Squalamine, a novel cationic steroid, specifically inhibits the brush-border Na+/H+ exchanger isoform NHE3

Squalamine, anendogenous molecule found in the liver and other tissues ofSqualus acanthias, hasantibiotic properties and causes changes in endothelial cell shape. Thelatter suggested that its potential targets might include transportproteins that control cell volume or cell shape. The effect of purifiedsqualamine was examined on clonedNa⁺/H⁺exchanger isoforms NHE1, NHE2, and NHE3 stably transfected in PS120fibroblasts. Squalamine (1-h pretreatment) decreased the maximalvelocity of rabbit NHE3 in a concentration-dependent manner (13, 47, and 57% inhibition with 3, 5, and 7 µg/ml, respectively) and alsoincreasedK'[H⁺]_i.Squalamine did not affect rabbit NHE1 or NHE2 function. The inhibitoryeffect of squalamine was 1) timedependent, with no effect of immediate addition and maximum effect with1 h of exposure, and 2) fullyreversible. Squalamine pretreatment of the ileum for 60 min inhibitedbrush-border membrane vesicleNa⁺/H⁺activity by 51%. Further investigation into the mechanism of squalamine's effects showed that squalamine required the COOH-terminal 76 amino acids of NHE3. Squalamine had no cytotoxic effect at theconcentrations studied, as indicated by monitoring lactate dehydrogenase release. These results indicate that squalamine 1) is a specific inhibitor of thebrush-border NHE isoform NHE3 and not NHE1 or NHE2,2) acts in a nontoxic and fullyreversible manner, and 3) has adelayed effect, indicating that it may influence brush-borderNa⁺/H⁺exchanger function indirectly, through an intracellular signaling pathway or by acting as an intracellular modulator.

     

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.

     

Age- and tissue-specific induction of NHE3 by glucocorticoids in the rat small intestine

Of the two known apical isoforms of theNa⁺/H⁺ exchanger (NHE) family, only the NHE3gene is regulated by glucocorticoids. The aim of these studies was toinvestigate the mechanisms underlying the effects of methylprednisolone(MP) on expression of NHE3 in the proximal and distal small intestineof suckling and adult rats. Immunoblots showed that the glucocorticoidresponsiveness in the proximal small intestine was greatest in sucklinganimals (NHE3/-actin: 0.43 ± 0.09 control vs. 1.57 ± 0.15 MP;P < 0.001), and responsiveness decreased with age with noeffect in adults (0.56 ± 0.14 vs. 0.64 ± 0.17). Distal smallintestine was responsive only in adult rats (0.49 ± 0.13 vs. 1.65 ± 0.09; P < 0.001). This pattern was confirmed at the mRNAlevel and by ²²Na⁺ uptake. Western blot and[³H]dexamethasone mesylate binding showed thatthe responsiveness of NHE3 to glucocorticoids is directly related tothe expression of glucocorticoid receptor (GR) in the small intestine.These studies suggest that loss and gain of glucocorticoidresponsiveness in the proximal and distal small intestine,respectively, are related to age- and segment-dependent expression of GR.

     

Regulation of apical membrane Na+/H+ exchangers NHE2 and NHE3 in intestinal epithelial cell line C2/bbe

We examined the regulation of theNa⁺/H⁺exchangers (NHEs) NHE2 and NHE3 by expressing them in human intestinalC2/bbe cells, which spontaneously differentiate and have little basalapical NHE activity. Unidirectional apical membrane²²Na⁺influxes were measured in NHE2-transfected (C2N2) and NHE3-transfected (C2N3) cells under basal and stimulated conditions, and their activities were distinguished as the HOE-642-sensitive and -insensitive components of5-(N,N-dimethyl)amiloride-inhibitableflux. Both C2N2 and C2N3 cells exhibited increased apical membrane NHEactivity under non-acid-loaded conditions compared with nontransfected control cells. NHE2 was inhibited by 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate and thapsigargin, was stimulatedby serum, and was unaffected by cGMP- and protein kinase C-dependent pathways. In contrast, NHE3 was inhibited by all regulatory pathways examined. Under acid-loaded conditions (which increase apical Na⁺ influx), NHE2 and NHE3exhibited similar patterns of regulation, suggesting that the secondmessenger effects observed were not secondary to effects on cell pH.Thus, in contrast to their expression in nonepithelial cells, NHE2 andNHE3 expressed in an epithelial cell line behave similarly toendogenously expressed intestinal apical membrane NHEs. We concludethat physiological regulation and function of epithelium-specific NHEsare dependent on tissue-specific factors and/or conditionalrequirements.

     

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.

