钠钾泵和钠氢交换体对心衰小鼠心肌细胞收缩力和钙瞬变的影响
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摘要
心力衰竭是一种复杂的临床症状群,是高血压、缺血性心肌病、心瓣膜病等众多心脏病的终末阶段,其发病率高、预后差,是世界性日趋严重的危害健康的主要问题。
强心苷(Cardiotonic steroid,Cs)用于治疗慢性充血性心力衰竭(Congestive heart failure,CHF)已有200多年历史,但其加强心肌收缩力的作用机制却一直是人们争论的焦点。一般认为:Cs治疗心衰是因为其抑制衰竭心肌细胞膜上的Na+, K+-ATP酶(NKA),使细胞内Na+增加,通过Na+/Ca2+交换,引起细胞内Ca2+的升高,并促进肌浆网对钙的摄取及随后的钙释放,使心肌收缩力随之增加。
钠氢交换体(Na+/H+ exchanger,NHE)作为细胞膜上的一种交换糖蛋白转运体,其功能主要是调节细胞内pH值、细胞容积以及对多种不同激素和丝裂原作出应答。细胞内酸化变构激活NHE,以1:1的比例将胞外的钠离子与胞内的氢离子进行交换。NHE的活化导致Na+内流,依次活化多种亚型PKC,最终改变基因表达和蛋白合成,促进肥厚的发生。最近Baartscheer报道,诱导压力/容积超负荷而引发家兔心肌肥厚并具有心衰体征一个月后,连续饲服NHE-1抑制剂两个月,可复原已形成的心肌肥厚、心力衰竭和离子、电生理改变。
Saini HK等报道,NHE在哇巴因诱导的心肌细胞胞内钙离子浓度([Ca2+]i)的增加中发挥着重要作用。然而,NHE抑制剂甲基-N-异丁基氨氯吡咪(MIA)在哇巴因存在情况下,对离体缺血再灌注心脏的正性肌力作用与其对静息或KCl去极化的心肌细胞的[Ca2+]i作用不一致,因此很难解释NKA与NHE之间的相互作用及两者在病理情况下发挥作用的机制。
本实验选用小鼠为研究对象,建立小鼠CHF模型,进一步观察在哇巴因存在或不存在情况下NHE抑制剂对小鼠正常(NC)和心衰心肌细胞(CHFC)的肌力作用和[Ca2+]i的影响,以探讨抑制NHE作为治疗心衰新方案的可能性。
目的:通过建立小鼠慢性充血性心力衰竭动物模型,同步检测MIA在哇巴因(ouabian)存在下对慢性心衰所致小鼠心肌细胞收缩及钙瞬变的影响,检测CHF小鼠心肌细胞膜NKAα1、α2亚基和NHE-1蛋白表达的改变,以探讨NKA和NHE-1影响慢性心衰小鼠心肌细胞肌力作用的机制。
方法:采用主动脉弓缩窄法建立小鼠CHF模型;以酶解法急性分离其左室心肌细胞,然后采用可视化单细胞动缘探测系统(Video-based motion edge-detection system)同步检测不同浓度OUA对单个小鼠心肌细胞舒张、收缩功能及钙瞬变的变化,并比较同一浓度OUA对正常和心衰小鼠的心肌细胞的变化。同步检测在OUA存在下,MIA对单个小鼠正常和衰竭心脏心肌细胞收缩力和钙瞬变的影响,并与单独使用MIA的作用进行比较。采用Western Blot方法检测小鼠NKAα1、α2亚基和NHE-1蛋白表达的改变。
结果:⑴OUA对小鼠NC和CHFC收缩力和细胞内钙的作用。若将给药前细胞收缩幅度、钙瞬变幅度及胞浆基础钙离子浓度标准化为100%,则0.5μM,1μM,5μM,10μM,50μM,100μM,500μM,1 mM的OUA使小鼠NC收缩幅度明显分别增大至(124.60±5.02)%、(133.02±5.65)%、(148.42±6.63)%、(174.14±6.29)%、(204.64±3.66)%、(228.50±10.87)%、(286.42±27.57)%和(288.80±23.82)%(p<0.01)。为探讨OUA对心肌细胞正性变力作用与Ca2+的关系,本研究同步检测了OUA对钙瞬变的影响。上述浓度的OUA可以使小鼠NC钙瞬变幅度分别增加为(119.26±4.30)%、(125.43±5.64)%、(126.10±6.77)%、(133.92±10.26)%、(139.72±6.43)%、(152.90±8.10)%、(157.38±11.53)%和(156.39±12.24)%(p<0.01);5μM,10μM,50μM,100μM,500μM,1 mM可以使小鼠NC胞浆基础钙离子浓度分别增加为(107.33±2.18)%、(110.82±2.32)%、(111.10±2.81)%、(112.24±2.56)%、(115.72±4.63)%和(116.70±3.80)%(p<0.01),但0.5μM,1μM OUA对NC胞浆基础钙离子浓度没有明显影响,分别为(103.64±2.26)%和(102.47±3.29)%(p>0.05);其中0.1μM OUA对细胞收缩幅度、钙瞬变幅度以及胞浆基础钙离子浓度均没有影响,分别为(104.59±2.35)%、(101.41±1.82)%和(99.62±2.23)%(p>0.05),而500μM和1 mM的OUA使NC很快出现毒性反应,表现为不规律收缩,细胞长度变短,甚至变为球形。而50μM的OUA使小鼠CHFC收缩幅度、钙瞬变幅度以及胞浆基础钙离子浓度分别明显增大至(351.71±90.6)%、(164.77±6.87)%和(117.67±7.70)%(p<0.01),较50μM OUA增加NC收缩幅度、钙瞬变幅度以及胞浆基础钙离子浓度的作用显著为强(p<0.01)。
⑵MIA对小鼠NC和CHFC收缩力和细胞内钙的作用。灌流0.1和1μM的MIA 5 min后,小鼠NC收缩幅度分别降低至(78.82±5.44)%和(46.93±7.36)%(p<0.01);钙瞬变幅度分别降低至(73.41±8.73)%和(49.59±4.01)%(p<0.01);但对胞浆基础钙离子浓度没有明显影响(p>0.05)。灌流0.1和1μM的MIA 15 min后,小鼠NC收缩幅度明显分别增加至(142.91±12.64)%和(204.36±27.22)%(p<0.01);钙瞬变幅度分别增加为(117.51±2.68)%和(132.51±4.30)%(p<0.01);胞浆基础钙离子浓度分别增加为(115.22±1.90)%和(126.93±3.97)%(p<0.01)。当正性肌力作用达到最大后,可使NC很快出现毒性反应,表现为细胞长度缩短,甚至变为球形。但是,对于小鼠CHFC,灌流0.1和1μM的MIA 5 min后,收缩幅度可分别增加至(117.01±1.11)%和(130.08±3.61)%(p<0.01);钙瞬变幅度分别增加为(104.64±2.18)%(p>0.05)和(113.38±2.61)%(p<0.01),但均对CHFC胞浆基础钙离子浓度没有明显影响,分别为(101.13±4.90)%和(104.21±2.49)%(p>0.05)。灌流0.1和1μM的MIA 15 min后,小鼠CHFC收缩幅度(104.55±3.89)%和(101.83±2.62)%、钙瞬变幅度(100.11±4.59)%和(97.27±3.72)%及胞浆基础钙离子浓度(103.63±6.91)%和(99.01±4.82)%均没有显著变化(p>0.05)。
⑶在低浓度哇巴因存在下MIA对小鼠NC和CHFC收缩力和细胞内钙的作用。与仅用1μM MIA灌流时相比,小鼠NC用10 nM OUA预先孵育10 min后,再灌流10 nM OUA +1μM MIA,使NC收缩力由原来5 min时降低为(46.93±7.36)%变为降低至(78.49±9.19)%(p<0.01),由原来15 min时增加至(204.36±27.22)%变为增加至(135.42±10.74)%(p<0.01);使NC钙瞬变幅度由原来5 min时降低为(49.59±4.01)%变为(78.51±10.83)%(p<0.01),由原来15 min时增加至(132.51±4.30)%变为增加至(108.64±2.68)%(p<0.01);5 min时对NC胞浆基础钙离子浓度没有明显影响,但15 min时由原来增加(126.93±3.97)%(p<0.01)变为无明显增加(103.11±2.07)%(p>0.05)。然而,小鼠CHFC用10 nM OUA预先孵育10 min后,再灌注10 nM OUA + 1μM MIA,与仅用1μM MIA时相比,OUA+ MIA可以使其收缩力由原来5 min时增加为(130.08±3.61)%变为增加至(183.14±23.77)%(p<0.01),由原来15 min时没有显著性变化(101.83±2.62)%(p>0.05)变为增加至(180.56±18.31)%(p<0.01);使CHFC钙瞬变幅度由原来5 min时增加至(113.38±2.61)%变为增加(155.37±4.89)%(p<0.01),由原来15 min时没有显著性变化(97.27±3.72)%(p>0.05)变为增加至(158.48±8.62)%(p<0.01);使CHFC胞浆基础钙离子浓度由原来5 min时(104.21±2.49)%和15 min时(99.01±4.82)%没有明显影响(p>0.05)分别变为增加为(115.19±1.98)%和(114.37±2.05)%(p<0.01)。
⑷小鼠NC和CHFC的NKAα1、α2亚基与NHE-1在蛋白水平的表达。与NC相比,CHFC的NKAα1、α2亚基以及NHE-1在蛋白水平的表达量均显著下降(p<0.01)。
