Cytosolic free calcium concentration and intracellular calcium distribution in lymphocytes from cystic fibrosis patients

Lymphocytes prepared from normal individuals and patients with cystic fibrosis (CF) were compared with regard to intercellular Ca²⁺ concentration, distribution, and handling. No difference between control and CF was found in the concentration of cytosolic free Ca²⁺ ($\text{98 ± 5}\text{vs}\text{102 ± 7}\text{nM}$), and no difference was observed in the kinetics with which control and CF cells restored cytoplasmic Ca²⁺ toward normal following a perturbation induced by cold-exposure. However, total intracellular Ca²⁺ is about 25% higher in CF lymphocytes than in control. Of this excess Ca²⁺, about 50% appears to be sequested in mitochondria. This suggests that some difference in Ca²⁺ handling does exist, but the significance of this cystic fibrosis remains to be determined.     

Slow oscillations of free intracellular calcium ion concentration in human fibroblasts responding to mechanical stretch

Calcium transients in single, human gingival fibroblasts were studied after mechanical stretching of flexible culture substrates. A model system was developed to reproducibly stretch and rapidly (< 1 sec) refocus cells in the same focal plane so that changes in the concentration of free intracellular calcium ions ([Ca²⁺]_i) were monitored without delay. Attached cells were grown on flexible bottom Petriperm dishes, loaded with fura-2/AM, and stretched by 1% or 2.8% of substrate area. The stretch caused no significant cell detachment or membrane lesions. A 1% stretch induced no calcium response, but a 2.8% stretch stimulated an initial calcium transient and the subsequent generation of [Ca²⁺]_i oscillations of up to 2,000 sec. At 1% stretch, there was no calcium response. Cell shape and plating time were important determinants in the calcium response to mechanical stimulation: the responder cells were small and round without long processes. Major calcium transients were inhibited completely by 5 mM EGTA or by 10 μM gadolinium ions, by 50 μM nifedipine, or 250 μM verapamil, suggesting an influx of calcium through stretch-activated (SA) channels and L-type calcium channels. Depolarization by high KCl (144 mM) in the extracellular medium enhanced the amplitude of calcium transients by 54%. Calcium oscillations were not inhibited by preincubation with thapsigargin, caffeine, cholera toxin, staurosporine or 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), indicating that IP₃ sensitive pools, IP₃ insensitive pools, G₅α subunits, and protein kinase C, respectively, were not involved in the generation of calcium oscillations. Pretreatment with genistein, a specific tyrosine kinase inhibitor or cytochalasin D, an inhibitor of actin polymerization, or pertussis toxin, an inhibitor of G_iα and G_oα subunits, completely abolished calcium transients and oscillations. These results indicate that Ca²⁺ flux due to mechanical stretching is likely mediated through SA ion channe s and is dependent on tyrosine kinases, pertussis toxin-sensitive subunits of G-proteins, and actin filaments. © 1994 Wiley-Liss, Inc.     

The effect of calcium ion concentration on myotube formation in vitro

The relationship of Ca^a+ concentration in in vitro tissue culture medium to the ability of the medium to support lizard and chick myotube formation was studied. Lizard myogenic cells (from lines established from the regenerating tail of the lizard, Anolis caroliensis) do not fuse in media with a Ca²⁺ concentration of below 650 μM; good fusion occurs at 1 750μM; and large anastomosing tubes result in media with concentrations of 2 750,μM. Chick myogenic cells from the limb of 11 day embryos do not fuse at Ca²⁺ concentrations below 260,μM, fuse well at 1 000 μM, and produce large anastomosing myotubes at concentrations above 1 700 μM. Colonies of myogenic cells from established lines plated at clonal densities in a medium with a 1 750 μM Ca²⁺ concentration grow more rapidly than those at 650 μM Ca²⁺; however, there is no increase in plating efficiency. Regardless of the Ca²⁺ concentration, lizard myogenic cells do not fuse until a large percentage of the cells in a colony have withdrawn from the mitotic cycle in Gl and entered GO. The similarities between in vivo and in vitro lizard myogenesis are discussed.     

