离体灌流大鼠心脏Na^＋／H^＋交换的意义

离体大鼠等容收缩心脏预灌流30min,再换用含20mmol/L NH_4Cl的K-H液灌流10min后,随机分为两组:(1)对照组(n=5): 恢复常规K-H液冲洗15min;(2)给药组(n=5):在常规K-H液冲洗时,加入0.5mmol/L amiloride以抑制Na~+/H~+交换。结果如下:预灌及NH_4Cl负荷阶段,两组血液动力学指标无明显差别;在冲洗时,对照组血液动力学指标逐渐恢复,而给药组心肌一直处于挛缩状态。对照组心肌组织Na~+、Ca~(2+)、K~+含量高于给药组;而心肌组织MDA及冠脉流出液中LDH活性则低于给药组。因此,酸负荷状态下,Na~+/H~+交换是pHi及生理活动恢复的重要调节机制,抑制这—过程,将加重心肌组织的损伤,以至于死亡。     

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     

Physiology and molecular biology of the renal Na/H antiporter

Summary The renal Na/H antiporter is involved in cell pH regulation and, predominantly in the proximal tubule, also mediates transcellular NaCl and NaHCO₃ transport. The transporter is regulated by systemic acid-base factors and hormones such as angiotensin II, PTH, glucocorticoids and alpha₂-adrenergics. The human gene of the Na/H antiporter has been cloned and sequenced recently.     

S3226, a novel inhibitor of Na+/H+ exchanger subtype 3 in various cell types

 Inhibition of Na⁺/H⁺ exchange (NHE) subtypes has been investigated in a study of the mouse fibroblast L cell line (LAP1) transfected with human (h) NHE1, rabbit (rb) NHE2, rat (rt) or human (h) NHE3 as well as an opossum kidney cell line (OK) and porcine renal brush-border membrane vesicles (BBMV). S3226 {3-[2-(3-guanidino-2-methyl-3-oxo-propenyl)-5-methyl-phenyl]-N-isopropylidene-2-methyl-acrylamide dihydro-chloride} was the most potent and specific NHE3 inhibitor with an IC₅₀ value of 0.02 μmol/l for the human isoform, whereas its IC₅₀ value for hNHE1 and rbNHE2 was 3.6 and @80 μmol/l, respectively. In contrast, amiloride is a weak NHE3 inhibitor (IC₅₀>100 μmol/l) with a higher affinity to hNHE1 and rbNHE2. Cariporide (4-isopropyl-3-methylsulphonyl-benzoyl-guanidine methane-sulphonate), which has an IC₅₀ for NHE3 of approximately 1 mmol/l, is a highly selective NHE1 inhibitor (0.08 μmol/l). Therefore, S3226 is a novel tool with which to investigate the physiological and pathophysiological roles of NHE3 in animal models. Received: 14 May 1998 / Received after revision: 29 June 1998 / Accepted: 2 July 1998     

High salt diet down-regulates proximal tubule Na+, K+-ATPase activity in Dahl salt-resistant but not in Dahl salt-sensitive rats: evidence of defective dopamine regulation

Nishi , A., Bertorello , A. M. & Aperia , A. 1992. High salt diet down-regulates proximal tubule Na⁺, K⁺-ATPase activity in Dahl salt-resistant but not in Dahl salt-sensitive rats; evidence of defective dopamine regulation. Acta Ptiysiol Scand 144 , 263–267. Received 26 July 1991, accepted 25 October 1991. ISSN 0001–6772. Department of Paediatrics, Karolinska Institute, Sweden We examined the regulation of Na⁺, K⁺-ATPase activity in proximal tubule segments during a high salt diet in prehypertensive Dahl salt-sensitive and salt-resistant rats. Rats were placed on normal salt or high salt diets (0.9% saline as drinking water). During the normal salt diet, Na⁺, K⁺-ATPase activity was not different between Dahl salt-sensitive and salt-resistant rats. After 2 days and 10 days on a high salt diet, Na⁺, K⁺-ATPase activity in Dahl salt-resistant rats significantly decreased when compared to Dahl salt-resistant rats on a normal salt diet (P < 0.01). The decreased Na⁺, K⁺-ATPase activity in Dahl salt-resistant rats during a high salt diet was reversed by treatment with an inhibitor of aromatic l -amino acid decarboxylase (dopamine synthesizing enzyme), benserazide. In contrast, Na⁺, K⁺-ATPase activity did not decrease during the high salt diet and benserazide had no effect on Na⁺, K⁺-ATPase activity in Dahl salt-sensitive rats. These results indicate that Dahl salt-sensitive rats do not have the capacity to down-regulate the proximal tubule Na⁺, K⁺-ATPase activity during a high salt diet. Indirect evidence suggests that the regulation of Na⁺, K⁺-ATPase activity by locally produced dopamine is absent in Dahl salt-sensitive rats.     

