The Arabidopsis thaliana salt tolerance gene SOS1 encodes a putative Na+/H+ antiporter

In Arabidopsis thaliana, the SOS1 (Salt Overly Sensitive 1) locus is essential for Na(+) and K(+) homeostasis, and sos1 mutations render plants more sensitive to growth inhibition by high Na(+) and low K(+) environments. SOS1 is cloned and predicted to encode a 127-kDa protein with 12 transmembrane domains in the N-terminal part and a long hydrophilic cytoplasmic tail in the C-terminal part. The transmembrane region of SOS1 has significant sequence similarities to plasma membrane Na(+)/H(+) antiporters from bacteria and fungi. Sequence analysis of various sos1 mutant alleles reveals several residues and regions in the transmembrane as well as the tail parts that are critical for SOS1 function in plant salt tolerance. SOS1 gene expression in plants is up-regulated in response to NaCl stress. This up-regulation is abated in sos3 or sos2 mutant plants, suggesting that it is controlled by the SOS3/SOS2 regulatory pathway.     

The plasma membrane Na+/H+ antiporter SOS1 interacts with RCD1 and functions in oxidative stress tolerance in Arabidopsis

The adverse effects of high salt on plants include Na(+) toxicity and hyperosmotic and oxidative stresses. The plasma membrane-localized Na(+)/H(+) antiporter SOS1 functions in the extrusion of toxic Na(+) from cells and is essential for plant salt tolerance. We report here that, under salt or oxidative stress, SOS1 interacts through its predicted cytoplasmic tail with RCD1, a regulator of oxidative-stress responses. Without stress treatment, RCD1 is localized in the nucleus. Under high salt or oxidative stress, RCD1 is found not only in the nucleus but also in the cytoplasm. Like rcd1 mutants, sos1 mutant plants show an altered sensitivity to oxidative stresses. The rcd1mutation causes a decrease in salt tolerance and enhances the salt-stress sensitivity of sos1 mutant plants. Several genes related to oxidative-stress tolerance were found to be regulated by both RCD1 and SOS1. These results reveal a previously uncharacterized function of a plasma membrane Na(+)/H(+) antiporter in oxidative-stress tolerance and shed light on the cross-talk between the ion-homeostasis and oxidative-stress detoxification pathways involved in plant salt tolerance.     

Regulation of myocardial Na+/H+ exchanger activity

The Na⁺/H⁺ exchanger is a plasma membrane protein, present in the myocardium, which removes intracellular protons and exchanges them with extracellular Na⁺. The protein comprises an N-terminal, hydrophobic, integral membrane domain that transports the ions and a C-terminal, hydrophilic region that regulates the N-terminal domain. The C-terminal domain has several sub-domains, including one region that binds calmodulin and another that is phosphorylated by protein kinases. The Na⁺/H⁺ exchanger is activated by angiotensin, endothelin and α1-adrenergic stimulation. These effectors increase phosphorylation of the C-terminal domain by protein kinases, and G proteins have been implicated in this, but their role remains to be defined. It has recently been shown that ischemia and other stimuli lead to an increased expression of the Na⁺/H⁺ exchanger in the myocardium. The role of this increased expression in the pathology of ischemia and reperfusion-mediated myocardial damage has yet to be determined. Recent evidence suggests that the Na⁺/H⁺ exchanger may play a key role in hypertrophy of the myocardium, and that its activation through G protein-coupled receptors may be important in mediating its effects. Received: 23 April 2001 / Accepted: 14 May 2001     

Regulation of the luminal Na+/H+ exchanger NHE3 by intracellular protein trafficking

The article summarizes some of the recent developments in the understanding of the mechanisms of regulation of the proximal tubule apical membrane Na+/H+ antiporter NHE3. NHE3 antiporter has a major role in HCO3- and NaCl reabsorption in the proximal tubule. NHE3 protein is associated with the regulatory factor NHERF which interacts with ezrin, an actin-binding protein. This multi-protein complex constitutes a link between a membrane protein, NHE3, and actin cytoskeleton. Cytoskeleton organization has a key role to control NHE3 activity under normal conditions. Pharmacological perturbations of actin polymerization interfere with NHE3 activity. Parathyroid hormone-induced NHE3 activity inhibition results first, from a protein kinase A-mediated phosphorylation without protein trafficking, and then from endocytosis involving dynamin. The stimulatory effect of systemic angiotensin II concentrations on NHE3 activity is protein kinase C-dependent and results, at least in part, from exocytic insertion of the protein in luminal membranes. It requires cytoskeleton integrity.     

