The Na+/H+ Exchanger Present in Trout Erythrocyte Membranes is Not Responsible for the Amiloride-insensitive Na+/Li+ Exchange Activity

The protein responsible for the Na⁺/Li⁺ exchange activity across the erythrocyte membrane has not been cloned or isolated. It has been suggested that a Na⁺/H⁺ exchanger could be responsible for the Na⁺/Li⁺ exchange activity across the erythrocyte membrane. Previously, we reported that in the trout erythrocyte, the Li⁺/H⁺ exchange activity (mediated by the Na⁺/H⁺ exchanger βNHE) and the Na⁺/Li⁺ exchange activity respond differently to cAMP, DMA (dimethyl-amiloride) and O₂. We concluded that the DMA insensitive Na⁺/Li⁺ exchange activity originates from a different protein. To further examine these findings, we measured Li⁺ efflux in fibroblasts expressing the βNHE as the only Na⁺/H⁺ exchanger. Moreover, the internal pH of these cells was monitored with a fluorescent probe. Our findings indicate that acidification of fibroblasts expressing the Na⁺/H⁺ exchanger βNHE, induces a Na⁺ stimulated Li⁺ efflux activity in trout erythrocytes. This exchange activity, however, is DMA sensitive and therefore differs from the DMA insensitive Na⁺/Li⁺ exchange activity. In these fibroblasts no significant DMA insensitive Na⁺/Li⁺ exchange activity was found. These results support the hypothesis that the trout erythrocyte Na⁺/Li⁺ exchange activity is not mediated by the Na⁺/H⁺ exchanger (βNHE) present in these membranes. Received: 6 December 1996/Revised: 11 August 1997     

Structure and analysis of the mouse Na+/H+ exchanger (NHE1) gene: Homology and conservation of splice sites

The Na⁺/H⁺ exchanger is a widely distributed integral membrane protein that is responsible for pH regulation in mammalian tissues. We have cloned and analyzed the NHE1 isoform of the mouse genomic Na⁺/H⁺exchanger. A clone from a mouse genomic library contained the NHE1 promoter region and the 5-untranslated region. It also contained the first 121 amino acids of the coding region of the Na⁺/H⁺ exchanger. A splice site occurred after amino acid 121, at the same region as in the human NHE1 gene. The deduced amino terminal coding sequence was 76 and 88% identical to the human and rat NHE1 sequences respectively. The 5-untranslated region was highly homologous to that of other species and two minicistrons contained in the human Na⁺/H⁺ exchanger were present in the mouse sequence. The results show that the deduced protein sequence of the mouse NHE1 gene has a high level of homology with other species and that the splice site of the first intron is conserved. These results suggest that the first large intron may play an important role in the NHE1 gene expression.     

Activation of Na/H Exchange in Erythrocytes of Frog Rana ridibunda

Transport of Na⁺ in isolated erythrocytes of the frog Rana ridibunda was studied using radioactive isotope ^{22 22}Na. Treatment of erythrocytes with -adrenergic agonist isoproterenol (ISP) or with a combination of ISP and phosphodiesterase blocker 3-isobutyl-methyl-xanthine (IBMX) did not affect the Na⁺ transport into the cells. These data indicated that cAMP-dependent protein kinase A did not participate in regulation of the Na⁺ transport into the frog erythrocytes. Incubation of erythrocytes with protein kinase C activator phorbol ester (PMA, 0.15 µM) led to a pronounced increase of ^{22 22}Na accumulation and intracellular Na⁺ concentration. These changes of the Na⁺ transport into the cells were completely blocked in the presence of 50 µM ethyl-isopropyl-amiloride (EIPA), a selective blocker of the NHE1-isoform of Na⁺/H⁺ exchanger. Hence, PMA produced activation of Na⁺/H⁺ exchange in frog erythrocytes. The unidirectional Na⁺ influx into erythrocytes amounted, on average, to 0.99 ± 0.12 and 147 ± 9 mmol/l cells/h for control and PMA-treated cells, respectively. The EIPA concentration producing a 50% inhibition of the PMA-induced Na⁺ influx (IC₅₀) was 0.28 µM. A high sensitivity of the frog Na/H exchanger to EIPA indicates its similarity with the mammalian NHE1 isoform. The obtained data for the first time clearly indicate an important role of PKC in Na/H exchange regulation in the frog red blood cells.     

