Quantitative autoradiography of [H]ouabain binding sites in rat brain

In vitro quantitative autoradiography was used to localize in rat brain binding sites for [³H]ouabain, an inhibitor of the Na⁺,K⁺-ATPase. High levels of [³H]ouabain binding sites were found in the superior and inferior colliculi, the mammillary nucleus, the interpeduncular nucleus, and in various divisions of the olfactory, auditory and somatomotor systems. The heterogeneous distribution of [³H]ouabain binding closely parallels the regional brain glucose consumption as determined by the [¹⁴C]deoxyglucose method. Lesion studies of the rat hippocampus using the excitotoxin, ibotenic acid, showed both a marked decrease of neuronal cell types on the injected side and a corresponding decrease in [³H]ouabain binding, indicating that some of the [³H]ouabain binding sites are localized to neurons. The close correlation between [³H]-ouabain binding and regional glucose utilization provides further evidence for a linkage between glucose utilization and the neuronal Na⁺,K⁺-ATPase.     

Neurostimulatory and antioxidative effects of L-deprenyl in aged rat brain regions

This study investigated the influence of chronically administered L-deprenyl on normal ageing-related parameters: multiple unit action potentials, the activities of the enzymes Na⁺,K⁺-adenosinetriphosphatase, glutathione-s-transferase and glutathione peroxidase, and the levels of lipid peroxidation products and lipofuscin contents in the brain regions (cerebral cortex, hippocampus, striatum and thalamus) of 24-month-old rats. The drug increased the activity of Na⁺,K⁺-ATPase and glutathione-s-transferase. The activity of glutathione peroxidase remained unaffected. The drug also increased the multiple unit action potentials activity. The levels of lipid peroxidation products were, however, decreased, and lipofuscin accumulation was diminished by the drug. The results essentially indicated that chronic treatment of rats with L-deprenyl significantly influenced the ageing-related alterations in: multiple unit action potentials, Na⁺,K⁺-adenosinetriphosphatase, glutathione-s-transferase, lipid peroxidation products and lipofuscin accumulation. These novel data on the effect of L-deprenyl on parameters of normal ageing provide new additional evidence concerning the anti-ageing therapeutic potential of L-deprenyl. This revised version was published online in July 2006 with corrections to the Cover Date.     

Antagonists of the Vasopressin V1 Receptor and of the β1-Adrenoceptor Inhibit Cytotoxic Brain Edema in Stroke by Effects on Astrocytes – but the Mechanisms Differ

Brain edema is a serious complication in ischemic stroke because even relatively small changes in brain volume can compromise cerebral blood flow or result in compression of vital brain structures on account of the fixed volume of the rigid skull. Literature data indicate that administration of either antagonists of the V1 vasopressin (AVP) receptor or the β₁-adrenergic receptor are able to reduce edema or infarct size when administered after the onset of ischemia, a key advantage for possible clinical use. The present review discusses possible mechanisms, focusing on the role of NKCC1, an astrocytic cotransporter of Na⁺, K⁺, 2Cl^- and water and its activation by highly increased extracellular K⁺ concentrations in the development of cytotoxic cell swelling. However, it also mentions that due to a 3/2 ratio between Na⁺ release and K⁺ uptake by the Na⁺,K⁺-ATPase driving NKCC1 brain extracellular fluid can become hypertonic, which may facilitate water entry across the blood-brain barrier, essential for development of edema. It shows that brain edema does not develop until during reperfusion, which can be explained by lack of metabolic energy during ischemia. V1 antagonists are likely to protect against cytotoxic edema formation by inhibiting AVP enhancement of NKCC1-mediated uptake of ions and water, whereas β₁-adrenergic antagonists prevent edema formation because β₁-adrenergic stimulation alone is responsible for stimulation of the Na⁺,K⁺-ATPase driving NKCC1, first and foremost due to decrease in extracellular Ca²⁺ concentration. Inhibition of NKCC1 also has adverse effects, e.g. on memory and the treatment should probably be of shortest possible duration.     