Regulation of the cardiac Na+/H+ exchanger in health and disease

The Na⁺ gradient produced across the cardiac sarcolemma by the ATP-dependent Na⁺-pump is a constant source of energy for Na⁺-dependent transporters. The plasma membrane Na⁺/H⁺ exchanger (NHE) is one such secondary active transporter, regulating intracellular pH, Na⁺ concentration, and cell volume. NHE1, the major isoform found in the heart, is activated in response to a variety of stimuli such as hormones and mechanical stress. This important characteristic of NHE1 is intimately linked to heart diseases, including maladaptive cardiac hypertrophy and subsequent heart failure, as well as acute ischemic-reperfusion injury. NHE1 activation results in elevation of pH and intracellular Na⁺ concentration, which potentially enhance downstream signaling cascades in the myocardium. Therefore, in addition to determining the mechanism underlying regulation of NHE1 activity, it is important to understand how the ionic signal produced by NHE1 is transmitted to the downstream targets. Extensive studies have identified many accessory factors that interact with NHE1. Here, we have summarized the recent progress on understanding the molecular mechanism underlying NHE1 regulation and have shown a possible signaling pathway leading to cardiac remodeling, which is initiated from NHE1. This article is part of a Special Issue entitled “Na⁺ Regulation in Cardiac Myocytes”.     

Regression of isoproterenol-induced cardiac hypertrophy by Na+/H+ exchanger inhibition

Cardiac hypertrophy is often associated with an increased sympathetic drive, and both in vitro and in vivo studies have demonstrated the development of cardiomyocyte hypertrophy in response to either alpha- or beta-adrenergic stimulation. Because an association between the Na+/H+ exchanger and cellular growth has been proposed, this study aimed to analyze the possible role of the antiporter in isoproterenol-induced cardiac hypertrophy. Isoproterenol alone (5 mg/kg IP once daily) or combined with a selective inhibitor of the Na+/H+ exchanger activity (3 mg x kg(-1) x d(-1) BIIB723) was given to male Wistar rats for 30 days. Sex- and age-matched rats that received 0.9% saline IP daily served as controls. Echocardiographic follow-up showed a 33% increase in left ventricular mass in the isoproterenol-treated group, whereas it did not increase in the isoproterenol+BIIB723-treated group. Heart weight-to-body weight ratio at necropsy was 2.44+/-0.11 in controls and increased to 3.35+/-0.10 (P<0.05) with isoproterenol, an effect that was markedly attenuated by BIIB723 (2.82+/-0.07). Intense cardiomyocyte enlargement and severe subendocardial fibrosis were found in isoproterenol-treated rats, and both effects were attenuated by BIIB723. Myocardial Na+/H+ exchanger activity and protein expression significantly increased in isoproterenol-treated rats compared with the control group (1.45+/-0.11 vs 0.91+/-0.05 arbitrary units, P<0.05). This effect was significantly reduced by BIIB723 (1.17+/-0.02, P<0.05). In conclusion, our results show that Na+/H+ exchanger inhibition prevented the development of isoproterenol-induced hypertrophy and fibrosis, providing strong evidence in favor of a key role played by the antiporter in this model of cardiac hypertrophy.     

