Na/H Exchanger 1 Research Articles

Deletion of Slc9a1 in Cx3cr1+ cells stimulated microglial subcluster CREB1 signaling and microglia-oligodendrocyte crosstalk

Microglial Na/H exchanger-1 (NHE1) protein, encoded by Slc9a1, plays a role in white matter demyelination of ischemic stroke brains. To explore underlying mechanisms, we conducted single cell RNA-seq transcriptome analysis in conditional Slc9a1 knockout (cKO) and wild-type (WT) mouse white matter tissues at 3 days post-stroke. Compared to WT, Nhe1 cKO brains expanded a microglial subgroup with elevated transcription of white matter myelination genes including Spp1, Lgals3, Gpnmb, and Fabp5. This subgroup also exhibited more acidic pHi and significantly upregulated CREB signaling detected by ingenuity pathway analysis and flow cytometry. Moreover, the Nhe1 cKO white matter tissues showed enrichment of a corresponding oligodendrocyte subgroup, with pro-phagocytosis and lactate shuffling gene expression, where activated CREB signaling is a likely upstream regulator. These findings demonstrate that attenuation of NHE1-mediated H+ extrusion acidifies microglia/macrophage and may underlie the stimulation of CREB1 signaling, giving rise to restorative microglia-oligodendrocyte interactions for remyelination.

Endurance training increases skeletal muscle Na/H+ exchanger1 (NHE1) and Na/HCO3 cotransporter1 (NBC1) gene and protein expressions in rats

The purpose of this research was to study the effects of endurance training on the expression of NHE1, NBC1 genes and proteins in rat muscle. Twenty male Wistar rats (n = 20), 4 weeks old and 93.7 ± 10.8 g, were selected and divided into the control and the training groups, randomly. Endurance training was performed at a relatively low speed and duration for 7 weeks in the first week, with durations of 20 m min−1 and 20 min, and gradually reached 30 m min−1 and 35 min in the last week. The NHE1, NBC1 gene and protein expressions were determined by Real-time PCR and Western Blot techniques, respectively. Our results indicated that in the training group, the expressions of NHE1 and NBC1 mRNA in EDL increased respectively, compared with the control group (P < 0.05). There was a significant increase in soleus NHE1 mRNA, but this difference was not significant in Soleus NBC1 mRNA in comparison to the control group (P > 0.05). The protein expressions of NHE1 in Soleus and EDL increased by 32% and 31%, respectively (P < 0.05). The protein expressions of NBC1 in Soleus and EDL were not significant compared to the control group (P > 0.05). The results show that endurance training with moderate intensity and volume could improve the ability of skeletal muscle pH adjustment through the H+ transport pathway independent of lactate. This increase depends on the specificity of the most important transporter and fiber type in pH adjustment.

The positive feedback loop of NHE1-ERK phosphorylation mediated by BRAFV600E mutation contributes to tumorigenesis and development of glioblastoma

The v-raf murine sarcoma viral oncogene homolog B1 (BRAF) activating mutation V600E (BRAFV600E) is involved in glioblastoma multiforme (GBM). Na/H exchanger 1 (NHE1), a main pH regulator affecting cell microenvironment, is hyper-expressed in GBM. However, the relationship between BRAFV600E signal pathway and NHE1 in GMB cells remains unclear. This study found that NHE1 was a downstream target of BRAFV600E and an upstream factor of extracellular signal-regulated kinase (ERK). In addition, there was a positive feedback loop between NHE1-ERK phosphorylation under regulation of BRAFV600E mutation contributing to the proliferation and invasion of GBM cells. Moreover, the proliferation and invasion abilities of BRAFV600E-mutant and BRAF wild type GBM cells were all suppressed by the NHE1 inhibitor, BRAFV600E inhibitor and combination of them. The inhibitory effect of combination of the two inhibitors was better than each single drug both in vitro and in vivo. Combination of BRAFV600E and NHE1 inhibitors could be considered as a new therapeutic regimen for GBM, especially for GBM with BRAFV600E.

Blocking NHE1 stimulates glioma tumor immunity by restoring OXPHOS function of myeloid cells.

