脱氧鬼臼毒素对美洲大蠊的毒力及几种酶系的影响

采用药膜法测试了脱氧鬼臼毒素对美洲大蠊Periplaneta americana初孵若虫的触杀活性,并测定了其对成虫中枢神经系统乙酰胆碱酯酶（AChE）和腺苷三磷酸酶(ATPase)离体活性的影响。结果表明：脱氧鬼臼毒素对美洲大蠊初孵若虫具有较强的毒杀活性,在接触时间24、48、72和96 h 的LC₅₀分别为26.26、4.68、1.51和0.62 μg/cm²;其对AChE没有明显影响; 对Na⁺ -K⁺ -ATPase有明显抑制作用,并存在浓度 效应关系,IC₅₀为44.9 μmol/L; 对Ca²⁺-Mg²⁺-ATPase表现出低剂量激活,高剂量抑制的现象。结果提示美洲大蠊的AChE不是脱氧鬼臼毒素靶标,而ATPase可能是脱氧鬼臼毒素的重要靶标之一。     

增效混剂对神经细胞钠通道的抑制作用

应用膜片钳技术,以MN-9D神经细胞为材料研究了溴氰菊酯及辛硫磷混剂的增效机理。膜片钳实验表明10^-5mol/L辛硫磷对Na⁺通道电流抑制作用很小,并随作用时间延长而逐步恢复。加药1 min Na⁺电流抑制率为6.99%,10 min为3.65%。10^-6 mol/L溴氰菊酯1 min抑制率为20.28%,10 min为21.43%。对蜚蠊中枢神经系统传导的动作电位抑制中时为53 min;10^-6mol/L溴氰菊酯与10^-5 mol/L辛硫磷混剂1 min抑制率为34.15%,10 min为36.69%,动作电位抑制中时为40 min,因此混剂可增强对Na⁺通道电流的抑制作用。通过Na⁺电流数据、尾电流衰减时间常数统计分析表明溴氰菊酯的修饰作用主要发生在关闭和静止状态的Na⁺通道,减缓通道的打开,延长通道关闭或失活状态。     

NaHCO3胁迫下星星草根中Ca2+与Ca2+-ATPase 的超微细胞化学定位

利用焦锑酸盐和磷酸铅沉淀技术分别对NaHCO₃胁迫条件下星星草(Puccinellia tenuiflora)根中Ca²⁺和Ca²⁺-ATPase 进行超微细胞化学定位研究, 旨在进一步探讨Ca²⁺在NaHCO₃胁迫诱导胞内信号转导过程中的作用, 以及Ca²⁺-ATPase活性定位变化与NaHCO₃胁迫下星星草抗盐碱能力的关系。结果表明: 在正常状态下, 根毛区细胞质内Ca²⁺较少, 主要位于质膜附近和液泡中, Ca²⁺-ATPase主要定位于质膜和液泡膜, 有一定活性。在0.448%NaHCO₃胁迫下, 根毛区细胞质中Ca²⁺增多, 液泡中Ca²⁺减少, 且主要集中于液泡膜附近, 质膜和液泡膜Ca²⁺-ATPase活性明显升高。在1.054%NaHCO₃胁迫下,细胞质中分布的Ca²⁺增多, 而液泡中Ca²⁺极少, Ca²⁺-ATPase活性也降低。以上结果表明, Ca²⁺亚细胞定位和Ca²⁺-ATPase活性变化在星星草响应NaHCO₃胁迫的信号传递过程中具有重要作用。     

