NHE Isoforms Research Articles

Suppression of Na+/H+ exchanger isoform-3 in human inflammatory bowel disease: Lack of reversal by 5′-aminosalicylate treatment

Objective. Na+/H+ exchanger isoform 3 (NHE-3) is responsible for net uptake of NaCl and water from the gastrointestinal (GI) tract. However, its status in human inflammatory bowel diseases (IBDs) such as ulcerative colitis (UC) and Crohn's disease (CD) remains poorly understood. The aim of this study was to investigate the underlying mechanism of NHE-3 isoform expression and its modulation by 5′-aminosalicylate in human CD and UC. Material and methods. Subjects were divided into three groups: 1) controls; 2) untreated/new IBD cases (n=13) and 3) 5′- aminosalicylate-treated IBD patients (n_13). Subjects presenting with abdominal pain but with endoscopically normal colons served as normal controls. Inflammation was confirmed by the level of myeloperoxidase (MPO) activity, malondialdehyde (MDA) concentrations and by histologic evaluation. Expressions of NHE-3 protein and mRNA, sodium pump activity and IL-1β and TNF-α mRNA were estimated in the colonic biopsies using ECL-Western blot analysis, reverse transcription-polymerase chain reaction (RT-PCR) and enzyme assays. Results. The level of NHE-3 protein and sodium pump activity was reduced (p<0.05) in both the untreated and treated CD and UC patients. NHE-3 mRNA was reduced only in CD patients but not in those with UC. The treatment reversed the symptoms, but levels of MPO activity, MDA concentration, IL-1β, TNF-α and infiltration of inflammatory cells remained high with the exception of IL-1β mRNA in the treated patients. Conclusions. NHE-3 suppression is regulated differentially in CD and UC, which together with suppression of sodium pump activity will reduce NaCl and water uptake from the colonic lumen. These findings suggest a role of TNF-α in the regulation of NHE-3 expression in IBD.

Tumor necrosis factor-{alpha} downregulates intestinal NHE8 expression by reducing basal promoter activity.

NHE8 transporter is a member of the sodium/hydrogen exchanger (NHE) family. This transporter protein is expressed at the apical membrane of epithelial cells of kidney and intestine and contributes to vectorial Na(+) transport in both tissues. Although NaCl absorption has been shown to be reduced in diarrhea associated with colitis and enteritis, little is known about the role of Na(+)/H(+) exchange and the involvement of NHE isoforms in the pathogenesis of inflammatory disorders and the mechanism of inflammation-associated diarrhea. This study investigated the role of NHE8 in the setting of inflammatory states. Jejunal mucosa was harvested from trinitrobenzene sulfonic acid (TNBS) colitis rats or lipopolysaccharide (LPS) rats for RNA extraction and brush-border membrane protein purification. The human NHE8 gene promoter was cloned from human genomic DNA and characterized in Caco-2 cells. The promoter was further used to study the mechanisms of TNF-alpha-mediated NHE8 expression downregulation in Caco-2 cells. Results from Western blot and real-time PCR indicated that NHE8 protein and mRNA were significantly reduced in TNBS rats and LPS rats. In Caco-2 cells, TNF-alpha produces similar reduction levels in the endogenous NHE8 mRNA expression observed in our in vivo studies. The downregulation of NHE8 expression mediated by TNF-alpha could be blocked by transcription inhibitor actinomycin D, suggesting the involvement of transcriptional regulation. Further studies indicated that the human NHE8 gene transcription could be activated by Sp3 transcriptional factor, and TNF-alpha inhibits human NHE8 expression by reducing Sp3 interaction at the minimal promoter region of the human NHE8 gene. In conclusion, our studies suggest that TNF-alpha decreases NHE8 expression in inflammation induced by TNBS and LPS, which may contribute to the diarrhea associated with inflammation.

