NHE Activity Research Articles

Effects of Na+ -H+ exchanger 1 knockdown on protein expression levels of ATP binding cassette transporter A1 and cholesterol efflux in hypoxic RAW264.7 cells

Objective To explore the effects of Na+ -H+ exchanger 1(NHE1) knockdown on ATP binding cassette transporter A1 (ABCA1) protein expression levels and cholesterol efflux in the hypoxic RAW264.7 cells. Methods The RAW264.7 cells were infected with lentiviral vectors expressing shRNA specific for NHE1(siNHE1) or scramble RNA (siNC). The expression of NHE1 at mRNA or protein level was detected by qRT-PCR and Western blotting respectively in the infected cells after 24 h in a hypoxia condition.In the meantime, the methods of SNARF-1, Fluo-4 NW andSuc-LLVY-aminoluciferin were employed to determine NHE1 activity, intracellular Ca2+ ([Ca2+ ]i) and calpain activity, respectively.Furthermore, ABCA1 protein levels were detected by Western blotting in the 24 h hypoxic cells.In parallel, the intracellular cholesterol content and cholesterol efflux were analyzed by the methods of combined enzymatic HLPC and 3H-cholesterol. Results The hypoxia condition versus the normoxia condition up-regulated NHE1 mRNA and protein expression level and activity by 2.48 folds, 1.28 folds and 61.96% (all P<0.05), and increased[Ca2+ ]i and calpain activity by 4.51 folds and 2.41 folds(all P<0.05). Whereas the NHE1 mRNA and protein expression and activity at the presence of hypoxia were inhibited by siNHE1 with the inhibition ratio of 84.95%, 60.75% and 66.44%, respectively (all P<0.05) and[Ca2+ ]i and calpain activity were reduced by 59.23% and 54.66% (P<0.05). Furthermore, the ABCA1 protein level was 61.67% lower in the hypoxic cells than in the normoxic cells (P<0.05), and siNHE1 was increased by 56.52%after treatment of Hypoxia.Hypoxia elevated intracellular total cholesterol and cholesterol ester by 74.57% and 101.81% (all P<0.05). Treatment with siNHE1 in the hypoxia condition can reduce total cholesterol and cholesterol ester by 34.24%及49.66%(all P<0.05). Hypoxia reduced the cholesterol efflux by 34.79%(P<0.05), which were partially reversed by siNHE1. Conclusions NHE1 might play an important role in hypoxia-induced ABCA1 protein attenuation and reverse cholesterol transport dysfunction through[Ca2+ ]i/calpain pathway. Key words: ATP binding cassette transporter A1; Na+ -H+ exchanger 1; Anoxia; Cholesterol

NHE1 has a notable role in metastasis and drug resistance of T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) represents a spectrum of hematological malignancies that affect human health. Metastasis and chemotherapeutic drug resistance are the primary causes of mortality in patients with T-ALL. Sodium-hydrogen antiporter 1 (NHE1) is established to serve a role in metastasis and drug resistance in numerous types of cancer; however, the function of NHE1 in T-ALL remains to be elucidated. Previously, the C-C-motif chemokine ligand 25 (CCL25) was identified to be involved in metastasis and drug resistance in the MOLT4 T-ALL cell line, as was the ezrin protein. The present study investigated the role of NHE1 in the metastasis of T-ALL using a Transwell assay and scanning electron microscopy, using MOLT4 cells as a model. The association between NHE1 and ezrin was assessed using laser scanning confocal microscopy. The effect of NHE1 on resistance to the chemotherapy drug doxorubicin (DOX) was also investigated using a cell viability and cytotoxicity assay. Expression of NHE1 increased following treatment with CCL25, accompanied by morphological changes in MOLT4 cells and the co-localization of NHE1 with ezrin. In addition, wild-type MOLT4 cells exhibited an increased polarization ability compared with NHE1- or ezrin-silenced cells. NHE1- or ezrin-silenced cells exhibited higher sensitivity to DOX compared with wild-type MOLT4 cells. In conclusion, the increased expression or activity of NHE1 may potentially be a poor prognostic indicator for human T-ALL.

