【作者】 李吉和；

【导师】 王永利；

【作者基本信息】 河北医科大学 ， 药理学， 2007， 博士

【摘要】 高血压病患病率高、致残率高、死亡率高,严重危害人类的健康。对人群心血管发病、死亡和危险因素监测的结果表明,高血压是心血管病的一个主要危险因素。高血压对心脏的影响主要包括心肌舒缩功能异常、心室肥厚、心力衰竭及冠心病等。临床研究证实高血压患者早期就可出现心脏舒、缩功能异常,而左心室肥厚和充血性心力衰竭的发病率随病人收缩压和舒张压的增加而增加,表明高血压时心功能的改变是一个渐进过程。一般认为,Ca2+是引起肌肉收缩和舒张的关键。高血压时心肌细胞内钙代谢及信号转导系统发生异常与舒缩功能改变、心肌肥厚及心室重构关系密切。但目前对其发生发展的病理生理机制还未清楚。心肌细胞膜上的Na+,K+-ATP酶主要调节Na+和K+的跨膜主动转运,也通过Na+/Ca2+交换间接调控着细胞内Ca2+的活动。高血压、激素水平、心肌肥厚和心衰影响Na+,K+-ATP酶α亚基的表达。目前对Na+,K+-ATP酶在高血压形成和发展过程中的动态变化和作用研究较少,对高血压时心肌细胞内钙代谢及舒缩功能改变发生发展的病理生理机制仍未探明。本研究采用一肾一夹(1k1c)和自发性高血压大鼠(SHR)两种高血压模型大鼠,对不同时期血压、心功能、心肌细胞收缩力与细胞内Ca2+、Na+的变化进行检测,同时测定Na+,K+-ATP酶的活性、α亚基的表达,从而探讨高血压发生发展过程中大鼠心肌细胞Na+,K+-ATP酶对心肌收缩力、钙瞬变的影响,为高血压的发病机制研究提供理论和实验依据。第一部分高血压对大鼠心肌细胞收缩性和钙瞬变的影响目的:通过测定不同阶段高血压大鼠心肌细胞舒缩功能、静息Na+浓度、钙瞬变和钙增敏作用,探讨高血压对心肌细胞收缩功能的影响及其可能机制。方法:选用4个月龄SD大鼠制作1k1c高血压大鼠模型,并于6月龄(即手术后2个月)、12月龄和18月龄时用于实验研究,SHR及对照组大鼠也选购相应月龄的动物进行匹配。采用鼠尾法测定两种高血压模型大鼠的尾动脉收缩压,采用生物信号定量分析系统实时监测高血压大鼠血流动力学指标,以湿重法测定两种高血压模型大鼠的左室重量指数,以酶解法急性分离左心室心肌细胞,采用可视化动缘探测系统同步检测心肌细胞舒缩功能、静息Na+浓度、钙瞬变和钙增敏作用,并比较6月龄、12月龄和18月龄1k1c高血压大鼠和SHR心肌细胞在上述指标方面的差别。结果:(1)1k1c模型大鼠术后2个月(即6月龄)后即形成高血压,总成模率为73%。在6、12及18月龄时,1k1c高血压大鼠和SHR的收缩压均显著高于同龄对照组,且SHR的收缩压均明显高于同龄1k1c模型大鼠(P < 0.05或0.01),SHR血压进一步明显升高的时间(6月龄)早于1k1c高血压大鼠(12月龄)。(2)对左室重量指数的测定结果表明,SHR和1k1c高血压模型大鼠在6、12和18月龄时,其左心室重量指数均明显高于同龄的对照大鼠,表明均已形成心肌肥厚,且SHR左心室肥厚的程度明显大于同龄1k1c高血压模型大鼠( P < 0.01)。但SHR心肌肥厚明显增大的时间(12月龄)早于1k1c高血压模型大鼠(18月龄)。这种两模型左心室重量指数变化与其收缩压变化一致的趋势,提示高血压所致大鼠心肌肥厚的时程及程度与其收缩压升高的时程和程度密切相关。(3)对两种高血压模型大鼠血流动力学的研究表明,在6、12及18月龄时, 1k1c高血压模型大鼠和SHR的LVEDP和-dp/dt均较同龄对照组大鼠显著升高,左心室充盈也持续增加。但1k1c高血压模型大鼠在12月龄时的-dp/dt大于6月龄,18月龄时又低于12月龄,而SHR在12、18月龄时的-dp/dt低于6月龄,提示1k1c高血压模型大鼠在12月龄时左心室舒张功能的代偿能力达到最大值,而SHR左心室舒张功能的代偿能力在6月龄时即达最大值。(4)对心肌细胞收缩力的测定结果表明,在6月龄时,1k1c高血压大鼠和SHR的Dep v和Peak h均高于同龄对照组大鼠, 1k1c高血压大鼠的Ret v也大于同龄对照组大鼠,但SHR的Ret v则低于同龄对照组大鼠,提示在高血压早期,1k1c高血压大鼠的心肌收缩功能和舒张功能均增强,而SHR仅收缩功能增强,其舒张功能则在6月龄时开始下降。这些结果与血流动力学的研究结果一致。(5)与心肌收缩力同步检测钙瞬变的结果表明,1k1c高血压大鼠和SHR在6月龄时,其DCa与同龄对照大鼠无明显差异,但在12、18月龄时均低于同龄对照组,也显著低于相应模型6月龄大鼠,提示6月龄的1k1c高血压大鼠和SHR仍处于高血压的代偿阶段,二者心肌细胞均以舒张期钙的改变为主。在6月龄时,1k1c高血压大鼠ΔCa2+与同龄对照大鼠无明显差异,而SHR则高于同龄对照组;在12、18月龄时两种模型大鼠的ΔCa2+均显著高于6个月组和同期对照组,说明随高血压时程的延长,1k1c高血压大鼠和SHR心肌细胞内ΔCa2+不断增加,可能是增加其心肌收缩力的主要代偿方式之一。从以上结果不难看出,在6月龄,1k1c高血压模型大鼠和SHR心肌细胞内钙瞬变与同龄对照组相比无明显改变,但1k1c高血压模型大鼠心肌细胞收缩力高于同龄对照组,而SHR心肌细胞收缩力则低于同龄对照组。在12、18月龄时,1k1c高血压模型大鼠和SHR心肌细胞内钙瞬变高于同期对照组,但二者收缩力反而逐渐降低。(6)心肌细胞内Na+浓度测定结果表明,在6月龄时,1k1c高血压模型大鼠和SHR心肌细胞内静息Na+浓度与同龄对照组无明显差异(p>0.05),在12、18月龄时,两种模型大鼠心肌细胞内静息Na+浓度均明显高于同龄对照组及各自的6月龄大鼠的心肌细胞内静息Na+浓度(P<0.05或0.01)。随着动物年龄从6月龄到18月龄的增长,对照组大鼠心肌细胞内静息Na+浓度无明显改变,而1k1c高血压大鼠和SHR则随年龄的增长,其心肌细胞内的钠荧光比值进一步逐渐升高(P<0.01),提示大鼠心肌细胞内的Na+水平升高也是高血压不断发展的结果。(7)心肌细胞钙敏感性测定表明,在6月龄时,1k1c高血压大鼠心肌细胞对外钙的收缩幅度-Fura2荧光强度比值关系曲线比同龄对照曲线明显左移,提示心肌细胞对钙的敏感性增强。但在12、18个月时,其心肌细胞对钙的敏感性则均显著降低;而SHR在6月龄时,其心肌细胞对钙的敏感性就已显著低于同龄对照组大鼠,且随高血压时间的延长,心肌细胞对钙敏感性呈继续下降趋势。这种高血压所致的心肌细胞对钙敏感性的降低,可能是两种12、18月龄模型鼠心肌细胞收缩力随着ΔCa2+不断增加反而均逐渐减小的原因之一。小结:高血压的发生和发展是个渐进的过程,随时间的延长,其收缩压升高和心肌肥厚程度逐渐增大,且1k1c模型大鼠血压升高的幅度和心肌肥厚的程度明显低于SHR。1k1c模型大鼠和SHR心肌细胞内钙瞬变幅值在6月龄时无明显改变,而在12、18月龄时明显升高;1k1c模型大鼠心肌细胞收缩力和钙敏感性在6月龄时均显著升高,12月龄后均明显降低,而SHR心肌细胞收缩力和钙敏感性均在6月龄时即显著下降。