Functional coupling of the downregulated in adenoma Cl−/base exchanger DRA and the apical Na+/H+exchangers NHE2 and NHE3

Non-nutrient-dependent salt absorption across the brush-border membrane of intestinal epithelial cells is primarily mediated by coupled apical Na(+)/H(+) (aNHE) and anion exchange transport, with the latter suspected of being mediated by DRA (downregulated in adenoma; SLC26A3) that is defective in congenital chloridorrhea. To investigate DRA in greater detail and determine whether DRA and NHE activities can be coupled, we measured (22)Na(+) and (36)Cl(-) uptake in Caco2BBE colon cells infected with the tet-off-inducible DRA transgene. Under basal conditions, DRA activity was low in normal and infected Caco2BBE cells in the presence of tetracycline, whereas NHE activities could be easily detected. When apical NHE activity was increased by transfection or serum-induced expression of the aNHE isoforms NHE2 and NHE3, increased (36)Cl(-) uptake was observed. Inhibition of DRA activity by niflumic acid was greater than that by DIDS as well as by the NHE inhibitor dimethylamiloride and the carbonic anhydrase inhibitor methazolamide. DRA activity was largely aNHE-dependent, whereas a component of DRA-independent aNHE uptake continued to be observed. Coupled aNHE and DRA activities were inhibited by increased cellular cAMP and calcium and were associated with synaptotagmin I-dependent, clathrin-mediated endocytosis. In summary, these data support the role of DRA in electroneutral NaCl absorption involving functional coupling of Cl(-)/base exchange and apical NHE.

Insulin inhibits Na+/H+exchange in vascular smooth muscle and endothelial cells in situ: involvement of H2O2and tyrosine phosphatase SHP-2

Insulin signals through several intracellular pathways. Here, we tested the hypothesis that insulin modulates Na+/H+ exchange (NHE) activity in vascular cells through H2O2-mediated inhibition of tyrosine phosphatase Src homology 2 domain containing tyrosine phosphatase 2 (SHP-2). We measured intracellular pH (pHi) in isolated mouse mesenteric arteries using fluorescence confocal and wide-field microscopy. In the absence of CO(2)/HCO(3)(-), removal of bath Na(+) produced endothelial acidification (DeltapHi = -0.71 +/- 0.12) inhibited by cariporide. Cariporide reduced endothelial steady-state pHi (DeltapHi=-0.28 +/- 0.08). Insulin and H2O2 acidified endothelial cells 0.2-0.3 pH units and reduced the acidification upon Na+ removal by approximately 65%. Cariporide abolished the effect of insulin and H2O2. In vascular smooth muscle cells, H2O2 produced intracellular acidification (DeltapHi = -0.48 +/- 0.06) as did high concentrations of insulin (DeltapHi = -0.03 +/- 0.01). NHE activity after an NH4+ prepulse was approximately 80% attenuated by H2O2 and approximately 40% by high insulin concentrations. H2O2 had no effect on Na+-HCO3- cotransport activity. NHE1 (slc9a1) was the only plasma membrane NHE isoform detected in mouse mesenteric arteries by RT-PCR analyses. In both cell types, polyethylene glycol catalase abolished the effect of insulin on pHi. Exposure to insulin increased the intracellular concentration of reactive oxygen species estimated with the fluorophore 5-(6)-chloromethyl-2',7'-dichlorodihydrofluorescein. The SHP-2 selective inhibitor NSC-87877 and protein tyrosine phosphatase (PTP) inhibitor IV reduced steady-state pHi up to 0.3 pH units and inhibited NHE activity 60-80%; when applied in combination with insulin or H2O2, no further effect was obtained. We conclude that NHE contributes to pHi regulation in arterial endothelial and smooth muscle cells in situ and is inhibited by insulin and H2O2. We propose that insulin signaling involves H2O2 and inhibition of PTP SHP-2.

Paradoxical resistance to myocardial ischemia and age-related cardiomyopathy in NHE1 transgenic mice: A role for ER stress?