结论:哇巴因能够浓度依赖性增加小鼠心肌细胞的基础钙离子浓度、钙瞬变幅度以及收缩力,且对衰竭心脏心肌细胞的作用较对正常心脏心肌细胞的作用显著为强,其原因可能与心衰后小鼠心肌细胞NKAα1和、α2亚基蛋白表达均有所降低有关。NHE-1抑制剂MIA对正常小鼠心肌细胞的收缩力和钙瞬变先减弱后增强,而对心衰小鼠心肌细胞则仅有短暂的正性肌力作用。低浓度哇巴因(10 nM)能够部分抵消MIA对正常小鼠心肌细胞的收缩和钙瞬变作用,但却显著增强了MIA对心衰小鼠心肌细胞的正性肌力作用和钙瞬变作用,提示NHE与NKA在调节心肌细胞收缩过程中存在密切的功能联系,且此调节取决于心肌细胞的机能状况,在正常心肌细胞二者呈协同作用,在心肌细胞发生心衰病理变化后,二者呈拮抗作用。另外,本研究也进一步提示,NHE-1抑制剂可作为强心苷类药物治疗心衰的辅助药具有研究开发前景。
Heart failure (HF) is a series of multiplicity clinical symptom, and the end stage of many cardiac disease, such as hypertension, ischemic cardiomyopathy, valvular disease and so on. The incidence and prevalence of heart failure (HF) is high and the prognosis of HF is rather poor. It is one of the most harmful problems which is increasingly severe.
Cardiotonic steroid (Cs) had been used for the congestive heart failure (CHF) for more than 200 years. But the mechanism of its positive inotropic effect is still controversial. It is generally believed that Cs produce positive inotropic actions in failure heart via inhibition of Na+, K+-ATPase (NKA) activities which leads to an increase in intracellular Na+ concentration ([Na+]i), and in turn raises intracellular Ca2+ concentration ([Ca2+]i) via Na+/Ca2+ exchange, thus increasing the uptake and subsequent release of Ca2+ by the sarcoplasmic reticulum (SR).
Na+/H+ exchanger (NHE) as a plasma membrane exchange glycoprotein transporter that functions in intracellular pH regulation, cell volume regulation, and cellular response to many different hormones and mitogens. Following allosteric activation by intracellular acidification, NHE exchanges extra- cellular sodium for intracellular hydrogen with stoichiometry of 1:1. Na+ influx via activation of Na+/H+ exchanger reactivates PKC in myocytes, which is one of the important signaling molecules in the development of the cardiac hypertrophic response. Recently, Baartscheer et al reported that two months of dietary treatment with the NHE-1 inhibitor cariporide caused regression of hypertrophy, heart failure and ionic and electrophysiological remodelling.
Saini HK et al reported NHE play a critical role in the ouabain-induced [Ca2+]i increase in cardiomyocytes. However, inotropic effect of methyl-n- isobutyl amiloride (MIA), a NHE inhibitor, in isolated ischemia-perfusion hearts was not consistent with its effect on [Ca2+]i in quiescent or KCl- depolarized cardiomyocytes exposed to ouabain. Therefore, it is hard to explain the interacton between NKA and NHE, and effect of them on the pathological condition. Thus mice were chosen as test animals. The mouse CHF models were developed. The present study is to observe the effects of the NHE on inotropic effects and change of [Ca2+]i of normal and CHF ventricular myocytes in the presence or obsence of ouabain in order to investigate inhibition of NHE as a potentiality treatment of CHF.
Objective: To detect the effects of NHE-1 on the contractile and calcium transient in the ventricular myocytes from heart failure mice through establishing CHF animal model and the effects of CHF on the expression of NKAα1 andα2 isoform and NHE-1 in protein level to identify the mechanism of the inotropic effect of NHE-1 and NKA in isolated mouse ventricular myocytes.
Methods: Mice CHF model was made by constricting aortic arch. Left ventricular myocytes were enzymatically isolated. Then the contractile and calcium transient of a single myocyte from normal and CHF mice were assessed by a video-based motion edge-detection system simultaneously. At the same time, effects of the same concentration of ouabian on inotropic effects and calcium transient in CHF mouse ventricular myocytes were compared with those in normal mouse ventricular myocytes. Then the experiments were repeated when the myocytes were preincubated with 10nM ouabain. The effects of MIA with ouabain on contractility and calcium transient were recorded and the results were compared with those of MIA-only. Then the expressions of NKAα1 andα2 isoform and NHE-1 in protein level were detected with Western blot.
