Potential Role of Cerebral Glutathione in the Maintenance of Blood-Brain Barrier Integrity in Rat

Using the model of glutathione (GSH) depletion, possible role of GSH in the maintenance of blood-brain barrier (BBB) integrity was evaluated in rats. Administration (ip) of GSH depletors, diethyl maleate (DEM, 1–4 mmol/kg), phorone (2–3 mmol/kg) and 2-cyclohexene-1-one (CHX, 1 mmol/kg), to male adults was found to deplete brain and liver GSH and increase the BBB permeability to micromolecular tracers (sodium fluorescein and [¹⁴C]sucrose) in a dose-dependent manner at 2h. However, BBB permeability to macromolecular tracers such as horseradish peroxidase and Evan's blue remained unaltered. It was also shown that observed BBB permeability dysfunction was associated with brain GSH depletion. A lower magnitude of BBB increase in rat neonates, as compared to adults, indicated a possible bigger role of GSH in the BBB function of mature brain. The treatment with N-acetylcysteine, methionine and GSH provided a partial to full protection against DEM-induced brain (microvessel) GSH depletion and BBB dysfunction; however, the treatment with -tocopherol, ascorbic acid and turmeric were not effective. Our studies showed that cerebral GSH plays an important role in maintaining the functional BBB integrity.     

洛伐他汀抑制Rho激酶信号通路增强人内皮一氧化氮合酶信使RNA稳定性

他汀类药物可上调内皮一氧化氮合酶（ENOS）的表达,并由甲羟戊酸（MVA）途径介导,但具体机制未完全阐明．本研究旨在探索洛伐他汀(LVT)上调ENOS表达的分子信号机制并明确同ENOS表达相关的顺式作用元件的定位．洛伐他汀通过减少细胞内固醇,如MVA和geranylgeranyl pyrophosphate （GGPP）,从而增加ENOS mRNA的稳定性．因GGPP是细胞内信号蛋白如Ras、Rho GTPase进行翻译后修饰和膜定位所必需的,因此很可能洛伐他汀的作用与细胞内信号途径有关．进一步的实验结果显示Rho激酶抑制剂 hydroxyfasudil和细胞松弛素D均可在转录后水平上调ENOS mRNA表达,表明Rho途径介导的细胞骨架状态在ENOS mRNA降解率的调控中起一定作用． 细胞转染实验证明调控ENOS mRNA 降解的顺式作用元件位于其mRNA序列上的3′未翻译区(3′UTR)和相邻的编码区．其中调控GGPP介导ENOS mRNA稳定性的顺式作用元件位于序列的3 751～4 606位点间;而hydroxyfasudil通过位于3 751～ 4 468位点间的顺式作用元件稳定ENOS mRNA;细胞松弛素D通过位于3 904～4 188位点间的元件稳定ENOS mRNA．洛伐他汀可能通过抑制Rho激酶途径稳定ENOS mRNA,此过程由位于mRNA序列上3′UTR及相邻编码区的多样顺式作用元件介导．另外,细胞骨架的空间构造也可影响ENOS mRNA的稳定性,此过程由位于其mRNA序列编码区的顺式作用元件介导．本研究为转录子稳定性调控机制的进一步研究提供了有力根据,或许可为心血管疾病的治疗提供新的分子靶点．     

雌性动物生殖系统中的一氧化氮

一氧化氮(nitric oxide,NO)属于无机自由基气体,作为一种特殊的生物传递信号分子,日益受到生命科学各领域的普遍重视。机体内的NO是由三种一氧化氮合酶(nitric oxide synthase,NOS)合成的。NOS在体内的分布极为广泛,几乎遍布机体的每一个系统。研究表明,生殖系统中的NO参与了卵泡的发育和成熟、胚胎的植入、妊娠的维持、分娩等许多生理过程。现就NO在雌性生殖系统中的作用进行阐述。     

Inhibition of Transforming Growth Factor-<Emphasis Type="Bold">β</Emphasis> Production in Brain Pericytes Contributes to Cyclosporin A-Induced Dysfunction of the Blood-Brain Barrier

