Ouabain及其受体Na^+/K^+-ATPase α_1、α_4抗体对人精子运动功能的影响

目的研究Ouabain及其受体Na+/K+-ATPase α1、α4抗体对正常人精子运动功能的影响。方法将60例正常人的精液上游法进行优化,其中30例与不同浓度Ouabain共孵育,在1,2,3,4h采用CASA检测精子运动参数;另外30例分别与Na+/K+-ATPase α1和α4抗体共同孵育,1h后同样方法检测。结果①与阴性对照组比较,优化后的精子与较高剂量Ouabain(1×10-5～1×10-2mol.L-1)共孵育后,活动率显著下降(P<0.05),a+b级精子所占比例显著下降(P<0.01);但各浓度组两参数差异无显著性;②与阴性对照组比较,较低剂量Ouabain(1×10-6和1×10-7mol·L-1)作用后,精子活动率和a+b级精子所占比例下降均不明显,两浓度组之间差异无显著性;③Ouabain作用后,精子其他运动参数如直线速度、鞭打频率、直线性、前向性、摆动性均未见显著变化。④Anti-α1和Anti-α4作用后的精子活动率均显著下降(均P<0.01);但两者之间差异无显著性;⑤Anti-α1和Anti-α4作用后的a+b级精子所占比例均显著下降(均P<0.01);且Anti-α4作用后降低的幅度明显大于Anti-α1(P<0.05);⑥Na+/K+-ATPase α1和α4抗体作用后,精子其他运动参数如直线速度、鞭打频率、直线性、前向性、摆动性均未见显著变化。结论Ouabain在体外能够降低正常人精子运动功能;Na+/K+-ATPase α1、α4抗体也能够降低精子运动功能,但α4抗体的作用更明显。

Ouabain对髓袢升支粗段管周膜50pS钾通道活性的影响

肾脏髓袢升支粗段（thick ascending limb,TAL）对NaCl的主动重吸收启动了髓质渗透梯度的形成,其动力来自管周膜上的钠-钾泵。50pS钾通道是管周膜上分布最多的钾通道,在NaCl的主动转运中发挥重要作用。50pS钾通道的活性是否受钠-钾泵活动的影响，目前尚不清楚。本实验利用单通道膜片钳技术，观察钠-钾泵抑制剂ouabain对髓袢升支粗段管周膜50pS钾通道活动的影响，探讨50pS钾通道是否在功能上与钠-钾泵偶联。

Ouabain在急性缺氧诱导心房钠尿肽分泌机制中的作用

目的观察急性缺氧(hypoxia)对心房钠尿肽(atria1 natriuretic peptide,ANP)分泌的影响,并探讨ouabain在急性缺氧诱导心房ANP分泌机制中的作用。方法取大耳白家兔30只。采用搏动的离体心房灌流模型做如下实验:①观察急性缺氧对心房ANP分泌、心房搏动压和心房搏出量的影响;②观察ouabain(3.0 mol/L)对心房ANP分泌、心房搏动压和心房搏出量的影响;③观察预处理ouabain(3.0 mol/L)后缺氧对心房ANP分泌影响;④将正常心房组织、单独处理ouaba-in(3.0 mol/L)及缺氧的心房组织用苏木精-伊红染色方法制备石蜡切片,光镜下观察组织形态学变化。结果①缺氧明显促进心房ANP的分泌,并显著抑制心房搏动压和心房搏出量(P<0.001,与对照循环比较);②ouabain明显增加ANP分泌的同时增强心房搏动压和心房搏出量(P<0.001,与对照循环比较);③ouabain能明显抑制缺氧诱导心房ANP分泌增加的效应,但并不完全阻断ANP分泌增加的效应(P<0.001,与缺氧循环比较);④与正常心房组织相比,ouabain使诱导心房肌细胞间质水肿;而缺氧不仅使心肌细胞间质水肿,且能引起心房肌细胞水肿。结论缺氧明显促进心房ANP的分泌,其作用与钠泵(Na+-K+/ATPase)活性抑制及心房肌细胞牵张刺激有关。