Background: Immunosuppressive tumor microenvironment (TME) in glioblastoma (GBM) is one of the contributing factors for failed immunotherapies. Therefore, there is an urgent need to better understand TME and to identify novel modulators of TME for more effective GBM therapies. We hypothesized that H+ extrusion protein Na/H exchanger 1 (NHE1) plays a role in dysregulation of glucose metabolism and immunosuppression of GBM. We investigated the efficacy of blockade of NHE1 activity in combination with temozolomide (TMZ) therapy in increasing anti-tumor immunity.Methods: Mouse syngeneic intracranial glioma model was used to test four treatment regimens: DMSO (Vehicle-control), TMZ, NHE1 specific inhibitor HOE642, or TMZ+HOE642 (T+H) combination. Ex vivo 1H/19Fluorine magnetic resonance imaging (MRI) with cell tracking agent Vsense was performed to monitor the infiltration of glioma-associated microglia/myeloid cells (GAMs). Glucose metabolism and transcriptome profiles were analyzed by Seahorse analyzer and bulk RNA-sequencing. The impact of selective Nhe1 deletion in GAMs on sensitivity to anti-PD-1 therapy was evaluated in transgenic NHE1 knockout (KO) mice.Results: Among the tested treatment regimens, the T+H combination therapy significantly stimulated the infiltration of GAMs and T-cells; up-regulated Th1 activation, and mitochondrial oxidative phosphorylation (OXPHOS) pathway genes, increased glucose uptake and mitochondrial mass, and decreased aerobic glycolysis in GAMs. Selective deletion of Nhe1 in Cx3cr1+ Nhe1 KO mice increased anti-tumor immunity and sensitivity to TMZ plus anti-PD-1 combinatorial therapy.Conclusions: NHE1 plays a role in developing glioma immunosuppressive TME in part by dysregulating glucose metabolism of GAMs and emerges as a therapeutic target for improving glioma immunity.

Burn-Induced Apoptosis of Pulmonary Microvascular Endothelial Cell is NHE1 Dependent and Regulated by PI3K-Akt and p38 MAPK Pathways.

Na/H exchanger 1 (NHE1) is a ubiquitously expressed protein on mammalian plasma membranes and involved in cell apoptosis and tissue injury. Our previous study found that NHE1 inhibition prevents burn-induced acute lung injury (ALI). However, the potential mechanism of NHE1 in burn-induced ALI is still unclear. This study investigated the role of NHE1 in burn-induced apoptosis of human pulmonary microvascular endothelial cells. Based on the western blot analyses, real-time PCR, fluorescence spectroscopy, and apoptosis analysis, we found that burn serum significantly induced NHE1 activation, promoted intracellular Na accumulation, and elevated apoptosis ratio. Inhibition of NHE1 with cariporide reversed burn-induced intracellular Na accumulation and cell apoptosis. Different doses of cariporide also significantly decreased Cai concentrations and calpain activity induced by burn serum. Furthermore, inhibition of PI3K contributed to the increase of NHE1 activation and cell apoptosis, whereas the inhibition of p38 MAPK led to inhibition of NHE1 activation and significant decreases of cell apoptosis. The data demonstrate that NHE1 activation facilitates burn-induced endothelial cell apoptosis, mediated by Ca-dependent pathway. PI3K-Akt and p38 MAPK were found to be upstream regulators of NHE1. This study provides new mechanisms underlying burn-induced ALI.

Role of Carbonic Anhydrase in the Regulation of Na‐H exchanger‐1 in Experimental Colitis

A role of carbonic anhydrase (CA) was investigated in the regulation of Na–H exchanger‐1 (NHE‐1) in experimental colitis. Trinitrobenzenesulphonic acid (TNBS) was administered intra‐rectally to induce colitis in rats, and the animals were sacrificed on day 6 post TNBS. The isoforms CA‐I and CA‐II were the abundant isoforms in the colon but expressed as minor ones in the ileum. Both CA isoforms, on co‐immunoprecipitation and confocal microscopy, appeared to be colocalized with the NHE‐1 isoform in the colon. Importantly, inflammation reduced the expression of these proteins in the colon, but not in the ileum. The reduction in the level of co‐immunoprecipitated CA isoforms was more pronounced as compared to their level of expression in the inflamed tissue. Ex‐vivo treatment of uninflamed colonic strips by TNF‐α reduced the expression of CA isoforms. The binding site of the CA‐II isoform was identified in a peptide (83 aa) selected from the NHE‐1 C‐terminus. However, the CA‐I isoform did not bind with the same peptide. Since the tissues used in this study expressed inflammatory markers, the present data suggest that the uncoupling of CA and NHE‐1 is due to inflammation. This uncoupling would lead to a reduction in the NHE‐1 activity and an accumulation of H+ intracellularly, which together cause acidosis and necrosis in the inflamed colon in the present model.Support or Funding InformationResearch Sector, Kuwait University, Kuwait (Grant # MB03/15)

