The epithelial Na(+) channel (ENaC) is related to the hypertonicity-induced Na(+) conductance in rat hepatocytes

The epithelial Na(+) channel (ENaC) is composed of the subunits alpha, beta, and gamma [Canessa et al., Nature 367 (1994) 463-467] and typically exhibits a high affinity to amiloride [Canessa et al., Nature 361 (1993) 467-470]. When expressed in Xenopus oocytes, conflicting results were reported concerning the osmo-sensitivity of the channel [Ji et al., Am. J. Physiol. 275 (1998) C1182-C1190; Hawayda and Subramanyam, J. Gen. Physiol. 112 (1998) 97-111; Rossier, J. Gen. Physiol. 112 (1998) 95-96]. Rat hepatocytes were the first system in which amiloride-sensitive sodium currents in response to hypertonic stress were reported [Wehner et al., J. Gen. Physiol. 105 (1995) 507-535; Wehner et al., Physiologist 40 (1997) A-4]. Moreover, all three ENaC subunits are expressed in these cells [B?hmer et al., Cell. Physiol. Biochem. 10 (2000) 187-194]. Here, we injected specific antisense oligonucleotides directed against alpha-rENaC into single rat hepatocytes in confluent primary culture and found an inhibition of hypertonicity-induced Na(+) currents by 70%. This is the first direct evidence for a role of the ENaC in cell volume regulation.     

cAMP-stimulated Na+ transport in H441 distal lung epithelial cells: role of PKA, phosphatidylinositol 3-kinase, and sgk1

H441 cells, a bronchiolar epithelial cell line, develop a cAMP-regulated benzamil-sensitive Na+ transport pathway on permeable supports (Itani OA, Auerbach SD, Husted RF, Volk KA, Ageloff S, Knepper MA, Stokes JB, Thomas CP. Am J Physiol Lung Cell Mol Physiol 282: L631-L641, 2002). To understand the molecular basis for the stimulation of Na+ transport, we delineated the role of specific intracellular pathways and examined the effect of cAMP on alphabetagamma-epithelial Na+ channel (ENaC) and sgk1 expression. Na+ transport increases within 5 min of cAMP stimulation and is sustained for >24 h. The sustained effect of cAMP on Na+ transport is abolished by LY-294002, an inhibitor of phosphatidylinositol 3-kinase, by H89, an inhibitor of PKA, or by SB-202190, an inhibitor of p38 MAP kinase. The sustained effect of cAMP was associated with increases in alpha-ENaC mRNA and protein but without a detectable increase in betagamma-ENaC and sgk1. The early effect of cAMP on Na+ transport is brefeldin sensitive and is mediated via PKA. These results are consistent with a model where the early effect of cAMP is to increase trafficking of Na+ channels to the apical cell surface whereas the sustained effect requires the synthesis of alpha-ENaC.     

Hypoxia reversibly inhibits epithelial sodium transport but does not inhibit lung ENaC or Na-K-ATPase expression

Hypoxia reduces alveolar liquid clearance and the nasal potential difference, a marker of airway epithelial sodium transport. The mechanisms underlying this impaired epithelial sodium transport in vivo remain uncertain. We hypothesized that epithelial sodium transport impaired by hypoxia would recover quickly with reoxygenation and that hypoxia decreases the expression of lung epithelial sodium channels and Na,K-ATPases. We studied adult rats exposed to normoxia, hypoxia (Fi(O(2)) = 0.1) for 24 h, or hypoxia followed by recovery in normoxia. Nasal potential differences decreased by 40% with hypoxia (P < 0.001), returning to baseline levels with reoxygenation. Lung Na,K-ATPase activity decreased by 40% with hypoxia (P = 0.003), recovering to baseline levels with reoxygenation. Lung expression of mRNA encoding for epithelial sodium channel (ENaC)-alpha, -beta, and -gamma or for Na,K-ATPase-alpha(1) did not change significantly with hypoxia or recovery nor did lung expression of ENaC-alpha, ENaC-beta, Na,K-ATPase-alpha(1), or Na,K-ATPase-beta(1) protein. We conclude that subacute exposure to moderate hypoxia reversibly impairs airway epithelial sodium transport and lung Na,K-ATPase activity but that those changes are not due to changes in the lung expression of sodium-transporting proteins.     