老年人高血压Ouabain抵抗现象与肾素-血管紧张素系统的关系及硝苯啶干预的效应

目的：探讨老年人高血压哇巴因（Ouabain）抵抗现象机制及其与肾素、血管紧张素系统（RAS）的关系。方法：47例高血压病（EH）病人，分为老年EH组和非老年EH组，选择25名健康老年人作对照组。采用放射免疫分析法测定血浆肾素活性（PRA）、血管紧张素Ⅱ（AⅡ）、血浆内源性类地高辛物质（EDLS）浓度、EDLS与红细胞结合率（RBC－D％）；测定加Ouabain后红细胞内Na+浓度（[Na＋]i）变化，计算出Ouabain敏感Na+外流速度常数(oKos)，反映总的Na+泵活性；红细胞[Na+]i用火焰光度法测定。老年EH组和非老年EH组服硝苯啶治疗4周后复查。结果：老年EH组和非老年EH组RBC－D％、oKos下降，EDLS升高．但老年EH组RBC－D％、PRA、AII明显低于非老年EH组，血浆EDLS和oKos高于非老年EH组，Na+泵活性呈现Ouabain抵抗现象。在老年EH组中，AII与收缩压、年龄和RBC－D％呈负相关，PRA与EDLS呈负相关；在非老年EH组中，血压与EDI上呈负相关。降压治疗后，老年EH病人PRA、AII、Na+泵活性升高，EDLS有下降趋势；中年EH者仅Na+泵活性升高，EDLS有升高趋势，PRA、AII无显著变化。结论：老年EH病人EDLS与细胞膜亲合力下降，呈现Ouabain抵抗现象，通过影响钠代谢抑制PRA、AII分泌。钙拮抗剂降压治疗可部分改善这种异常。

Ouabain诱导沙鼠蜗外电诱发听性脑干反应变化研究

目的沙鼠耳蜗圆窗应用毒毛旋花甙G(Ouabain),观察沙鼠蜗外电诱发听性脑干反应(EABR)的变化。方法显微镜下将浸有50μl Ouabain(1 mM)的明胶海绵置于沙鼠耳蜗圆窗膜表面,观察应用Ouabain 24 h、96 h后听觉脑干反应(ABR)、耳蜗微音电位(CM)、蜗外电诱发听性脑干反应(EABR)阈值变化。结果沙鼠圆窗应用Ouabain 24h、96 h后ABR、EABR阈值明显升高,差异具有统计学意义(P<0.05),CM阈值无明显变化,差异无统计学意义(P>0.05)。结论沙鼠圆窗膜表面应用50μl Ouabain(1 mM)24 h、96 h后可导致沙鼠ABR、EABR阈值明显升高,而CM阈值无明显变化。

老年人高血压呈现Ouabain抵抗现象及非洛地平干预的影响

目的 :探讨老年人高血压Ouabain抵抗现象机制及钙拮抗剂非洛地平的疗效。方法 :47例高血压病 (Es sentialHypertension ,EH)病人 ,分为老年EH组和非老年EH组 ,选择 2 0 0名健康老年人作对照组。采用放射免疫分析法测定血浆内源性类洋地黄物质 (Endogenousdigitalis -likecompound ,EDLC)浓度、EDLC与红细胞结合率 (RBC -D % ) ;测定加哇巴因 (Ouabain)后红细胞内 [Na+]i 变化 ,计算出Ouabain敏感Na+外流速度常数 (theratecontentofouabainsensitivesodiumefflux ,oKos/h ,反映总的Na+泵活性 ) ;红细胞内 [Na+]i 用火焰光度法测定。老年EH组和非老年EH组服非洛地平治疗 3周后复查。结果 :老年EH组和非老年EH组RBC-D %、oKos下降 ,EDLC升高 ,但老年EH组RBC -D %明显低于非老年EH组 ,血浆EDLC和oKos高于非老年EH组 ,Na+泵呈现Ouabain抵抗现象。非洛地平降压治疗后 ,老年EH病人Na+泵活性升高 ,EDLC下降 ;非老年EH者仅Na+泵活性升高 ,EDLC有升高趋势 ,两组 [Na+]i 治疗前后无显著变化。结论 :老年EH病人EDLC与细胞膜亲合力下降 ,Na+泵呈现Ouabain抵抗现象。钙拮抗剂非洛地平降压治疗可部分改善这种异常。