Uncoupling of Carbonic Anhydrase from Na-H exchanger-1 in Experimental Colitis: A Possible Mechanistic Link with Na-H Exchanger.

In this study, we investigated a mechanistic link between Na–H exchanger-1 (NHE-1) and carbonic anhydrase (CA) in experimental colitis induced in the rats by intrarectal administration of trinitrobenzenesulphonic acid (TNBS). Western blot analysis showed CA-I and CA-II as the major isoforms and CA-IV as a minor one in the colon, and they all are expressed as minor isoforms in the ileum. Co-immunoprecipitation and confocal immunofluorescence microscopy showed colocalization of NHE-1 with CA-I and CA-II, but not with CA-IV. TNBS significantly reduced the levels of NHE-1 and CA protein isoforms in the colon, but not in the uninflamed ileum. A similar reduction profile of the expression of CA isozymes was also obtained in ex vivo treatment of normal colon strips with TNF-α. The level of uncoupling as detected by co-immunoprecipitation was significantly more pronounced. A peptide (83 aa) from the NHE-1 C-terminus demonstrated binding of CA-II only, but not of the CA-I or CA-IV isoform. Furthermore, the profile of inflammatory test markers confirmed inflammation in the tissue used. These findings taken together suggest an inflammation-induced uncoupling of CA and NHE-1, which might be a putative mechanism for reducing the activity of NHE-1 in experimental colitis. This uncoupling might lead to an intracellular accumulation of H+, resulting in acidosis and necrosis in the inflamed colon.

Induction of apoptosis and differentiation by Na/H exchanger 1 modulation in acute myeloid leukemia cells

We investigated the effect of the modulation of Na/H exchanger 1 (NHE1) on apoptosis, differentiation, and chemoresistance in acute myeloid leukemia (AML) cells to evaluate the possibility of NHE1 modulation as a novel therapeutic strategy for AML. The pHi of leukemia cell lines except KG1a was higher than that of normal bone marrow mononuclear cells (BM MNCs). Notably, in K562, cytarabine (AraC)-resistant OCI-AML2, and primary leukemia cells, pHi was significantly higher than that of normal BM MNCs. Western blotting and real-time quantitative PCR confirmed that the increased NHE1 expression was responsible for the higher pHi. Specifically, compared to CD34+CD38+ leukemia cells, the mean fluorescence intensity of NHE1 was significantly higher in CD34+CD38− leukemic stem cells. The out of range in pHi by treatment with an NHE inhibitor, the amiloride analogue 5-(N,N-hexamethylene) amiloride (HMA), or an NHE activator, phorbol 12-myristate 13-acetate (PMA), resulted in dose- and time-dependent inhibition of leukemia cell proliferation. PMA induced CD14+ differentiation of leukemia cells, whereas HMA induced cell cycle arrest at the G1 phase. HMA could induce apoptosis of leukemia cells even in AraC-resistant cells and showed an additive effect on apoptosis in AraC-sensitive cells. Our result revealed that AML cells prefer more alkalic intracellular moiety than normal BM MNCs following increased NHE1 expression and that NHE1 modulation can induce apoptosis and differentiation of AML cells. These findings imply that NHE1 is a potential target in cytotoxic or differentiation-induction treatment for AML.

Amelioration of Lipopolysaccharide-Induced Acute Lung Injury in Rats by Na-H Exchanger-1 Inhibitor Amiloride Is Associated with Reversal of ERK Mitogen-Activated Protein Kinase.

