旱黑口服液对衰老小鼠心、脑细胞膜Na+-K+-ATPase、Ca2+-Mg2+-ATPase活性的影响

目的观察旱黑口服液对D-gal衰老小鼠心肌细胞膜、脑细胞膜上Na -K -ATP,ase、Ca2 -Mg2 -ATPase活性的影响,探讨旱黑口服液在延缓衰老方面的效应机制。方法以D-gal致亚急性衰老小鼠为模型,同时给予旱黑口服液治疗,6周后测定心、脑细胞膜上Na -K -ATPase、Ca2 -ATPase活性。结果与空白组比较,衰老组心、脑细胞膜上Na -K -ATPase、Ca2 -Mg2 -ATPase活性降低,经旱黑口服液治疗后心、脑细胞膜上Na -K -ATPase、Ca2 -Mg2 -ATPase活性活性明显提高。结论旱黑口服液具有提高D-gal致衰老小鼠心、脑细胞膜上Na -K -ATPase、Ca2 -Mg2 -ATPase活性,延缓衰老的作用。     

人肝再生增强因子相互作用蛋白基因的克隆

目的：寻找与人肝再生增强因子(hALR)相互作用的蛋白质．探讨其在肝再生过程中的分子生物学机制。方法：采用酵母双杂交系统，以hALR作诱饵蛋白筛选预转化的人肝cDNA文库，对阳性克隆进行生物信息学分析。结果：筛选出6组与hALR具有特异性相互作用的蛋白基因，分别是：血清白蛋白、金属硫蛋白、Na／K-ATPase、硒蛋白P和两个未知功能基因的cDNA序列。结论：初步克隆了与hALR相互作用蛋白基因，为以后深入研究这些蛋白质与hALR之间的相互作用，进一步揭示hALR的作用机制奠定了基础。     

GST Pull-down实验鉴定NF-κB相互作用多肽

目的 体外鉴定NF-κB相互作用多肽与p50和p65间的相互作用。方法 首先构建GST-作用多肽融合蛋白的原核表达载体pET-42a/polypeptide及NF-κB p50和p65亚基保守结构域的原核表达载体pET22b／p50和pET22b／p65，并在大肠杆菌E．coli BL-21中诱导表达，后进行GST pull-down实验验证多肽与NF-κB p50和p65的结合效应。结果 经诱导表达获得了可溶性的GST-多肽融合蛋白和具有DNA结合活性的p50、p65蛋白，GST pull-down实验证实3条多肽与p50发生特异地相互作用。结论 证实3条多肽在体外能与p50发生物理性的相互作用，这为获得靶向NF-κB的功能拮抗多肽工作奠定了基础。     

东莨菪碱对兔脑缺血再灌注Na+-K+-ATPase活性的影响

目的 :观察再灌注损伤时脑组织Na -K -ATPase活性的改变及东莨菪碱对缺血再灌注脑组织Na -K -ATPase活性的影响。方法 :以选择性头部低温为阳性对照。通过低压低灌法造成完全性脑缺血模型。将 30只兔分为 :假手术组 (Ⅰ组 ) ,缺血组 (Ⅱ组 ) ,缺血再灌注组 (Ⅲ组 ) ,低温治疗组 (Ⅳ组 ) ,东莨菪碱治疗组 (Ⅴ组 ) ,东莨菪碱低温治疗组 (Ⅵ组 )。结果 :脑组织Na -K -ATPase活性以Ⅲ组最低 ;Ⅱ、Ⅳ、Ⅴ、Ⅵ组的Na -K -ATPase活性显著高于Ⅲ组 (P分别 <0 0 5或 <0 0 1) ,与Ⅰ组无显著差异 (P >0 0 5 )。Ⅳ、Ⅴ、Ⅵ 3组间的Na -K -ATPase活性无显著差异 (P >0 0 5 )。结论 :脑组织Na -K -ATPase对缺血损伤不敏感 ,对再灌注损伤敏感 ;东莨菪碱和低温一样 ,能保护缺血后再灌注脑组织Na -K -ATPase活性 ,但东莨菪碱和低温无明显的协同作用     