Sustained Intracellular Acidosis Triggers the Na⁺/H⁺ Exchager-1 Activation in Glutamate Excitotoxicity.

The Na+/H+ exchanger-1 (NHE-1) is a ubiquitously expressed pH-regulatory membrane protein that functions in the brain, heart, and other organs. It is increased by intracellular acidosis through the interaction of intracellular H+ with an allosteric modifier site in the transport domain. In the previous study, we reported that glutamate-induced NHE-1 phosphorylation mediated by activation of protein kinase C-β (PKC-β) in cultured neuron cells via extracellular signal-regulated kinases (ERK)/p90 ribosomal s6 kinases (p90RSK) pathway results in NHE-1 activation. However, whether glutamate stimulates NHE-1 activity solely by the allosteric mechanism remains elusive. Cultured primary cortical neuronal cells were subjected to intracellular acidosis by exposure to 100 μM glutamate or 20 mM NH4Cl. After the desired duration of intracellular acidosis, the phosphorylation and activation of PKC-β, ERK1/2 and p90RSK were determined by Western blotting. We investigated whether the duration of intracellular acidosis is controlled by glutamate exposure time. The NHE-1 activation increased while intracellular acidosis sustained for >3 min. To determine if sustained intracellular acidosis induced NHE-1 phosphorylation, we examined phosphorylation of NHE-1 induced by intracellular acidosis by transient exposure to NH4Cl. Sustained intracellular acidosis led to activation and phosphorylation of NHE-1. In addition, sustained intracellular acidosis also activated the PKC-β, ERK1/2, and p90RSK in neuronal cells. We conclude that glutamate stimulates NHE-1 activity through sustained intracellular acidosis, which mediates NHE-1 phosphorylation regulated by PKC-β/ERK1/2/p90RSK pathway in neuronal cells.

DJ-1/Park7 Sensitive Na+ /H+ Exchanger 1 (NHE1) in CD4+ T Cells.

DJ-1/Park7 is a redox-sensitive chaperone protein counteracting oxidation and presumably contributing to the control of oxidative stress responses and thus inflammation. DJ-1 gene deletion exacerbates the progression of Parkinson's disease presumably by augmenting oxidative stress. Formation of reactive oxygen species (ROS) is paralleled by activation of the Na+ /H+ exchanger 1 (NHE1). ROS formation in CD4+ T cells plays a decisive role in regulating inflammatory responses. In the present study, we explored whether DJ-1 is expressed in CD4+ T cells, and affects ROS production as well as NHE1 in those cells. To this end, DJ-1 and NHE1 transcript, and protein levels were quantified by qRT-PCR and Western blotting, respectively, intracellular pH (pHi ) utilizing bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) fluorescence, NHE activity from realkalinization after an ammonium pulse, and ROS production utilizing 2',7' -dichlorofluorescin diacetate (DCFDA) fluorescence. As a result DJ-1 was expressed in CD4+ T cells. ROS formation, NHE1 transcript levels, NHE1 protein, and NHE activity were higher in CD4+ T cells from DJ-1 deficient mice than in CD4+ T cells from wild type mice. Antioxidant N-acetyl-cysteine (NAC) and protein tyrosine kinase (PTK) inhibitor staurosporine decreased the NHE activity in DJ-1 deficient CD4+ T cells, and blunted the difference between DJ-1-/- and DJ-1+/+ CD4+ T cells, an observation pointing to a role of ROS in the up-regulation of NHE1 in DJ-1-/- CD4+ T cells. In conclusion, DJ-1 is a powerful regulator of ROS production as well as NHE1 expression and activity in CD4+ T cells. J. Cell. Physiol. 232: 3050-3059, 2017. © 2016 Wiley Periodicals, Inc.

