Characterization of Na+/H+ Exchanger Isoform (NHE1, NHE2 and NHE3) Expression in Prairie Dog Gallbladder

Gallbladder Na⁺ absorption is linked to gallstone formation in prairie dogs. Na⁺/H⁺ exchange (NHE) is one of the major Na⁺ absorptive pathways in gallbladder. In this study, we measured gallbladder Na⁺/H⁺ exchange and characterized the NHE isoforms expressed in prairie dogs. Na⁺/H⁺ exchange activity was assessed by measuring amiloride-inhibitable transepithelial Na⁺ flux and apical ²²Na⁺ uptake using dimethylamiloride (DMA). HOE-694 was used to determine NHE2 and NHE3 contributions. Basal J ^Na _ms was higher than J ^Na _sm with J ^Na _net absorption. Mucosal DMA inhibited transepithelial Na⁺ flux in a dose-dependent fashion, causing J ^Na _ms equal to J ^Na _sm and blocking J ^Na _net absorption at 100 μm. Basal ²²Na⁺ uptake rate was 10.9 ± 1.0 μmol · cm⁻²· hr⁻¹ which was inhibited by ∼43% by mucosal DMA and ∼30% by mucosal HOE-694 at 100 μm. RT-PCR and Northern blot analysis demonstrated expression of mRNAs encoding NHE1, NHE2 and NHE3 in the gallbladder. Expression of NHE1, NHE2 and NHE3 polypeptides was confirmed using isoform-specific anti-NHE antibodies. These data suggest that Na⁺/H⁺ exchange accounts for a substantial fraction of gallbladder apical Na⁺ entry and most of net Na⁺ absorption in prairie dogs. The NHE2 and NHE3 isoforms, but not NHE1, are involved in gallbladder apical Na⁺ uptake and transepithelial Na⁺ absorption. Received: 9 February 2001/Revised: 11 April 2001     

Two Histidine Residues in the Juxta-Membrane Cytoplasmic Domain of Na+/H+ Exchanger Isoform 3 (NHE3) Determine the Set Point

Histidine residues in Na+/H+ exchangers are believed to participate in proton binding and influence the Na+/H+ exchanger activity. In the present study, the function of three highly conserved histidines in the juxtamembrane cytoplasmic domain of NHE3 was studied. His-479, His-485, and His-499 were mutated to Leu, Gln or Asp and expressed in an Na+/H+ exchanger null cell line and functional consequences on Na+/H+ exchange kinetics were characterized. None of the histidines were essential for NHE3 activity, with all mutated NHE3 resulting in functional exchangers. However, the mutation in His-475 and His-499 significantly lowered NHE3 transport activity, whereas the mutation in H485 showed no apparent effect. In addition, the pH profiles of the H479 and H499 mutants were shifted to a more acidic region, and lowered its set point, the intracellular pH value above which the Na+/H+ exchanger becomes inactive, by approximately 0.3-0.6 pH units. The changes in set point by the mutations were further shifted to more acidic values by ATP depletion, indicating that the mechanism by which the mutations on the histidine residues altered the NHE3 set point differs from that caused by ATP depletion. We suggest that His-479 and His-499 are part of the H+ sensor, which is involved in determining the sensitivity to the intracellular H+ concentration and Na+/H+ exchange rate.     

Structural and Functional Analysis of Transmembrane Segment VI of the NHE1 Isoform of the Na+/H+ Exchanger

