钠氢交换体1在病理性心肌肥厚大鼠心肌组织及血浆中的表达

目的：研究异丙肾上腺素诱导的病理性心肌肥厚大鼠心肌组织及血浆中钠氢交换体1（sodium—hydrogen exchanger1,NHE—1）的表达,探讨NHE1在心肌肥厚发生和发展中的作用。方法：30只雄性SD大鼠随机并平均分为2组：病理性心肌肥厚组和对照组,每组15只,病理性心肌肥厚组（以下简称ISO组）予以ISO（异丙肾上腺素）连续每日以20、10和5mg／kg的剂量递减皮下注射,再以3mg／kg的剂量维持皮下注射7d,对照组予相同剂量生理盐水皮下注射。给药结束后进行心脏超声检测左室舒张末径（LVEDD）、左室收缩末径（LVESD）、室间隔厚度（IVST）、短轴缩短率（FS）、左室射血分数（LVEF）。分别测定各组大鼠体重（Bw）、心室重量（VW）、左心室重量（LVW）,计算心室重量指数VWI（VW／BW）、左心室重量指数LVWI（LVW／BW）。取血检测血浆中NHE．1的浓度,并取心肌组织观察病理形态学特征,用免疫组化法检测心肌组织中NHE—1的表达量。结果：与对照组相比,ISO组大鼠LVEF、IVST显著增加（P〈0．05）,LVESD明显降低（P〈0．05）,VWI、LVWI明显增加（P〈0．01）,血浆NHE—1浓度明显升高（P〈0．01）,心肌组织NHE-1表达增多（P〈0．01）。结论：NHE-1可能在病理性心肌肥厚的发生和发展过程中起着重要作用。     

钠氢交换体1在慢性心衰大鼠心肌组织及血浆中的表达

目的:观察钠氢交换体1(sodium-hydrogen exchanger 1,NHE-1)在慢性心衰大鼠心肌组织及血浆中的表达,探讨NIlE-1在慢性心衰发生和发展中的作用.方法:40只雄性SD大鼠随机分为心袁组30只和对照组10只,心衰组予以阿霉素(ADR)2.5mg/kg/w,腹腔注射,共6周,对照组予相同剂量生理盐水腹腔注射.6周后进行心脏超声检测左室舒张末径(LVEDO)、左室收缩末径(LVESD)、室间隔厚度(IVST)、左室后壁厚度(LVPWT)、短轴缩短率(FS),左室射血分数(LVEF).分别测定各组大鼠体重(BW)、心室重量(VW)、左心室重量(LVW),计算心室重量指数VWI(VW/BW)、左心室重量指数LVWI(LVW/BW).取血检测血浆中NHE-1的浓度,并取心肌观察病理形态学特征,用免疫组化法检测心肌组织中NHE-1的表达量.结果:与对照组相比,心衰组大鼠LVEF、FS明显下降(P<0.01),LVEDD、LVESD显著扩大(P<0.01),VWI、LVWI明显增加(P<0.01),血浆N HE-1浓度显著升高(P<0.01),心肌组织中NHE-1蛋白表达量明显升高(P<0.01).结论:提示NHE-1可能在慢性心衰的发生和发展过程中起着重要作用.     