Targeting V-ATPase in primary human monocytes by archazolid potently represses the classical secretion of cytokines due to accumulation at the endoplasmic reticulum

The macrolide archazolid inhibits vacuolar-type H(+)-ATPase (V-ATPase), a proton-translocating enzyme involved in protein transport and pH regulation of cell organelles, and potently suppresses cancer cell growth at low nanomolar concentrations. In view of the growing link between inflammation and cancer, we investigated whether inhibition of V-ATPase by archazolid may affect primary human monocytes that can promote cancer by sustaining inflammation through the release of tumor-promoting cytokines. Human primary monocytes express V-ATPase, and archazolid (10–100 nM) increases the vesicular pH in these cells. Archazolid (10 nM) markedly reduced the release of pro-inflammatory (TNF-α, interleukin-6 and -8) but also of anti-inflammatory (interleukin-10) cytokines in monocytes stimulated with LPS, without affecting cell viability up to 1000 nM. Of interest, secretion of interleukin-1β was increased by archazolid. Comparable effects were obtained by the V-ATPase inhibitors bafilomycin and apicularen. The phosphorylation of p38 MAPK and ERK-1/2, Akt, SAPK/JNK or of the inhibitor of NFκB (IκBα) as well as mRNA expression of IL-8 were not altered by archazolid in LPS-stimulated monocytes. Instead, archazolid caused endoplasmic reticulum (ER) stress response visualized by increased BiP expression and accumulation of IL-8 (and TNF-α) at the ER, indicating a perturbation of protein secretion. In conclusion, by interference with V-ATPase, archazolid significantly affects the secretion of cytokines due to accumulation at the ER which might be of relevance when using these agents for cancer therapy.     

穴位电刺激对大鼠急性运动后腓肠肌收缩功能的影响

目的探讨穴位电刺激疗法对大鼠急性运动后腓肠肌收缩功能的影响。方法 45只SD大鼠,随机分成三组,适应性游泳训练1星期后,进行急性运动（2 h的游泳）,全麻下取腓肠肌,应用差速离心法提取SR,检测肌质网（SR）钙泵（Ca2＋-ATPase）、Ca2＋浓度;30只SD大鼠,随机分成正常对照组、急性运动模型组、穴位电刺激治疗组三组,运动后制备坐骨神经-腓肠肌标本,在SMUP-E型生物信息处理系统上观察腓肠肌最大收缩力。结果模型组大鼠腓肠肌SR Ca2＋-ATPase的活力明显低于对照组（P〈0.05）,治疗组则明显高于模型组（P〈0.05）;模型组大鼠腓肠肌SR Ca2＋浓度明显高于对照组（P〈0.05）,治疗组则明显低于模型组（P〈0.01）,治疗组与对照组差异无统计学意义（P〉0.05）;模型组大鼠腓肠肌的最大收缩力明显小于对照组（P〈0.05）,而治疗组则明显大于模型组（P〈0.05）。结论一次性急性游泳（2 h）大鼠腓肠肌SR的Ca2＋-ATPase活性明显下降,Ca2＋浓度显著升高,腓肠肌的最大收缩力下降;穴位电刺激则可以明显提高大鼠腓肠肌SR Ca2＋-ATPase的活力,降低SR内Ca2＋浓度,增强大鼠腓肠肌的最大收缩力。这可能是穴位电刺激疗法延缓运动疲劳的机理之一。     