Effects of cardiotonic steroids on isolated perfused kidney and NHE3 activity in renal proximal tubules

Cardiotonic steroids (CS) are known as modulators of sodium and water homeostasis. These compounds contribute to the excretion of sodium under overload conditions due to its natriuretic property related to the inhibition of the renal Na+/K+-ATPase (NKA) pump α1 isoform. NHE3, the main route for Na+ reabsorption in the proximal tubule, depends on the Na+ gradient generated by the NKA pump. In the present study we aimed to investigate the effects of marinobufagin (MBG) and telocinobufagin (TBG) on the renal function of isolated perfused rat kidney and on the inhibition of NKA activity. Furthermore, we investigated the mechanisms for the cardiotonic steroid-mediated natriuretic effect, by evaluating and comparing the effects of bufalin (BUF), ouabain (OUA), MBG and TBG on NHE3 activity in the renal proximal tubule in vivo. TBG significantly increased GFR, UF, natriuresis and kaliuresis in isolated perfused rat kidney, and inhibits the activity of NKA at a much higher rate than MBG. By stationary microperfusion technique, the perfusion with BUF, OUA, TBG or MBG promoted an inhibitory effect on NHE3 activity, whereas BUF was the most effective agent, and demonstrated a dose-dependent response, with maximal inhibition at 50nM. Furthermore, our data showed the role of NKA-Src kinase pathway in the inhibition of NHE3 by CS. Finally, a downstream step, MEK1/2-ERK1/2 was also investigated, and, similar to Src inhibition, the MEK1/2 inhibitor (U0126) suppressed the BUF effect. Our findings indicate the involvement of NKA-SRc-Kinase-Ras-Raf-ERK1/2 pathway in the downregulation of NHE3 by cardiotonic steroids in the renal proximal tubule, promoting a reduction of proximal sodium reabsorption and natriuresis.

Phosphorylation of NHE3-S719 regulates NHE3 activity through the formation of multiple signaling complexes.

Casein kinase 2 (CK2) binds to the NHE3 C-terminus and constitutively phosphorylates a downstream site (S719) that accounts for 40% of basal NHE3 activity. The role of CK2 in regulation of NHE3 activity in polarized Caco-2/bbe cells was further examined by mutation of NHE3-S719 to A (not phosphorylated) or D (phosphomimetic). NHE3-S719A but not -S719D had multiple changes in NHE3 activity: 1) reduced basal NHE3 activity-specifically, inhibition of the PI3K/AKT-dependent component; 2) reduced acute stimulation of NHE3 activity by LPA/LPA5R stimulation; and 3) reduced acute inhibition of NHE3 activity-specifically, elevated Ca2+ related (carbachol/Ca2+ ionophore), but there was normal inhibition by forskolin and hyperosmolarity. The S719A mutant had reduced NHE3 complex size, reduced expression in lipid rafts, increased BB mobile fraction, and reduced binding to multiple proteins that bind throughout the NHE3 intracellular C-terminus, including calcineurin homologous protein, the NHERF family and SNX27 (related PDZ domains). These studies show that phosphorylation of the NHE3 at a single amino acid in the distal part of the C-terminus affects multiple aspects of NHE3 complex formation and changes the NHE3 lipid raft distribution, which cause changes in specific aspects of basal as well as acutely stimulated and inhibited Na+/H+ exchange activity.

Glucose enhances rotavirus enterotoxin-induced intestinal chloride secretion.

Rotavirus causes severe diarrhea in small children and is typically treated using glucose-containing oral rehydration solutions; however, glucose may have a detrimental impact on these patients, because it increases chloride secretion and presumably water loss. The rotavirus enterotoxin nonstructural protein 4 (NSP4) directly inhibits glucose-mediated sodium absorption. We examined the effects of NSP4 and glucose on sodium and chloride transport in mouse small intestines and Caco-2 cells. Mouse small intestines and Caco-2 cells were incubated with NSP4114-135 in the presence/absence of glucose. Absorption and secretion of sodium and chloride, fluid movement, peak amplitude of intracellular calcium fluorescence, and expression of Ano1 and sodium-glucose cotransporter 1 were assessed. NHE3 activity increased, and chloride secretory activity decreased with age. Net chloride secretion increased, and net sodium absorption decreased in the intestines of 3-week-old mice compared to 8-week-old mice with NSP4. Glucose increased NSP4-stimulated chloride secretion. Glucose increased NSP4-stimulated increase in short-circuit current measurements (I sc) and net chloride secretion. Ano1 cells with siRNA knockdown showed a significant difference in I sc in the presence of NSP4 and glucose without a significant difference in peak calcium fluorescence intracellular when compared to non-silencing (N.S.) cells. The failure of glucose to stimulate significant sodium absorption was likely due to the inhibition of sodium-hydrogen exchange and sodium-glucose cotransport by NSP4. Since glucose enhances intestinal chloride secretion and fails to increase sodium absorption in the presence of NSP4, glucose-based oral rehydration solutions may not be ideal for the management of rotaviral diarrhea.