Sarcolemmal Na +/H + exchanger (NHE) activity, which is provided by the NHE isoform 1 (NHE1), has been implicated in ischemia/reperfusion-induced myocardial injury in animal models and humans, on the basis of studies with pharmacological NHE1 inhibitors. We generated a transgenic (TG) mouse model with cardiac-specific over-expression of NHE1 to determine whether this would be sufficient to increase myocardial susceptibility to ischemia/reperfusion-induced injury. TG mouse hearts exhibited increased sarcolemmal NHE activity and normal morphology and function. Surprisingly, they also showed reduced susceptibility to ischemia/reperfusion-induced injury, as reflected by improved functional recovery and smaller infarcts. Such protection was sustained in the presence of NHE1 inhibition with zoniporide, indicating a mechanism that is independent of sarcolemmal NHE activity. Immunoblot analysis revealed accumulation of immature NHE1 protein as well as marked upregulation of both cytoprotective (78/94 kDa glucose-regulated proteins, calreticulin, protein disulfide isomerase) and pro-apoptotic (C/EBP homologous protein) components of the endoplasmic reticulum (ER) stress response in TG myocardium. With increasing age, NHE1 TG mice exhibited increased myocyte apoptosis, developed left ventricular contractile dysfunction, underwent cardiac remodelling and died prematurely. Our findings indicate that: (1) Cardiac-specific NHE1 over-expression induces the ER stress response in mouse myocardium, which may afford protection against ischemia/reperfusion-induced injury despite increased NHE activity; (2) Ageing NHE1 TG mice exhibit myocyte apoptosis, cardiac remodelling and failure, likely as a result of sustained ER stress; (3) The pluripotent effects of the ER stress response may confound studies that are based on the chronic over-expression of complex proteins in myocardium.

IRBIT, Inositol 1,4,5-Triphosphate (IP3) Receptor-binding Protein Released with IP3, Binds Na+/H+ Exchanger NHE3 and Activates NHE3 Activity in Response to Calcium

Calcium (Ca2+) is a highly versatile second messenger that regulates various cellular processes. Previous studies showed that elevation of intracellular Ca2+ regulates the activity of Na+/H+ exchanger 3 (NHE3). However, the effect of Ca2+-dependent signaling on NHE3 activity varies depending on cell types. In this study, we report the identification of IP3 receptor-binding protein released with IP3 (IRBIT) as a NHE3 interacting protein and its role in regulation of NHE3 activity. IRBIT bound to the carboxyl-terminal domain of NHE3, which is necessary for acute regulation of NHE3. Ectopic expression of IRBIT resulted in Ca2+-dependent activation of NHE3 activity, whereas silencing of endogenous IRBIT resulted in inhibition of NHE3 activity. Ca2+-dependent stimulation of NHE3 activity was dependent on the binding of IRBIT to NHE3. Previously Ca2+-dependent inhibition of NHE3 was demonstrated in the presence of NHERF2. Co-expression of IRBIT was able to reverse the NHERF2-dependent inhibition of NHE3. We also showed that IRBIT-dependent activation of NHE3 involves exocytic trafficking of NHE3 to the plasma membrane and this activation was blocked by inhibition of calmodulin (CaM) or CaM-dependent kinase II. These results suggest that the overall effect of Ca2+ on NHE3 activity is balanced by IRBIT-dependent activation and NHERF2-dependent inhibition.

Steady-state Function of the Ubiquitous Mammalian Na/H Exchanger (NHE1) in Relation to Dimer Coupling Models with 2Na/2H Stoichiometry

We describe the steady-state function of the ubiquitous mammalian Na/H exchanger (NHE)1 isoform in voltage-clamped Chinese hamster ovary cells, as well as other cells, using oscillating pH-sensitive microelectrodes to quantify proton fluxes via extracellular pH gradients. Giant excised patches could not be used as gigaseal formation disrupts NHE activity within the patch. We first analyzed forward transport at an extracellular pH of 8.2 with no cytoplasmic Na (i.e., nearly zero-trans). The extracellular Na concentration dependence is sigmoidal at a cytoplasmic pH of 6.8 with a Hill coefficient of 1.8. In contrast, at a cytoplasmic pH of 6.0, the Hill coefficient is <1, and Na dependence often appears biphasic. Results are similar for mouse skin fibroblasts and for an opossum kidney cell line that expresses the NHE3 isoform, whereas NHE1−/− skin fibroblasts generate no proton fluxes in equivalent experiments. As proton flux is decreased by increasing cytoplasmic pH, the half-maximal concentration (K1/2) of extracellular Na decreases less than expected for simple consecutive ion exchange models. The K1/2 for cytoplasmic protons decreases with increasing extracellular Na, opposite to predictions of consecutive exchange models. For reverse transport, which is robust at a cytoplasmic pH of 7.6, the K1/2 for extracellular protons decreases only a factor of 0.4 when maximal activity is decreased fivefold by reducing cytoplasmic Na. With 140 mM of extracellular Na and no cytoplasmic Na, the K1/2 for cytoplasmic protons is 50 nM (pH 7.3; Hill coefficient, 1.5), and activity decreases only 25% with extracellular acidification from 8.5 to 7.2. Most data can be reconstructed with two very different coupled dimer models. In one model, monomers operate independently at low cytoplasmic pH but couple to translocate two ions in “parallel” at alkaline pH. In the second “serial” model, each monomer transports two ions, and translocation by one monomer allosterically promotes translocation by the paired monomer in opposite direction. We conclude that a large fraction of mammalian Na/H activity may occur with a 2Na/2H stoichiometry.