钠氢交换体1在星形细胞瘤中的表达及其与恶性程度的关系*

目的：检测人星形细胞瘤和正常脑组织中钠氢交换体1（ NHE1）的表达差异及其与恶性程度的关系,探 讨星形细胞瘤增殖、生长的分子机制。方法：收集人星形细胞瘤标本51 例,低、高级别星形细胞瘤组织分别为 22 例、29 例,以肿瘤周围相对正常脑组织作为对照。用H-E 染色进行诊断和分级,免疫组织化学和免疫印迹检 测肿瘤组织与正常脑组织中NHE1表达变化。结果：NHE1主要分布在对照组神经元和少量星形胶质细胞胞膜上; 在肿瘤组织中,NHE1分布在低级别星形细胞瘤细胞膜上,并强烈表达于高级别星形细胞瘤的胞质和胞膜上。与 对照组相比较,在低级别和高级别星形细胞瘤组织中NHE1表达上调,其中,恶性程度较高的高级别肿瘤相对于 恶性程度低的低级别肿瘤,NHE1的表达更为强烈。结论：NHE1在人星形细胞瘤组织中表达增强,其强度变化 与肿瘤的恶性程度有关。     

Na+-H+ exchange is not important for pancreatic HCO-3 secretion in the pig

Veel , T., Villanger , O., Holthe , M.R., Cragoe jr ., E.J. & Reder , M.G. 1992. Na⁺-H exchange is not important for pancreatic HCO^-₃ secretion in the pig. Acta Physiol Scand 144 , 239–246. Received 13 September 1991, accepted 14 November 1991. ISSN 0001–6772. University of Oslo, Institute for Experimental Medical Research and Surgical Department, Ullevaal Hospital, Oslo, Norway. Pancreatic inter- and intralobular duct cells extrude H⁺-ions to interstitial fluid when they secrete HCO^-₃ to pancreatic juice. This study assesses the potential importance of Na⁺-H⁺-ion exchange for H⁺-ion extrusion and secretion of HCO^-₃ using the Na⁺-H⁺ exchange blockers amiloride and hexamethylene-amiloride. Intracellular pH (pH,) in inter- and intralobular pancreatic duct epithelium was measured using BCECF fluorescence. H⁺-ion efflux was measured using a NH₄Cl prepulse, acid-loading technique. In HCO^-₃-free media, pH₁ recovery following acid loading was blocked by amiloride (10^-4 m) and hexamethylene-amiloride (10^-6 m) , demonstrating amiloride-and hexamethylene-amiloride-sensitive Na⁺-H⁺ exchange. However, 5 × 10^-6 M hexamethylene-amiloride did not reduce secretin-dependent pancreatic HCO, secretion in vivo. Maximal H⁺-efflux through Na⁺-H⁺ exchange was 1.5 ± 0.2μmol min^-1 ml cell volume^-l, i.e. less than 1 % of estimated net H⁺-ion efflux during HCO^-₃ secretion. Conclusion: amiloride- and hexamethylene amiloride sensitive Na⁺-H⁺ exchange is not important for secretin-dependent pancreatic HCO^-₃ secretion in the pig. Other mechanisms for H⁺ extrusion dominate.     

