca²⁺]_i,其机制可能与其促进肌浆网钙离子释放有关。     

Salusins对大鼠离体心脏及心肌细胞的生物学效应的研究

目的:研究新发现的心血管活性肽salusin的心脏及心肌细胞的生物学效应。方法:利用Langendorff装置灌流的成年大鼠离体心脏及原代培养的乳鼠心肌细胞,测定心功能和心肌细胞[⁴⁵Ca²⁺]摄入及[³H]-亮氨酸([³H]-Leu)掺入量。结果:10^-12-10^-7mol/L salusin-α及salusin-β对成年大鼠离体心脏功能无明显影响;但10^-10-10^-6mol/L salusin-α及salusin-β均呈浓度依赖性地促进心肌细胞[⁴⁵Ca²⁺]摄入及[³H]-Leu掺入,其效应在10^-8mol/L时达峰值;salusin-α及salusin-β对心肌细胞[⁴⁵Ca²⁺]摄入的效应可被钙通道阻断剂尼卡地平(nicardipine)所拮抗,且与内皮素有协同效应。 Salusin-α及salusin-β对心肌细胞[³H]-Leu掺入的效应可被尼卡地平、钙调磷酸酶抑制剂FK506、丝裂原活化蛋白激酶(MAPK)阻断剂PD₉₈₀₅₉及蛋白激酶C(PKC)阻断剂chelerthine chloride等不同程度抑制。Salusin-β对心肌细胞 摄入的效应强于salusin-α,但[³H]-Leu掺入的效应两者之间无显著性差异。结论:Salusin-α及salusin-β对成年大鼠离体心脏功能无直接效应,但能促进乳鼠心肌细胞钙摄入及蛋白合成,其效应可能与钙通道、钙调神经磷酸酶、MAPK和PKC等信号转导途径有关。Salusin可能具有心肌生长、肥大的调节的作用。     

丹参对抗缺氧/复氧引起的大鼠心室肌细胞收缩和细胞内钙的改变

目的:观察丹参在常氧和缺氧/复氧过程中对心肌细胞收缩和电刺激诱导的细胞内钙([Ca²⁺]_i)瞬态的影响。方法: 采用酶解分离成年大鼠心室肌细胞化学缺氧模型, 用视频跟踪计算机系统和细胞内双波长钙荧光系统分别观察心肌细胞收缩力学和[Ca²⁺]_i等指标。结果:丹参(1-9 g/L)处理后降低心肌细胞最大收缩和舒张速率、收缩幅度以及电刺激诱导的[Ca²⁺]_i幅度, 且呈剂量依赖性。缺氧后, 与对照相比细胞收缩力和钙瞬态幅度降低、舒张末细胞长度缩短、舒张末钙水平增高;复氧后细胞收缩力、钙瞬态幅度和舒张末钙水平有所回复, 但不能达对照水平。用3 g/L的丹参处理后, 缺氧/复氧引起的心肌细胞最大收缩和舒张速率、收缩幅度和电刺激诱导的[Ca^2＋]_i幅度高于单纯缺氧组, 舒张末[Ca²⁺]_i水平低于单纯缺氧组。结论:丹参可对抗缺氧/复氧引起的大鼠心室肌细胞收缩力降低和细胞内动态和静态钙的变化。     

虎杖甙对心肌细胞钙的调节作用

目的:观察虎杖甙(polydatin,PD)对大鼠心肌细胞内游离钙浓度([Ca²⁺]i)的影响,以探讨PD增强心肌细胞收缩性的作用机制。方法:用荧光染料Fluo-3-AM标记细胞,在粘附式细胞仪上测定细胞[Ca²⁺]i的变化。结果:给予PD后心肌[Ca²⁺]i升高。但不同细胞群的[Ca²⁺]i升幅不一致。10min后有些细胞[Ca²⁺]i仅升至起始浓度的111.80%±2.22%,有些细胞内钙则急升到最初的224.00%±24.33%。当先用EGTA或维拉帕米分别预孵育10min后再加PD,细胞[Ca²⁺]i则呈持续下降趋势,10min后[Ca²⁺]i下降至起始浓度的53.00%±9.20%和52.00%±7.07%。给予TTX预处理后再加PD,[Ca²⁺]i也下降为起始值的72.67%±12.70%。与单纯PD作用相比都有显著差异。结论:PD可通过增加心肌[Ca²⁺]i而增强心肌收缩性,其作用可能与钙、钠通道开放有关。     