第二部分强心苷对高血压大鼠心肌细胞收缩性和钙瞬变的影响目的:用毒毛旋花子苷原(strophanthidin, Str)抑制Na+,K+-ATP酶后,观察不同时期高血压大鼠心肌细胞舒缩功能、静息Na+浓度、钙瞬变和钙敏感性的变化,探讨Na+,K+-ATP酶与高血压的关系及其在高血压形成和发展过程中的作用。方法:选用4个月龄SD大鼠制作1k1c高血压大鼠模型,并于6月龄(即手术后2个月)、12月龄和18月龄时用于实验研究,SHR及对照组大鼠也选购相应月龄的动物进行匹配。以酶解法急性分离左心室心肌细胞,采用可视化动缘探测系统同步检测6月龄、12月龄和18月龄正常对照大鼠、1k1c高血压大鼠模型及SHR心肌细胞舒缩功能、静息Na+浓度、钙瞬变和钙增敏作用,并观察不同浓度强心苷毒毛旋花子苷原对这些指标的影响。结果:(1)Str 1、10、50和100μmol?L-1均可剂量依赖性显著增大6月龄、12月龄和18月龄的正常对照大鼠、1k1c高血压模型大鼠和SHR心肌细胞收缩幅度(P < 0.05)。而且在6月龄大鼠,Str对1k1c模型大鼠和SHR心肌细胞收缩的增强幅度明显高于对正常心肌细胞收缩幅度的增强作用,对1k1c模型大鼠心肌细胞收缩的增强作用明显高于SHR心肌细胞的收缩幅度。但随着大鼠年龄的增长,在正常对照大鼠,Str各浓度增强其心肌细胞收缩幅度的作用不变,而在1k1c模型大鼠和SHR,Str各浓度增强其心肌细胞收缩幅度的作用呈年龄依赖性减弱,提示高血压的发展可显著降低心肌细胞对Str的敏感性。(2)用1mM Str灌流后,6月龄的1k1c高血压大鼠和SHR心肌细胞内Na+浓度增加值与同龄对照组大鼠无明显差异,但在12和18月龄时不仅较同龄对照组大鼠心肌细胞内Na+浓度明显增加,也均较各自6月龄大鼠的心肌细胞内Na+浓度有大幅度提高(P<0.01),并在12月龄时均达各自的最大值。在12、18月龄时,SHR心肌细胞内Na+浓度均高于同期1k1c高血压大鼠,且随高血压时间的延长细胞内钠含量逐渐升高。(3)Str 1、10、50和100μmol?L-1均可剂量依赖性显著增大6月龄、12月龄和18月龄的正常对照大鼠、1k1c高血压模型大鼠和SHR心肌细胞钙瞬变幅度(P < 0.05)。Str对三种月龄的1k1c模型大鼠和SHR心肌细胞钙瞬变的增强幅度明显高于对正常心肌细胞钙瞬变幅度的增强作用,但对1k1c模型大鼠和SHR心肌细胞钙瞬变的增强作用无明显差别,随着大鼠年龄的增长,Str各浓度对各年龄段正常对照大鼠心肌细胞钙瞬变的增强作用均未改变。而在1k1c模型大鼠和SHR,Str各浓度增强其心肌细胞钙瞬变幅度的作用仅呈减弱趋势。(4)结果显示,在6月龄时,正常对照大鼠、1k1c高血压模型大鼠和SHR心肌细胞的收缩幅度-荧光比率曲线均无明显变化,仅1k1c高血压模型大鼠心肌细胞的收缩幅度-荧光比率曲线轻微右移,但并无显著性差别。在12月龄时,正常对照大鼠心肌细胞的收缩幅度-荧光比率曲线无明显改变,而1k1c高血压模型大鼠和SHR心肌细胞的收缩幅度-荧光比率曲线明显左移,说明Str对12月龄1k1c高血压模型大鼠和SHR心肌细胞均具有钙增敏作用。在18月龄时,正常对照大鼠心肌细胞的收缩幅度-荧光比率曲线无明显改变,而1k1c高血压模型大鼠和SHR心肌细胞的收缩幅度-荧光比率曲线明显左移,说明Str对18月龄1k1c高血压模型大鼠和SHR心肌细胞均具有钙增敏作用。小结: Na+,K+-ATP酶的特异性抑制剂毒毛旋花子苷原可明显增强高血压大鼠心肌细胞收缩性和钙瞬变,也能显著提高高血压大鼠心肌细胞对钙的敏感性和细胞内Na+浓度,且其增强心肌细胞收缩幅度和钙瞬变作用呈年龄依赖性减弱,提示高血压的发展可显著降低心肌细胞对Str的敏感性。第三部分高血压对大鼠心肌细胞Na泵活性及其α亚基表达的影响目的:检测高血压大鼠心肌细胞Na+, K+ - ATP酶活性和其三个α亚基的表达,探讨Na+, K+ - ATP在高血压影响大鼠心肌细胞收缩性过程中的可能作用及机制。方法:选用4个月龄SD大鼠制作1k1c高血压大鼠模型,并于6月龄(即手术后2个月)、12月龄和18月龄时用于实验研究,SHR及对照组大鼠也选购相应月龄的动物进行匹配。用酶解法急性分离6、12和18月龄1k1c高血压大鼠和SHR的左心室心肌细胞,分别用于提取心肌细胞膜和膜蛋白,用无机磷法测定高血压对心肌细胞膜Na+, K+ - ATP酶活性的影响,用Western blot检测高血压对心肌细胞Na+, K+-ATP酶α1、α2及α3亚基蛋白表达的改变。结果:(1)对Na+,K+-ATP酶活性的测定结果显示:在6月龄时,1k1c高血压大鼠和SHR心肌细胞Na+,K+-ATP酶活性均明显高于同龄对照组大鼠(P<0.05),在12、18月龄时,1k1c模型大鼠和SHR心肌细胞的Na+,K+-ATP酶活性均显著低于同龄对照组大鼠(P<0.05或0.01)。它们的心肌细胞Na+,K+-ATP酶活性,对照组大鼠无论在6月龄、12月龄还是在18月龄时,均无明显差别;而1k1c高血压大鼠和SHR在12、18月龄时,它们的心肌细胞Na+,K+-ATP酶活性均显著低于它们各自在6月龄时的测定值(P<0.01)。这些结果提示,大鼠高血压的发展与其心肌细胞Na+,K+-ATP酶的功能改变密切相关。(2)Western blot检测结果表明,在6月龄时,1k1c高血压大鼠心肌细胞Na+,K+-ATP酶α1、α2和α3亚基表达均无明显变化(p>0.05),但SHR心肌细胞Na+,K+-ATP酶的三个α亚基表达均比同期对照明显增加(P<0.05或0.01)。与同龄对照组大鼠相比,在12月龄时,1k1c高血压大鼠心肌细胞α1亚基和α2亚基的表达无明显改变(p>0.05),仅α3亚基的表达明显增加(P<0.01);而SHR除α2亚基表达无明显改变外,其心肌细胞Na+,K+-ATP酶α1和α3亚基表达均显著高于同龄对照大鼠(P<0.05或0.01)。在18月龄时,1k1c高血压大鼠心肌细胞α1亚基表达较同龄对照大鼠明显减少(P<0.01),α2亚基表达无明显改变(p>0.05),而α3亚基表达则比同龄对照大鼠显著增强(P<0.01),而SHR心肌细胞Na+,K+-ATP酶α1和α2亚基表达较同龄对照大鼠明显减少,α3亚基表达则显著升高(P<0.05或0.01)。随着动物年龄从6月龄到18月龄的增长,对照组大鼠心肌细胞的Na+,K+-ATP酶α1、α2和α3亚基表达均无明显变化(p>0.05);1k1c高血压大鼠心肌细胞Na+,K+-ATP酶α1亚基表达在18月龄时显著降低(P<0.01),α2亚基的表达无明显改变(p>0.05),但α3亚基的表达呈年龄依赖性增加(P<0.01);SHR心肌细胞Na+,K+-ATP酶α1和α2亚基表达呈年龄依赖性减少(P<0.05),而α3亚基表达则呈年龄依赖性增加(P<0.01)。这些研究结果提示,大鼠高血压的发展主要与其心肌细胞Na+,K+-ATP酶α1和α2亚基表达减少和α3亚基表达增加有关。