法舒地尔对异丙肾上腺素诱导大鼠心肌肥厚的干预作用及其机制

目的：研究法舒地尔对异丙肾上腺素诱导大鼠心肌肥厚的影响及其机制。方法：除正常对照组外,其它SD大鼠均皮下注射异丙肾上腺素（Iso,5 mg/kg）建立心肌肥厚模型。大鼠随机分为4组：正常对照组、Iso模型组、法舒地尔低剂量组（Fas,5 mg/kg,i.p）和法舒地尔高剂量组（Fas,20 mg/kg,i.p）,连续给药8周。给药结束后,血流动力学检测大鼠心率（HR）、左心室收缩压（LVSP）、左心室末舒张压（LVEDP）和左室压力变化最大速率（±dp/dt_max）;分别测定大鼠体重（BW）,心脏重量（HW）,并计算HW/BW;大鼠心肌HE、Masson染色观察组织病理学改变;免疫组化法观察大鼠心肌组织ERK1、ERK2蛋白表达,RT-PCR观察ERK1、ERK2 mRNA的表达。结果：Iso模型组HR和LVEDP明显升高,LVSP和±dp/dt_max明显下降;HW/BW增大;心肌细胞体积变大,排列紊乱,胶原纤维增生;左心室组织ERK1、ERK2蛋白与mRNA表达上调。法舒地尔不同剂量干预后,心脏收缩和舒张能力得到改善,心指数明显下降,心肌细胞体积变小,纤维化减少,ERK1/2 mRNA表达下调,心肌组织损害均得到不同程度改善。结论：ERK1/2信号通路活化参与了异丙肾上腺素诱导的心肌肥厚,法舒地尔对异丙肾上腺素诱导的心肌肥厚具有明显改善作用,这可能与法舒地尔阻断ERK1、ERK2通路活化有关。     

心肌细胞内质网应激介导APPswe/PS1dE9转基因成年小鼠发生心肌肥厚的研究

目的:探讨内质网应激在APPswe/PS1dE9转基因成年小鼠发生心肌肥厚中的作用。方法:选择APPswe/PS1dE9转基因成年小鼠及野生型(WT)成年小鼠各10只,分别为转基因实验组及对照组、野生型实验组及对照组,每组各5只,实验组背部皮下注射低剂量异丙肾上腺素(ISO)(2 mg/kg),每日1次,连续4周;对照组注射同等体积的生理盐水,4周后麻醉各组小鼠,描记体表心电图,计算心率,测量体重后处死,取出心脏,测量心脏质量及左室质量,计算左心室重量指数(LVW/BW)及全心重量比(HW/BW)。用于HE染色法测定心肌细胞病理形态学变化,应用Western Blot方法及免疫组织化学方法检测内质网应激相关蛋白即GRP78、JNK、P-JNK及CaMKⅡ的表达。结果:APPswe/PS1dE9转基因小鼠在低剂量的ISO诱导后较WT小鼠心脏明显肥大,APPswe/PS1dE9转基因实验组较转基因对照组及WT小鼠实验组及对照组HW/BW及LVW/BW显著增加(P0.05);HE染色可见APPswe/PS1dE9转基因实验组较转基因对照组及WT实验组、对照组心肌细胞直径明显变大,细胞密度变小,毛细血管密度减少,细胞间质增多,细胞间距变大。Western blot结果显示APPswe/PS1dE9转基因实验组GRP78、p-JNK及CaMKⅡ的表达较转基因对照组及WT两组明显增加(P0.05,P0.01),而转基因对照组与WT两组无明显差异。免疫组化结果显示APPswe/PS1dE9转基因实验组GRP78及CaMKⅡ蛋白在心肌细胞胞浆中表达阳性率80%,明显高于野生型实验组(40%)(P0.05),两组对照组表达均为阴性;转基因实验组p-JNK表达为90%,JNK表达为30%,其它三组均为阴性。结论:APPswe/PS1dE9转基因成年小鼠在较低剂量的ISO诱导后较WT实验组更容易出现心肌肥厚,内质网应激参与了APPswe/PS1dE9转基因成年小鼠心肌肥厚的发生。     