     

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     

Na+/H+ exchangers (NHE1-3) have similar turnover numbers but different percentages on the cell surface

NHE1, NHE2, andNHE3 are well-characterized cloned members of the mammalianNa⁺/H⁺exchanger (NHE) gene family. Given the specialized function and regulation of NHE1, NHE2, and NHE3, we compared basal turnover numbersof NHE1, NHE2, and NHE3 measured in the same cell system: PS120fibroblasts lacking endogenous NHEs. NHE1, NHE2, and NHE3 were epitopetagged with vesicular stomatitis virus glycoprotein (VSVG). Thefollowing characteristics were determined on the same passage of cellstransfected with NHE1V, NHE2V, or NHE3V:1) maximal reaction velocity(V_max) by²²Na⁺uptake and fluorometery, 2) totalamount of NHE protein by quantitative Western analysis with internalstandards of VSVG-tagged maltose-binding protein, and3) cell surface expression by cellsurface biotinylation. Cell surface expression (percentage of totalNHE) was 88.8 ± 3.5, 64.6 ± 3.3, 20.0 ± 2.6, and 14.0 ± 1.3 for NHE1V, 85- and 75-kDa NHE2V, and NHE3V, respectively. Despitethese divergent cell surface expression levels, turnover numbers forNHE1, NHE2, and NHE3 were similar (80.3 ± 9.6, 92.1 ± 8.6, and99.2 ± 9.1 s¹, whenV_max wasdetermined using ²²Na uptake at22°C and 742 ± 47, 459 ± 16, and 609 ± 39 s¹ whenV_max wasdetermined using fluorometry at 37°C). These data indicate that, inthe same cell system, intrinsic properties that determine turnovernumber are conserved among NHE1, NHE2, and NHE3.

     

NHE3-dependent cytoplasmic alkalinization is triggered by Na(+)-glucose cotransport in intestinal epithelia

Cytoplasmic pH (pH_i) was evaluated duringNa⁺-glucose cotransport in Caco-2 intestinal epithelialcell monolayers. The pH_i increased by 0.069 ± 0.002 within 150 s after initiation of Na⁺-glucosecotransport. This increase occurred in parallel with glucose uptake andrequired expression of the intestinal Na⁺-glucosecotransporter SGLT1. S-3226, a preferential inhibitor ofNa⁺/H⁺ exchanger (NHE) isoform 3 (NHE3),prevented cytoplasmic alkalinization after initiation ofNa⁺-glucose cotransport with an ED₅₀ of 0.35 µM, consistent with inhibition of NHE3, but not NHE1 or NHE2. Incontrast, HOE-694, a poor NHE3 inhibitor, failed to significantlyinhibit pH_i increases at <500 µM.Na⁺-glucose cotransport was also associated with activationof p38 mitogen-activated protein (MAP) kinase, and the p38 MAP kinase inhibitors PD-169316 and SB-202190 prevented pH_i increasesby 100 ± 0.1 and 86 ± 0.1%, respectively. Conversely,activation of p38 MAP kinase with anisomycin induced NHE3-dependentcytoplasmic alkalinization in the absence of Na⁺-glucosecotransport. These data show that NHE3-dependent cytoplasmic alkalinization occurs after initiation of SGLT1-mediatedNa⁺-glucose cotransport and that the mechanism of this NHE3activation requires p38 MAP kinase activity. This coordinatedregulation of glucose (SGLT1) and Na⁺ (NHE3) absorptiveprocesses may represent a functional activation of absorptiveenterocytes by luminal nutrients.

     

Half-lives of plasma membrane Na+/H+ exchangers NHE1-3: plasma membrane NHE2 has a rapid rate of degradation

The Na⁺/H⁺ exchangers NHE2 and NHE3 areinvolved in epithelial Na⁺ and HCOabsorption. To increase insights into the functions of NHE2 vs. NHE3,we compared their cellular processing with each other and with thehousekeeping isoform NHE1. Using biotinylated exchanger, we determinedthat the half-life of plasma membrane NHE2 was short (3 h) comparedwith that of NHE1 (24 h) and NHE3 (14 h) in both PS120 fibroblasts andCaco-2 cells. NHE2 transport and plasma membrane levels were reduced by3 h of Brefeldin A treatment, whereas NHE1 was unaffected. NHE2was degraded by the lysosomes but not proteosomes, as demonstrated byincreasing levels of endocytosed NHE2 protein after inhibition of thelysosomes, but not with proteosome inhibition. Unlike that of NHE3,basal NHE2 transport activity was not affected by phosphatidylinositol 3-kinase inhibition and did not appear to be localized in the juxtanuclear recycling endosome. Therefore, for NHE2, protein degradation and/or protein synthesis probably play important roles inits basal and regulated states. These results suggest fundamental differences in the cellular processing and trafficking of NHE2 andNHE3. These differences may underlie the specialized roles that theseexchangers play in epithelial cells.