1. The present study was designed to clarify whether brain pericytes and pericyte-derived transforming growth factor-β1 (TGF-β1) participate in cyclosporin A (CsA)-induced dysfunction of the blood-brain barrier (BBB). 2. The presence of brain pericytes markedly aggravated CsA-increased permeability of MBEC4 cells to sodium fluorescein and accumulation of rhodamine 123 in MBEC4 cells. 3. Exposure to CsA significantly decreased the levels of TGF-β1 mRNA in brain pericytes in pericyte co-cultures. Treatment with TGF-β1 dose-dependently inhibited CsA-induced hyperpermeability and P-glycoprotein dysfunction of MBEC4 cells in pericyte co-cultures. 4. These findings suggest that an inhibition of brain pericyte-derived TGF-β1 contributes to the occurrence of CsA-induced dysfunction of the BBB.     

一氧化氮对小麦叶片老化过程的调节

用不同浓度(0.05、0.10、0.20、0.50 mmol/L)的外源一氧化氮(nitric oxide,NO)供体硝普钠(sodiumnitroprusside,SNP)处理正常生长小麦(Triticum aestivum L.)叶片(二叶一心期时全展第一叶).结果显示低浓度SNP(0.05、0.10、0.20 mmol/L)可以明显降低叶片H2O2和MDA的水平,其中0.10 mmol/LSNP的作用最为明显;而较高浓度SNP(0.50 mmol/L)则作用相反.进一步采用0.10 mmol/L SNP处理不同叶位的小麦叶片(四叶一心期),结果表明低浓度NO对不同老化阶段中叶片的H2O2、O-2和MDA累积都有缓解作用,并明显减缓叶绿素、可溶性叶蛋白尤其是Rubisco的降解,有效延缓了叶片的老化进程.在完整叶绿体体外老化实验中也发现,不同浓度SNP(0.05、0.10、0.20、0.50、1.00、5.00 mmol/L)的作用同样表现双重性,其中0.20 mmol/L SNP对膜结构及Rubisco保护作用最明显.上述结果证实,低浓度外源NO可延缓小麦叶片的老化过程,并可能与其降低叶片活性氧(ROS)水平及缓解氧化损伤有关.     

一氧化氮对小麦叶片老化过程的调节

用不同浓度(0．05、0．10、0．20、0．50mmol／L)的外源一氧化氮(nitric oxide,NO)供体硝普钠(sodium nitroprusside,SNP)处理正常生长小麦(Triticum aestivum L．)叶片(二叶一心期时全展第一叶)。结果显示低浓度SNP(0．05、0．10、0．20mmol／L)可以明显降低叶片H2O2和MDA的水平,其中0．10mmol／L SNP的作用最为明显;而较高浓度SNP(0．50mmol／L)则作用相反。进一步采用0．10mmol／L SNP处理不同叶位的小麦叶片(四叶一心期),结果表明低浓度NO对不同老化阶段中叶片的H2O2、O2^7和MDA累积都有缓解作用,并明显减缓叶绿素、可溶性叶蛋白尤其是Rubisco的降解,有效延缓了叶片的老化进程。在完整叶绿体体外老化实验中也发现,不同浓度SNP(0．05、0．10、0．20、0．50、1．00、5．00mmol／L)的作用同样表现双重性,其中0．20mmol／L SNP对膜结构及Rubisco保护作用最明显。上述结果证实,低浓度外源NO可延缓小麦叶片的老化过程,并可能与其降低叶片活性氧(ROS)水平及缓解氧化损伤有关。     

On the mechanism of cerebral accumulation of cholestanol in patients with cerebrotendinous xanthomatosis

