心肌Na^＋，K^＋-ATP酶在抑郁症时的活性及mRNA表达

目的 探讨Na+,K+-ATP酶在抑郁症发病中的作用及其和单胺类神经递质之间的关系.方法 对SD大鼠进行慢性随机刺激建立抑郁症模型,检测大鼠心肌Na+,K+-ATP酶的活性及mRNA表达,海马组织及血液中去甲肾上腺素(NA)和5-羟色胺代谢物5-羟吲哚乙酸(5-HIAA)水平.结果 抑郁症大鼠心肌Na+,K+-ATP酶的转录水平下调(P＜0.01);酶的活性明显降低(P＜0.01);海马及血浆中的NA及5-HIAA水平显著下降(P＜0.01).结论 推测抑郁症时神经体液因素的变化可能是引起心肌细胞Na+,K+-ATP酶分子变化的原因之一,该酶在抑郁症的发病机制中有一定的生理和病理意义.     

可视化动缘探测系统同步检测慢性心衰对豚鼠心肌细胞舒、缩功能和钙瞬变的影响

目的建立豚鼠慢性充血性心力衰竭(CHF)模型,采用可视化动缘探测系统同步检测慢性心衰对豚鼠心肌细胞舒缩功能及钙瞬变的影响。方法采用有无呼吸困难、左心室质量与体质量比值、肺质量与体质量比值、室壁厚度及血流动力学指标判断,以降主动脉缩窄法建立豚鼠CHF模型成功后,以酶解法急性分离其左室心肌细胞,用可视化动缘探测系统同步检测慢性心衰豚鼠心肌细胞收缩力和钙瞬变的变化,并与正常豚鼠心肌细胞进行比较。结果降主动脉缩窄8 wk后,出现呼吸困难的豚鼠,其左室收缩压及舒张末压显著性增高,左室压上升和下降最大速率降低;左心室质量与体质量比值、肺质量与体质量比值及室壁厚度明显增高,呈现明显的心衰指征;心衰心肌细胞与正常细胞相比,其收缩幅度、收缩和舒张速度均明显减弱;舒张期细胞内钙升高,钙瞬变幅度减小,舒张期钙减少50%所需时间延长,但收缩期钙及细胞内钙达峰时间无明显改变。结论降主动脉缩窄8 wk后,豚鼠呼吸困难是判断CHF模型形成的指标;慢性心衰可使心肌细胞收缩力下降,钙瞬变幅度减小,这为探讨慢性心衰的发生机制提供了实验依据。     

氯胺酮抗惊厥的作用机制

目的观察氯胺酮对惊厥小鼠不同脑区Na+,K+-ATP酶、Ca2+-ATP酶以及NOS酶活性的影响,探讨氯胺酮抗惊厥作用的中枢机制。方法昆明种小鼠随机分成空白组、生理盐水(NS)组、氯胺酮25mg·kg-1(KetⅠ)和50mg·kg-1(KetⅡ)组。空白组直接断头取脑。其余各组分别ip相应药物,5min后ip士的宁1.5mg·kg-1诱发惊厥,观察惊厥小鼠行为学变化,并于给士的宁30min后断头,用分光光度计法测定皮层额、顶、枕脑区的Na+,K+-ATP酶、Ca2+-ATP酶和NOS酶活性。结果氯胺酮组明显降低动物死亡率,KetⅡ组惊厥持续期较KetⅠ组明显缩短。与空白组相比,NS组和KetⅠ组Na+,K+-ATP酶、Ca2+-ATP酶活性均有降低,KetⅡ组顶、枕区Na+,K+-ATP和Ca2+-ATP酶活性维持在正常水平;KetⅡ组TNOS酶活性降低约1/3(P<0.05),各组对iNOS活性均无影响。结论氯胺酮抗惊厥作用机制可能与增加大脑皮层顶枕区的Na+,K+-ATP和Ca2+-ATP酶活性、降低cNOS酶活性有关。     

