Evaluation of the osmoregulatory function of taurine in brain cells

Summary Homogenous primary cultures of mouse astrocytes and cortical neurons were used to clarify the role of taurine in ion and osmoregulation in the CNS. This study indicates that both neurons and glial cells have uptake systems for taurine. The cell water content does not change during loading of cells with taurine. Chemical analysis indicates that part of the accumulated taurine is metabolized and that the product(s) are stored in the cells. Extracellular taurine (1 mM) has no effect on K⁺, Na⁺, Cl^-, or Ca²⁺ movements in astrocytes. However, astrocytes loaded to a taurine content which corresponds a concentration of 60 mM (corresponds to normal mouse cortex levels) show a 50% reduction in their K⁺ accumulation by carriers and a 100% increase in Ca²⁺ turnover rates. Movements of Ca²⁺ and K⁺ are involved in neurotransmission. It appears that taurine stored in glial cells, has an important effect on ion homeostasis in the CNS and may act indirectly on neuronal excitability.     

Synthesis,uptake and release of taurine in astrocytes treated with 8-Br-cAMP

Taurine is one of the most abundant free amino acids in the mammalian central nervous system, where it is crucial for proper development. Moreover, taurine has been related with epilepsy, as it can reduce or prevent seizures. It is also a neuroprotectant in other experimental conditions. Glial cultures were analysed to determine the changes in taurine synthesis and traffic that occur in a more differentiated state of these cells. The cultures were treated with 8-Br-cAMP, an analogue of cAMP that induces differentiation in astrocytes. We observed an increase in immunoreactivity for GFAP, as well as an alteration in uptake-release kinetics in these cells. Moreover, we noted an increase in taurine levels and in cysteine sulfinic decarboxylase, which is the rate-limiting enzyme in taurine synthesis. The data indicate that taurine synthesis and traffic kinetics vary according to the differentiation state of the astrocytes. Thus, our results highlight the importance of astrocytes in modulating taurine levels in the brain.     

Untersuchung der Nierenfunktion mit Hilfe isolierter Zellen

Summary After summarizing the progress which has been made with regard to the isolation and characterization of homogeneous cell populations from the kidney, a brief survey of current techniques available for the analysis of intracellular parameters is given. Special emphasis is thereby placed on the use of electron probe X-ray microanalysis to determine intracellular elements and on in vivo nuclear magnetic resonance to define metabolic pathways in isolated cells. These methods have been applied to study ion and substrate fluxes in isolated collecting duct cells and the response of these cells to changes in osmolality of the extracellular medium. This response involves initially fast water movements accompanied by changes in intracellular sodium and chloride but not potassium concentration. Longterm adaptation is achieved by the adjustment of the intracellular concentration of organic osmolytes such as sorbitol, myoinositol, glycerophosphorylcholine, and betaine through changes in the rate of efflux of these metabolites from the cell. In the last section the effect of experimentally induced diabetes mellitus on the osmoregulation in isolated collecting ducts is described.

Wir danken Frau D. Mägdefessel für das Schreiben des Manuskriptes, Herrn F. Sieland für die Fotoarbeiten und Herrn F. Dräger für das Erstellen der Zeichnungen.     

牛磺酸降温时腹中隔,下丘脑精氨酸加压素含量的变化

目的：探讨牛磺酸的降温机制。方法：建立家兔ＥＴ发热模型，观察侧脑室灌注牛磺酸对家兔体温的影响和用放射免疫分析法检测腹中隔、下丘脑区ＡＶＰ含量的变化。结果：牛磺酸具有降低家兔ＥＴ发热和正常体温的作用，伴有腹中隔ＡＶＰ含量明显增加（Ｐ＜００１）；而下丘脑ＡＶＰ含量变化不明显（Ｐ＞００５）。结论：牛磺酸的降温作用可能与腹中隔ＡＶＰ的含量增加有关     

