Nongenomic regulation by aldosterone of the epithelial NHE3 Na(+)/H(+) exchanger

The relevance of nongenomic pathways to regulation of epithelial function by aldosterone is poorly understood. Recently, we demonstrated that aldosterone inhibits transepithelial HCO₃^– absorption in the renal medullary thick ascending limb (MTAL) through a nongenomic pathway. Here, we examined the transport mechanism(s) responsible for this regulation, focusing on Na⁺/H⁺ exchangers (NHE). In the MTAL, apical NHE3 mediates H⁺ secretion necessary for HCO₃^– absorption; basolateral NHE1 influences HCO₃^– absorption by regulating apical NHE3 activity. In microperfused rat MTALs, the addition of 1 nM aldosterone rapidly decreased HCO₃^– absorption by 30%. This inhibition was unaffected by three maneuvers that inhibit basolateral Na⁺/H⁺ exchange and was preserved in MTALs from NHE1 knockout mice, ruling out the involvement of NHE1. In contrast, exposure to aldosterone for 15 min caused a 30% decrease in apical Na⁺/H⁺ exchange activity over the intracellular pH range from 6.5 to 7.7, due to a decrease in V_max. Inhibition of HCO₃^– absorption by aldosterone was not affected by 0.1 mM lumen Zn²⁺ or 1 mM lumen DIDS, arguing against the involvement of an apical H⁺ conductance or apical K⁺-HCO₃^– cotransport. These results demonstrate that aldosterone inhibits HCO₃^– absorption in the MTAL through inhibition of apical NHE3, and identify NHE3 as a target for nongenomic regulation by aldosterone. Aldosterone may influence a broad range of epithelial transport functions important for extracellular fluid volume and acid-base homeostasis through direct regulation of this exchanger. thick ascending limb; acid-base transport; epithelial Na⁺ transport; kidney     

The apical Na(+)/H(+) exchanger isoform NHE3 is regulated by the actin cytoskeleton.

The epithelial isoform of the Na(+)/H(+) exchanger, NHE3, associates with at least two related regulatory factors called NHERF1/EBP50 and NHERF2/TKA-1/E3KARP. These factors in addition interact with the cytoskeletal protein ezrin, which in turn binds to actin. The possible linkage of NHE3 with the cytoskeleton prompted us to test the effect of actin-modifying agents on NHE3 activity. Cytochalasins B and D and latrunculin B, which interfere with actin polymerization, induced a profound inhibition of NHE3 activity. The effect was isoform-specific inasmuch as the "housekeeping" exchanger NHE1 was virtually unaffected. Cytoskeletal disorganization was associated with a subcellular redistribution of NHE3, which accumulated at sites where actin aggregated, suggesting a physical interaction of exchangers with the cytoskeleton. An interaction was further suggested by the co-sedimentation of a detergent-insoluble fraction of NHE3 with the actin cytoskeleton. Inhibition of transport was not due to diminution in the number of transporters at the plasmalemma. Functional analyses of NHE1/NHE3 chimeras revealed that the cytoplasmic domain of NHE3 conferred sensitivity to cytochalasin B. Progressive carboxyl-terminal and internal deletions of the cytoplasmic region of NHE3 indicated that the region between residues 650 and 684 is critical for this response. This region overlaps with the domain reported to interact with NHERF and also contains a putative ezrin-binding site; hence, it likely plays a role in interactions with the cytoskeleton.     

IFN-gamma downregulates expression of Na(+)/H(+) exchangers NHE2 and NHE3 in rat intestine and human Caco-2/bbe cells

Diarrhea associated with inflammatory bowel diseases has traditionally been attributed to stimulated secretion. The purpose of this study was to determine whether chronic stimulation of intestinal mucosa by interferon-gamma (IFN-gamma) affects expression and function of the apical membrane Na(+)/H(+) exchangers NHE2 and NHE3 in rat intestine and Caco-2/bbe (C2) cells. Confluent C2 cells expressing NHE2 and NHE3 were treated with IFN-gamma for 2, 24, and 48 h. Adult rats were injected with IFN-gamma intraperitoneally for 12 and 48 h. NHE2 and NHE3 activities were measured by unidirectional (22)Na influx across C2 cells and in rat brush-border membrane vesicles. NHE protein and mRNA were assessed by Western and Northern blotting. IFN-gamma treatment of C2 monolayers caused a >50% reduction in NHE2 and NHE3 activities and protein expression. In rats, region-specific, time- and dose-dependent reductions of NHE2 and NHE3 activities, protein expression, and mRNA were observed after exposure to IFN-gamma. Chronic exposure of intestinal epithelial cells to IFN-gamma results in selective downregulation of NHE2 and NHE3 expression and activity, a potential cause of inflammation-associated diarrhea.     

