Na~+-H~+交换蛋白的分子生物学及其对心血管系统的作用

Na~+-H~+交换蛋白(NHE)是细胞膜上的一种糖蛋白,分为4型。NHE-1型主要在心肌细胞上表达,参与胞内pH值、细胞容量、离子转运、细胞增殖的调节。NHE也参与某些心血管疾病的调节,如心肌缺血-再灌注损伤等。因此NHE抑制剂作为心肌保护药,具有潜在性应用前景。     

复方中药注射液对小白鼠艾氏腹水癌细胞膜表面的(Na~+-K~+)-ATP酶和微绒毛影响的电镜观察

本实验应用电镜细胞化学和扫描电镜技术,观察到小白鼠艾氏腹水癌细胞在复方中药注射液的连续作用下,膜表面(Na~+-K~+)-ATP酶活性减弱,微绒毛减退等变化,同时观察到该复方中药注射液对癌细胞增殖的抑制作用,抑瘤率可达87%,癌细胞增殖和(Na~+-K~+)-ATP酶活性及微绒毛的多少有平行关系。讨论了该复方中药抑制癌细胞增殖与膜表面(Na~+-K~+)-ATP酶活性和微绒毛变化的关系。     

常压氧对缺血再灌注大鼠大脑皮层1型Na~+/H~+交换蛋白的影响

目的:研究常压氧(NBO)对缺血再灌注大鼠脑组织1型Na+/H+交换蛋白(NHE1)水平的影响。方法:采用线栓法构建大脑中动脉缺血(MCAO)大鼠模型,术后2 h进行神经功能评分。MCAO模型构建成功者进行再灌注,随机分为MCAO组和NBO组,NBO组于再灌注后给予常压氧治疗。各组大鼠分别在NBO治疗后0、2、4、6、12、24 h处死,取脑皮层组织进行HE染色后观察脑组织形态结构的改变,并采用免疫荧光法及Western Blot法检测脑组织NHE1蛋白和缺氧诱导因子-1α(HIF-1α)的蛋白表达水平。结果:与假手术组相比,模型组神经功能评分显著增高(P 0. 05); HE染色结果显示MCAO组和NBO组大鼠缺血2 h时大脑皮层组织均无明显病理结构改变,随着再灌注时间的延长,MCAO组大鼠大脑皮层组织病理改变逐渐增加,NBO组大鼠的脑组织病理改变明显减轻;免疫荧光检查和Western Blot检测结果显示MCAO组和NBO组在起始2 h内,大脑皮层组织NHE1和HIF-1α蛋白表达水平没有明显变化,随着再灌注时间的延长,MCAO组NHE1蛋白和HIF-1α蛋白表达水平逐渐增加,NBO组NHE1蛋白和HIF-1α蛋白表达水平稍有增加,但是其表达水平明显低于MCAO组(P 0. 05)。结论:常压氧疗法可以下调缺血再灌注后大脑皮层组织NHE1蛋白和HIF-1α蛋白的表达,减轻大鼠脑缺血再灌注损伤,促进大鼠神经功能恢复,具有脑损伤保护作用。     

哌唑嗪对原发性高血压病人红细胞Ca~(2+),Mg~(2+)-ATP酶和钙调素的影响

本实验观察了原发性高血压(EH)病人红细胞Ca~(2+)Mg~(2+)-ATP酶活性,红细胞和血浆钙调素(CaM)的变化及哌唑嗪对其影响。结果表明:EH病人红细胞基础和最大Ca~(2+),Mg~(2+)-ATP酶活性显著下降,并与血压呈显著负相关;红细胞CaM活性也显著降低并与最大Ca~(2+),Mg~(2+)-ATP酶活性呈显著正相关;经哌唑嗪治疗后,EH病人红细胞Ca~(2+),Mg~(2+)-ATP酶活性和CaM变化不显著,但血浆CaM有所降低。提示EH病人红细胞钙代谢异常与外周阻力增高有密切关系,哌唑嗪对细胞和体液CaM有不同影响。     

侧脑室注射山莨菪碱对钙离子降压效应的影响

侧脑室注入氯化钙(200微克/kg),可使正常家兔的动脉血压明显降低,而侧脑室注入钙拮抗剂异搏停(100微克/kg)或山莨菪碱(200微克/kg)均可抵消钙离子(Ca~(2+))引起的低血压。这一结果提示,山莨菪碱对抗Ca~(2+)在中枢降压效应的机制可能和异搏停一样,是钙拮抗作用。     