Protein Kinase B/Akt Phosphorylates and Inhibits the Cardiac Na +/ H + Exchanger NHE1

Sarcolemmal Na(+)/H(+) exchanger (NHE) activity is mediated by NHE isoform 1 (NHE1), which is subject to regulation by protein kinases. Our objectives were to determine whether NHE1 is phosphorylated by protein kinase B (PKB), identify any pertinent phosphorylation site(s), and delineate the functional consequences of such phosphorylation. Active PKBalpha phosphorylated in vitro a glutathione S-transferase (GST)-NHE1 fusion protein comprising amino acids 516 to 815 of the NHE1 carboxyl-terminal regulatory domain. PKBalpha-mediated phosphorylation of GST-NHE1 fusion proteins containing overlapping segments of this region localized the targeted residues to the carboxyl-terminal 190 amino acids (625 to 815) of NHE1. Mass spectrometry and phosphorylation analysis of mutated (Ser-->Ala) GST-NHE1 fusion proteins revealed that PKBalpha-mediated phosphorylation of NHE1 occurred principally at Ser648. Far-Western assays demonstrated that PKBalpha-mediated Ser648 phosphorylation abrogated calcium-activated calmodulin (CaM) binding to the regulatory domain of NHE1. In adult rat ventricular myocytes, adenovirus-mediated expression of myristoylated PKBalpha (myr-PKBalpha) increased cellular PKB activity, as confirmed by increased glycogen synthase kinase 3beta phosphorylation. Heterologously expressed myr-PKBalpha was present in the sarcolemma, colocalized with NHE1 at the intercalated disc regions, increased NHE1 phosphorylation, and reduced NHE1 activity following intracellular acidosis. Conversely, pharmacological inhibition of endogenous PKB increased NHE1 activity following intracellular acidosis. Our data suggest that NHE1 is a novel PKB substrate and that its PKB-mediated phosphorylation at Ser648 inhibits sarcolemmal NHE activity during intracellular acidosis, most likely by interfering with CaM binding and reducing affinity for intracellular H(+).

Structural and Functional Characterization of Transmembrane Segment IX of the NHE1 Isoform of the Na+/H+ Exchanger

The Na(+)/H(+) exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH by removing one intracellular H(+) in exchange for one extracellular Na(+). It has a large N-terminal membrane domain of 12 transmembrane segments and an intracellular C-terminal regulatory domain. We characterized the cysteine accessibility of amino acids of the putative transmembrane segment IX (residues 339-363). Each residue was mutated to cysteine in a functional cysteineless NHE1 protein. Of 25 amino acids mutated, 5 were inactive or nearly so after mutation to cysteine. Several of these showed aberrant targeting to the plasma membrane and reduced expression of the intact protein, whereas others were expressed and targeted correctly but had defective NHE1 function. Of the active mutants, Glu(346) and Ser(351) were inhibited >70% by positively charged [2-(trimethylammonium)-ethyl]methanethiosulfonate but not by anionic [2-sulfonatoethyl]methanethiosulfonate, suggesting that they are pore lining and make up part of the cation conduction pathway. Both mutants also had decreased affinity for Na(+) and decreased activation by intracellular protons. The structure of a peptide representing amino acids 338-365 was determined by using high resolution NMR in dodecylphosphocholine micelles. The structure contained two helical regions (amino acids Met(340)-Ser(344) and Ile(353)-Ser(359)) kinked with a large bend angle around a pivot point at amino acid Ser(351). The results suggest that transmembrane IX is critical with pore-lining residues and a kink at the functionally important residue Ser(351).