Cl- interference with the epithelial Na+ channel ENaC

The cystic fibrosis transmembrane conductance regulator (CFTR) is a protein kinase A and ATP-regulated Cl- channel that also controls the activity of other membrane transport proteins, such as the epithelial Na+ channel ENaC. Previous studies demonstrated that cytosolic domains of ENaC are critical for down-regulation of ENaC by CFTR, whereas others suggested a role of cytosolic Cl- ions. We therefore examined in detail the anion dependence of ENaC and the role of its cytosolic domains for the inhibition by CFTR and the Cl- channel CLC-0. Coexpression of rat ENaC with human CFTR or the human Cl- channel CLC-0 caused inhibition of amiloride-sensitive Na+ currents after cAMP-dependent stimulation and in the presence of a 100 mM bath Cl- concentration. After activation of CFTR by 3-isobutyl-1-methylxanthine and forskolin or expression of CLC-0, the intracellular Cl- concentration was increased in Xenopus oocytes in the presence of a high bath Cl- concentration, which inhibited ENaC without changing surface expression of alpha beta gammaENaC. In contrast, a 5 mM bath Cl- concentration reduced the cytosolic Cl- concentration and enhanced ENaC activity. ENaC was also inhibited by injection of Cl- into oocytes and in inside/out macropatches by exposure to high cytosolic Cl- concentrations. The effect of Cl- was mimicked by Br-, Br-, NO3(-), and I-. Inhibition by Cl- was reduced in trimeric channels with a truncated COOH terminus of betaENaC and gammaENaC, and it was no longer detected in dimeric alpha deltaCbeta ENaC channels. Deletion of the NH2 terminus of alpha-, beta-, or gammaENaC, mutations in the NH2-terminal phosphatidylinositol bisphosphate-binding domain of betaENaC and gammaEnaC, and activation of phospholipase C, all reduced ENaC activity but allowed for Cl(-)-dependent inhibition of the remaining ENaC current. The results confirm a role of the carboxyl terminus of betaENaC for Cl(-)-dependent inhibition of the Na+ channel, which, however, may only be part of a complex regulation of ENaC by CFTR.     

Oxygen induction of epithelial Na(+) transport requires heme proteins

Fetal distal lung epithelial (FDLE) cells exposed to a postnatal O(2) concentration of 21% have higher epithelial Na(+) channel (ENaC) mRNA levels and Na(+) transport relative to FDLE cells grown in a fetal O(2) concentration of 3%. To investigate the mechanism of this process, FDLE monolayers were initially cultured in 3% O(2), and then some were switched to a 21% O(2) environment. Incubation of FDLE cells with the iron chelator deferoxamine, CoCl(2), NiCl(2), or an inhibitor of heme synthesis prevented or diminished the O(2) induction of amiloride-sensitive short-circuit current in FDLE cells. Similarly, defer- oxamine and cobalt prevented O(2)-induced ENaC mRNA expression. Exposure of FDLE cells grown under hypoxic conditions to carbon monoxide increased both ENaC mRNA expression and amiloride-sensitive short-circuit current. We therefore concluded that induction of ENaC mRNA expression and amiloride-sensitive Na(+) transport in FDLE cells by a physiological increase in O(2) concentration seen at birth requires iron and heme proteins.     