外源性3’,5’环腺苷酸对肝癌-H_(22)腹水型癌细胞Mg~(++),(Na~+-K~+)-ATP酶、磷酸二酯酶影响的观察

本实验通过外源性3′,5′环腺苷酸和茶碱对小白鼠肝癌-H_(22)腹水型癌细胞的连续作用,观察到癌细胞的增殖受到明显抑制,伴随有癌细胞膜表面Mg~(++)-ATP酶、(Na~+-K~+)-ATP酶以及磷酸二酯酶活性明显下降;酶活性变化与膜表面微绒毛的变化,有一定平行关系。     

血管活性肠肽对人脐静脉内皮细胞Ca2+及其膜通道的调控

目的　观察血管活性肽 (VIP)对人脐静脉内皮细胞 (HUVEC)膜上Ca2 + 通道及胞浆内游离Ca2 +([Ca2 + ]i)的调控作用。方法　应用共聚焦激光扫描显微术 (CLSM)和膜片钳单通道记录技术对体外培养HU VEC胞浆内 [Ca2 + ]i、膜上Ca2 + 通道开放情况进行观察。结果　VIP促使HUVEC胞膜上Ca2 + 通过道的开放概率和胞浆内 [Ca2 + ]i 均明显升高。结论　IVP对HUVEC胞浆内 [Ca2 + ]i的调节可能除通过细胞内储存的Ca2 +释放外还依赖细胞膜上Ca2 + 通道开放 ,从而达到其调节效应     

大鼠正中视前核注射血管紧张素Ⅱ对近球小管Na~+,K~+-ATPase活性的影响

目的:探讨大鼠正中视前核(MnPO)的血管紧张素Ⅱ(AngⅡ)对肾近球小管Na~+, K~+-ATPase活性的作用及其途径.方法:SD大鼠MnPO注射AngⅡ,注射后15、 45、 60、 75、 105、 145min取肾,分离单根近球小管,测定Na~+, K~+-ATPase活性;注射后取血测定血清的内源性洋地黄样物质(EDLS).结果:与MnPO注射人工脑脊液(aCSF)相比,注射AngⅡ后60min,血清EDLS水平达峰值,近球小管Na~+, K~+-ATPase活性降到最低值,注射后105min,近球小管Na~+, K~+-ATPase的活性和血清EDLS水平分别恢复到对照水平.MnPO注射AngⅡ后145min内的EDLS浓度与其近球小管Na~+, K~+-ATPase活性呈显著负相关.结论:AngⅡ作用于MnPO可抑制近球小管的Na~+, K~+-ATPase活性;AngⅡ在MnPO是通过AT1受体介导的;AngⅡ在MnPO通过AT1受体介导,远距离抑制近球小管Na~+, K~+-ATPase活性的途径,可能与EDLS释放增多有关.     

Zn~(2+)预处理对离体大鼠缺血再灌注损伤心肌的保护作用

目的 :观察Zn2 + 预处理诱导金属硫蛋白 (MT)在心肌细胞的表达及对离体大鼠缺血再灌注损伤心肌的保护作用。方法 :3 2只Sprague Dawley大鼠随机分为对照组及实验组 ,实验组按腹腔注射ZnSO4 到建立再灌注模型的时间分为E1 2h,E2 4h及E4 8h组 ,每组各 8只。检测LDH ,CK、ATP含量及心功能指标 (LVSP与±dp/dtmax) ,用1 0 9Cd/血红素饱和法测量心肌组织中金属硫蛋白的含量。结果 :E2 4h及E4 8h组MT表达量较对照组高 (P <0 .0 1) ,E1 2h组虽有表达 ,但其与对照组间差异无显著性。E2 4h及E4 8h组LDH与CK较其它二组低 ,而ATP较其它二组高 ,心功能指标较其它二组改善。E1 2h组及对照组之间相应各指标 (LDH ,CK、ATP、LVSP与±dp/dtmax)间差异无显著性 (P >0 .0 5 )。结论 :Zn2 + 预处理可诱导MT在心肌细胞的表达而对离体大鼠缺血再灌注损伤心肌具有保护作用。     