Transepithelial resistance can be regulated by the intestinal brush-border Na(+)/H(+) exchanger NHE3

Initiation of intestinal Na⁺-glucose cotransport results intransient cell swelling and sustained increases in tight junction permeability. Since Na⁺/H⁺ exchange has beenimplicated in volume regulation after physiological cell swelling, wehypothesized that Na⁺/H⁺ exchange might also berequired for Na⁺-glucose cotransport-dependent tightjunction regulation. In Caco-2 monolayers with activeNa⁺-glucose cotransport, inhibition ofNa⁺/H⁺ exchange with 200 µM5-(N,N-dimethyl)- amiloride induced 36 ± 2% increases in transepithelial resistance (TER). Evaluation using multiple Na⁺/H⁺ exchange inhibitors showed thatinhibition of the Na⁺/H⁺ exchanger 3 (NHE3)isoform was most closely related to TER increases. TER increases due toNHE3 inhibition were related to cytoplasmic acidification becausecytoplasmic alkalinization with 5 mM NH₄Cl prevented bothcytoplasmic acidification and TER increases. However, NHE3 inhibitiondid not affect TER when Na⁺-glucose cotransport wasinhibited. Myosin II regulatory light chain (MLC) phosphorylationdecreased up to 43 ± 5% after inhibition ofNa⁺/H⁺ exchange, similar to previous studiesthat associate decreased MLC phosphorylation with increased TER afterinhibition of Na⁺-glucose cotransport. However, NHE3inhibitors did not diminish Na⁺-glucose cotransport. Thesedata demonstrate that inhibition of NHE3 results in decreased MLCphosphorylation and increased TER and suggest that NHE3 may participatein the signaling pathway of Na⁺-glucosecotransport-dependent tight junction regulation.

     

Second mutations rescue point mutant of the Na(+)/H(+) exchanger NHE1 showing defective surface expression

We studied the effect of point mutation within the putative 11th transmembrane domain (TM11) of the Na(+)/H(+) exchanger NHE1 on the plasma membrane expression. Of the 19 mutants tested, two mutants (Tyr454 or Arg458 replaced by Cys) were retained in the endoplasmic reticulum. Interestingly, Y454C was expressed on the cell surface when one of the endogenous cysteine residues at position 8, 133, 421, or 477 was substituted with alanine. Random mutagenesis at Cys8 and its surrounding residues in the cytosolic N-tail revealed that replacement of Cys8 with Ala was the only identified single residue mutation that rescued Y454C. These results suggest that the abnormal conformation of the region of TM11 containing the Y454C mutation is compensated by the second mutation within other domains such as the N-tail. This approach may provide evidence for the interdomain interaction in NHE1.     

A novel topology model of the human Na(+)/H(+) exchanger isoform 1

The membrane topology of the human Na(+)/H(+) exchanger isoform 1 (NHE1) was assessed by substituted cysteine accessibility analysis. Eighty-three cysteine residues were individually introduced into a functional cysteineless NHE1, and these mutants were expressed in the exchanger-deficient PS120 cells. The topological disposition of introduced cysteines was determined by labeling with a biotinylated maleimide in the presence or absence of preincubation with the membrane-impermeable sulfhydryl reagent, 2-trimethylammoniumethyl-methanethiosulfonate in streptolysin O-permeabilized or nonpermeabilized cells. We proposed a new model for the topology of NHE1 that is significantly different from the model derived from hydropathy analysis. In this model, NHE1 is composed of 12 transmembrane segments (TMs) with the N and C termini located in the cytosol. The large, last extracellular loop in the membrane domain of the original model was suggested to comprise an intracellular loop, a new transmembrane segment (TM11), and an extracellular loop in the new model. Interestingly, cysteines at 183 and 184 and at 324 and 325 mapped to intracellular loops connecting TMs 4 and 5 (IL2) and TMs 8 and 9 (IL4), respectively, were accessible to sulfhydryl reagents from the outside. Furthermore, exchange activities of two mutants, R180C and Q181C, within IL2 were markedly inhibited by external MTSET. These data suggest that part of IL2 or IL4 may be located in a pore-lining region that is accessible from either side of the membrane and involved in ion transport.     