Results:⑴The effects of ouabain on the contractility and calcium transient of NC or CHFC from mice. The extent of cell shortening, ampitude of calcium transient and basal level of [Ca2+]i was normalized using the value before ouabain perfusion as 100%. In NC, OUA 0.5μM, 1μM, 5μM, 10μM, 50μM, 100μM, 500μM, and 1 mM increased the extent of cell shortening by (124.60±5.02)%, (133.02±5.65)%, (148.42±6.63)%, (174.14±6.29)%, (204.64±3.66)%, (228.50±10.87)%, (286.42±27.57)%, and (288.80±23.82)% (p< 0.01); The concentration of OUA above increased the ampitude of calcium transient by (119.26±4.30)%, (125.43±5.64)%, (126.10±6.77)%, (133.92±10.26)%, (139.72±6.43)%, (152.90±8.10)%, (157.38±11.53)%, and (156.39±12.24)% (p<0.01); OUA 5μM, 10μM, 50μM, 100μM, 500μM, and 1 mM increased basal level of [Ca2+]i by (107.33±2.18)%, (110.82±2.32)%, (111.10±2.81)%, (112.24±2.56)%, (115.72±4.63)%, and (116.70±3.80)% (p< 0.01), while OUA 0.5μM and, 1μM had no effect on basal level of [Ca2+]i, which are (103.64±2.26)% and (102.47±3.29)% (p>0.05), respectively; 0.1μM OUA had no effect on the extent of cell shortening, ampitude of calcium transient and basal level of [Ca2+]i, which are (104.59±2.35)%, (101.41±1.82)% and (99.62±2.23)% (p>0.05),respectively; and OUA 500μM and 1 mM caused the cell irregular contraction, shorter length even became to“round-up”. In CHFC, OUA 50μM increased extent of cell shortening, ampitude of calcium transient and basal level of [Ca2+]i by (351.71±90.6)%, (164.77±6.87) % and (117.67±7.70)% (p<0.01); At the same concentration, effect of Str was more potent in CHFC than in NC.
⑵The effects of MIA on the contractility and calcium transient of NC or CHFC from mice. In NC, after 5 min perfusion MIA 0.1 and 1μM decreased the extent of cell shortening by (78.82±5.44)% and (46.93±7.36)% (p<0.01); and decreased the ampitude of calcium transient by (73.41±8.73)% and (49.59±4.01)% (p<0.01); but had no effect on basal level of [Ca2+]i (p>0.05). After 15 min perfusion MIA 0.1 and 1μM increased the extent of cell shortening by (142.91±12.64)% and (204.36±27.22)% (p<0.01), increased the ampitude of calcium transient by (117.51±2.68)% and (132.51±4.30)% (p<0.01), and increased basal level of [Ca2+]i by (115.22±1.90)% and (126.93±3.97)% (p<0.01). When the positive inotropic effect reach the maximal value, MIA caused the shorter length even became to“round-up”. In CHFC, however, after 5min perfusion MIA 0.1 and 1μM increased the extent of cell shortening by (117.01±1.11)% and (130.08±3.61)% (p<0.01), increased the ampitude of calcium transient by (104.64±2.18)% (p>0.05) and (113.38±2.61)% (p<0.01), and had no effect on basal level of [Ca2+]i, (101.13±4.90)% and (104.21±2.49)% (p>0.05). In CHFC, after 15min perfusion MIA 0.1 and 1μM had no effect on the extents of cell shortening, which were (104.55±3.89)% and (101.83±2.62)%, the ampitudes of calcium transient, which were (100.11±4.59)% and (97.27±3.72)%, and basal levels of [Ca2+]i, which were (103.63±6.91)% and (99.01±4.82)% (p>0.05), respectively.
⑶The effects of MIA on the contractility and calcium transient of NC or CHFC from mice in the present of low concentration of ouabain. In NC, after preincubation with 10 nM OUA for 10 min, 10 nM OUA+1μM MIA was perfused. Compared with the effect of MIA in NC, OUA+MIA decreased the extent of cell shortening form (46.93±7.36)% to (78.49±9.19)% (p<0.01) in 5 min perfusion, increased the extent of cell shortening form (204.36±27.22)% to (135.42±10.74)% (p<0.01) in 15 min perfusion; and decreased the ampitude of calcium transient from (49.59±4.01)% to (78.51±10.83)% (p<0.01) in 5 min perfusion, increased the ampitude of calcium transient from (132.51±4.30)% to (108.64±2.68)% (p<0.01) in 15 min perfsion; and had no effect on basal level of [Ca2+]i in 5 min perfusion, while increased basal level of [Ca2+]i from (126.93±3.97)% (p<0.01) to (103.11±2.07)% (p>0.05), which had no significant difference compared with those before perfusion. In CHFC, however, after preincubation with 10 nM OUA for 10 min, 10 nM OUA+1μM MIA was perfused. Compared with the effect of MIA in NC, OUA+MIA increased the extent of cell shortening form (130.08±3.61)% to (183.14±23.77)% (p<0.01) in 5 min perfusion, and from significant difference to increased the extent of cell shortening by (180.56±18.31)% in 15 min perfusion; and increased the ampitude of calcium transient from (113.38±2.61)% to (155.37±4.89)% (p<0.01) in 5 min perfusion, and from no significant difference (97.27±3.72)% (p>0.05) to increased the ampitude of calcium transient by (158.48±8.62)% (p<0.01) in 15 min perfusion; OUA+ MIA increased basal level of [Ca2+]i from no significant difference (104.21± 2.49)% and (99.01±4.82)% (p>0.05) to increased by (115.19±1.98)% and (114.37±2.05)% (p<0.01), respectively, after 5 min and 15 min perfusion.⑷The expression of NKAα1 andα2 isoform and NHE-1 in protein level in ventricular myocytes from mice with CHF. Compared with NC, the protein level of NKAα1 andα2 isoform and NHE-1 were decreased in CHFC (p<0.01).
Conclusions: Ouabain increases the extent of cell shortening, ampitude of calcium transient and basal level of [Ca2+]i. The reason why effects of OUA is more potent in CHFC than in NC may be related to that the more protein level of NKAα1 andα2 isoform are decreased in CHFC than in NC. MIA enhances the contractility and calcium transient followed by a weaken effect; whlie MIA moderately increased the inotropy and calcium transient of CHFC. Low concentration of OUA can partially abolish MIA-induced the changes in NC; however, MIA-induced changes in inotropic effect and [Ca2+]i were augmented by OUA in CHFC. It is prompted that there is a colsed relationship between NHE and NKA on the process of regulating cadiocytes, and the regulation is dependent on the condition of cadiocytes. In NC, NHE and NKA showed synergistic action, while in CHFC, they showed antagonistic effect. In addition, our study also suggest that research and development of the inhibitor of NHE-1 have the prospect of as adjunctive therapy with Cs to treat CHF.