The Na⁺/H⁺ exchanger isoform 1 is a ubiquitously expressed integral membrane protein. It resides on the plasma membrane of cells and regulates intracellular pH in mammals by extruding an intracellular H⁺ in exchange for one extracellular Na⁺. We characterized structural and functional aspects of the transmembrane segment (TM) VI (residues 227–249) by using cysteine scanning mutagenesis and high resolution NMR. Each residue of TM VI was mutated to cysteine in the background of the cysteineless NHE1 protein, and the sensitivity to water-soluble sulfhydryl-reactive compounds (2-(trimethylammonium)ethyl)methanethiosulfonate (MTSET) and (2-sulfonatoethyl)methanethiosulfonate (MTSES) was determined for those residues with significant activity remaining. Three residues were essentially inactive when mutated to Cys: Asp²³⁸, Pro²³⁹, and Glu²⁴⁷. Of the remaining residues, proteins with the mutations N227C, I233C, and L243C were strongly inhibited by MTSET, whereas amino acids Phe²³⁰, Gly²³¹, Ala²³⁶, Val²³⁷, Ala²⁴⁴, Val²⁴⁵, and Glu²⁴⁸ were partially inhibited by MTSET. MTSES did not affect the activity of the mutant NHE1 proteins. The structure of a peptide representing TM VI was determined using high resolution NMR spectroscopy in dodecylphosphocholine micelles. TM VI contains two helical regions oriented at an approximate right angle to each other (residues 229–236 and 239–250) surrounding a central unwound region. This structure bears a resemblance to TM IV of the Escherichia coli protein NhaA. The results demonstrate that TM VI of NHE1 is a discontinuous pore-lining helix with residues Asn²²⁷, Ile²³³, and Leu²⁴³ lining the translocation pore.     

Stimulation of Na+/H+ Exchanger Isoform 1 Promotes Microglial Migration

Regulation of microglial migration is not well understood. In this study, we proposed that Na⁺/H⁺ exchanger isoform 1 (NHE-1) is important in microglial migration. NHE-1 protein was co-localized with cytoskeletal protein ezrin in lamellipodia of microglia and maintained its more alkaline intracellular pH (pH_i). Chemoattractant bradykinin (BK) stimulated microglial migration by increasing lamellipodial area and protrusion rate, but reducing lamellipodial persistence time. Interestingly, blocking NHE-1 activity with its potent inhibitor HOE 642 not only acidified microglia, abolished the BK-triggered dynamic changes of lamellipodia, but also reduced microglial motility and microchemotaxis in response to BK. In addition, NHE-1 activation resulted in intracellular Na⁺ loading as well as intracellular Ca²⁺ elevation mediated by stimulating reverse mode operation of Na⁺/Ca²⁺ exchange (NCX_rev). Taken together, our study shows that NHE-1 protein is abundantly expressed in microglial lamellipodia and maintains alkaline pH_i in response to BK stimulation. In addition, NHE-1 and NCX_rev play a concerted role in BK-induced microglial migration via Na⁺ and Ca²⁺ signaling.     

Na+/H+ Exchanger Isoform 1-Induced Osteopontin Expression Facilitates Cardiomyocyte Hypertrophy

Enhanced expression and activity of the Na⁺/H⁺ exchanger isoform 1 (NHE1) has been implicated in cardiomyocyte hypertrophy in various experimental models. The upregulation of NHE1 was correlated with an increase in osteopontin (OPN) expression in models of cardiac hypertrophy (CH), and the mechanism for this remains to be delineated. To determine whether the expression of active NHE1-induces OPN and contributes to the hypertrophic response in vitro, cardiomyocytes were infected with the active form of the NHE1 adenovirus or transfected with OPN silencing RNA (siRNA-OPN) and characterized for cardiomyocyte hypertrophy. Expression of NHE1 in cardiomyocytes resulted in a significant increase in cardiomyocyte hypertrophy markers: cell surface area, protein content, ANP mRNA and expression of phosphorylated-GATA4. NHE1 activity was also significantly increased in cardiomyocytes expressing active NHE1. Interestingly, transfection of cardiomyocytes with siRNA-OPN significantly abolished the NHE1-induced cardiomyocyte hypertrophy. siRNA-OPN also significantly reduced the activity of NHE1 in cardiomyocytes expressing NHE1 (68.5±0.24%; P<0.05), confirming the role of OPN in the NHE1-induced hypertrophic response. The hypertrophic response facilitated by NHE1-induced OPN occurred independent of the extracellular-signal-regulated kinases and Akt, but required p90-ribosomal S6 kinase (RSK). The ability of OPN to facilitate the NHE1-induced hypertrophic response identifies OPN as a potential therapeutic target to reverse the hypertrophic effect induced by the expression of active NHE1.     