The epithelial Na(+)/H(+) exchanger, NHE3, is internalized through a clathrin-mediated pathway

Trafficking of the Na(+)/H(+) exchanger isoform 3 (NHE3) between sub-apical vesicles and apical membrane of epithelial cells is a suggested mechanism of regulation of NHE3 activity. When epitope-tagged NHE3 was stably expressed in NHE-deficient Chinese hamster ovary cells, a sizable fraction was found in recycling endosomes. This system was used to analyze the mechanism of endocytosis of NHE3. Immunofluorescence and radiolabeling experiments showed that inhibition of clathrin-mediated endocytosis using hypertonicity, acid treatment, or K(+) depletion inhibited internalization of NHE3. Moreover, transient transfection of an inhibitory mutant of dynamin (DynS45N) blocked the clathrin-mediated uptake of transferrin, as well as the endocytosis of NHE3. In ileal villus cells, endogenous NHE3 was also found to co-purify with isolated clathrin-coated vesicles, thereby confirming their association in native tissues. The role of COP-I subunits in the intracellular traffic of NHE3 was evaluated using ldlF cells, which bear a temperature-sensitive mutation in the epsilon-COP subunit. At the permissive temperature, NHE3 distributed normally, whereas at the restrictive temperature, which induces rapid degradation of epsilon-COP, NHE3 was still internalized, but its subcellular distribution was altered. These results indicate that endocytosis of NHE3 occurs primarily via clathrin-coated pits and vesicles and that normal intracellular trafficking of NHE3 involves an epsilon-COP-dependent step.     

Na(+)/H(+) exchanger NHE1 as plasma membrane scaffold in the assembly of signaling complexes

The plasma membrane Na⁺/H⁺ exchanger NHE1 has an established function in intracellular pH and cell volume homeostasis by catalyzing electroneutral influx of extracellular Na⁺ and efflux of intracellular H⁺. A second function of NHE1 as a structural anchor for actin filaments through its direct binding of the ezrin, radixin, and moesin (ERM) family of actin-binding proteins was recently identified. ERM protein binding and actin anchoring by NHE1 are necessary to retain the localization of NHE1 in specialized plasma membrane domains and to promote cytoskeleton-dependent processes, including actin filament bundling and cell-substrate adhesions. This review explores a third function of NHE1, as a plasma membrane scaffold in the assembly of signaling complexes. Through its coordinate functions in H⁺ efflux, actin anchoring, and scaffolding, we propose that NHE1 promotes protein interactions and activities, assembles signaling complexes in specialized plasma membrane domains, and coordinates divergent signaling pathways. hydrogen ion efflux; intracellular pH; molecular scaffold     

C-terminal domains of Na(+)/H(+) exchanger isoform 3 are involved in the basal and serum-stimulated membrane trafficking of the exchanger

When expressed either in polarized epithelial cells or in fibroblasts, two Na(+)/H(+) exchanger isoforms, NHE1 and NHE3, have different subcellular distributions. Using a quantitative cell surface biotinylation technique, we found PS120 cells target approximately 90% of mature NHE1 but only 14% of NHE3 to the cell surface, and this pattern occurs irrespective of NHE protein expression levels. In this study, we examined surface fractions of NHE3 C-terminal truncation mutants to identify domains involved in the targeting of NHE3. Removing the C-terminal 76 amino acids doubled surface fractions to 30% of total and doubled the V(max) from 1300 to 2432 microM H(+)/s. Removal of another 66 amino acids increased surface levels to 55% of total with an increase in the V(max) to 5794 microM H(+)/s. Surface fractions did not change with a further 105 amino acid truncation. We postulated that inhibition of the basal recycling of NHE3 could result in the surface accumulation of the NHE3 truncations. Accordingly, we found that, unlike wild-type NHE3, the truncations were shown to internalize poorly and were not affected by PI3 kinase inhibition. However, while the truncations demonstrated reduced basal recycling, they retained the same serum response as full-length NHE3, with a mobilization of approximately 10% of total NHE to the surface. We conclude that basal recycling of NHE3 is controlled by endocytic determinants contained within its C-terminal 142 amino acids and that serum-mediated exocytosis is independently regulated through a different part of the protein.     