小结:随着高血压的发展,大鼠心肌细胞膜Na+, K+ - ATP酶的活性先升高后减弱。在高血压早期,大鼠心肌细胞Na+,K+-ATP酶α1、α2、α3亚基的表达均增强;而SHR发展到12月龄和1k1c高血压大鼠发展到18月龄时,均表现为心肌细胞Na+,K+-ATP酶α1和α2亚基表达逐渐减少和α3亚基的表达逐渐增加。这些结果提示,大鼠心肌细胞Na+,K+-ATP酶在高血压的发生与发展过程中有着重要作用。结论1.高血压的发生和发展是个渐进的过程,随时间的延长,两种高血压大鼠的收缩压升高、心肌肥厚和钙瞬变的程度逐渐增大,但其收缩力和钙敏感性却逐渐降低,表明心肌细胞收缩性大小由心肌细胞的钙瞬变和钙敏感性共同决定。2.随着大鼠年龄的增长,Na+,K+-ATP酶的特异性抑制剂毒毛旋花子苷原(Str)各浓度增强正常大鼠心肌细胞收缩幅度的作用不变,而在高血压模型大鼠,Str各浓度增强其心肌细胞收缩幅度的作用则呈时间依赖性减弱,表明高血压的发展可降低心肌细胞对Str的敏感性,提示高血压所致的心肌细胞对Str敏感性的改变,可能是通过影响Na+,K+-ATP酶功能介导的,推测与高血压的发展逐步降低了心肌Na+,K+-ATP酶对Str的亲和力有关。3.随着高血压的发展,两种高血压大鼠心肌细胞膜Na+, K+ - ATP酶的活性先升高后减弱,与Na+,K+-ATP酶α1和α2亚基的表达先增加后降低变相一致;但其α3亚基的表达则呈时间依赖性增加。这些结果提示,大鼠心肌细胞Na+,K+-ATP酶在高血压的发生与发展过程中有着重要作用。更多还原

【Abstract】 Hypertension is a main risk factor for human with high attack rate, mutilation and mortality rate. The influence of hypertension on heart includes disfunction of contraction and diastolic, ventricular hypertrophy, cardiac failure and coronary artery disease. The change of heart function is a gradual course with the disfunction of contraction and diastole at earlier stage of hypertension and the left ventricular hypertrophy and cardiac failure with increasing systolic blood pressure (SBP) and diastolic pressure (DP). Generally, Ca2+ is a key factor for muscle contraction and diastole. The disfunction of calcium metabolism and signal transduction system are closely related to the change of contraction and diastole, myocardial hypertrophy and cardiac ventricle reconstitution. But the physiological mechanism is not knownNa+, K+-ATPase in the myocyte mainly regulates the active transport of Na+ and K+ across membrane, and also regulate Ca2+ indirectly by Na+/Ca2+ exchange. The expression of Na+, K+-ATPaseα-isoform is impacted by hypertension, the level of hormone, myocardial hypertrophy and cardiac failure. The present study, carried out mostly on the change of the activity of Na+, K+-ATPase, the inotropy of myocyte, Ca2+ and Na+, the expression ofαisoform of Na+, K+-ATPase by two different models of the hypertensive rats, is to explore the role of the Na+, K+-ATPase in the alteration of myocyte contractibility induced by hypertension.I. Effects of hypertension on the contractility and calcium transient in the rat ventricular myocytesObjective : To approach the effects and possible mechanisms of hypertension on the contractility of myocytes by detemining the systolic and diastolic function, the Na+ concentration, Ca2+ transient and the sensitivity of myocytes to Ca2+ in different phases of hypertension development .Methods : Choosing 4-month-old rats were used to prepare the hypertensive models of 1k1c rat (the rat with one kidney and the constriction of one renal artery). These rats were used in the research at 6 months old (2 months after operating), 12 months old and 18 months old. The SHR and control rats were chosen by corresponding age. The caudilis arterial pressure were measured by the rat caudae, the index of hemodynamics on hypertensive