多炔类化合物对美洲大蠊的触杀活性及对乙酰胆碱酯酶和腺苷三磷酸酶活性的影响

采用药膜法测定了人工合成的11个多炔类化合物对美洲大蠊Periplaneta americana初孵若虫的触杀活性。结果表明,当化合物处理浓度为20 μg/cm²时,致死率达70%以上的有: 化合物2 (1-叔丁基-4-羟甲基-丁二炔)、化合物9 (1-苯甲基-4-甲基-丁二炔)和化合物10 (O-炔丙基硫代磷酸二乙酯)。经毒力测定,化合物9和化合物10的LC₅₀分别为3.91 μg/cm²和1.50 μg/cm²。化合物2、化合物7 (1-苯基-4-邻硝基苯基-丁二炔)和化合物10对美洲大蠊乙酰胆碱酯酶(AChE)具有抑制活性,抑制率分别为12.00%、27.24%和62.22%。化合物2和化合物10对Na⁺-K⁺-ATPase具有抑制活性,抑制率分别为44.55%和31.44%;而化合物5 (1-苯基-4-（3,4-亚甲基二氧）苯基-丁二炔)和化合物6 (1-苯基-4-间硝基苯基-丁二炔)对该酶具有激活活性,激活率分别为24.98%和20.99%。化合物2、化合物4 （1-苯基-4-对甲氧基苯基-丁二炔）和化合物7对Ca²⁺-Mg²⁺-ATPase具有抑制活性,抑制率分别为49.02%、38.53%和35.32%, 其他化合物对该酶具有激活活性,其中激活活性最高的为化合物5,激活率达81.12%。     

马蔺体内主要阳离子含量月变化及分布

在自然盐碱生境下,通过测定不同月份土壤和马蔺体内主要阳离子Na⁺、K⁺、Ca²⁺、Mg²⁺的含量,研究了主要阳离子的吸收、转运变化及其在马蔺体内的分布.结果表明: 不同月份马蔺体内阳离子含量变动很大.在6月以后,随着马蔺的生长, Na⁺、K⁺、Ca²⁺和Mg²⁺4种离子在植物体内累积量逐渐增加.其中,根中Ca²⁺、Na⁺含量峰值出现在7月,分别为2.30%和0.51%,K⁺、Mg²⁺的含量峰值分别出现在9、10月,分别为0.27%和0.28%;叶片中Na⁺含量在7月达到最大值(0.57%);K⁺、Ca²⁺和Mg²⁺在8月分别达到1.30%、2.69%和0.47%.与Na⁺相比, 7、8月时马蔺对K⁺的选择吸收能力较低,但转运能力较强.马蔺对所测离子有很强的富集能力,各种离子在植物体内的含量都明显高于土壤背景值,且不同部位对离子的利用和累积能力不同,马蔺对各阳离子的累积主要集中在地上30 cm到地下40 cm范围内.马蔺地上部分平均单株K⁺、Na⁺、Ca²⁺和Mg²⁺含量分别是地下部分的9.11、4.07、0.98和2.27倍.     

蜂毒肽的溶血作用与红细胞膜上两种酶活性变化的关系

从蜂毒肽作用于红细胞膜上的Na^＋-K^＋-ATPase和葡萄糖-6-磷酸脱氢酶(G-6-PD)活性变化的角度,利用分光光度法测定酶活性,研究蜂毒肽与红细胞及膜作用过程中可能的靶点,讨论了蜂毒肽溶血过程与RBC膜上2种酶活性的变化．结果发现,蜂毒肽抑制RBC膜上酶活性的主要模式为附着/插入质膜与游离态并存模式,附着/插入质膜中的作用大于游离态的作用．Na^＋-K^＋-ATPase的K⁺结合位点是蜂毒肽的1个作用靶点．蜂毒肽插膜过程与其对此酶的作用随时间延长同步发生．蜂毒肽通过作用于葡萄糖-6-磷酸和NADP使G-6-PD的催化受到缓慢抑制,蜂毒肽形成四聚体的程度与酶活性密切相关．EDTA抑制蜂毒肽聚集,干扰蜂毒肽作用于G-6-P,蜂毒肽作用于底物G-6-P及辅酶NADP的生化机理相似,蜂毒肽抑制作用与G-6-PD的结构无关.     

盐胁迫下构树幼苗各器官中K+、Ca2+、Na+和Cl-含量分布及吸收特征

将当年生构树幼苗置于含有不同浓度（04、1、2、3、4 g·kg^-1）NaCl的土壤中,研究其生物量积累、叶片细胞质膜透性和K⁺、Ca²⁺、Na⁺、Cl^-等离子的吸收、分布及运输,并观察盐害症状.结果表明：构树幼苗的叶片质膜透性随着NaCl浓度的增加和胁迫时间的延长而升高,根冠比随NaCl浓度的升高而增加,大于3 g·kg^-1的土壤盐胁迫对构树叶片的质膜透性及植株的生物量积累影响显著.构树幼苗各器官中Na⁺和Cl^-含量随土壤NaCl浓度升高而显著增加,K⁺和Ca²⁺则随之降低,叶片各离子含量均明显高于根和茎.说明盐胁迫影响根系对K⁺和Ca²⁺的吸收,并抑制了它们向地上部分的选择性运输,使叶和茎的K⁺和Ca²⁺含量下降.构树通过吸收积累Na⁺和Cl^-抵御土壤盐分带来的渗透胁迫,但过量的Na⁺和Cl^-积累会造成单盐毒害.作为抗盐性较高的非盐生植物,构树地上部分的拒盐作用不显著.     