Basic fibroblast growth factor stimulates surface expression and activity of Na(+)/H(+) exchanger NHE3 via mechanism involving phosphatidylinositol 3-kinase

Na(+)/H(+) exchanger NHE3 is a plasma membrane (PM) protein, which contributes to Na(+) absorption in the intestine. Growth factors stimulate NHE3 via phosphatidylinositol 3-kinase (PI3-K), but mechanism of this process is not clear. To examine the hypothesis that growth factors stimulate NHE3 by modulating NHE3 recycling, and that PI3-K participates in this mechanism, we used PS120 fibroblasts expressing a fusion protein of NHE3 and green fluorescent protein. At steady state, approximately 25% of cellular NHE3 content was expressed at PM. Inhibition of PI3-K decreased PM expression of NHE3, which correlated with retention of the exchanger in recycling endosomal compartment. In contrast, basic fibroblast growth factor (bFGF) increased PM expression of NHE3, which was associated with a 2-fold increase in rate constant for exit of the exchanger from the recycling compartment. Qualitatively similar effects of bFGF were observed in cells pretreated with PI3-K inhibitors, but their magnitude was only approximately 50% of that in intact cells. These data suggest that: (i) bFGF stimulates NHE3 by increasing PM expression of the exchanger; (ii) PI3-K mediates PM expression of NHE3 in both basal and bFGF-stimulated conditions, and (iii) not all of the effects of bFGF on NHE3 expression are mediated by PI3-K, suggesting additional regulatory mechanisms.     

Increased Na(+)/H(+) exchanger isoform 1 activity in spontaneously hypertensive rats: lack of mutations within the coding region of NHE1

Enhanced Na(+)/H(+) exchange, measured as amiloride derivative-sensitive Na(+) and H(+) fluxes in cells with a preliminary acidified cytoplasm (Deltamu(H+)-induced Na(+)/H(+) exchange), is one of the most prominent intermediate phenotypes of altered vascular smooth muscle cell (VSMC) function in spontaneously hypertensive rats (SHR). Analysis of Na(+)/H(+) exchange in F(2) hybrids of SHR and normotensive rats seems to be the most appropriate approach in the search for the genetic determinants of abnormal activity of this carrier. However, the measurement of Deltamu(H+)-induced Na(+)/H(+) exchange is hardly appropriate for precise analysis of the carrier's activity in VSMC derived from several hundred F(2) hybrids. To overcome this problem, we compared the rate of (22)Na influx under baseline conditions and in Na(+)-loaded (ouabain-treated) VSMC. The dose-dependency of the rate of Deltamu(H+)-induced H(+) efflux as well as of (22)Na influx in control and ouabain-treated cells on ethylisopropylamiloride (EIPA) concentration were not different (K(0.5) approximately 0.3 microM), suggesting that these ion transport pathways are mediated by the same carrier. EIPA-sensitive (22)Na influx in Na(+)-loaded cells was approximately 6-fold higher than in ouabain-untreated VSMC and was increased by 50-70% in two different substrains of SHR. About the same increment of EIPA-sensitive (22)Na influx in Na(+)-loaded VSMC was observed in 5- to 6-week-old SHR (an age at which hypertension has not yet developed) as well as in stroke-prone SHR (SHRSP) with severe hypertension, indicating that the heightened activity of Na(+)/H(+) exchange is not a consequence of long-term blood pressure elevation. To examine whether or not the augmented activity of Na(+)/H(+) exchange in SHR is caused by mutation of NHE1, i.e. the only isoform of this carrier expressed in VSMC, we undertook single-stranded conformational polymorphism analysis of 23 NHE1 cDNA fragments from SHR and SHRSP and sequencing of the 456-2421 NHE1 cDNA fragment. This study did not reveal any mutation in the entire coding region of NHE1. The lack of mutation in the coding region of NHE1 indicates that the augmented activity of the ubiquitous Na(+)/H(+) exchanger in primary hypertension is caused by altered regulation of carrier turnover number or/and its plasma membrane content.     

Mammalian NHE2 Na(+)/H+ exchanger mediates efflux of potassium upon heterologous expression in yeast

Na(+)/H+exchangers form a broad family of transporters that mediate opposing fluxes of alkali metal cations and protons across cell membranes. They play multiple roles in different organisms (protection from toxic cations, regulation of cell volume or pH). Rat NHE2 exchanger was expressed in a Saccharomyces cerevisiae mutant strain lacking its own exporters of alkali metal cations. Though most of the overexpressed NHE2 remained entrapped in the secretory pathway, part of it reached the plasma membrane and mediated K+ efflux from the yeast. We demonstrate for the first time that a mammalian Na(+)/H+ exchanger transports alkali metal cations in yeast in the opposite direction than in mammalian cells, and that the substrate specificity of the rat NHE2 exchanger is limited only to potassium cations upon expression in yeast cells.     