High capacity Na+/H+ exchange activity in mineralizing osteoblasts

Osteoblasts synthesize bone in polarized groups of cells sealed by tight junctions. Large amounts of acid are produced as bone mineral is precipitated. We addressed the mechanism by which cells manage this acid load by measuring intracellular pH (pHi) in non‐transformed osteoblasts in response to weak acid or bicarbonate loading. Basal pHi in mineralizing osteoblasts was ～7.3 and decreased by ～1.4 units upon replacing extracellular Na⁺ with N‐methyl‐D ‐glucamine. Loading with 40 mM acetic or propionic acids, in normal extracellular Na⁺, caused only mild cytosolic acidification. In contrast, in Na⁺‐free solutions, weak acids reduced pHi dramatically. After Na⁺ reintroduction, pHi recovered rapidly, in keeping with Na⁺/H⁺ exchanger (NHE) activity. Sodium‐dependent pHi recovery from weak acid loading was inhibited by amiloride with the Ki consistent with NHEs. NHE1 and NHE6 were expressed strongly, and expression was upregulated highly, by mineralization, in human osteoblasts. Antibody labeling of mouse bone showed NHE1 on basolateral surfaces of all osteoblasts. NHE6 occurred on basolateral surfaces of osteoblasts mainly in areas of mineralization. Conversely, elevated HCO alkalinized osteoblasts, and pH recovered in medium containing Cl^?, with or without Na⁺, in keeping with Na⁺‐independent Cl^?/HCO exchange. The exchanger AE2 also occurred on the basolateral surface of osteoblasts, consistent with Cl^?/HCO exchange for elimination of metabolic carbonate. Overexpression of NHE6 or knockdown of NHE1 in MG63 human osteosarcoma cells confirmed roles of NHE1 and NHE6 in maintaining pHi. We conclude that in mineralizing osteoblasts, slightly basic basal pHi is maintained, and external acid load is dissipated, by high‐capacity Na⁺/H⁺ exchange via NHE1 and NHE6. J. Cell. Physiol. 226: 1702–1712, 2011. © 2010 Wiley‐Liss, Inc.     

Synthesis and Na+/H+ Exchanger‐1 Inhibitory Activity of Substituted (Quinolinecarbonyl)guanidine Derivatives

The Na⁺/H⁺ exchanger (NHE) is a protein expressed in many mammalian cell types. It is involved in intracellular pH (pH_i) homeostasis by exchanging extracellular Na⁺ for intracellular H⁺. To date, nine NHE isoforms (NHE1–NHE9) have been identified. NHE1 is the most predominant isoform expressed in mammalian cardiac muscle. A novel series of substituted (quinolinecarbonyl)guanidine derivatives were designed and synthesized as NHE inhibitors. Most compounds can inhibit NHE1‐mediated platelet swelling in a concentration‐dependent manner, among which compound 7f was the most active and more potent than cariporide. Furthermore, compound 7f has also been demonstrated to exhibit the in vivo cardioprotective effects against SD rat myocardial ischemic‐reperfusion injury superior to those of cariporide.     

Overexpression of the NHE1 isoform of the Na+/H+ exchanger causes elevated apoptosis in isolated cardiomyocytes after hypoxia/reoxygenation challenge