The most serious consequence of sterol 27-hydroxylase deficiency in humans [cerebrotendinous xanthomatosis (CTX)] is the development of cholestanol-containing brain xanthomas. The cholestanol in the brain may be derived from the circulation or from 7alpha-hydroxylated intermediates in bile acid synthesis, present at 50- to 250-fold increased levels in plasma. Here, we demonstrate a transfer of 7alpha-hydroxy-4-cholesten-3-one across cultured porcine brain endothelial cells (a model for the blood-brain barrier) that is approximately 100-fold more efficient than the transfer of cholestanol. Furthermore, there was an efficient conversion of 7alpha-hydroxy-4-cholesten-3-one to cholestanol in cultured neuronal and glial cells as well as in monocyte-derived macrophages of human origin. It is concluded that the continuous intracellular production of cholestanol from a bile acid precursor capable of rapidly passing biomembranes, including the blood-brain barrier, is likely to be of major importance for the accumulation of cholestanol in patients with CTX. Such a mechanism also fits well with the observation that treatment with chenodeoxycholic acid, which normalizes the level of the bile acid precursor, results in a reduction of cholestanol-containing xanthomas even in the brain.     

Functional expression of the P-glycoproteinmdr in primary cultures of bovine cerebral capillary endothelial cells

The P-glycoproteinmdr is expressed not only in tumoral cells, but also in nontransformed cells, including the specialized endothelial cells of brain capillaries which build up the blood-brain barrier. Since all previously identified blood-brain barrier markers are rapidly lost when cerebral capillary endothelial cells are maintained in primary culture, we have investigated whether P-glycoprotein (P-gp) would follow the same rule, in order to address the influence of the cerebral environment on the specific P-gp expression in the brain endothelium. As compared to freshly isolated purified cerebral capillaries, P-glycoprotein was detected by immunochemistry at a high level in 5–7 day primary cultures. In our culture conditions, P-glycoprotein was immunodetected at a lower molecular weight than that found in freshly isolated capillaries. Enzymatic deglycosylation led to the same 130 kDa protein for both fresh and cultured samples, suggesting that P-gp post-translational modifications were altered in primary cultures. However, studies on the uptake and efflux of the P-gp substrate [³H]vinblastine, and on the effect of variousmdr reversing agents on the uptake and efflux, clearly indicated that the efflux pump function of the P-glycoprotein was maintained in primary cultures of bovine cerebral capillary endothelial cells. P-Glycoprotein may thus represent the first blood-brain barrier marker which is maintained in cerebral endothelial cells cultured in the absence of factors originating from the brain parenchyma.Abbreviations BBB blood-brain barrier - BCEC brain capillary endothelial cells - -GT -glutamyltranspeptidase - HBSS Hank's balanced salt solution - Mab monoclonal antibody - mdr multidrug resistance - P-gp P-glycoprotein     

一氧化氮在植物中的生理功能

一氧化氮是植物体内一种重要的活性分子,它对植物的种子萌发、生长发育、气孔运动、呼吸作用以及抗逆反应等生理过程起重要的调节作用,与植物激素存在密切关系。现对一氧化氮在植物中的生理功能进行综述。     

Uptake and Efflux of Quinacrine, a Candidate for the Treatment of Prion Diseases, at the Blood-Brain Barrier

1. A clinical trial of quinacrine in patients with Creutzfeldt-Jakob disease is now in progress. The permeability of drugs through the blood-brain barrier (BBB) is a determinant of their therapeutic efficacy for prion diseases. The mechanism of quinacrine transport across the BBB was investigated using mouse brain endothelial cells (MBEC4). 2. The permeability of quinacrine through MBEC4 cells was lower than that of sodium fluorescein, a BBB-impermeable marker. The basolateral-to-apical transport of quinacrine was greater than its apical-to-basolateral transport. In the presence of P-glycoprotein (P-gp) inhibitor, cyclosporine or verapamil, the apical-to-basolateral transport of quinacrine increased. The uptake of quinacrine by MBEC4 cells was enhanced in the presence of cyclosporine or verapamil. 3. Quinacrine uptake was highly concentrative, this event being carried out by a saturable and carrier-mediated system with an apparent Km of 52.1 microM. Quinacrine uptake was insensitive to Na+-depletion and changes in the membrane potential and sensitive to changes in pH. This uptake was decreased by tetraethylammonium and cimetidine, a substrate and an inhibitor of organic cation transporters, respectively. 4. These findings suggest that quinacrine transport at the BBB is mediated by the efflux system (P-gp) and the influx system (organic cation transporter-like machinery).     