毒毛旋花子苷元对豚鼠心室肌细胞内钙浓度的影响

观察毒毛旋花子苷元(strophanthidin, Str)对分离豚鼠心室肌细胞内游离钙浓度(［Ca²⁺］_i)的影响。酶解分离豚鼠心室肌细胞, 用Fluo 3-AM负载, 激光共聚焦显微镜法测定单个豚鼠心室肌细胞［Ca²⁺］_i的荧光密度。Str可浓度依赖性地升高［Ca²⁺］_i, Str (10 μmol·L^-1)在［Ca²⁺］_i升高达峰值时, 可使细胞挛缩, 而Str (1和10 nmol·L^-1)对细胞形态无影响。TTX、 尼索地平或升高细胞外钙可影响Str (1和100 nmol·L^-1)对［Ca²⁺］_i的升高作用,而对Str (10 μmol·L^-1)无明显影响。在外液中加入ryanodine或去除细胞外钙, 则3个检测浓度的Str升高［Ca²⁺］_i作用均被明显抑制。在无K⁺、 无Na⁺液中, 10 μmol·L^-1 Str升高［Ca²⁺］_i的作用减弱, 而Str (1和100 nmol·L^-1)升高［Ca²⁺］_i的作用无明显影响。加入TTX、 尼索地平或增加细胞外的钙离子浓度, 则3个检测浓度Str的作用均受到影响。提示低浓度Str对［Ca²⁺］_i的升高作用与抑制Na⁺、K⁺-ATP酶活性无关, 而与促进L-型钙通道和TTX敏感性钠通道的“slip-mode”钙电导有关; 高浓度Str升高［Ca²⁺］_i的作用则是抑制Na⁺、K⁺-ATP酶的结果。此外, Str对［Ca²⁺］_i的升高作用还与直接作用于ryanodine受体促进内钙释放有关。     

旋覆代赭汤对RE模型大鼠食管黏膜Na+-K+-ATP酶及 Ca2+-Mg2+-ATP酶的影响

目的: 观察旋覆代赭汤对反流性食管炎(RE)模型大鼠食管黏膜组织Na⁺-K⁺-ATP酶及Ca²⁺-Mg²⁺-ATP酶活性的影响,探讨RE食管黏膜细胞能量代谢障碍与黏膜损伤的关系以及旋覆代赭汤治疗RE的作用机制。方法: 将96只雄性Wistar大鼠随机分成6组,即假手术组、模型组、旋覆代赭汤组高、中、低剂量组(24,12,6 g·kg^-1)及西药对照组(奥美拉唑10 mg·kg^-1+2 mg·kg^-1莫沙必利),每组16只。采用"食管-十二指肠端侧吻合术"制备胃及十二指肠液混合RE模型。从术后第3天开始,假手术组、模型组给予生理盐水灌胃,药物治疗组分别给予旋覆代赭汤高、中、低剂量、西药(奥美拉唑10 mg·kg^-1+莫沙必利2 mg·kg^-1)灌胃,连续7天,记录大鼠体重变化及死亡情况,于第8天处死大鼠留取标本,观察食管下段黏膜大体及病理组织学变化;化学法检测食管黏膜组织Na⁺-K⁺-ATP和Ca²⁺-Mg²⁺-ATP酶活性。结果: 与假手术组比,模型组大鼠体重明显减轻(P<0.05),食管黏膜肉眼及镜下病理改变明显,评分增高(P<0.05);食管黏膜组织Na⁺-K⁺-ATP酶及Ca²⁺-Mg²⁺-ATP酶活性显著降低(P<0.05);与模型组及西药组比,旋覆代赭汤高、中剂量组大鼠死亡率明显降低(P<0.05):与模型组比,旋覆代赭汤高、中剂量组、西药组肉眼及病理评分显著降低(P<0.05);食管黏膜组织Na⁺-K⁺-ATP酶及Ca²⁺-Mg²⁺-ATP酶活性明显提高(P<0.05)。结论: 旋覆代赭汤能明显提高RE大鼠食管黏膜细胞膜Na⁺-K⁺-ATP 酶及Ca²⁺-Mg²⁺-ATP酶活性, 从而保持食管黏膜细胞完整性,减轻黏膜损伤。     

乙醇对生物膜功能和细胞结构的影响

按10mL/kg 剂量,每天给雄性 Wistar 大鼠分别于腹腔注射2%、5%、20%和40%无水乙醇水溶液。实验期为8天。结果表明大鼠 RBC 膜的荧光偏振度下降,Na~ ,K~ -ATP 酶活性增加。用光镜和 TEM 观察,在大鼠的组织和细胞中可见有出血、水肿、脂滴、变形,线粒体肿胀、RER 扩张,膜破裂和脱粒等。乙醇和生物膜的相互作用,可引起大鼠膜功能明显改变和一定的损伤。     