BARD1 expression during spermatogenesis is associated with apoptosis and hormonally regulated

The BRCA1-binding RING-finger domain protein BARD1 may act conjointly with BRCA1 in DNA repair and in ubiquitination, but it may also induce apoptosis in a BRCA1-independent manner. In this study, we have investigated BARD1 expression during spermatogenesis. In contrast with BRCA1, which is expressed only in meiotic spermatocytes and early round spermatids, BARD1 is expressed during all stages of spermatogenesis. However, while spermatogonia expressed full-length BARD1 mRNA, later stages of spermatocyte precursors express predominantly a novel, shorter splice form BARD1beta. BARD1beta lacks the BRCA1-interacting RING finger but maintains its proapoptotic activity. Consistently, BRCA1 can counteract the proapoptotic activity of full-length BARD1 but not of BARD1beta. Several lines of evidence suggest that BARD1 is involved in proapoptotic signaling in testis: i) both BARD1 isoforms are mostly found in cells that stain positive for TUNEL, Bax, and activated caspase 3; ii) BARD1beta, capable of inducing apoptosis even in the presence of BRCA1, is specifically expressed in BRCA1-positive later stages of spermatogenesis; iii) antiapoptotic hormonal stimulation leads to BARD1 downregulation; and iv) BARD1 expression is associated with human pathologies causing sterility due to increased germ cell death. Our data suggest that full-length BARD1 might be involved in apoptotic control in spermatogonia and primary spermatocytes, while a switch to the BRCA1-independent BARD1beta might be necessary to induce apoptosis in BRCA1-expressing meiotic spermatocytes and early round spermatids.     

Kidney Na+,K(+)-ATPase is associated with moesin

Na+,K(+)-ATPase is a ubiquitous plasmalemmal membrane protein essential for generation and maintenance of transmembrane Na+ and K+ gradients in virtually all animal cell types. Activity and polarized distribution of renal Na+,(+)-ATPase appears to depend on connection of ankyrin to the spectrin-based membrane cytoskeleton as well as on association with actin filaments. In a previous study we showed copurification and codistribution of renal Na+,K(+)-ATPase not only with ankyrin, spectrin and actin, but also with two further peripheral membrane proteins, pasin 1 and pasin 2. In this paper we show by sequence analysis through mass spectrometry as well as by immunoblotting that pasin 2 is identical to moesin, a member of the FERM (protein 4.1, ezrin, radixin, moesin) protein family, all members of which have been shown to serve as cytoskeletal adaptor molecules. Moreover, we show that recombinant full-length moesin as well as its FERM domain bind to Na+,K(+)-ATPase and that this binding can be inhibited by an antibody specific for the ATPase activity-containing cytoplasmic loop (domain 3) of the Na+,K(+)-ATPase alpha-subunit. This loop has been previously shown to be a site essential for ankyrin binding. These observations indicate that moesin might not only serve as direct linker molecule of Na+,K(+)-ATPase to actin filaments but also modify ankyrin binding at domain 3 of Na+,K(+)-ATPase in a way similar to protein 4.1 modifying the binding of ankyrin to the cytoplasmic domain of the erythrocyte anion exchanger (AE1).     

PRL-1 PTPase expression is developmentally regulated with tissue-specific patterns in epithelial tissues

The mechanisms controlling tyrosine phosphorylation of cellular proteins are important in the regulation of many cellular processes, including development and differentiation. Protein tyrosine phosphatases (PTPases) may be as important as protein tyrosine kinases (PTKs) in these processes. PRL-1 is a distinct PTPase originally identified as an immediate-early gene in liver regeneration whose expression is associated with growth in some tissues but with differentiation in others. We now demonstrate that the PRL-1 protein is expressed during development in a number of digestive epithelial tissues. It is expressed at variable time points in the developing intestine, but its expression is limited to the developing villus enterocytes. In the gastric epithelium, PRL-1 expression in the adult is restricted to zymogen cells. PRL-1 is also expressed in the developing liver and esophagus and in the epithelia of the kidney and lung. In each of these contexts, the expression of PRL-1 is associated with terminal differentiation, suggesting that it may play a role in this important developmental process.     