基于Akt/mTOR/NF-κB信号通路研究地龙提取物对病理性心肌肥厚大鼠的保护作用

基于Akt/mTOR/NF-κB信号通路,探讨地龙提取物对病理性心肌肥厚的保护作用及机制。30只Wistar大鼠随机分为假手术组、模型组、地龙低剂量组(400 mg/kg)、地龙高剂量组(800 mg/kg)和卡托普利对照组(50 mg/kg),每组6只;采用腹主动脉缩窄术建立病理性心肌肥厚模型,假手术组除不结扎外,与模型组相同。地龙各剂量组大鼠按照体重腹腔注射给药,假手术组和模型组给予等体积生理盐水,每天1次,连续3周。小动物超声观察大鼠心脏射血分数(ejection fraction, EF)和短轴缩短率(short axis shortening rate, FS)的变化;ELISA测定大鼠血清TNF-α含量;HE染色观察心肌组织病理变化;qRT-PCR检测心肌肥大基因(ANP、BNP和β-MHC)及促炎因子(TNF-α、IL-6)的表达水平;免疫印迹法测定Akt/mTOR/NF-κB通路蛋白的表达水平。结果表明,与模型组相比,地龙各剂量组和卡托普利组大鼠的EF、FS升高,心脏质量指数(HW、HW/BW及LVW/BW)降低,心肌肥大因子和促炎因子水平下调,且Akt/mTOR/NF...     

丹参酚酸B通过抑制PI3K/AKT/mTOR通路促进自噬减轻大鼠心肌纤维化的研究

目的:研究丹参酚酸B(SA-B)能否通过抑制PI3K/AKT/mTOR通路促进自噬,从而减轻大鼠心肌纤维化。方法:选用SD大鼠40只,完全随机化分为对照组、模型组、低剂量SA-B治疗组和高剂量SA-B治疗组,采用皮下注射异丙肾上腺素(ISO)构建大鼠心肌纤维化模型。低、高剂量SA-B治疗组在造模同时灌喂丹参酚酸B水溶液,对照组和模型组分别灌胃等体积0.9%生理盐水。测定心重指数(HW/BW)和左心室重指数(LVW/BW);ELISA法测定心肌中Ⅰ型、Ⅲ型胶原水平;Western blot检测自噬相关蛋白PI3K、AKT、p-AKT、mTOR、Beclin1、LC3-Ⅱ水平;大鼠心肌HE染色评估心肌纤维化程度。结果:与对照组比较,模型组中大鼠的心重指数、左心室重指数和心肌中Ⅰ型、Ⅲ型胶原的水平升高(P0.05),HE染色结果提示心肌组织发生明显的纤维化。模型组大鼠心肌细胞中的自噬相关蛋白PI3K、AKT、p-AKT、mTOR表达升高,Beclin1、LC3-Ⅱ表达较对照组明显降低(P0.05)。SA-B组中心重指数、左心室重指数和心肌中Ⅰ型、Ⅲ型胶原的水平明显降低,HE染色未见明显纤维化病灶,其自噬相关蛋白PI3K、AKT、p-AKT、mTOR表达降低,Beclin1、LC3-Ⅱ表达较模型组明显升高(P0.05)。结论:丹参酚酸B能够抑制ISO所致的大鼠心肌纤维化,且具有剂量依耐性,其机制与抑制PI3K/AKT/mTOR传导通路促进细胞自噬密切相关。     