rats were monitored by quantified system analysis, left ventricular mass index were determined by wet weight, the left ventricle myocyte were acutely isolated by enzymolysis method, and the changes of the systolic and diastolic function, the rest Na+ concentration, Ca2+ transient and the sensitivity of myocytes to Ca2+ were observed by video-motion edge-detection system. In the meantime, the distinction of above-mentioned indices of 6-, 12- and 18-month-old rats in different hypertensive models were compared. The rest Na+ concentration in myocyte were observed by video-motion edge-detection system.Results:(1)Hypertension in 1k1c rats was induced at two months after operating, i.e. at 6 months old. The achievement ratio was 73%. The SBP of 1k1c rats and SHR at 6, 12 and 18 months old were higher than those of control rats with the same age, and the SBP of SHR were higher than that of 1k1c rats with the same age(P<0.05 or 0.01). The elevating time of the SHR systolic pressure (at 6 months old) was earlier than that of 1k1c rats (at 12 months old).(2)The left ventricular mass index of 1k1c rat and SHR at 6, 12 and 18 months old were higher than those of control rats with the same age, indicating the formation of myocardial hypertrophy. Furthermore, The degree of myocardial hypertrophy in SHR were obviously greater than those in 1k1c rats (P<0.01). Nevertheless, the obvious accrescent time of myocardial hypertrophy in SHR (at 12 months old) was earlier than those in 1k1c rats (at 18 months old). It is indicated that the process and degree of myocardial hypertrophy in rats induced by hypertension is highly correlated with the elevation of systolic blood pressure.(3)In both hypertensive models, LVEDP, dp/dt and -dp/dt increased significantly in 6, 12, and 18-month old rats, compared with control rats with the same age. But dp/dt and -dp/dt in 1k1c rats at 12 months old were higher than that at 6 months old, while that at 18 months old is lower than at 12 months old. And the -dp/dt in SHR at 12 and 18 months old were lower than that at 6-month-old ones. Above trends indicate that the maximal values of compensation of the diastolic and contractile function were achieved in 12-month-old 1k1c rats and in 6-month-old SHR.(4)Dep v and Peak of rat myocytes from both models were higher than those from control rats with the same age. Ret v of rat myocytes from 1k1c rats exceed that of control rats with the same age, but Ret v of SHR is lower than that of rat myocytes from control rats with the same age. This effect indicates that in the earlier period of hypertension, the diastolic and contractile function of myocytes in 1k1c rats all enhanced. But in SHR rats, only contractile function enhanced, and the diastolic function started to decrease at 6 months old. The above results were same as those observed in hemodynamic research.