周期性张应变对面颌部肌细胞Na+/K+-ATPaseα亚单位蛋白表达的影响

目的:研究周期性张应变对面颌肌细胞Na+/K+-ATPaseα亚单位蛋白表达的影响以确定其作用及机制.方法:在建立面颌肌细胞的力学刺激-细胞体外培养模型的基础上,采用Western blot法分析周期性张应变对Na+/K+-ATPase α1和α2亚单位蛋白表达的影响,加力组分别给予1、2、12、24和48h的力学刺激,施加力值为15％的细胞形变,频率为10cycles/min.以静态组为对照组.对照组及实验组各包含4个实验样本.Western blot检测Na+/K+-ATPaseα1和α2亚单位蛋白的表达.结果:α1亚单位的蛋白表达量除加力1h组与对照组之间、加力24 h与48 h之间无统计学差异外,其余各组之间以及各组与对照组之间均有显著的统计学意义.α2亚单位蛋白表达量除加力24 h与48 h组之间无统计学差异外,其余各组之间以及各组与对照组之间均有显著的统计学意义.结论:在一定时间范围内,周期性张应变可刺激α1和α2亚单位蛋白表达增加,随作用时间的延长蛋白表达受抑制.提示在肌能力的刺激下,面颌肌细胞的相关酶蛋白的功能及表达将发生适应性改建,但其功能亚基的调控机制可能不同.这为选择不同的方法和手段进行临床干预提供了理论依据,因而具有重要的参考意义.     

周期性张应力对面颌肌细胞Na+/K+-ATPase的影响

目的:研究周期性张应力对Na+/K+-ATPase功能活性及其表达的影响,明确Na+/K+-ATPase在功能矫形中面颌肌肉适应性改建的生物和分子机制.方法:构建面颌肌细胞体外培养-力学刺激模型;应用多通道细胞牵张应力加载系统,对面颌肌细胞施加不同时段的张应力刺激,测定Na+/K+-ATPase的功能活性;运用实时荧光定量PCR研究周期性张应力刺激对Na+/K+-ATPase功能亚基α亚单位mRNA的影响.结果:Na+/K+-ATPaseα1,α2亚单位随着加力时间延长,表达增强,同对照组比较,呈一致性上调(P＜0.001);细胞加力1小时,α1的mRNA表达不受影响;加力2小时后,α1和α2的mRNA表达呈现逐渐增强趋势,48 h时达到最大值.张应力刺激对α2亚单位的mRNA表达似乎更为敏感,加力lh时α2亚单位的mRNA表达水平即增加,增加量约为对照组的37.74％,具有显著的统计学意义.结论:周期性的机械牵张作用于培养的骨骼肌细胞,可诱导α1和α2亚单位mRNA的表达量增加;α1和α2亚单位对周期性张应力刺激的作用时间反应不同,α2亚单位的反应可能更为敏感.周期性张力刺激的增加所产生的压力可能是转录调节的主要因素;周期性张应力对骨骼肌细胞Na+/K+-ATPase水解亚的调节作用不同,可能在面颌肌肉对功能矫形力的适应性改建中具有重要作用.     

拟南芥内膜Na+,K+/H+反向转运体研究进展

拟南芥内膜Na,K~+/H~+反向转运体(Endosomal NHX)的亚细胞定位、离子转运特性及生物学功能阐释取得了重要进展。拟南芥内膜Na~+,K~+/H~+反向转运体包含AtNHX5和AtNHX6两个成员,它们的氨基酸序列相似性为78.7%。研究表明,AtNHX5和AtNHX6具有功能冗余,它们都定位在高尔基体(Golgi)、反面高尔基体管网状结构(TGN)、内质网(ER)和液胞前体(PVC),参与调控耐盐胁迫、pH平衡和K~+平衡等。有报道显示内膜NHXs跨膜结构域存在能够调控自身离子活性的酸性保守氨基酸残基,对其自身功能具有决定性作用。最新研究结果表明,拟南芥内膜NHXs影响囊泡运输和蛋白存贮,并参与生长素介导的植物生长和发育。文中主要对拟南芥内膜NHXs的亚细胞定位、离子转运、功能及应用进展进行了概述。     