miRNA在异丙肾上腺素诱导大鼠心肌肥厚中的表达及生物信息学分析*

目的:探讨miRNAs(miR199a-5P、miR206、miR133a-3P、miR499-5P)在异丙肾上腺素(ISO)诱导大鼠心肌肥厚模型组中的表达变化;并运用生物信息学方法分析相关的主要信号通路及分子机制。方法:将16只SD雄性大鼠随机分为2组:对照组和ISO模型组,模型组给予ISO(1 mg/kg)诱导心肌肥厚模型,对照组给予等量生理盐水,均采用背部皮下多点注射。连续给药10 d后采用超声心动图测量舒张期室间隔厚度(IVSd)、舒张期左室后壁厚度(LVPWd)、左室舒张末期内径(LVDd)及心脏收缩功能(EF%);称量心脏重量(HW)、大鼠体重(BW),并计算心脏/体重比(HW/BW);心肌组织HE染色,Image J分析软件测量心肌细胞表面积;RT-qPCR检测大鼠心肌组织中4种miRNAs的表达情况。运用Targetscan、miRDB、miRwalk 数据库预测大鼠4种miRNAs可能的靶基因,FunRich软件分析预测靶基因相关的信号通路。结果:与正常组相比,模型组IVSd、LVPWd增厚,LV增大,EF%明显降低;HW、HW/BW增加;模型组心肌细胞体积明显增大,排列紊乱,细胞表面积增加;模型组miR199a-5P、miR206表达上调(P＜0.05);miR133a-3P、miR499-5P表达下调(P＜0.05)。应用生物信息学预测4种miRNAs的靶基因可能参与心肌肥厚相关的信号通路主要有:VEGF/VEGFR信号通路、ErbB受体信号通路等。结论:ISO诱导心肌肥厚导致miRNAs表达的改变,生物信息学预测4种miRNAs参与心肌肥厚相关的靶基因及其主要信号通路,这些研究为心肌肥厚的调控机制及其防治措施提供了新思路。     

卡托普利防治心肌胆厚的效应及其机理探讨

目的和方法：应用大鼠腹主动脉狭窄心肌厚模型,观察血管紧张素转换酶抑制剂卡托普利防治心肌肥厚的作用,以及该作用与心肌组织内儿茶酚胺、氧自由基、相关离子代谢之间的关系。结果：①应用卡托普利后,大鼠HW、LVW、HW／BW、LVW／BW各指标与心肌肥厚组比较均明显降低;②卡托普利可明显抑制心肌NE、DA含量的降低,E含量的增高;③卡托普利可增强心肌组织SOD和GSH－Px活性,减少LPO的生成;④卡托普利可明显抑制心肌Na^ 、Ca^2 含量的升高和心肌K^＋含量的降低。结论：卡托普利能有效防治心肌肥厚的发生,其作用机理不仅与卡托普利直接抑制体内RAS代谢有关,而且还与它调整和改善了心脏局部儿荷酚、氧自由基、相关离子代谢密切相关。     

PI3K和Akt蛋白在异丙肾上腺素所致大鼠心肌肥厚中的表达

目的研究异丙肾上腺素(ISO)致大鼠心肌肥厚中PI3K和Akt在心肌组织中的表达,为探讨心肌肥厚的信号转导机制和逆转心肌肥厚提供形态学资料.方法健康成年SD大鼠20只,随机分为实验组、对照组,每组10只.实验组给予异丙肾上腺素处理.1周后处死大鼠,取心肌组织,常规石蜡切片,HE染色,观察心肌组织的病理变化,测量心肌肥厚指标;免疫组织化学染色和免疫荧光染色,检测p-PI3K和p-Akt的表达及分布.结果实验组大鼠心肌肥厚指标与对照组相比均明显升高;免疫组织化学检测显示,实验组心肌组织p-PI3K和p-Akt蛋白表达面积和平均光密度较对照组高.免疫荧光检测实验组心肌组织p-PI3K和p-Akt蛋白表达较对照组高.结论小剂量持续给予 ISO 能建立大鼠心肌肥厚模型;p-PI3K和p-Akt蛋白表达均与心肌肥厚的发生和发展过程相关,PI3K/Akt信号通路激活,可能是导致心肌肥厚的机制之一.     