Endogenous Ouabain in Hypertensive Disorder Complicating Pregnancy

Expression of endogenous ouabain in placenta and the concentrations of serum ET-1 and NO were examined in 30 patients with hypertensive disorder complicating pregnancy （HDCP） and 30 healthy pregnant women to investigate the effect of endogenous ouabain on HDCP. Compared with the healthy pregnant group, the expression of endogenous ouabain dramatically increased in the HDCP groups （P〈0.01）. There was a significantly positive correlation between the expression of en- dogenous ouabain with ET-1 （r= 0.5567, P〈0.01）, while the correlation of endogenous ouabain and NO was significantly negative （r=-0.6895, P〈0.01）. As expected, the correlation between ET-1 and NO was negative （r=-0.7796, P〈0.01）. ET-1 concentrations of maternal and cord sera in HDCP groups were significantly higher in comparison with healthy pregnant group （P〈0.01）. On the contrast, NO concentrations were much lower in the maternal and cord sera of HDCP groups as compared with healthy pregnant group （P〈0.01）. Our data suggest that endogenous ouabain is directly involved in the nosogenesis of HDCP, with accompanying decreased NO and the elevated of ET-1.

老年人高血压呈现Ouabain抵抗现象及非洛地平干预的影响

目的：探讨老年人高血压Ｏｕａｂａｉｎ 抵抗现象机理及钙拮抗剂非洛地平对其影响。方法：４２ 例高血压（ＥＨ）病人， 分为老年ＥＨ 组和非老年ＥＨ组。采用放射免疫分析法测定血浆肾素活性（ＰＲＡ）、血管紧张素Ⅱ（ＡｎｇⅡ）、血浆内源性类地高辛物质（ＥＤＬＳ）浓度、ＥＤＬＳ与红细胞结合率（ＲＢＣ－Ｄ％ ）；测定加哇巴因（ｏｕａｂａｉｎ）后红细胞内［Ｎａ＋ ］ｉ变化，计算出ｏｕａｂａｉｎ 敏感Ｎａ＋ 外流速度常数（ｏＫｏｓ，反映总的Ｎａ＋ 泵活性）；［Ｎａ＋ ］ｉ用火焰光度法测定。ＥＨ组服非洛地平治疗３周后复查。结果：老年ＥＨ组ＲＢＣ－Ｄ％ 、ＰＲＡ、ＡｎｇⅡ明显低于非老年ＥＨ组，血浆ＥＤＬＳ和ｏＫｏｓ 高于非老年ＥＨ 组，Ｎａ＋ 泵活性呈现Ｏｕａｂａｉｎ 抵抗现象。在老年ＥＨ 组中，ＡｎｇⅡ与年龄和ＲＢＣ－Ｄ％ 呈负相关，ＰＲＡ与ＥＤＬＳ呈负相关。降压治疗后，老年ＥＨ病人ＰＲＡ、ＡｎｇⅡ、Ｎａ＋ 泵活性升高，非老年ＥＨ者仅Ｎａ＋ 泵活性升高，ＥＤＬＳ、ＰＲＡ、ＡｎｇⅡ无显著变化。结论：老年ＥＨ 病人ＥＤＬＳ与细胞膜亲合力下降，呈现Ｏｕａｂａｉｎ 抵抗现象，通过影响钠代谢抑制ＰＲＡ、ＡｎｇⅡ分泌。

Mechanisms of antinociceptive effects of ouabain in combination with neostigmine in the rat