玉郎伞两种黄酮单体对心肌细胞缺氧/复氧损伤的保护作用

目的研究从玉郎伞(Yulangsan,YLS)中首次分离的两种黄酮单体对体外培养大鼠乳鼠心肌细胞缺氧/复氧损伤的保护作用,并初步探讨其作用机制。方法建立体外培养大鼠乳鼠心肌细胞缺氧/复氧损伤模型,倒置显微镜下观察加入YLS两种单体的含药血清(10%、5%、2.5%)后,各组缺氧/复氧心肌细胞形态学和搏动频率的变化;以MTT法检测各组细胞的存活率;用ELISA法测定心肌细胞Na+,K+-ATP酶、Ca2+,Mg2+-ATP酶活性及细胞培养上清液中总超氧化物歧化酶(T-SOD)、乳酸脱氢酶(LDH)、一氧化氮合酶(NOS)活性和丙二醛(MDA)含量。结果与模型组相比,YLS两种单体含药血清的高、中剂量均能明显增加心肌细胞的存活率,增强Na+,K+-ATP酶、Ca2+,Mg2+-ATP酶活性,降低细胞培养上清液LDH、NOS活性、MDA含量,提高T-SOD活性并呈剂量依赖性(P<0.05)。结论两种YLS单体对体外培养大鼠乳鼠心肌细胞缺氧/复氧损伤具有保护作用,其机制可能与清除自由基、抑制心肌细胞Ca2+超载有关。     

抗心衰药——Istaroxime

Istaroxime(PST-2744; Debio-0614)是一种新型的、具有抑制Na /K -ATP酶和激动肌浆网钙泵双重作用的抗心衰药.其对Na /K -ATP酶的抑制导致心肌细胞内钙离子浓度升高,从而促进心肌细胞的收缩、使心跳强度增加.     

低浓度毒毛旋花子苷原对离体豚鼠衰竭心脏的影响

目的　研究低浓度毒毛旋花子苷原 (strophanthidin ,Str)对离体衰竭心脏心功能及心肌细胞膜Na+,K+ ATP酶活性的影响。方法　Langendorff离体心脏灌流装置制备戊巴比妥钠心衰模型 ,八道生理记录仪测定不同浓度Str对心功能的影响 ,无机磷法测定各组心肌细胞膜Na+,K+ ATP酶活性。结果　Str 1× 10 -9～ 1× 10 -7mol·L-1均能不同程度地持续增加衰竭心脏的心率、左室收缩压及左室收缩的最大速率 ,但 1× 10 -7mol·L-1对Na+,K+ ATP酶活性无明显抑制 ,1× 10 -10 ～ 1× 10 -8mol·L-1则可升高Na+,K+ ATP酶的活性 (P <0 0 5或P <0 0 1)。 1× 10 -6 ～ 1× 10 -4 mol·L-1可使心功能指标先升高、后降低 ,且伴有心脏收缩不规则和心律失常 ,也可剂量依赖性地抑制Na+,K+ ATP酶活性 (P <0 0 1)。结论　高浓度Str的正性肌力及伴有的心脏毒性作用是通过抑制Na+,K+ ATP酶实现的 ;而低浓度的正性肌力作用则和Na+,K+ ATP酶抑制无关     

甲基葡糖苷对豚鼠离体心房的正性肌力作用

目的　观察甲基葡糖苷对豚鼠离体心房的正性肌力作用 ,并探讨其作用机制。方法　采用豚鼠离体左右心房 ,测定药物对心房肌收缩力、右心房心率 ,以及对静息后收缩和正阶梯现象的影响 ,并测定大鼠心肌细胞膜Na+ K+ ATP酶活性。结果　甲基葡糖苷显著增强心房肌收缩力 ,减慢右心房心率 ,且呈剂量依赖性 ;能明显增强左心房静息后收缩和正阶梯现象 ,并能显著抑制大鼠心肌细胞膜Na+ K+ ATP酶活性。结论　甲基葡糖苷具有加强心房肌收缩力 ,降低右心房心率的作用 ,其正性变力作用可能与抑制心肌细胞膜Na+ K+ ATP酶活性 ,促进心肌细胞外钙内流和内钙释放有关     

松果菊苷对过氧化氢损伤的PC12细胞的保护作用及机制研究

目的研究松果菊苷(echinacoside,ECH)对过氧化氢(H2O2)诱导的PC12细胞损伤的保护作用及机制。方法用终浓度为0.4 mmol.L-1的H2O2损伤PC12细胞,测定细胞存活率和Na+,K+-ATP酶的活性;用激光共聚焦法(LSCM)检测线粒体膜电位(MMP);用RT-PCR法检测Bcl-2和p53 mRNA表达量的变化。结果10 mg.L-1松果菊苷可以提高H2O2损伤的PC12细胞存活率和Na+,K+-ATP酶的活力、升高线粒体膜电位、降低p53 mRNA水平但增高Bcl-2 mRNA水平(P<0.05或P<0.01)。结论松果菊苷可保护H2O2诱导的PC12细胞损伤,升高MMP、下调p53mRNA和上调Bcl-2 mRNA可能是其作用机制。     