窒息新生儿脐血D-二聚体水平和红细胞膜Na~+-K~+-ATP酶活性变化

目的:分析窒息新生儿脐血pH、D-二聚体水平及红细胞膜Na+-K+-ATP酶活性变化。方法 :选择临产过程出现急性胎儿窘迫孕妇40例,其剖宫娩出新生儿以1min Apgar评分确定为正常者20例(窘迫组),出现窒息者20例(窒息组);另选无急性胎儿窘迫、同样剖宫娩出的正常新生儿20例作为对照组。取各组脐动脉血,血气分析仪检测pH值,免疫比浊法检测D-二聚体水平,定磷法检测红细胞膜Na+-K+-ATP酶活性。比较各组上述指标的差异。结果:方差分析显示,各组脐动脉血pH值、D-二聚体水平和红细胞膜Na+-K+-ATP活性差异均有统计学意义(P0.05)。窒息组脐动脉血pH值明显低于对照组和窘迫组(P均0.05),D-二聚体水平显著高于对照组和窘迫组(P均0.05),红细胞膜Na+-K+-ATP酶活性显著低于对照组与窘迫组(P均0.05);窘迫组pH值和红细胞膜Na+-K+-ATP酶活性显著低于对照组(P均0.05)。结论 :窒息新生儿纤溶和红细胞膜泵功能检测结果,可为新生儿窒息治疗措施的选择提供实验依据。     

缺碘致甲状腺功能减退大鼠肾脏抗氧化能力降低

目的研究甲减大鼠肾脏抗氧化能力的变化,探讨甲状腺激素水平对肾脏的影响。方法Wistar大鼠随机分2组:甲减组(HT)和对照组(Control)。2组大鼠通过不同碘含量饮食复制甲减动物模型;肾脏形态定量学观察;比色法测定肾脏组织丙二醛(MDA)含量、谷胱甘肽过氧化物酶(GPx)、超氧化物歧化酶(SOD)及生物膜Na+,K+-ATPase活性;RT-PCR方法测定肾脏Na+,K+-ATPaseα1亚基的基因表达。结果与对照组比较,HT组FT3、FT4、TT3、TT4显著降低(P<0.01);肾小球萎缩;MDA含量和GPx活性显著升高(P<0.05),而SOD和Na+,K+-ATPase活性显著降低(P<0.05);Na+,K+-ATPaseα1亚基的基因表达下降(P<0.05)。结论甲状腺激素不足可导致肾脏抗氧化能力下降,肾小球萎缩。     

空调环境中豚鼠红细胞膜Na~+-K~+-ATP酶、SOD、MDA、GSH-Px的变化及意义

目的:通过动物实验研究空调环境中红细胞膜Na+-K+-ATP酶、超氧化物歧化酶(SOD)、丙二醛(MDA)、谷胱甘肽过氧化酶(GSH-PX)的变化,为探讨空调环境对红细胞变形性改变的机制提供实验依据。方法:将豚鼠分为实验组和对照组,对照组(12只):豚鼠在通风房间中自由饮水和进食;实验组(12只):豚鼠在温度为22℃空调房间内饲养。15天后,用心脏穿刺法采集两组豚鼠血液5ml,肝素抗凝,制备红细胞膜。检测红细胞膜MDA的含量以及Na+-K+-ATP酶、SOD、GSH-PX的活力。结果:空调环境中豚鼠的红细胞膜Na+-K+-ATP酶、SOD、GSH-PX活力比对照组显著下降(P<0.01),MDA的含量比对照组显著增加(P<0.01)。结论:空调环境中红细胞膜Na+-K+-ATP酶、SOD、GSH-PX活力下降、MDA含量增加,因而红细胞变形力降低。     

糖尿病大鼠心肌磷酸受纳蛋白基因表达和肌浆网 Ca2+-ATPase活性的变化

目的：观察糖尿病(DM)大鼠心肌磷酸受纳蛋白（PLB）基因表达和肌浆网 Ca2+-ATPase活性的变化及其与心功能的关系。方法:复制糖尿病大鼠模型，分别于4、6、8周后对糖尿病组和对照组进行左心室血流动力学检测，测定心肌PLB mRNA转录水平以及蛋白表达水平变化，检测心肌肌浆网 Ca2+-ATPase活性。结果:糖尿病大鼠心肌PLB mRNA转录和蛋白表达水平4周时与正常大鼠无明显差异，6周时和8周时明显高于正常大鼠；肌浆网 Ca2+-ATPase活性4周时无明显改变，6周时和8周时明显低于正常大鼠；糖尿病大鼠4周时LVSP、LVEDP、±dp/dtmax与正常大鼠无明显差异，6周、8周时LVSP、±dp/dtmax显著降低，LVEDP显著升高。结论：糖尿病大鼠心肌PLB表达水平升高，肌浆网 Ca2+-ATPase活性降低，引起心功能下降。     