Immunolocalization of anion exchanger AE2, Na(+)/H(+) exchangers NHE1 and NHE4, and vacuolar type H(+)-ATPase in rat pancreas.

We have studied the expression and localization of several H(+) and HCO(3)(-) transporters, whose presence in the rat pancreas is still unclear. The Cl(-)/HCO(3)(-) exchanger AE2, the Na(+)/H(+) exchangers NHE1 and NHE4, and the 31-kD and 70-kD vacuolar H(+)-ATPase (V-ATPase) subunits were detected by immunoblotting and immunocytochemical techniques. Immunoblotting of plasma membranes with transporter-specific antibodies revealed protein bands at approximately 160 kD for AE2, at approximately 90 kD and approximately 103 kD for NHE1 and NHE4, respectively, and at 31 kD and 70 kD for V-ATPase. NHE1 and NHE4 were further identified by amplification of isoform-specific cDNA using RT-PCR. Immunohistochemistry revealed a basolateral location of AE2, NHE1, and NHE4 in acinar cells. In ducts, NHE1 and NHE4 were basolaterally located but no AE2 expression was detected. V-ATPase was detected in zymogen granules (ZGs) by immunogold labeling, and basolaterally in duct cells by immunohistochemistry. The data indicate that NHE1 and NHE4 are co-expressed in rat pancreatic acini and ducts. Basolateral acinar AE2 could contribute to Cl(-) uptake and/or pH regulation. V-ATPase may be involved in ZG fusion/exocytosis and ductal HCO(3)(-) secretion. The molecular identity of the ductal Cl(-)/HCO(3)(-) exchanger remains unclear.     

Human Na(+)/H(+) exchanger isoform 6 is found in recycling endosomes of cells, not in mitochondria

Since thediscovery of the first intracellular Na⁺/H⁺exchanger in yeast, Nhx1, multiple homologs have been cloned andcharacterized in plants. Together, studies in these organismsdemonstrate that Nhx1 is located in the prevacuolar/vacuolarcompartment of cells where it sequesters Na⁺ into thevacuole, regulates intravesicular pH, and contributes to vacuolarbiogenesis. In contrast, the human homolog of Nhx1, Na⁺/H⁺ exchanger isoform 6 (NHE6), has beenreported to localize to mitochondria when transiently expressed as afusion with green fluorescent protein. This result warrantsreevaluation because it conflicts with predictions from phylogeneticanalyses. Here we demonstrate that when epitope-tagged NHE6 istransiently expressed in cultured mammalian cells, it does notcolocalize with mitochondrial markers. It also does not colocalize withmarkers of the lysosome, late endosome, trans-Golgi network,or Golgi cisternae. Rather, NHE6 is distributed in recyclingcompartments and transiently appears on the plasma membrane. Theseresults suggest that, like its homologs in yeast and plants, NHE6 is anendosomal Na⁺/H⁺ exchanger that may regulateintravesicular pH and volume and contribute to lysosomal biogenesis.

     

Effect of azathioprine on Na(+)/H(+) exchanger activity in dendritic cells

Azathioprine is a powerful immunosuppressive drug, which is partially effective by interfering with the maturation and function of dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity. DCs are stimulated by bacterial lipopolysaccharides (LPS), which trigger the formation of reactive oxygen species (ROS), paralleled by activation of the Na(+)/H(+) exchanger. The carrier is involved in the regulation of cytosolic pH, cell volume and migration. The present study explored whether azathioprine influences Na(+)/H(+) exchanger activity in DCs. DCs were isolated from murine bone marrow, cytosolic pH (pH(i)) was estimated utilizing 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF-AM) fluorescence, Na(+)/H(+) exchanger activity from the Na(+)-dependent realkalinization following an ammonium pulse, cell volume from forward scatter in FACS analysis, ROS production from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, TNFα release utilizing ELISA, and migration utilizing transwell migration assays. Exposure of DCs to lipopolysaccharide (LPS, 1 μg/ml) led to a transient increase of Na(+)/H(+) exchanger activity, an effect paralleled by ROS formation, increased cell volume, TNFα production and stimulated migration. Azathioprine (10 μM) did not significantly alter the Na(+)/H(+) exchanger activity, cell volume and ROS formation prior to LPS exposure but significantly blunted the LPS-induced stimulation of Na(+)/H(+) exchanger activity, ROS formation, cell swelling, TNFα production and cell migration. In conclusion, azathioprine interferes with the activation of dendritic cell Na(+)/H(+) exchanger by bacterial lipopolysaccharides, an effect likely participating in the anti-inflammatory action of the drug.