Direct measurement of the increase in intracellular free calcium ion concentration in response to the action of complement.

1. The effect of rabbit anti-(pigeon erythrocyte) antibodies plus human complement on the concentration of intracellular free Ca2+ in sealed pigeon erythrocyte ''ghosts'' was investigated with the photoprotein obelin. 2. The addition of human serum, as a source of complement, to ''ghosts'' coated with antibody caused a rapid increase in intracellular free Ca2+ after a lag of 20-40 s, as detected by an increase in obelin luminescence. 3. The increase in obelin luminescence could not be explained by release of obelin into the medium. It was also Ca2+-dependent in that extracellular EGTA abolished the effect and intracellular EGTA inhibited it and required the complete terminal complex (C56789). No effect was seen with C5678. 4. The concentration of intracellular free Ca2+ before addition of complement was approx. 0.3 microM. This increased to a maximum of 5-30 microM after complement addition and then remained constant for at least 1-2 min. 5. Antibody plus complement induced a rapid increase in 42K+ efflux and an inhibition of cyclic AMP formation. 6. When partially purified complement components (C5b-9) were used in ''reactive lysis'' it was possible to inhibit the release of macromolecules from pigeon erythrocyte ''ghosts'' by extracellular EGTA. 7. It was concluded that the increase in intracellular free Ca2+ concentration caused by anti-cell antibody plus complement occurred before cell lysis and may be involved in the mechanism of complement-induced cell injury.     

The regulation of extramitochondrial free calcium ion concentration by rat liver mitochondria

The mechanism whereby rat liver mitochondria regulate the extramitochondrial concentration of free Ca(2+) was investigated. At 30 degrees C and pH7.0, mitochondria can maintain a steady-state pCa(2+) (0) (the negative logarithm of the free extramitochondrial Ca(2+) concentration) of 6.1 (0.8mum). This represents a true steady state, as slight displacements in pCa(2+) (0) away from 6.1 result in net Ca(2+) uptake or efflux in order to restore pCa(2+) (0) to its original value. In the absence of added permeant weak acid, the steady-state pCa(2+) (0) is virtually independent of the Ca(2+) accumulated in the matrix until 60nmol of Ca(2+)/mg of protein has been taken up. The steady-state pCa(2+) (0) is also independent of the membrane potential, as long as the latter parameter is above a critical value. When the membrane potential is below this value, pCa(2+) (0) is variable and appears to be governed by thermodynamic equilibration of Ca(2+) across a Ca(2+) uniport. Permeant weak acids increase, and N-ethylmaleimide decreases, the capacity of mitochondria to buffer pCa(2+) (0) in the region of 6 (1mum-free Ca(2+)) while accumulating Ca(2+). Permeant acids delay the build-up of the transmembrane pH gradient as Ca(2+) is accumulated, and consequently delay the fall in membrane potential to values insufficient to maintain a pCa(2+) (0) of 6. The steady-state pCa(2+) (0) is affected by temperature, incubation pH and Mg(2+). The activity of the Ca(2+) uniport, rather than that of the respiratory chain, is rate-limiting when pCa(2+) (0) is greater than 5.3 (free Ca(2+) less than 5mum). When the Ca(2+) electrochemical gradient is in excess, the activity of the uniport decreases by 2-fold for every 0.12 increase in pCa(2+) (0) (fall in free Ca(2+)). At pCa(2+) (0) 6.1, the activity of the Ca(2+) uniport is kinetically limited to 5nmol of Ca(2+)/min per mg of protein, even when the Ca(2+) electrochemical gradient is large. A steady-state cycling of Ca(2+) through independent influx and efflux pathways provides a model which is kinetically and thermodynamically consistent with the present observations, and which predicts an extremely precise regulation of pCa(2+) (0) by liver mitochondria in vivo.     

