Na+/H+ exchanger (NHE) in the basolateral membrane is encoded by NHE-1 mRNA in LLC-PK1 clone 4 cells

Several isoforms of Na⁺/H⁺ exchanger (NHE-1–5) have been identified. LLC-PK1 clone 4 (CL4) expresses the amiloride-sensitive type of NHE predominantly in the basolateral membrane, which is believed to be NHE-1. It is not clear whether CL4 expresses NHE in the apical membrane and which side of NHE is encoded by the NHE-1 mRNA. Using acidified CL4 cells on the filter membrane, we examined Na⁺-dependent pH recovery of the apical and basolateral membranes separately. Na⁺ applied to the apical membrane recovered cell pH. Na⁺-dependent pH recovery in the apical membrane was not inhibited by SITS, DIDS, or contralateral amiloride. Li⁺ but not K⁺, chol⁺, or NMG⁺ could replace Na⁺. These data are consistent with the presence of NHE in the apical membrane. Transfection with an antisense oligonucleotide corresponding to the 5′ terminal site of NHE-1 cDNA of CL4 decreased NHE activity in the basolateral membrane but not in the apical membrane. We conclude that CL4 expresses NHE activities in both apical and basolateralmembranes and that NHE-1 mRNA encodes NHE only in the basolateral membrane. J. Cell. Physiol. 171:318–324, 1997. © 1997 Wiley-Liss, Inc.     

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.     

Localization and characterization of transport-related elements in the plasma membrane of turtle bladder epithelial cells

A mixed membrane preparation obtained from turtle bladder epithelial cells contains (Na⁺ + K⁺)-ATPase, adenylate cyclase and protein kinase, which interact with ouabain, norepinephrine and cyclic AMP, respectively. When such a preparation is obtained from bladders which had been preexposed to serosal fluids containing the tritiated form of 4,4′-diisothiocyano-2,2′-disulfonic stilbene, the subsequently isolated membrane proteins are enriched in tritium as well as in the afore-mentioned enzymes, none of which is inhibited. Freeflow electrophoresis separates the mixed membrane preparation into two distinguishable groups: one, construed as apical membranes, is enriched in norepinephrine-sensitive adenylate cyclase and cyclic AMP-sensitive protein kinase; the other, construed as basal-lateral membranes, is enriched in ouabain-sensitive ATPase and 4,4′-diisothiocyano-2,2′-disulfonic stilbene-binding proteins.The physiological counterparts of these enzymatically defined membrane markers are the mucosal sidedness of the transport effects of norepinephrine and cyclic AMP derivatives and the serosal sidedness of the transport effects of ouabain and disulfonic stilbenes in the intact turtle bladder. The discreteness and ion selectivity of each membrane-bound, transport-related element are discussed in relation to the corresponding characteristics of each transport process in vivo; the possibility of regulation of anion transport by adenylate cyclase-protein kinase system is also discussed.     

Cl− transport in apical plasma membrane vesicles isolated from bovine tracheal epithelium

The Cl^? transport properties of the luminal border of bovine tracheal epithelium have been investigated using a highly purified preparation of apical plasma membrane vesicles. Transport of Cl^? into an intravesicular space was demonstrated by (1) a linear inverse correlation between Cl^? uptake and medium osmolarity and (2) complete release of accumulated Cl^? by treatment with detergent. The rate of Cl^? uptake was highly temperature-sensitive and was enhanced by exchange diffusion, providing evidence for a carrier-mediated transport mechanism. Transport of Cl^? was not affected by the ‘loop’ diuretic bumetanide or by the stilbene-derivative anion-exchange inhibitors SITS (4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid) and DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonic acid). In the presence of the impermeant cation, tetramethylammonium (TMA⁺), uptake of Cl^? was minimal; transport was stimulated equally by the substitution of either K⁺ or Na⁺ for TMA⁺. Valinomycin in the presence of K⁺ enhanced further Cl^? uptake, while amiloride reduced Na⁺-stimulated Cl^? uptake towards the minimal level observed with TMA⁺. These results suggest the following conclusions: (1) the tracheal vesicle membrane has a finite permeability to both Na⁺ and K⁺; (2) the membrane permeability to the medium counterion determines the rate of Cl^? uptake; (3) Cl^? transport is not specifically coupled with either Na⁺ or K⁺; and, finally (4) Cl^? crosses the tracheal luminal membrane via an electrogenic transport mechanism.     