竹节参皂苷IVa减轻异丙肾上腺素诱导的小鼠心肌纤维化作用机制研究

摘要 目的：探讨竹节参皂苷（IVa）减轻异丙肾上腺素（ISO）诱导的小鼠心肌纤维化作用机制。方法：白变种实验室老鼠（Balb/C）小鼠40只并随机分为4组：正常对照组（n=10）、ISO模型组（n=10）、IVa低剂量组（n=10）、IVa高剂量组（n=10）。采用皮下注射ISO构建小鼠心肌纤维化模型,IVa剂量组在建模同时给予IVa治疗,正常组给予等量生理盐水。采用马松（Masson）三色标准和HE染色方法分析评估心脏组织形态学和胶原沉积。采用小麦胚芽凝集素（WGA）染色法测定心肌细胞面积。蛋白免疫印迹试验检测细胞自噬相关标志物（LC3-Ⅱ、Beclin1和p62）和腺苷单磷酸活化蛋白激酶（AMPK）/哺乳动物雷帕霉素靶蛋白（mTOR）/自噬激活激酶1（ULK1）信号通路相关标志物。采用酶联免疫吸附法（ELISA）检测血清中血管紧张素II（Ang II）和I型前胶原羧基末端肽（PICP）的含量。结果：高剂量IVa（15mg/kg）治疗后,HW/BW和LVW/BW较ISO模型组升高,而血清中Ang II和PICP含量降低。IVa以剂量依赖的方式减轻心肌细胞在心脏组织中的损伤。皮下注射ISO后,心肌间质内胶原沉积明显,IVa治疗后胶原沉积明显减少。IVa可有效降低ISO诱导的小鼠心肌细胞面积大小。IVa能有效抑制ISO诱导的LC3-Ⅱ和Beclin1蛋白降低,减少p62蛋白增多。AMPK直接磷酸化ULK1（Ser555）,通过抑制mTOR磷酸化间接抑制ULK1（Ser757）磷酸化,均参与了ISO诱导的心肌纤维化小鼠自噬活性的降低;此外,IVa低剂量组和IVa高剂量组均显著增加了AMPK磷酸化,并抑制了mTOR磷酸化,降低了ULK1(Ser757)磷酸化。结论：IVa通过AMPK/mTOR/ULK1途径激活自噬,减轻了ISO诱导的心肌纤维化。表明IVa是一种潜在的抗心肌纤维化候选药物,是治疗心脏病的潜在药物靶点。     

Normalization of the calcineurin pathway underlies the regression of hypertensive hypertrophy induced by Na+/H+ exchanger-1 (NHE-1) inhibition

Na+/H+ exchanger-1 (NHE-1) inhibition induces cardiac hypertrophy regression and (or) prevention in several experimental models, although the intracellular events involved remain unclarified. We aimed to determine whether the calcineurin/NFAT pathway mediates this effect of NHE-1 inhibitors. Spontaneously hypertensive rats (SHR) with cardiac hypertrophy were treated with the NHE-1 inhibitors cariporide and BIIB723 for 30 days. Wistar rats served as normotensive controls. Their hearts were studied by echocardiography, immunoblotting, and real-time RT-PCR. Cytoplasmic Ca2+ and calcineurin Abeta expression were measured in cultured neonatal rat ventricular myocytes (NRVM) stimulated with endothelin-1 for 24 h. NHE-1 blockade induced cardiac hypertrophy regression (heart mass/body mass=3.63+/-0.07 vs. 3.06+/-0.05 and 3.02+/-0.13 for untreated vs. cariporide- and BIIB723-treated SHR, respectively; p<0.05) and decreased myocardial brain natriuretic peptide, calcineurin Abeta, and nuclear NFAT expressions. Echocardiographic evaluation demonstrated a reduction in left ventricular wall thickness without changes in cavity dimensions or a significant decrease in blood pressure. NHE-1-inhibitor treatment did not affect myocardial stiffness or endocardial shortening, but increased mid-wall shortening, suggesting that a positive inotropic effect develops after hypertrophy regression. Cariporide normalized the increased diastolic Ca2+ and calcineurin Abeta expression observed in ET-1-stimulated NRVM. In conclusion, our data suggest that inactivation of calcineurin/NFAT pathway may underlie the regression of cardiac hyper-trophy induced by NHE-1 inhibition.     