Protein mediated regulation of the NHE1 isoform of the Na+/H+ exchanger in renal cells. A regulatory role of Hsp90 and AKT kinase

Na+/H+ exchanger isoform one (NHE1) is a pH regulatory protein that is present in renal tissues and serves to remove protons from within cells and protect against intracellular acidification. NHE1 has a large 315 amino acid cytosolic regulatory domain that regulates the catalytic membrane domain. We examined protein-mediated regulation of NHE1 through the cytosolic domain. Affinity chromatography with the C-terminus of NHE1 yielded a number of NHE1 binding proteins including 14-3-3 protein, heat shock proteins (Hsp90 and Hsp70) and Na+/K+ ATPase. We confirmed that 14-3-3 and heat shock proteins bind to or regulate NHE1 but could not confirm that Na+/K+ ATPase binds to the intact protein. The Hsp90 inhibitor 17-AAG decreased NHE1 activity and NHE1 phosphorylation in MDCK cells but did not decrease protein levels. Additionally, 17-AAG decreased phospho-AKT levels. Direct inhibition of AKT with MK2206 decreased NHE1 activity, though this effect was not additive with the effect of 17-AAG. The results demonstrate that in renal cells, NHE1 is associated with several regulatory proteins including Hsp90, and that Hsp90 affects its function possibly through altered phosphorylation of the protein via the AKT kinase.

PDZ domain-dependent regulation of NHE3 protein by both internal Class II and C-terminal Class I PDZ-binding motifs

NHE3 directly binds Na+/H+ exchanger regulatory factor (NHERF) family scaffolding proteins that are required for many aspects of NHE3 regulation. The NHERFs bind both to an internal region (amino acids 586-660) of the NHE3 C terminus and to the NHE3 C-terminal four amino acids. The internal NHERF-binding region contains both putative Class I (-592SAV-) and Class II (-595CLDM-) PDZ-binding motifs (PBMs). Point mutagenesis showed that only the Class II motif contributes to NHERF binding. In this study, the roles in regulation of NHE3 activity of these two PBMs were investigated, revealing the following findings. 1) Interaction occurred between these binding sites because mutation of either removed nearly all NHERF binding. 2) Mutations in either significantly reduced basal NHE3 activity. Total and percent plasma membrane (PM) NHE3 protein expression was reduced in the C-terminal but not in the internal PBD mutation. 3) cGMP- and Ca2+-mediated inhibition of NHE3 was impaired in both the internal and the C-terminal PBM mutations. 4) There was a significant reduction in half-life of the PM pool of NHE3 in only the internal PBM mutation but no change in total NHE3 half-life in either. 5) There were some differences in NHE3-associating proteins in the two PBM mutations. In conclusion, NHE3 binds to NHERF proteins via both an internal Class II PBM and C-terminal Class I PBM, which interact. The former determines NHE3 stability in the PM, and the latter determines total expression and percent PM expression.

Extracellular acidosis and very low [Na+ ] inhibit NBCn1- and NHE1-mediated net acid extrusion from mouse vascular smooth muscle cells.