Membrane conductances of principal cells in Malpighian tubules of Aedes aegypti

Two-electrode voltage clamp (TEVC) methods were used to explore conductive transport pathways in principal cells, the dominant cell type in Malpighian tubules of the yellow fever mosquito. The basolateral membrane of principal cells had a voltage (V_bl) of -85.1 mV in 49 principal cells under control conditions. Measures of the input resistance R_pc together with membrane fractional resistance yielded estimates of the conductance of the basolateral membrane (g_bl = 1.48 μS) and the apical membrane (g_a = 3.13 μS). K⁺ channels blocked by barium accounted for 0.94 μS of g_bl. Estimates of transference numbers yielded the basolateral membrane Na⁺ conductance of 0.24 μS, leaving 0.30 μS (20%) of g_bl unaccounted. The secretagogue db-cAMP (0.1 mM), a known activator of the basolateral membrane Na⁺ conductance, significantly depolarized V_bl to -65.0 mV and significantly increased g_bl from 1.48 μS to 2.47 μS. The increase was blocked with amiloride (1 mM), a known blocker of epithelial Na⁺ transport. The inhibition of metabolism with di-nitrophenol significantly depolarized V_bl to -9.7 mV and significantly increased R_pc from 391.6 kΩ to 2612.5 kΩ. Similar results were obtained with cyanide, but it remains unclear whether the large increases in R_pc stem from the uncoupling of epithelial cells and/or the shutdown of conductive transport pathways in basolateral and apical membranes. Our results indicate that the apical membrane of principal cells is more than twice as conductive as the basolateral membrane. Partial ionic conductances suggest the rate-limiting step for transepithelial Na⁺ secretion at the basolateral membrane.     

Amiloride and guggulsterone suppression of esophageal cancer cell growth in vitro and in nude mouse xenografts

Esophageal adenocarcinoma is increasing in the US and Western countries and frequent gastresophageal reflux or gastresophageal reflux disease carrying gastric acid and bile acid could contribute to esophageal adenocarcinogenesis. This study was designed to detect the expression of gastric acid-inducing gene Na＋/H＋ exchanger-1 （NHE-1） ex vivo and then to explore targeting of NHE-1 expression or activity to control esophageal cancer cell viability in vitro and in nude mouse xenografts. The data showed that NHE-1 was highly expressed in esophageal adenocarcinoma tissues （66 of 101 cases [65.3%｜, but not in normal esophageal squamous cell epithelium （1 of 26 cases [3.8~0]）. Knockdown of NHE-1 expression using NHE-1 shRNA or inhibition of NHE-1 activity using the NHE-1 inhibitor amiloride suppressed viability and induced apoptosis in esophageal cancer cells. Molecularly, amiloride inhibited expression of cyclooxygenase-2 and matrix metallopeptidase-9 but not NHE-1 mRNA in esophageal cancer cells. A combination of amiloride and guggulsterone （a natural bile acid receptor inhibitor） showed more than additive effects in suppressing esophageal cancer cell growth in vitro and in nude mouse xenografts. This study suggests that inhibition of NHE-1 expression or activity or combination of amiloride and guggulsterone could be useful in control of esophageal adenocarcinoma.     

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.     

Fibronectin stimulates migration through lipid raft dependent NHE-1 activation in mouse embryonic stem cells: Involvement of RhoA,Ca/CaM,and ERK

Background

Extracellular matrix (ECM) components and intracellular pH (pH_i) may serve as regulators of cell migration in various cell types.

