在ts-RSV LA90细胞转化过程中钙流变化的研究

本文以ts-RSV LA90细胞为模型,用放射性同位素示踪技术测定了通过细胞质膜的~(45)Ca~(2+)流水平;同时用钙指示剂Indo-1 AM和光学多道分析仪测定了胞内[Ca~(2+)]_i,初步研究了Ca~(2+)流和[Ca~(2+)]_i在v-src基因引起细胞转化过程中的动态变化。结果表明LA90细胞质膜上~(45)Ca~(2+)流的改变是细胞转化过程中可以检测到的早期事件之一,转化状态(33℃)细胞的~(45)Ca~(2+)流大于正常状态(40℃)的,细胞从正常到转化(40℃→33℃)的25分钟内~(45)Ca~(2+)流就有明显增大。TMB-8可以抑制转化引起的~(45)Ca~(2+)流出的增大,小牛血清可以刺激正常状态细胞的~(45)Ca~(2+)流出增大,~(45)Ca~(2+)流出与温度有一定依赖关系;细胞转化引起的~(45)Ca~(2+)流入增大,可被异博定抑制,~(45)Ca~(2+)流入不受温度的影响。LA90细胞[Ca~(2+)]_i在转化早期有明显升高,并维持在较正常细胞高2—3倍的水平,A23187-Br可提高正常LA90细胞[Ca~(2+)]_i,[Ca~(2+)]_i不受温度的影响。从质膜上~(45)Ca~(2+)流和[Ca~(2+)]_i的增大说明转化细胞虽然对胞外Ca~(2+)浓度依赖性下降,但维持增殖及转化状态仍然需要一定的胞外Ca~(2+),并通过提高质膜Ca~(2+)流入和释放内源性Ca~(2+),使转化细胞[Ca~(2+)]_i维持在较高水平上。LA90细膜质膜上~(45)Ca~(2+)流和[Ca~(2+)]_i的增大在细胞转化中起着重大作用。     

血管紧张素Ⅱ增加新生鼠脑细胞胞浆Ca~(2+)浓度

本文应用荧光钙测定技术观察了血管紧张素Ⅱ(AⅡ)对新生Wistar鼠脑细胞胞浆Ca~(2+)浓度([Ca~(2+)]_i)的影响。结果表明:血管紧张素Ⅱ在1nmol/L—1μmol/L浓度下可诱导新生鼠脑细胞[Ca~(2+)]_i增加,具量效关系。在无外Ca~(2+)存在对,其增加幅度有所减少。上述效应可被血管紧张素Ⅱ拮抗剂Saralasin所阻断,并呈剂量依赖关系。上述结果提示,血管紧张素Ⅱ可激活血管紧张素AⅡ受体,增加脑细胞[Ca~(2+)]_i,该效应通过细胞内Ca~(2+)释放和细胞外Ca~(2+)内流两条适径实现,前者的作用是主要的。     

L-型钙通道自身调控异常参与缺血再灌注心肌钙超载的形成

钙超载作为心肌缺血再灌注损伤的重要机制之一,其形成原因与治疗策略一直是研究的热点。心肌遭受缺血再灌注后,参与细胞内钙循环的L-型电压依赖钙通道(L-type voltage-dependent calcium channel,L-VDCC)、肌浆网钙ATP酶2a(sarco/endoplasmic reticulum ATPase 2a,SERCA2a)和受磷蛋白(phospholamban,PLB)、Ryanodine受体2(RyR2)、Na~+/Ca~(2+)交换体、Na~+/H~+交换体等多种蛋白功能异常,导致舒张期[Ca~(2+)]_i上升,钙瞬变幅度降低,细胞出现钙超载。[Ca~(2+)]_i升高的过程大致可分为两个阶段:早期的[Ca~(2+)]_i升高过程(部分由钙通道介导)和晚期的[Ca~(2+)]_i升高过程(主要由Na~+/Ca~(2+)交换体介导)。L-VDCC活性增加参与钙超载的形成,但是L-VDCC蛋白在缺血再灌注过程中的分子变化机制尚不清楚。L-VDCC通道调控方式包括两类:自身调节和外源性调节,其中外源性调节蛋白PKG和PKA的调控不能解释细胞水平的L-VDCC活性增加现象,而在缺血再灌注过程中,钙依赖的失活(calcium-dependent inactivation,CDI)效应减弱、钙依赖的易化(calcium-dependent facilitation,CDF)效应增强、羧基远端部分肽链(distal carboxy terminus,DCT)的抑制效应减弱,这三种自身调节机制的改变引起L-VDCC活性的增加。因此,可以认为L-VDCC通道自身调控异常参与缺血再灌注损伤中心肌细胞钙超载的形成。     