应用酵母双杂交系统发现hALR与Na,K-ATPase间的相互作用

目的:采用酵母双杂交系统寻找与人肝再生增强因子(hALR)相互作用的蛋白质, 探讨ALR的作用机理。方法: 构建hALR诱饵质粒pGBKT7-hALR, 醋酸锂法转化AH109酵母菌, 转化菌在SD/-Trp-His培养基上培养及滤纸法β一半乳糖苷酶活性检测排除自身激活作用后, 与人肝cDNA文库质粒预转化的酵母菌Y187进行接合试验, 接合产物在QDO培养基上筛选, 阳性克隆进一步在含X-α-Gal的QDO平板上鉴定, X-α-Gal活性呈阳性的克隆, 进行PCR及酶切鉴定排除完全相同克隆, 进一步进行回交试验排除假阳性, 对阳性克隆进行序列测定和生物信息学分析。结果:得到数个阳性克隆, 序列分析结果表明其中1个阳性克隆是Na⁺, K⁺-ATPaseβ亚基部分基因, 长669bp, 3'端非编码区224bp, 编码区长445bp, 编码Na⁺, K⁺-ATPaseβ亚基C端的147个氨基酸残基。结论:应用酵母双杂交系统筛选出Na⁺, K⁺-ATPase与hALR具有相互作用。     

Na~+-K~+-ATP酶α1亚基DR多肽单克隆抗体的制备和应用

目的制备钠钾ATP酶(Na~+-K~+-ATPase)α1亚基DR区段(897DVEDSYGQQWTYEQR911)的单克隆抗体(mAb)并鉴定其活性。方法以合成的DR-钥孔戚血蓝素(KLH)为免疫原,免疫BALB/c小鼠,通过细胞融合技术建立能稳定分泌抗DR的杂交瘤细胞株,制备Na~+-K~+-ATPaseα1亚基DR mAb。ELISA测定杂交瘤细胞上清中mAb效价,斑点免疫杂交、Western blot法及免疫荧光细胞化学染色法检测单克隆的特异性,采用Senso LyteFDP蛋白磷酸酶定量试剂盒检测Na~+-K~+-ATPase活性,高糖细胞损伤实验检测DR mAb对高糖引起细胞损伤的保护作用。结果成功制备3株稳定分泌抗体的阳性细胞株。选择强阳性DRm217细胞株,制备腹水进一步验证。免疫荧光细胞化学染色、免疫斑点杂交及Western blot法结果显示,DRm217 mAb能够特异性结合在细胞膜表面,识别Na~+-K~+-ATPase的DR多肽。DRm217能够提高Na~+-K~+-ATPase活性,对高糖引起的细胞损伤具有一定的保护作用。结论成功制备了针对Na~+-K~+-ATPase DR多肽的mAb。     

Decreased myocardial Na+-K+-ATPase activity in one-kidney, one-clip hypertensive rats

We have previously shown that Na+-K+ pump activity (ouabain-sensitive 86Rb uptake) is decreased in vascular tissue of animals with various forms of low renin hypertension. In the present study we measured Na+-K+-ATPase activity, the energy source for Na+-K+ pumping, in membrane fractions prepared from myocardial tissue of rats with chronic one-kidney, one-clip hypertension and their one-kidney normotensive controls. Membranes were prepared by two independent methods: microsomal fractions (method 1) and fractions prepared by the hypotonic LiBr method of Dhalla et al. (method 2). In membranes prepared from left ventricles of the hypertensive rats (by method 1) Na+-K+-ATPase activity was decreased, Mg2+-ATPase activity was increased, and the sialic acid content and 5'-nucleotidase activity (two putative membrane markers) were unchanged relative to the control rats. The sensitivity of cardiac Na+-K+-ATPase to inhibition by ouabain was also unchanged. Na+-K+-ATPase activity was also decreased in the right ventricles (method 1) of these hypertensive rats, suggesting that this defect is probably not pressure related. In membranes prepared from the left ventricles of the hypertensive rats by method 2, Na+-K+-ATPase activity was again reduced, whereas the Mg2+-ATPase and 5'-nucleotidase activities were unchanged relative to the controls. These studies suggest that myocardial Na+-K+-ATPase activity is suppressed in rats with this low renin form of hypertension and the possible effect of this suppression on myocardial contractile activity is discussed.     