Methods

The Oris migration assay was used to assess the effect of fibronectin (FN) on cell motility. The Na⁺/H⁺ exchanger (NHE)-1 activity was evaluated by measuring pH_i and [²²Na⁺] uptake. To examine activated signaling molecules, western blot analysis and immunoprecipitation was performed.

Results

ECM components (FN, laminin, fibrinogen, and collagen type I) increased [²²Na⁺] uptake, pH_i, and cell migration. In addition, FN-induced increase of cell migration was inhibited by NHE-1 inhibitor amiloride or NHE-1-specific siRNA. FN selectively increased the mRNA and protein expression of NHE-1, but not that of NHE-2 or NHE-3. FN binds integrin β1 and subsequently stimulates caveolin-1 phosphorylation and Ca^2 + influx. Then, NHE-1 is phosphorylated by RhoA and Rho kinases, and Ca^2 +/calmodulin (CaM) signaling elicits complex formation with NHE-1, which is enriched in lipid raft/caveolae microdomains of the plasma membrane. Activation of NHE-1 continuously induces an increase of [²²Na⁺] uptake and pH_i. Finally, NHE-1-dependent extracellular signal-regulated kinase (ERK) 1/2 phosphorylation enhanced matrix metalloproteinase-2 (MMP-2) and filamentous-actin (F-actin) expression, partially contributing to the regulation of embryonic stem cells (ESCs) migration.

Conclusions

FN stimulated mESCs migration and proliferation through NHE-1 activation, which were mediated by lipid raft-associated caveolin-1, RhoA/ROCK, and Ca^2 +/CaM signaling pathways.

General significance

The precise role of NHE in the modulation of ECM-related physiological functions such as proliferation and migration remains poorly understood. Thus, this study analyzed the relationship between FN and NHE in regulating the migration of mouse ESCs and their related signaling pathways.     

A new model for fish ion regulation: identification of ionocytes in freshwater- and seawater-acclimated medaka (Oryzias latipes)

The ion regulation mechanisms of fishes have been recently studied in zebrafish (Danio rerio), a stenohaline species. However, recent advances using this organism are not necessarily applicable to euryhaline fishes. The euryhaline species medaka (Oryzias latipes), which, like zebrafish, is genetically well categorized and amenable to molecular manipulation, was proposed as an alternative model for studying osmoregulation during acclimation to different salinities. To establish its suitability as an alternative, the present study was conducted to (1) identify different types of ionocytes in the embryonic skin and (2) analyze gene expressions of the transporters during seawater acclimation. Double/triple in situ hybridization and/or immunocytochemistry revealed that freshwater (FW) medaka contain three types of ionocyte: (1) Na⁺/H⁺ exchanger 3 (NHE3) cells with apical NHE3 and basolateral Na⁺-K⁺-2Cl^? cotransporter (NKCC), Na⁺-K⁺-ATPase (NKA) and anion exchanger (AE); (2) Na⁺-Cl^? cotransporter (NCC) cells with apical NCC and basolateral H⁺-ATPase; and (3) epithelial Ca²⁺ channel (ECaC) cells [presumed accessory (AC) cells] with apical ECaC. On the other hand, seawater (SW) medaka has a single predominant ionocyte type, which possesses apical cystic fibrosis transmembrane conductance regulator (CFTR) and NHE3 and basolateral NKCC and NKA and is accompanied by smaller AC cells that express lower levels of basolateral NKA. Reciprocal gene expressions of decreased NHE3, AE, NCC and ECaC and increased CFTR and NKCC in medaka gills during SW were revealed by quantative PCR analysis.     