Neutrophil intracellular pH and Na+/H+ exchanger activity in pre-eclampsia

Elevated Na(+)/H(+) exchanger activity and intracellular acidosis have previously been demonstrated in white blood cells isolated from women who have suffered from a pre-eclamptic pregnancy. The mechanisms underlying this abnormality and the implications in pre-eclamptic pregnancies are, at present, unclear. In this study, we used neutrophils from third trimester pre-eclamptic patients and third trimester normotensive pregnant controls to determine Na(+)/H(+) exchanger isoform-1 (NHE-1) activity and intracellular pH. This was performed using a well-validated technique involving flurometry and a pH sensitive dye, 2,7'Bis-(carboxyethyl) 5.6 carboxyfluorescein acetomethyl ester (BCECF-AM). Time course experiments were performed to assess the contribution of plasma factors to intracellular pH measurements. Plasma digoxin-like factor (DLF) was assessed in both patients and normotensive controls. Neutrophil intracellular pH was significantly lower in the pre-eclamptic patients (7.15 +/- 0.050) compared with the normotensive pregnant controls (7.36 +/- 0.027; P<.001). NHE-1 activity (in mmol/L/min) was significantly higher in the pre-eclamptics (32.4 +/- 1.9) compared with the normotensive neutrophils (27.1 +/- 1.6; P =.038). Times course experiments showed that mean pre-eclamptic intracellular pH increased from 7.11 +/- 0.049 to 7.25 +/- 0.043 after 2 hours of incubation. DLF, measured as amount of inorganic phosphate liberated from adenosine triphosphate (ATP), was significantly lower when plasma from the pre-eclamptic patients was incubated with the enzyme compared with plasma from the normotensive pregnant women (54.9 +/- 2.6 nmol/mL plasma v 63.91 +/- 1.7 nmol/mL plasma, n = 6, P =.018 unpaired Student's t test). The results suggest that elevated NHE-1 activity and intracellular acidosis are intermediate phenotypes in women who have pre-eclampsia. Intracellular pH may have been affected by plasma as shown in the time course experiments. DLF, an inhibitor of Na(+)/K(+)ATPase, may contribute to this intracellular acidosis in pre-eclamptic neutrophils.     

Oxidative stress causes renal angiotensin II type 1 receptor upregulation, Na+/H+ exchanger 3 overstimulation, and hypertension

Oxidative stress modulates angiotensin (Ang) II type 1 receptor (AT(1)R) expression and function. Ang II activates renal Na(+)/H(+) exchanger 3 (NHE3) to increase sodium reabsorption, but the mechanisms are still elusive. In addition, the upregulation of AT(1)R during oxidative stress could promote sodium retention and lead to an increase in blood pressure. Herein, we investigated the mechanism of Ang II-mediated, AT(1)R-dependent renal NHE3 regulation and effect of oxidative stress on AT(1)R signaling and development of hypertension. Male Sprague-Dawley rats received tap water (control) or 30 mmol/L of l-buthionine-sulfoximine, an oxidant, with and without 1 mmol/L of Tempol, an antioxidant, for 3 weeks. l-Buthionine-sulfoximine-treated rats exhibited oxidative stress and high blood pressure. Incubation of renal proximal tubules with Ang II caused significantly higher NHE3 activation in l-buthionine-sulfoximine-treated rats compared with control. The activation of NHE3 was sensitive to AT(1)R blocker and inhibitors of phospholipase C, tyrosine kinase, janus kinase 2 (Jak2), Ca(2+)-dependent calmodulin (CaM), and Ca(2+) chelator. Also, incubation of proximal tubules with Ang II caused Jak2-dependent CaM phosphorylation, which led to Jak2-CaM complex formation and increased Jak2-CaM interaction with NHE3. The activation of these signaling molecules was exaggerated in l-buthionine-sulfoximine-treated rats, whereas Tempol normalized the AT(1)R signaling. In conclusion, Ang II activates renal proximal tubular NHE3 through novel pathways that involve phospholipase C and an increase in intracellular Ca(2+), Jak2, and CaM. In addition, oxidative stress exaggerates Ang II signaling, which leads to overstimulation of renal NHE3 and contributes to an increase in blood pressure.     