The electroneutral Na+ , HCO3- cotransporter NBCn1 and Na+ /H+ exchanger NHE1 regulate acid-base balance in vascular smooth muscle cells (VSMCs) and modify artery function and structure. Pathological conditions - notably ischaemia - can dramatically perturb intracellular (i) and extracellular (o) pH and [Na+ ]. We examined effects of low [Na+ ]o and pHo on NBCn1 and NHE1 activity in VSMCs of small arteries. We measured pHi by 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein-based fluorescence microscopy of mouse mesenteric arteries and induced intracellular acidification by NH4+ prepulse technique. NBCn1 activity - defined as Na+ -dependent, amiloride-insensitive net base uptake with CO2 /HCO3- present - was inhibited equally when pHo decreased from 7.4 (22mm HCO3-/5% CO2 ) by metabolic (pHo 7.1/11mm HCO3-: 22±8%; pHo 6.8/5.5mm HCO3-: 61±7%) or respiratory (pHo 7.1/10% CO2 : 35±11%; pHo 6.8/20% CO2 : 56±7%) acidosis. Extracellular acidosis more prominently inhibited NHE1 activity - defined as Na+ -dependent net acid extrusion without CO2 /HCO3- present - at both pHo 7.1 (45±9%) and 6.8 (85±5%). Independently of pHo , lowering [Na+ ]o from 140 to 70mm reduced NBCn1 and NHE1 activity <20% whereas transport activities declined markedly (25-50%) when [Na+ ]o wasreduced to 35mm. Steady-state pHi decreased more during respiratory(ΔpHi /ΔpHo =71±4%) than metabolic (ΔpHi /ΔpHo =30±7%) acidosis. Extracellular acidification inhibits NBCn1 and NHE1 activity in VSMCs. NBCn1 is equivalently inhibited when pCO2 is raised or [HCO3-]o decreased. Lowering [Na+ ]o inhibits NBCn1 and NHE1 markedly only below the typical physiological and pathophysiological range. We propose that inhibition of Na+ -dependent net acid extrusion at low pHo protects against cellular Na+ overload at the cost of intracellular acidification.

CK2 Phosphorylation of NHE3-S 719 Regulates NHE3 Activity by Organizing the C-Terminus to form Multiple Signaling Complexes and Establish Lipid Raft Distribution

CK2 Phosphorylation of NHE3-S 719 Regulates NHE3 Activity by Organizing the C-Terminus to form Multiple Signaling Complexes and Establish Lipid Raft Distribution

Na+ /H+ exchanger NHE1 and NHE2 have opposite effects on migration velocity in rat gastric surface cells.

Following superficial injury, neighbouring gastric epithelial cells close the wound by rapid cell migration, a process called epithelial restitution. Na+/H+ exchange (NHE) inhibitors interfere with restitution, but the role of the different NHE isoforms expressed in gastric pit cells has remained elusive. The role of the basolaterally expressed NHE1 (Slc9a1) and the presumably apically expressed NHE2 (Slc9a2) in epithelial restitution was investigated in the nontransformed rat gastric surface cell line RGM1. Migration velocity was assessed by loading the cells with the fluorescent dye DiR and following closure of an experimental wound over time. Since RGM1 cells expressed very low NHE2 mRNA and have low transport activity, NHE2 was introduced by lentiviral gene transfer. In medium with pH 7.4, RGM1 cells displayed slow wound healing even in the absence of growth factors and independently of NHE activity. Growth factors accelerated wound healing in a partly NHE1‐dependent fashion. Preincubation with acidic pH 7.1 stimulated restitution in a NHE1‐dependent fashion. When pH 7.1 was maintained during the restitution period, migratory speed was reduced to ∼10% of the speed at pH 7,4, and the residual restitution was further inhibited by NHE1 inhibition. Lentiviral NHE2 expression increased the steady‐state pHi and reduced the restitution velocity after low pH preincubation, which was reversible by pharmacological NHE2 inhibition. The results demonstrate that in RGM1 cells, migratory velocity is increased by NHE1 activation, while NHE2 activity inhibit this process. A differential activation of NHE1 and NHE2 may therefore, play a role in the initiation and completion of the epithelial restitution process.

Enhanced Reactive Oxygen Species Production, Acidic Cytosolic pH and Upregulated Na+/H+ Exchanger (NHE) in Dicer Deficient CD4+ T Cells