     

Incubation of OKP cells in low-K+ media increases NHE3 activity after early decrease in intracellular pH

Chronichypokalemia increases the activity of proximal tubule apical membraneNa⁺/H⁺antiporter NHE3. The present study examined the effect ofthe incubation of OKP cells (an opossum kidney, clone P cell line) incontrol medium {K⁺ concn([K⁺]) = 5.4 mM} or low-K⁺ medium([K⁺] = 2.7 mM) onNHE3. The activity of an ethylisopropyl amiloride-resistant Na⁺/H⁺antiporter, whose characteristics were consistent with those ofNHE3, was increased inlow-K⁺ cells beginning at 8 h.NHE3 mRNA and NHE3 protein abundance were increased 2.2-fold and 62%,respectively, at 24 h but not at 8 h. After incubation inlow-K⁺ medium, intracellular pH(pH_i) decreased by 0.27 pH units(maximum at 27 min) and then recovered to the control level.Intracellular acidosis induced by 5 mM sodium propionate increasedNa⁺/H⁺antiporter activity at 8 and 24 h. Herbimycin A, a tyrosine kinase inhibitor, blocked low-K⁺- andsodium propionate-induced activation of theNa⁺/H⁺antiporter at 8 and 24 h. Our results demonstrate thatlow-K⁺ medium causes an earlydecrease in pH_i, which leads to anincrease in NHE3 activity via a tyrosine kinase pathway.     

Cloning and expression of the Na+/H+ exchanger from Amphiuma RBCs: resemblance to mammalian NHE1

The cDNAencoding theNa⁺/H⁺exchanger (NHE) from Amphiumaerythrocytes was cloned, sequenced, and found to be highly homologous to the human NHE1 isoform (hNHE1), with 79% identity and 89%similarity at the amino acid level. Sequence comparisons with otherNHEs indicate that the Amphiumatridactylum NHE isoform 1 (atNHE1) islikely to be a phylogenetic progenitor of mammalian NHE1. The atNHE1protein, when stably transfected into the NHE-deficient AP-1 cell line(37), demonstrates robustNa⁺-dependent proton transportthat is sensitive to amiloride but not to the potent NHE1 inhibitorHOE-694. Interestingly, chimeric NHE proteins constructed by exchangingthe amino and carboxy termini between atNHE1 and hNHE1 exhibited drugsensitivities similar to atNHE1. Based on kinetic, sequence, andfunctional similarities between atNHE1 and mammalian NHE1, we proposethat the Amphiuma exchanger shouldprove to be a valuable model for studying the control of pH and volumeregulation of mammalian NHE1. However, low sensitivity of atNHE1 to theNHE inhibitor HOE-694 in both nativeAmphiuma red blood cells (RBCs) and intransfected mammalian cells distinguishes this transporter from itsmammalian homologue.     

Functional upregulation of H+-ATPase by lethal acid stress in cultured inner medullary collecting duct cells

The response ofH⁺-ATPase to lethal acid stress isunknown. A mutant strain (called NHE2d) was derived from cultured inner medullary collecting duct cells (mIMCD-3 cells) following three cyclesof lethal acid stress. Cells were grown to confluence on coverslips,loaded with2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, andmonitored for intracellular pH(pH_i) recovery from an acid load. The rate of Na⁺-independentpH_i recovery from an acid load inmutant cells was approximately fourfold higher than in parent cells(P < 0.001). TheNa⁺-independentH⁺ extrusion was ATP dependent and K⁺ independent and wascompletely inhibited in the presence of diethylstilbestrol, N, N'-dicyclohexylcarbodiimide,or N-ethylmaleimide. Theseresults indicate that theNa⁺-independentH⁺ extrusion in cultured medullarycells is mediated via H⁺-ATPaseand is upregulated in lethal acidosis. Northern hybridization experiments demonstrated that mRNA levels for the 16- and 31-kDa subunits of H⁺-ATPase remainedunchanged in mutant cells compared with parent cells. We propose thatlethal acid stress results in increased H⁺-ATPase activity in innermedullary collecting duct cells. Upregulation ofH⁺-ATPase could play a protectiverole against cell death in severe intracellular acidosis.