Hypotonicity stimulates translocation of ICln in neonatal rat cardiac myocytes

 Cell volume expansion stimulates the efflux of solutes, including the amino acid taurine, to accomplish a regulatory volume decrease (RVD). One protein that may play a role in taurine efflux is the cytosolic protein ICln. In rat neonatal cardiac myocytes under isotonic conditions, ICln is found predominantly (greater than 90%) in the cytosol. However, after cell volume expansion by exposure to hypotonic medium, ICln rapidly translocates to the particulate fraction (the Triton X-114-insoluble fraction). After 2 min in hypotonic medium the percentage of ICln in the particulate fraction increases to 30%, 46% at 5 min, 40% at 10 min, and 25% at 30 min. The time course of this response is similar to that of hypotonicity-stimulated taurine efflux. Hypotonicity-stimulated taurine efflux as well as ICln translocation parallel the reduction in medium osmolarity. As osmolarity decreases, taurine efflux and ICln movement increase. The movement of ICln from the particulate back to the cytosolic fraction is accelerated when volume-expanded cells are returned to isotonic medium. When ICln is analyzed under non-denaturing conditions, a dimer is detected in the particulate fraction of volume-expanded cells, along with the monomer. This dimer is not detected in the cytosol. Treatment of the particulate fraction from volume-expanded cells with the lyotropic agent KSCN caused release of ICln but not Na-K-ATPase into the soluble fraction, indicating that translocated ICln associates with membranes in the particulate fraction rather than inserting into them. Received: 31 October 1997 / Received after revision and accepted: 23 March 1998     

Increase in taurine content before onset of seizures induced by a glutamate decarboxylase inhibitor

Summary The concentration of taurine was measured in 15 brain regions of the rabbit before the onset of convulsions induced by the potent glutamate decarboxylase inhibitor methoxypyridoxine. A significant rise in taurine content was observed in the hippocampus, putamen, caudate nucleus, frontal cortex, thalamus and hypothalamus. GABA levels determined from the same tissue samples were all significantly reduced. An unaffected taurine synthesis coupled with blocked transport to the blood is considered as a possible explanation for this taurine increase.     

侧脑室灌注牛磺酸对家兔db—cAMP性发热的影响

将30只新西兰白兔随机均分成3组,第一组二丁酰腺苷环一磷酸 牛磺酸,第二组生理盐水 牛磺酸和第三组二丁酰腺苷环一磷酸 生理盐水。采用侧脑室埋管、侧脑室注射和灌注技术,把牛磺酸和二丁酰腺苷环一磷酸等被试物注入动物脑室。结果发现,在中枢注射二丁酰腺苷环一磷酸导致单相长热程的过程中,侧脑室注入牛磺酸能明显抑制二丁酰腺苷环一磷酸性发热。3组的6小时体温反应指数分别为7.10±2.44、0.21±1.58和12.47±4.60(P<0.01)。本文还讨论了牛磺酸抑热的可能机制。     

牛磺酸对大鼠异丙肾上腺素心肌损伤的保护作用

大鼠皮下注射异丙肾上腺素(40mg/kg/日)造成心肌损伤模型,发现心肌组织钙含量和心肌AGTⅡ水平显著增加,并伴有严重的心肌损伤。若同时注射牛磺酸(200mg/kg/日)或疏甲丙辅酸(1mg/kg/日)均可拮抗异丙肾上腺素引起的心肌钙增加及AGTⅡ水平升高,并可缓解心肌组织病理损伤。结果提示牛磺酸抑制心肌AGTⅡ可能是其拮抗异丙肾上腺素心肌损伤的重要机制之一。     

Effective T-cell recall responses require the taurine transporter Taut

T-cell activation and the subsequent transformation of activated T cells into T-cell blasts require profound changes in cell volume. However, the impact of cell volume regulation for T-cell immunology has not been characterized. Here we studied the role of the cell-volume regulating osmolyte transporter Taut for T-cell activation in Taut-deficient mice. T-cell mediated recall responses were severely impaired in taut(-/-) mice as shown with B16 melanoma rejection and hapten-induced contact hypersensitivity. CD4(+) and CD8(+) T cells were unequivocally located within peripheral lymph nodes of unprimed taut(-/-) mice but significantly decreased in taut(-/-) compared with taut(+/+) mice following in vivo activation. Further analysis revealed that Taut is critical for rescuing T cells from activation-induced cell death in vitro and in vivo as shown with TCR, superantigen, and antigen-specific activation. Consequently, reduction of CD4(+) and CD8(+) T cells in taut(-/-) mice upon antigen challenge resulted in impaired in vivo generation of T-cell memory. These findings disclose for the first time that volume regulation in T cells is an element in the regulation of adaptive immune responses and that the osmolyte transporter Taut is crucial for T-cell survival and T-cell mediated immune reactions.     