Background Na-H exchanger-1 (NHE-1) is expressed in the lung of rats. Accumulating evidence shows that Na-H exchangers are involved in inflammation. Amiloride, an inhibitor of NHE-1, inhibits the activation of macrophages and endothelial cells and reduces their production of cytokines. Since these processes have been implicated in acute lung injury (ALI) induced by lipopolysaccharide (LPS), we examined the protective effect of amiloride on ALI induced by LPS in rats. Material and Methods ALI in specific pathogen-free male Sprague-Dawley rats was induced by an intravenous injection of 6 mg/kg LPS. Amiloride pretreated rats received an intravenous injection of 10 mg/kg amiloride 30 min before the administration of LPS. Controls received normal saline in a similar manner. All animals were sacrificed 6 h after LPS or normal saline administration. The degree of ALI was assessed by wet-to-dry weight ratio (W/D) and lung histological examination. Neutrophilic infiltration was determined by myeloperoxidase (MPO) activity in lung tissue. Concentrations of total protein (TP), tumor necrosis factor-alpha (TNF-α), and macrophage inflammatory protein-2 (MIP-2) in bronchoalveolar lavage fluid (BALF) were also measured. Expression of NHE-1 and mitogen-activated protein kinase (MAPK) p38, p-p38, ERK, and p-ERK was evaluated by western blot analysis. Results Pretreatment with amiloride significantly reduced the increase in W/D, ALI score, lung tissue MPO activity, concentrations of TP, TNF-α, and MIP-2 in BALF, resulting in attenuation of ALI induced by LPS. Meanwhile, levels of NHE-1 and p-ERK proteins were reversed, whereas that of p-p38 was not. Conclusions These findings suggest that NHE-1 inhibitor amiloride could attenuate ALI induced by LPS in rats. This effect is mediated through reversal of ERK.

Mechanism of Na‐H Exchanger‐1 Expression in Experimental Colitis: Role of Carbonic Anhydrase

Na-H exchanger-1 (NHE-1) plays an important role in the neutral uptake of NaCl and water from GI-tract, and is suppressed in inflammatory bowel diseases (IBD). It is believed to be regulated by interaction of carbonic anhydrase (CA) with its C-terminal domain. Therefore in this study we investigated a role of CA in experimental colitis induced in rats by intra-rectal administration of trinitrobenzene sulphonic acid (TNBS). There are multiple CA isoforms which are expressed and regulated differently in various tissues. The isoforms CA-I and -II are cytoplasmic while the CA-IV is a membrane bound isoform. These isoforms are reported to express in the colon. In this study therefore we examined the level of CA isoforms -I, -II and -IV in colonic tissues taken from day 5 post colitis by western blot analysis. Colon was inflamed as indicated by elevation of MPO activity and tissue histology. There was suppression of protein levels of NHE-1, CA-1, -2 and -4 isoforms in the inflamed colon as compared to the non colitic controls. The levels of actin used as an internal control however remained unchanged in colitis. Since NHE-1 is fueled by protons which are produced by CA, a concomitant suppression of CA expression in inflamed colon suggests that NHE-1 is regulated by CA in colitis. Our findings discount the role of CA in contributing to an acidic pH of luminal contents reported in the IBD patients. Acknowledgment: A financial support from the Kuwait University is gratefully acknowledged.

Inhibition of NHE1 Induced Apoptosis in Cytarabine Resistant Leukemia Cell Lines and Primary Leukemia Cells from AML Patients, Which Showed Increased Intracellular pH and NHE1 Activity