Control of free cytoplasmic calcium by intracellular pH in rat lymphocytes

Activation of Na⁺-H⁺ exchange in rat thymocytes was found to be followed by an increase in free cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i). We determined whether the change in [Ca²⁺]_i was secondary to the uptake of Na⁺, or to the cytoplasmic alkalinization that result from activation of the antiport. Increasing intracellular [Na⁺] by treating the cells with ouabain or gramicidin failed to affect [Ca²⁺]_i. In contrast, procedures that increased the cytoplasmic pH, such as addition of monensin or NH₃, significantly elevated [Ca²⁺]_i. These results suggest an important role of cytoplasmic pH in the control of [Ca²⁺]_i in lymphocytes.     

Cold-induced cytosolic free calcium ion concentration changes in wheat

Relatively little is known about changes in the cytosolic free calcium ion concentration ([Ca²⁺]_c) in monocotyledonous plants. Therefore, we produced transgenic winter wheat lines stably expressing the calcium-sensitive photoprotein aequorin constitutively in the cytosol. [Ca²⁺]_c was detected in vivo by luminometry, and [Ca²⁺]_c elevations were imaged at video rate. Experiments with the transgenic seedlings focused on potential changes in [Ca²⁺]_c during cold exposure. Temperature-induced changes in [Ca²⁺]_c were found to be more dependent on the change in temperature (dT dt⁻¹) than on the absolute value of temperature. [Ca²⁺]_c increased only at cooling rates higher than 8°C min⁻¹, indicating that an overall cellular [Ca²⁺]_c increase is of minor relevance as a signal for cold acclimation in wheat under ecological conditions. The results are discussed with regard to the so-called ‘calcium signature hypothesis’.     

白藜芦醇降低大鼠心室肌细胞内游离钙浓度

实验旨在研究白藜芦醇(resveratrol)对大鼠心室肌细胞内钙浓度(intracellular calcium concentratoin,[Ca2+]i)的影响.应用激光共聚焦显微镜技术记录心室肌细胞内的钙荧光强度.结果表明在正常台氏液和无钙台氏液中,白藜芦醇(15～60μmol/L)呈浓度依赖性地降低[Ca2+]i.蛋白酪氨酸磷酸酶抑制剂正钒酸钠(sodium orthovanadate,1.0 mmol/L)和L型Ca2+通道激动剂Bay K8644(10 μmol/L)可部分抑制正常台氏液中白藜芦醇的效应.但NO合酶阻断剂L-NAME(1.0 mmol/L)对白藜芦醇的作用无影响.白藜芦醇也能明显抑制无钙台氏液中由低浓度ryanodine(1.0 nmol/L)引起的[Ca2+]i增加.当细胞外液钙浓度由1 mmol/L增加到10 mmol/L而诱发心室肌细胞钙超载时,部分心室肌细胞产生可传播的钙波,白藜芦醇(60 μmol/L)可降低钙波的传播速度和持续时间,最终阻断钙波.结果提示,白藜芦醇能够降低心室肌细胞内游离钙浓度,此作用可能与其抑制电压依赖性Ca2+通道、酩氨酸激酶和肌浆网内钙释放有关.     

Platelet activating factor raises intracellular calcium ion concentration in macrophages