Sarcolemmal Na+-K+-ATPase activity in diabetic rat heart

Heart sarcolemmal membranes were isolated by the hypotonic shock-LiBr treatment from rats with chronic diabetes induced by a streptozotocin (65 mg/kg, iv) injection. Sarcolemmal Mg2+-dependent ATPase activity was elevated, whereas 5'-nucleotidase and K+-p-nitrophenylphosphatase activities in diabetic heart were depressed in comparison to control preparations. Although patent Na+-K+-ATPase and patent ouabain-sensitive Na+-K+-ATPase activities were unaltered, latent Na+-K+-ATPase activities, as determined in membranes after alamethicin or deoxycholate treatments, were found to be significantly depressed in diabetic animals. A depression in the latent Na+-K+-ATPase activity in diabetic preparations was also observed in membranes prepared by the sucrose density gradient method. Insulin-treated diabetic rats were observed to have normalized latent Na+-K+-ATPase activities. Total phospholipid content did not differ, but cholesterol content of the sarcolemmal membranes was significantly increased in diabetic heart preparations. Sarcolemmal Na+-K+-ATPase activity in diabetic heart was more resistant to treatments with filipin, an agent known to bind with cholesterol residues. These results suggest that chronic experimental diabetes is associated with some defects in sarcolemmal enzymatic activities and composition.     

Regulation of renal Na+-K+-ATPase in the rat by adrenal steroids

The effects of single and multiple injections of aldosterone and dexamethasone on renal Na+-K+-ATPase, in vitro renal gluconeogenesis, and urinary electrolyte excretion were examined in adrenalectomized rats in a dose-dependent manner. Single maximal and supramaximal doses of aldosterone (defined by the effect of electrolyte excretion) had no effect on Na+-K+-ATPase or gluconeogenesis. By contrast, a single administration of dexamethasone (in a dose range that increased fasting blood sugar, stimulated renal gluconeogenesis, and had no mineralocorticoid effects) yielded clear-cut activation of Na+-K+-ATPase. Multiple submaximal doses of dexamethasone produced quantitatively similar stimulation of Na+-K+-ATPase and gluconeogenesis. Multiple supramaximal doses of aldosterone stimulated Na+-K+-ATPase and gluconeogenesis, but maximal and submaximal doses of the hormone were without effect. Aldosterone had no effect on hepatic Na+-K+-ATPase or gluconeogenesis. These results suggest that activation of renal Na+-K+-ATPase can be considered a putative glucocorticoid (not mineralocorticoid) effect. Renal Na+-K+-ATPase activation by chronic aldosterone treatment may be mediated by glucocorticoid receptor sites and, hence, may not represent a genuine mineralocorticoid effect.     

Dopamine-induced inhibition of Na+-K+-ATPase activity requires integrity of actin cytoskeleton in opossum kidney cells