Background: Na/H exchanger 1 (NHE1), an important participant in the precise regulation system of intracellular pH (pHi), is known to be involved in pathological processes such as cell transformation, maintenance and active progression of the neoplastic process. Some studies have showed that leukemic cells showed higher pHi than normal cells, and NHE1 inhibitor could induce acidification and apoptosis of the leukemic cells. In this study, we tried to elucidate the role of NHE1 in leukemic cells according to cytarabine (AraC) resistance.Materials and Methods: Two human AML cell lines, AraC sensitive (AS)-OCI-AML2 cells and AraC resistant (AR)-OCI-AML2 cells, primary leukemic cells from AML patients, and normal bone marrow mononuclear cells (BMMNC) from healthy donor were analyzed. The pH-sensitive fluorescent dye, 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) was used to measure pHi and NHE1 activity. The fluorescent ratio of the 490/440 nm was calibrated intracellularly. The expression of NHE1 was measured by qRT-PCR and western blot analysis. To inhibit the NHE1, the amiloride analogue, 5-(N,N-hexamethylene) amiloride (HMA) (10 µM, 20 µM, 30 µM) was used.Results: To confirmed AraC sensitivity, cell lines were treated with 10 µM AraC for 24 hours, and apoptosis fraction in AS-OCI-AML2 cells and AR-OCI-AML2 cells were 53.1±7.2 % and 4.0±0.8 %, respectively. The pHi of AR-OCI-AML2 cells was significantly higher than AS-OCI-AML2 cells (7.839±0.033 vs. 7.589±0.129, P=0.045) and BMMNC (7.839±0.033 vs. 7.578±0.035, P=0.083), and these differences were associated with higher NHE1 activity. Compared AS-OCI-AML2 cells, AR-OCI-AML2 cells showed significantly higher NHE1 expression by western blot analysis (Figure 1), and NHE1 mRNA levels (0.039±0.014 vs. 1.565±0.070, P<.001) by qRT-PCR. Treatment with HMA (20 µM) could induce apoptosis both on AS-OCI-AML2 cells (26.9±2.8%) and AR-OCI-AML2 cells (37.4±18.8%). Interestingly, induction of apoptosis by HMA was dose-dependent both in AS-OCI-AML2 cells and AR-OCI-AML2 cells, and higher concentration of HMA (30 µM) could induce apoptosis on most of AR-OCI-AML2 cells (68.7±20.2%). Co-treatment experiment with 10 µM AraC and 20 µM HMA in AS-OCI-AML2 cells showed additive effect on inducing apoptosis (AraC vs. HMA vs. HMA+AraC = 53.1±12.4 vs. 53.1±12.4 vs. 67.20±4.3%, Figure 2), but in AR-OCI-AML2 cells, co-treatment did not show additional or synergistic effect on inducing apoptosis (AraC vs. HMA vs. HMA+AraC = 4.0±0.1 vs. 27.1±2.2 vs. 28.1±2.0%, Figure 2). As in the cell lines, primary leukemia cells from patients with AraC resistance showing higher pHi and NHE activity than those from patients without. HMA could induce apoptosis on primary cell lines regardless AraC sensitivity.Conclusions: In this study, we first showed that NHE1 inhibition could induce apoptosis in leukemia cells regardless AraC sensitivity. Apoptotic activity was related with higher pHi and NHE activity in AraC resistant cell lines and primary leukemic cells. NHE inhibition induced apoptosis may be independent with AraC induced apoptosis. The heterogeneity in pHi and NHE activity within leukemic cells may be related to alteration in drug delivery machinery or dormant status of leukemia cells. Further experimental and clinical studies are needed to elucidate the therapeutic application of NHE1 inhibitor to AraC resistant AML. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Transport, cilia, and PKD: must we in (cyst) on interrelationships? Focus on “Increased Na+/H+ exchanger activity on the apical surface of a cilium-deficient cortical collecting duct principal cell model of polycystic kidney disease”

<i>Transport, cilia, and PKD: must we in (cyst) on interrelationships?</i> Focus on “Increased Na⁺/H⁺ exchanger activity on the apical surface of a cilium-deficient cortical collecting duct principal cell model of polycystic kidney disease”

Mechanism of Regulation of Na-H Exchanger in Inflammatory Bowel Disease: Role of TLR-4 Signaling Mechanism