Structural and Functional Analysis of Transmembrane XI of the NHE1 Isoform of the Na+/H+ Exchanger

The Na⁺/H⁺ exchanger isoform 1 is a ubiquitously expressed integral membrane protein that regulates intracellular pH in mammals by extruding an intracellular H⁺ in exchange for one extracellular Na⁺. We characterized structural and functional aspects of the critical transmembrane (TM) segment XI (residues 449-470) by using cysteine scanning mutagenesis and high resolution NMR. Each residue of TM XI was mutated to cysteine in the background of the cysteine-less protein and the sensitivity to water-soluble sulfhydryl reactive compounds MTSET ((2-(trimethylammonium) ethyl)methanethiosulfonate) and MTSES ((2-sulfonatoethyl) methanethiosulfonate) was determined for those residues with at least moderate activity remaining. Of the residues tested, only proteins with mutations L457C, I461C, and L465C were inhibited by MTSET. The activity of the L465C mutant was almost completely eliminated, whereas that of the L457C and I461C mutants was partially affected. The structure of a peptide representing TM XI (residues Lys⁴⁴⁷-Lys⁴⁷²) was determined using high resolution NMR spectroscopy in dodecylphosphocholine micelles. The structure consisted of helical regions between Asp⁴⁴⁷-Tyr⁴⁵⁴ and Phe⁴⁶⁰-Lys⁴⁷¹ at the N and C termini of the peptide, respectively, connected by a region with poorly defined, irregular structure consisting of residues Gly⁴⁵⁵-Gly⁴⁵⁹. TM XI of NHE1 had a structural similarity to TM XI of the Escherichia coli Na⁺/H⁺ exchanger NhaA. The results suggest that TM XI is a discontinuous helix, with residue Leu⁴⁶⁵ contributing to the pore.The mammalian Na⁺/H⁺ exchanger isoform 1 (NHE1)⁴ is a ubiquitous integral membrane protein that regulates intracellular pH. It mediates removal of a single intracellular proton in exchange for an extracellular sodium ion (1). NHE1 has many functions aside from protection of cells from intracellular acidification (2). It promotes cell growth and differentiation (3), regulates sodium fluxes and cell volume after challenge by osmotic shrinkage (4), and has been demonstrated to be involved in modulating cell motility (5). In addition its activity is important in invasiveness of neoplastic breast cancer cells (6). NHE1 also plays critical roles in heart disease. It has a contributing role in heart hypertrophy and in the damage that occurs during ischemia and reperfusion. Inhibition of NHE1 with Na⁺/H⁺ exchanger inhibitors protects the myocardium during various disease states (7-10).NHE1 is composed of two general regions, an N-terminal membrane domain of ∼500 amino acids and a C-terminal regulatory domain of ∼315 amino acids (1, 8). The membrane domain is responsible for ion movement and an analysis of topology by cysteine scanning accessibility suggested it has 3 membrane-associated segments and 12 integral transmembrane segments (11) (Fig. 1A). The mechanism of transport of the membrane domain is of great interest both from a scientific viewpoint and in the design of improved NHE1 inhibitors that may be necessary for clinical use (1). In this regard, we have recently characterized the functionally important residues and the structure of both TM IV and TM VII. Prolines 167 and 168 of TM IV were critical to NHE1 function (12) and cysteine-scanning mutagenesis was used to show that Phe¹⁶¹ is a pore lining residue critical to transport. Analysis of the structure