The present study evaluated the importance of the association between Na+-K+-ATPase and the actin cytoskeleton on dopamine-induced inhibition of Na+-K+-ATPase activity. The approach used measures the transepithelial transport of Na+ in monolayers of opossum kidney (OK) cells, when the Na+ delivered to Na+-K+-ATPase was increased at the saturating level by amphotericin B. The maximal amphotericin B (1.0 microg mL-1) induced increase in short-circuit current (Isc) was prevented by ouabain (100 microM) or removal of apical Na+. Dopamine (1 microM) applied from the apical side significantly decreased (29 +/- 5% reduction) the amphotericin B-induced increase in Isc, this being prevented by the D1-like receptor antagonist SKF 83566 (1 microM) and the protein kinase C (PKC) inhibitor chelerythrine (1 microM). Exposure of OK cells to cytochalasin B (1 microM) or cytochalasin D (1 microM), inhibitors of actin polymerization, from both cell sides reduced by 31 +/- 4% and 36 +/- 3% the amphotericin B-induced increase in Isc and abolished the inhibitory effect of apical dopamine (1 microM), but not that of the PKC activator phorbol-12,13-dibutyrate (PDBu; 100 nM). Colchicine (1 microM) failed to alter the inhibitory effects of dopamine. The relationship between Na+-K+-ATPase and the concentration of extracellular Na+ showed a Michaelis-Menten constant (Km) of 44.1 +/- 13.7 mM and a Vmax of 49.6 +/- 4.8 microA cm-2 in control monolayers. In the presence of apical dopamine (1 microM) or cytochalasin B (1 microM) Vmax values were significantly (P < 0.05) reduced without changes in Km values. These results are the first, obtained in live cells, showing that the PKC-dependent inhibition of Na+-K+-ATPase activity by dopamine requires the integrity of the association between actin cytoskeleton and Na+-K+-ATPase.     

Renal Na+-K+-ATPase in renin release

The effects of ouabain and furosemide on renin secretion, renal function, and renal Na+-K+-ATPase were investigated in anesthetized dogs. Furosemide (2 mg/kg) induced significant diuresis, natriuresis, an increase in renal blood flow (RBF), and a fivefold increase in renin secretory rate (RSR), but no changes in glomerular filtration rate (GFR). Infusion of ouabain (1 microgram . kg-1 . min-1) into one renal artery during furosemide diuresis increased fractional sodium excretion from 22 +/- 2 to 30 +/- 3% from the ipsilateral kidney but did not change urine flow, RBF, or GFR, whereas RSR fell to control values (698 +/- 203 to 137 +/- 43). When ouabain preceded furosemide, the rise in RBF and RSR induced by furosemide was abolished but sodium excretion increased. Ouabain infused in vivo inhibited Na+-K+-ATPase in microsomal fractions from cortex (34%) and medulla (27%) as compared with control. Neither saline nor furosemide exerted any effect on Na+-K+-ATPase. Moreover, the effect of ouabain alone on Na+-K+-ATPase was not different from that of ouabain plus furosemide. No changes in Mg2+-ATPase were detected in any of the experiments. These results indicate that inhibition of renal Na+-K+-ATPase abolishes furosemide-induced renin secretion despite potentiation of the natriuretic effect of the diuretic. It is apparent that the level of activity of Na+-K+-ATPase is of prime importance for renin secretion. In addition, ouabain may act directly on the juxtaglomerular cells to inhibit renin secretion.     

Mechanism of activation of renal Na+-K+-ATPase in the rat: effects of reduction of renal mass

The mechanism of activation of renal Na+-K+-ATPase was studied in rats 2 wk after unilateral nephrectomy. The increase in enzyme specific activity was confined to the outer medulla and occurred without changes in the cellular contents of RNA or protein. Enzyme activation was accompanied by increases in the levels of the phosphorylated intermediate with little or no change in the apparent turnover numbers of the reaction. The specific activity of the ouabain-sensitive p-nitrophenylphosphatase also increased by uninephrectomy but to a larger extent than did Na+-K+-ATPase. Kinetic studies demonstrated an increase in Vmax for ATP, sodium, and potassium, and small increases in Km for ATP and K1/2 for potassium. There was no change in the activation energies or phase transition temperature to indicate alterations in the membrane environment of Na+-K+-ATPase. Adrenalectomy did not adversely affect activation. These results indicate that activation of renal Na+-K+-ATPase after reduction of renal mass occurs mainly by an increase in the number of sodium pump sites.     