Peritoneal cells from thioglycollate-stimulated mice were allowed to adhere to coverglasses for 2 h to give a dense monolayer of adherent cells greater than 95% of which were macrophages. After incubation with the tetra-acetoxymethyl ester of quin2, coverglasses were rinsed with Ca2+-free saline, oriented at a 45 degree angle in square cuvettes containing a magnetically driven stir bar, and analyzed for changes in quin2 fluorescence in a spectrofluorimeter. Such fluorescence, taken as an indication of intracellular calcium ion concentration ([Ca2+]i), increased as exogenous calcium ion concentration ([Ca2+]o) was raised to 1 mM. At [Ca2+]o approximately equal to 10 microM, [Ca2+]i = 72 +/- 14 nM (n = 26); at [Ca2+]o = 1 mM, [Ca2+]i = 140-220 nM, levels not increased by N, N, N', N'-tetrakis (2-pyridylmethyl) ethylenediamine, a membrane-permeant chelator of heavy metals than can quench quin2. Addition of mouse alpha + beta fibroblast interferon, lipopolysaccharide, thrombin, collagen, vasopressin, ADP, compound 48/80, or U46619 did not change [Ca2+]i. However, addition of platelet activating factor (PAF) (2-20 ng/ml) raised [Ca2+]i by 480 nM within 1 min if [Ca2+]o = 1 mM. In the presence of 5 mM EGTA, PAF raised [Ca2+]i by 25 nM. This suggests that PAF causes influx of exogenous Ca2+, as well as releasing some Ca2+ from intracellular stores. Consistent with these results, when PAF was added to 1 mM Ca2+ in the presence of 100 microM Cd2+ or Mn2+ to block Ca2+ influx, [Ca2+]i increased by only intermediate amounts; at the times of such dampened peak response, [Ca2+]i could be raised within 1 min to normal PAF-stimulated levels by chelation of the exogenous heavy metals with diethylenetriaminepentaacetic acid. Normal PAF responses were observed in the presence of indomethacin. The lowest dose of PAF observed to raise [Ca2+]i was 0.1 ng/ml. Response of [Ca2+]i to 2-20 ng/ml PAF was transient, and second applications had no effect. The PAF response also was seen in cell suspensions. These results suggest that an increase in [Ca2+]i may be an early event in PAF activation of macrophages.     

The effects of isoproterenol on intracellular calcium concentration

beta-Adrenergic agonist, isoproterenol (ISO), is a potent relaxant of tracheal smooth muscle and inhibits carbachol-induced contraction. The effect of ISO on intracellular free Ca2+ concentration ([Ca2+]i) was examined in bovine tracheal smooth muscle strips, employing aequorin as Ca2+ indicator. Surprisingly, 10 microM ISO induces a 5-fold increase in [Ca2+]i which then gradually declines but still remains higher than basal after 1 h of stimulation. The ISO-induced increase in [Ca2+]i is dose-dependent, and the ED50 is approximately 50 nM. The ISO-induced increase in [Ca2+]i is inhibited by a beta-receptor blocker, propranolol, not by an alpha-blocker, phentolamine. The ISO-induced rise in [Ca2+]i is dependent on extracellular Ca2+. Forskolin, an adenylate cyclase activator, and vasoactive intestinal peptide, which is known to stimulate adenylate cyclase via a specific receptor in this tissue, have similar effects on [Ca2+]i, suggesting that a rise in cyclic AMP concentration mediates this effect of ISO on [Ca2+]i. Pretreatment of muscle with 10 microM ISO inhibits both the initial Ca2+ transient and the contractile response induced by 0.3 microM carbachol. Conversely, in carbachol-pretreated muscle strips, addition of ISO causes a fall rather than a rise in [Ca2+]i, and an inhibition of contraction. These results indicate that ISO has effects on cellular Ca2+ metabolism at more than a single site in bovine tracheal smooth muscle, that these effects are different in control and carbachol-pretreated muscle, and that the relaxing effect of ISO is not due solely to its effect on Ca2+ metabolism.     

Measurement of the cytosolic free calcium ion concentration of individual lymphocytes by microfluorometry using quin 2 or fura-2