Background: MicroRNAs (miRNAs) negatively regulate gene expression at a post-transcriptional level. Dicer, a cytoplasmic RNase III enzyme, is required for the maturation of miRNAs from precursor miRNAs. Dicer, therefore, is a critical enzyme involved in the biogenesis and processing of miRNAs. Several biological processes are controlled by miRNAs, including the regulation of T cell development and function. T cells generate reactive oxygen species (ROS) with parallel H+ extrusion accomplished by the Na+/H+-exchanger 1 (NHE1). The present study explored whether ROS production, as well as NHE1 expression and function are sensitive to the lack of Dicer (miRNAs deficient) and could be modified by individual miRNAs. Methods: CD4+ T cells were isolated from CD4 specific Dicer deficient (DicerΔ/Δ) mice and the respective control mice (Dicerfl/fl). Transcript and protein levels were quantified with RT-PCR and Western blotting, respectively. For determination of intracellular pH (pHi) cells were incubated with the pH sensitive dye bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) and Na+/H+ exchanger (NHE) activity was calculated from re-alkalinization after an ammonium pulse. Changes in cell volume were measured using the forward scatter in flow cytometry, and ROS production utilizing 2',7' -dichlorofluorescin diacetate (DCFDA) fluorescence. Transfection of miRNA-control and mimics in T cells was performed using DharmaFECT3 reagent. Results: ROS production, cytosolic H+ concentration, NHE1 transcript and protein levels, NHE activity, and cell volume were all significantly higher in CD4+ T cells from DicerΔ/Δ mice than in CD4+ T cells from Dicerfl/fl mice. Furthermore, individual miR-200b and miR-15b modify pHi and NHE activity in Dicerfl/fl and DicerΔ/Δ CD4+ T cells, respectively. Conclusions: Lack of Dicer leads to oxidative stress, cytosolic acidification, upregulated NHE1 expression and activity as well as swelling of CD4+ T cells, functions all reversed by miR-15b or miR-200b.

CD95-Mediated Proton Regulation.

The Na+/H+ exchanger NHE1 is at the crossroads of a large diversity of signaling pathways, whose activation modifies the cooperative response of the transporter to intracellular H+ ions. Here we show how the activation of the Na+/H+ exchanger NHE1 by the cleaved ligand of CD95 can be measured. We demonstrate two different methods designed to set intracellular pH at precise values. Then we show how these can be coupled to fast kinetics of lithium transport, which will enable to measure the NHE1 activity like for an enzyme, because they will yield rates of transport.

Dematin and Adducin Tether Sodium-Hydrogen Exchanger, NHE1, to Erythrocyte Membrane Cytoskeleton

Dematin is a critical component of the membrane junctional complex in red blood cells. It tethers the spectrin cytoskeleton proteins to the membrane and its genetic deletion in mice causes dissociation of the spectrin, actin and β-adducin from the membrane resulting in the collapse of the red blood cells (RBCs). As dematin lacks a transmembrane domain, it is still unclear how this critical component of the junctional complex is anchored to the RBC membrane. Our previous studies have shown that the multi-transmembrane glucose transporter-1 (GLUT1) interacts with dematin and β-adducin in human RBCs, suggesting a potential role for GLUT1 in recruiting dematin to the membrane. However, as mouse RBCs do not express a GLUT1 homologue, an equivalent membrane receptor for dematin and/or adducin in mice remains to be determined. Using multiple in vitro and in vivo biochemical assays, here we demonstrate that the ubiquitously expressed plasma membrane Na+/H+ exchanger, NHE1 (Slc9a1), is one of the receptors for dematin and β-adducin in mature mouse red blood cells. NHE1 directly interacts with the core domain of dematin. Moreover, the dematin headpiece domain mutant S381E, which binds to the core domain with a higher affinity than the wild type, abolished the biochemical interaction between dematin and NHE1. This observation suggests that NHE1 and dematin headpiece domain compete for the same binding site(s) on the core domain. Furthermore, this finding highlights a molecular mechanism whereby an intermolecular switch of dematin regulates its interaction with NHE1 by phosphorylation. Dematin and β-adducin directly interact with NHE1 at its membrane-proximal cytoplasmic domain, which in turn regulates NHE1 activity in response to growth factor stimuli and intracellular pH alterations. Accordingly,we observed an increased cellular sodium content in erythrocytes of dematin headpiece and adducin double knockout mice (DAKO), suggesting a higher NHE1 activity in DAKO erythrocytes. Unlike GLUT1, NHE1 is expressed in both mouse and human RBCs. Thus, our results provide a novel mechanism for linking NHE1 to membrane skeleton and multiple cell signaling pathways through dematin and adducin (Figure 1). Since NHE1 is one of the major regulators of intracellular pH and hypertonic stress, our findings raise the possibility that the dematin-adducin-NHE1 complex may modulate these functions in RBCs as well as in other cell types with broad impact on the regulation of the actin cytoskeleton and cell migration. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Phosphorylation and subcellular localization of Na+/H+ exchanger isoform 3 (NHE3) are associated with altered gallbladder absorptive function after formation of cholesterol gallstones.