一种菊酯类复合剂对黑胸散白蚁体内CarEs和Ca-ATPase活性的影响

以黑胸散白蚁Reticulitermes chinensis Snyder为试验材料,研究了一种白蚁防治复合剂中的主要成分对白蚁体内羧酸酯酶（CarEs）和钙 腺苷三磷酸酶（Ca-ATPase）的影响。结果表明：氯菊酯在终浓度为1.66×10^-4 mol·L^-1以下时,对羧酸酯酶无明显抑制作用;八氯二丙醚和壬基酚聚氧乙烯醚在此浓度下都对羧酸酯酶表现出明显抑制作用,其IC₅₀分别为7.1148×10^-5 mol·L^-1和7.3373×10^-4 mol·L^-1;氯菊酯对Ca-ATPase表现出较强抑制作用,IC₅₀为5.11×10^-7 mol·L^-1。认为Ca-ATPase是黑胸散白蚁体内拟除虫菊酯类杀虫剂作用的主要靶标之一。     

金属离子和脲对白蜡虫碱性磷酸酶的影响

各种金属离子及脲对白蜡虫Ericerus pela (Chavannes)碱性磷酸酶的活性有不同的影响。从白蜡虫雌成虫中分离纯化得到碱性磷酸酶,加入各种不同浓度的金属离子及脲测定酶的活力。一价金属离子Na⁺、K⁺、Li⁺对酶活力没有影响。碱土金属离子Ca²⁺、Mg²⁺、Ba²⁺对酶有激活作用,激活作用的大小顺序依次为Ca²⁺、Ba²⁺、Mg²⁺。第一过渡金属离子中,Mn²⁺、Co²⁺、Ni²⁺对酶有激活作用,而Zn²⁺、Cu²⁺有抑制作用。重金属离子Cd²⁺、Pb²⁺对酶有抑制作用。Ca²⁺激活作用表现为非竞争性激活效应。Cu²⁺抑制作用表现为非竞争性抑制效应。脲对碱性磷酸酶有变性失活作用,按脲浓度可分为低于3 mol/L和高于3 mol/L两种类型。低浓度的脲对白蜡虫碱性磷酸酶的活性抑制的动力学表现为混合型效应。     

不同森林群落类型溪流水化学特征的季节动态

以小兴安岭凉水自然保护区内的阔叶红松林、云冷杉林和落叶松人工林为研究对象, 于2006年3—10月, 分析了其溪流水化学特征的动态变化. 结果表明: 不同月份3种森林群落溪流水的主要阳离子含量均表现为 Ca²⁺>Na⁺>K⁺>Mg²⁺, 主要阴离子含量均为HCO₃^->SO₄^2->NO₃^->Cl^-;不同群落类型的主要离子含量影响显著, 3种森林群落溪流水中Na⁺、Ca²⁺、Mg²⁺、Fe²⁺和Fe³⁺平均含量为云冷杉林>落叶松人工林>阔叶红松林, 而K⁺为落叶松人工林>云冷杉林>阔叶红松林; 主要阴离子平均含量均以落叶松人工林溪流水中为最高.     