HCO3− Secretion by Murine Nasal Submucosal Gland Serous Acinar Cells during Ca2+-stimulated Fluid Secretion

Airway submucosal glands contribute to airway surface liquid (ASL) composition and volume, both important for lung mucociliary clearance. Serous acini generate most of the fluid secreted by glands, but the molecular mechanisms remain poorly characterized. We previously described cholinergic-regulated fluid secretion driven by Ca2+-activated Cl− secretion in primary murine serous acinar cells revealed by simultaneous differential interference contrast (DIC) and fluorescence microscopy. Here, we evaluated whether Ca2+-activated Cl− secretion was accompanied by secretion of HCO3−, possibly a critical ASL component, by simultaneous measurements of intracellular pH (pHi) and cell volume. Resting pHi was 7.17 ± 0.01 in physiological medium (5% CO2–25 mM HCO3−). During carbachol (CCh) stimulation, pHi fell transiently by 0.08 ± 0.01 U concomitantly with a fall in Cl− content revealed by cell shrinkage, reflecting Cl− secretion. A subsequent alkalinization elevated pHi to above resting levels until agonist removal, whereupon it returned to prestimulation values. In nominally CO2–HCO3−-free media, the CCh-induced acidification was reduced, whereas the alkalinization remained intact. Elimination of driving forces for conductive HCO3− efflux by ion substitution or exposure to the Cl− channel inhibitor niflumic acid (100 μM) strongly inhibited agonist-induced acidification by >80% and >70%, respectively. The Na+/H+ exchanger (NHE) inhibitor dimethylamiloride (DMA) increased the magnitude (greater than twofold) and duration of the CCh-induced acidification. Gene expression profiling suggested that serous cells express NHE isoforms 1–4 and 6–9, but pharmacological sensitivities demonstrated that alkalinization observed during both CCh stimulation and pHi recovery from agonist-induced acidification was primarily due to NHE1, localized to the basolateral membrane. These results suggest that serous acinar cells secrete HCO3− during Ca2+-evoked fluid secretion by a mechanism that involves the apical membrane secretory Cl− channel, with HCO3− secretion sustained by activation of NHE1 in the basolateral membrane. In addition, other Na+-dependent pHi regulatory mechanisms exist, as evidenced by stronger inhibition of alkalinization in Na+-free media.

Calcineurin B Homologous Protein 3 Promotes the Biosynthetic Maturation, Cell Surface Stability, and Optimal Transport of the Na+/H+ Exchanger NHE1 Isoform

Calcineurin B homologous protein (CHP) 1 and 2 are Ca(2+)-binding proteins that modulate several cellular processes, including cytoplasmic pH by positively regulating plasma membrane-type Na(+)/H(+) exchangers (NHEs). Recently another CHP-related protein, termed tescalcin or CHP3, was also shown to interact with the ubiquitous NHE1 isoform, but seemingly suppressed its activity. However, the precise physical and functional nature of this association was not examined in detail. In this study, biochemical and cellular studies were undertaken to further delineate this relationship. Glutathione S-transferase-NHE1 fusion protein pulldown assays revealed that full-length CHP3 binds directly to the proximal juxtamembrane C-terminal region (amino acids 505-571) of rat NHE1 in the same region that binds CHP1 and CHP2. The interaction was further validated by coimmunoprecipitation and coimmunolocalization experiments using full-length CHP3 and wild-type NHE1 in transfected Chinese hamster ovary AP-1 cells. Simultaneous mutation of four hydrophobic residues within this region ((530)FLDHLL(535)) to either Ala, Gln, or Arg (FL-A, FL-Q, or FL-R) abrogated this interaction both in vitro and in intact cells. The NHE1 mutants were sorted properly to the cell surface but showed markedly reduced (FL-A) or minimal (FL-R and FL-Q) activity. Interestingly, and contrary to an earlier finding, ectopic coexpression of CHP3 up-regulated the cell surface activity of wild-type NHE1. This stimulation was not observed with the CHP3 binding-defective mutants. Mechanistically, overexpression of CHP3 did not alter the H(+) sensitivity of wild-type NHE1 but rather promoted its biosynthetic maturation and half-life at the cell surface, thereby increasing the steady-state abundance of functional NHE1 protein.