of TM IV showed that TM IV is composed of one region of β-turns, an extended middle region including Pro¹⁶⁷-Pro¹⁶⁸, and a helical region (13). TM VII was much more typical of a transmembrane helix although it was interrupted with a break in the helix at the functionally critical residues Gly²⁶¹-Glu²⁶² (14).Open in a separate windowFIGURE 1.Models of the Na⁺/H⁺ exchanger. A, simplified topological model of the transmembrane domain of the NHE1 isoform of the Na⁺/H⁺ exchanger as described earlier (11). EL, extracellular loop; IL, intracellular loop. B, model of amino acids present in TM XI.Another important TM segment of the Na⁺/H⁺ exchanger is TM XI (Fig. 1B). Several different lines of evidence have suggested that it is critical to NHE1 function. A recent study generated chimeras of NHE1 from various species and found that a region including TM XI was important in determining NHE1 inhibitor sensitivity (15). More specifically, mutagenesis of several amino acids of TM XI has shown that it is likely involved in either ion transport or proper targeting to the plasma membrane. Two mutants in TM XI, Y454C and R458C, are retained in the endoplasmic reticulum (16). In addition, mutation of Gly⁴⁵⁵ and Gly⁴⁵⁶ in TM XI shift the pH_i dependence of the exchanger to the alkaline side, whereas mutation of Arg⁴⁴⁰ in intracellular loop 5 at the N-terminal end of TM XI shifts the pH_i dependence to make it more acidic (17, 18). Also, the structure of the bacterial Na⁺/H⁺ exchanger NhaA has been elucidated. Both TM IV and TM XI play a critical role forming an assembly that cross, with each being a helix, an extended polypeptide and a short helix (19). We found that TM IV of NHE1 has a similar structure and function to that of TM IV of NhaA (2, 13), leaving open the possibility that TM XI of NHE1 is also similar in structure and function to TM XI of NhaA.For these reasons, we undertook a systematic examination of the structural and functional aspects of TM XI of the NHE1 isoform of the Na⁺/H⁺ exchanger. The sequence of human TM XI of NHE1 is ⁴⁴⁹QFIIAYGGLRGAIAFSLGYLLD⁴⁷⁰. In this study we use cysteine scanning mutagenesis and site-specific mutagenesis to identify and characterize critical pore lining residues of the protein. We also use nuclear magnetic resonance (NMR) spectroscopy to characterize the structure of a synthetic peptide representing TM XI in dodecylphosphocholine (DPC) micelles. Evidence has suggested that TM segments of membrane proteins possess all the structural information required to form their higher order structures in their amino acid sequence (20). This has been demonstrated in earlier studies on membrane protein segments such as the cystic fibrosis transmembrane conductance regulator (21), a fungal G-protein-coupled receptor (22), bacteriorhodopsin (23, 24), and rhodopsin (25), where it was shown that isolated TM segments from membrane proteins had structures in good agreement with the segments of the entire protein. Also, the use of DPC micelles has been shown to be an excellent membrane mimetic environment for these studies (26, 27). Our study identifies Leu⁴⁶⁵ as contributing to the pore of the protein and shows that the structure of TM XI consists of two helices corresponding to Asp⁴⁴⁷-Tyr⁴⁵⁴ and Phe⁴⁶⁰-Lys⁴⁷¹ at the N and C termini, respectively, connected by a flexible region at residues 455-459. The structure of TM XI was similar to the x-ray structure of TM XI of NhaA.     