     

Acid incubation causes exocytic insertion of NHE3 in OKP cells

Incubation of opossum kidney clone P (OKP) cells in acid media(pH 6.8) causes activation of Na⁺/H⁺ exchanger3 (NHE3) at 6, 12, and 24 h. OKP cell NHE3 protein abundance was increased by 45% at 24 h of acid incubation but wasunaffected at 3-12 h. By contrast, apical membrane NHE3, measured by surface biotinylation, increased approximately twofold at 6, 12, and24 h, mirroring the increase in activity. Acid incubation caused a76% increase in exocytic insertion of NHE3 into the apical membranebut had no effect on endocytic internalization at 6 h. LatrunculinB, an inhibitor of microfilament organization, inhibited theacid-induced increases in apical membrane NHE3, exocytic insertion ofNHE3, and NHE3 activity at 6 h. These studies demonstrate two mechanisms for acid-induced increases in NHE3 activity. Beginning at6 h, there is an increase in apical membrane NHE3 that is due tostimulated exocytic insertion and is required for increased NHE3activity. At 24 h, there is an additional increase in total cellular NHE3.

     

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     

NHE2 contains subdomains in the COOH terminus for growth factor and protein kinase regulation

The cloned epithelial cell-specificNa⁺/H⁺exchanger (NHE) isoform NHE2 is stimulated by fibroblast growth factor(FGF), phorbol 12-myristate 13-acetate (PMA), okadaic acid (OA), andfetal bovine serum (FBS) through a change in maximal velocity of thetransporter. In the present study, we used COOH-terminal truncationmutants to delineate specific domains in the COOH terminus of NHE2 that are responsible for growth factor and/or protein kinase regulation. Five truncation mutants (designated by the amino acid number at thetruncation site) were stably expressed in NHE-deficient PS120 fibroblasts. The effects of PMA, FGF, OA, FBS, and W-13 [aCa²⁺/calmodulin (CaM)inhibitor] were studied. Truncation mutant E2/660, but notE2/573, was stimulated by PMA. OA stimulated E2/573 but not E2/540. FGFstimulated E2/540 but not E2/499. The most truncated mutant, E2/499,was stimulated by FBS. W-13 stimulated the basal activity of thewild-type NHE2. However, W-13 had no effect on E2/755. By monitoringthe emission spectra of dansylated CaM fluorescence, we showed thatdansylated CaM bound directly to a purified fusion protein ofglutathione S-transferase and the last87 amino acids of NHE2 in aCa²⁺-dependent manner, with astoichiometry of 1:1 and a dissociation constant of 300 nM. Our results showed that the COOH terminus of NHE2 is organized intoseparate stimulatory and inhibitory growth factor/protein kinaseregulatory subdomains. This organization of growth factor/proteinkinase regulatory subdomains is very similar to that of NHE3,suggesting that the tertiary structures of the putative COOH termini ofNHE2 and NHE3 are very similar despite the minimal amino acid identityin this part of the two proteins.     

Acute inhibition of brain-specific Na(+)/H(+) exchanger isoform 5 by protein kinases A and C and cell shrinkage

Little is known of the functional properties of the mammalian,brain-specific Na⁺/H⁺ exchanger isoform 5 (NHE5). Rat NHE5 was stably expressed in NHE-deficient PS120 cells, andits activity was characterized using the fluorescent pH-sensitive dye2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. NHE5was insensitive to ethylisopropyl amiloride. The transport kinetics displayed a simple Michaelis-Menten relationship for extracellular Na⁺ (apparent K_Na = 27 ± 5 mM) and a Hill coefficient near 3 for the intracellularproton concentration with a half-maximal activity at an intracellularpH of 6.93 ± 0.03. NHE5 activity was inhibited by acute exposureto 8-bromo-cAMP or forskolin (which increases intracellular cAMP byactivating adenylate cyclase). The kinase inhibitor H-89 reversed thisinhibition, suggesting that regulation by cAMP involves a proteinkinase A (PKA)-dependent process. In contrast, 8-bromo-cGMP did nothave a significant effect on activity. The protein kinase C (PKC)activator phorbol 12-myristrate 13-acetate inhibited NHE5, and the PKCantagonist chelerythrine chloride blunted this effect. Activity wasalso inhibited by hyperosmotic-induced cell shrinkage but wasunaffected by a hyposmotic challenge. These results demonstrate thatrat brain NHE5 is downregulated by activation of PKA and PKC and bycell shrinkage, important regulators of neuronal cell function.

     

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.     