Modulation of Na+‐H+ exchange isoforms NHE1 and NHE3 by insulin‐like growth factor‐1 in isolated bovine articular chondrocytes

Incubation with serum modulates the transporters that regulate intracellular pH (pH(i)) in articular chondrocytes, upregulating acid extrusion by Na(+)-H(+) exchange (NHE). There is stimulation of NHE1, together with induction of NHE3 activity. These isoforms exhibit differential responses to components of mechanical load experienced by chondrocytes during joint loading. The identity of the component(s) of serum responsible is unknown. A possibility, however, is insulin-like growth factor-1 (IGF-1), present in normal cartilage and found at enhanced levels in osteoarthritic tissue. In the present study, the effects of IGF-1 on pH(i) regulation have been characterized using fluorescence measurements of bovine articular chondrocytes, and the sensitivity of pH(i) regulation to hyperosmotic shock and raised hydrostatic pressure determined. For cells isolated in the absence of IGF-1, pH(i) recovery following acidification was predominantly mediated by NHE1. Recovery was enhanced when cells were incubated for 18 h with 20 ng mL(-1) IGF; this effect represented increased acid extrusion by NHE1, supplemented by NHE3 activity. NHE3 activity was not detected in IGF-1-treated cells that had been incubated with the protein synthesis inhibitor cycloheximide, although NHE1 activity was unaffected. In the absence of IGF-1, suspension in hyperosmotic solutions or raised hydrostatic pressure enhanced pH(i) recovery of acidified cells. This response was missing in cells incubated with IGF-1. Unresponsiveness to hyperosmotic shock represented inhibition of NHE3 activity, and was prevented using the protein kinase A inhibitor KT5720. For raised hydrostatic pressure, a decrease in NHE1 activity was responsible, and was prevented by the protein kinase C inhibitor chelerythrine.

Effect of chronic inhibition of converting enzyme on proximal tubule acidification

The acute effect of angiotensin-converting enzyme inhibition (ACEi) on proximal convoluted tubule (PCT) function is well documented. However, the effect of chronic treatment is less known. The aim of this work was to evaluate the effect of chronic ACEi on PCT acidification (J(HCO(3)(-))). Rats received enalapril (10 mg.kg(-1).day(-1), added to the drinking water) during 3 mo. Micropuncture experiments were performed to measure the effect of chronic ACEi on J(HCO(3)(-)). Nitric oxide (NO.) synthesis in kidney cortex homogenates was assessed by quantifying the conversion of [(14)C]-L-arginine to [(14)C]-L-citrulline. Western blot analysis was performed to determine the abundances of V-H(+)ATPase and NHE3 isoform of the Na(+)/H(+) exchanger in proximal brush-border membrane vesicles (BBMV). Enalapril treatment induced an approximately 50% increase in J(HCO(3)(-)). Luminal perfusion with ethyl-isopropyl amiloride (EIPA) 10(-4)M or bafilomycin 10(-6)M decreased J(HCO(3)(-)) by approximately 60% and approximately 30%, respectively, in both control and enalapril-treated rats. The effect of EIPA and bafilomycin on absolute J(HCO(3)(-)) was larger in enalapril-treated than in control rats. Acute inhibition of NO. synthesis with N(G)-nitro-L-arginine methyl ester abolished the enalapril-induced increase in J(HCO(3)(-)). Cortex homogenates from enalapril-treated rats displayed a 46% increase in nitric oxide synthase (NOS) activity compared with those from untreated animals. Enalapril treatment did not affect the abundances of NHE3 and V-H(+)ATPase in BBMV. Our results suggest that PCT acidification is increased during chronic ACEi probably due to an increase in NO. synthesis, which would stimulate Na(+)/H(+) exchange and electrogenic proton transport.