强心苷(Cardiotonic steroid,Cs)用于治疗慢性充血性心力衰竭(Congestive heart failure,CHF)已有200多年历史,但其加强心肌收缩力的作用机制却一直是人们争论的焦点。一般认为:Cs治疗心衰是因为其抑制衰竭心肌细胞膜上的Na+, K+-ATP酶(NKA),使细胞内Na+增加,通过Na+/Ca2+交换,引起细胞内Ca2+的升高,并促进肌浆网对钙的摄取及随后的钙释放,使心肌收缩力随之增加。
钠氢交换体(Na+/H+ exchanger,NHE)作为细胞膜上的一种交换糖蛋白转运体,其功能主要是调节细胞内pH值、细胞容积以及对多种不同激素和丝裂原作出应答。细胞内酸化变构激活NHE,以1:1的比例将胞外的钠离子与胞内的氢离子进行交换。NHE的活化导致Na+内流,依次活化多种亚型PKC,最终改变基因表达和蛋白合成,促进肥厚的发生。最近Baartscheer报道,诱导压力/容积超负荷而引发家兔心肌肥厚并具有心衰体征一个月后,连续饲服NHE-1抑制剂两个月,可复原已形成的心肌肥厚、心力衰竭和离子、电生理改变。
Saini HK等报道,NHE在哇巴因诱导的心肌细胞胞内钙离子浓度([Ca2+]i)的增加中发挥着重要作用。然而,NHE抑制剂甲基-N-异丁基氨氯吡咪(MIA)在哇巴因存在情况下,对离体缺血再灌注心脏的正性肌力作用与其对静息或KCl去极化的心肌细胞的[Ca2+]i作用不一致,因此很难解释NKA与NHE之间的相互作用及两者在病理情况下发挥作用的机制。
本实验选用小鼠为研究对象,建立小鼠CHF模型,进一步观察在哇巴因存在或不存在情况下NHE抑制剂对小鼠正常(NC)和心衰心肌细胞(CHFC)的肌力作用和[Ca2+]i的影响,以探讨抑制NHE作为治疗心衰新方案的可能性。
目的:通过建立小鼠慢性充血性心力衰竭动物模型,同步检测MIA在哇巴因(ouabian)存在下对慢性心衰所致小鼠心肌细胞收缩及钙瞬变的影响,检测CHF小鼠心肌细胞膜NKAα1、α2亚基和NHE-1蛋白表达的改变,以探讨NKA和NHE-1影响慢性心衰小鼠心肌细胞肌力作用的机制。
方法:采用主动脉弓缩窄法建立小鼠CHF模型;以酶解法急性分离其左室心肌细胞,然后采用可视化单细胞动缘探测系统(Video-based motion edge-detection system)同步检测不同浓度OUA对单个小鼠心肌细胞舒张、收缩功能及钙瞬变的变化,并比较同一浓度OUA对正常和心衰小鼠的心肌细胞的变化。同步检测在OUA存在下,MIA对单个小鼠正常和衰竭心脏心肌细胞收缩力和钙瞬变的影响,并与单独使用MIA的作用进行比较。采用Western Blot方法检测小鼠NKAα1、α2亚基和NHE-1蛋白表达的改变。
结果:⑴OUA对小鼠NC和CHFC收缩力和细胞内钙的作用。若将给药前细胞收缩幅度、钙瞬变幅度及胞浆基础钙离子浓度标准化为100%,则0.5μM,1μM,5μM,10μM,50μM,100μM,500μM,1 mM的OUA使小鼠NC收缩幅度明显分别增大至(124.60±5.02)%、(133.02±5.65)%、(148.42±6.63)%、(174.14±6.29)%、(204.64±3.66)%、(228.50±10.87)%、(286.42±27.57)%和(288.80±23.82)%(p<0.01)。为探讨OUA对心肌细胞正性变力作用与Ca2+的关系,本研究同步检测了OUA对钙瞬变的影响。上述浓度的OUA可以使小鼠NC钙瞬变幅度分别增加为(119.26±4.30)%、(125.43±5.64)%、(126.10±6.77)%、(133.92±10.26)%、(139.72±6.43)%、(152.90±8.10)%、(157.38±11.53)%和(156.39±12.24)%(p<0.01);5μM,10μM,50μM,100μM,500μM,1 mM可以使小鼠NC胞浆基础钙离子浓度分别增加为(107.33±2.18)%、(110.82±2.32)%、(111.10±2.81)%、(112.24±2.56)%、(115.72±4.63)%和(116.70±3.80)%(p<0.01),但0.5μM,1μM OUA对NC胞浆基础钙离子浓度没有明显影响,分别为(103.64±2.26)%和(102.47±3.29)%(p>0.05);其中0.1μM OUA对细胞收缩幅度、钙瞬变幅度以及胞浆基础钙离子浓度均没有影响,分别为(104.59±2.35)%、(101.41±1.82)%和(99.62±2.23)%(p>0.05),而500μM和1 mM的OUA使NC很快出现毒性反应,表现为不规律收缩,细胞长度变短,甚至变为球形。而50μM的OUA使小鼠CHFC收缩幅度、钙瞬变幅度以及胞浆基础钙离子浓度分别明显增大至(351.71±90.6)%、(164.77±6.87)%和(117.67±7.70)%(p<0.01),较50μM OUA增加NC收缩幅度、钙瞬变幅度以及胞浆基础钙离子浓度的作用显著为强(p<0.01)。
⑵MIA对小鼠NC和CHFC收缩力和细胞内钙的作用。灌流0.1和1μM的MIA 5 min后,小鼠NC收缩幅度分别降低至(78.82±5.44)%和(46.93±7.36)%(p<0.01);钙瞬变幅度分别降低至(73.41±8.73)%和(49.59±4.01)%(p<0.01);但对胞浆基础钙离子浓度没有明显影响(p>0.05)。灌流0.1和1μM的MIA 15 min后,小鼠NC收缩幅度明显分别增加至(142.91±12.64)%和(204.36±27.22)%(p<0.01);钙瞬变幅度分别增加为(117.51±2.68)%和(132.51±4.30)%(p<0.01);胞浆基础钙离子浓度分别增加为(115.22±1.90)%和(126.93±3.97)%(p<0.01)。当正性肌力作用达到最大后,可使NC很快出现毒性反应,表现为细胞长度缩短,甚至变为球形。但是,对于小鼠CHFC,灌流0.1和1μM的MIA 5 min后,收缩幅度可分别增加至(117.01±1.11)%和(130.08±3.61)%(p<0.01);钙瞬变幅度分别增加为(104.64±2.18)%(p>0.05)和(113.38±2.61)%(p<0.01),但均对CHFC胞浆基础钙离子浓度没有明显影响,分别为(101.13±4.90)%和(104.21±2.49)%(p>0.05)。灌流0.1和1μM的MIA 15 min后,小鼠CHFC收缩幅度(104.55±3.89)%和(101.83±2.62)%、钙瞬变幅度(100.11±4.59)%和(97.27±3.72)%及胞浆基础钙离子浓度(103.63±6.91)%和(99.01±4.82)%均没有显著变化(p>0.05)。
⑶在低浓度哇巴因存在下MIA对小鼠NC和CHFC收缩力和细胞内钙的作用。与仅用1μM MIA灌流时相比,小鼠NC用10 nM OUA预先孵育10 min后,再灌流10 nM OUA +1μM MIA,使NC收缩力由原来5 min时降低为(46.93±7.36)%变为降低至(78.49±9.19)%(p<0.01),由原来15 min时增加至(204.36±27.22)%变为增加至(135.42±10.74)%(p<0.01);使NC钙瞬变幅度由原来5 min时降低为(49.59±4.01)%变为(78.51±10.83)%(p<0.01),由原来15 min时增加至(132.51±4.30)%变为增加至(108.64±2.68)%(p<0.01);5 min时对NC胞浆基础钙离子浓度没有明显影响,但15 min时由原来增加(126.93±3.97)%(p<0.01)变为无明显增加(103.11±2.07)%(p>0.05)。然而,小鼠CHFC用10 nM OUA预先孵育10 min后,再灌注10 nM OUA + 1μM MIA,与仅用1μM MIA时相比,OUA+ MIA可以使其收缩力由原来5 min时增加为(130.08±3.61)%变为增加至(183.14±23.77)%(p<0.01),由原来15 min时没有显著性变化(101.83±2.62)%(p>0.05)变为增加至(180.56±18.31)%(p<0.01);使CHFC钙瞬变幅度由原来5 min时增加至(113.38±2.61)%变为增加(155.37±4.89)%(p<0.01),由原来15 min时没有显著性变化(97.27±3.72)%(p>0.05)变为增加至(158.48±8.62)%(p<0.01);使CHFC胞浆基础钙离子浓度由原来5 min时(104.21±2.49)%和15 min时(99.01±4.82)%没有明显影响(p>0.05)分别变为增加为(115.19±1.98)%和(114.37±2.05)%(p<0.01)。
⑷小鼠NC和CHFC的NKAα1、α2亚基与NHE-1在蛋白水平的表达。与NC相比,CHFC的NKAα1、α2亚基以及NHE-1在蛋白水平的表达量均显著下降(p<0.01)。