牛磺酸减轻内毒素诱导的大鼠心肌损伤

目的:探讨牛磺酸对内毒素(即脂多糖,LPS)诱导的大鼠心肌损伤的影响。方法:健康雄性Sprague-Dawley(SD)大鼠30只随机分为3组:正常对照组、内毒素模型组及牛磺酸处理组。正常对照组和内毒素模型组大鼠尾静脉注射生理盐水,牛磺酸处理组大鼠尾静脉注射牛磺酸(100 mg/kg),2 h后,内毒素模型组和牛磺酸处理组大鼠腹腔注射LPS(10 mg/kg),正常对照组大鼠腹腔注射生理盐水。注射内毒素6 h后,采集血样品和心肌组织,检测血清超氧化物歧化酶(SOD)活性、丙二醛(MDA)含量、肿瘤坏死因子α(TNF-α)及白细胞介素6(IL-6)水平;光镜下观察心肌形态学变化;Western blot检测心肌组织磷酸化核因子κB(p-NF-κB)、环氧合酶2(COX-2)、TNF-α、IL-6及血红素加氧酶1(HO-1)的表达。结果:与正常对照组比较,内毒素模型组大鼠血清SOD活性及心肌组织HO-1表达明显降低(P0.01),血清MDA、TNF-α和IL-6水平明显升高(P0.01),心肌组织p-NF-κB、COX-2、TNF-α及IL-6水平明显升高(P0.01)。与内毒素模型组比较,牛磺酸处理组大鼠血清MDA、TNF-α和IL-6水平明显降低(P0.01),牛磺酸处理明显降低心肌组织COX-2、TNF-α、IL-6及p-NF-κB水平(P0.01),血清SOD活性及心肌组织HO-1表达明显提高(P0.01)。组织学观察显示内毒素模型组大鼠心肌组织有炎症细胞浸润,心肌纤维排列疏松不规则,而正常对照组和牛磺酸处理组大鼠心肌纤维排列整齐规则。结论:牛磺酸预处理能减轻内毒素诱导的心肌损伤,其机制可能通过HO-1/CO信号下调p-NF-κB/COX-2而发挥作用。     

牛磺酸对百草枯中毒大鼠肾损伤的作用及机制研究

目的:探讨不同剂量牛磺酸对百草枯中毒大鼠肾脏氧化应激和炎症反应的影响。方法:选取48只雄性SD大鼠随机分为阴性对照组、百草枯染毒组、百草枯染毒+小剂量牛磺酸组和百草枯染毒+大剂量牛磺酸组。采用生化检测仪检测大鼠血清肌酐及尿素氮水平;比色法检测血标本中丙二醛(malondialdehyde,MDA)和超氧化物歧化酶(superoxide dismutase,SOD)水平评估氧化应激状态;另以ELISA法检测血标本中IL-6及细胞间黏附分子1(ICAM-1)水平评估炎症反应;DHE荧光探针检测肾脏活性氧簇(reactive oxygen species,ROS)水平;Western blot法检测大鼠肾脏标本丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)中p-P38 MAPK、p-JNK和pERK1/2的蛋白水平;并以real-time PCR法检测大鼠肾脏内TNF-α、TGF-β1及IL-6的mRNA水平。结果:百草枯中毒大鼠血清肌酐及尿素氮增高,牛磺酸干预后降低了中毒大鼠的肌酐及尿素氮水平,且大剂量牛磺酸组的肌酐及尿素氮水平更低。牛磺酸的干预不仅明显减轻了肾脏组织的氧化应激与炎症反应,同时也降低了百草枯中毒大鼠肾脏的MAPK活性。结论:牛磺酸干预能减轻百草枯中毒大鼠的肾损伤,其作用机制可能与下调了肾脏MAPK活性、减轻了肾脏氧化应激及炎症反应有关。     