血管平滑肌细胞的钙动力学

血管平滑肌细胞外的Ca~(2+)通过多种通道进入细胞内。Ca~(2+)通道的本质是镶嵌在膜脂质双分子层中的糖蛋白,神经介质和药物可影响Ca~(2+)通道的功能。靠近胞膜的肌质网和胞膜内侧面的高亲和性Ca~(2+)结合位点是血管平滑肌细胞内储存和释放Ca~(2+)的主要部位。胞浆[Ca~(2+)]增高后在钙调蛋白的介导下引起血管收缩。高血压等血管性疾病的发生与其平滑肌细胞的钙动力学异常有关。     

钙离子研究进展

一、钙离子振荡的时间空间分布通常有两个平行的系统调节着细胞内Ca~(2+)的释放。在大多数可兴奋细胞,细胞外钙由电压敏感钙通道进入细胞,使胞内游离钙[Ca~(2+)]_i有一个初期上升,后者使内质网上的ryanodine敏感钙通道开放,将Ca~(2+)释放到胞浆内。而在许多非可兴奋细胞,细胞表面受体由G蛋白介导     

强声暴露对巴马小型猪海马细胞内Ca~(2+)变化及其信号通道的影响

声音对神经系统有重要影响,本研究旨在探讨噪音或高强度声音刺激对神经系统的影响及其机制。将听力正常的巴马小型猪随机分为正常对照组与强声暴露组。强声暴露组的巴马小型猪暴露于中低频强声(900 Hz-142 dB SPL)环境中15min,暴露结束后即刻分离出海马组织。用Fluo-4探针观察海马组织细胞内Ca~(2+)浓度([Ca~(2+)]_i)的变化,用real-time PCR和Western blot分别检测Ca~(2+)受体、L-型Ca~(2+)通道α2/δ1亚基、PKC和PI3K的mRNA和蛋白表达,用DAPI染色法观察细胞核形态变化。结果显示,相对对照组,强声暴露组小型猪海马组织细胞[Ca~(2+)]_i明显增加,L-型Ca~(2+)通道α2/δ1亚基、PKC和PI3K mRNA表达上调,Ca~(2+)受体和PKC蛋白表达显著上调。此外,强声暴露引起海马组织细胞核出现肿胀变形等损伤样改变。以上结果提示,强声暴露可以通过激活海马组织PKC信号通路,引起[Ca~(2+)]_i上调,最终导致海马组织内细胞的损伤。本研究结果不仅揭示了强声引起神经损伤的可能机制,同时为防护强声对神经系统造成的损伤提供了新的思路。     

用Fura-2测定缺氧时海马细胞内游离钙离子浓度的变化

本文用Fura-2荧光测定技术直接监测了缺氧时大鼠海马细胞内游离钙离子浓度[Ca~(2+)]_1的变化。实验发现,缺氧可使海马细胞[Ca~(2+)]_1显著增高,并且在缺氧过程中其增高呈现明显的时相性变化。在去除细胞外钙的情况下,缺氧仍能使[Ca~(2+)]_1增高,仅增高幅度有所降低;另外[Ca~(2+)]_1不再出现时相性变化特征。结果提示,胞外Ca~(2+)的内流以及内源Ca~(2+)的释放均参与了缺氧所致海马细胞[Ca~(2+)]_1的增高过程,缺氧时[Ca~(2+)]_1升高的时相性变化为胞外Ca~(2+)内流引起。     

钙通道与钙释放通道

1.Ca~(2+)的重要生理作用胞内游离钙浓度([Ca~(2+)])的变化调节着细胞的代谢、基因表达等细胞共有的活动,以及始动兴奋、收缩或出胞分泌以及激活和失活离子通道等细胞不同的反应。[Ca~(2+)]的升高主要依赖于胞外钙经质膜上的钙通道内流或/和胞内储存钙的释放。释放的内钙也是藉细胞器膜的钙释放通道进入胞浆。可见通道启闭活动的正常是维持[Ca~(2+)]正常的一个重要保证。2.离子通道及其分类离子通道是贯穿于质膜或细胞器膜的大分子蛋白质,其中央形成能通过离子的亲水性孔道(pores)。离子的跨膜转运是通过膜上通道蛋白的功能来完     