结论:哇巴因能够浓度依赖性增加小鼠心肌细胞的基础钙离子浓度、钙瞬变幅度以及收缩力,且对衰竭心脏心肌细胞的作用较对正常心脏心肌细胞的作用显著为强,其原因可能与心衰后小鼠心肌细胞NKAα1和、α2亚基蛋白表达均有所降低有关。NHE-1抑制剂MIA对正常小鼠心肌细胞的收缩力和钙瞬变先减弱后增强,而对心衰小鼠心肌细胞则仅有短暂的正性肌力作用。低浓度哇巴因(10 nM)能够部分抵消MIA对正常小鼠心肌细胞的收缩和钙瞬变作用,但却显著增强了MIA对心衰小鼠心肌细胞的正性肌力作用和钙瞬变作用,提示NHE与NKA在调节心肌细胞收缩过程中存在密切的功能联系,且此调节取决于心肌细胞的机能状况,在正常心肌细胞二者呈协同作用,在心肌细胞发生心衰病理变化后,二者呈拮抗作用。另外,本研究也进一步提示,NHE-1抑制剂可作为强心苷类药物治疗心衰的辅助药具有研究开发前景。
Heart failure (HF) is a series of multiplicity clinical symptom, and the end stage of many cardiac disease, such as hypertension, ischemic cardiomyopathy, valvular disease and so on. The incidence and prevalence of heart failure (HF) is high and the prognosis of HF is rather poor. It is one of the most harmful problems which is increasingly severe.
Cardiotonic steroid (Cs) had been used for the congestive heart failure (CHF) for more than 200 years. But the mechanism of its positive inotropic effect is still controversial. It is generally believed that Cs produce positive inotropic actions in failure heart via inhibition of Na+, K+-ATPase (NKA) activities which leads to an increase in intracellular Na+ concentration ([Na+]i), and in turn raises intracellular Ca2+ concentration ([Ca2+]i) via Na+/Ca2+ exchange, thus increasing the uptake and subsequent release of Ca2+ by the sarcoplasmic reticulum (SR).
Na+/H+ exchanger (NHE) as a plasma membrane exchange glycoprotein transporter that functions in intracellular pH regulation, cell volume regulation, and cellular response to many different hormones and mitogens. Following allosteric activation by intracellular acidification, NHE exchanges extra- cellular sodium for intracellular hydrogen with stoichiometry of 1:1. Na+ influx via activation of Na+/H+ exchanger reactivates PKC in myocytes, which is one of the important signaling molecules in the development of the cardiac hypertrophic response. Recently, Baartscheer et al reported that two months of dietary treatment with the NHE-1 inhibitor cariporide caused regression of hypertrophy, heart failure and ionic and electrophysiological remodelling.
Saini HK et al reported NHE play a critical role in the ouabain-induced [Ca2+]i increase in cardiomyocytes. However, inotropic effect of methyl-n- isobutyl amiloride (MIA), a NHE inhibitor, in isolated ischemia-perfusion hearts was not consistent with its effect on [Ca2+]i in quiescent or KCl- depolarized cardiomyocytes exposed to ouabain. Therefore, it is hard to explain the interacton between NKA and NHE, and effect of them on the pathological condition. Thus mice were chosen as test animals. The mouse CHF models were developed. The present study is to observe the effects of the NHE on inotropic effects and change of [Ca2+]i of normal and CHF ventricular myocytes in the presence or obsence of ouabain in order to investigate inhibition of NHE as a potentiality treatment of CHF.
Objective: To detect the effects of NHE-1 on the contractile and calcium transient in the ventricular myocytes from heart failure mice through establishing CHF animal model and the effects of CHF on the expression of NKAα1 andα2 isoform and NHE-1 in protein level to identify the mechanism of the inotropic effect of NHE-1 and NKA in isolated mouse ventricular myocytes.
Methods: Mice CHF model was made by constricting aortic arch. Left ventricular myocytes were enzymatically isolated. Then the contractile and calcium transient of a single myocyte from normal and CHF mice were assessed by a video-based motion edge-detection system simultaneously. At the same time, effects of the same concentration of ouabian on inotropic effects and calcium transient in CHF mouse ventricular myocytes were compared with those in normal mouse ventricular myocytes. Then the experiments were repeated when the myocytes were preincubated with 10nM ouabain. The effects of MIA with ouabain on contractility and calcium transient were recorded and the results were compared with those of MIA-only. Then the expressions of NKAα1 andα2 isoform and NHE-1 in protein level were detected with Western blot.