Analysis of Na+/Ca2+ exchanger knockout mice

The Na(+)/Ca(2+) exchanger (NCX) on the plasma membrane is thought to be the main calcium extrusion system from the cytosol to the extracellular space in many mammalian excitable cells including cardiac myocytes. However, the precise roles of NCX are still unclear because of lack of its specific inhibitors. We generated NCX1-deficient mice by gene targeting to determine the in vivo function of the exchanger. Homozygous mutant died at 9.5 days post coitum. Embryonic hearts did not beat and cardiac myocytes showed apoptosis. These results suggest that NCX1 is required for heart beats and survival of cardiac myocytes in embryos. Heterozygous mutant mice were viable and indistinguishable from wild type mice. mRNA and protein levels in the heart of heterozygous mutant were half as much as wild type mice. In response to pressure overload, mutant mice showed better systolic and diastolic relaxation functions than wild type mice. Intracellular Ca(2+) measurement revealed an increase in calcium content of cytoplasm and sarcoplasmic reticulum (SR) and RNA analysis revealed preserved SR Ca(2+) ATPase expression in the ventricle of mutant mice. These results suggest that NCX plays an important role in cardiac performance in these pathological situations.     

Upregulation of myocardial Na+/H+ exchanger induced by chronic treatment with a selective inhibitor

Rats exposed to prolonged administration of the NHE-1 inhibitor cariporide showed enhanced activity of the exchanger in cardiac tissue, as assessed by the rise in the steady-state pHi value in the absence of bicarbonate (7.15+/-0.01 in control vs 7.49+/-0.06 and 7.41+/-0.05 in cariporide-treated for 1 or 2 months, respectively, P<0.05). In the presence of bicarbonate, the change in pHi was blunted due to a compensatory activation of acid loading pHi regulatory mechanisms. The enhancement of NHE activity disappeared after 1 week of the inhibitor withdrawal. The kinetic analysis of H+ fluxes after an acid load revealed an increased net H+ efflux (JH+) at any given pHi value and an alkaline shift of the apparent "set-point" of the exchanger (from 7.11+/-0.02 to 7.38+/-0.04,P <0.05) in treated rats. In the presence of the PKC inhibitor chelerythrine, the "set-point" of the exchanger was normalized in the cariporide-treated rats while JH+ at acidic pHi values persisted elevated. Cardiac NHE-1 mRNA levels and protein expression were increased in cariporide-treated rats. In addition to the increased protein expression after the treatment, the normalization of the augmented "set-point" by chelerythrine suggests an increased turnover rate of the units through a PKC dependent pathway. These data demonstrate that long-term treatment with the NHE-1 inhibitor cariporide enhances the antiporter activity in cardiac tissue through an increase of the number and turnover of functional units. This finding deserves further experimental and clinical evaluations to consider whether it would be advisable a gradual withdrawal of prolonged NHE inhibition to avoid an enhanced response when the exchanger is stimulated.     

Sarcolemmal Na+/H+ exchanger activity and expression in human ventricular myocardium

OBJECTIVES: To determine sarcolemmal Na+/H+ exchanger (NHE) activity and expression in human ventricular myocardium. BACKGROUND: Although the sarcolemmal NHE has been implicated in various physiological and pathophysiological phenomena in animal studies, its activity and expression in human myocardium have not been studied. METHODS: Ventricular myocardium was obtained from unused donor hearts with acute myocardial dysfunction (n = 5) and recipient hearts with chronic end stage heart failure (n = 11) through a transplantation program. Intracellular pH (pHi) was monitored in enzymatically isolated single ventricular myocytes by microepifluorescence. As the index of sarcolemmal NHE activity, the rate of H+ efflux at a pHi of 6.90 J(H6.9)) was determined after the induction of intracellular acidosis in bicarbonate-free medium. Na+/H+ exchanger isoform 1 (NHE1) expression in ventricular myocardium was determined by immunoblot analysis. RESULTS: Human ventricular myocytes exhibited readily detectable sarcolemmal NHE activity after the induction of intracellular acidosis, and this activity was suppressed by the NHE1-selective inhibitor HOE-642 (cariporide) at 1 micromol/L. Sarcolemmal NHE activity of myocytes was significantly greater in recipient hearts (JH6.9 = 1.95+/-0.18 mmol/L/min) than it was in unused donor hearts (J(H6.9 = 1.06+/-0.15 mmol/L/min). In contrast, NHE1 protein was expressed in similar abundance in ventricular myocardium from both recipient and unused donor hearts. CONCLUSIONS: Sarcolemmal NHE activity of human ventricular myocytes arises from the NHE1 isoform and is inhibited by HOE-642. Sarcolemmal NHE activity is significantly greater in recipient hearts with chronic end-stage heart failure than it is in unused donor hearts, and this difference is likely to arise from altered posttranslational regulation.     