Na+/H+ exchanger regulatory factor 1 expression levels in blood and tissue predict breast tumour clinical behaviour

Bellizzi A, Mangia A, Malfettone A, Cardone R A, Simone G, Reshkin S J & Paradiso A
(2011) Histopathology 58, 1086–1095
Na ⁺ /H ⁺ exchanger regulatory factor 1 expression levels in blood and tissue predict breast tumour clinical behaviour Aims: Several studies have demonstrated that Na⁺/H⁺ exchanger regulatory factor 1 (NHERF1) protein, which is overexpressed and heterogeneously distributed in different stages of breast cancer, could be used as a tumour marker for prognosis in molecular detection strategies. We observed that tumour‐infiltrated lymphocytes in the tumour tissue display a high level of NHERF1 staining, in contrast to those present in the contiguous non‐involved tissue. Hypothesizing that cancer cells elicit a specific T‐cell response associated with the characteristics of the solid tumour, our aim was to evaluate NHERF1 in peripheral lymphocytes from healthy donors and breast cancer patients. Methods and results: NHERF1 levels were analysed in 55 breast cancer patients and 40 healthy donors, and these levels were compared with clinical pathological features. NHERF1 was overexpressed in circulatory peripheral lymphocytes from patients as compared with those from healthy subjects. Furthermore, in both circulatory lymphocytes and tissues, NHERF1 was positively associated with tumour grade, Nottingham Prognostic Index and oestrogen receptor, whereas there was no association with other clinical parameters in either tissue. Conclusions: We propose that NHERF1 measurements in circulatory lymphocytes of breast cancer patients may be a valid method for the prediction of breast cancer occurrence and prognosis, and may have value in the management of cancer patients.     

Selective inhibition of the Na+/H+ exchanger type 3 activates CO2/H+-sensitive medullary neurones

Hypercapnia as well as lowered intracellular pH (pH_i) increase the bioelectric activity of CO₂/H⁺-sensitive neurones (VLNcs) of the ventrolateral medulla oblongata. Here we describe that immunoreactive Na⁺/H⁺ exchanger (NHE3) is present in ventrolateral neurones from medullary organotypic cultures (obex level). To test whether VLNcs can be acidified and thereby activated by inhibition of NHE3, we used the novel high-affinity NHE3-inhibitors S1611 and S3226. Both drugs raised the firing rates of VLNcs to at least 150% of the control values, and depolarized membrane potential by up to 15 mV at concentrations (0.5–1 μmol/l) suitable for selective inhibition of NHE3. The changes in bioelectric activity strongly resembled the responses to hypercapnia (PCO₂: 60–100 mmHg). In BCECF-AM-loaded cultures a subfraction of ventrolateral VLNcs was found to be intracellularly acidified by 0.05–0.1 pH units following treatment with S1611; the time course of this acidification was similar to that evoked by hypercapnia. All drug effects were sustained and readily reversible upon washing. Non-CO₂/H⁺-responsive medullary neurones as well as hippocampal CA3 neurones were unaffected by up to 20 μmol/l S1611. It is concluded that the selective inhibition of NHE3 acidifies and activates CO₂/H⁺-sensitive neurones within the ventrolateral medulla oblongata. Received: 12 February 1999 / Received after revision and accepted: 15 April 1999     

Characterization of the Na+/H+ exchanger in human choriocarcinoma (BeWo) cells

 We examined the expression and activity of the Na⁺/H⁺ exchanger in the human choriocarcinoma BeWo cell line. When treated with methotrexate, these cells differentiated from cytotrophoblast-like cells to enlarged multinucleate syncytiotrophoblast-like cells. There was no change in the apparent K _m for Na⁺ between undifferentiated and differentiated cells. However, differentiated cells could transport more than five times the proton flux of undifferentiated cells. There was no difference in the Hill coefficient between undifferentiated and differentiated cells. However, the maximal flux (J _max) for undifferentiated cells was higher than that for differentiated cells. Inhibition of Na⁺/H⁺ exchange activity by an amiloride analog and Hoe694 revealed a sensitive and a resistant component in both differentiated and undifferentiated cells. Northern blot analysis and immunocytochemistry suggested that the sensitive component was due to the NHE1 isoform of the protein while the resistant component was due to the NHE3 isoform. The NHE1 isoform was localized to the brush border membrane of BeWo cells and Western blot analysis showed that the NHE1 protein was more abundant in brush border membranes from differentiated BeWo cells compared to undifferentiated cells. The results show that BeWo cells contain the NHE1 and NHE3 isoforms of the Na⁺/H⁺ exchanger and that the NHE1 isoform is primarily localized to the brush border membrane. Received: 25 July 1996 / Received after revision: 25 Novembber 1996 / Accepted: 3 December 1996     