Changes in Na+-K+-ATPase activity influence cell attachment to fibronectin

Most vital cellular functions aredependent on a fine-tuned regulation of intracellular ion homeostasis.Here we have demonstrated, using COS cells that were untransfected ortransfected with wild-type rat ouabain-resistantNa⁺-K⁺-ATPase, that partial inhibition ofNa⁺-K⁺-ATPase has a dramatic influence oncell attachment to fibronectin. Ouabain dose-dependently decreasedattachment in untransfected cells and in cells expressing wild-typeNa⁺-K⁺-ATPase, but not in cells expressingouabain-insensitive Na⁺-K⁺-ATPase, whereasinhibition of Na⁺-K⁺-ATPase by loweringextracellular K⁺ concentration decreased attachment in allthree cell types. Thirty percent inhibition ofNa⁺-K⁺-ATPase significantly attenuatedattachment. Na⁺-K⁺-ATPase inhibition caused asustained increase in the intracellular Ca²⁺ concentrationthat obscured Ca²⁺ transients observed in untreated cellsduring attachment. Inhibitors of Ca²⁺ transporterssignificantly decreased attachment, but inhibition ofNa⁺/H⁺ exchanger did not. Ouabain reduced focaladhesion kinase autophosphorylation but had no effect on cell surfaceintegrin expression. These results suggest that the level ofNa⁺-K⁺-ATPase activity strongly influences cellattachment, possibly by an effect on intracellular Ca²⁺.

     

Caloxin: a novel plasma membrane Ca2+ pump inhibitor

Plasma membrane (PM) Ca²⁺ pump is aCa²⁺-Mg²⁺-ATPase that expels Ca²⁺from cells to help them maintain low concentrations of cytosolic Ca²⁺. There are no known extracellularly acting PMCa²⁺ pump inhibitors, as digoxin and ouabain are forNa⁺ pump. In analogy with digoxin, we define caloxins asextracellular PM Ca²⁺ pump inhibitors and describe caloxin2A1. Caloxin 2A1 is a peptide obtained by screening a random peptidephage display library for binding to the second extracellular domain(residues 401-413) sequence of PM Ca²⁺ pump isoform1b. Caloxin 2A1 inhibits Ca²⁺-Mg²⁺-ATPase inhuman erythrocyte leaky ghosts, but it does not affect basalMg²⁺-ATPase or Na⁺-K⁺-ATPase in theghosts or Ca²⁺-Mg²⁺-ATPase in the skeletalmuscle sarcoplasmic reticulum. Caloxin 2A1 also inhibitsCa²⁺-dependent formation of the 140-kDa acid-stableacylphosphate, which is a partial reaction of this enzyme. Consistentwith inhibition of the PM Ca²⁺ pump in vascularendothelium, caloxin 2A1 produces an endothelium-dependent relaxationthat is reversed byN^G-nitro-L-arginine methyl ester.Thus caloxin 2A1 is a novel PM Ca²⁺ pump inhibitor selectedfor binding to an extracellular domain.

     

高胆固醇血症病人的红细胞膜ATP酶活性变化

研究表明高胆固醇血症病人的红细胞膜Na⁺-K⁺-ATP酶和Ca²⁺-Mg²⁺-ATP酶活性均降低,并且血浆总胆固醇和低密度脂蛋白-胆固醇浓度与这两种酶活性呈高度负相关,而血浆高密度脂蛋白-胆固醇浓度与Na⁺-K⁺-ATP酶活性呈正相关。这些变化的研究,对于进一步探讨动脉粥样硬化的发生机制及其防治可能具有重要意义。     

Ouabain potentiates the activation of ERK1/2 by carbachol in parotid gland epithelial cells; inhibition of ERK1/2 reduces Na(+)-K(+)-ATPase activity

The Na⁺-K⁺-ATPase and the ERK1/2 pathway appear to be linked in some fashion in a variety of cells. The Na⁺-K⁺-ATPase inhibitor ouabain can promote ERK1/2 activation. This activation involves Src, intracellular Ca²⁺ concentration ([Ca²⁺]_i) elevation, reactive oxygen species (ROS) generation, and EGF receptor (EGFR) transactivation. In contrast, ERK1/2 can mediate changes in Na⁺-K⁺-ATPase activity and/or expression. Thus signaling between ERK1/2 and Na⁺-K⁺-ATPase can occur from either direction. Whether such bidirectionality can occur within the same cell has not been reported. In the present study, we have demonstrated that while ouabain (1 mM) produces only a small (50%) increase in ERK1/2 phosphorylation in freshly isolated rat salivary (parotid acinar) epithelial cells, it potentiates the phosphorylation of ERK1/2 by submaximal concentrations of carbachol, a muscarinic receptor ligand that initiates fluid secretion. Although ERK1/2 is only modestly phosphorylated when cells are exposed to 1 mM ouabain or 10^–6 M carbachol, the combination of these agents promotes ERK1/2 phosphorylation to near-maximal levels achieved by a log order carbachol concentration. These effects of ouabain are distinct from Na⁺-K⁺-ATPase inhibition by lowering extracellular K⁺, which promotes a rapid and large increase in ERK1/2 phosphorylation. ERK1/2 potentiation by ouabain (EC₅₀ 100 µM) involves PKC, Src, and alterations in [Ca²⁺]_i but not ROS generation or EGFR transactivation. In addition, inhibition of ERK1/2 reduces Na⁺-K⁺-ATPase activity (measured as stimulation of QO₂ by carbachol and the cationophore nystatin). These results suggest that ERK1/2 and Na⁺-K⁺-ATPase may signal to each other in each direction under defined conditions in a single cell type. protein kinase C; intracellular Ca²⁺ concentration; muscarinic receptor; ₁-subunit; potassium removal     