内向整流钾通道阻滞剂BaCl_2引起大鼠冠状动脉收缩的机制

为了探讨内向整流钾通道(inward rectifier K~+channels,K_(ir))阻滞剂BaCl_2引起大鼠冠状动脉(rat coronary artery,RCA)收缩的作用机制,本研究采用离体微血管环张力记录法观察BaCl_2引起的RCA收缩对细胞内Ca~(2+)([Ca~(2+)]_i)释放和细胞外Ca~(2+)([Ca~(2+)]_o)内流的依赖性,并通过抑制剂实验探讨其作用机制。结果显示,静息状态下,BaCl_2(0.1~1.0 mmol/L)浓度依赖性地收缩离体RCA,最大收缩幅度为(5.69±1.07)m N,与KCl(60 mmol/L)收缩幅度相近;BaCl_2在无钙液中所引起的收缩占其总收缩的(35.44±6.72)%,复钙进一步引起(64.56±5.94)%的收缩;钙通道阻滞剂硝苯地平(0.3μmol/L)、环氧合酶抑制剂吲哚美辛(100μmol/L)、细胞外信号调节激酶ERK1/2抑制剂PD98059(10μmol/L)和氯通道阻滞剂尼氟灭酸(100μmol/L)分别使BaCl_2引起的RCA最大收缩幅度降低(87.82±5.43)%(P0.01)、(73.23±5.47)%(P0.01)、(75.69±7.94)%(P0.01)和(83.24±7.69)%(P0.01)。上述实验结果表明,BaCl_2引起RCA收缩依赖于[Ca~(2+)]_i释放和[Ca~(2+)]_o内流,并提示该过程与增加前列腺素类物质合成、钙通道和氯通道激活及ERK1/2通路有关。     

胡萝卜及其愈伤组织细胞质Ca~(2 )水平分析的研究

为测定植物细胞质内[Ca~(2 )]_i,对胡萝卜(Daucus carota var.sativa DC.)原生质体制备介质做了改进,并在正常生理条件下,用温和的、非损伤性的方法将Ca~(2 )荧光指示剂indo-1 K~ 和fura-2 K~ 导入该原生质体,能很好地标记细胞质内的游离Ca~(2 )。在此基础上,用显微荧光光度单波法测定被标记原生质体单个细胞胞质[Ca~(2 )]_i。结果表明:被indo-1 K~ 标记的胡萝卜及其愈伤组织的原生质体[Ca~(2 )]_i分别为88.3nmol/L和263.0nmol/L;fura-2 K~ 标记的分别为99.9nmol/L和255.5nmol/L。由此可见,脱分化的、处在细胞周期中的愈伤组织细胞质中[Ca~(2 )]_i远高于分化了的、处于静息态的胡萝卜细胞。此外,为了确认测量的可靠性,对两种Ca~(2 )荧光指示剂分别做了体外校正,证明其线性相关。     

Increase in cytoplasmic Ca2+ and stimulation of calcitonin secretion from human medullary thyroid carcinoma cells by the gastrin-releasing peptide.

Examination was made of the effects of gastrin-releasing peptide (GRP) on human medullary thyroid carcinoma cells (TT cells). GRP stimulated calcitonin(CT) release in a concentration-dependent manner at 0.1-1000 nmol/l. On adding forskolin along with GRP, CT release was greater than by GRP alone. The stimulatory effect of A23187 was not additive. Intracellular free calcium concentration ([Ca2+]i) was measured for individual TT cells loaded with fura-2. The addition of GRP caused a rapid and transient rise in [Ca2+]i in a concentration-dependent manner followed by a sustained increase in [Ca2+]i. In the medium without Ca2+, this sustained increase did not occur and the concentration of CT release from TT cells by GRP was reduced by approximately a half. GRP would thus appear to be importantly involved in the regulation of thyroid C cell function through modulation of [Ca2+]i.     