(5)DCa of rat myocytes from two models at 6-months old were not obviously different from those from control rats, but was lower at 12 and18 months old than that of control group at the same age and that of corresponding model group at 6 months old. These results indicated that 6-month-old 1k1c rat and SHR occupy the compensation stage and the main change is diastolic Ca2+. ?Ca2+ in 1k1c rats at 6-month old have no obvious discrepancy with the same age control group, those of SHR were higher than those in control rats with the same age, those of both 1k1c rat and SHR in 12 and18 month old exceeded those in 6-months group and in control group with the same age. These results indicated that ?Ca2+in 1k1c rats and SHR increased with in the development of hypertensive course, which was likely to be one of the main compensative modes of myocardial contractility.Judged from above results, it is obviously that Ca2+ transient in rat myocytes from two models at 6-months old was not significantly different from the control group, the myocardial contractility of myocytes in 1k1c rat was higher than that in control rats with the same age and lower than that in SHR. Both of Ca2+ transient in myocytes from 1k1c rat and SHR were higher than that in control rats with the same age, but myocardial contractility decreased gradually.(6)Resting Na+ concentration in rat myocytes of two models at 6-months old was not obviously different from the control group(p>0.05), those at 12 and 18-months old were higher than those in control rats with the same age and at 6-month-old model group (P<0.05 or 0.01). The resting Na+ concentration in myocytes of control rats had no change from 6-month old to 18-month old, but the [Na+]i in rat myocytes from two models increased gradually with the age (P<0.01). It was indicated that the level of [Na+]i was increased by hypertension.(7)The contractile amplitude-fluorescence intensity curve in 6-months old 1k1c rats shift left comparing with that in control rats with the same age, which indicated that the sensitivity of myocyte to Ca2+ enhanced. The sensitivity in both 12 and 18-month-old rats decreased. The sensitivity in 6-month-old rats was significantly lower than that in control rats with the same age, and decreased with the prolongation of hypertension duration. The depressed sensitivity of myocyte to Ca2+ induced by hypertension could be one of causes in gradual decline of myocyte contractility by the increasing ofΔCa2+ in 12 and 18-month-old 1k1c rats and SHR.Conclusion: The development of hypertension is a gradual course. The degree of SBP elevation and myocardial hypertrophy increased gradually with temporal prolongation. The degrees of them in 1k1c rats were significantly lower than those in SHR. The amplitudes of Ca2+ transient did not change in 1k1c rats and SHR at two 6-month-old, but enhanced at 12 and 18-month old. The myocardial hypertrophy and sensitivity of myocte to Ca2+ in 1k1c rats all increased at 6-months old but decreased at 12-months old, while both in SHR all started to decrease