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.     

Severe acute respiratory syndrome coronavirus 2 may cause liver injury via Na+/H+ exchanger

The liver has many significant functions, such as detoxification, the urea cycle, gluconeogenesis, and protein synthesis. Systemic diseases, hypoxia, infections, drugs, and toxins can easily affect the liver, which is extremely sensitive to injury. Systemic infection of severe acute respiratory syndrome coronavirus 2 can cause liver damage. The primary regulator of intracellular p H in the liver is the Na⁺/H⁺ exchanger(NHE). Physiologically, NHE protects hepatocytes from ap...     

Immunolocalisation of NHE3-like immunoreactivity in the gills of the rainbow trout (Oncorhynchus mykiss) and the blue-throated wrasse (Pseudolabrus tetrious)

Na⁺/H⁺ exchange has been implicated in models of ion transport across the branchial epithelium of marine and freshwater fishes. In this preliminary study, we present immunohistochemical data using a polyclonal antibody raised against NHE3 which show NHE3-like immunoreactivity (IR) in the gills from a freshwater and a marine teleost species. In both species, branchial epithelial cells demonstrating NHE3-like IR were localised predominantly to the junction between the filament and the secondary lamellae. However, there was a marked difference in the morphology of the NHE3-like immunoreactive epithelial cells between the species. This morphological difference between the species suggests functional differences in the exchanger, which may be related to marine versus freshwater environments.     

Exercise-induced regulation of phospholemman (FXYD1) in rat skeletal muscle: implications for Na+/K+-ATPase activity

Background: Na⁺/K⁺‐ATPase activity is upregulated during muscle exercise to maintain ionic homeostasis. One mechanism may involve movement of α‐subunits to the outer membrane (translocation). Aim: We investigated the existence of exercise‐induced translocation and phosphorylation of phospholemman (PLM, FXYD1) protein in rat skeletal muscle and exercise‐induced changes in V_max and K_m for Na⁺ of the Na⁺/K⁺‐ATPase. Methods: Two membrane fractionation methods and immunoprecipitation were used. Results: Both fractionation methods revealed a 200–350% increase in PLM in the sarcolemma after 30 min of treadmill running, while the phosphorylation of Ser‐68 of PLM appeared to be unchanged. Exercise did not change V_max or K_m for Na⁺ of the Na⁺/K⁺‐ATPase in muscle homogenate, but induced a 67% increase in V_max in the sarcolemmal giant vesicle preparation; K_m for Na⁺ remained constant. The main part of the increase in V_max is related to a 36–53% increase in the level of α‐subunits; the remainder may be related to increased PLM content. Similar results were obtained with another membrane purification method. In resting muscle, 29% and 32% of α₁‐ and α₂‐subunits, respectively, were co‐immunoprecipitated by PLM antibodies. In muscle homogenate prepared after exercise, immunoprecipitation of α₁‐subunits was increased to 227%, whereas the fraction of precipitated α₂ remained constant. Conclusion: Exercise translocates PLM to the muscle outer membrane and increases its association with mainly the α₁‐subunit, which may contribute to the increased V_max of the Na⁺/K⁺‐ATPase.     