OX-LDL及辛伐他汀对平滑肌细胞PKC活性和胞内Ca2+的影响

目的:探讨OX-LDL是否对大鼠主动脉平滑肌细胞(ASMC)蛋白激酶C(PKC)活性和胞浆内游离钙([Ca²⁺]i)水平有影响。方法:应用γ-[³²P]-ATP磷酸转移法和Fluo-3/Am荧光负载、流式细胞术分别检测PKC活性和胞内([Ca²⁺]i)水平。结果:OX-LDL呈剂量依赖方式促进ASMCPKC总活性增加,并可引起ASMC中PKC发生浆膜的转移。胞浆内[Ca²⁺]i以2个时相升高,即快速相和持续相。而辛伐他汀能明显抑制OX-LDL引起的ASMC中PKC活性的浆膜转移,并显著降低持续相胞浆内钙水平,而对快速相无影响。结论:OX-LDL能引起ASMC内信号通路PKC及[Ca²⁺]i的动态变化,二者密切相关。     

心肌缺血再灌注损伤时细胞核膜钙泵功能的改变

目的:研究心肌缺血再灌注损伤时心肌细胞核膜钙泵(Ca²⁺-ATPase)功能的改变。方法:采用大鼠心肌缺血再灌注模型,密度梯度离心分离纯化细胞核,定磷法测定Ca-ATPase活性,[⁴⁵Ca²⁺]直接测定核钙摄取。结果:缺血再灌注损伤动物血浆MDA和FFA显著高于正常大鼠(P<0.01),再灌注心肌细胞核膜Ca-ATPase在[Ca²⁺]i较低时,酶活力低于正常细胞,而在[Ca²⁺]i较高浓度50μmol/L时酶活力高于正常细胞。对[⁴⁵Ca²⁺]摄取的变化与Ca-ATPase活性变化相似。结论:心肌缺血再灌注损伤时核膜Ca²⁺-ATPase功能发生了改变。     

低氧后心肌钙瞬变变化与β-肾上腺素受体脱敏的关系

目的:慢性低氧时,心脏对β-肾上腺素受体激动的反应出现脱敏现象,细胞内钙[Ca²⁺]_i瞬变的幅度降低、时程延长,本研究观察上述变化在低氧时发生和发展的时间过程和对应关系。方法:在年龄对等的正常及慢性低氧1d、3d、1周、2周、3周、4周和8周的大鼠,分别分离正常及慢性低氧条件下的心室肌细胞,以Fura-2为[Ca²⁺]_i的指示剂,用光谱荧光法测定心肌细胞的[Ca²⁺]_i瞬变及其对心肌β-受体激动后反应的变化。结果:在低氧1d、3d、1周等时间内,电刺激引起的[Ca²⁺]_i瞬变、咖啡因引起的[Ca²⁺]_i瞬变及电刺激引起的[Ca²⁺]_i瞬变对β-受体激动剂异丙肾上腺素的增加反应无明显变化;在低氧2周以上时,电刺激引起的[Ca²⁺]_i瞬变的幅度开始降低,而时程开始延长,其对异丙肾上腺素的反应也开始降低。咖啡因引起的[Ca²⁺]_i瞬变幅度也开始降低;在低氧3周和4周时,上述变化程度逐渐加重;在低氧8周时各参数的变化有所恢复,但与4周时的变化程度无明显差异。结论:低氧2-4周时,心脏的β-肾上腺素受体发生脱敏现象,脱敏的机制与3种调节[Ca²⁺]_i瞬变的蛋白质:L-型钙通道、ryanodine受体操纵的钙通道和钙泵等活性的降低有关,后者也可能是低氧时心脏功能降低的重要机制。低氧4-8周时,心脏对低氧产生了适应和代偿。     