The mammalian Na⁺/H⁺ exchanger isoform 1 (NHE1) is a ubiquitously expressed membrane protein that regulates intracellular pH in the myocardium and other tissues. NHE1 is an important mediator of myocardial damage that occurs after ischemia–reperfusion injury. It has also been implicated in apoptotic damage in many tissues and its expression and activity are elevated in disease states in the myocardium. In this study, we examined the effect of additional exogenous NHE1 expression on isolated cardiomyocytes susceptibility to ischemia/reperfusion damage. Exogenous NHE1 elevated Na⁺/H⁺ exchanger expression and activity when introduced into isolated cardiomyocytes through an adenoviral system. Isolated cardiomyocytes were subjected to simulated ischemia and reperfusion after infection with either control or NHE1-containing adenovirus. Cells were placed into an anaerobic chamber and effects of NHE1 expression after hypoxia/reoxygenation were examined. Hypoxia/reoxygenation increased caspase-3-like activity in controls, and the effect was greatly magnified in cells expressing NHE1 protein. It also elevated the percentage of apoptotic cardiomyocytes, which was also aggravated by expression of NHE1 protein. Hypoxia/reoxygenation also increased phospho-ERK levels. Elevated NHE1 expression was coincidental with increased expression of the ER stress protein, protein disulfide isomerase (PDI) and calreticulin (CRT). Our results demonstrate that increased NHE1 protein expression makes cells more susceptible to damage induced by hypoxia/reoxygenation in isolated cardiomyocytes. They suggest that elevated NHE1 in cardiovascular disease could predispose the human myocardium to enhanced apoptotic damage.     

Functional Characterization of Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Exchangers in Primary Cultures of Prairie Dog Gallbladder

Gallbladder Na⁺ absorption is linked to gallstone formation in prairie dogs. We previously reported Na⁺/H⁺ exchanger (NHE1-3) expression in native gallbladder tissues. Here we report the functional characterization of NHE1, NHE2 and NHE3 in primary cultures of prairie dog gallbladder epithelial cells (GBECs). Immunohistochemical studies showed that GBECs grown to confluency are homogeneous epithelial cells of gastrointestinal origin. Electron microscopic analysis of GBECs demonstrated that the cells form polarized monolayers characterized by tight junctions and apical microvilli. GBECs grown on Snapwells exhibited polarity and developed transepithelial short-circuit current, I_sc, (11.6 ± 0.5 µA · cm^–2), potential differences, V_t (2.1 ± 0.2 mV), and resistance, R_t (169 ± 12 · cm²). NHE activity in GBECs assessed by measuring dimethylamiloride-inhibitable ²²Na⁺ uptake under a H⁺ gradient was the same whether grown on permeable Snapwells or plastic wells. The basal rate of ²²Na⁺ uptake was 21.4 ± 1.3 nmol · mg prot^–1 · min^–1, of which 9.5 ± 0.7 (~45%) was mediated through apically-restricted NHE. Selective inhibition with HOE-694 revealed that NHE1, NHE2 and NHE3 accounted for ~6%, ~66% and ~28% of GBECs total NHE activity, respectively. GBECs exhibited saturable NHE kinetics (V_max 9.2 ± 0.3 nmol · mg prot^–1 · min^–1; K_m 11.4 ± 1.4 mM Na⁺). Expression of NHE1, NHE2 and NHE3 mRNAs was confirmed by RT-PCR analysis. These results demonstrate that the primary cultures of GBECs exhibit Na⁺ transport characteristics similar to native gallbladder tissues, suggesting that these cells can be used as a tool for studying the mechanisms of gallbladder ion transport both under physiologic conditions and during gallstone formation.     

Kinetic properties of sodium transport pathways in the river lamprey <Emphasis Type="Italic">Lampetra fluviatilis</Emphasis> erythrocytes

To activate Na⁺/H⁺ exchange, intracellular pH (pH_i) of erythrocytes of the river lamprey Lampetra fluviatilis were changed from 6 and 8 using nigericin. The Na⁺/H⁺ exchanger activity was estimated from the values of amiloride-sensitive components of Na⁺ (²²Na) inflow or of H⁺ outflow from erythrocytes. Kinetic parameters of the carrier functioning were determined by using Hill equation. Dependence of Na⁺ and H⁺ transport on pH_i value is described by hyperbolic function with the Hill coefficient value (n) close to 1. Maximal rate of ion transport was within the limits of 9–10 mmol/l cells/min, and the H⁺ concentration producing the exchanger 50% activation amounted to 0.6–1.0 μM. Stimulation of H⁺ outcome from acidified erythrocytes (pH_i 5.9) with increase of H⁺ concentration in the incubation medium is described by Hill equation with n value of 1.6. Concentration Na⁺ for the semimaximal stimulation of H⁺ outcome amounted to 10 mM. The obtained results indicate the presence in lamprey erythrocytes of only binding site for H⁺ from the cytoplasm side and the presence of positive cooperativity in Na⁺-binding from the extracellular side of the Na⁺/H⁺ exchanger. Na⁺ efflux from cells in the Na⁺-free medium did not change at a 10-fold increase of H⁺ concentration in the incubation medium. The presented data indicate differences of kinetic properties of the lamprey erythrocyte Na⁺/H⁺ exchanger and of this carrier isoforms in mammalian cells. In intact erythrocytes the dependence of the amiloride-sensitive Na⁺ inflow on its concentration in the medium is described by Hill equitation with n 1.6. The Na⁺ concentration producing the 50% transport activation amounted to 39 mM and was essentially higher as compared with that in acidified erythrocytes. These data confirm conception of the presence of two amiloride-sensitive pathways of Na+ transport in lamprey erythrocytes.     