Na+/H+ exchanger type 1 is a receptor for pathogenic subgroup J avian leukosis virus

Subgroup J avian leukosis virus (ALV-J) is a recently identified avian oncogenic retrovirus responsible for severe economic losses worldwide. In contrast with the other ALV subgroups, ALV-J predominantly induces myeloid leukosis in meat-type chickens. Despite significant homology with the other ALV subgroups across most of the genome, the envelope protein of ALV-J (EnvJ) shares low homology with the others. Pathogenicity and myeloid leukosis induction map to the env gene of ALV-J. A chimeric protein composed of the surface domain of EnvJ fused to the constant region of a rabbit IgG and mass spectrometry were used to identify the chicken Na(+)/H(+) exchanger type 1 (chNHE1) as a binding protein for ALV-J. Flow cytometry analysis and coprecipitation experiments demonstrated a specific interaction between EnvJ and chNHE1. When introduced into nonpermissive human 293T cells and quail QT6 cells, chNHE1 conferred susceptibility to EnvJ-mediated infection. Furthermore, 293T cells expressing chNHE1 fused with 293T cells expressing EnvJ in a low-pH-dependent manner. Together, these data identify chNHE1 as a cellular receptor for the highly pathogenic ALV-J.     

Role of the cardiac Na+/H+ exchanger during ischemia and reperfusion

The coupled exchanger theory describes one of the central mechanisms of damage in the ischemic heart. The theory proposes that anaerobic glycolysis produces lactate and protons and that the protons can leave the cardiac cell on the cardiac Na+/H+ exchanger (NHE1). The subsequent rise in [Na+]i stimulates the cardiac Na+/Ca2+ exchanger (NCX) and results in an increase in [Ca2+]i which promotes myocardial cell damage. Although the general features of this theory are widely accepted, there is dispute about some aspects, specifically whether the NHE1 remains active during ischemia or not. We review the evidence on this issue and conclude that NHE1 is substantially inhibited during ischemia. This issue is central to the design of a clinical trial of NHE1 inhibitors in the treatment of human cardiac ischemia and the existing clinical trials are considered in this light.     

Salt-sensitive hypertension, Na+/Ca2+ exchanger, and vascular smooth muscle

Hypertension is the most common chronic disease and is the leading risk factor for death caused by stroke, myocardial infarction, and end-stage renal failure. The critical importance of excess salt intake in the pathogenesis of hypertension is widely recognized. However, the molecular mechanisms underlying salt-sensitive hypertension remain obscure. Recent studies using selective Na(+)/Ca(2+) exchanger (NCX) inhibitors and genetically engineered mice provide compelling evidence that salt-sensitive hypertension is triggered by Ca(2+) entry through NCX type 1 (NCX1) in arterial smooth muscle. Cardiotonic steroids, such as endogenous ouabain, which may contribute to the pathogenesis of salt-sensitive hypertension, seem to be necessary for NCX1-mediated hypertension. These findings have enabled us to explain how high salt intake leads to hypertension and further to describe the potential of vascular NCX1 as a new therapeutic or diagnostic target for salt-sensitive hypertension.     

钠/氢交换体在非风湿性心脏病慢性心房颤动病人心房肌表达的研究

电重构和结构重构是心房颤动 (房颤 )的两个最重要特征 ,被认为是房颤自身延续性发作和恢复窦性心律后心房收缩功能抑制的原因[1 ,2 ] 。最近研究表明钠 /氢交换体 (Na+ /H+ exchanger) 1型 (NHE1 )参与心房短期电重构和结构重构[3,4 ] 。本研究旨在阐明NHE1在慢性房颤患者心房肌的表达情况。资料和方法　 2 0 0 1年 6月～ 1 2月在浙江大学医学院附属第一医院行心脏手术的非风湿性心脏病患者 2 4例 ,其中窦性心律和慢性房颤 (房颤时间 >1年 )患者各 1 2例。所有患者心功能均为Ⅱ～Ⅲ级 (NYHA分级 ) ,均无高血压、糖尿病、冠心病 (部分…