We investigated the role of toll-like receptor-4 (TLR-4) signal transduction in the regulation of Na-H exchanger-1 isoform (NHE-1) in ulcerative colitis (UC). Colonic biopsies from control and UC patients were selected from four groups: controls (group 1), untreated UC patients (group 2), UC patients treated with 5'-aminosalicylic acid (5'-ASA) plus steroid (group 3), and UC patients treated with 5'-ASA plus azathioprine (AZA) (group 4). Patients presenting with abdominal pain (n = 13) and a normal colon on endoscopy served as controls. NHE-1, TLR-4, MyD88, NFkB and actin protein levels were estimated using Western blot analysis and sodium pump activity (PNPase) by a spectrophotometeric method. Myeloperoxidase (MPO) activity and histologic evaluation confirmed inflammation. PNPase activity decreased significantly (P < 0.05) in the untreated UC patients as compared to the controls or treated UC groups 3 and 4. There was a significant decrease of NHE-1 and a significant increase (P < 0.05) of TLR-4, MyD88 and NFkB protein levels in the untreated UC or 5'-ASA plus steroid treated UC patients as compared to the controls. NHE-1, TLR-4, MyD88 and NFkB protein levels were not significantly different in 5'-ASA plus AZA treated biopsies as compared to controls. The level of actin remained unaltered. Inflammatory cells' infiltration and MPO activity increased significantly in the untreated UC, but was significantly lower in the treated UC groups 3 and 4 (P < 0.05). These findings suggest that NHE-1 in UC is regulated by NFkB induced through TLR-4 and MyD88 signaling mechanism. These findings identify TLR-4 as a putative therapeutic target for IBD.

Effect of amiloride pretreatment on lipopolysaccharide-induced acute lung injury in rats

Objective To investigate the effect of amiloride pretreatment on the acute lung injury (ALI)induced by lipopolysaccharide (LPS) in rats. Methods Thirty-two pathogen-free male SD rats weighing 200-250 g were randomly divided into 4 groups (n = 8 each); group Ⅰ received iv normal saline (group C); group Ⅱ ALI received iv LPS 6 mg/kg (group ALI); group Ⅲ received iv amiloride 10 mg/kg (group A) and group Ⅳ received amiloride 10 mg/kg iv 30 min before iv LPS ( group AL). The animals were killed by exsanguination at 6 h after iv LPS infusion. The lungs were immediately removed. Microscopic examination of lung tissue was performed. The left lung was lavaged. The total protein (TP), TNF-α and macrophage inflammatory protein-2 (MIP-2)concentrations in broncho-alveolar lavage fluid (BALF) were measured. The W/D weight ratio and the myeloperoxidase (MPO) activity and expression of Na-H exchanger-1 ( NHE1 ), p38MAPK and extracellular signal-regulated kinase (ERK) in lung tissue were determined. Results LPS significantly increased ALI score (0 = slightest, 4 = severest), W/D lung weight ratio, TP, TNF-α and MIP-2 concentrations in BALF and MPO activity and the expression of NHE1, p38MAPK and ERK in the lung as compared with. control group. Amiloride pretreatment significantly attenuated LPS-induced changes except p38MAPK expression. Conclusion Pretreatment with amiloride can attenuate LPS-induced ALI by inhibition of ERK activation. Key words: Amiloride; Respiratory distress syndrome, adult; Endotoxemia; Pretreatment

Elevated Hydrostatic Pressure Activates Sodium‐Hydrogen Exchanger‐1 in Rat Optic Nerve Head Astrocytes

ObjectiveOptic nerve head (ONH) astrocytes are thought to involve in the degeneration of ganglion cells and remodeling of surrounding extracellular matrix in glaucoma. Elevated intraocular pressure (IOP) is the main glaucoma risk factor. Here we report altered sodium hydrogen exchange (NHE) activity in cells exposed to elevated hydrostatic pressure .MethodsHydorstatic pressure (HP) of 15 mmHg was applied to the cells in a 20 cm glass cylinder filled with the culture medium equilibrated with 5% CO2/95% air for 2h. Proteins were analyzed by Western blot and cytoplasmic pH (pHi) was measured by BCECF ratiometry.ResultsElevated HP causes phosphorylation of ERK1/2, ribosomal S6 kinase (p90RSK), and Na‐H exchanger‐1 (NHE1). The MEK/ERK inhibitor UO126 abolished phosphorylation of NHE1 and p90RSK as well as ERK1/2. Although baseline pHi was unaltered in pressure treated cells, the rate of pHi recovery from ammonium chloride acidification was four fold higher. Dimethylamiloride (DMA), an NHE inhibitor, abolished and U0126 partially inhibited the increased rate of pHi recovery in pressure treated astrocytes.ConclusionsThe findings suggest elevated HP caused NHE‐1 activation through phosphorylation following activation of ERK1/2 and p90RSK. These events may lead to the long term changes of protein expression known to occur in pressure‐stressed astrocytes.FundingNIH Grant EY014069

