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     

Serum- and glucocorticoid-induced kinase 3 in recycling endosomes mediates acute activation of Na+/H+ exchanger NHE3 by glucocorticoids

Na(+)/H(+) exchanger 3 (NHE3) is the major Na(+) transporter in the intestine. Serum- and glucocorticoid-induced kinase (SGK) 1 interacts with NHE regulatory factor 2 (NHERF2) and mediates activation of NHE3 by dexamethasone (Dex) in cultured epithelial cells. In this study, we compared short-term regulation of NHE3 by Dex in SGK1-null and NHERF2-null mice. In comparison to wild-type mice, loss of SGK1 or NHERF2 significantly attenuated regulation of NHE3 by Dex but did not completely obliterate the effect. We show that transfection of SGK2 or SGK3 in PS120 cells resulted in robust activation of NHE3 by Dex. However, unlike SGK1 or SGK2, SGK3 rapidly activated NHE3 within 15 min of Dex treatment in both PS120 and Caco-2bbe cells. Immunofluorescence analysis showed that SGK3 colocalized with NHE3 in recycling endosomes, whereas SGK1 and SGK2 were diffusely distributed. Mutation of Arg-90 of SGK3 disrupted the endosomal localization of SGK3 and delayed NHE3 activation. Activation of SGK3 and NHE3 by Dex was dependent on phosphoinositide 3-kinase (PI3K) and phosphoinositide-dependent kinase 1 (PDK1), and Dex induced translocation of PDK1 to endosomes. Our study identifies SGK3 as a novel endosomal kinase that acutely regulates NHE3 in a PI3K-dependent mechanism.     

SGK1 activates Na+-K+-ATPase in amphibian renal epithelial cells

Serum- and glucocorticoid-induced kinase 1 (SGK1) is thought to be an important regulator of Na⁺ reabsorption in the kidney. It has been proposed that SGK1 mediates the effects of aldosterone on transepithelial Na⁺ transport. Previous studies have shown that SGK1 increases Na⁺ transport and epithelial Na⁺ channel (ENaC) activity in the apical membrane of renal epithelial cells. SGK1 has also been implicated in the modulation of Na⁺-K⁺-ATPase activity, the transporter responsible for basolateral Na⁺ efflux, although this observation has not been confirmed in renal epithelial cells. We examined Na⁺-K⁺-ATPase function in an A6 renal epithelial cell line that expresses SGK1 under the control of a tetracycline-inducible promoter. The results showed that expression of a constitutively active mutant of SGK1 (SGK1^T_S425D) increased the transport activity of Na⁺-K⁺-ATPase 2.5-fold. The increase in activity was a direct consequence of activation of the pump itself. The onset of Na⁺-K⁺-ATPase activation was observed between 6 and 24 h after induction of SGK1 expression, a delay that is significantly longer than that required for activation of ENaC in the same cell line (1 h). SGK1 and aldosterone stimulated the Na⁺ pump synergistically, indicating that the pathways mediated by these molecules operate independently. This observation was confirmed by demonstrating that aldosterone, but not SGK1^T_S425D, induced an 2.5-fold increase in total protein and plasma membrane Na⁺-K⁺-ATPase ₁-subunit abundance. We conclude that aldosterone increases the abundance of Na⁺-K⁺-ATPase, whereas SGK1 may activate existing pumps in the membrane in response to chronic or slowly acting stimuli. sodium transport; serum- and glucocorticoid-induced kinase; A6 cells; sodium pump     