腺相关病毒载体介导的PLB基因反义RNA对大鼠心肌细胞肌浆网Ca2+-ATPase活性和［Ca2+］i的作用

目的：探讨腺相关病毒载体介导的磷酸受纳蛋白（PLB）反义RNA对肌浆网Ca2+-ATPase活性以及细胞内钙浓度（［Ca2+］i）的作用。 方法： 构建PLB反义RNA的重组腺相关病毒载体（rAAV-asPLB）和携带报告基因LacZ的重组腺相关病毒载体（rAAV-LacZ）。分别转染培养的大鼠心肌细胞，测定PLB mRNA和蛋白质表达，检测肌浆网Ca2+-ATPase活性和［Ca2+］i。 结果： RT-PCR和Western blotting显示，感染rAAV-asPLB的心肌细胞的PLB mRNA和蛋白质表达低于正常对照和感染rAAV-LacZ组；而Ca2+-ATPase活性大于正常对照和感染rAAV-LacZ组。激光共聚焦显微镜检测显示，静息状态时，rAAV-asPLB感染组［Ca2+］i降低；异丙肾上腺素刺激后，各组［Ca2+］i均升高，但是rAAV-asPLB感染组［Ca2+］i变化幅度大。 结论： 腺相关病毒介导的PLB反义RNA对大鼠心肌细胞PLB表达具有抑制作用，增强Ca2+-ATPase活性，降低静息状态的［Ca2+］i ，增加异丙肾上腺素刺激后［Ca2+］i 的变化幅度。     

异搏停和山莨菪碱对大鼠被动型Heymann肾炎病变的影响

本实验应用抗大鼠肾小管抗原的抗血清制做大鼠被动型Ｈｅｙｍａｎｎ肾炎（ＰＨＮ）动物模型，并选用异搏停和山莨菪碱进行处理，观察两药对其病变的影响，结果表明：异搏停及山莨菪碱处理的大鼠尿蛋白均明显低于ＰＨＮ组，病理组织损伤亦有一定程度的改善。提示：上述两药均对大鼠ＰＨＮ病变有一定的治疗作用。     

Intracellular Na+ measurements in smooth muscle using SBFI – changes in [Na+], Ca2+ and force in normal and Na+-loaded ureter

 Our understanding of the control and effects of intracellular [Na⁺] ([Na⁺]_i) in intact smooth muscle is limited by the lack of data concerning [Na⁺]_i. The initial aim of this work was therefore to investigate the suitability of using the Na⁺-sensitive fluorophore SBFI in intact smooth muscle. We find this to be a good method for measuring [Na⁺]_i in ureteric smooth muscle. Resting [Na⁺]_i was found to be around 10 mM and rose to 25 mM when the Na⁺-K⁺-ATPase was inhibited by ouabain. This relatively low [Na⁺]_i in the absence of Na⁺-K⁺-ATPase suggests that other cellular processes, such as Na⁺-Ca²⁺ exchange, play a role in maintaining [Na⁺]_i under these conditions. Simultaneous measurements of [Na⁺]_i or [Ca²⁺] _i and force showed that Na⁺-Ca²⁺ exchange can play a functional role in ureteric smooth muscle. We found that the greater the driving force for Na⁺ exit and hence Ca²⁺ entry, the larger the contraction. In addition the Na⁺-Ca²⁺ exchanger activity under these conditions was found to be pH sensitive: acidification reduced the contraction and concomitant changes in [Ca²⁺] and [Na⁺]_i. We conclude that SBFI is a useful method for monitoring [Na] in smooth muscle and that Na⁺-Ca²⁺ exchange may play a functional role in the ureter. Received: 26 August 1997 / Received after revision: 27 October 1997 / Accepted: 28 October 1997     

654—2对家兔红细胞膜Na^＋—K^＋—ATPase活性及血浆LPO含量的影响

日本大耳兔9只,其中6～8月龄5只,2～2.5年龄4只。均喂以2％654-2溶液1ml/天(即20mg)共43天。结果不分年龄统计,LPO含最下降0.827±1.048 nmol/ml(P<0.05)。Na~+-K~+-ATPase活性增加0.057±0.074μmol Pi/mg pro/hr(P<0.05)。认为654-2可能有抗衰老作用,并与普鲁卡因比较,讨论了其降低LPO的可能途径。     

Effect of dehydration on apical Na+-H+ exchange activity and Na+-dependent sugar transport in brush-border membrane vesicles isolated from chick intestine