Positive feedback between Cdc42 activity and H+ efflux by the Na-H exchanger NHE1 for polarity of migrating cells

A fundamental feature of cell polarity in response to spatial cues is asymmetric amplification of molecules generated by positive feedback signaling. We report a positive feedback loop between the guanosine triphosphatase Cdc42, a central determinant in eukaryotic cell polarity, and H+ efflux by Na-H+ exchanger 1 (NHE1), which is necessary at the front of migrating cells for polarity and directional motility. In response to migratory cues, Cdc42 is not activated in fibroblasts expressing a mutant NHE1 that lacks H+ efflux, and wild-type NHE1 is not activated in fibroblasts expressing mutationally inactive Cdc42-N17. H+ efflux by NHE1 is not necessary for release of Cdc42–guanosine diphosphate (GDP) from Rho GDP dissociation inhibitor or for the membrane recruitment of Cdc42 but is required for GTP binding by Cdc42 catalyzed by a guanine nucleotide exchange factor (GEF). Data indicate that GEF binding to phosphotidylinositol 4,5–bisphosphate is pH dependent, suggesting a mechanism for how H+ efflux by NHE1 promotes Cdc42 activity to generate a positive feedback signal necessary for polarity in migrating cells.

Characterization of ion channel and transporter mRNA expressions in isolated gill chloride and pavement cells of seawater acclimating eels

Ion channels and transporters (i.e. cystic fibrosis transmembrane regulator (CFTR), inward rectifier potassium channel (eKir), Na/K-ATPase, Na/K/Cl 2 co-transporter (NKCC), aquaporin-3 (AQP-3), and Na/H exchanger-1 (NHE-1)) are known to be expressed in gill epithelia of teleost fish. Owing to the anatomical complexity of gill structures, their temporal expression profile in seawater acclimating gill pavement (PVCs) and chloride cells (CCs) are limited. In this study, we isolated the gill PVCs and CCs from seawater acclimating Japanese eels to address the issue. In the gill epithelia of freshwater adapted eels, CCs expressed the highest mRNA and/or protein levels of Na/K-ATPase, NKCC, and eKir as demonstrated by real-time PCR and/or immunohistochemical staining. AQP-3 mRNA was highly expressed in freshwater PVCs and its protein was in general expressed in all gill cells. The NHE-1 transcripts were expressed in similar levels in both PVCs and CCs. CFTR mRNA transcript was almost undetectable in all the freshwater gill cell samples. Seawater acclimation induced the transcript and/or protein levels of Na/K-ATPase, NKCC, CFTR, and eKir in CCs. The upregulation and the coexpression of these transporters in CCs suggested their cohort function in mediating Na +, K +, and Cl − transport. The expression of CFTR was found to be tightly regulated as its expression was restricted only in “seawater CCs”. AQP-3 transcript and protein levels in PVCs reduced significantly during the acclimation. Interestingly immunocytochemical (ICC) staining of seawater gill epithelia revealed that AQP-3 immunoreactivities were mainly localized in seawater CCs. In the acclimation, there was no significant reduction of NHE-1 mRNA in both PVCs and CCs, however its protein level dropped significantly in the seawater condition. The present study is the first to demonstrate the activation of the mRNA transcripts for the ion channels and transporters in isolated gill CCs during seawater acclimation. The activating mechanism is found to be confined primarily in CCs. These results indicated that in addition to the increase in size and number of CCs, the molecular remodeling and the functional plasticity of CCs were essential in the ion transport process during seawater acclimation.