T1872 Alterations in Transporters and Regulators At the Base of Mouse Colonic Crypts After Genetic Disruption of the NHE2 Isoform Na +/H + Exchanger

T1872 Alterations in Transporters and Regulators At the Base of Mouse Colonic Crypts After Genetic Disruption of the NHE2 Isoform Na +/H + Exchanger

Na+/H+ exchanger inhibition modifies dopamine neurotransmission during normal and metabolic stress conditions

Na(+)/H(+) exchanger (NHE) proteins are involved in intracellular pH and volume regulation and may indirectly influence neurotransmission. The abundant NHE isoform 1 (NHE1) has also been linked to brain cell damage during metabolic stress. It is not known, however, whether NHE1 or other NHE isoforms play a role in striatal dopamine (DA) neurotransmission under normal or metabolic stress conditions. Our study tested the hypothesis that NHE inhibition with cariporide mesilate (HOE-642) modifies striatal DA overflow and DAergic terminal damage in mice caused by the mitochondrial inhibitor malonate. We also explored the expression of NHE1-5 in the striatum and substantia nigra. Reverse microdialysis of HOE-642 elicited a transient elevation followed by a reduction in DA overflow accompanied by a decline in striatal DA content. HOE-642 pre-treatment diminished the malonate-induced DA overflow without reducing the intensity of the metabolic stress or subsequent DAergic axonal damage. Although NHE isoforms 1-5 are expressed in the striatum and midbrain, NHE1 protein was not co-located on nigrostriatal DAergic neurons. The absence of NHE1 co-location on DAergic neurons suggests that the effects of HOE-642 on striatal DA overflow are either mediated via NHE1 located on other cell types or that HOE-642 is acting through multiple NHE isoforms.

Protons extruded by NHE1: Digestive or glue?

Many physiological and pathophysiological processes, such as embryogenesis, immune defense, wound healing, or metastasis, are based on cell migration and invasion. The activity of the ubiquitously expressed NHE1 isoform of the plasma membrane Na +/H + exchanger is one of the requirements for directed locomotion of migrating cells. The mechanisms by which NHE1 is involved in cell migration are multiple. NHE1 contributes to cell migration by affecting the cell volume, by regulating the intracellular pH and thereby the assembly and activity of cytoskeletal elements, by anchoring the cytoskeleton to the plasma membrane, by the organization of signal transduction and by regulating gene expression. The present review focuses on two additional, extracellular mechanisms by which NHE1 activity contributes to cell migration and invasion. Protons extruded by the NHE1 lead to local, extracellular acidification which, on the one hand, can create pH optima needed for the activity of proteinases at invadopodia/podosomes necessary for extracellular matrix digestion and, on the other hand, facilitates cell/matrix interaction and adhesion at the cell front.

Identification of an NHE8 ortholog in the gills of the anadromous sea lamprey Petromyzon marinus

The maintenance of internal pH homeostasis is critical to the survival of all organisms. The transmembrane exchange of hydrogen ions for sodium ions is seen across all phyla. Na+/H+exchangers (NHEs) are a family of transmembrane proteins that facilitate this exchange. To date, 10 NHE isoforms (NHE1–10) have been characterized in mammals. Epithelial isoforms NHE2 and NHE3 have been previously identified in the gills of fishes from a wide diversity of classes, where they are thought to play a role in the maintenance of systemic pH. A recent study has described the localization of NHE8 to both the organelles and apical membrane of mouse proximal tubule epithelial cells. These studies suggest that similar to the epithelial isoform NHE3, NHE8 may have the ability to recycle to the plasma membrane. The NHE8 clade appears to have originated in slime mold suggesting that NHE8 isoform could be the precursor to the development of the plasma membrane recycling NHE isoform NHE3. Single orthologs of NHE8 have been identified in the genomes of invertebrates, fish and mammals. However, the NHE8 group is not as well characterized. In this study we have identified an NHE8 ortholog in the gills of the primitive vertebrate, the anadromous sea lamprey, the NHE8 cDNA shares a high homology to other known NHE8 sequences. Tissue distribution PCR suggests that the transcripts of the lamprey NHE8 are present in multiple tissues: gill, heart, stomach, liver, muscle and intestine. The functional role of NHE8 in the lamprey is yet to be determined.