Results:⑴The effects of ouabain on the contractility and calcium transient of NC or CHFC from mice. The extent of cell shortening, ampitude of calcium transient and basal level of [Ca2+]i was normalized using the value before ouabain perfusion as 100%. In NC, OUA 0.5μM, 1μM, 5μM, 10μM, 50μM, 100μM, 500μM, and 1 mM increased the extent of cell shortening by (124.60±5.02)%, (133.02±5.65)%, (148.42±6.63)%, (174.14±6.29)%, (204.64±3.66)%, (228.50±10.87)%, (286.42±27.57)%, and (288.80±23.82)% (p< 0.01); The concentration of OUA above increased the ampitude of calcium transient by (119.26±4.30)%, (125.43±5.64)%, (126.10±6.77)%, (133.92±10.26)%, (139.72±6.43)%, (152.90±8.10)%, (157.38±11.53)%, and (156.39±12.24)% (p<0.01); OUA 5μM, 10μM, 50μM, 100μM, 500μM, and 1 mM increased basal level of [Ca2+]i by (107.33±2.18)%, (110.82±2.32)%, (111.10±2.81)%, (112.24±2.56)%, (115.72±4.63)%, and (116.70±3.80)% (p< 0.01), while OUA 0.5μM and, 1μM had no effect on basal level of [Ca2+]i, which are (103.64±2.26)% and (102.47±3.29)% (p>0.05), respectively; 0.1μM OUA had no effect on the extent of cell shortening, ampitude of calcium transient and basal level of [Ca2+]i, which are (104.59±2.35)%, (101.41±1.82)% and (99.62±2.23)% (p>0.05),respectively; and OUA 500μM and 1 mM caused the cell irregular contraction, shorter length even became to“round-up”. In CHFC, OUA 50μM increased extent of cell shortening, ampitude of calcium transient and basal level of [Ca2+]i by (351.71±90.6)%, (164.77±6.87) % and (117.67±7.70)% (p<0.01); At the same concentration, effect of Str was more potent in CHFC than in NC.
⑵The effects of MIA on the contractility and calcium transient of NC or CHFC from mice. In NC, after 5 min perfusion MIA 0.1 and 1μM decreased the extent of cell shortening by (78.82±5.44)% and (46.93±7.36)% (p<0.01); and decreased the ampitude of calcium transient by (73.41±8.73)% and (49.59±4.01)% (p<0.01); but had no effect on basal level of [Ca2+]i (p>0.05). After 15 min perfusion MIA 0.1 and 1μM increased the extent of cell shortening by (142.91±12.64)% and (204.36±27.22)% (p<0.01), increased the ampitude of calcium transient by (117.51±2.68)% and (132.51±4.30)% (p<0.01), and increased basal level of [Ca2+]i by (115.22±1.90)% and (126.93±3.97)% (p<0.01). When the positive inotropic effect reach the maximal value, MIA caused the shorter length even became to“round-up”. In CHFC, however, after 5min perfusion MIA 0.1 and 1μM increased the extent of cell shortening by (117.01±1.11)% and (130.08±3.61)% (p<0.01), increased the ampitude of calcium transient by (104.64±2.18)% (p>0.05) and (113.38±2.61)% (p<0.01), and had no effect on basal level of [Ca2+]i, (101.13±4.90)% and (104.21±2.49)% (p>0.05). In CHFC, after 15min perfusion MIA 0.1 and 1μM had no effect on the extents of cell shortening, which were (104.55±3.89)% and (101.83±2.62)%, the ampitudes of calcium transient, which were (100.11±4.59)% and (97.27±3.72)%, and basal levels of [Ca2+]i, which were (103.63±6.91)% and (99.01±4.82)% (p>0.05), respectively.
⑶The effects of MIA on the contractility and calcium transient of NC or CHFC from mice in the present of low concentration of ouabain. In NC, after preincubation with 10 nM OUA for 10 min, 10 nM OUA+1μM MIA was perfused. Compared with the effect of MIA in NC, OUA+MIA decreased the extent of cell shortening form (46.93±7.36)% to (78.49±9.19)% (p<0.01) in 5 min perfusion, increased the extent of cell shortening form (204.36±27.22)% to (135.42±10.74)% (p<0.01) in 15 min perfusion; and decreased the ampitude of calcium transient from (49.59±4.01)% to (78.51±10.83)% (p<0.01) in 5 min perfusion, increased the ampitude of calcium transient from (132.51±4.30)% to (108.64±2.68)% (p<0.01) in 15 min perfsion; and had no effect on basal level of [Ca2+]i in 5 min perfusion, while increased basal level of [Ca2+]i from (126.93±3.97)% (p<0.01) to (103.11±2.07)% (p>0.05), which had no significant difference compared with those before perfusion. In CHFC, however, after preincubation with 10 nM OUA for 10 min, 10 nM OUA+1μM MIA was perfused. Compared with the effect of MIA in NC, OUA+MIA increased the extent of cell shortening form (130.08±3.61)% to (183.14±23.77)% (p<0.01) in 5 min perfusion, and from significant difference to increased the extent of cell shortening by (180.56±18.31)% in 15 min perfusion; and increased the ampitude of calcium transient from (113.38±2.61)% to (155.37±4.89)% (p<0.01) in 5 min perfusion, and from no significant difference (97.27±3.72)% (p>0.05) to increased the ampitude of calcium transient by (158.48±8.62)% (p<0.01) in 15 min perfusion; OUA+ MIA increased basal level of [Ca2+]i from no significant difference (104.21± 2.49)% and (99.01±4.82)% (p>0.05) to increased by (115.19±1.98)% and (114.37±2.05)% (p<0.01), respectively, after 5 min and 15 min perfusion.⑷The expression of NKAα1 andα2 isoform and NHE-1 in protein level in ventricular myocytes from mice with CHF. Compared with NC, the protein level of NKAα1 andα2 isoform and NHE-1 were decreased in CHFC (p<0.01).
Conclusions: Ouabain increases the extent of cell shortening, ampitude of calcium transient and basal level of [Ca2+]i. The reason why effects of OUA is more potent in CHFC than in NC may be related to that the more protein level of NKAα1 andα2 isoform are decreased in CHFC than in NC. MIA enhances the contractility and calcium transient followed by a weaken effect; whlie MIA moderately increased the inotropy and calcium transient of CHFC. Low concentration of OUA can partially abolish MIA-induced the changes in NC; however, MIA-induced changes in inotropic effect and [Ca2+]i were augmented by OUA in CHFC. It is prompted that there is a colsed relationship between NHE and NKA on the process of regulating cadiocytes, and the regulation is dependent on the condition of cadiocytes. In NC, NHE and NKA showed synergistic action, while in CHFC, they showed antagonistic effect. In addition, our study also suggest that research and development of the inhibitor of NHE-1 have the prospect of as adjunctive therapy with Cs to treat CHF.