The Vacuolar Na+/H+ Antiporter Gene SsNHX1 from the Halophyte Salsola soda Confers Salt Tolerance in Transgenic Alfalfa (Medicago sativa L.)

A putative vacuolar Na⁺/H⁺ antiporter gene (SsNHX1) was isolated from the halophyte Salsola soda using the rapid amplification of cDNA ends method. Highly conserved regions of plant vacuolar Na⁺/H⁺ antiporter, including amiloride-binding domain, NHE (Na⁺/H⁺ exchange) domain, and 12 transmembrane segments, were found in the deduced amino acid sequence of SsNHX1. Multiple alignments of vacuolar Na⁺/H⁺ antiporters showed that SsNHX1 shared high identity with other plant vacuolar Na⁺/H⁺ antiporters. Phylogenetic relationship analysis indicated that SsNHX1 was clustered into the vacuolar Na⁺/H⁺ antiporter group. Taken together, these results suggest that SsNHX1 is a new member of the vacuolar Na⁺/H⁺ antiporter family. The effective expression of SsNHX1 in alfalfa (Medicago sativa L.) enhanced the salt tolerance of transgenic alfalfa which could grow in high concentrations of NaCl (up to 400 mM) over 50 days. This was the highest level of salt tolerance reported in transgenic plants. A further analysis of the physiological characteristics of transgenic and wild-type plants, including the Na⁺ and K⁺ contents, superoxide dismutase activity, the rate of electrolyte leakage, and the proline content, showed that large amounts of Na⁺ in the cytoplasm of leaves were transported into vacuoles by the exogenous Na⁺/H⁺ antiporter, which averted the toxic effects of Na⁺ to the cell of transgenic alfalfa.     

Expression and characterization of the Na+/H+ exchanger in the mammalian myocardium

We examined two expression systems for studying the Na⁺/H⁺ exchanger in the mammalian myocardium. Mammalian NHE1 with a hemagglutinin (HA) tag and was cloned behind the alpha myosin heavy chain promoter. Transgenic mice were made with wild type NHE1 protein or with a hyperactive NHE1 protein mutated at the calmodulin-binding domain. Three lines of transgenic mice were made of each cDNA with expression levels of each type varying from high to low. Higher levels and activity of the Na⁺/H⁺ exchanger were associated with decreased long-term survival of mice, and with dilated or hypertrophic cardiomyopathy. The exogenous NHE1 protein was present in freshly made cardiomyocytes from transgenic mice, however, expression from the alpha myosin heavy chain promoter declined rapidly and little exogenous NHE1 was apparent on the fourth day after cardiomyocyte isolation. To express NHE1 protein in isolated cardiomyocytes, we transferred a mutated form of the protein into an adenoviral expression system. Infection of neonatal rat cardiomyocytes resulted in robust expression of the exogenous NHE1 protein. The mutant form of the NHE1 protein could be distinguished from the endogenous Na⁺/H⁺ exchanger by its resistance to inhibition by amiloride analogs. Our results suggest that for in vivo studies on intact hearts and animals, expression in transgenic mice is an appropriate system, however for long-term studies on cardiomyocytes, this model is inappropriate due to waning expression from the alpha myosin heavy chain promoter. Therefore, infection by adenovirus is a superior system for long-term studies on cardiomyocytes in culture.     