Role of SGK1 in nitric oxide inhibition of ENaC in Na+-transporting epithelia

Several studies have shown that nitric oxide (NO) inhibits Na⁺ transport in renal and alveolar monolayers. However, the mechanisms by which NO alters epithelial Na⁺ channel (ENaC) activity is unclear. Therefore, we examined the effect of applying the NO donor drug L-propanamine 3,2-hydroxy-2-nitroso-1-propylhidrazino (PAPA-NONOate) to cultured renal epithelial cells. A6 and M1 cells were maintained on permeable supports in medium containing 1.5 µM dexamethasone and 10% bovine serum. After 1.5 µM PAPA-NONOate was applied, amiloride-sensitive short-circuit current measurements decreased 29% in A6 cells and 44% in M1 cells. This differed significantly from the 3% and 19% decreases in A6 and M1 cells, respectively, treated with control donor compound (P < 0.0005). Subsequent application of PAPA-NONOate to amiloride-treated control (no NONOate) A6 and M1 cells did not further decrease transepithelial current. In single-channel patch-clamp studies, NONOate significantly decreased ENaC open probability (P_o) from 0.186 ± 0.043 to 0.045 ± 0.009 (n = 7; P < 0.05) without changing the unitary current. We also showed that aldosterone significantly decreased NO production in primary cultures of alveolar type II (ATII) epithelial cells. Because inducible nitric oxide synthase (iNOS) coimmunoprecipitated with the serum- and glucocorticoid-inducible kinase (SGK1) and both proteins colocalized in the cytoplasm (as shown in our studies in mouse ATII cells), SGK1 may also be important in regulating NO production in the alveolar epithelium. Our study also identified iNOS as a novel SGK1 phosphorylated protein (at S733 and S903 residues in miNOS) suggesting that one way in which SGK1 could increase Na⁺ transport is by altering iNOS production of NO. aldosterone; epithelial sodium channel; serum- and glycocorticoid-inducible kinase     

Membrane androgen receptor sensitive Na+/H+ exchanger activity in prostate cancer cells

Membrane androgen receptors (mAR) are expressed in several tumors. mAR activation by testosterone albumin conjugates (TAC) suppresses tumor growth and migration. mAR signaling involves phosphoinositide-3-kinase (PI3K) and Rho-associated protein kinase (ROCK). PI3K stimulates serum- and glucocorticoid-inducible kinase SGK1, which in turn activates Na⁺/H⁺-exchangers (NHE). In prostate cancer cells cytosolic pH (pH_i) was determined utilizing 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein-fluorescence and NHE-activity utilizing Na⁺-dependent cytosolic realkalinization following an ammonium pulse. TAC (100 nM) significantly increased pH_i and NHE-activity, effects abrogated by NHE1-inhibitor cariporide (10 μM), SGK1-inhibitors EMD638683 (50 μM) and GSK650349 (10 μM) and ROCK-inhibitors Y-27632 (10 μM) and fasudil (100 μM). TAC treatment rapidly and significantly increased cell volume and actin polymerization, effects abolished in the presence of cariporide. Thus, mAR-activation activates cariporide-sensitive Na⁺/H⁺-exchangers, an effect requiring SGK1 and ROCK activity.     

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.

     

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.

     

Extracellular Na+ inhibits Na+/H+ exchange: cell shrinkage reduces the inhibition

Na⁺/H⁺ exchangers (NHE) are ubiquitous transporters participating in regulation of cell volume and pH. Cell shrinkage, acidification, and growth factors activate NHE by increasing its sensitivity to intracellular H⁺ concentration. In this study, the kinetics were studied in dog red blood cells of Na⁺ influx through NHE as a function of external Na⁺ concentration ([Na⁺]_o). In cells in isotonic media, [Na⁺]_o inhibited Na⁺ influx >40 mM. Osmotic shrinkage activated NHE by reducing this inhibition. In cells in isotonic media + 120 mM sucrose, there was no inhibition, and influx was a hyperbolic function of [Na⁺]_o. The kinetics of Na⁺-inhibited Na⁺ influx were analyzed at various extents of osmotic shrinkage. The curves for inhibited Na⁺ fluxes were sigmoid, indicating more than one Na⁺ inhibitory site associated with each transporter. Shrinkage significantly increased the Na⁺ concentration at half-maximal velocity of Na⁺-inhibited Na⁺ influx, the mechanism by which shrinkage activates NHE. erythrocytes; cell volume regulation; amiloride; kinetics of sodium ion influx     

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.

     

Stimulation of astrocyte Na+/H+ exchange activity in response to in vitro ischemia depends in part on activation of ERK1/2