The cytosolic free calcium ion concentration ([Ca2+]i) of individual lymphocytes was measured by microfluorometry with dual excitation wavelengths using quin 2 for fura-2. Fura-2 was a more suitable fluorescent Ca2+ indicator than quin 2 for measurements of single cells because of the standard curve calibrated for fura-2 had a good linearity, and the standard deviation (SD) of the value of the intensity ratio of fura-2-loaded cells was much smaller than that of quin 2-loaded cells. The [Ca2+]i in quiescent lymphocytes was about 1 x 10(-7) M, and an increase in the [Ca2+]i was observed within a few minutes of ionomycin, protein A, phorbol myristate acetate (PMA) or concanavalin A (Con A) stimulation. Ionomycin-induced proliferation occurred when the initial [Ca2+]i was approximately 3 x 10(-7) M or greater. The increase in the [Ca2+]i induced by Con A occurred transiently, and another rise in the [Ca2+]i was observed in the stage prior to the S-phase. These results indicate that Ca2+ is necessary for stimulated lymphocytes to enter the cell cycle and S-phase.     

胍丁胺对大鼠心室肌细胞内游离钙浓度的影响

本研究旨在观察胍丁胺 (agmatine ,Agm)对分离大鼠心室肌细胞内游离钙浓度 ( [Ca2 +]i)的影响。用酶解方法分离大鼠心室肌细胞 ,用Fluo 3 AM负载 ,然后用激光共聚焦法测定单个心室肌细胞 [Ca2 +]i 的荧光强度 (fluorescenceintensity ,FI) ,结果以FI或相对荧光强度 (F/F0 % )表示。实验结果表明 ,在正常台氏液 (含钙 1 0mmol/L)和无钙台氏液中 ,单个大鼠心室肌细胞的荧光密度分别为 12 8 8± 13 8和 119 6± 13 6,两者无差异。Agm 0 1、1和 10mmol/L浓度依赖性地显著降低细胞的钙浓度 ;在正常台氏液中加入EGTA 3mmol/L ,Agm同样降低细胞的钙浓度。KCl 60mmol/L ,PE 3 0 μmol/L ,和Bay K 864 410 μmol/L均升高心室肌细胞的[Ca2 +]i。Agm同样降低高浓度KCl、Bay K 864 4和PE诱发的心室肌细胞 [Ca2 +]i 升高。当细胞外液钙浓度由 1mmol/L增加到 10mmol/L时 ,诱发心室肌细胞钙超载 ,同时部分心室肌细胞产生可传播的钙波 (Ca2 +wave) ,Agm 1mmol/L降低钙波的传播速度和持续时间 ,最终阻断钙波。以上结果提示 ,Agm对心室肌细胞的胞浆[Ca2 +]i具有抑制作用 ,此作用通过阻断电压依赖性钙通道而实现 ;并可能与抑制大鼠心室肌细胞内钙释放有关     

Intracellular free calcium concentration in lymphocytes of patients with muscular dystrophies

Intracellular free calcium concentration [( Ca2+]i) of human peripheral blood lymphocytes was determined by fluorescence spectroscopic measurements with quin2 in patients with different types of muscular dystrophy and in controls. The [Ca2+]i level in lymphocytes showed a significant increase in adult type (facioscapulohumeral and limb-girdle) muscular dystrophies, while it showed a decrease in Duchenne dystrophy as compared to the values of age- and sex-matched controls. The data obtained suggest an alteration in the effectiveness of the calcium pump in lymphocytes and may represent a sign of generalized membrane damage in these hereditary muscle diseases.     