Na+ Sensitivity of ROMK1 K+ Channel: Role of the Na+/H+ Antiporter

To examine the extracellular Na⁺ sensitivity of a renal inwardly rectifying K⁺ channel, we performed electrophysiological experiments on Xenopus oocytes or a human kidney cell line, HEK293, in which we had expressed the cloned renal K⁺ channel, ROMK1 (Kir1.1). When extracellular Na⁺ was removed, the whole-cell ROMK1 currents were markedly suppressed in both the oocytes and HEK293 cells. Single-channel ROMK1 activities recorded in the cell-attached patch on the oocyte were not affected by removal of Na⁺ from the pipette solution. However, macro-patch ROMK1 currents recorded on the oocyte were significantly suppressed by Na⁺ removal from the bath solution. A blocker of Na⁺/H⁺ antiporters, amiloride, largely inhibited the Na⁺ removal-induced suppression of whole-cell ROMK1 currents in the oocytes. The pH-insensitive K80M mutant of ROMK1 was much less sensitive to Na⁺ removal. Na⁺ removal was found to induce a significant decrease in intracellular pH in the oocytes using H⁺-selective microelectrodes. Coexpression of ROMK1 with NHE3, which is a Na⁺/H⁺ antiporter isoform of the kidney apical membrane, conferred increased sensitivity of ROMK1 channels to extracellular Na⁺ in both the oocytes and HEK293 cells. Thus, it is concluded that the ROMK1 channel is regulated indirectly by extracellular Na⁺, and that the interaction between NHE transporter and ROMK1 channel appears to be involved in the mechanism of Na⁺ sensitivity of ROMK1 channel via regulating intracellular pH. Received: 13 April 1999/Revised: 15 July 1999     

The role of ion antiporters in the maintenance of intracellular pH in rat vascular smooth muscle cells

Vascular smooth muscle intracellular pH is maintained by the Na⁺/H⁺ and Cl^–/HCO ₃ ^– antiporters. The Na⁺/H⁺ exchanger is a major route of H⁺ extrusion in most eukaryotic cells and is present in vascular smooth muscle cells in a similar capacity. It extrudes H^– into the extracellular space in exchange for Na⁺. The Cl^–/HCO ₃ ^– exchanger plays an analogous role to lower the pH of vascular smooth muscle cells when increases in intracellular pH occur. Its activity has also been demonstrated in A7r5 and A10 vascular smooth muscle cells. The Na⁺/H⁺ exchanger is regulated by a number of agents which act through inositol trisphosphate/diacylglycerol, to stimulate the antiporter. Calcium-calmodulin dependent protein kinase may also activate the antiporter in vivo. Phosphorylation of the Cl^–/HCO ₃ ^– exchanger has also been observed but its physiological role is not known. Both these antiporters exist in the plasma membrane as integral proteins with free acidic cytoplasmic termini. These regions may be important in sensing changes in intracellular pH, to which these antiporters respond.Abbreviations CaM Calmodulin - DCCD Dicylohexyl-Carbodiimide - DG Diacylglycerol - DIDS-4 4-Diisthiocyanostilbene-2,2-Disulfonic Acid - IP₃ Inositol Trisphosphate - PKC protein Kinase C - SITS-4 4-Acetamido-4-Isothiocyanstilbene-2,2-Disulfonate - VSMC Vascular Smooth Muscle Cell     