钙离子在半边旗提取物6F的细胞毒及诱导HL-60细胞DNA片段化中的作用

目的:研究蕨类植物半边旗提取物6F对HL-60细胞内游离钙浓度([Ca²⁺]_i)及Bcl-2蛋白表达的影响, Ca²⁺与6F细胞毒作用及诱导细胞DNA片段化的关系。方法:用荧光探针Fura-2/AM标记细胞内游离Ca²⁺, 在荧光分光光度计上测[Ca²⁺]_i;流式细胞仪检测Bcl-2的表达;噻唑蓝(MTT)法测定细胞成活率;二苯胺法测DNA片段化形成率。结果:6F作用后, HL-60细胞内[Ca²⁺]_i显著升高, 呈明显的时间剂量效应关系;6F降低Bcl-2的表达;于培养基中加2mmol/LCa²⁺、或加1mmol/LEDTA络合细胞外钙, 或加4μmol/L钙通道A₂₃₁₈₇升高细胞内钙浓度, 均可增强6F的细胞毒作用, 但均不影响6F诱导细胞DNA片段化程度。加入250μmol/LZn²⁺可显著降低6F所致DNA片段化率, 并可增强6F的细胞毒作用。结论:化合物6F可显著升高HL-60细胞[Ca²⁺]_i, 推测与6F降低Bcl-2的表达有关;6F诱导HL-60细胞DNA片段化可能是通过Ca²⁺-非依赖性DNA酶的作用所致。     

白细胞介素-2改变心肌细胞钙瞬态的频率依赖性

目的:观察白细胞介素-2(IL-2)对大鼠心肌细胞内钙处理能力的影响。方法:采用细胞内双波长钙荧光系统检测稳定状态及不同电刺激频率下单个心肌细胞钙瞬态的变化。结果:在稳定状态(0.2Hz)下, 2×10⁵U/LIL-2抑制心肌细胞钙瞬态的幅度和峰值, 使舒张末钙水平升高, 钙瞬态下降相时间常数延长。细胞外钙浓度为1.25mmol/L时, 心肌细胞舒张末钙水平、钙瞬态的幅度均随刺激频率(0.2-1.0Hz)的增高而增高。IL-2抑制了这种正性的频率依赖关系, 当将外钙浓度增加至2.5mmol/L时并不能改变其抑制作用。咖啡因诱导的内贮钙释放也呈现正性的频率依赖关系, 2×10⁵U/LIL-2抑制其频率依赖关系。在细胞外钙浓度为1.25mmol/L时, 两组间的钙瞬态机械恢复特性无差异, 当外钙为2.5mmol/L时, IL-2组的恢复率减慢。结论:IL-2通过抑制肌浆网内贮钙的释放而抑制了细胞内钙瞬态的正性频率依赖关系。     

The nuclear envelope of resting C6 glioma cells is able to release and uptake Ca2+ in the absence of chemical stimulation

 Many agonists evoke events in the cell nucleus through the control of Ca²⁺ signals. Recent studies using isolated nuclei have indicated that the nuclear envelope is a store for nuclear Ca²⁺. However, the release of Ca²⁺ directly from the nuclear envelope in living cells has never been reported. In the present study, we have investigated the changes of Ca²⁺ signals at the cyto-nucleoplasmic interface of rat C6 glioma cells using confocal microscopy. Digital imaging indicates that fluo-3, a Ca²⁺-sensitive fluorescent probe, was concentrated in or around the nuclear envelope. Our experiments also revealed that C6 cells at rest produced spontaneous Ca²⁺ spikes in the absence of chemical stimulation. The amplitude of the repetitive Ca²⁺ spikes was higher at the nuclear envelope than in the whole cell or cytosol. After image subtraction, circular rims of Ca²⁺ release and uptake were seen at the outer boundary of the nucleus. When the cells were treated with thapsigargin (2 μM), a specific Ca²⁺-ATPase inhibitor, a long-lasting Ca²⁺ release was observed at the nuclear envelope. Moreover, most of the released Ca²⁺ was directed inwardly to the nucleoplasm with little outward diffusion. Our results thus indicate: (1) that the nuclear envelope is a Ca²⁺ store that possesses the ability to discharge and sequestrate Ca²⁺; and (2) the Ca²⁺-releasing channels are present in the inner nuclear membrane. Received: 21 July 1997 / Received after revision: 22 September 1997 / Accepted: 23 September 1997     