牛磺酸对大鼠脑缺血再灌注损伤时细胞凋亡的影响

目的：研究牛磺酸对脑缺血再灌注损伤中细胞凋亡的影响。方法：建立大鼠全脑缺血再灌注模型，使用ＤＮＡ片段原位末端标记和流式细胞仪观察了Ｔａｕ治疗后细胞凋亡的变化。结论：Ｔａｕ对脑缺血再灌汪损伤中神经细胞的保护作用可能部分由于降低细胞凋亡的发生。     

雷帕霉素对小鼠星形胶质细胞体外诱导凋亡的影响

目的:探讨雷帕霉素(rapamycin,Rapa)对小鼠星形胶质细胞体外凋亡的影响。方法:无菌分离并体外培养C57BL/6J幼鼠脑组织星形胶质细胞。通过MTT比色法测定并分析Rapa浓度对幼鼠星形胶质细胞存活的影响;SYTOXGreen荧光染色联合荧光酶标仪检测并分析Rapa对H2O2、ionomycin、deferorxamine等诱导剂作用一定时间内细胞存活的影响;Di OC6(3)染色分析Rapa在H2O2氧化应激损伤条件下对星形胶质细胞线粒体膜电势的影响;分别采用H2DCFDA和Mito SOXTMRed荧光染色联合流式细胞术检测Rapa预适应对星形胶质细胞ROS生成以及线粒体内ROS含量的影响。结果:Rapa能促进H2O2以及ionomycin联合deferorxamine损伤作用下的星形胶质细胞的存活,对线粒体膜电势有保护作用,可降低H2O2损伤作用下星形胶质细胞ROS的产生并可以维持胞内线粒体ROS的含量在较低水平。结论:Rapa能够减少细胞内ROS的生成量并降低胞内线粒体内ROS水平;能够减轻H2O2对细胞线粒体膜的损伤破坏,维护线粒体膜电势的稳定性,进而对氧化应激损伤介导细胞凋亡有一定的抑制作用。     

星形胶质细胞在脑缺血诱导的炎症反应中的作用及其机制

脑缺血诱发的炎症反应在急性期可引发脑水肿,挤压缺血灶周围正常脑组织,从而加重神经功能损伤;而在卒中恢复期,对神经组织进行修复具有重要保护作用。近年研究发现星形胶质细胞(Ast)也参与脑缺血后的炎症反应,通过产生抑炎/促炎因子及形成胶质瘢痕/胶质限制对脑组织兼具保护和损伤双重作用,多条信号通路参与这一过程,此外,其与小胶质细胞协同作用也越来越受到重视。靶向调控星形胶质细胞调节脑缺血后炎症反应、促进康复为缺血性卒中治疗和新药研发指明了新的方向。     

Changes in plasma concentration of taurine in stroke

Taurine is critical for proper brain functioning. Increase in plasma taurine concentration has already been shown in many diseases [1,2,5,10,12,14,17,22,25,47]. The plasma concentrations of taurine in 60 patients, suffering from stroke, were compared with that of 54 healthy volunteers. The plasma samples of the patients were obtained three times in the first five days of hospitalization. A Student's t-test showed a significant difference (P<0.0001) between the plasma concentrations of taurine of the patients group (136.5±8.2mmol/L) and the control group (41.9±2.5mmol/L). In addition, a one-way repeated measures ANOVA test showed that the mean plasma concentration of taurine in the patients during the first five days of hospitalization declined significantly from 136.9±8.2mmol/L in the first day of hospitalization to 120.1±5.9mmol/L on the third day and 110.2±7.0mmol/L by the fifth day (P>0.05). The plasma concentration of taurine was increased in the patients with stroke probably because of brain tissue damage. Although, according to the result of the study, mean plasma taurine concentration in stroke patients declined during five days of hospitalization. Further studies are needed to introduce taurine as a biomarker of recovery in stroke.     