Effect of Sodium Nitroprusside on pHi Recovery in Porcine Non‐pigmented Ciliary Epithelium

ObjectiveAtriopeptin, acting through cGMP, inhibits NHE1 in the bovine ocular non‐pigmented ciliary epithelium (NPE)1. Here we examined NHE isoforms and tested whether SNP, a NO donor and a stimulator of cGMP, alters intracellular pH (pHi) responses.MethodsNHE isoforms in native and cultured NPE were detected by immunohistochemistry and immunoblot. BCECF fluorescence imaging was used to study the effect of drugs on pHi recovery after an acid load.ResultsNative and cultured NPE expressed NHE1 and NHE4 isoforms. pHi recovery in NPE was Na‐dependent and had DIDS and amiloride sensitive components. SNP inhibited pHi recovery concentration‐dependently. L‐arginine, the NOS substrate, also slowed pHi recovery. Dimethyl amiloride (DMA), a potent inhibitor of NHE, caused substantial inhibition of pHi recovery at 100μM. In cells exposed to both SNP and DMA, the degree of inhibition of pHi recovery was similar to that elicited by SNP alone. The cyclic GMP analog 8‐pCPT‐cGMP (10μM) mimicked the SNP effect. The inhibitory effect of SNP on pHi recovery was suppressed by 1μM H‐8, an inhibitor of cGMP‐dependent protein kinase.ConclusionPrimary cultures of porcine NPE express NHE isoforms found in native cells. NO donors slow pHi recovery through inhibition of NHE. The effect of NO donors on NHE involves a cGMP‐PKG signal transduction pathway.Funding: NIH‐EY06915

BBB Na/H exchange: plasma membrane distribution of NHE1 and NHE2 isoforms and stimulation by arginine vasopressin

Cerebral edema formation in ischemic stroke involves increased activity of blood‐brain barrier (BBB) Na transporters through mechanisms not well understood. Na/H exchange inhibitors can reduce brain edema in rat models of ischemic stroke. We have found previously that cerebral microvascular endothelial cells (CMEC) express both NHE1 and NHE2 isoforms of the Na/H exchanger and that CMEC exchanger activity is stimulated by the ischemic factors hypoxia, aglycemia and arginine vasopressin (AVP). In the present study we evaluated BBB in situ for the presence of NHE isoforms and further, investigated the mechanism by which AVP stimulates the BBB Na/H exchanger. Western blot analysis and immunoelectron microscopy were used to evaluate NHE isoforms in freshly isolated rat cerebral microvessels and perfusion‐fixed rat brain, respectively. In addition, Na/H exchange activity of CMEC was evaluated by microspectrofluorometry, using the pH‐sensitive dye BCECF. NHE1 and NHE2 isoforms were found in the microvessels and in BBB in situ, with both isoforms predominantly in the luminal BBB membrane (70% for NHE1, 75% for NHE2). CMEC Na/H exchange activity was increased 2.3‐fold by AVP and 2.5‐fold by the V1 agonist Orn VP (both 100 nM, 5 min) but not by a V2 agonist (DDAVP). Our findings suggest that AVP stimulates luminal BBB NHE1 and/or NHE2 activity via a V1 AVP receptor pathway.Supported by NINDS and AHA Western States Affiliate

Ligand mediated protein‐protein interaction between the angiotensin receptor type AT1 and the human NHE6 isoform

The Renin Angiotensin System (RAS) plays a vital role in the regulation of blood pressure and fluid homeostasis. RAS is regulated through the hormone Angiotensin II (Ang II), a powerful vasoconstrictor that serves as a regulator of hypertension and other cardiovascular diseases. Involvement of Ang II in the regulation of sodium levels by activation of the Na+/H+ Exchangers (NHE) has been demonstrated in many tissues. Of the seven known NHE isoforms, only two, NHE1 and NHE6, exist in heart tissue. The activities of the NHE6 protein remain unclear. To analyze the conditions leading to activation of the NHE6 protein in mammalian cells, the human MCF‐7 cell line was used. The Heameagglutinin‐tagged NHE6 plasmid was introduced to the MCF‐7 cells via transient transfection. The transfected cells were exposed to Ang II and results implicated that the presence of Ang II was cardinal for strong interaction between AT1 and NHE6. It was ascertained that MCF‐7 co‐transfected with AT2 and Heameagglutinin‐tagged NHE6, also required the presence of Ang II for activation. Further investigation revealed that tyrosine and threonine phosphorylation of NHE6 occurred when AT1 was activated by ligand binding. In summary, our results insinuate Ang II activates NHE6 through the AT1 receptor and therefore may play a role in the development of the pathophysiological conditions leading to the onset of cardiac ischemia.