Amelioration of experimental colitis by Na-H exchanger-1 inhibitor amiloride is associated with reversal of IL-1β and ERK mitogen-activated protein kinase

ObjectiveNa-H exchanger-1 (NHE-1) is induced in experimental colitis. It has not yet been established whether its inhibition ameliorates colitis. The effects of amiloride, an inhibitor of NHE-1, on colitis were examined in this study. Levels of mitogen-activated protein (MAP) kinases ERK, p38 and interleukin 1β which participate in intestinal inflammation were also examined in the colonic smooth muscle of rats with colitis.Material and methodsColitis was induced in Sprague-Dawley male rats by intrarectal administration of trinitrobenzenesulphonic acid (TNBS) and treated daily with amiloride (3, 5, and 10 mg/kg b.w. (body-weight), orally) starting 1 h before induction of colitis. The animals were sacrificed on day 5 post-TNBS. Controls received phosphate buffered saline in a similar manner.ResultsThe highest dose of amiloride (10 mg/kg) was lethal. The lowest dose (3 mg/kg) was tolerated and was used in this study. Amiloride significantly reversed the colitis-reduced contractility and induction of MPO activity, NHE-1, IL-1β and ERK, but not of p38 in inflamed colonic smooth muscle. Splenomegaly, increased colonic mass and decreased sodium pump activity were significantly reversed by amiloride treatment. There was no recovery of b.w. loss in the treated colitic animals. Urine output was increased, whereas food and water intake remained unchanged following amiloride treatment.ConclusionsThese findings suggest that the beneficial effects of NHE-1 inhibition in experimental colitis are mediated through IL-1β and ERK MAP kinase.

Regulation of Na/H exchanger-1 in gastroesophageal reflux disease: possible interaction of histamine receptor.

Na/H exchanger-1 (NHE-1) isoform regulates intracellular pH and protects the esophageal mucosa. These functions are compromised in gastroesophageal reflux disease (GERD); however, the role and the underlying mechanism of NHE-1 regulation are obscure. To address this issue, NHE-1 protein and mRNA levels were measured by ECL western blot analysis and a semiquantitative RT-PCR using alpha-actin as an internal control. Ouabain-sensitive and K-stimulated p-nitrophenylphosphatase activity was measured as a marker of the sodium pump. The level of NHE-1 protein and mRNA and sodium pump activity was increased in GERD patients with or without esophagitis. Interestingly, myeloperoxidase (MPO) activity and infiltration of inflammatory cells were significantly increased in the GERD patients with esophagitis as compared to the GERD without esophagitis and the normal controls. This induction of NHE-1 and sodium pump was reversed in the GERD patients taking an H2 blocker, but not in those taking antacids. The internal control alpha-actin did not change under these conditions. Yield of total RNA and crude microsomal proteins was also statistically similar in all the test groups. These findings demonstrate that induction of NHE-1 is regulated posttranscriptionally through a possible interaction of histamine receptor. Induction of NHE-1 and sodium pump activity together might be a mechanism underlying the GERD pathogenesis and suggests a significance of NHE-1 inhibition in the treatment of GERD.

The role of sodium/hydrogen exchange in the teratogenic response to acetazolamide

ABSTRACT Administration of acetazolamide to pregnant rodents leads to postaxial forelimb ectrodactyly, generally right‐sided, in the offspring. We previously postulated that this uniquely localized malformation was caused by a reduction of intracellular PH(pHi) in the embryonic limb bud cells. The hypothesis is supported by numerous lines of evidence including the exacerbation of forelimb ectrodactyly by co‐administration of amiloride. One action of amiloride is to inhibit the activity of Na+/H+ exchangers (NHEs), the major mechanism by which most eukaryotic cells regulate their internal pH. A mouse without this exchange mechanism should theoretically be hypersensitive to acetazolamide‐induced postaxial forelimb ectrodactyly because the limb bud cells should be less able to recover from reduction of intracellular pH. To test this hypothesis we created a targeted knockout of NHE‐1 (Na+/H+ Exchanger‐1), to which we administered acteazolamide. The frequency and/or severity of forelimb ectrodactyly was no worse in NHE‐1 null fetuses than in wild type or heterozygous littermates. We interpret this negative result to suggest that there are compensatory pathways by which embryonic cells can rid themselves of excess protons. We examined this idea by co‐administration of acetazolamide and DIDS. This latter agent inhibits CI‐/HCO3 exchange, a common mechanism for regulation of pHi against acidosis. Although malformation frequency and severity were high after this combination treatment, NHE‐1 null embryos were not more affected with postaxial fore‐limb ectrodactyly than their heterozygous and wild type littermates. We presume that embryonic limb bud cells possess another pathway through which they can regulate cytoplasmic proton concentration under acidotic conditions.