at 6 months old.II. Effects of cardiac glycosides on the contractility and Ca2+ transient of myocytes from hypertensive ratsObjective:To explore the relationship between the Na+-K+-ATPase and hypertension and the role of the Na+-K+-ATPase in the process of hypertension development by observing the effects of strophanthidin (Str), a specific blocker of Na+,K+-ATPase, on the contractility and Ca2+ transient of myocyte, the Na+ concentration in myocytes, and the sensitivity of myocytes to Ca2+ at different stages of hypertension development in 1k1c rats and SHR.Methods: Choosing 4-month-old rats were used to prepare hypertensive models of 1k1c rat. These rats were used in the research at 6 months old (2 months after operating), 12 months old and 18 months old. The SHR and control rats were chosen by corresponding age. The left ventricle myocyte were acutely isolated by enzymolysis method, and the effects of Str on the contractility and Ca2+ transient of myocyte, the Na+ concentration in myocytes, and the sensitivity of myocytes to Ca2+ at different stages of hypertension development in 1k1c rats and SHR were observed by video-motion edge-detection system.Results:(1)Str 1, 10, 50 and 100μmol?L-1 increased concentration-dependently the amplitude of myocyte contraction in all rats, either normal control rats, 1k1c rat, or SHR, at 6, 12 and 18 months old (P<0.05). The augmented amplitudes of myocyte contraction by Str in 1k1c rats and SHR at 6 months old were obviously higher than those in control rats with the same age. Meanwhile, the enlarged effects of Str on the amplitude of myocyte contraction in 1k1c rats were higher than those in SHR. With the growth of rats, the augmented amplitudes of myocyte contraction by different concentrations of Str did not altered in the control rats, but were decreased concentration-dependently in 1k1c rat and SHR, which indicated that the sensitivity of myocytes to Str was obviously decreased with the development of hypertension.(2)Perfused with 1mM Str, resting Na+ concentration in rat myocytes from two hypertensive models were not obviously different from the control rats at 6-months old (p>0.05), but higher at 12 and 18-months old than those in the corresponding control rats and two hypertensive model rats at 6-month-old (P<0.01), and reached to the maximal value respectively at 12-months old. The resting Na+ concentration at 12 and 18-months old were higher than that of corresponding SHR, and step up gradually with development of hypertension.(3)Str 1、10、50 and 100μmol?L-1 concentration-dependently increased the amplitudes of myocyte Ca2+ transientin in all groups at 6, 12 and 18 months old (P < 0.05). The augmented amplitude of 6, 12 and 18-month-old 1k1c rats and SHR were obviously higher than that of control group, but there were no difference between 1k1c rat and SHR. With the growth of rats, different str concentration had the same effect on the control group, but decreased concentration-dependently on 1k1c rat and SHR.