We recently reported that Na⁺/H⁺ exchanger isoform 1 (NHE1) activity in astrocytes is stimulated and leads to intracellular Na⁺ loading after oxygen and glucose deprivation (OGD). However, the underlying mechanisms for this stimulation of NHE1 activity and its impact on astrocyte function are unknown. In the present study, we investigated the role of the ERK1/2 pathway in NHE1 activation. NHE1 activity was elevated by 75% in NHE1^+/+ astrocytes after 2-h OGD and 1-h reoxygenation (REOX). The OGD/REOX-mediated stimulation of NHE1 was partially blocked by 30 µM PD-98059. Increased expression of phosphorylated ERK1/2 was detected in NHE1^+/+ astrocytes after OGD/REOX. Moreover, stimulation of NHE1 activity disrupted not only Na⁺ but also Ca²⁺ homeostasis via reverse-mode operation of Na⁺/Ca²⁺ exchange. OGD/REOX led to a 103% increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in NHE1^+/+ astrocytes in the presence of thapsigargin. Inhibition of NHE1 activity with the NHE1 inhibitor HOE-642 decreased OGD/REOX-induced elevation of [Ca²⁺]_i by 73%. To further investigate changes of Ca²⁺ signaling, bradykinin-mediated Ca²⁺ release was evaluated. Bradykinin-mediated intracellular Ca²⁺ transient in NHE1^+/+ astrocytes was increased by 84% after OGD/REOX. However, in NHE1^–/– astrocytes or NHE1^+/+ astrocytes treated with HOE-642, the bradykinin-induced Ca²⁺ release was increased by only 34%. Inhibition of the reverse mode of Na⁺/Ca²⁺ exchange abolished OGD/REOX-mediated Ca²⁺ rise. Together, our data suggest that ERK1/2 is involved in activation of NHE1 in astrocytes after in vitro ischemia. NHE1-mediated Na⁺ accumulation subsequently alters Ca²⁺ homeostasis via Na⁺/Ca²⁺ exchange. intracellular pH; cortical astrocytes; sodium/calcium exchange; intracellular sodium ion     

Effect of glucocorticoids on C3 gene expression by the A549 human pulmonary epithelial cell line.

The third component of C, C3, is the key opsonin of the C cascade and is produced locally within the lung by pulmonary epithelial cells, macrophages, and fibroblasts. Because glucocorticoids regulate the maturation and expression of several physiologically important genes in pulmonary epithelial cells, we examined the effects of glucocorticoids on C3 mRNA expression and C3 synthesis by the human pulmonary epithelial cell line, A549. Treatment with dexamethasone enhanced C3 production in a time- and dose-dependent fashion such that concentrations of dexamethasone greater than or equal to 0.001 microM significantly increased C3 production on day 3 of culture. Natural glucocorticoids, corticosterone, cortisol, and 11-deoxycortisol also increased C3 concentrations in A549 supernatants. Both cycloheximide and the glucocorticoid receptor antagonist, RU486, individually inhibited the effect of dexamethasone on C3 production. Northern analysis demonstrated that the steady state 5.2-kb C3 message increased in A549 cells within 10 h of treatment with dexamethasone. RU486 inhibited the effect of dexamethasone on C3 mRNA expression. The integrity of the C3 thiolester bond, as measured by [3H]iodoacetic acid titration and hemolytic assay, was not disrupted by dexamethasone. We conclude that glucocorticoids such as dexamethasone enhance the expression of C3 mRNA and increase the production of functionally active C3 by A549 cells by a mechanism that is mediated by the intracellular glucocorticoid receptor.     

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     

ETB receptor activation leads to activation and phosphorylation of NHE3

In OKP cellsexpressing ET_B endothelinreceptors, activation ofNa⁺/H⁺antiporter activity by endothelin-1 (ET-1) was resistant to low concentrations of ethylisopropyl amiloride, indicating regulation ofNa⁺/H⁺exchanger isoform 3 (NHE3). ET-1 increased NHE3 phosphorylation incells expressing ET_B receptors butnot in cells expressing ET_Areceptors. Receptor specificity was not due to demonstrable differencesin receptor-specific activation of tyrosine phosphorylation pathways orinhibition of adenylyl cyclase. Phosphorylation was associated with adecrease in mobility on SDS-PAGE, which was reversed by treatingimmunoprecipitated NHE3 with alkaline phosphatase. Phosphorylation wasfirst seen at 5 min and was maximal at 15-30 min. Phosphorylationwas maximal with 10⁹ MET-1. Phosphorylation occurred on threonine and serine residues atmultiple sites. In summary, ET-1 induces NHE3 phosphorylation in OKPcells on multiple threonine and serine residues.ET_B receptor specificity, timecourse, and concentration dependence are all similar betweenET-1-induced increases in NHE3 activity and phosphorylation, suggestingthat phosphorylation plays a key role in activation.