Mechanism of action of the new cardiotonic suphan on calcium exchange in cardiomyocytes

Effects of suphan, a new cardiotonic agent containing succinyl tryptophan, on the entry of Ca²⁺ into rat cardiomyocytes, its intracellular compartmentalization, and its exit from these cells were evaluatedin vitro. It was found that the recorded sulfan-induced rise of intracellular calcium was due to Ca²⁺ entering the cell via L-type calcium channels, and that a reversible reduction of its concentration in the sarcoplasm occurred through its accumulation in the sarcoplasmic reticulum and was blocked by the specific Ca²⁺-ATPase inhibitor thapsigargin (10 μM). Suphan did not alter the activity of Na⁺/Ca²⁺ exchange in a concentration range of 5–150 μg/ml. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 122, No. 7, pp. 57–59, July, 1996     

How Does the Ca2+-paradox Injury Induce Contracture in the Heart?—A Combined Study of the Intracellular Ca2+ Dynamics and Cell Structures in Perfused Rat Hearts—

The calcium (Ca²⁺)-paradox injury of the heart, induced by restoration of extracellular Ca²⁺ after its short-term depletion, is known to provoke cardiomyocyte contracture. However, undetermined is how the Ca²⁺-paradox provokes such a distinctive presentation of myocytes in the heart. To address this, we imaged sequential intracellular Ca²⁺ dynamics and concomitant structures of the subepicardial ventricular myocytes in fluo3-loaded, Langendorff-perfused rat hearts produced by the Ca²⁺ paradox. Under rapid-scanning confocal microscopy, repletion of Ca²⁺ following its depletion produced high-frequency Ca²⁺ waves in individual myocytes with asynchronous localized contractions, resulting in contracture within 10 min. Such alterations of myocytes were attenuated by 5-mM NiCl₂, but not by verapamil, SEA0400, or combination of ryanodine and thapsigargin, indicating a contribution of non-specific transmembrane Ca²⁺ influx in the injury. However, saponin-induced membrane permeabilization of Ca²⁺ showed no apparent contracture despite the emergence of high-frequency Ca²⁺ waves, indicating an essential role of myocyte-myocyte and myocyte-extracellular matrix (ECM) mechanical connections in the Ca²⁺ paradox. In immunohistochemistry Ca²⁺ depletion produced separation of the intercalated disc that expresses cadherin and dissipation of β-dystroglycan located along the sarcolemma. Taken together, along with the trans-sarcolemmal Ca²⁺ influx, disruption of cell-cell and cell-ECM connections is essential for contracture in the Ca²⁺-paradox injury.     

The role of sarcoplasmic reticulum and sarcoplasmic reticulum Ca2+-ATPase in the smooth muscle tone of the cat gastric fundus