Inverse Relationship Between Membrane Lipid Fluidity and Activity of Na+/H+ Exchangers, NHE1 and NHE3, in Transfected Fibroblasts

This report presents a study of the effects of the membrane fluidizer, benzyl alcohol, on NHE isoforms 1 and 3. Using transfectants of an NHE-deficient fibroblast, we analyzed each isoform separately. An increase in membrane fluidity resulted in a decrease of ≈50% in the specific activities of both NHE1 and NHE3. Only V _max was affected; K _Na was unchanged. This effect was specific, as Na⁺, K⁺, ATPase activity was slightly stimulated. Inhibition of NHE1 and NHE3 was reversible and de novo protein synthesis was not required to restore NHE activity after washout of fluidizer. Inhibition kinetics of NHE1 by amiloride, 5-(N,N-dimethyl)amiloride (DMA), 5-(N-hexamethyl)amiloride (HMA) and 5-(N-ethyl-N-isopropyl)amiloride (EIPA) were largely unchanged. Half-maximal inhibition of NHE3 was also reached at approximately the same concentrations of amiloride and analogues in control and benzyl alcohol treated, suggesting that the amiloride binding site was unaffected. Inhibition of vesicular transport by incubation at 4°C augmented the benzyl alcohol inhibition of NHE activity, suggesting that the fluidizer effect does not solely involve vesicle trafficking. In summary, our data demonstrate that the physical state of membrane lipids (fluidity) influences Na⁺/H⁺ exchange and may represent a physiological regulatory mechanism of NHE1 and NHE3 activity. Received: 23 January 1997/Revised: 1 August 1997     

Expression and sub cellular localization of the sodium hydrogen exchanger isoform-1 in rat tissues: A possible functional relevance

In an attempt to understand the mechanism underlying the tissue-dependent function, the expression of NHE-1 protein and its sub cellular localization was examined in the rat GI-tract and other tissues. Rat NHE-1 polyclonal antibodies were raised in rabbits using a NHE-1 fusion protein antigen. The antibodies recognized a 110 kD protein in rats and mice, but not in human or rabbit RBCs. Colon, stomach, brain, spleen and kidney expressed NHE-1 protein abundantly, whereas the skeletal muscle the least abundant. Ouabain-sensitive-K⁺-stimulated p-nitrophenylphosphatase (PNPPase), the partial activity of the sodium pump and alkaline phosphatase (Apase) were used as the markers of the basolateral and apical membranes. NHE-1 was detected predominantly in the PNPPase enriched membrane fractions, but was also detected in the apical membrane enriched fractions in the kidney cortex, jejunum and colon at a lower level. NHE-1 was detected in the plasma membrane enriched fractions from the skeletal muscle and ventricle. Immunofluorescence data showed a similar localization pattern of NHE-1 in the colon and kidney sections. These findings suggest that NHE-1 is localized both on the apical and basolateral membrane. In view of its similar sub cellular localization in the GI-tract and kidney, but a different level of expression, might suggest that the level of protein, but not the sub cellular distribution is important to regulate its tissue-dependent function.     

Morphofunctional modifications in gill mitochondria-rich cells of Mozambique tilapia transferred from freshwater to 70% seawater, detected by dual observations of whole-mount immunocytochemistry and scanning electron microscopy

Acute responses of gill mitochondria-rich (MR) cells to direct transfer from freshwater to 70% seawater were examined in a euryhaline teleost Mozambique tilapia (Oreochromis mossambicus). Scanning electron microscopic (SEM) observations revealed that apical openings of MR cells were morphologically classified into an apical pit, a convex apical surface, a concave apical surface, and a transitory apical surface. Meanwhile, in whole-mount immunocytochemistry with anti-Na⁺/K⁺-ATPase (NKA), T4 antibody (detecting apical Na⁺/Cl^? cotransporter (NCC) and basolateral Na⁺/K⁺/2Cl^? cotransporter (NKCC)), and anti-Na⁺/H⁺ exchanger-3 (NHE3), NKA-immunoreactive MR cells were functionally classified into immature cells without both NKCC/NCC and NHE3 (type I), ion-absorptive cells with apical NCC (type II), those with apical NHE3 (type III), and ion-secretory cells with basolateral NKCC (type IV). Dual observations of whole-mount immunocytochemistry and SEM clearly showed morphofunctional alterations in MR cells. After transfer to 70% seawater, type-II MR cells with a convex surface or pit closed their apical openings to suspend ion absorption. Type-III MR cells with a concave surface or pit were transformed into type-IV MR cells with an enlarged pit, via a transitory surface. Our findings indicate functional plasticity of type-III/IV MR cells to switch ion-transport functions, whereas type-II MR cells are considered to be specific for freshwater adaptation.     