Influence of thyroid hormones on transport function and Na+-K+-ATPase activity in the rat choroid plexus

The sympathomimetic stimulation of choroid plexus transport and secretion in rat seems to be mediated by beta-adrenergic receptors. In the present report the effect of induced changes in thyroid function on transport mechanisms in the rat choroid plexus was studied. Following induction of hyperthyroidism (treatment with T3 for 10 days) the tissue/medium ratio (T/M) for choline uptake in choroid plexus in vitro decreased significantly by 68%. The Na+-K+-ATPase activity showed a statistically significant increase of about 16%. Also following cervical sympathectomy, T3 caused a reduction of the T/M for choline, to the same level as in the non-sympathectomized animals, while the effect of T3 on the Na+-K+-ATPase activity was changed into a 22% decrease. Hypothyroidism (administration of PTU in the drinking water) had little or no effect on the uptake and accumulation of choline in the choroid plexus. The Na+-K+-ATPase activity was reduced by 40%, in contrast to the stimulating effect of T3. There is, hence, reason to believe that the transport of choline in the choroid plexus is only partly regulated by adrenergic mechanisms acting via Na+-K+-ATPase. The major effect of T3 on the choline uptake may be exerted by a mechanism different from the ATPase activity and not involving adrenergic receptors.     

Na+-K+-ATPase inhibition stimulates PGE release in guinea pig taenia coli.

Inhibition of the plasma membrane enzyme Na+-K+-ATPase by ouabain zero extracellular K+, or low extracellular Na+, markedly augmented prostaglandin E release from the guinea pig taenia coli. Data suggest this phenomenon may be linked directly to Na+-K+-ATPase or Na+ pump activities, or changes in intracellular K+ concentration. The augmented prostaglandin E release was not due to changes in intracellular Na+, Ca2+, pH, or membrane potential, resulting from Na+ pump inhibition. The characteristics of the plasma membrane may exert a control on prostaglandin E release in this smooth muscle.     

Mechanism of activation of renal Na+-K+-ATPase in the rat: effects of potassium loading

The mechanism of activation of Na+-K+-ATPase after chronic potassium loading has been investigated in the rat kidney. Potassium loading stimulated the specific activity of Na+-K+-ATPase in the cortex and medulla of the kidney. This effect was not accompanied by a generalized increase in the cellular contents of RNA and proteins and could not be accounted for by an effect of potassium loading on renal growth. Enzyme induction does not appear to be mediated by changes in the endogenous levels of glucocorticoid or thyroid hormones. Evidence obtained from investigation of the partial reactions (Pi intermediate, ouabain-sensitive pNPPase) of the Na+-K+-ATPase enzymatic reaction is consistent with the interpretation that chronic potassium loading in the rat increases the number of enzyme units (Na+ pumps) in the cortex of the kidney. Analysis of the kinetic parameters (Km, K1/2, Vmax, Hill coefficients) of the enzymatic reaction indicates that K+ loading has little or no effect on the kinetic properties (affinity, cooperativity) of the stimulated transport enzyme.     

Extracellular matrix activity and caveolae events contribute to cell surface receptor activation that leads to MAP kinase activation in response to UV irradiation in cultured human keratinocytes

Activation of cell surface components has been implicated in the activation of downstream signaling cascade in response to UV irradiation, and yet the identity and the interaction of those components have been scantly documented. Accumulating evidence indicates that caveolae encapsulating caveolins is the location for those interactions. We found in cultured human keratinocytes that UV irradiation induced both caveolin-1 and EGFR phosphorylation. Filipin, a caveolae disruptive agent, inhibited UV-induced caveolin-1 activation. Na+-K+-ATPase catalyzes active transport of Na+ and K+ across plasma membrane of mammalian cells, inactivation of which has recently been shown to be involved in the activation of signal transduction pathways including MAP kinase cascade. We found in this study that UV inactivated Na+-K+-ATPase in time-dependent manner, Na+-K+-ATPase activity started to decrease 5 min post UV irradiation and reduced to 60% of its original activity within 1 h. Pretreatment with Flipin and MMP inhibitor recovered Na+-K+-ATPase activity lost by UV irradiation. ECIS analysis indicated that both EGF treatment and UV irradiation increased membrane electric activity which was inhibited by MMP inhibitor and Filipin. Further study showed that pretreatment of human keratinocytes with MMP inhibitor or Filipin inhibited UV-induced phosphorylation of p38 and JNK, which was however not observed in LnCap cells, a prostate cancer cell line lacking caveolin-1. UV irradiation also induced ectodomain shedding of HB-EGF in a time-dependent manner in keratinocytes. Collectively, we conclude that UV-induced MAP kinase activation is mediated by cell surface receptor activation due to the matrix activity and membrane caveolae function and inactivation of Na+-K+-ATPase.