NHE-1 participates in isoproterenol-induced downregulation of SERCA2a and development of cardiac remodeling in rat hearts

Impaired Ca(2+) handling is one of the main characteristics in heart failure patients. Recently, we reported abnormal expressions of Ca(2+)-handling proteins in isoproterenol (ISO)-induced hypertrophied rat hearts. On the other hand, Na(+)/H(+) exchanger (NHE)-1 inhibitor has been demonstrated to exert beneficial effects in ischemic-reperfusion injury and in the development of cardiac remodeling. The aims of the present study are to investigate the role of NHE-1 on Ca(2+) handling and development of cardiac hypertrophy in ISO-infused rats. Male Wistar rats were randomly divided into vehicle [control (CTL)] and ISO groups without or with pretreatment with a selective NHE-1 inhibitor, BIIB-723. ISO infusion for 1 wk significantly increased the ratios of heart to body weight and left ventricle (LV) to body weight and collagen accumulation. All of these increases were antagonized by coadministration with BIIB-723. The ISO-induced significant increase in LV wall thickness was suppressed significantly (P < 0.05) by BIIB-723. ISO-induced decreases in cardiac stroke volume and a total mechanical energy per beat index, systolic pressure-volume area at midrange LV volume, were normalized by BIIB-723. The markedly higher expression of NHE-1 protein in the ISO group than that in CTL group was suppressed (P < 0.05) by BIIB-723. Surprisingly, ISO induced downregulation of the important Ca(2+)-handling protein sarcoplasmic reticulum Ca(2+)-ATPase 2a, the expression of which was also normalized by BIIB-723 without changes in phosphorylated phospholamban (PLB)/PLB expression. We conclude that NHE-1 contributes to ISO-induced abnormal Ca(2+) handling associated with cardiac hypertrophy. Inhibition of NHE-1 ameliorates cardiac Ca(2+)-handling impairment and prevents the development of cardiac dysfunction in ISO-infused rats.     

The cardiac Na-H exchanger: a key downstream mediator for the cellular hypertrophic effects of paracrine, autocrine and hormonal factors.

The major mechanism by which the heart cell regulates intracellular pH is the Na(+)-H(+) exchanger (NHE) with the NHE-1 isoform as the primary cardiac subtype. Although NHE-1 has been implicated in mediating ischemic injury, more recent evidence implicates the antiporter as a key mediator of hypertrophy, which is produced by various autocrine, paracrine and hormonal factors such as endothelin-1, angiotensin II, and alpha(1) adrenoceptor agonists. These agonists activate the antiporter via phosphorylation-dependent processes. NHE-1 inhibition is likely conducive to attenuating the remodelling process after myocardial infarction. These effects probably occur independently of infarct size reduction and involve attenuation of subsequent postinfarction heart failure. As such, inhibitors of NHE offer substantial promise for clinical development that will attenuate acute responses to myocardial postinfarction and chronic pos t infarction, which evolve toward heart failure. The regulation of NHE-1 is discussed as is its potential role in mediating cardiomyocyte hypertrophy.     

Mechanism of garlic (Allium sativum) induced reduction of hypertension in 2K-1C rats: a possible mediation of Na/H exchanger isoform-1

Garlic causes reduction in blood pressure (BP), however the role of Na/H exchanger (NHE) which mediates hypertension and related tissue-damage is poorly understood. In this study the effect of an established dose of raw garlic extract was investigated on the expression of NHE-1 and -3 and sodium pump activity in a 2K-1C model of hypertension in rats. 2K-1C animals showed high BP, increased serum concentration of PGE2 and TxB2, hypertrophy of the unclipped kidneys, but not in the clipped kidneys In addition, NHE-1 and NHE-3 isoforms were increased in both the 2K-1C kidneys, whereas alpha-actin was increased in the clipped but not in unclipped kidneys. Sodium pump activity was decreased in the clipped kidneys, but remained unchanged in the unclipped kidneys. Garlic treatment reduced the induction of NHE-1 only in the unclipped 2K-1C kidneys, whereas garlic treatment increased the sodium pump activity in both the 2K-1C kidneys. These findings demonstrate that the antihypertensive action of garlic is associated with a reversal of NHE-1 induction in the unclipped kidneys. Induction of NHE isoforms together with a reduced sodium pump activity might cause necrosis in the 2K-1C clipped kidneys due to cellular retention of Na+. On the other hand, activation of sodium pump by garlic extract in the kidneys should reduce intracellular Na+ concentration and normalize BP. These findings signify the use of garlic in the treatment of hypertension.     