Na^＋,K^＋-ATPase亚单位表达的研究进展

以往研究已发现Na^＋,K^＋-ATPase含有α、β和γ亚单位。为了对三种亚单位有一个较为全面的认识,现对亚单位的基本结构、研究简况、生理及病理功能、表达调节等基本情况作一综述。     

周期性张应力对面颌肌细胞Na+/K+-ATPase功能活性影响

目的:本研究在成功构建面颌肌细胞体外培养一力学刺激模型的基础上,探讨机械张应力对细胞内Na+水平和面颌肌细胞Na+/K+-ATPase功能活性的影响.方法:本实验采用Blua法,对SD大鼠乳鼠面颌肌细胞进行体外原代培养、鉴定并绘制生长曲线;取第3代细胞接种于细胞加力板上,采用Forcel四点弯曲加力装置,对细胞分别施加1h、2h、4h、8h、12h、16h、24 h和48 h的张应力刺激,后测定细胞内Na+水平变化和Na+/K+-ATPase功能活性改变.结果:(1)Na+水平变化:与对照(不加力)组相比,加力1h细胞内Na+显著增加(P＜0.05),并随加力时间延长逐渐降低,加力至12h时降到正常水平,再延长加力时间Na+无明显变化.(2) Na+/K+-ATPase功能活性变化:分别施加不同时间的周期性张应力后,Na泵的活性在加力8小时后才开始增加(0.5725mmolPi/mgPr.hr),随加力时间延长进一步增加,48小时后达到最大值(0.8963mmolPi/mgPr.h).结论:周期性张应力刺激会引起细胞内Na+的改变,并可以增加骨骼肌细胞Na+/K+-ATPase的活性.     

大鼠单根近端肾小管Na+-K+-ATPase活性测定方法的改进

本研究旨在对Doucet等报道的定量检测大鼠单根近端肾小管Na^＋-K^＋-ATPase活性方法进行改进。取经过Ⅱ型胶原酶消化的大鼠肾脏皮质组织,在体视显微镜下手工分离单根近端肾小管,并测量其长度,经低渗和冻融处理后与[γ-^32P]ATP共同孵育,液闪法检测从[γ-^32P]ATP解离出的^32Pi,采用修正后的公式计算Na^＋-K^＋-ATPase活性。改良法与Doucet等的方法比较,测定单根近端肾小管Na^＋-K^＋-ATPase活性无显著性差异（P〉0．05）。改进后的方法节省试剂,操作简便、省时。     

周期性张应变对面颌部肌细胞Na~＋/K~＋-ATPaseα亚单位蛋白表达的影响

目的：研究周期性张应变对面颌肌细胞Na＋/K＋-ATPaseα亚单位蛋白表达的影响以确定其作用及机制。方法：在建立面颌肌细胞的力学刺激--细胞体外培养模型的基础上,采用Western blot法分析周期性张应变对Na＋/K＋-ATPaseα1和α2亚单位蛋白表达的影响,加力组分别给予1、2、12、24和48h的力学刺激,施加力值为15%的细胞形变,频率为10cycles/min。以静态组为对照组。对照组及实验组各包含4个实验样本。Western blot检测Na＋/K＋-ATPaseα1和α2亚单位蛋白的表达。结果：α1亚单位的蛋白表达量除加力1h组与对照组之间、加力24 h与48 h之间无统计学差异外,其余各组之间以及各组与对照组之间均有显著的统计学意义。α2亚单位蛋白表达量除加力24 h与48 h组之间无统计学差异外,其余各组之间以及各组与对照组之间均有显著的统计学意义。结论：在一定时间范围内,周期性张应变可刺激α1和α2亚单位蛋白表达增加,随作用时间的延长蛋白表达受抑制。提示在肌能力的刺激下,面颌肌细胞的相关酶蛋白的功能及表达将发生适应性改建,但其功能亚基的调控机制可能不同。这为选择不同的方法和手段进行临床干预提供了理论依据,因而具有重要的参考意义。     