钠氢交换蛋白的研究进展

钠氢交换蛋白是一类存在于细胞膜表面的离子转运泵蛋白家族.它负责将细胞内H 与胞外Na 按照1:1的比例进行交换来调控细胞内pH的动态平衡,影响细胞的容积、运动、分化、凋亡和营养吸收,从而参与许多复杂的生理和病理过程.迄今为止,钠氢交换蛋白家族已发现有9个成员,各亚型间具有结构相似性和组织分布特异性.深入研究NHE的结构、功能及基因表达调控,将为人和哺乳动物的营养生理、疾病治疗提供新的思路和方法.     

Regulation of Intestinal Epithelial Brush Border Na+/H+ Exchanger Isoforms, NHE2 and NHE3, in C2bbe Cells

Until recently, studies to characterize the intestinal epithelial Na⁺/H⁺ exchangers had to be done in nonepithelial, mutated fibroblasts. In these cells, detection of any Na⁺/H⁺ exchange activity requires prior acid loading. Furthermore, most of these experiments used intracellular pH changes to measure NHE activity. Because changes in pH _i only approximate Na⁺/H⁺ exchange activity, and may be confounded by alterations in buffering capacity and/or non-NHE contributions to pH regulation, we have used ²²[Na] unidirectional apical to cell uptake to measure activities specific to NHE2 or NHE3. Furthermore, we performed these measurements under basal, nonacid-stimulated conditions to avoid bias from this nonphysiological experimental precondition. Both brush border NHEs, when expressed in the well-differentiated, intestinal villuslike Caco-2 subclone, C2bbe (C2), localize to the C2 apical domain and are regulated by second messengers in the same way they are regulated in vivo. Increases in intracellular calcium and cAMP inhibit both isoforms, while phorbol ester affects only NHE3. NHE2 inhibition by cAMP and Ca⁺⁺ involves changes to both K _Na and V _max . In contrast, the same two second messengers inhibit NHE3 by a decrease in V _max exclusively. Phorbol ester activation of protein kinase C alters both V _max and K _Na of NHE3, suggesting a multilevel regulatory mechanism. We conclude that NHE2 and NHE3, in epithelial cells, are basally active and are differentially regulated by signal transduction pathways. Received: 28 January 1999/Revised: 18 May 1999     

The Na+/H+ Exchanger NHE6 in the Endosomal Recycling System Is Involved in the Development of Apical Bile Canalicular Surface Domains in HepG2 Cells

Polarized epithelial cells develop and maintain distinct apical and basolateral surface domains despite a continuous flux of membranes between these domains. The Na⁺/H⁺exchanger NHE6 localizes to endosomes but its function is unknown. Here, we demonstrate that polarized hepatoma HepG2 cells express an NHE6.1 variant that localizes to recycling endosomes and colocalizes with transcytosing bulk membrane lipids. NHE6.1 knockdown or overexpression decreases or increases recycling endosome pH, respectively, and inhibits the maintenance of apical, bile canalicular plasma membranes and, concomitantly, apical lumens. NHE6.1 knockdown or overexpression has little effect on the de novo biogenesis of apical surface domains. NHE6.1 knockdown does not inhibit basolateral-to-apical transcytosis of bulk membrane lipids, but it does promote their progressive loss from the apical surface, leaving cells unable to efficiently retain bulk membrane and bile canalicular proteins at the apical surface. The data suggest that a limited range of endosome pH mediated by NHE6.1 is important for securing the polarized distribution of membrane lipids at the apical surface and maintenance of apical bile canaliculi in HepG2 cells and hence cell polarity. This study underscores the emerging role of the endosomal recycling system in apical surface development and identifies NHE6 as a novel regulatory protein in this process.     

Unique Regulation of Human Na+/H+ Exchanger 3 (NHE3) by Nedd4-2 Ligase That Differs from Non-primate NHE3s

Na⁺/H⁺ exchanger NHE3 expressed in the intestine and kidney plays a major role in NaCl and HCO₃⁻ absorption that is closely linked to fluid absorption and blood pressure regulation. The Nedd4 family of E3 ubiquitin ligases interacts with a number of transporters and channels via PY motifs. A comparison of NHE3 sequences revealed the presence of PY motifs in NHE3s from human and several non-human primates but not in non-primate NHE3s. In this study we evaluated the differences between human and non-primate NHE3s in ubiquitination and interaction with Nedd4-2. We found that Nedd4-2 ubiquitinated human NHE3 (hNHE3) and altered its expression and activity. Surprisingly, rat NHE3 co-immunoprecipitated Nedd4-2, but its expression and activity were not altered by silencing of Nedd4-2. Ubiquitination by Nedd4-2 rendered hNHE3 to undergo internalization at a significantly greater rate than non-primate NHE3s without altering protein stability. Insertion of a PY motif in rabbit NHE3 recapitulated the interaction with Nedd4-2 and enhanced internalization. Thus, we propose a new model where disruption of Nedd4-2 interaction elevates hNHE3 expression and activity.     