 The current work examines the effect of 4 days of water deprivation on Na⁺-H⁺ exchange and Na⁺-sugar cotransport systems in brush-border membrane vesicles isolated from either the jejunum, ileum or the colon of the chick. Apical Na⁺-H⁺ exchange activity was evaluated by measuring the pH-gradient-dependent Na⁺ uptake. The contribution of the Na⁺-H⁺ exchangers NHE2 and NHE3 to total Na⁺-H⁺ exchange activity was evaluated from their sensitivity to the amiloride-related drug HOE694. Dehydration increased plasma aldosterone levels from 12 to 70 pg/ml and also the activities of both Na⁺-H⁺ exchange and Na⁺-dependent sugar transport in the three intestinal regions tested. Na⁺-independent sugar transport was not modified by 4 days of water deprivation. In the ileum and colon the increase in Na⁺-H⁺ exchange activity was due to an increase in NHE2 activity, whereas in the jejunum it was due to an increase in both NHE2 and NHE3. Since we have previously reported that chronic Na⁺ depletion increases plasma aldosterone levels and NHE2 activity in ileum and colon, decreased small and large intestine Na⁺-sugar cotransport activity and had no effect on jejunal apical Na⁺-H⁺ exchange activity, it can be concluded that: (1) aldosterone does not regulate intestinal Na⁺-dependent sugar transport, and (2) the regulation of jejunal Na⁺-H⁺ exchange activity differs from that of either the ileum or the colon. Received: 31 October 1997 / Received after revision: 17 December 1997 / Accepted: 8 January 1998     

三种莨菪药对急性肾衰大鼠血栓素及前列环素代谢的影响

本文研究了急性肾衰时血栓素及前列环素代谢的改变，观察了樟柳碱，东莨菪碱及山莨菪碱对这些改变的影响。结果表明，肾衰后24小时，左肾皮质及髓质血栓素B2（TXB2）及6－酮－前列腺素F1α（6KF）均明显增高，6KF与TXB2比值无明显改变；肾衰后48小时，左肾皮质及髓质TXB2明显增高，6KF增高不明显，6KF与TXB2比值明显降低。三种药物对肾组织TXB2及6KF无明显影响．但均可提高血液中6KF含量，使6KF与TXB2比例增加。其作用以东莨菪碱最明显，其次是樟柳碱及山莨菪碱。结果说明，肾组织TXB2和6KF比例的变化可能是急性肾衰维持期肾组织血流量持续降低，肾功能恶化的重要因素。东莨菪碱及樟柳碱可提高血液中前列环素含量，这可能有助于肾组织血流量的恢复。     

Expression and function of Na+/Ca2+ exchangers 1 and 3 in human macrophages and monocytes

The Na⁺/Ca²⁺ exchanger (NCX) is a membrane transporter that can switch Na⁺ and Ca²⁺ in either direction to maintain the homeostasis of intracellular Ca²⁺. Three isoforms (NCX1, NCX2, and NCX3) have been characterized in excitable cells, e.g. neurons and muscle cells. We examined the expression of these NCX isoforms in primary human lung macrophages (HLM) and blood monocytes. NCX1 and NCX3, but not NCX2, are expressed in HLM and monocytes at both mRNA and protein levels. Na⁺‐free medium induced a significant increase in intracellular calcium concentration ([Ca²⁺]_i) in both cell types. This response was completely abolished by the NCX inhibitor 5‐(N‐4‐chlorobenzyl)‐20,40‐dimethylbenzamil (CB‐DMB). Moreover, inhibition of NCX activity during Ca²⁺‐signaling induced by histamine caused a delay in restoring baseline [Ca²⁺]_i. Na⁺‐free medium induced TNF‐α expression and release in HLM comparable to that caused by LPS. TNF‐α release induced by Na⁺‐free medium was blocked by CB‐DMB and greatly reduced by RNAi‐mediated knockdown of NCX1. These results indicate that human macrophages and monocytes express NCX1 and NCX3 that operate in a bidirectional manner to restore [Ca²⁺]_i, to generate Ca²⁺‐signals, and to induce TNF‐α production. Therefore, NCX may contribute to regulate Ca²⁺ homeostasis and proinflammatory functions in human macrophages and monocytes.     