A stable Na+/H+ antiporter of thermophilic bacterium PS3

As a first step in the isolation of a stable Na⁺/H⁺ antiporter, its reaction in sonicated membrane vesicles of thermophilic bacterium PS3 has been characterized. The sonicated vesicles showed quenching of quinacrine fluorescence in either NADH oxidation or ATP hydrolysis. The quenching was reversed by the addition of Na⁺, Li⁺, Mn²⁺, Cd²⁺, and Co²⁺, but not of choline⁺ or Ca²⁺, regardless of their counter anions.²²Na⁺ was taken up into the vesicles by NADH oxidation, and the²²Na⁺ uptake was inhibited by the addition of an uncoupler. H⁺ release was observed on addition of Na⁺ to sonicated vesicles. The magnitude of the pH difference across the membrane induced by NADH oxidation was constant at pH 7.0 to 9.1, but the Na⁺/H⁺ antiport was affected by the pH of the medium (optimum pH=8.5). TheK _m 's of the antiporter for Na⁺ and Li⁺ were 2.5 and 0.1 mM, respectively, but theV _max values for the two ions were the same at pH 8.0. In the presence of Li⁺, no further decrease of fluorescence quenching was observed on addition of Na⁺ andvice versa. The Na⁺/H⁺ antiporter activity in PS3 was stable at 70°C, and the optimum temperature for activity was 55–60°C. In contrast to mesophilic cation/H⁺ antiporters, this antiporter was not inhibited by a thiol reagent.Abbreviations Tricine N-tris(hydroxymethyl)methylglycine - MOPS morpholinopropane sulfonic acid - TMAHO tetramethylammonium hydroxide - DCCD N,N-dicyclohexylcarbodiimide - FCCP carbonyl cyanidep-trifluoromethoxyphenylhydrazone - H⁺ — ATPase proton-translocating adenosine triphosphatase - electrochemical proton gradient across membrane - electrochemical Na⁺ gradient across membrane - pH pH difference across membrane     

Stimulation of human cheek cell Na+/H+ antiporter activity by saliva and salivary electrolytes: amplification by nigericin

Proton-dependent, ethylisopropylamiloride (EIPA)-sensitive Na⁺ uptake (Na⁺/H⁺ antiporter) studies were performed to examine if saliva, and ionophores which alter cellular electrolyte balance, could influence the activity of the cheek cell Na⁺/H⁺ antiporter. Using the standard conditions of 1 mmol/1 Na⁺, and a 65:1 (inside:outside) proton gradient in the assay, the uniport ionophores valinomycin (K⁺) and gramicidin (Na⁺) increased EIPA-sensitive Na⁺ uptake by 177% (p < 0.01) and 227% (p < 0.01), respectively. The dual antiporter ionophore nigericin (K⁺-H⁺) increased EIPA-sensitive Na⁺ uptake by 654% (p < 0.01), with maximal Na⁺ uptake achieved by 1 min and at an ionophore concentration of 50 mol/l, with an EC ₅₀ value 6.4 mol/l. Preincubation of cheek cells with saliva or the low molecular weight (MW) components of saliva (saliva activating factors, SAF) for 2 h at 37°C, also significantly stimulated EIPA-sensitive Na⁺ uptake. This stimulation could be mimicked by pre-incubation with 25 mmol/l KCl or K⁺-phosphate buffer. Pre-incubating cheek cells with SAF and the inclusion of 20 mol/1 nigericin in the assay, produced maximum EIPA-sensitive Na⁺ uptake. After pre-incubation with water, 25 mmol/1 K⁺-phosphate or SAF, with nigericin in all assays, the initial rate of proton-gradient dependent, EIPA-sensitive Na⁺ uptake was saturable with respect to external Na⁺ with K_m values of 0.9, 1.7, and 1.8 mmol/l, and V _max values of 13.4, 25.8, and 31.1 nmol/mg protein/30 sec, respectively. With 20 mol/1 nigericin in the assay, Na⁺ uptake was inhibited by either increasing the [K⁺]_o in the assay, with an ID ₅₀ of 3 mmol/l. These results indicate that nigericin can facilitate K⁺ _i exchange for H⁺ _o and the attending re-acidification of the cheek cell amplifies IINa+ uptake via the Na⁺/H⁺ antiporter. The degree of stimulation of proton-dependent, EIPA-sensitive Na⁺ uptake is therefore dependent, in part, on the intracellular K⁺ _i.     

Cytoplasmic acidification and activation of Na+/H+ exchange during regulatory volume decrease in Ehrlich ascites tumor cells

Summary Ehrlich ascites tumor cells undergoing regulatory volume decrease (RVD) exhibit cytoplasmic acidification as measured by an intracellular fluorescent pH indicator. The acidification results in an activation of the Na⁺/H⁺ exchanger. The intracellular pH set point for the activation is estimated to be around 7.0. The activation of the Na⁺/H⁺ exchanger leads to an incomplete RVD. In support of this conclusion, amiloride and Na⁺-free medium, known to limit the Na⁺/H⁺ exchange, indeed enhance the RVD response. Intracellular acidification and activation of Na⁺/H⁺ exchange may be a general response of cells undergoing RVD.     