BACKGROUND： It has been previously shown that intrathecal administration of either ouabain or neosdgmine can produce antinociceptive effects. Moreover, ouabain and neostigmine are differently associated with acetylcholine. OBJECTIVE： It has been hypothesized that intrathecal administration of ouabain, in combination with neostigmine, can produce antinociceptive synergistic effects. Atropine, as a competitive antagonist, was pre-injected to verify the mechanisms of action. DESIGN, TIME AND SETTING： This study was a randomized, controlled, animal experiment, performed at the State Key Laboratory of Oncology in Southern China between May 2006 and February 2007. MATERIALS： A total of 102 healthy, adult, Sprague Dawley rats were included. Ouabain and neostigmine （Sigma, USA）, as well as atropine （Tanabe Seiyaku, Japan）, were also used. METHODS： Varied doses of ouabain, neostigmine, and a combination of the two were intrathecally injected into rats. Six rats were allotted for each dose group. Intrathecal pretreatment with atropine was tested 10 minutes prior to intrathecal administration of neostigmine or the combination of ouabain and neostigmine. MAIN OUTCOME MEASURES： Tail-flick tests were performed to measure tail-flick latency （seconds） prior to and after administration. The response in the tail-flick test was expressed as the percentage of maximum possible effect （% MPE）, where % MPE = [tail-flick latency after administration （seconds） -mean baseline value for tail-flick latency]/[ 10 seconds - the mean baseline value for tail-flick latency （seconds）] x 100%. RESULTS： Rat spinal intrathecal administration of either ouabain or neostigmine alone produced antinociceptive effects in a dose-dependent manner. Intrathecally administration of neostigmine （0.05, 0.1, 0.3 μg ） in combination with ouabain （1 μ g ） produced enhanced antinociceptive effects, with a % MPE of 29%, 78%, and 95%, respectively （P 〈 0.05）. Intrathecally administration of 0.3μg neostigmine （% MPE： 45%）, in combination with 1 μ g ouabain （% MPE： 27%） produced potent antinociceptive effects （% MPE： 95%）. Intrathecally pre-injected atropine antagonized the antinociceptive effects of neostigmine （3 μg）, or a combination of ouabain （1 μg） and neostigmine （0.3 μg） （P 〈 0.01）. CONCLUSION： Rat spinal intrathecal administration of either ouabain or neostigmine alone produced dose-dependent andnociceptive effects. Ouabain enhanced the antinociceptive effects of neostigmine. Atropine antagonized the antinociceptive effects of neostigmine or the combination of ouabain and neostigmine. This occurs possibly due to the fact that atropine is a competitive antagonist of the muscarinic acetylcboline receptors.

EXPERIMENTAL STUDY ON THE ROLES OF ENDOGENOUS OUABAIN IN THE DEVELOPMENT OF HYPERTENSION

Objective To explore the relationship between endogenous ouabain and the pathogenesis of hypertension. Methods ① Sixteen Sprague Dawley (SD) rats were selected and randomly divided into two groups, and then the rats were injected with ouabain in dosage of 20μg/kg.d and normal saline (NS), respectively. The indirect systolic blood pressure of all the rats were recorded once a week. ② Twenty five 1k1c (one kidney and one clipped) hypertensive rats were established and injected randomly with anti ouabain antibody, normal rabbit IgG and normal saline, respectively. The direct blood pressure of all the 1klc hypertensive rats were recorded by cannulated carotid artery for 3h after administration. Results The systolic blood pressure of rats injected with ouabain began to increase 2 weeks after ouabain administration and increased significantly at 6 weeks compared with NS group (17.7±1.2)kPa vs (15.4±1.1)kPa, P <0.01).Anti ouabain antibody could significantly decrease the blood pressure of 1k1c rats, while the normal rabbit IgG could not. Conclusion The results of this study indicate that endogenous ouabain might be one of the pathogenetic factors of hypertension.

Relationship between Ouabain and Asthenozoospermia

A growing number of researches have shown that ouabain can regulate mammalian sperm function and male reproduction by modulating the sperm motility, capacitation and acrosome reaction in vitro. This study further examined the relationship between ouabain and asthenozoospermia. In this study, the rat was intraperitoneally injected with ouabain at different concentrations（low-dose ouabain group： 12.5 μg/kg body weight per day, and high-dose ouabain group： 25 μg/kg body weight per day） for 30 days to establish the asthenozoospermia model. The sperms from 60 males with normal fertility were incubated with ouabain of gradient concentrations（10-7–10-2mol/L） for 4 h. The sperm motility was evaluated under a microscope. Moreover, the endogenous ouabain（EO） level was determined in seminal plasma of mild or severe asthenozoospermia patients and males with normal fertility by competitive inhibition ELISA. The results showed that the sperm motility was significantly diminished in the rats treated with different concentrations of ouabain. The number of motile sperms（grades a and b） was decreased greatly in a time- and dose-dependent manner in 10-5–10-2mol/L ouabain groups（P0.01）, while no obvious change in sperm motility was observed in 10-7–10-6mol/L groups even for 4-h incubation（P0.05）. Furthermore, the EO level was significantly increased in asthenozoospermia patients as compared with that in males with normal fertility（25.27±1.71 μg/L in mild asthenozoospermia patients, 26.52±1.82 μg/L in severe asthenozoospermia patients, 19.31±1.45 μg/L in normal fertility men）（P0.01）. In conclusion, rat asthenozoospermia was successfully established by intraperitoneal injection of ouabain, and 10-5mol/L ouabain was sufficient enough to inhibit sperm motility in vitro. Moreover, EO, a normal constituent of seminal plasma, was highly expressed in asthenozoospermia males as compared with normal fertility ones.