Modulation of epithelial sodium channel (ENaC) expression in mouse lung infected with Pseudomonas aeruginosa

Background

The intratracheal instillation of Pseudomonas aeruginosa entrapped in agar beads in the mouse lung leads to chronic lung infection in susceptible mouse strains. As the infection generates a strong inflammatory response with some lung edema, we tested if it could modulate the expression of genes involved in lung liquid clearance, such as the α, β and γ subunits of the epithelial sodium channel (ENaC) and the catalytic subunit of Na⁺-K⁺-ATPase.

Methods

Pseudomonas aeruginosa entrapped in agar beads were instilled in the lung of resistant (BalB/c) and susceptible (DBA/2, C57BL/6 and A/J) mouse strains. The mRNA expression of ENaC and Na⁺-K⁺-ATPase subunits was tested in the lung by Northern blot following a 3 hours to 14 days infection.

Results

The infection of the different mouse strains evoked regulation of α and β ENaC mRNA. Following Pseudomonas instillation, the expression of αENaC mRNA decreased to a median of 43% on days 3 and 7 after infection and was still decreased to a median of 45% 14 days after infection (p < 0.05). The relative expression of βENaC mRNA was transiently increased to a median of 241%, 24 h post-infection before decreasing to a median of 43% and 54% of control on days 3 and 7 post-infection (p < 0.05). No significant modulation of γENaC mRNA was detected although the general pattern of expression of the subunit was similar to α and β subunits. No modulation of α₁Na⁺-K⁺-ATPase mRNA, the catalytic subunit of the sodium pump, was recorded. The distinctive expression profiles of the three subunits were not different, between the susceptible and resistant mouse strains.

Conclusions

These results show that Pseudomonas infection, by modulating ENaC subunit expression, could influence edema formation and clearance in infected lungs.     

S-adenosyl-L-homocysteine hydrolase is necessary for aldosterone-induced activity of epithelial Na(+) channels

The A6 cell line was used to study the role ofS-adenosyl-L-homocysteine hydrolase (SAHHase) inthe aldosterone-induced activation of the epithelial Na⁺channel (ENaC). Because aldosterone increases methylation of severaldifferent molecules, and because this methylation is associated withincreased Na⁺ reabsorption, we tested the hypothesis thataldosterone increases the expression and activity of SAHHase protein.The rationale for this work is that general methylation may be promotedby activation of SAHHase, the only enzyme known to metabolize SAH, apotent end-product inhibitor of methylation. Although aldosteroneincreased SAHHase activity, steroid did not affect SAHHase expression.Antisense SAHHase oligonucleotide decreased SAHHaseexpression and activity. Moreover, this oligonucleotide, as well as apharmacological inhibitor of SAHHase, decreased aldosterone-inducedactivity of ENaC via a decrease in ENaC open probability. The kineticsof ENaC in cells treated with antisense plus aldosterone were similarto those reported previously for the channel in the absence of steroid. This is the first report showing that active SAHHase, in part, increases ENaC open probability by reducing the transition rate fromopen states in response to aldosterone. Thus aldosterone-induced SAHHase activity plays a critical role in shifting ENaC from a gatingmode with short open and closed times to one with longer open andclosed times.

     

Conductive Na+ transport in fetal lung alveolar apical membrane vesicles is regulated by fatty acids and G proteins