引文
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18 Bondarenko A, Svichar N, Chesler M. Role of Na+/H+ and Na2+/Ca2+ exchange in hypoxia-related acute astrocyte death. Glia, 2005, 49: 143- 152
19 Orchard CH, Kentish JC. Effects of changes of pH on the contractile function of cardiac muscle. Am J Physiol Cell Physiol, 1990, 258:C967- 981
20 Lago J, Alfonso A, Botana LM et al. Ouabain-induced enhancement of rat mast cells response. Modulation by protein phosphorylation and intracellular pH. Cell Signal, 2001, 13: 515-524
21 Mellergard P, Ouyang YB, Siesjo BK. Intracellular pH regulation in cultured rat astrocytes in CO2/HCO3--containing media. Exp Brain Res, 1993, 95: 371-380
22 Mandal A, Delamere NA, Shahidullah M. Ouabain-induced stimulation of sodium-hydrogen exchange in rat optic nerve astrocytes. Am J Physiol Cell Physiol, 2008, 295: C100-110
23 Sisczkowski M, Ng LL. Phorbol ester activation of the rat vascular myocyte Na+/H+ exchanger isoform 1. Hypertension, 1996, 27:859-866
24 Wang H, Silva NL and Lucchesi PA et al. Phosphorylation and regulation of the Na+/H+ exchanger through mitogenactivated protein kinase. Biochemistry, 1997,29: 9151-9158
25 Moor AN, Fliegel L. Protein kinase mediated regulation of the Na+/H+ exchanger in the rat myocardium by mitogen-activated protein kinase dependent pathways. J Biol Chem, 1999, 274: 22985-22992
26 Wallert MA, Frohlich O.α1-Adrenergic stimulation of the Na+/H+ exchange in cardiac myocytes. Am J Physiol, 1992, 263: C1096-1102
27 Puceat M, Vassort G. Neurohumoral modulation of intracellular pH in the heart. Cardiovasc Res, 1995, 29: 178-183
28 Yamazaki T, Komuro I, Kudoh S et al. Role of ion channels and exchanger in mechanical stretch-induced cardiomyocyte hypertrophy. Circ Res, 1998, 82: 430-437
1 Malo ME, Fliegel L. Physiological role and regulation of the Na+/H+ exchanger. Can J Physiol Pharmacol, 2006; 84: 1081-1095
2 Orlowski J, and Grinstein S. Na+/H+ exchangers of mammalian cells. J. Biol. Chem., 1997, 272, 22373-22376
3 Karmazyn M, Gan T, Humphreys RA et al. The myocardial Na+/H+ exchange. Structure, regulation, and its role in heart disease. Circ Res, 1999, 85: 777-786
4 Cardone RA, CasavolaV and Reshkin SJ. The role of disturbed pH dynamics and the Na+/H+ exchanger in metastasis. Nat. Rev. Cancer, 2005, 5: 786-795
5 Hayasaki KY, Naya N, Shimamura T.Endothelin generating pathway through endothelin1-31 in human cultured bronchial smooth muscle cells. British Journal of Pharmacology, 1999, 127: 1415-1421
6 Avkiran M, Marber MS. Na+/H+ exchange inhibitors for cardio- protective therapy: progress, problems and prospects. J Am Coll Cardiol, 2002, 39: 747-53
7 Phillis JW, Pilitsis JG, O'Regan MH. The potential role of the Na+/H+ exchanger in ischemia/reperfusion injury of the central nervous system. p. 177-189 In: Karmazyn M, Avkiran M, Fliegel L, editors. The Na+/H+ Exchanger, From Molecular to Its Role in Disease. Boston: Kluwer Academic Publishers; 2003, 318 pp
8 Karmazyn M. Therapeutic potential of Na+/H+ exchange inhibitors for the treatment of heart failure. Expert Opin Investig Drugs, 2001, 10: 835-843
9 Gazmuri RJ, Hoffner E and Kalcheim J et al. Myocardial protection during ventricular fibrillation by reduction of proton-driven sarcolemmal sodium influx. J Lab Clin Med, 2001, 137: 43-55
10 Orlowski J and Grinstein S. Diversity of the mammalian sodium/ proton exchanger SLC9 gene family. Pflugers Arch, 2004, 447: 549-565
11 Nakamura N, Tanaka S and Teko Y et al. Four Na+/H+ exchanger isoforms are distributed to Golgi and post-Golgi compartments and are involved in organelle pH regulation. J. Biol. Chem., 2005, 280: 1561-1572
12 Khadilkar A, Iannuzzi P and Orlowski J. Identification of sites in the second exomembrane loop and ninth transmembrane helix of the mammalian Na+/H+ exchanger important for drug recognition and cation translocation. J. Biol. Chem., 2001, 276: 43792-43800
13 Orlowski J, Kandasamy RA and Shull GE. Molecular cloning of putative members of the Na+/H+ exchanger gene family. J. Biol. Chem., 1992, 267: 9331-9339
14 Noel J, Roux D and Pouyssegur J. Differential localization of Na+/H+ exchanger isoforms (NHE1 and NHE3) in polarized epithelial cell lines. J. Cell Sci., 1996, 109: 929-939
15 Baird NR, Orlowski J and Szabo EZ et al. Molecular cloning, genomic organization, and functional expression of Na+/H+ exchanger isoform 5 (NHE5) from human brain. J. Biol. Chem., 1999, 274: 4377-4382
16 Attaphitaya S, Park K and Melvin JE. Molecular cloning and functional expression of a rat Na+/H+ exchanger (NHE5) highly expressed in brain. J. Biol. Chem., 1999, 274: 4383-4388
17 Brett CL, Wei Y and Donowitz M et al. Human Na+/H+ exchanger isoform 6 is found in recycling endosomes of cells, not in mitochondria. Am. J. Physiol. Cell Physiol., 2002, 282: C1031-C1041
18 Numata M and Orlowski J. Molecular cloning and characterization of a novel (Na+, K+)/H+ exchanger localized to the trans-Golgi network. J. Biol. Chem., 2001, 276: 17387-17394
19 Putney LK, Denker SP and Barber DL. The changing face of the exchanger, NHE1: structure, regulation, and cellular actions. Annu Rev Pharmacol Toxicol, 2002, 42: 527-552
20 Aharonovitz O, Zaun HC and Balla T et al. Intracellular pH regulation by Na+/H+ exchange requires phosphatidylinositol 4,5-bisphosphate. J Cell Biol, 2000, 150: 213-224
21 Lin X and Barber DL. A calcineurin homologous protein inhibits GTPase-stimulated Na+/H+ exchange. Proc Natl Acad Sci USA, 1996, 93: 12631-12636
22 Pang T, Su X and Wakabayashi S et al. Calcineurin homologous protein as an essential cofactor for Na+/H+ exchangers. J Biol Chem, 2001, 276: 17367-17372
23 Denker SP, Huang DC and Orlowski J et al. Direct binding of the Na+/H+ exchanger NHE1 to ERM proteins regulates the cortical cytoskeleton and cell shape independently of H+ translocation. Mol Cell, 2000, 6: 1425- 1436
24 Takahashi E, Abe J and Gallis B et al. p90(RSK) is a serum-stimulated Na+/ H+ exchanger isoform-1 kinase. Regulatory phosphorylation of serine 703 of Na+/H+ exchanger isoform-1. J Biol Chem, 1999, 274: 20206-20214