Repression of the K+ Uptake and Cation-Stimulated ATPase Activity Associated with the Plasma Membrane-Enriched Fraction of Cucumber Roots Due to Ca2+ Starvation

The uptake of K⁺ by cucumber plants decreased markedly duringCa²⁺ starvation. A plasma membrane-enriched fraction, judgedfrom the distribution of marker enzymes, was prepared from controland Ca²⁺-starved roots. The Mg²⁺- and K⁺-Mg²⁺-ATPase activitiesassociated with the plasma membrane-enriched fraction of controlroots were maxima at pH 6.5. Various monovalent cations andpotassium salts of monovalent anions stimulated Mg²⁺-ATPaseactivity. Vanadate, DES and DCCD inhibited K⁺- Mg²⁺-ATPase activity.Of the divalent cations and phosphate esters tested, Mg²⁺ andATP were most effective for the stimulation of ATPase by K⁺,whereas Ca²⁺ was ineffective in replacing Mg²⁺. Mg²⁺- and K⁺-Mg²⁺-ATPase activities associated with the plasmamembrane enriched fraction of Ca²⁺-starved roots were much lowerthan those of control roots. K_m values of K⁺-Mg²⁺-ATPase forATP were comparable for control and Ca²⁺-starved roots. The K⁺-stimulated activity of Mg²⁺-ATPase in Ca²⁺-starved rootswas approximately one fourth that of the control, whereas therate of stimulation was only slightly lower in Ca²⁺-starvedroots. (Received May 9, 1984; Accepted September 17, 1984)     

Ca2+ mediates the effect of inhibition of Na+-K+-ATPase on the basolateral K+ channels in the rat CCD

We investigated the effect ofinhibiting Na⁺-K⁺-ATPase on the basolateral18-pS K⁺ channel in the cortical collecting duct (CCD) ofthe rat kidney. Inhibiting Na⁺-K⁺-ATPase withstrophanthidin decreased the activity of the 18-pS K⁺channel and increased the intracellular Ca²⁺ to 420 nM.Removal of extracellular Ca²⁺ abolished the effect ofstrophanthidin. When intracellular Ca²⁺ was raised with 5 µM ionomycin or A-23187 to 300, 400, and 500 nM, the activity of the18-pS K⁺ channel in cell-attached patches fell by 40, 85, and 96%, respectively. To explore the mechanism ofCa²⁺-induced inhibition, the effect of 400 nMCa²⁺ on channel activity was studied in the presence ofcalphostin C, an inhibitor of protein kinase C, or KN-93 and KN-62,inhibitors of calmodulin-dependent kinase II. Addition of calphostin Cor KN-93 or KN-62 failed to block the inhibitory effect of highconcentrations of Ca²⁺. This suggested that the inhibitoryeffect of high concentrations of Ca²⁺ was not mediated byprotein kinase C or calmodulin-dependent kinase II pathways. To examinethe possibility that the inhibitory effect of high concentrations ofCa²⁺ was mediated by the interaction of nitric oxide withsuperoxide, we investigated the effect of 400 nM Ca²⁺ onchannel activity in the presence of 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron) orN-nitro-L-arginine methyl ester.Pretreatment of the tubules with 4,5-dihydroxy-1,3-benzenedisulfonicacid or N-nitro-L-arginine methylester completely abolished the inhibitory effect of 400 nMCa²⁺ on channel activity. Moreover, application of4,5-dihydroxy-1,3-benzenedisulfonic acid reversed the inhibitory effectof strophanthidin. We conclude that the effect of inhibitingNa⁺-K⁺-ATPase is mediated by intracellularCa²⁺ and the inhibitory effect of high concentrations ofCa²⁺ is the result of interaction of nitric oxide with superoxide.