(4)In 6-month-old rats, the contraction amplitude-fluorescence intensity curve of all groups didn’t change, only that of 1k1c rat shifted right slightly, but there was no obvious difference.In 12-month-old rats, the contraction amplitude-fluorescence intensity curves of both in 1k1c rats and SHR shifted left, while that of control group didn’t change, which indicated that Str could enhance the sensitivity of myocyte to Ca2+ in 1k1c rat and SHR.In 18-month-old rats, the contraction amplitude-fluorescence intensity curves of both 1k1c rat and SHR shifted left, while that of control group didn’t change, which indicated that Str could enhance the sensitivity of myocyte to Ca2+ in 1k1c rat and SHR.Conclusion: Str which is a specific Na+,K+-ATPase blocker not only enhances the contractility and Ca2+ transient of myocytes from hypertensive rats, but also increases the sensitivity of myocyte to Ca2+ and the Na+ concentration in myocytes. Furthermore, the effects of Str enhancing the contractility and Ca2+ transient are decreased in age-dependent manner, which indicate the sensitivity of myocytes to Str obviously decreased following the develpment of hypertension.III The effects of hypertension on the activity of Na pump and expressions ofαisoform in rat myoctyteObjectives:To detect the activity of Na+, K+-ATPase and the expressions of its threeαisoforms in hypertensive rats and study the role and underlying mechanism of Na+, K+-ATPase in the alteration of rat myocyte contractility induced by hypertension.Methods:Choosing 4-month-old rats were used to prepare hypertensive models of 1k1c rat. These rats were used in the research at 6 months old (2 months after operating), 12 months old and 18 months old. The SHR and control rats were chosen by corresponding age. The left ventricle myocyte were acutely isolated by enzymolysis method, Na+, K+-ATPase activity was measured through spectrophotometry method,The expression and distribution of the Na+, K+-ATPaseαisofroms were determined with Western blot.Results:(1) At 6 months old, the activity of Na+,K+-ATPase in mycoytes from 1k1c hypertensive rats was significantly higher (P<0.05) than those from SHR and control rats. At 12 and 18 months old, the activities of Na+,K+-ATPase in mycoytes from both 1k1c and SHR rats were significantly lower (P<0.05 or 0.01) than that from control rats. In control rats, there is no significant difference in activity of Na+,K+-ATPase among 6, 12, and 18 months old. While in 1k1c and SHR rats, the activity of Na+,K+-ATPase at 12 and 18 months old is significantly lower than those at 6 months old (P<0.01). The above results indicates that there changes of Na+,K+-ATPase function are highly correlated with development of hypertension.