Role of Nitric Oxide in Pathogenesis Underlying Ischemic Cerebral Damage

1. Based upon the intriguing report that nitric oxide synthase (NOS) inhibitor dose-dependently reverses N-methyl-D-aspartate (NMDA)-induced neurotoxicity observed in primary cortical cell cultures, many laboratories have investigated whether NOS inhibition is beneficial as a treatment for cerebral ischemia.2. Although the results are variable, it is likely thought that nitric oxide plays a key role in pathomechanism underlying ischemic brain damage.3. We review the experimental studies on effects of NOS inhibition on cerebral ischemia and measuring nitric oxide produced in the brain subjected to cerebral ischemia.4. Finally, the possibility of NOS inhibitors as a therapeutical tool is discussed.     

Growth of brain microvessel endothelial cells on collagen gels: Applications to the study of blood-brain barrier physiology and CNS inflammation

Summary Brain microvessel endothelial cells (BMEC) exhibit the tendency to migrate through 3.0-vm pore semipermeable inserts and establish monolayers on both apical and basal filter surfaces. This can potentially lead to complications in accurately assessing a wide variety of physiologic parameters uniquely associated with these cells. To avoid this problem, we have explored growing BMEC on Transwell filters coated with hydrated collagen gels. BMEC seeded on such gels grow as a monolayer until confluency, but do not invade the subendothelial collagen matrix or the underlying support filter. Furthermore, BMEC grown in this manner exhibit biochemical, morphologic, and electrophysiologic properties reflective of the endothelial cells that comprise the blood-brain barrier in vivo. Although the collagen gel acts as an impenetrable barrier to BMEC, and thus ensures the growth of only a single layer of cells, it nevertheless can be infiltrated by monocytes that have been stimulated by a chemotaxin to undergo diapedesis. Thus, growing BMEC on collagen gel-coated Transwells has broad applications for the in vitro study of both blood-brain barrier physiology as well as the mechanisms underlying central nervous system inflammation.     

利用微电子细胞传感器阵列动态监测内皮屏障功能

内皮在保持血管的动态平衡中起着至关重要的作用,而内皮屏障的功能损伤是很多炎症反应的重要特征。利用实时细胞电子分析系统体外研究脐静脉内皮的屏障功能,从细胞的贴附、生长到融合状态,实时监测了微电子细胞传感器阵列上细胞阻抗的动态变化。α-凝血酶以剂量依赖的方式显著诱导单层内皮细胞阻抗快速下降,而后缓慢回升。相应地,α-凝血酶显著引起细胞单层对荧光标记葡聚糖通透性的改变和F-肌动蛋白细胞骨架的重分布。结果表明,实时细胞阻抗系统可能成为一种体外研究血管内皮细胞形态和屏障功能的有力的手段。     

Preliminary Characterization of Glial-Secreted Factors Responsible for the Induction of High Electrical Resistances Across Endothelial Monolayers in a Blood-Brain Barrier Model

Factors secreted by C6 glioma cells which induce electrical resistances across endothelial monolayers in an in vitro blood-brain barrier model have been partially characterised for the first time. These transendothelial electrical resistances (TEERs) were only evident when cell-free conditioned medium derived from C6 glioma cells was applied to the basolateral surfaces of confluent ECV304 or ECV304-9 cells which are both human umbilical vein endothelial cell lines (HUVEC). Electrical resistance values as high as 600 ohm. sq cm were obtained with this blood-brain barrier model and ultrafiltration techniques suggest that any factor(s) in the conditioned medium responsible for these TEERs have molecular masses of less than 1000 Da. Enzymic proteolysis and heat treatment carried out on the conditioned medium failed to inhibit its effect on the HUVEC monolayers suggesting that these C6 cell-secreted factors are unlikely to be proteins.     