Myocardial Na+/H+ exchanger-1 (NHE1) content is decreased by exercise training

The effect of exercise training on myocardial Na⁺/H⁺ exchanger-1 (NHE1) protein expression was examined. Adult female Sprague–Dawley rats were randomly divided into sedentary (S; n?=?8) and exercised (E; n?=?9) groups. Twenty-four hours after the last exercise bout, hearts were weighed and connected to an isolated perfused working heart apparatus for evaluation of cardiac functional performance. Heart weight and heart weight/body weight from E rats was significantly increased by 7.1 and 7.2 % (P?<?0.05), respectively, compared with S hearts. The E hearts displayed 15 % greater cardiac output and 35 % external cardiac work compared with the S group at both low and high workloads (P?<?0.05 for both parameters). Left ventricular tissue from the same hearts was homogenized and NHE1 and Na⁺/Ca²⁺ exchanger (NCX) content determined by Western blotting. E hearts had a 38 % (P?<?0.001) reduction in NHE1 content related to S hearts, and there was no difference in NCX content between groups. Cytochrome c oxidase activity in plantaris increased by 100 % (P?<?0.05) and was assessed as a marker of mitochondria content and to verify training status. Our data indicate that exercise training at an intensity that results in cardiac hypertrophy and improved performance is accompanied by decreased NHE1 content in heart.     

Characterization of the Na+/H+ Exchanger in the Luminal Membrane of the Distal Nephron

In the rabbit as well as the rat, a Na⁺/H⁺ exchanger is expressed in the apical membrane of both the proximal and distal tubules of the renal cortex. Whereas the isoform derived from the proximal tubule has been extensively studied, little information is available concerning the distal luminal membrane isoform. To better characterize the latter isoform, we purified rabbit proximal and distal tubules, and examined the ethylpropylamiloride (EIPA)-sensitive ²²Na uptake by the luminal membrane vesicles from the two segments. The presence of 100 μm EIPA in the membrane suspension decreased the 15 sec Na⁺ uptake to 75.70 ± 4.70% and 50.30 ± 2.23% of the control values in vesicles from proximal and distal tubules, respectively. The effect of EIPA on 35 mm Na⁺ uptake was concentration dependent, with a IC₅₀ of 700 μm and 75 μm for the proximal and distal luminal membranes. Whereas the proximal tubule membrane isoform was insensitive to cimetidine and clonidine up to a concentration of 2 mm, the 35 mm Na⁺ uptake by the distal membrane was strongly inhibited by cimetidine (IC₅₀ 700 μm) and modestly inhibited by clonidine (IC₅₀ 1.6 mm). The incubation of proximal tubule suspensions with 1 mm (Bu₂) cAMP decreased the 15-sec EIPA-sensitive Na⁺ uptake by the brush border membranes to 24.1 ± 2.38% of the control values. Unexpectedly, the same treatment of distal tubules enhanced this uptake by 46.5 ± 10.3%. Finally, incubation of tubule suspensions with 100 nm phorbol 12-myristate 13-acetate (PMA) decreased the exchanger activity to 58.6 ± 3.04% and 79.7 ± 3.21% of the control values in the proximal and distal luminal membranes, respectively. In conclusion, the high sensitivity of the distal luminal membrane exchanger to various inhibitors, and its stimulation by cAMP-dependent protein kinase A, indicate that this isoform differs from that of the proximal tubule and probably corresponds to isoform 1. Received: 6 March 1998/Revised: 6 July 1998     