铜、镉及其复合作用下中华大蟾蜍蝌蚪Na+-K+-ATPase酶的变化

采用动物毒理实验法,研究了铜、镉及其复合污染对中华大蟾蜍(Bufo gargarizans)蝌蚪Na -K -ATPase酶活性的影响。结果表明,在0.0298mg/LCu2 和1.14mg/LCd2 作用下,蝌蚪Na -K -ATPase酶活性极显著高于对照组(P<0.01)。随着Cu2 、Cd2 暴露浓度的增加,蝌蚪Na -K -ATPase酶活性又逐渐降低,甚至被抑制。铜、镉复合污染在Na -K -ATPase酶水平上表现出一定的协同作用。     

空泡膜类型H~+-ATPase的研究进展

真核细胞内空泡细胞器,如高尔基体、内质网、溶酶体等,膜上存在的质子泵ATPase 与线粒体类型的质子泵 ATPase 类似.近几年对该类型 H~+-ATPase 的结构、作用机制进行了深入的研究,证明这是一类新型质子泵,在进化的过程中与线粒体类型的 H~+-ATPase 有密切的亲缘关系.     

高等植物Na+ 吸收、转运及细胞内Na+ 稳态平衡研究进展

盐胁迫是影响农业生产的重要环境因素之一。本文对植物Na+吸收的机制和途径、Na+在植物体内的长距离转运以及细胞内Na+稳态平衡的研究进展进行了概述。参与植物Na+吸收与转运的蛋白和通道可能包括HKT、LCT1、AKT和NSCC等。其中, HKT是植物体内普遍存在的一类转运蛋白, 能够介导Na+的吸收, 其结构中的带电氨基酸残基对于其离子选择性有着非常明显的影响。LCT1是从小麦中发现的一类能够介导低亲和性阳离子吸收的蛋白, 然而在典型的土壤Ca2+浓度下LCT1并不能发挥吸收Na+的功能。AKT家族的成员在高盐环境下可能也参与了Na+的吸收。目前虽然还没有克隆到编码NSCC蛋白的基因, 但是NSCC作为植物吸收Na+的主要途径的观点已被广泛接受。SOS1和HKT参与了Na+在根部与植株地上部的长距离转运过程, 它们在木质部和韧皮部的Na+装载和卸载中发挥重要作用, 从而影响植物的抗盐性。另外, 由质膜Na+/H+逆向转运蛋白SOS1、蛋白激酶SOS2以及Ca2+结合蛋白SOS3组成的SOS复合体对细胞的Na+稳态具有重要的调节作用, 单子叶和双子叶植物之间的这种调节机制在结构和功能上具有保守性。SOS复合体与其它位于质膜或液泡膜上的Na+/H+逆向转运蛋白以及H+泵一起调节着细胞的Na+稳态。     

Inward-directed current generated by the Na+,K+ pump in Na+- and K+-free medium

In Na⁺- and K⁺-free solution, an inward-directed current can be detected in Xenopus oocytes, which is inhibited by cardic glycosides and activated by ATP. Therefore, it is assumed to be generated by the Na⁺, K⁺ pump. At negative membrane potentials, the pump current increases with more negative potentials and with increasing [H⁺] in the external medium. This current is not observed when Mg²⁺ instead of Ba²⁺ is the only divalent cation present in the bath medium, and it does not depend on whether Na⁺ or K⁺ is present internally. At 5 to 10 mM Na⁺ externally, maximum pump-generated current is obtained while no current can be detected in presence of physiological [Na⁺]. It is suggested that in low-Na⁺ and K⁺-free medium the Na⁺, K⁺ pump molecule can either form a conductive pathway that is permeable to Ba²⁺ or protons or operate in its conventional transport mode accepting Ba²⁺ as a K⁺ congener. A reversed pump mode or an electrogenic uncoupled Na⁺-efflux mode is excluded.     