Modulation of vincristine sensitivity of human kidney tumor cells by pharmacological agents interfering with intracellular signals. No apparent relationship to changes in cytoplasmic Ca2+ or pH

The effect of substances proposed to modulate intracellular signal systems on growth and sensitivity to vincristine in the human kidney tumor cell line ACHN was investigated and related to changes in cytoplasmic free Ca2+ concentration ([Ca2+]i) and cytoplasmic pH (pHi). Presence during culture of the protein kinase C (PKC) activator 12-O-tetradecanoyl phorbol 13-acetate (TPA) had no effect on cell growth but significantly increased the EC50 concentration for vincristine inhibited cell growth. There was no indication for endogenous PKC activity being responsible for basal vincristine insensitivity since it was not affected by the PKC inhibitor H-7. The Ca2+ ionophore ionomycin tended to increase cell growth and induced vincristine resistance, whereas the calmodulin inhibitor W-7 had opposite effects. Presence during culture of the adenylate cyclase activator forskolin did not affect basal cell growth but dose-dependently made the cells more sensitive to vincristine. The modulators of vincristine sensitivity had no immediate effect on pHi, whereas after 3 days of incubation ionomycin and forskolin tended to increase pHi. Ionomycin and forskolin induced an immediate increase in [Ca2+]i which remained after 3 days only for ionomycin, whereas TPA decreased [Ca2+]i, a change which tended to remain after 3 days of incubation. It is concluded that perturbation of the intracellular signal system may affect both cell growth and cytotoxic drug sensitivity. However, there is no apparent relationship between immediate or late changes in [Ca2+]i and pHi and vincristine sensitivity.     

Effects of acetylcholine, TSH and other stimulators on intracellular calcium concentration in dog thyroid cells

The intracellular free calcium concentration, [Ca2+]i, has been measured in dog thyroid cells using the fluorescent Ca2+-indicator, quin2. Acetylcholine or its non-hydrolyzable analog, carbamylcholine rapidly increased [Ca2+]i by 40 +/- 4% (mean +/- SE) over the basal level of 81 +/- 2 nM. This increase was totally abolished by atropine, a muscarinic cholinergic receptor blocker, but was not influenced by verapamil, a voltage dependent-calcium channel blocker. Depletion of extracellular Ca2+ by the addition of EGTA, diminished but did not abolish the response to carbamylcholine. These data suggest that cholinergic effectors increase [Ca2+]i by mobilization of Ca2+ from intracellular stores rather than from an influx of Ca2+. Addition of TSH, isoproterenol, phorbol ester, dibutyryl cyclic GMP or cyclic AMP did not elicit any change in [Ca2+]i suggesting that their action may not involve any mobilization of intracellular Ca2+. These data provide direct evidence that in the thyroid cell, cholinergic agents act via their receptors to cause a rapid increase in [Ca2+]i, which may mediate their metabolic effects.     

Evidence of cyclic AMP-independent action of glucagon on calcium mobilization in rat hepatocytes

Glucagon increases the cytoplasmic free calcium concentration as measured by aequorin bioluminescence. It has been proposed by Wakelam et al. (Nature 323 (1986) 68-71) that low concentrations of glucagon mobilize calcium from an intracellular pool by causing polyphosphoinositide breakdown. To identify whether cyclic AMP mediates changes in the cytoplasmic free calcium concentration ([Ca2+]c) induced by glucagon, the effects of forskolin and exogenous cyclic AMP on [Ca2+]c were compared with that of glucagon in aequorin-loaded hepatocytes. Although the magnitudes of the [Ca2+]c responses to 250 microM forskolin and 1 mM 8-bromo cyclic AMP were identical to that of 5 nM glucagon, these two agents induced a more prolonged elevation of [Ca2+]c. Glucagon-induced elevation of [Ca2+]c was accompanied by a smaller increase in cyclic AMP than that induced by forskolin. When the cyclic AMP response to glucagon was potentiated by an inhibitor of phosphodiesterase, 3-isobutyl-1-methylxanthine, the glucagon-induced increase in [Ca2+]c was not affected. Conversely, when the cyclic AMP response to glucagon was reduced by pretreatment of the cells with angiotensin II, glucagon-induced changes in [Ca2+]c were rather enhanced. Furthermore, vasopressin potentiated glucagon-induced changes in [Ca2+]c despite the reduction of the cyclic AMP response to glucagon. In the presence of 1 microM extracellular calcium, angiotensin II did not enhance glucagon-induced changes in [Ca2+]c. These results suggest that at least part of the action of 5 nM glucagon on calcium mobilization is independent of cyclic AMP.     