Circular smooth muscle strips isolated from cat gastric fundus were studied in order to understand whether the sarcoplasmic reticulum (SR) and SR Ca²⁺-ATPase could play a role in the regulation of the muscle tone. Cyclopiazonic acid (CPA), a specific inhibitor of SR Ca²⁺-ATPase, caused a significant and sustained increase in muscle tone, depending on the presence of extracellular Ca²⁺. Nifedipine and cinnarizin only partially suppressed the CPA-induced tonic contraction. Bay K 8644 antagonized the relaxant effect of nifedipine in CPA-contracted fundus. Nitric-oxide-releasing agents sodium nitroprusside and 3-morpholino-sydnonimine completely suppressed the CPA-induced tonic contraction. The blockers of Ca²⁺-activated K⁺ channels, tetraethylammonium, charybdotoxin and/or apamin, decreased the contractile effect of CPA. Vanadate increased the tone but did not change significantly the effect of CPA. CPA exerted its contractile effect even when Ca²⁺ influx was triggered through the Na⁺/Ca²⁺ exchanger and the other Ca²⁺ entry pathways were blocked. Thapsigargin, another specific SR Ca²⁺-ATPase inhibitor, also increased the muscle tone. The effect of thapsigargin was completely suppressed by sodium nitroprusside and 3-morpholino-sydnonimine and partially by nifedipine. In conclusion, under conditions when the SR Ca²⁺-ATPase is inhibited, the tissue develops a strong tonic contraction and a large part of this is mediated by Ca²⁺ influx presumably via nifedipine-sensitive Ca²⁺ channels. This study suggests the important role of SR Ca²⁺-ATPase in the modulation of the muscle tone and the function of SR as a “buffer barrier” to Ca²⁺ entry in the cat gastric fundus smooth muscle. Received: 10 August 1995/Received after revision: 9 November 1995/Accepted: 10 November 1995     

Acetylcholine-induced Ca2+ oscillations are modulated by a Ca2+ regulation of InsP3R2 in rat portal vein myocytes

Oscillations of cytosolic Ca²⁺ levels are believed to have important roles in various metabolic and signalling processes in many cell types. Previously, we have demonstrated that acetylcholine (ACh) evokes Ca²⁺ oscillations in vascular myocytes expressing InsP₃R1 and InsP₃R2, whereas transient responses are activated in vascular myocytes expressing InsP₃R1 alone. The molecular mechanisms underlying oscillations remain to be described in these native smooth muscle cells. Two major hypotheses are proposed to explain this crucial signalling activity: (1) Ca²⁺ oscillations are activated by InsP₃ oscillations; and (2) Ca²⁺ oscillations depend on the regulation of the InsP₃R by both InsP₃ and Ca²⁺. In the present study, we used a fluorescent InsP₃ biosensor and revealed that ACh induced a transient InsP₃ production in all myocytes. Moreover, steady concentrations of 3F-InsP₃, a poorly hydrolysable analogue of InsP₃, and pharmacological activation of PLC evoked Ca²⁺ oscillations. Increasing cytosolic Ca²⁺ inhibited the ACh-induced calcium oscillations but not the transient responses and strongly reduced the 3F-InsP₃-evoked Ca²⁺ response in oscillating cells but not in non-oscillating cells. These results suggest that, in native vascular myocytes, ACh-induced InsP₃ production is transient and Ca²⁺ oscillations depend on a Ca²⁺ modulation of InsP₃R2. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Maxi K+ channels in the basolateral membrane of the exocrine frog skin gland regulated by intracellular calcium and pH

With the single-channel patch-clamp technique we have identified Ca²⁺-sensitive, high-conductance (maxi) K⁺ channels in the basolateral membrane (BLM) of exocrine gland cells in frog skin. Under resting conditions, maxi K⁺ channels were normally quiescent, but they were activated by muscarinic agonists or by high serosal K⁺. In excised inside-out patches and with symmetrical 140 mmol/l K⁺, single-channel conductance was 200 pS and the channel exhibited a high selectivity for K⁺ over Na⁺. Depolarization of the BLM increased maxi K⁺ channel activity. Increasing cytosolic free Ca²⁺ (by addition of 100 nmol/l thapsigargin to the bathing solution of cell-attached patches also increased channel activity, whereas thapsigargin had no effect when added to excised inside-out patches. An increase in cytosolic free Ca²⁺ directly activated channel activity in a voltage-dependent manner. Maxi K⁺ channel activity was sensitive to changes in intracellular pH, with maximal activity at pH 7.4 and decreasing activities following acidification and alkalinization. Maxi K⁺ channel outward current was reversibly blocked by micromolar concentrations of Ba²⁺ from the cytosolic and extracellular site, and was irreversibly blocked by micromolar concentrations of charybdotoxin and kaliotoxin from the extracellular site in outside-out patches.