异氟醚对离体大鼠心肌缺血/再灌注损伤的影响

目的研究麻醉药异氟醚对缺血/再灌注心肌功能和代谢、氧自由基及ATP酶活性的影响。方法SD大鼠56只,随机分为7小组,每组8只。采用Langendorff离体大鼠心脏模型。按给药方式又分为两大组,各组记录平衡后,给药后(或续灌15min)复灌末左室收缩压(LVSP)、左室舒张末期压(LVEDP)、左室发展压(LVDP)、左室压力升高或降低最大速率(±dp/dtmax)、心率(HR)、冠脉流量(CF)。实验结束后测定心肌超氧化物歧化酶(SOD)活性、心肌丙二醛(MDA)含量、高能磷酸盐(ATP)含量、Na+,K+-ATP酶、Ca2+-ATP酶活性。结果异氟醚组明显降低LVDP、+dp/dt,升高LVEDP(P<0.05);缺血/再灌注后,异氟醚组的LVDP分别恢复到基础值的57%、61%、59%、77%,+dp/dt分别恢复到基础值的45%、50%、46%、52%;与再灌末对照组相比,差异具有显著性;异氟醚组能提高心肌ATP含量,缺血后心肌ATP下降较慢,复灌后恢复较快;复灌后异氟醚组SOD活性较高,MDA生成量下降(P<0.05);异氟醚能提高再灌后心肌Na+,K+-ATP酶活性(P<0.05)。结论异氟醚对心肌收缩功能和Ca2+-ATP酶活性具有一定的抑制作用,能明显促进心肌功能与代谢的恢复,提高冠脉流量、心肌Ca2+-ATP酶及Na+,K+-ATP酶活性。     

d-α-生育酚对乙醇引发的肝脏氧化应激的影响

目的观察d-α-生育酚对乙醇诱发的小鼠肝脏氧化应激的影响并探讨其机制。方法昆明种小鼠每天给予2.4g.kg-1bw乙醇后,再给予3个不同剂量的d-α-生育酚(25、50、100mg·kg-1bw.d-1),同时设正常对照组和乙醇对照组(乙醇2.4g·kg-1bw.d-1),连续灌胃60d后,测定肝脏超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、Na+,K+-ATP酶(Na+,K+-ATPase)、Ca2+,Mg2+-ATP酶(Ca2+,Mg2+-ATPase)、谷胱甘肽-S转移酶(GST)活力及丙二醛(MDA)的含量。结果小鼠摄入乙醇60d后,肝脏氧化应激水平明显增高。除Ca2+,Mg2+-ATPase以外,其余各项指标与正常对照组相比,差异均有显著性(P<0.05)。50mg·kg-1bw.d-1d-α-生育酚干预后,与乙醇对照组相比,GSH-Px、SOD、GST、Na+,K+-ATPase活力明显升高,MDA含量显著下降。其中,GSH-Px、Na+,K+-ATPase、MDA等3个指标在25mg·kg-1bw.d-1d-α-生育酚干预时亦出现同样的变化。结论适宜剂量的d-α-生育酚可以通过直接抑制脂质过氧化对乙醇诱发的肝脏氧化应激起保护作用。     

蜂毒肽对豚鼠心肌线粒体Na~ 、K~ -ATP酶的影响(英文)

目的:研究蜂毒肽对豚鼠心肌线粒体Na~ ,K~ -ATP酶活性的影响。方法:应用光电比色法测定Na~ ,K~ -ATP酶活性。以Line-weaver-burk双倒数作图法分析酶动力学参数。结果:蜂毒肽抑制心肌Na~ ,K~ -ATP酶表现为浓度和时间依赖性,IC_(50)为2.60μmol/L。酶动力学研究表明,蜂毒肽对Na~ ,K~ -ATP酶有抑制作用,对K~ 表现为竞争性结合,对Na~ 及ATP表现为非竞争性结合。结论:蜂毒肽对心肌Na~ ,K~ -ATP酶有抑制作用,其抑制机制主要是与K~ 竞争结合酶外侧的K~ 结合位点。     

Chronic administration of amiodarone does not affect Na+/Ca2+ exchange current in guinea pig cardiac ventricular myocytes