THE CHARACTERISTICS OF ENDOGENOUS OUABAIN SECRETION FROM CULTURED BOVINE ADRENOCORTICAL CELLS

Objective To compare the characteristics or endogenous ouabaln(EO) secretion with the other adrenocortical hormones and determine the effects of angiotensin I (Ang I ), and ad reno corticotrophin (ACTH ) on the secretion or EO. Methods EO was measured by radioimmunoassay from primary cultured bovine adrenocotical cells (BAC). Results oouabain was determined in the media or cultured BAC. Both EO and aldosterone secretion were decreased from the 6uter to inner layer or the cultured adrenal cortex, and the responses to Ang I and ACTH were hlgher than that in the mld layer (P <o. o5) and inner layer (P <o. o1 ). Cortisol secretion was activated by Ang I or ACTH was slgnificantly higher in the mid layer and in the inner layer than that in the outer layer. The tlme-course experlment showed that the gradually rising amounts or aldosterone and cortisol could be determined during the continuous incubation to 48h wlth or without Ang I or ACTH. However, EO did not increase continuously arter 24h or incubation in the basal secreting sltuation and arter 12h of lncubatiou in the stimulating situation by Ang,or ACTH. There were obvlous drops in aldosterone and cortisol secretlou from 3rd day during a 21 day-perlod cell culture, but the peak secretion of ouabain was in 7th day. Conclusion it suggests that the secretory mechanism might be different between EO and aldosterone or cortisol. Also, Ang I and ACTR might be involved in the regulation of Eo secretion.

EXPRESSION OF ENDOGENOUS OUABAIN IN MULTIPLE ADRENAL TUMORS

Objective To explore expression of endogenous ouabain(EO) in multiple adrenal tumors.Methods Thirty one cases of adrenal tumors and 6 cases of healthy adrenal tissues were selected. The expression of EO in the adrenal tissue was detected with immunohistochemical streptavidin peroxadase conjugated(SP) method.Results Most of EO positive products were localized in cytoplasm of the zona reticularis of human adrenal cortex, and positive products showed to be fine granular. There was no positive signal in the medulla. EO showed on diffused positive in patients with pheochromocytoma accompanied high blood pressure[SBP:(165.22±7.61) mmHg, DBP:(105.52±4.26) mmHg], but there were negative in ones with normative blood pressure[SBP:(118.52±4.58) mmHg, DBP:(83±3.60) mmHg]. The expression of EO was positive in all adrenocortical hyperplasic, adenoma and carcinoma, no matter its high or normative blood pressure. The degree of expression of EO in adrenal tissues was related to the level of BP.Conclusion Expression of endogenous ouabain(EO) in healthy adrenal tissue and adrenal tumors was a valuable morphological and pathophysiological clue for the research on ouabain.

Ouabain诱导沙鼠听觉电生理变化研究

目的沙鼠耳蜗圆窗应用毒毛旋花甙G（Ouabain），观察沙鼠听觉电生理变化。方法显微镜下将浸有100μl毒毛旋花甙G（Ouabain）1mM的明胶海绵置于沙鼠耳蜗圆窗膜表面，观察应用Ouabain5。12h后沙鼠听性脑干反应（auditorybrainstemresponse，ABR）、耳蜗微音电位（cochlearmicrophoniC，CM）、复合动作电位（compoundactionpotential，CAP）的变化。结果应用Ouabain5h：ABR阈值明显升高（P〈0．05），且波形不典型；CM阈值无明显变化（P〉0．05）；CAP阈值明显升高（P〈0．05）：应用Ouabain12h：ABR消失；CM阈值无明显变化（P〉0．05）；CAP波形消失（P〈0．05）。结论沙鼠圆窗膜表面应用Ouabain（5、12h）导致沙鼠CAP与ABR阈值升高甚至波形消失，而CM阈值不变。为建立听神经病动物模型提供了较好的研究方法。