Vanadyl ions stimulate K+ uptake into isolated perfused rat liver via the Na+/K+-pump by a tyrosine kinase-dependent mechanism

 Vanadium salts mimic most metabolic effects of insulin in vitro. We report here that vanadyl sulfate (VOSO₄) and sodium vanadate (NaVO₃) stimulate net K⁺ uptake in isolated perfused rat liver. Stimulation was evident at low concentrations of vanadyl ions (range 1–20 μM) and occurred within minutes following the addition of VOSO₄. By comparison with VOSO₄, insulin had less of a stimulatory effect on K⁺ uptake. Ouabain prevented the activating effect of VOSO₄ on K⁺ uptake. Following a VOSO₄ challenge, measured intracellular Na⁺ concentration ([Na⁺]_i) fell (control, 17.1 ± 1.2; VOSO₄-treated, 13.0 ± 1.1 mmol·g^–1 wet weight, P = 0.027). The results indicate that active K⁺ uptake via the Na⁺/K⁺-ATPase was stimulated by vanadyl ions. An indirect mechanism due to changes in [Na⁺]_i can be excluded. The tyrosine kinase inhibitor genistein was found to inhibit stimulation of K⁺ by vanadyl and vanadate ions which are known inhibitors of phosphotyrosine phosphatases. We conclude that stimulation of active K⁺ influx involves a tyrosine kinase. Possible mechanisms include phosphorylation at tyrosine residues and direct activation of the Na⁺/K⁺-ATPase, or phosphorylation of other proteins that regulate the activity or number of pumps in the cells. Received: 8 July 1997 / Received after revision: 3 November 1997 / Accepted: 11 November 1997     

Evidence for the presence of a Na+-H+ exchanger in the endolymphatic sac epithelium of guinea-pigs

 The intracellular pH (pH_i) of epithelial cells from the endolymphatic sac (ES) of the guinea-pig was measured microfluorometrically with the pH-sensitive fluorescent dye, 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) to examine the presence of a Na⁺-H⁺ exchanger (NHE) in the ES epithelial cells. pH_i recovery from acid loading with an NH₄ ⁺-prepulse in a nominally HCO₃-free solution was dependent on extracellular Na⁺ ([Na⁺]_o) and was inhibited by amiloride and its analogue ethylisopropylamiloride (EIPA), suggesting that a decreased pH_i induced by an acute acid load may be equilibrated by a NHE. In the steady-state, amiloride had no effect on pH_i, indicating that the NHE activity is low at the resting pH_i. However, the intracellular acidification induced by the removal of [Na⁺]_o was inhibited by the simultaneous application of amiloride. H⁺-efflux rate (J _H, mean activity of NHE), which was calculated as the product of the recovery rate (dpH_i/dt) from the acid loading and the intrinsic buffering capacity (β_i) at the corresponding pH_i, was decreased as pH_i was increased. The concentration/response curve for the inhibition of initial J _H by EIPA revealed an apparent 50% inhibitory constant (K _i ) of 0.85 μM. Kinetic analysis of initial J _H as a function of [Na⁺]_o revealed a Michaelis-Menten constant (K _m) of 24.14 mM for Na⁺-dependent H⁺ efflux. The results indicate that NHE in the ES epithelium belongs to an amiloride-sensitive subtype. Received: 5 August 1997 / Received after revision and accepted: 26 February 1998     

人肝再生增强因子在体外可与Na+-K+-ATPase 特异性结合

目的： 用体外下拉实验证明人肝再生增强因子（hALR）与其相互作用蛋白Na+-K+-ATPase的体外结合作用。方法：将Na+-K+-ATPase β亚基部分基因片段定向克隆到pGEX-4T-1 中, 转化大肠杆菌DH5α，IPTG 诱导, 获得Na+-K+-ATPase β亚基部分蛋白与谷胱甘肽（GST）的融合表达，经GST偶联的琼脂糖珠纯化, 以GST下拉实验检测其与hALR蛋白的体外直接相互作用。结果：还原型SDS-PAGE 显示GST- Na+-K+-ATPase 融合蛋白泳道有hALR蛋白单体和二聚体条带，Western blotting 结果进一步证实为hALR蛋白。结论：Na+-K+-ATPase可在体外与hALR蛋白特异地结合。