We investigated chronic effects of amiodarone on Na+/Ca2+ exchange current (INCX) and on the level of Na+/Ca2+ exchanger (NCX1) mRNA in guinea pig ventricular myocytes using the whole-cell clamp technique and RT-PCR analysis, respectively. Guinea pigs were intraperitoneally injected with 80 mg/kg per day of amiodarone or the vehicle (saline) for 1 or 4 weeks. Single ventricular cells were isolated from the hearts of both groups of animals. Action potential duration at 90% repolarization level was prolonged to 143% and 165% of the control values by treatment with amiodarone for 1 and 4 weeks, respectively. INCX density and the level of NCX1 mRNA were not significantly changed by chronic treatment with amiodarone. The level of thyroid hormone (T4) within the blood was not changed by the treatments. These results suggest that chronic treatment with amiodarone does not affect the Na+/Ca2+ exchanger, with respect to the level of its mRNA and current density in guinea pig ventricular myocytes.     

Trimetazidine inhibits Na+,K(+)-ATPase activity, and overdrive hyperpolarization in guinea-pig ventricular muscles.

The effect of trimetazidine on Na+,K(+)-ATPase activity or the Na+,K+ pump was studied in guinea pig ventricular muscles with the use of biochemical and electrophysiological methods. The effect of trimetazidine on enzyme activity was compared with that in the liver, jejunum and kidney obtained from the same species. Na+,K(+)-ATPase activity in the heart and liver was significantly and concentration dependently decreased by trimetazidine (above 1.5 x 10(-5) M). Even the highest concentration (1.5 x 10(-4) M) of trimetazidine failed to decrease the Na+,K(+)-ATPase activity in the jejunum and kidney. The membrane potential was recorded in the ventricular muscle with a microelectrode. The hyperpolarization which followed 1-min overdrive stimulation (3.3 Hz) was decreased by trimetazidine (1.5 x 10(-4) M), but the depolarization during the stimulation was not affected by this drug. Ouabain, a potent Na+,K+ pump inhibitor, markedly decreased the overdrive hyperpolarization and increased the depolarization during the stimulation (10(-7), 5 x 10(-7), 10(-6) M). Therefore, the effect of trimetazidine and ouabain on the Na+,K+ pump-mediated alteration in the resting potential is different, suggesting that trimetazidine has additional direct membrane effects, e.g. a decrease in K+ conductance. In conclusion, trimetazidine inhibits Na+,K(+)-ATPase activity and thus the Na+,K+ pump in the ventricular muscles but with an inhibitory effect about 300 times less than that of ouabain. Trimetazidine inhibited the Na+,K(+)-ATPase in the liver as well, but not that in jejunum and kidney.     

Prednisolone-3, 20-bisguanylhydrazone: Na+, K+-ATPase inhibition and positive inotropic action.

The relationship between two known actions of prednisolone-3, 20-bisguanylhydrazone (PBGH); Na+, K+-ATPase inhibition and positive inotropic effects, was investigated. In electrically driven left atrial preparations of guinea pig heart, the positive inotropic action of PBGH was not affected by beta-adrenergic or histamine antagonists. Pretreatment of animals with reserpine also failed to influence the positive inotropic action of PBGH. Inotropic concentrations of PBGH inhibited Na+, K+-ATPase and the ATP-dependent binding of (3/)-ouabain to Na+, K+- ATPase preparations in vitro. Additionally, ouabain-sensitive 86Rb uptake, an estimate of sodium pump activity was inhibited when sodium-loaded ventricular slices were obtained from Langendorff preparations at the peak inotropic response to PBGH. Guinea pig heart was highly sensitive to PBGH to the positive inotropic action, the inhibition of Na+, K+ -ATPase and (3H)-ouabain binding, whereas rat, rabbit and dog heart were markedly less sensitive. These findings suggest that the mechanism of the positive inotropic action of PBGH resembles that of ouabain and probably involves Na+, K+ -ATPase inhibition, although the mode of interaction of these steroids with Na+, K+ -ATPase may be different.     