Na+-independent Lysine Transport in Human Intestinal Caco-2 Cells

The nature of transepithelial and cellular transport of the dibasic amino acid lysine in human intestinal epithelial Caco-2 cells has been characterized. Intracellular accumulation of lysine across both the apical and basolateral membranes consists of a Na⁺-independent, membrane potential-sensitive uptake. Na⁺-independent lysine uptake at the basolateral membrane exceeds that at the apical membrane. Lysine uptake consists of both saturable and nonsaturable components. Na⁺-independent lysine uptake at both membranes is inhibited by lysine, arginine, alanine, histidine, methionine, leucine, cystine, cysteine and homoserine. In contrast, proline and taurine are without inhibitory effects at both membranes. Fractional Na⁺-independent lysine efflux from preloaded epithelial layers is greater at the basolateral membrane and shows trans-stimulation across both epithelial borders by lysine, arginine, alanine, histidine, methionine, and leucine but not proline and taurine. Na⁺-independent lysine influx (10 μm) in the presence of 10 mm homoserine shows further concentration dependent inhibition by lysine. Taken together, these data are consistent with lysine transport being mediated by systems b^o,+, y⁺ and a component of very low affinity (nonsaturable) at both membranes. The relative contribution to lysine uptake at each membrane surface (at 10 μm lysine), normalized to total apical uptake (100%), is apical b^o,+ (47%), y⁺ (27%) and the nonsaturable component (26%), and basal b^o,+ (446%), y⁺ (276%) and the nonsaturable component (20%). Northern analysis shows hybridization of Caco-2 poly(A)⁺RNA with a human rBAT cDNA probe. Received: 3 July 1995/Revised: 6 February 1996     

NKCC1 and NHE1 are abundantly expressed in the basolateral plasma membrane of secretory coil cells in rat, mouse, and human sweat glands

In isolated sweat glands, bumetanide inhibits sweat secretion. The mRNA encoding bumetanide-sensitive Na⁺-K⁺-Cl^– cotransporter (NKCC) isoform 1 (NKCC1) has been detected in sweat glands; however, the cellular and subcellular protein localization is unknown. Na⁺/H⁺ exchanger (NHE) isoform 1 (NHE1) protein has been localized to both the duct and secretory coil of human sweat duct; however, the NHE1 abundance in the duct was not compared with that in the secretory coil. The aim of this study was to test whether mRNA encoding NKCC1, NKCC2, and Na⁺-coupled acid-base transporters and the corresponding proteins are expressed in rodent sweat glands and, if expressed, to determine the cellular and subcellular localization in rat, mouse, and human eccrine sweat glands. NKCC1 mRNA was demonstrated in rat palmar tissue, including sweat glands, using RT-PCR, whereas NKCC2 mRNA was absent. Also, NHE1 mRNA was demonstrated in rat palmar tissue, whereas NHE2, NHE3, NHE4, electrogenic Na⁺-HCO₃^– cotransporter 1 NBCe1, NBCe2, electroneutral Na⁺-HCO₃^– cotransporter NBCn1, and Na⁺-dependent Cl^–/HCO₃^– exchanger NCBE mRNA were not detected. The expression of NKCC1 and NHE1 proteins was confirmed in rat palmar skin by immunoblotting, whereas NKCC2, NHE2, and NHE3 proteins were not detected. Immunohistochemistry was performed using sections from rat, mouse, and human palmar tissue. Immunoperoxidase labeling revealed abundant expression of NKCC1 and NHE1 in the basolateral domain of secretory coils of rat, mouse, and human sweat glands and low expression was found in the coiled part of the ducts. In contrast, NKCC1 and NHE1 labeling was absent from rat, mouse, and human epidermis. Immunoelectron microscopy demonstrated abundant NKCC1 and NHE1 labeling of the basolateral plasma membrane of mouse sweat glands, with no labeling of the apical plasma membranes or intracellular structures. The basolateral NKCC1 of the secretory coils of sweat glands would most likely account for the observed bumetanide-sensitive NaCl secretion in the secretory coils, and the basolateral NHE1 is likely to be involved in Na⁺-coupled acid-base transport. bumetanide; eccrine glands; immunohistochemistry; immunoblotting     