染料木素抗大鼠心肌肥厚作用及其与ATPase活性的关系

目的:观察染料木素(Gen)对异丙肾上腺素(iso)所致大鼠心肌肥厚的保护作用及其对心肌组织Na+-K+-ATPase,Ca2+-Mg2+-ATPase活性及羟脯氨酸含量的影响。方法:0.2 g·L-1的iso 5 mL·kg-1·d-1,背部皮下注射,连续10 d,建立大鼠心肌肥厚模型。造模第2天开始给予不同浓度的染料木素、二甲基亚砜或NS,连续14 d,末次给药后禁食12 h,称体重,麻醉,取心脏,称全心及左心室质量,计算全心质量指数及左心室质量指数,测定心肌组织Na+-K+-ATPase,Ca2+-Mg2+-ATPase活性及羟脯氨酸(Hyp)含量。结果:模型组全心质量指数及左心室质量指数分别为(3.30±0.24),(2.55±0.14)mg·kg-1,与正常对照组相比显著升高(P<0.001);心肌组织Na+-K+-ATPase和Ca2+-Mg2+-ATPase活力为(1.81±0.25),(1.00±0.22)U·mg-1,与正常对照组相比活性显著降低(P<0.01);Hyp含量为(0.80±0.05)mg·g-1,与正常对照组相比含量显著升高(P<0.001)。与模型组相比,染料木素高、低剂量组(0.3,0.1μmol.kg-1)全心质量指数分别为(2.83±0.22),(2.97±0.19)mg·kg-1;左心室质量指数分别为(2.32±0.16),(2.34±0.13)mg·kg-1均显著降低(P<0.01或P<0.05);高剂量组心肌组织Na+-K+-ATPase(2.58±0.40)U·mg-1及Ca2+-Mg2+-ATPase(1.35±0.22)U·mg-1活性升高(P<0.01或P<0.05),染料木素高、低剂量组心肌组织Hyp含量分别为(0.48±0.11),(0.57±0.14)mg·g-1显著降低(P<0.01或P<0.05)。结论:染料木素可通过提高Na+-K+-ATPase及Ca2+-Mg2+-ATPase的活性,降低Hyp含量,从而抑制心脏纤维化,起抗心肌肥厚作用。     

白藜芦醇对心肌缺血再灌注过程中线粒体的保护

目的:探讨白藜芦醇对心肌缺血再灌注损伤过程中Ca²⁺-ATPase及HSP70表达的影响。方法:健康雄性SD大鼠40只,采用随机数字量表法分为4组(假手术组、缺血再灌注组、缺血预适应组、白藜芦醇组),采用结扎冠状动脉前降支制备心肌缺血再灌注损伤模型,于结扎前15 min及再灌注前1 min经舌下静脉注射白藜芦醇10 mg·kg^-1。提取心肌线粒体,紫外可见光分光光度计测定线粒体中SDH、Na⁺-K⁺-ATPase、Ca²⁺-ATPase活性;荧光分光光度计测定线粒体中游离钙、mPTP开放度及跨膜电位;RT-PCR及Western blot检测心肌组织HSP70、Ca²⁺-ATPase mRNA及蛋白质的表达。结果:白藜芦醇与缺血再灌注组及缺血预适应组相比,线粒体中SDH、Na⁺-K⁺-ATPase、Ca²⁺-ATPase的活性明显提高(P<0.05或P<0.01);线粒体内钙离子浓度明显下降(P<0.05或P<0.01)、mPTP开放程度减小(P<0.01)、线粒体膜电位增高(P<0.01);Ca²⁺-ATPase、HSP70在 mRNA或蛋白水平上的表达均有所增加(P<0.05或P<0.01)。结论:白藜芦醇可能通过增加心肌组织中Ca²⁺-ATPase、HSP70 mRNA和蛋白的表达,提高线粒体Ca²⁺-ATPase的活性,产生对大鼠心肌缺血再灌注损伤时线粒体的保护作用。