Comparative study of renal sodium transport between ouabain-hypertensive rats and ouabain-nonhypertensive rats

Objective: To compare renal sodium transport, using fractional excretions of lithium(FEii)as a marker of proximal tubule sodium reabsorption, between hypertensive and non-hypertensive ouabaintreated rats and further to elucidate the role of ouabain in pathogenesis of hypertension. Methods:Thirty male Sprague-Dawley rats weighting 180-200 g were randomly divided into normal control group and ouabain treated group. Rats were infused with 1 ml/kg · d normal saline or 27.8 μg/kg · d ouabain intraperitoneally once a day respectively. Systolic blood pressure (SBP), heart rate and body weight were recorded weekly. Rats were sacrificed 6 weeks after treatment. Blood and 24-hour urine sample were collected to measure the serum and urinary concentration of sodium, trace lithium and creatinine. Endogenous creatinine clearance rate (Ccr), fractional excretions of sodium (FENa), fractional excretions of lithium (FELi) and fractional reabsorption of sodium in the postproximal tubules (FDRNa) were calculated.Ouabain levels of plasma and renal tissue, plasma renin activity, angiotensin Ⅱ and aldosterone concentration were determined. Results: 65% of the ouabain-treated rats achieved significantly higher SBP after 4weeks, compared with that of the saline control groups or self baseline (P＜0. 01). But in the other 35%of the ouabain-treated rats, their SBP was similar with control group during the experiment (P＞0. 05).The body weight, heart rate and food intake between the 3 groups were no significant differences (P＞0.05). FELi and FDRNa were significantly lower in ouabain-hypertensive group compared with ouabain-nonhypertensive group and control group(P＜0.01 and P＜0.05). The FELi and FDRNa of ouabain-nonhypertensive groups were similar with control group(P＞0.05). Ccr and FENa were comparable between the 3 groups (P＞0. 05). Plasma and renal tissue ouabain levels, plasma renin activity, angiotensin Ⅱ and aldosterone contents in ouabain-hypertensive rats were comparable with ouabain-nonhypertensive rats. Conclusion: Increase of proximal tubule sodium reabsorption play an important role in the pathogenesis of ouabain-hypertensive rats. The change of renal sodium transport may result from regulation to renal Na+ ,K+-ATPase by ouabain.

Ouabain-resistant phenomenon and changes of transmembrane ion transport in essential hypertensives with a family history of hypertension

Objective: To investigate the role of heredity in the ouabain-resistant phenomenon and the rela tionship between ouabain-resistance and transmembrane ion transport in essential hypertensives. Methods:A total of 52 essential hypertensives were investigated. of the patients, 23 were with a family history of hyper tension (FH+ group) and 29 were without (FH- group). Other 25 normotensives were employed to serve as the controls (control group). The percentage of 125I-digoxin binding to red blood cells (RBC-D% ) and plasma endogenous digoxin-like substance (EDLS) were measured with radioimmunoassay, 45Ca2+ influx in ATP-de pleted red cells by liquid scintillation counting. The rate constant of ouabain-sensitive sodium efflux (°Kos, h-1) was analyzed as half the increase in erythrocyte Na+ concentration during incubation with ouabain for 2 h. The maximal rate (Vmax) of red cell Na+/H+ exchange was determined as the influx promoted by an out ward H+ gradient then calculated. Results:The ouabain-sensitive Na+ efflux and RBC-D% were significantly lower but the levels of plasma EDLS and 45Ca2+ influx significantly higher in both FH+ and FH groups than in the control group. The plasma EDLS and ouabain-sensitive Na+ efflux were significantly higher but Ca2+ innux lower in FH+ group than in FH- group. Positive correlation was found between RBc-D% and ca2+ in flux in FH+ group. Conclusion: Ouabain-resistant phenomenon is related to the heredity of hypertension.The decrease in affinity of EDLS for membrane affects the transmembrane ion transport, which may partici pate in the pathogenesis of salt-sensitive hypertension.