Acute activation of NHE3 by dexamethasone correlates with activation of SGK1 and requires a functional glucocorticoid receptor

Glucocorticoids stimulate the intestinal absorption of Na⁺ and water partly by regulation of the Na⁺/H⁺ exchanger 3 (NHE3). Previous studies have shown both genomic and nongenomic regulation of NHE3 by glucocorticoids. Serum and glucocorticoid-inducible kinase 1 (SGK1) has been shown to be part of this cascade, where phosphorylation of NHE3 by SGK1 initiates the translocation of NHE3 to the cell surface. In the present work, we examined a series of changes in SGK1 and NHE3 induced by glucocorticoids using human colonic Caco-2 and opossum kidney cells. We found that dexamethasone rapidly stimulated SGK1 mRNAs, but a significant change in protein abundance was not detected. Instead, there was an increase in SGK1 kinase activity as early as at 2 h. An increase in NHE3 protein abundance was not detected until 12 h of dexamethasone exposure, although the transport activity was significantly stimulated at 4 h. These data demonstrate that the changes of SGK1 precede those of NHE3. Chronic regulation (24 h) of NHE3 was blocked completely by prevention of protein synthesis with cycloheximide or actinomycin D and by the glucocorticoid receptor blocker RU486. The acute effect of dexamethasone was similarly abrogated by RU486, but was insensitive to cycloheximide and actinomycin D. Similarly, the stimulation of SGK1 activity by dexamethasone was blocked by RU486 but not by actinomycin D. Together, these data show that the acute effect of glucocorticoids on NHE3 is mediated by a glucocorticoid receptor dependent mechanism that activates SGK1 in a nongenomic manner. Na⁺/H⁺ exchanger 3; serum and glucocorticoid-inducible kinase 1     

Cloning and characterization of a type III Na-dependent phosphate cotransporter from mouse intestine

Intestinal and renalabsorption of inorganic phosphate (P_i) is critical forphosphate homeostasis in mammals. We have isolated a cDNA that encodesa type III Na-dependent phosphate cotransporter from mouse smallintestine (mPit-2). The nucleotide sequence of mPit-2 predicts aprotein of 653 amino acids with at least 10 putative transmembranedomains. Kinetic studies, carried out in Xenopus oocytes,showed that mPit-2 cRNA induces significant Na-dependent P_iuptake with an apparent Michaelis constant (K_m)for phosphate of 38 µM. The transport of phosphate by mPit-2 isinhibited at high pH. Northern blot analysis demonstrated the presenceof mPit-2 mRNA in various tissues, including intestine, kidney, heart,liver, brain, testis, and skin. The highest expression of mPit-2 in the intestine was found in the jejunum. In situ hybridization revealed thatmPit-2 mRNA is expressed throughout the vertical crypt-villus axis ofthe intestinal epithelium. The presence of mPit-2 in the mouseintestine and its unique transport characteristics suggest thatmultiple Na-dependent cotransporters may contribute to phosphate absorption in the mammalian small intestine.

     

Protein kinase D inhibits plasma membrane Na+/H+ exchanger activity

The regulation of plasma membraneNa⁺/H⁺exchanger (NHE) activity by protein kinase D (PKD), a novel proteinkinase C- and phorbol ester-regulated kinase, was investigated. Todetermine the effect of PKD on NHE activity in vivo, intracellular pH(pH_i) measurements were made inCOS-7 cells by microepifluorescence using the pH indicator cSNARF-1.Cells were transfected with empty vector (control), wild-type PKD, orits kinase-deficient mutant PKD-K618M, together with green fluorescentprotein (GFP). NHE activity, as reflected by the rate of acid efflux(J_H), wasdetermined in single GFP-positive cells following intracellularacidification. Overexpression of wild-type PKD had no significanteffect on J_H(3.48 ± 0.25 vs. 3.78 ± 0.24 mM/min in control atpH_i 7.0). In contrast,overexpression of PKD-K618M increasedJ_H (5.31 ± 0.57 mM/min at pH_i 7.0;P < 0.05 vs. control). Transfectionwith these constructs produced similar effects also in A-10 cells,indicating that native PKD may have an inhibitory effect on NHE in bothcell types, which is relieved by a dominant-negative action ofPKD-K618M. Exposure of COS-7 cells to phorbol ester significantlyincreased J_H in control cells but failed to do so in cells overexpressing either wild-type PKD (due to inhibition by the overexpressed PKD) or PKD-K618M(because basal J_Hwas already near maximal). A fusion protein containing the cytosolicregulatory domain (amino acids 637-815) of NHE1 (the ubiquitousNHE isoform) was phosphorylated in vitro by wild-type PKD, but with lowstoichiometry. These data suggest that PKD inhibits NHE activity,probably through an indirect mechanism, and represents a novel pathwayin the regulation of the exchanger.