NO在植物中的调控作用

一氧化氮(NO)是一种易扩散的生物活性分子,是生物体内重要的信号分子.植物细胞通过NO合酶、硝酸还原酶、或非生化反应途径产生NO.NO参与植物生长发育调控和对生物与非生物环境胁迫的应答反应,大量证据表明NO是植物防御反应中的关键信使,其信号转导机制也受到越来越多的关注.本文主要通过讨论NO的产生、对植物生长周期的影响、在植物代谢中的信号调节以及参与细胞凋亡来阐述NO在植物中的作用.     

一种实时检测剪切力引起培养的大鼠动脉内皮细胞内一氧化氮含量的新方法

建立一个稳定和实时检测在不同剪切力作用下内皮细胞内一氧化氮含量的方法。利用流动小室建立内皮细胞剪切模型 ,在内皮细胞用DAF FM染色后 ,用Zeiss荧光共聚焦显微镜和ICCD摄象头检测细胞内的荧光强度。DAF FM的荧光强度可以反映一氧化氮的胞内含量。剪切力引起内皮细胞合成一氧化氮增加 ,并且这种作用是随着剪切力的增加而增加。剪切力的作用被一氧化氮合酶抑制剂L NAME全部抑制 ,被无Ca2 缓冲液部分抑制。这个方法可以实时反映一氧化氮含量的变化 ,可以用来研究剪切力引起一氧化氮变化的机制以及用来评价内皮细胞对剪切力的反应特性     

Stretch in Brain Microvascular Endothelial Cells (cEND) as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying mechanisms involved in it are useful for treatment. There are both in vivo and in vitro methods available for this purpose. In vivo models can mimic actual head injury as it occurs during TBI. However, in vivo techniques may not be exploited for studies at the cell physiology level. Hence, in vitro methods are more advantageous for this purpose since they provide easier access to the cells and the extracellular environment for manipulation.Our protocol presents an in vitro model of TBI using stretch injury in brain microvascular endothelial cells. It utilizes pressure applied to the cells cultured in flexible-bottomed wells. The pressure applied may easily be controlled and can produce injury that ranges from low to severe. The murine brain microvascular endothelial cells (cEND) generated in our laboratory is a well-suited model for the blood brain barrier (BBB) thus providing an advantage to other systems that employ a similar technique. In addition, due to the simplicity of the method, experimental set-ups are easily duplicated. Thus, this model can be used in studying the cellular and molecular mechanisms involved in TBI at the BBB.     

Alterations in Transendothelial Electrical Resistance by Vasoactive Agonists and Cyclic AMP in a Blood-Brain Barrier Model System

We have previously reported that the co-culture of endothelial and glioma cell lines provides an in vitro model for investigating properties of the blood-brain barrier (BBB). To characterise the model system further we have investigated the effects of vasoactive substances implicated in increases in BBB permeability. Additionally, we have also examined whether activation of cyclic AMP signalling pathways, which elevate cerebral endothelial cell barrier function, similarly modulate our model system. ATP, histamine, bradykinin, and serotonin significantly decreased model BBB transendothelial electrical resistance and manipulations which elevate cyclic AMP enhanced culture resistance. These data indicate that our model BBB system responds in a manner characteristic of cerebral microvascular endothelial cells and the BBB in vivo. These data further emphasize the usefulness of our model system.     

Improved Method for the Preparation of a Human Cell-based,Contact Model of the Blood-Brain Barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.     

Glucose Transporter 1, Distribution in the Brain and in Neural Disorders: Its Relationship With Transport of Neuroactive Drugs Through the Blood-Brain Barrier

Facilitative glucose transport is mediated by one or more of the members of the closely related glucose transporter (GLUT) family. Thirteen members of the GLUT family have been described thus far. GLUT1 is a widely expressed isoform that provides many cells with their basic glucose requirement. It is also the primary transporter across the blood-brain barrier. This review describes the distribution and expression of GLUT1 in brain in different pathophysiological conditions including Alzheimers disease, epilepsy, ischemia, or traumatic brain injury. Recent investigations show that GLUT1 mediates the transport of some neuroactive drugs, such as glycosylated neuropeptides, low molecular weight heparin, and d-glucose derivatives, across the blood-brain barrier as a delivery system. By utilizing such highly specific transport mechanisms, it should be possible to establish strategies to regulate the entry of candidate drugs.