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.     

The Detailed Localization Pattern of Na+/K+/2Cl? Cotransporter Type 2 and Its Related Ion Transport System in the Rat Endolymphatic Sac

The endolymphatic sac (ES) is a part of the membranous labyrinth. ES is believed to perform endolymph absorption, which is dependent on several ion transporters, including Na⁺/K⁺/2Cl⁻ cotransporter type 2 (NKCC-2) and Na⁺/K⁺-ATPase. NKCC-2 is typically recognized as a kidney-specific ion transporter expressed in the apical membrane of the absorptive epithelium. NKCC-2 expression has been confirmed only in the rat and human ES other than the kidney, but the detailed localization features of NKCC-2 have not been investigated in the ES. Thus, we evaluated the specific site expressing NKCC-2 by immunohistochemical assessment. NKCC-2 expression was most frequently seen in the intermediate portion of the ES, where NKCC-2 is believed to play an important role in endolymph absorption. In addition, NKCC-2 expression was also observed on the apical membranes of ES epithelial cells, and Na⁺/K⁺-ATPase coexpression was observed on the basolateral membranes of ES epithelial cells. These results suggest that NKCC-2 performs an important role in endolymph absorption and that NKCC-2 in apical membranes and Na⁺/K⁺-ATPase in basolateral membranes work coordinately in the ES in a manner similar to that in renal tubules. (J Histochem Cytochem 58:759–763, 2010)     

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     

Studies on the subcellular distribution of (Na+ + K+)-ATPase,K+-stimulated ATPase and HCO3−-stimulated ATPase activities in malpighian tubules of Locusta migratoria L.

The present study confirms previous reports of the presence of (Na⁺ + K⁺)-ATPase and anion-stimulated ATPase activity in Malpighian tubules of Locusta. In addition, the presence of a K⁺-stimulated, ouabain-insensitive ATPase activity has been identified in microsomal fractions. Differential and sucrose density-gradient centrifugation of homogenates has been used to separate membrane fractions which are rich in mitochondria, apical membranes and basolateral membranes; as indicated by the presence of succinate dehydrogenase and the presence or absence of non-specific alkaline phosphatase activity, respectively. Relatively high specific (Na⁺ + K⁺)-ATPase activity was associated with the basolateral membrane-rich fractions with only low levels of this activity being associated with the apical membrane-rich preparation. K⁺-stimulated ATPase activity was also associated, predominantly, with the basolateral membrane-rich fractions. However, comparison of the distribution of this activity with that of the (Na⁺ + K⁺)-ATPase suggests that the two enzymes did not co-separate. The possibility that the K⁺-stimulated ATPase was not associated with the basolateral plasma membrane is discussed.Anion-stimulated ATPase activity was found in the apical and basolateral membrane-rich fractions and in the fraction contaning mainly mitochondria. Nevertheless, the fact that this bicarbonate-stimulated activity did not co-separate with succinate dehydrogenase activity suggests that it was not exclusively mitochondrial in origin. These results are consistent with physiological studies indicating a basolateral (Na⁺ + K⁺)-ATPase but do not support the K⁺-stimulated ATPase as a candidate for the apical electrogenic pump. The possible role of the bicarbonate-stimulated ATPase activity in ion transport across both the basolateral and apical cell membranes is discussed.