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2 Hayasaki-KajiwaraY, KitanoY, Iwasaki T et al. Na+ influx via Na+/H+ exchange activates protein kinase C isozymesδandεin cultured neonatal rat cardiac myocytes. J Mol Cell Cardiol, 1999, 31:1559-72
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17 Wajima T, Beguier B, Yaguchi M. Effects of cariporide (HOE642) on myocardial infarct size and ventricular arrhythmias in a rat ischemia/ reperfusion model: comparison with other drugs. Pharmacology, 2004, 70: 68-70
18 Bondarenko A, Svichar N, Chesler M. Role of Na+/H+ and Na2+/Ca2+ exchange in hypoxia-related acute astrocyte death. Glia, 2005, 49: 143- 152
19 Orchard CH, Kentish JC. Effects of changes of pH on the contractile function of cardiac muscle. Am J Physiol Cell Physiol, 1990, 258:C967- 981
20 Lago J, Alfonso A, Botana LM et al. Ouabain-induced enhancement of rat mast cells response. Modulation by protein phosphorylation and intracellular pH. Cell Signal, 2001, 13: 515-524
21 Mellergard P, Ouyang YB, Siesjo BK. Intracellular pH regulation in cultured rat astrocytes in CO2/HCO3--containing media. Exp Brain Res, 1993, 95: 371-380
22 Mandal A, Delamere NA, Shahidullah M. Ouabain-induced stimulation of sodium-hydrogen exchange in rat optic nerve astrocytes. Am J Physiol Cell Physiol, 2008, 295: C100-110
23 Sisczkowski M, Ng LL. Phorbol ester activation of the rat vascular myocyte Na+/H+ exchanger isoform 1. Hypertension, 1996, 27:859-866
24 Wang H, Silva NL and Lucchesi PA et al. Phosphorylation and regulation of the Na+/H+ exchanger through mitogenactivated protein kinase. Biochemistry, 1997,29: 9151-9158
25 Moor AN, Fliegel L. Protein kinase mediated regulation of the Na+/H+ exchanger in the rat myocardium by mitogen-activated protein kinase dependent pathways. J Biol Chem, 1999, 274: 22985-22992
26 Wallert MA, Frohlich O.α1-Adrenergic stimulation of the Na+/H+ exchange in cardiac myocytes. Am J Physiol, 1992, 263: C1096-1102
27 Puceat M, Vassort G. Neurohumoral modulation of intracellular pH in the heart. Cardiovasc Res, 1995, 29: 178-183
28 Yamazaki T, Komuro I, Kudoh S et al. Role of ion channels and exchanger in mechanical stretch-induced cardiomyocyte hypertrophy. Circ Res, 1998, 82: 430-437
1 Malo ME, Fliegel L. Physiological role and regulation of the Na+/H+ exchanger. Can J Physiol Pharmacol, 2006; 84: 1081-1095
2 Orlowski J, and Grinstein S. Na+/H+ exchangers of mammalian cells. J. Biol. Chem., 1997, 272, 22373-22376
3 Karmazyn M, Gan T, Humphreys RA et al. The myocardial Na+/H+ exchange. Structure, regulation, and its role in heart disease. Circ Res, 1999, 85: 777-786
4 Cardone RA, CasavolaV and Reshkin SJ. The role of disturbed pH dynamics and the Na+/H+ exchanger in metastasis. Nat. Rev. Cancer, 2005, 5: 786-795
5 Hayasaki KY, Naya N, Shimamura T.Endothelin generating pathway through endothelin1-31 in human cultured bronchial smooth muscle cells. British Journal of Pharmacology, 1999, 127: 1415-1421
6 Avkiran M, Marber MS. Na+/H+ exchange inhibitors for cardio- protective therapy: progress, problems and prospects. J Am Coll Cardiol, 2002, 39: 747-53
7 Phillis JW, Pilitsis JG, O'Regan MH. The potential role of the Na+/H+ exchanger in ischemia/reperfusion injury of the central nervous system. p. 177-189 In: Karmazyn M, Avkiran M, Fliegel L, editors. The Na+/H+ Exchanger, From Molecular to Its Role in Disease. Boston: Kluwer Academic Publishers; 2003, 318 pp
8 Karmazyn M. Therapeutic potential of Na+/H+ exchange inhibitors for the treatment of heart failure. Expert Opin Investig Drugs, 2001, 10: 835-843
9 Gazmuri RJ, Hoffner E and Kalcheim J et al. Myocardial protection during ventricular fibrillation by reduction of proton-driven sarcolemmal sodium influx. J Lab Clin Med, 2001, 137: 43-55
10 Orlowski J and Grinstein S. Diversity of the mammalian sodium/ proton exchanger SLC9 gene family. Pflugers Arch, 2004, 447: 549-565
11 Nakamura N, Tanaka S and Teko Y et al. Four Na+/H+ exchanger isoforms are distributed to Golgi and post-Golgi compartments and are involved in organelle pH regulation. J. Biol. Chem., 2005, 280: 1561-1572
12 Khadilkar A, Iannuzzi P and Orlowski J. Identification of sites in the second exomembrane loop and ninth transmembrane helix of the mammalian Na+/H+ exchanger important for drug recognition and cation translocation. J. Biol. Chem., 2001, 276: 43792-43800
13 Orlowski J, Kandasamy RA and Shull GE. Molecular cloning of putative members of the Na+/H+ exchanger gene family. J. Biol. Chem., 1992, 267: 9331-9339
14 Noel J, Roux D and Pouyssegur J. Differential localization of Na+/H+ exchanger isoforms (NHE1 and NHE3) in polarized epithelial cell lines. J. Cell Sci., 1996, 109: 929-939
15 Baird NR, Orlowski J and Szabo EZ et al. Molecular cloning, genomic organization, and functional expression of Na+/H+ exchanger isoform 5 (NHE5) from human brain. J. Biol. Chem., 1999, 274: 4377-4382
16 Attaphitaya S, Park K and Melvin JE. Molecular cloning and functional expression of a rat Na+/H+ exchanger (NHE5) highly expressed in brain. J. Biol. Chem., 1999, 274: 4383-4388
17 Brett CL, Wei Y and Donowitz M et al. Human Na+/H+ exchanger isoform 6 is found in recycling endosomes of cells, not in mitochondria. Am. J. Physiol. Cell Physiol., 2002, 282: C1031-C1041
18 Numata M and Orlowski J. Molecular cloning and characterization of a novel (Na+, K+)/H+ exchanger localized to the trans-Golgi network. J. Biol. Chem., 2001, 276: 17387-17394
19 Putney LK, Denker SP and Barber DL. The changing face of the exchanger, NHE1: structure, regulation, and cellular actions. Annu Rev Pharmacol Toxicol, 2002, 42: 527-552
20 Aharonovitz O, Zaun HC and Balla T et al. Intracellular pH regulation by Na+/H+ exchange requires phosphatidylinositol 4,5-bisphosphate. J Cell Biol, 2000, 150: 213-224
21 Lin X and Barber DL. A calcineurin homologous protein inhibits GTPase-stimulated Na+/H+ exchange. Proc Natl Acad Sci USA, 1996, 93: 12631-12636
22 Pang T, Su X and Wakabayashi S et al. Calcineurin homologous protein as an essential cofactor for Na+/H+ exchangers. J Biol Chem, 2001, 276: 17367-17372
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