整合素介导小鼠卵内钙离子增加

为了研究整合素是否作为跨膜信号传递受体介导小鼠卵[Ca^2 ]i的变化并探讨其机制。本实验采用甘-精-甘-天冬-丝-脯(GLY-ARG-GLY-ASP-SER-PRO,RGD肽)、纤连蛋A(fibronectin,Fn)及抗整合素α6、β1的单克隆抗体作用于负载了钙探针Fluo-3／AM的去透明带小鼠卵,用激光共聚焦显微镜检测小鼠卵的荧光强度以反映卵[Ca^2 ];用无钙液替代有钙液、或用酪氨酸激酶抑制剂或蛋白激酶C的抑制剂预先作用于卵,然后再观察RGD肽所致卵[Ca^2 ]i的变化。结果显示整合素配体RGD肽或Fn作用于去透明带小鼠卵可引起卵[Ca^2 ]i增加,增加的程度与精子作用相似;去除培养液中的Ca^2 后,再用RGD肽、Fn作用仍可引起卵[Ca^2 ]i增加：用功能性的抗小鼠整合素α6、β1的单克隆抗体也可引起不同程度的卵[Ca^2]i增加,尤其以抗小鼠整合素α6、β1单克隆抗体的作用明显;用酪氨酸激酶抑制剂预先作用于鼠卵,RGD肽或精子作用都不再引起卵[Ca^2 ]i增加;蛋白激酶C抑制剂预先作用鼠卵,RGD肽及Fn也不再引起卵[Ca^2 ]i增加。实验证明。小鼠卵膜整合素与其配体结合可使卵内贮存钙离子释放,引起卵[Ca^2 ]i增加这一卵激活的早期事件;整合素介导小鼠卵激活需要酪氨酸激酶信号转导途径的参与;蛋白激酶C也参与了整合素介导的卵激活。     

脱氢紫堇碱对正常和低氧豚鼠心肌细胞内钙的影响

目的 :探讨脱氢紫堇碱 (dehydeocorydaline,DHC)及维拉帕米 (verapamil,Ver)对豚鼠心肌细胞内游离钙浓度([Ca2 + ] i)变化的影响。方法 :采用离体豚鼠心脏Langendorff法灌注 ,用荧光指示剂方法 (Fure 2 /AM)标记心肌([Ca2 + ] i)变化。观察低氧后心肌 [Ca2 + ] i 的变化。结果 :①正常氧状态心肌 [Ca2 + ] i 均值为 (1 2 0 .5± 8.3)nmol/L(n =2 0 ) ;②正常氧条件下 ,DHC、Ver均使心肌 [Ca2 + ] i 明显下降。 (3)低氧状态下 ,心肌 [Ca2 + ] i 增加与缺氧时间(程度 )直线相关 (r=0 .98)。④DHC对低氧后心肌 [Ca2 + ] i 增加明显减缓。结论 :DHC在正常氧、低氧条件下阻止心肌细胞内钙超载 ,我们认为DHC可能提高心肌细胞的自我保护作用     

腺苷减少豚鼠心室肌细胞内游离钙浓度

目的：研究腺苷对豚鼠心室肌细胞内游离钙浓度（[Ca^2＋]i）的影响并探讨其可能机制。方法：用激光共聚焦显微镜探测细胞内游离钙浓度,结果用相对荧光强度（（FI-FI0）／FI0,％;FI0：对照;FI：给药）表示。结果：①在正常台氏液和无钙台氏液中,腺苷（10,50,100μmol／L）浓度依赖性地降低[Ca^2＋];。②含30mmol／L KCl的台氏液（高钾台氏液）能够增加[Ca^2＋]i。腺苷（10,50,100μmol／L）能够显著抑制KCl引起的[Ca^2＋]i的增加。③预先应用选择性腺苷AI受体拮抗剂DPCPX（1μmol／L）,可大部分取消腺苷（100μmol／L）在高钾台氏液中的作用。腺苷（100μmol／L）在高钾台氏液的作用也可被预先应用一氧化氮（No）合酶抑制剂L-NAME（1mmol／L）所部分减弱。④腺苷（100μmol／L）能明显抑制无钙台氏液中由低浓度ryanodine引起的[Ca^2＋];增加。⑤当细胞外液钙浓度由1mmol／L增加到10mmol／L而诱发心室肌细胞钙超载时,部分心室肌细胞产生可传播的钙波,腺苷（100μmol／L）可降低钙波发生的频率和持续时间,最终阻断钙波并降低[Ca^2＋];。结论：腺苷可通过抑制外钙内流和减少肌浆网内钙释放从而降低[Ca^2＋],其减少外钙内流可能是由于腺苷A1受体介导的电压依赖性Ca^2＋通道的抑制,NO可能参与这一过程。