     

Functional significance of channels and transporters expressed in the inner ear and kidney

A number of ion channels and transporters are expressed in both the inner ear and kidney. In the inner ear, K⁺ cycling and endolymphatic K⁺, Na⁺, Ca²⁺, and pH homeostasis are critical for normal organ function. Ion channels and transporters involved in K⁺ cycling include K⁺ channels, Na⁺-2Cl^–-K⁺ cotransporter, Na⁺/K⁺-ATPase, Cl^– channels, connexins, and K⁺/Cl^– cotransporters. Furthermore, endolymphatic Na⁺ and Ca²⁺ homeostasis depends on Ca²⁺-ATPase, Ca²⁺ channels, Na⁺ channels, and a purinergic receptor channel. Endolymphatic pH homeostasis involves H⁺-ATPase and Cl^–/HCO₃^– exchangers including pendrin. Defective connexins (GJB2 and GJB6), pendrin (SLC26A4), K⁺ channels (KCNJ10, KCNQ1, KCNE1, and KCNMA1), Na⁺-2Cl^–-K⁺ cotransporter (SLC12A2), K⁺/Cl^– cotransporters (KCC3 and KCC4), Cl^– channels (BSND and CLCNKA + CLCNKB), and H⁺-ATPase (ATP6V1B1 and ATPV0A4) cause hearing loss. All these channels and transporters are also expressed in the kidney and support renal tubular transport or signaling. The hearing loss may thus be paralleled by various renal phenotypes including a subtle decrease of proximal Na⁺-coupled transport (KCNE1/KCNQ1), impaired K⁺ secretion (KCNMA1), limited HCO₃^– elimination (SLC26A4), NaCl wasting (BSND and CLCNKB), renal tubular acidosis (ATP6V1B1, ATPV0A4, and KCC4), or impaired urinary concentration (CLCNKA). Thus, defects of channels and transporters expressed in the kidney and inner ear result in simultaneous dysfunctions of these seemingly unrelated organs. cochlea; vestibular labyrinth; stria vascularis; deafness; renal tubule     

Moderate-to-severe ischemic conditions increase activity and phosphorylation of the cerebral microvascular endothelial cell Na+-K+-Cl- cotransporter

Brain edema that forms during the early stages of stroke involves increased transport of Na⁺ and Cl^– across an intact blood-brain barrier (BBB). Our previous studies have shown that a luminal BBB Na⁺-K⁺-Cl^– cotransporter is stimulated by conditions present during ischemia and that inhibition of the cotransporter by intravenous bumetanide greatly reduces edema formation in the rat middle cerebral artery occlusion model of stroke. The present study focused on investigating the effects of hypoxia, which develops rapidly in the brain during ischemia, on the activity and expression of the BBB Na⁺-K⁺-Cl^– cotransporter, as well as on Na⁺-K⁺-ATPase activity, cell ATP content, and intracellular volume. Cerebral microvascular endothelial cells (CMECs) were assessed for Na⁺-K⁺-Cl^– cotransporter and Na⁺-K⁺-ATPase activities as bumetanide-sensitive and ouabain-sensitive ⁸⁶Rb influxes, respectively. ATP content was assessed by luciferase assay and intracellular volume by [³H]-3-O-methyl-D-glucose and [¹⁴C]-sucrose equilibration. We found that 30-min exposure of CMECs to hypoxia ranging from 7.5% to 0.5% O₂ (vs. 19% normoxic O₂) significantly increased cotransporter activity as did 7.5% or 2% O₂ for up to 2 h. This was not associated with reduction in Na⁺-K⁺-ATPase activity or ATP content. CMEC intracellular volume increased only after 4 to 5 h of hypoxia. Furthermore, glucose and pyruvate deprivation increased cotransporter activity under both normoxic and hypoxic conditions. Finally, we found that hypoxia increased phosphorylation but not abundance of the cotransporter protein. These findings support the hypothesis that hypoxia stimulation of the BBB Na⁺-K⁺-Cl^– cotransporter contributes to ischemia-induced brain edema formation. edema; blood-brain barrier; bumetanide; cell volume