Regulation of cytosolic Ca2+ in clonal human muscle cell cultures

Human muscle cells were grown in culture and clonally selected for fusion potential. The concentration of cytoplasmic ionized calcium, [Ca2+]i, was measured in monolayers of fused myotubes using the Ca2+ indicator indo-1. The contributions of independent routes of Ca2+ influx and efflux to/from the cytoplasm on [Ca2+]i were investigated. The resting [Ca2+]i was 170-190 nM in different cell clones. Acetylcholine increased [Ca2+]i by about 2-fold in the presence of absence of extracellular Ca2+. Cell depolarization by K+ elevated [Ca2+]i about 3-fold, and this increase was largely dependent on extracellular Ca2+. Replacing Na+ by N-methylglucammonium+ raised [Ca2+]i greater than 5-fold, and 50% of this increase was dependent on extracellular Ca2+. All these increases in [Ca2+]i were transient, returning to basal [Ca2+]i within 2 min. It is concluded that cells in culture [Ca2+]i can be elevated transiently by acetylcholine through Ca2+ release from intracellular stores, and by K through Ca2+ influx. The return to basal [Ca2+]i is due to Na+/Ca2+ exchange and Ca2+-ATPase activity.     

Protein kinase C-activated calcium channel in the osteoblast-like clonal osteosarcoma cell line UMR-106

The effects of protein kinase C stimulation on free cytosolic Ca2+ [( Ca2+]i) were studied in Fura 2-loaded UMR-106 cells. Stimulation of the protein kinase C with the tumor-promoting phorbol esters 12-O-tetradecanoylphorbol 13-acetate (TPA) and phorbol 12,13-diacetate or 1-oleoyl-2-acetylglycerol was followed by an increase in [Ca2+]i. The protein kinase C-induced increase in [Ca2+]i has a lag period, the duration of which was dependent on the stimulant and medium Ca2+ concentrations. With 2 microM TPA, the rise in [Ca2+]i peaked within 1.5 min, after which [Ca2+]i returned partially toward base line. The increase in [Ca2+]i was absolutely dependent on the presence of medium Ca2+ and was inhibited by the Ca2+ channel blockers nicardipine and verapamil. Cell stimulation also results in Ca2+ release from intracellular pool(s) which appears to be mediated by a Ca2+-dependent Ca2+ release mechanism. The reduction in [Ca2+]i was due to channel inactivation. Pretreatment of the cells with 1 nM TPA, 2 units/ml parathyroid hormone (PTH), or 15 microM forskolin blocked the effect of 2 microM TPA on [Ca2+]i. TPA and PTH were more potent inhibitors than was forskolin. The properties of this channel are compared to the cAMP-independent PTH-stimulated Ca2+ channel present in these cells.     

Mechanisms by which extracellular ATP and UTP stimulate the release of prostacyclin from bovine pulmonary artery endothelial cells.