Differential role of Na+/H+ exchange isoforms NHE-1 and NHE-2 in a rat cortical collecting duct cell line

The Na+/H+ exchanger (NHE) constitutes a gene family containing several isoforms that display different membrane localization and are involved in specialized functions. Although basolateral NHE-1 activity was described in the cortical collecting duct (CCD), the localization and function of other NHE isoforms is not yet clear, This study examines the expression, localization, and regulation of NHE isoforms in a rat cortical collecting duct cell line (RCCD1) that has previously been shown to be a good model of CCD cells. Present studies demonstrate the presence of NHE-1 and NHE-2 isoforms, but not NHE-3 and NHE-4, in RCCD1 cells. Cell monolayers, grown on permeable filters, were placed on special holders allowing independent access to apical and basolateral compartments. Intracellular pH (pHi) regulation was spectrofluorometrically studied in basal conditions and after stimulation by NH4Cl acid load or by a hyperosmotic shock. In order to differentiate the roles of NHE-1 and NHE-2, we have used HOE-694, an inhibitor more selective for NHE-1 than for NHE-2. The results obtained strongly suggest that NHE-1 and NHE-2 are expressed in the basolateral membrane but that they have different roles: NHE-1 is responsible for pHi recovery after an acid load and NHE-2 is mainly involved in steady-state pHi and cell volume regulation.     

Regulation of Na+/H+ exchanger 1 allosteric balance by its localization in cholesterol- and caveolin-rich membrane microdomains

The Na+/H+ exchanger 1, which plays an essential role in intracellular pH regulation in most tissues, is also known to be a key actor in both proliferative and apoptotic processes. Its activation by H+ is best described by the Monod-Wyman-Changeux model: the dimeric NHE-1 oscillates between a low and a high affinity conformation, the balance between the two forms being defined by the allosteric constant L(0). In this study, influence of cholesterol- and caveolin-rich microdomains on NHE-1 activity was examined by using cholesterol depleting agents, including methyl-beta-cyclodextrin (MBCD). These agents activated NHE-1 by modulating its L(0) parameter, which was reverted by cholesterol repletion. This activation was associated with NHE-1 relocation outside microdomains, and was distinct from NHE-1 mitogenic and hormonal stimulation; indeed MBCD and serum treatments were additive, and serum alone did not change NHE-1 localization. Besides, MBCD activated a serum-insensitive, constitutively active mutated NHE-1 ((625)KDKEEEIRK(635) into KNKQQQIRK). Finally, the membrane-dependent NHE-1 regulation occurred independently of Mitogen Activated Protein Kinases, especially Extracellular Regulated Kinase activation, although this kinase was activated by MBCD. In conclusion, localization of NHE-1 in membrane cholesterol- and caveolin-rich microdomains constitutes a novel physiological negative regulator of NHE-1 activity.     