H+ and HCO3? Transporters in Human Salivary Ducts. An Immunohistochemical Study

The presence and cellular distribution of key H⁺ and HCO₃ ^– transport proteins was studied in human salivary ducts. Immunofluorescence and immunoperoxidase light microscopy was applied, using specific antibodies against the NHE1 and NHE3 isoforms of the Na⁺H⁺ exchanger, against the 31 and 70kDa subunits of the vacuolar H⁺-ATPase and against the electrogenic Na⁺-HCO₃ ^– cotransporter. The results show basolateral NHE1 and apical NHE3 in human submandibular, parotid and sublingual duct cells. Vacuolar H⁺-ATPase was found predominantly in the apical membrane of parotid, submandibular and sublingual duct cells, although it was absent in certain parotid striated duct cells. The Na⁺-HCO₃ ^– cotransporter was predominantly expressed in the apical membrane of parotid and sublingual striated ducts, and intracellularly distributed in the distal parts of the gland tree and in submandibular ducts. The results indicate that HCO₃ ^– transport properties of salivary ducts may vary not only between gland and species, but even in different duct segments of the same gland as well.     

��Pix Up-regulates Na+/H+ Exchanger 3 through a Shank2-mediated Protein-Protein Interaction

Na⁺/H⁺ exchanger 3 (NHE3) plays an important role in neutral Na⁺ transport in mammalian epithelial cells. The Rho family of small GTPases and the PDZ (PSD-95/discs large/ZO-1) domain-based adaptor Shank2 are known to regulate the membrane expression and activity of NHE3. In this study we examined the role of βPix, a guanine nucleotide exchange factor for the Rho GTPase and a strong binding partner to Shank2, in NHE3 regulation using integrated molecular and physiological approaches. Immunoprecipitation and pulldown assays revealed that NHE3, Shank2, and βPix form a macromolecular complex when expressed heterologously in mammalian cells as well as endogenously in rat colon, kidney, and pancreas. In addition, these proteins co-segregated at the apical surface of rat colonic epithelial cells, as detected by immunofluorescence staining. When expressed in PS120/NHE3 cells, βPix increased membrane expression and basal activity of NHE3. Interestingly, the effects of βPix on NHE3 were abolished by cotransfection with dominant-negative Shank2 mutants and by treatment with Clostridium difficile toxin B, a Rho GTPase inhibitor, indicating that Shank2 and Rho GTPases are involved in βPix-mediated NHE3 regulation. Knockdown of endogenous βPix by RNA interference decreased Shank2-induced increase of NHE3 membrane expression in HEK 293T cells. These results indicate that βPix up-regulates NHE3 membrane expression and activity by Shank2-mediated protein-protein interaction and by activating Rho GTPases in the apical regions of epithelial cells.     

Different ionic conditions prompt NHE2 and NHE3 translocation to the plasma membrane

We tested whether NHE3 and NHE2 Na⁺/H⁺ exchanger isoforms were recruited to the plasma membrane (PM) in response to changes in ion homeostasis. NHE2-CFP or NHE3-CFP fusion proteins were functional Na⁺/H⁺ exchangers when transiently expressed in NHE-deficient PS120 fibroblasts. Confocal morphometry of cells whose PM was labeled with FM4-64 measured the fractional amount of fusion protein at the cell surface. In resting cells, 10-20% of CFP fluorescence was at PM and stable over time. A protocol commonly used to activate the Na⁺/H⁺ exchange function (NH₄-prepulse acid load sustained in Na⁺-free medium), increased PM percentages of PM NHE3-CFP and NHE2-CFP. Separation of cellular acidification from Na⁺ removal revealed that only NHE3-CFP translocated when medium Na⁺ was removed, and only NHE2-CFP translocated when the cell was acidified. NHE2/NHE3 chimeric proteins demonstrate that the Na⁺-removal response element resides predominantly in the NHE3 cytoplasmic tail and is distinct from the acidification response sequence of NHE2.