Extracellular ATP and UTP caused increases in the concentration of cytoplasmic free calcium ([Ca2+]i) and the intracellular level of inositol 1,4,5-trisphosphate (IP3), a second messenger for calcium mobilization, prior to the release of prostacyclin (PGI2) from cultured bovine pulmonary artery endothelial (BPAE) cells. The agonist specificity and dose-dependence were similar for nucleotide-mediated increases in IP3 levels, [Ca2+]i and PGI2 release. An increase in [Ca2+]; and PGI2 release was observed after addition of ionomycin, a calcium ionophore, to BPAE cells incubated in a calcium-free medium. The addition of ATP to the ionomycin-treated cells caused no further increase in [Ca2+]i or PGI2 release. The inability of ATP to cause an increase in [Ca2+]i or PGI2 release in ionomycin-treated cells was apparently due to the ionomycin-dependent depletion of intracellular calcium stores since the subsequent addition of extracellular calcium caused a significant increase in both [Ca2+]i and PGI2 release. Introduction of BAPTA, a calcium buffer, into BPAE cells inhibited ATP-mediated increases in [Ca2+]i and PGI2 release, further evidence that PGI2 release is dependent upon an increase in [Ca2+]i. The increase in [Ca2+]i elicited by ATP apparently caused the activation of a calmodulin-dependent phospholipase A2 since trifluoperazine, an inhibitor of calmodulin, and quinacrine, an inhibitor of phospholipase A2, prevented the stimulation of PGI2 release by ATP. Furthermore, ATP caused the specific hydrolysis of [14C]arachidonyl-labeled phosphatidylcholine and the generation of free arachidonic acid, the rate-limiting substrate for PGI2 synthesis, prior to the release of PGI2 from BPAE cells. These findings suggest that the increase in PGI2 release elicited by ATP and UTP is at least partially dependent upon a phospholipase C-mediated increase in [Ca2+]i and the subsequent activation of a phosphatidylcholine-specific phospholipase A2. ATP analogs modified in the adenine base or phosphate moiety caused PGI2 release with a rank order of agonist potency of adenosine 5'-O-(2-thiodiphosphate) (ADP beta S) greater than 2-methylthioATP (2-MeSATP) greater than ATP, whereas alpha, beta methyleneATP and beta, gamma methyleneATP had no effect on PGI2 release.     

The calcium mobilizing tumor promoting agent, thapsigargin elevates the platelet cytoplasmic free calcium concentration to a higher steady state level. A possible mechanism of action for the tumor promotion

The ability of the platelet agonists thapsigargin (Tg) and thrombin to elevate the cytoplasmic free calcium level ([Ca2+]i) was examined. Both agonists induced a transient increase of [Ca2+]i with a different time-course, however. Thus, the maximal [Ca2+]i was reached 15 sec and 2 min after stimulation with thrombin and Tg, respectively. The thrombin induced rise of [Ca2+]i was reversible, which indicates that active calcium sequestration and/or extrusion is operating. Tg affected [Ca2+]i in a divergent manner, thus, [Ca2+]i was stabilized on a elevated level without initial formation of a pronounced peak. The decline in [Ca2+]i observed after thrombin stimulation was not impaired by the calmodulin binding drug trifluoperazine but it was strongly reduced by vanadate, which suggests the active calcium transport systems to be insensitive to calmodulin. We put forward the hypothesis that the tumor promoting activity of Tg is attributable to its ability to stabilize [Ca2+]i on a new elevated steady state level.     

The basal level of intracellular calcium gates the activation of phosphoinositide 3-kinase-Akt signaling by brain-derived neurotrophic factor in cortical neurons

Brain-derived neurotrophic factor (BDNF) mediates survival and neuroplasticity through the activation of phosphoinositide 3-kinase-Akt pathway. Although previous studies suggested the roles of mitogen-activated protein kinase, phospholipase C-gamma-mediated intracellular calcium ([Ca2+]i) increase, and extracellular calcium influx in regulating Akt activation, the cellular mechanisms are largely unknown. We demonstrated that sub-nanomolar BDNF significantly induced Akt activation in developing cortical neurons. The TrkB-dependent Akt phosphorylation at S473 and T308 required only phosphoinositide 3-kinase, but not phospholipase C and mitogen-activated protein kinase activity. Blocking NMDA receptors, L-type voltage-gated calcium channels, and chelating extracellular calcium by EGTA failed to block BDNF-induced Akt phosphorylation. In contrast, chelating [Ca2+]i by 1,2-bis(o-aminophenoxy)ethane-N,N,N ',N '-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) abolished Akt phosphorylation. Interestingly, sub-nanomolar BDNF did not stimulate [Ca2+]i increase under our culture conditions. Together with that NMDA- and membrane depolarization-induced [Ca2+]i increase did not activate Akt, we conclude that the basal level of [Ca2+]i gates BDNF function. Furthermore, inhibiting calmodulin by W13 suppressed Akt phosphorylation. On the other hand, inhibition of protein phosphatase 1 by okadaic acid and tautomycin rescued Akt phosphorylation in BAPTA-AM and W13-treated neurons. We further demonstrated that the phosphorylation of phosphoinositide-dependent kinase-1 did not correlate with Akt phosphorylation at T308. Our results suggested novel roles of basal [Ca2+]i, rather than activity-induced calcium elevation, in BDNF-Akt signaling.