(2) At 6 months old, the expressions ofα1,α2 andα3 isoforms of Na+,K+ -ATPase in 1k1c rats did not change significantly (p>0.05), while those in SHR rats increased significantly comparing with the corresponding period control rats (P<0.05 or 0.01).At 12 months old, the expressions ofα1 andα2 isoforms of Na+,K+-ATPase in 1k1c rats did not change significantly (p>0.05), only that ofα3 isoforms increased (P<0.01), while those ofα1 andα3 isoforms in SHR rats increased significantly comparing with the corresponding period control group (P<0.05 or 0.01).At 18 months old, expressions ofα1 isoforms of Na+,K+-ATPase in 1k1c rats decreased, that ofα2 isoforms did not change significantly (p>0.05), and that ofα3 isoforms increased (P<0.01), while those ofα1 andα2 isoforms in SHR rats decreased significantly andα3 isoforms increased comparing with the corresponding period control group (P<0.05 or 0.01).Going up from six months to 18 months, expressions ofα1、α2 andα3 isoforms of control group did not change significantly (p>0.05). The expression ofα3 isoforms in 1k1c rats at 18-month old decreased (P<0.01),α2 isoforms had no change (p>0.05)andα3 isoforms increased age-dependently(P<0.01). The expression ofα1 andα2isoforms of SHR decreased and that ofα3isoforms increased age-dependently(P<0.01). The above result implied that the development of hypertension is related with the decreasingα1 andα2 isoforms and increasingα3 isoforms.Conclusion:With development of hypertension, the activity of Na+, K+-ATPase increases but subsequently decreases. In the earlier period of hypertension, the expressions ofα1,α2 andα3 isoforms all enhance. In 12-month-old SHR and 18-month-old 1k1c rats, the expressions ofα1 andα2 isoform all decrease and the expression ofα3 isoform increases gradually. These results imply that Na+,K+-ATPase play an important role in the development of hypertension. Summary1.The development of hypertension is a gradual course. The elevated degrees of SBP and myocardial hypertrophy and the amplitude of Ca2+ transient are gradually increased, but the contractility of myocyte and sensitivity of myocyte to Ca2+ are gradually decreased, with the development of hypertension in SHR and 1k1c rats, indicating that both amplitude of Ca2+ transient and the sensitivity of myocyte to Ca2+ are responsible to the contractility of myocyte.2.With the development of hypertension, the elevated amplitude of myocyte contraction induced by strophanthidin, a specific Na+,K+-ATPase blocker, is decreased in ventricular myocytes from SHR and 1k1c rats in an age-dependent manner, but does not change in those from normal control rats. These results indicate that the development of hypertension may decrease the sensitivity of myocytes to strophanthidin by lowering the affinity of Na+, K+-ATPase to strophanthidin.3.With development of hypertension, the activity of Na+, K+-ATPase increases but subsequently decreases, which is consistent with the expressions ofα1 andα2 isoforms. But the expression ofα3 isoform increases gradually. These results imply that Na+,K+-ATPase play an important role in the development of hypertension.更多还原