Dimerization is crucial for the function of the Na+/H+ exchanger NHE1

The Na(+)/H(+) exchanger 1 (NHE1) exists as a homo-dimer in the plasma membranes. In the present study, we have investigated the functional significance of the dimerization, using two nonfunctional NHE1 mutants, surface-expression-deficient G309V and transport-deficient E262I. Biochemical and immunocytochemical experiments revealed that these NHE1 mutants are capable of interacting with the wild-type NHE1 and, thus, forming a heterodimer. Expression of G309V retained the wild-type NHE1 to the ER membranes, suggesting that NHE1 would first form a dimer in the ER. On the other hand, expression of E262I markedly reduced the exchange activity of the wild-type NHE1 through an acidic shift in the intracellular pH (pH(i)) dependence, suggesting that dimerization is required for exchange activity in the physiological pH(i) range. However, a dominant-negative effect of E262I was not detected when exchange activity was measured at acidic pH(i), implying that one active subunit is sufficient to catalyze ion transport when the intracellular H(+) concentration is sufficiently high. Furthermore, intermolecular cysteine cross-linking at extracellular position Ser(375) with a bifunctional sulfhydryl reagent dramatically inhibited exchange activity mainly by inducing the acidic shift of pH(i) dependence and abolished extracellular stimuli-induced activation of NHE1 without causing a large change in the affinities for extracellular Na(+) or an inhibitor EIPA. Because monofunctional sulfhydryl regents had no effect, it is likely that cross-linking inhibited the activity of NHE1 by restricting a coupled motion between the two subunits during transport. Taken together, these data support the view that dimerization of two active subunits are required for NHE1 to possess the exchange activity in the neutral pH(i) range, although each subunit is capable of catalyzing transport in the acidic pH(i) range.     

Membrane curvature alters the activation kinetics of the epithelial Na+/H+ exchanger, NHE3

The epithelial Na(+)/H(+) exchanger, NHE3, was found to activate slowly following an acute cytosolic acidification. The sigmoidal course of activation could not be explained by the conventional two-state model, which postulates that activation results from protonation of an allosteric modifier site. Instead, mathematical modeling predicted the existence of three distinct states of the exchanger: two different inactive states plus an active form. The interconversion of the inactive states is rapid and dependent on pH, whereas the conversion between the second inactive state and the active conformation is slow and pH-independent but subject to regulation by other stimuli. Accordingly, exposure of epithelial cells to hypoosmolar solutions activated NHE3 by accelerating this latter transition. The number of surface-exposed exchangers and their association with the cytoskeleton were not affected by hypoosmolarity. Instead, NHE3 is activated by the membrane deformation, a result of cell swelling. This was suggested by the stimulatory effects of amphiphiles that induce a comparable positive (convex) deformation of the membrane. We conclude that NHE3 exists in multiple states and that different physiological parameters control the transitions between them.     

Quantitative contribution of NHE2 and NHE3 to rabbit ileal brush-border Na+/H+ exchange

Intestinal neutral NaCl absorption, which is made up ofbrush-border (BB)Na⁺/H⁺exchange linked to BBCl/HCO₃exchange, is up- and downregulated as part of digestion and diarrhealdiseases. Glucocorticoids stimulate ileal NaCl absorption and BBNa⁺/H⁺exchange. Intestinal BB contains twoNa⁺/H⁺exchanger isoforms, NHE2 and NHE3, but their relative roles in rabbitileal BBNa⁺/H⁺exchange has not been determined. A technique to separate the contribution of NHE2 and NHE3 to ileal BBNa⁺/H⁺exchange activity was standardized by using an amiloride-related compound, HOE-694. Under basal conditions, both NHE2 and NHE3 contribute ~50% to ilealNa⁺/H⁺exchange. Glucocorticoids (methylprednisolone) increase BBNa⁺/H⁺exchange (2.5 times) but increase only ileal NHE3 activity (4.1 times),without an effect on NHE2 activity. Thus ileal BBNa⁺/H⁺exchange in animals treated with glucocorticoids is 69% via NHE3. Aquantitative Western analysis for NHE3 was developed, using as aninternal standard a fusion protein of the COOH-terminal 85 amino acidsof NHE3 and maltose binding protein. Glucocorticoid treatment increasedthe amount of BB NHE3. The quantitative Western analysis showed thatNHE3 makes up 0.018% of ileal BB protein in control rabbits and0.042% (2.3 times as much) in methylprednisolone-treated rabbits.Methylprednisolone treatment did not alter the amount of ileal BB NHE2protein. NHE3 turnover number was estimated to be 458 cycles/s underbasal conditions and 708 cycles/s in glucocorticoid-treated ileum. Thusmethylprednisolone stimulates ileal BBNa⁺/H⁺exchange activity only by an effect on NHE3 and not on NHE2; it does soprimarily by increasing the amount of BB NHE3, although it alsoincreases the NHE3 turnover number.