牛磺酸对大鼠脑缺血再灌注损伤时内皮素变化的影响

为了探讨牛磺酸对缺血－再灌注损伤的保护机理，在大鼠脑缺血－再灌注模型上观察牛磺酸治疗效果，发现牛磺酸（６００ｍｇ／ｋｇｉ．ｐ．）能明显减轻脑组织的损伤和钙超载，增加脑组织牛磺酸的含量，抑制血浆和脑脊液内皮素水平的增加。在离体孵育的大鼠脑血管条上发现牛磺酸呈剂量依赖地抑制缺氧引起的内皮素释放。以上结果提示牛磺酸抑制内皮素释放可能是其防治脑缺血－再灌注损伤的机理之一。     

Significant apoptosis rather autophagy predominates in astrocytes caused by Toxocara canis larval excretory-secretory antigens

Background/purposeToxocariasis is a worldwide parasitic zoonosis and mainly caused by Toxocara canis. Humans can be infected by accidental ingestion of T. canis embryonated ova through contacting with contaminated food, water, or encapsulated larvae in paratenic hosts' viscera or meat. Since humans are the paratenic host of T. canis, the wandering and neuroinvasive larvae can cause mechanical tissue damage and the excretory-secretory antigens (TcES Ag) might induce neuroinflammatory responses in the brain. Human cerebral toxocariasis (CT) has been reported to cause several neurological symptoms and may develop into neurodegenerative diseases. However, the roles of astrocytes involved in the pathogenesis of CT remained largely unclear.MethodsThis study intended to investigate the cytotoxic effects of TcES Ag on astrocytes as assessed by apoptosis and autophagy expression.ResultsOur results showed TcES Ag treatment reduced cell viability and caused morphological changes. Expressions of autophagy associated proteins including Beclin 1, phosphor-mTOR and LC3-Ⅱ were not significantly changed; however, p62 as well as the cell survival protein, mTOR, was concomitantly decreased in TcES Ag treatment. Significantly accelerated cleaved caspase-3 and cytochrome c expression as well as enhanced caspase-9 and caspase-8 activation were found in astrocytes with TcES Ag treatment. Caspase-3 activity and apoptotic cells numbers were also increased as detected by fluorescence microscopy.ConclusionWe concluded that TcES Ag may trigger astrocytes apoptosis predominantly through intrinsic and extrinsic pathways rather autophagy, revealing a novel role of astrocytes in the pathogenesis of CT.     

Formation of inositol phosphates mediated by M3 muscarinic receptors in type-1 and type-2 astrocytes from neonatal rat cerebral cortex

Muscarinic receptor subtype in type-1 and type-2 astrocytes from rat neonalal cerebral cortex was examined for carbachol-elicited inositol phosphate (IP) formation. The formation of carbachol-elicited IP was inhibited by various muscarinic antagonists in the following relative order of potency: 4-DAMP atropine pirenzepine > AF-DX 116. This pharmacological profile suggests that the activation of the M₃ muscarinic receptor subtype is responsible for the stimulation of IP formation in both astrocytes.     

过氧化氢对乳鼠心肌细胞内游离钙浓度的影响及牛磺酸的拮抗作用

目的:研究过氧化氢(H₂O₂)对心肌细胞i的影响,以及牛磺酸对H₂O₂诱导钙超载的拮抗作用。方法:采用SD大鼠乳鼠进行心肌细胞培养,实验分4组:①正常对照组;②H₂O₂组:加入终浓度为100μmol/L的H₂O₂;③H₂O₂＋牛磺酸(同时)组:牛磺酸30mmol/L与H₂O₂100μmol/L同时加入;④H₂O₂＋牛磺酸(先后)组:先加入终浓度为100μmol/L的H₂O₂,2min后再加20mmol/L的牛磺酸。以Fluo-3/AM荧光指示剂负载,应用激光共聚焦显微镜技术,分别于加入H₂O₂后即刻与15min,检测i变化。结果:对照组心肌细胞内荧光强度和荧光光密度值较低。H₂O₂加入后即刻,细胞内荧光光密度值开始增加,15min后细胞内荧光强度和荧光光密度值显著高于对照组(P＜0.05)。而H₂O₂＋牛磺酸(同时)组细胞内荧光光密度值显著低于H₂O₂组(P＜0.05vsH₂O₂组);H₂O₂＋牛磺酸(先后)组细胞内荧光光密度值显著低于H₂O₂组(P＜0.05vsH₂O₂组)。结论:H₂O₂可引起心肌细胞内钙超载;牛磺酸能显著减轻H₂O2诱导的心肌细胞内Ca²⁺超载。