Determination of basolateral Na(+)/H(+) exchange activity in MDCK cells using a multiwell-multilabel reader

Na(+)/H(+) exchangers (NHE) are important membrane transport proteins involved in transepithelial transport and cellular pH homeostasis. NHE-1, important for cellular pH and volume homeostasis, is expressed in the basolateral membrane of epithelial cells. We evaluated the use of a multiwell-multilabel reader to investigate basolateral NHE-1 in confluent MDCK cells and compared the results with data obtained using an imaging system equipped with a filter perfusion system. Using the multiwell-multilabel reader we obtained virtually the same values for steady-state pH and NHE-1 activity under control conditions compared to the imaging system. With both setups Na(+)-dependent pH recovery after an acid load occurred virtually only after basolateral addition of Na(+). Furthermore, Na(+)-dependent pH recovery was reduced by >80% in the presence of the NHE-1 inhibitor HOE642. In addition, we detected an almost identical increase of NHE-1 activity with the two methods after stimulation of protein kinase C using phorbol myristate acetate. In summary, our data indicate that multiwell-multilabel readers are suitable to investigate physiology and regulation of basolateral NHE. Thus, multiwell-multilabel readers offer a valid and convenient alternative to investigate basolateral transporters.     

Hypertonicity activates Na+/H+ exchange through Janus kinase 2 and calmodulin

The type 1 sodium-hydrogen exchanger (NHE-1) is a ubiquitous electroneutral membrane transporter that is activated by hypertonicity in many cells. NHE-1 may be an important pathway for Na(+) entry during volume restoration, yet the molecular mechanisms underlying the osmotic regulation of NHE-1 are poorly understood. In the present study we conducted a screen for important signaling molecules that could be involved in hypertonicity-induced activation of NHE-1 in CHO-K1 cells. Hypertonicity rapidly activated NHE-1 in a concentration-dependent manner as assessed by proton microphysiometry and by measurements of intracellular pH on a FLIPR (fluorometric imaging plate reader). Inhibitors of Ca(2+)/calmodulin (CaM) and Janus kinase 2 (Jak2) attenuated this activation, whereas neither calcium chelation nor inhibitors of protein kinase C, the Ras-ERK1/2 pathway, Src kinase, and Ca(2+)/calmodulin-dependent enzymes had significant effects. Hypertonicity also resulted in the rapid tyrosine phosphorylation of Jak2 and STAT3 (the major substrate of Jak2) and CaM. Phosphorylation of Jak2 and CaM were blocked by AG490, an inhibitor of Jak2. Immunoprecipitation studies showed that hypertonicity stimulates the assembly of a signaling complex that includes CaM, Jak2, and NHE-1. Formation of the complex could be blocked by AG490. Thus, we propose that hypertonicity induces activation of NHE-1 in CHO-K1 cells in large part through the following pathway: hypertonicity --> Jak2 phosphorylation and activation --> tyrosine phosphorylation of CaM --> association of CaM with NHE-1 --> NHE-1 activation.     

Na(+)/H(+) exchange inhibition reduces hypertrophy and heart failure after myocardial infarction in rats

We investigated the effect of sodium/hydrogen exchange inhibition (NHE-1) on hypertrophy and heart failure after coronary artery ligation (CAL) in the rat. Animals were subjected to occlusion (or sham) of the left main coronary artery and immediately administered a control diet or one consisting of the NHE-1 inhibitor cariporide for 13-15 wk. Hearts were separated by small [30% of LV) infarcts. CAL depressed change in left ventricular increase in pressure over time (LV +dP/dt) in small and large infarct groups by 18.8% (P < 0.05) and 34% (P < 0.01), respectively, whereas comparative values for the cariporide groups were 8.7% (not significant) and 23.1% (P < 0.01), respectively. LV end-diastolic pressure was increased by 1,225% in the control large infarct group but was significantly reduced to 447% with cariporide. Cariporide also significantly reduced the degree of LV dilation in animals with large infarcts. Hypertrophy, defined by tissue weights and cell size, was reduced by cariporide, and shortening of surviving myocytes was preserved. Infarct sizes were unaffected by cariporide, and the drug had no influence on either blood pressure or the depressed inotropic response of infarcted hearts to dobutamine. These results suggest an important role for NHE-1 in the progression of heart failure after myocardial infarction.