Palytoxin Acts on Na+,K+-ATPase but not Nongastric H+,K+-ATPase

Palytoxin (PTX) opens a pathway for ions to pass through Na,K-ATPase. We investigate here whether PTX also acts on nongastric H,K-ATPases. The following combinations of cRNA were expressed in Xenopus laevis oocytes: Bufo marinus bladder H,K-ATPase α₂- and Na,K-ATPase β₂-subunits; Bufo Na,K-ATPase α₁- and Na,K-ATPase β₂-subunits; and Bufo Na,K-ATPase β₂-subunit alone. The response to PTX was measured after blocking endogenous Xenopus Na,K-ATPase with 10 μm ouabain. Functional expression was confirmed by measuring ⁸⁶Rb uptake. PTX (5 nm) produced a large increase of membrane conductance in oocytes expressing Bufo Na,K-ATPase, but no significant increase occurred in oocytes expressing Bufo H,K-ATPase or in those injected with Bufo β₂-subunit alone. Expression of the following combinations of cDNA was investigated in HeLa cells: rat colonic H,K-ATPase α₁-subunit and Na,K-ATPase β₁-subunit; rat Na,K-ATPase α₂-subunit and Na,K-ATPase β₂-subunit; and rat Na,K-ATPase β₁- or Na,K-ATPase β₂-subunit alone. Measurement of increases in ⁸⁶Rb uptake confirmed that both rat Na,K and H,K pumps were functional in HeLa cells expressing rat colonic HKα₁/NKβ₁ and NKα₂/NKβ₂. Whole-cell patch-clamp measurements in HeLa cells expressing rat colonic HKα₁/NKβ₁ exposed to 100 nm PTX showed no significant increase of membrane current, and there was no membrane conductance increase in HeLa cells transfected with rat NKβ₁- or rat NKβ₂-subunit alone. However, in HeLa cells expressing rat NKα₂/NKβ₂, outward current was observed after pump activation by 20 mm K⁺ and a large membrane conductance increase occurred after 100 nm PTX. We conclude that nongastric H,K-ATPases are not sensitive to PTX when expressed in these cells, whereas PTX does act on Na,K-ATPase.     

Studies of the antigenic properties of the catalytic and glycoprotein subunits of Na+,K+-ATPase

Antibodies were raised against isolated, delipidated catalytic [alpha] and glycoprotein [beta] subunits of the Na+,K+-dependent ATPase purified from lamb kidney medulla. The specificity of each antiserum was confirmed by agar double-diffusion precipitation, immunoelectrophoresis, and polyacrylamide gel electrophoresis. A solid phase adsorption assay was also employed to determine antibody binding titers and to further test the specificity of these antisera. Antibodies raised to the alpha subunit had a strong reactivity and similar titer values for both the holoenzyme and the alpha subunit and a low-affinity cross-reactivity with the beta subunit. In contrast, beta-subunit-directed antibodies had little reactivity or binding with the holoenzyme and a low-affinity cross-reactivity with the alpha subunit. Competition binding studies revealed that about 80% of the alpha-subunit-specific antibodies bound to the holoenzyme, indicating that similar sets of antigenic sites are exposed in the lipid-embedded holoenzyme complex and in the isolated alpha subunit. Competition binding studies also suggest that the subunit cross-reactivities of the antisera may not result from simple contamination of the respective antigens, but that there may be partial homologies of some antigenic sites. In addition, the beta-directed antibodies had no effect on Na+,K+-ATPase activity, while the alpha-directed antibodies were effective inhibitors of activity. This indicates that at least some functionally important antigenic sites of the alpha subunit may be unaltered by its isolation and delipidation.     