     

Effect of angiotensin-II on renal Na+/H+ exchanger-NHE3 and NHE2

The purpose of the present study was to determine the effect of angiotensin II (A-II) on membrane expression of Na+/H+ exchange isoforms NHE3 and NHE2 in the rat renal cortex. A-II (500 ng/kg per min) was chronically infused into the Sprague-Dawley rats by miniosmotic pump for 7 days. Arterial pressure and circulating plasma A-II level were significantly increased in A-II rats as compared to control rats. pH-dependent uptake of 22Na+ study in the presence of 50 microM HOE-694 revealed that Na+ uptake mediated by NHE3 was increased approximately 88% in the brush border membrane from renal cortex of A-II-treated rats. Western blotting showed that A-II increased NHE3 immunoreactive protein levels in the brush border membrane of the proximal tubules by 31%. Northern blotting revealed that A-II increased NHE3 mRNA abundance in the renal cortex by 42%. A-II treatment did not alter brush border NHE2 protein abundance in the renal proximal tubules. In conclusion, chronic A-II treatment increases NHE3-mediated Na+ uptake by stimulating NHE3 mRNA and protein content.     

Osmotic Stimulation of the Na+/H+ Exchanger NHE1: Relationship to the Activation of Three MAPK Pathways

The Na⁺/H⁺ exchanger (NHE) becomes activated by hyperosmolar stress, thereby contributing to cell volume regulation. The signaling pathway(s) responsible for the shrinkage-induced activation of NHE, however, remain unknown. A family of mitogen-activated protein kinases (MAPK), encompassing p42/p44 Erk, p38 MAPK and SAPK, has been implicated in a variety of cellular responses to changes in osmolarity. We therefore investigated whether these kinases similarly signal the hyperosmotic activation of NHE. The time course and osmolyte concentration dependence of hypertonic activation of NHE and of the three sub-families of MAPK were compared in U937 cells. The temporal course and dependence on osmolarity of Erk and p38 MAPK activation were found to be similar to that of NHE stimulation. However, while pretreatment of U937 cells with the kinase inhibitors PD98059 and SB203580 abrogated the osmotic activation of Erk and p38 MAPK, respectively, it did not prevent the associated stimulation of NHE. Thus, Erk1/2 and/or p38 MAPK are unlikely to mediate the osmotic regulation of NHE. The kinetics of NHE activation by hyperosmolarity appeared to precede SAPK activation. In addition, hyperosmotic activation of NHE persisted in mouse embryonic fibroblasts lacking SEK1/MKK4, an upstream activator of SAPK. Moreover, shrinkage-induced activation of NHE still occurred in COS-7 cells that were transiently transfected with a dominant-negative form of SEK1/MKK4 (SEK1/MKK4-A/L) that is expected to inhibit other isoforms of SEK as well. Together, these results demonstrate that the stimulation of NHE and the activation of Erk, p38 MAPK and SAPK are parallel but independent events. Received: 27 November 2000/Revised: 20 March 2001