We have characterised G protein and fatty acid regulation of the Na⁺ conductance in purified apical membrane vesicles prepared from late gestation fetal guinea-pig lung. Addition of 100 μM GTPγS or βγ-methylene-GTP, irreversible G protein activators, stimulated conductive ²²Na⁺ uptake (ratio of experimental to control 1.35±0.02 and 1.34±0.05, respectively). Conversely, the addition of GDPβS, an irreversible G protein inhibitor, reduced conductive ²²Na⁺ uptake from 1.00 (control) to 0.79±0.04. A range of saturated (myristic, palmitic, stearic), monounsaturated (elaidic, oleic) and polyunsaturated (linoleic, arachidonic) fatty acids all stimulated conductive ²²Na⁺ uptake, by between 1.18±0.05 to 1.56±0.13 over the control. Both arachidonic acid and GTPγS-dependent stimulation were abolished in the presence of 10 μM amiloride. The non-metabolisable analogue of arachidonic acid, eicosa-5,8,11,14-tetraynoic acid also stimulated conductive ²²Na⁺ uptake. Furthermore, addition of indomethacin and nordihydroguairetic acid, inhibitors of cyclooxygenase and lipoxygenase pathways of arachidonate metabolism respectively, did not affect the arachidonic acid stimulation suggesting a direct effect of fatty acid upon the Na⁺ channel. Since mepacrine (50 μM), a phospholipase A₂ inhibitor, did not affect the GTPγS-stimulated conductive ²²Na⁺ uptake, and inhibition of G protein turnover by GDPβS did not attenuate the arachidonic acid response, we conclude that these two regulatory pathways modulate alveolar Na⁺ transport directly and independently of each other.     

The putative plasma membrane Na(+)/H(+) antiporter SOS1 controls long-distance Na(+) transport in plants

The salt tolerance locus SOS1 from Arabidopsis has been shown to encode a putative plasma membrane Na(+)/H(+) antiporter. In this study, we examined the tissue-specific pattern of gene expression as well as the Na(+) transport activity and subcellular localization of SOS1. When expressed in a yeast mutant deficient in endogenous Na(+) transporters, SOS1 was able to reduce Na(+) accumulation and improve salt tolerance of the mutant cells. Confocal imaging of a SOS1-green fluorescent protein fusion protein in transgenic Arabidopsis plants indicated that SOS1 is localized in the plasma membrane. Analysis of SOS1 promoter-beta-glucuronidase transgenic Arabidopsis plants revealed preferential expression of SOS1 in epidermal cells at the root tip and in parenchyma cells at the xylem/symplast boundary of roots, stems, and leaves. Under mild salt stress (25 mM NaCl), sos1 mutant shoot accumulated less Na(+) than did the wild-type shoot. However, under severe salt stress (100 mM NaCl), sos1 mutant plants accumulated more Na(+) than did the wild type. There also was greater Na(+) content in the xylem sap of sos1 mutant plants exposed to 100 mM NaCl. These results suggest that SOS1 is critical for controlling long-distance Na(+) transport from root to shoot. We present a model in which SOS1 functions in retrieving Na(+) from the xylem stream under severe salt stress, whereas under mild salt stress it may function in loading Na(+) into the xylem.     

Impairment of transalveolar fluid transport and lung Na(+)-K(+)-ATPase function by hypoxia in rats.

We examined whether hypoxic exposure in vivo would influence transalveolar fluid transport in rats. We found a significant decrease in alveolar fluid clearance of the rats exposed to 10% oxygen for 48 h. Terbutaline did not stimulate alveolar fluid clearance, and alveolar fluid cAMP levels were lower than those determined in normoxia experiment. Hypoxia did not influence the alveolar fluid lactate dehydrogenase levels, Evans blue dye fluid-to-serum concentration ratio, or lung wet-to-dry weight ratio, indicating no significant change in the permeability of alveolar-capillary barrier. Histological examination showed no significant fluid accumulation into the interstitium and the alveolar space. Hypoxia did not reduce lung ATP content; however, we found significant decrease in Na(+)-K(+)-ATPase hydrolytic activity in lung tissue preparations and isolated alveolar type II cells. Our data indicate that hypoxic exposure in vivo impairs transalveolar fluid transport, and this impairment is related to the decrease in alveolar epithelial Na(+)-K(+)-ATPase hydrolytic activity but is not secondary to the alteration of cellular energy source.     