Ion pumps in polarized cells: sorting and regulation of the Na+, K+- and H+, K+-ATPases

The physiologic function of an ion transport protein is determined, in part, by its subcellular localization and by the cellular mechanisms that modulate its activity. The Na(+),K(+)-ATPase and the H(+),K(+)-ATPases are closely related members of the P-type family of ion transporting ATPases. Despite their homology, these pumps are sorted to different domains in polarized epithelial cells, and their enzymatic activities are subject to distinct regulatory pathways. The molecular signals responsible for these properties have begun to be elucidated. It appears that a complex array of inter- and intramolecular interactions govern trafficking, distribution, and catalytic capacities of these proteins.     

Vanadate sensitivity of Na+, K+-ATPase from Schistosoma mansoni and its modulation by Na+, K+ and Mg2+

Vanadate inhibitory effects on Na+, K+-ATPases from carcass of Schistosoma mansoni and from lamb kidney outer medulla were compared in the presence of various concentrations of Na+, K+ and Mg2+. Depending on the ionic conditions, the schistosomal Na+, K+-ATPase was 2.4- to 175-fold less sensitive to vanadate than the lamb kidney enzyme. In 100 mM Na+, 3 mM K+ and 3 mM Mg2+, schistosomal Na+, K+-ATPase was surprisingly resistant to vanadate (I50 = 944 microM). The difference in vanadate sensitivity between schistosomal and lamb Na+, K+-ATPases may be due to a species difference in the efficacy of Na+, K+ and Mg2+ in promoting conformational changes between E1 and E2 forms of the enzyme.     

Na+, K+-ATPase of the canine mesenteric artery

Na+,K+-ATPase, the enzymatic moiety that operates as the electrogenic sodium-potassium pump of the cell plasma membrane, is inhibited by cardiac glycosides, and this specific interaction of a drug with an enzyme has been considered to be responsible for digitalis-induced vascular smooth muscle contraction. Although studies aimed at localization, isolation, and measurement of the Na+,K+-ATPase activity (or Na+, K- pump activity) indicate its presence in vascular smooth muscle sarcolemma, its characterization as the putative vasopressor receptor site for cardiac glycosides has depended on pharmacological studies of vascular response in vivo and on isolated artery contractile responses in vitro. More recently, radioligand-binding studies using [3H]ouabain have aided in the characterization of drug-enzyme interaction. Such studies indicate that in canine superior mesenteric artery (SMA), Na+,K+-ATPase is the only specific site of interaction of ouabain with resultant inhibition of the enzyme. The characteristics of [3H]ouabain binding to this site are similar to those of purified or partially purified Na+,K+-ATPase of other tissues, which suggests that if Na+,K+-ATPase inhibition is causally related to digitalis-mediated effects on vascular smooth muscle contraction, then therapeutic concentrations of cardiac glycosides could act to cause SMA vasoconstriction. The additional finding from radioligand-binding studies that Na+,K+-ATPase exists in much smaller quantities (density of sites per cell) in SMA than in either heart or kidney may have implications concerning its physiological, biochemical or pharmacological role in modulating vascular muscle tone.     

Simplified Digitalis-like Compounds acting on Na+, K+-ATPase

Digitalis compounds are used in the treatment of congestive heart failure as positive inotropic agents; their action is mainly due to the inhibition of Na+,K+-ATPase. A well-known drawback is their arrhythmogenic potential. Attempts to find safer digitalis-like compounds by means of molecular simplifications of the typical 5β,14β-steroidal skeleton, which appeared in the medicinal chemistry literature from 1990 until 2002, are briefly reviewed. Several novel achievements were obtained in order to better understand the requisites of the digitalis binding site on Na+, K+-ATPase. Only minor simplification, such as cleavage of the D ring of the digitalis skeleton, could preserve the desired inotropic activity, while highly simplified digitalis-like compounds failed to give sufficiently high inotropic potency, even in the presence of a powerful pharmacophore, such as the O-aminoalkyloxime group.