Activation of Ca2+-sensitive Cl– current by reverse mode Na+/Ca2+ exchange in rabbit ventricular myocytes

 We investigated how Ca²⁺-sensitive transient outward current, I _to(Ca), is activated in rabbit ventricular myocytes in the presence of intracellular Na⁺ (Na⁺ _i) using the whole-cell patch-clamp technique at 36°C. In cells dialysed with Na⁺-free solutions,the application of nicardipine (5 μM) to block L-type Ca²⁺ current (I _Ca) completely inhibited I _to(Ca). In cells dialysed with a [Na⁺]_i≥5 mM, however, I _to(Ca) could be observed after blockade of I _Ca, indicating the activity of an I _Ca-independent component. The amplitude of I _Ca-independent I _to(Ca) increased with voltage in a [Na⁺]_i-dependent manner. The block of Ca²⁺ release from the sarcoplasmic reticulum by caffeine, ryanodine or thapsigargin blocked I _Ca-independent I _to(Ca). In Ca²⁺-free bath solution I _to(Ca) was completely abolished. The application of 2 mM Ni²⁺ or the newly synthesized compound KBR7943, a selective blocker of the reverse mode of Na⁺/Ca²⁺ exchange, or perfusion with pipette solution containing XIP (10 μM), a selective blocker of the exchanger, blocked I _Ca-independent I _to(Ca). From these results we conclude that, in the presence of Na⁺ _i, I _to(Ca) can be activated via Ca²⁺-induced Ca²⁺ release triggered by Na⁺/Ca²⁺ exchange operating in the reverse mode after blockade of I _Ca. Received: 20 January 1998 / Received after revision: 6 July 1998 / Accepted: 25 July 1998     

Regulation of Post-Tetanic Increases in the Cholinosensitivity of Neurons in the Common Snail by the Na,K Pump: The Role of Na/Ca Exchange and Calcium Mobilization

Neuroscience and Behavioral Physiology -     

Role of anions in the regulation of cell volume and intracellular pH in perfused rat mandibular salivary gland

 To investigate the role of Cl^– in the regulation of the basolateral transporters of salivary acinar cells, we have measured cell volume and intracellular pH (pH_i) in perfused rat mandibular glands using proton NMR spectroscopy and BCECF fluorometry respectively. When perfusate Cl^– was replaced by glucuronate, isethionate, methylsulphate, nitrate or thiocyanate, cell volume decreased slowly by about 15% over a 10-min period. Replacement with bromide, which substitutes for Cl^– on the Na⁺-K⁺-2Cl^– cotransporter, caused only a small (4%) reduction in cell volume. Replacement of Cl^– by glucuronate, isethionate or methylsulphate evoked a biphasic increase in pH_i consisting of a rapid initial increase followed by a slower secondary rise whose time course was similar to that of cell shrinkage. As judged by the effects of HCO₃ ^– omission, 100 μM 4,4’-diisothiocyanatostilbene-2,2’-disulphonic acid (DIDS) and 1 mM amiloride, the initial rise in pH_i was due to Cl^–/HCO₃ ^– exchange while the secondary rise resulted from activation of Na⁺/H⁺ exchange. Although replacement of Cl^– by nitrate or thiocyanate also caused cell shrinkage, these substituting anions were less effective in activating the exchanger. Therefore, while the upregulation of the exchanger following Cl^– replacement may be due in part to cell shrinkage, there is also evidence for the involvement of an anion-sensitive regulatory mechanism. This would be consistent with the hypothesis that both changes in cell volume and in intracellular Cl^– concentration contribute to the up-regulation of the exchanger following muscarinic stimulation. Received: 12 November 1996 / Received after revision: 11 August 1997 / Accepted: 18 August 1997     

急性肾衰大鼠肾阻抗容积脉波的改变及三种莨菪药的治疗作用

用自己建立的阻抗法描记大鼠肾容积脉波，研究油酸致急性肾衰时肾阻抗容积脉波的改变及樟柳碱、东莨菪碱、山莨菪碱对这些改变的影响。结果表明：左肾动脉注射油酸后１０ｍｉｎ、１ｈ、６ｈ出现低平及平顶波，波高及流入容积速度明显低于注射前及生理盐水对照组。三种莨菪药治疗后，低平及平顶波明显减少或消失，波高及流入容积速度明显提高。结果说明：急性肾衰早期肾内脉动血管弹性降低，紧张度增高，周围阻力增加，肾组织血液灌流量降低；三种莨菪药均可改善肾内脉动血管的功能，提高肾组织血液灌流量