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。     

甲基莲心碱对心肌细胞I_(Na)、I_(Ca-L)及稳态外向K~+电流的影响

目的　为证明甲基莲心碱（ Ｎｅｆｅｒｉｎｅ, Ｎｅｆ） 对单个心肌细胞离子通道电流的影响及抗心律失常作用的离子通道机制。方法　采用全细胞记录膜片钳技术,记录了 Ｎｅｆ 对培养大鼠心室肌细胞钠电流（ Ｉ Ｎａ） 及豚鼠心室肌细胞动作电位、 Ｉ Ｎａ 、 Ｌ 型钙电流（ Ｉ Ｃａ Ｌ） 及稳态外向 Ｋ＋ 电流的影响。结果　 Ｎｅｆ ３０ ,１００ μｍｏｌ· Ｌ－ １ 明显抑制培养大鼠心室肌细胞 Ｉ Ｎａ ,分别从对照水平的（３４ ±０７） ｎ Ａ 降至（２１ ±０５） 和（０４ ±０２） ｎ Ａ。 Ｎｅｆ １０ μｍｏｌ· Ｌ－ １ 可降低豚鼠心室肌细胞动作电位振幅、静息电位,延长动作电位时程。 Ｎｅｆ １０ ,３０ μｍｏｌ· Ｌ－ １ 分别使豚鼠心室肌细胞 Ｉ Ｎａ 及 Ｉ Ｃａ Ｌ从给药前的（７９ ±２１） ｎ Ａ 和（６８９ ±２４３） ｐ Ａ 降至（４０ ±１４） 、（１３ ±０６） ｎ Ａ和（３７４ ±１７２） 、（１０９ ±６７） ｐ Ａ。 Ｎｅｆ １０ μｍｏｌ· Ｌ－ １ 还抑制 Ｉ Ｎａ 、稳态外向 Ｋ＋ 电流和 Ｉ Ｃａ Ｌ的 Ｉ Ｖ 曲线并使后者的峰值电流电位略右移。结论　 Ｎｅｆ 有钠、 Ｌ 型钙通道阻滞作用并抑制稳态外向 Ｋ＋ 电流,这些可解释     

Blocking of sodium and potassium ion-dependent adenosine triphosphatase-α1 with ouabain and vanadate suppresses cell-cell fusion during RANKL-mediated osteoclastogenesis

To examine the possible enrolment of Na(+)/K(+)-ATPase during osteoclast differentiation, Na(+)/K(+)-ATPase inhibitors, including ouabain and vanadate, were used in this study. These inhibitors significantly inhibited cell-cell fusion of RAW264.7 cells and bone marrow cells induced by RANKL. Interestingly, in response to RANKL-stimulation, ouabain and vanadate decreased the number of large TRAP+ osteoclasts in the culture of RAW264.7 cells, as well as bone marrow cells. In contrast, the number of small TRAP+ osteoclasts either increased in RAW264.7 cells or were otherwise less affected in bone marrow cells than large TRAP+ osteoclasts. Large TRAP+ osteoclasts are defined as having ≥ 10 nuclei/cell and having more potency in bone resorption than small multinuclear osteoclasts with <9 nuclei/cell. Na(+)/K(+)-ATPase α1 and β2 mRNAs were detected in sRANKL-stimulated RAW264.7 cells. Moreover, real-time quantitative PCR showed that ouabain and vanadate suppressed the RANKL-dependent induction of the osteoclast fusion-promotion molecule DC-STAMP at the mRNA level. Finally, and importantly, RNAi-mediated suppression of Na(+)/K(+)-ATPase α1 resulted in a diminished number of large TRAP+ osteoclasts in the sRANKL-stimulated RAW264.7 cells, along with the decreased level of DC-STAMP mRNA expression. These findings strongly suggest that blockage of the Na(+)/K(+)-ATPase α1 subunit by ouabain or vanadate caused the inhibition of RANKL-induced cell-cell fusion, resulting in the generation of large osteoclasts through suppression of DC-STAMP expression. Thus, in addition to its known function of sodium and potassium ion exchange during bone resorption by mature osteoclasts, this study has revealed a novel molecular role of the Na(+)/K(+)-ATPase α1 subunit in osteoclastogenesis.     

Cardenolide analogues. 7. Synthesis and biological activity of some new steroidal guanylhydrazones.

The synthesis, proof of structure, and biological activity of some new steroidal 17beta-formyl guanylhydrazones are described. The guanylhydrazones of nondigitalis-like steroids inhibited myocardial Na+,K+-ATPase but had only a depressant effect on myocardial contractility. By comparison, the corresponding guanylhydrazone of a digitalis-like steroid gave a positive inotropic effect in concentrations that also inhibited Na+,K+-ATPase. The nondigitalis-like guanylhydrazones also inhibited membrane Mg2+-ATPase and this may infer that the compounds act nonspecifically by membrane stabilization rather than by interaction with stereoselective receptors. Biological activity was determined in the guinea pig.