Na+/H+ exchanger isoform 3 (NHE3) dysfunction is thought to contribute to the altered gallbladder absorption that occurs in cholesterol gallstone disease, but the mechanism is unknown. The current study was undertaken to examine the expression, phosphorylation, and subcellular localization of NHE3 in gallbladder epithelium cells (GBECs) of male C57BL/6 mice on a control or lithogenic diet. Thirty-six 8-week-old male C57BL/6 mice were randomly assigned to receive a high cholesterol diet or a regular diet for 8weeks. Gallstone formation was recorded. Gallbladder bile cholesterol, phospholipid, and total bile acids were examined. RT-PCR was used to measure NHE3 mRNA expression. NHE3 protein expression and subcellular localization were examined by Western blotting and immunofluorescence microscopy, respectively. Gallstones were formed in all mice fed the lithogenic diet. Despite higher NHE3 mRNA expression in gallbladders of the mice on the lithogenic diet than in those on the control diet, there was no significant difference in expression of total NHE3 protein. However, a higher level of NHE3 phosphorylated at serine-552 (P-NHE3) was seen on the lithogenic diet. In immunofluorescence studies, NHE3 protein was expressed both on the apical membrane and in the cytoplasm of mouse GBEC. This pattern of subcellular distribution of NHE3 strongly corroborates an exchanger trafficking mechanism in NHE3 activity regulation in mouse GBEC. We conclude that increased phosphorylation of NHE3 following gallstone formation leads to turnover of the exchanger, resulting in decreased gallbladder concentrating function.

A Histidine Cluster in the Cytoplasmic Domain of the Na-H Exchanger NHE1 Confers pH-sensitive Phospholipid Binding and Regulates Transporter Activity

The Na-H exchanger NHE1 contributes to intracellular pH (pHi) homeostasis in normal cells and the constitutively increased pHi in cancer. NHE1 activity is allosterically regulated by intracellular protons, with greater activity at lower pHi However, the molecular mechanism for pH-dependent NHE1 activity remains incompletely resolved. We report that an evolutionarily conserved cluster of histidine residues located in the C-terminal cytoplasmic domain between two phosphatidylinositol 4,5-bisphosphate binding sites (PI(4,5)P2) of NHE1 confers pH-dependent PI(4,5)P2 binding and regulates NHE1 activity. A GST fusion of the wild type C-terminal cytoplasmic domain of NHE1 showed increased maximum PI(4,5)P2 binding at pH 7.0 compared with pH 7.5. However, pH-sensitive binding is abolished by substitutions of the His-rich cluster to arginine (RXXR3) or alanine (AXXA3), mimicking protonated and neutral histidine residues, respectively, and the RXXR3 mutant had significantly greater PI(4,5)P2 binding than AXXA3. When expressed in cells, NHE1 activity and pHi were significantly increased with NHE1-RXXR3 and decreased with NHE1-AXXA3 compared with wild type NHE1. Additionally, fibroblasts expressing NHE1-RXXR3 had significantly more contractile actin filaments and focal adhesions compared with fibroblasts expressing wild type NHE1, consistent with increased pHi enabling cytoskeletal remodeling. These data identify a molecular mechanism for pH-sensitive PI(4,5)P2 binding regulating NHE1 activity and suggest that the evolutionarily conserved cluster of four histidines in the proximal cytoplasmic domain of NHE1 may constitute a proton modifier site. Moreover, a constitutively activated NHE1-RXXR3 mutant is a new tool that will be useful for studying how increased pHi contributes to cell behaviors, most notably the biology of cancer cells.