Regulation of the Na(+)-dependent and the Na(+)-independent polyamine transporters in renal epithelial cells (LLC-PK1)

We have studied the regulation of the Na(+)-dependent and Na(+)-independent polyamine transport pathways in the renal LLC-PK1 cell line. Most of the experiments were performed in the presence of 5 mM DL-2-difluoromethylornithine (DFMO) in order to inhibit the cellular synthesis of polyamines. The activity of both transporters as measured by putrescine uptake was increased by growth-promoting stimuli and decreased by exogenous polyamines. The time course of the increase in uptake activity induced by fetal calf serum could be fitted by a single exponential, and the process was three times faster for the Na(+)-dependent than for the Na(+)-independent transporter. Maximum activity was reached after more than 24 h. This increase could be inhibited by actinomycin D and by cycloheximide. Other growth-promoting stimuli, such as subconfluent cell density, as well as growth factors also induced an increase in the transport activity. Particularly, there was a marked stimulation of the Na(+)-dependent pathway by epidermal growth factor in combination with insulin. On the other hand, the transport activity decayed very rapidly upon addition of exogenous polyamines (t1/2 less than 60 min). The diamine putrescine was much less effective in this respect than the polyamines spermidine and spermine. The non-metabolizable substrate methylglyoxal bis(guanylhydrazone) did not induce a decay of the transport activity, but it protected the Na(+)-dependent pathway against the polyamine-induced decay. Inhibition of the protein synthesis by cycloheximide did not induce a rapid decrease of the transport activity; neither did it affect the polyamine-induced decay. These observations suggest that this polyamine-induced decay is not owing to an inhibitory effect on the rate of synthesis of the transporters, but rather to a degradation or an inactivation of the transporters. The polyamine-induced decay slowed down at lower cell density. This effect was particularly pronounced for the Na(+)-dependent transporter. Since the uptake of polyamines was increased at low cell density, the decreased rate of decay in this condition pleads against a simple mechanism of transinhibition by the substrate. In conclusion, both transport pathways were similarly affected by the regulatory parameters, but the Na(+)-dependent transporter was more rapidly and more effectively regulated. The numerous interacting regulatory steps furthermore suggest a physiological role for these transporters, such as an involvement in urinary polyamine disposal.     

K(+) channels regulate ENaC expression via changes in promoter activity and control fluid clearance in alveolar epithelial cells

Active Na(+) absorption by alveolar ENaC is the main driving force of liquid clearance at birth and lung edema resorption in adulthood. We have demonstrated previously that long-term modulation of KvLQT1 and K(ATP) K(+) channel activities exerts sustained control in Na(+) transport through the regulation of ENaC expression in primary alveolar type II (ATII) cells. The goal of the present study was: 1) to investigate the role of the α-ENaC promoter, transfected in the A549 alveolar cell line, in the regulation of ENaC expression by K(+) channels, and 2) to determine the physiological impact of K(+) channels and ENaC modulation on fluid clearance in ATII cells. KvLQT1 and K(ATP) channels were first identified in A549 cells by PCR and Western blotting. We showed, for the first time, that KvLQT1 activation by R-L3 (applied for 24h) increased α-ENaC expression, similarly to K(ATP) activation by pinacidil. Conversely, pharmacological KvLQT1 and K(ATP) inhibition or silencing with siRNAs down-regulated α-ENaC expression. Furthermore, K(+) channel blockers significantly decreased α-ENaC promoter activity. Our results indicated that this decrease in promoter activity could be mediated, at least in part, by the repressor activity of ERK1/2. Conversely, KvLQT1 and K(ATP) activation dose-dependently enhanced α-ENaC promoter activity. Finally, we noted a physiological impact of changes in K(+) channel functions on ERK activity, α-, β-, γ-ENaC subunit expression and fluid absorption through polarized ATII cells. In summary, our results disclose that K(+) channels regulate α-ENaC expression by controlling its promoter activity and thus affect the alveolar function of fluid clearance.