Ouabain inhibits anchorage-independent growth in human lung cancer cells via integrinαvβ3 reduction

Physiological effects of ouabain (Fig. 1A), a human endogenous hormone, have been intensively investigated nowadays.Recent studies demonstrate anticancer activity of ouabain in sensitization of apoptosis and suppression of migration in lung cancer cells (1,2)Focusing on metastatic process, the ability of cancer cells to grow in an anchorage-independent condition determines the success of cancer metastasis [3].

EFFECTS OF OUABAIN AND DIGOXIN ON THE GENE EXPRESSION OFSODIUM PUMP α-SUBUNIT ISOFORM IN AORTIC SMOOTH MUSCLE OF RATS

Objective To compare the effects or ouabain and digoxin on both the systolic blood pressure and sodium pump a-subunit isoforms gene expression in the aortic smooth muscle of rats. Methods Normal Sprague- Dawley rats were injected with ouabain (20μg·kg-1·d-l,i. p), digoxin (32 μg·kg-1·d-1, i. p)and normal saline once a day, respectively, and indirect systolic blood pressure was recorded once a week. Six weeks later,all the rats were killed and sodium pump α1-,α2-,and α3-subunit mRNA levels were detected in the aortic smooth muscle with reverse transcription polymerase chain reaction（RT-PCR） method. Results The systolic blood pressure of rats infused with ouabain increased significantly at the end of week 6 [l32. 6±9.0 mmHg （1 mmHg = 0. 133 kPa） vs 115. 7 ± 8. 2 mmHg, P <0.01],while no difference of blood pressure was found between digoxin group and NS group （P>0.05）. The expression or sodium pump α-subunit isoforms in aortic smooth muscle was regulated by either ouabain or digox- in:both ouabain and digoxin increased α1- and α3-subunit expression, α2-subunit decreased in digoxin group but un- changed in ouabain group. Conclusion These results suggest that both ouabain and digoxin could regulate sodium pump α-subunit isoform expression, which might be related to the physiological roles or endogenous ouabain and might be responsible for the difference between the pharmacological and toxicological effects or ouabain and digoxin, including their effects on blood pressure.

Gene Expression Analysis of Human Vascular Endothelial Cells Treated by Ouabain in Pathological Concentration

Objectives To study the geneexpression of human vascular endothelial cells (HU-VEC) treated by ouabain in pathological concentra-tion. Methods The response of endothelial cells toouabain of 1.8 nmol/L was explored with a comple-mentary DNA microarray representing 8 464 differenthuman genes. Results The results of mRNA profilesanalysis indicated that 129 of the genes were differ-ently expressed, 26 were upregulated. ConclusionsThe pathological role of ouabain on HUVEC may beinvolved in the controlling of DNA transcription、pro-tein translation、metabolism and signal transduction.

Ouabain-Induced Auditory Nerve Degeneration in Congenic Ly5.1 Mice

The Ly5.1 mouse,also termed B6.SJL-Ptprca Pepcb/BoyJ,is a congenic strain widely used as a recipient in animal studies of bone marrow transplant.Our previous study documented that a majority of type I spiral ganglion neurons (SGNs) in the apical turns of Ly5.1 mice are unmyelinated and aggregate into neuronal clusters,similar to the spiral ganglion in the human ear.Ouabain,a well known Na-K ATPase inhibitor,has been shown to induce neuronal degeneration in a variety of neural tissues including the adult gerbil and CBA/CaJ mouse spiral ganglion.Here,functional and pathological changes of the auditory nerves in young-adult Ly5.1 mice were examined at 3,7 and 14 days after ouabain exposure.Similar to observations in CBA/CaJ mice,ouabain application selectively removed type I SGNs,resulting in an immense decline of the auditory nerve function.Hyperplasia of glial cells was seen in the injured auditory nerves at 7 days after ouabain exposure.Our data indicate that the 'human-like' features of unmyelinated type I SGNs have no protective impact on the fate of SGNs after ouabain exposure.Cells incorporating bromodeoxyuridine (BrdU) and expressing Sox2 were also counted in the auditory nerves of control and ouabain-treated ears.The number of Sox2+ glial cells significantly increased at 3 and 7 days post-treatment.Interestingly,the highest density of BrdU+ cells appeared in the apical turn of the injured auditory nerve shortly after ouabain exposure,suggesting that the pattern of SGN loss at the apical turn in Ly5.1 mouse may have some impact on the reaction of non-neuronal cells in response to acute ototoxic drug exposure in the auditory nerve.