Alkaline Cytosolic pH and High Sodium Hydrogen Exchanger 1 (NHE1) Activity in Th9 Cells

CD4+ T helper 9 (Th9) cells are a newly discovered Th cell subset that produce the pleiotropic cytokine IL-9. Th9 cells can protect against tumors and provide resistance against helminth infections. Given their pivotal role in the adaptive immune system, understanding Th9 cell development and the regulation of IL-9 production could open novel immunotherapeutic opportunities. The Na+/H+ exchanger 1 (NHE1; gene name Slc9α1)) is critically important for regulating intracellular pH (pHi), cell volume, migration, and cell survival. The pHi influences cytokine secretion, activities of membrane-associated enzymes, ion transport, and other effector signaling molecules such as ATP and Ca2+ levels. However, whether NHE1 regulates Th9 cell development or IL-9 secretion has not yet been defined. The present study explored the role of NHE1 in Th9 cell development and function. Th cell subsets were characterized by flow cytometry and pHi was measured using 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein-acetoxymethyl ester (BCECF-AM) dye. NHE1 functional activity was estimated from the rate of realkalinization following an ammonium pulse. Surprisingly, in Th9 cells pHi and NHE1 activity were significantly higher than in all other Th cell subsets (Th1/Th2/Th17 and induced regulatory T cells (iTregs)). NHE1 transcript levels and protein abundance were significantly higher in Th9 cells than in other Th cell subsets. Inhibition of NHE1 by siRNA-NHE1 or with cariporide in Th9 cells down-regulated IL-9 and ATP production. NHE1 activity, Th9 cell development, and IL-9 production were further blunted by pharmacological inhibition of protein kinase Akt1/Akt2. Our findings reveal that Akt1/Akt2 control of NHE1 could be an important physiological regulator of Th9 cell differentiation, IL-9 secretion, and ATP production.

Sodium/proton exchanger isoform 1 regulates intracellular pH and cell proliferation in human ovarian cancer

Cancer cells generate protons (H+) that are extruded to the extracellular medium mainly via the Na+/H+ exchanger 1 (NHE1), which regulates intracellular pH (pHi) and cell proliferation. In primary cultures of human ascites-derived ovarian cancer cells (haOC) we assayed whether NHE1 was required for pHi modulation and cell proliferation. Human ovary expresses NHE1, which is higher in haOC and A2780 (ovarian cancer cells) compared with HOSE cells (normal ovarian cells). Basal pHi and pHi recovery (following a NH4Cl pulse) was higher in haOC and A2780, compared with HOSE cells. Zoniporide (NHE1 inhibitor) caused intracellular acidification and pHi recovery was independent of intracellular buffer capacity, but reduced in NHE1 knockdown A2780 cells. Zoniporide reduced the maximal proliferation capacity, cell number, thymidine incorporation, and ki67 (marker of proliferation) fluorescence in haOC cells. SLC9A1 (for NHE1) amplification associated with lower overall patient survival. In conclusion, NHE1 is expressed in human ovarian cancer where it has a pro-proliferative role. Increased NHE1 expression and activity constitute an unfavourable prognostic factor in these patients.

Pathophysiology of Intestinal Na+/H+ exchange.

Several members of the SLC9A family of Na+/H+ exchangers are expressed in the gut, with varying expression patterns and cellular localization. Not only do they participate in the regulation of basic epithelial cell functions, including control of transepithelial Na+ absorption, intracellular pH (pHi), cell volume, and nutrient absorption, but also in cellular proliferation, migration, and apoptosis. In addition, they modulate the extracellular milieu to facilitate other nutrient absorption and to regulate the intestinal microbial microenvironment. Na+/H+ exchangers are frequent targets of inhibition in gastrointestinal pathologies, either by intrinsic factors (eg, bile acids, inflammatory mediators) or infectious agents and associated microbial toxins. Based on emerging evidence, disruption of Na+/H+ exchange activity via impaired expression or function of respective isoforms may contribute not only to local and systemic electrolyte imbalance, but also to the disease severity via multiple mechanisms. Here, we review the current state of knowledge about the roles Na+/H+ exchangers play in the pathogenesis of disorders of diverse origin and affecting a range of gastrointestinal tissues.