aldosterone and amiloride alter enac abundance in vascular...

CELL AND MOLECULAR PHYSIOLOGY

Aldosterone and amiloride alter ENaC abundancein vascular endothelium

Kristina Kusche-Vihrog & Katja Sobczak &

Nadine Bangel & Marianne Wilhelmi &Volodymyr Nechyporuk-Zloy & Albrecht Schwab &

Hermann Schillers & Hans Oberleithner

Received: 16 May 2007 /Revised: 29 August 2007 /Accepted: 30 August 2007# Springer-Verlag 2007

Abstract The amiloride-sensitive epithelial sodium chan-nel (ENaC) is usually found in the apical membrane ofepithelial cells but has also recently been described invascular endothelium. Because little is known about theregulation and cell surface density of ENaC, we studiedthe influence of aldosterone, spironolactone, and amilorideon its abundance in the plasma membrane of humanendothelial cells. Three different methods were applied,single ENaC molecule detection in the plasma membrane,quantification by Western blotting, and cell surfaceimaging using atomic force microscopy. We found thataldosterone increases the surface expression of ENaCmolecules by 36% and the total cellular amount by 91%.The aldosterone receptor antagonist spironolactone pre-vents these effects completely. Acute application ofamiloride to aldosterone-pretreated cells led to a declineof intracellular ENaC by 84%. We conclude that, invascular endothelium, aldosterone induces ENaC expres-sion and insertion into the plasma membrane. Uponfunctional blocking with amiloride, the channel disappearsfrom the cell surface and from intracellular pools,indicating either rapid degradation and/or membranepinch-off. This opens new perspectives in the regulationof ENaC expressed in the vascular endothelium.

Keywords HUVEC . Atomic force microscopy .

Quantum dots . Plasmamembrane . Ion channel .

Spironolactone

Introduction

The amiloride-sensitive epithelial sodium channel (ENaC)has predominantly been described in the apical plasmamembrane of epithelia. There, the ion channel mediatesthe first step of active sodium reabsorption in kidney,colon, lung, and sweat glands [15]. It is involved in theregulation of the blood pressure and might be responsiblefor the progression of hypertensive diseases [28]. Recently,ENaC has been identified in human endothelial cells [9, 16,33] where its specific functions are yet unclear.

ENaC consists of three subunits named α, β, and γ [8]and is highly conserved throughout the vertebrate order.The channel is characterized by two transmembrane seg-ments and a large extracellular domain [7]. The regulationof ENaC is tissue specific and mediated by the mineralo-corticoid hormone aldosterone and aldosterone-inducedproteins, e.g., the serum- and glucocorticoid-regulatedkinase 1 (sgk1) [42, 43]. Thus, different proteins and anumber of extracellular factors interact directly or indirectlywith the channel [3, 14]. Although the kidney is known tobe the major target for aldosterone, there is accumulatingevidence for its influence on nonepithelial sites such asbrain, heart, and blood vessels [10, 31, 40]. Besides theclassical genomic pathway, aldosterone also acts at anongenomic level. This mechanism is rapid and involvesthe activation of second messenger pathways [13]. The fastaldosterone action was also reported for human umbilicalvenous endothelial cells (HUVEC), where the hormoneinduces cell swelling within minutes [34]. In addition, it

Pflugers Arch - Eur J PhysiolDOI 10.1007/s00424-007-0341-0

K. Kusche-Vihrog (*) :K. Sobczak :N. BangelInstitute of Animal Physiology, University of Muenster,Hindenburgplatz 55,48143 Muenster, Germanye-mail: [email protected]

M. Wilhelmi :V. Nechyporuk-Zloy :A. Schwab :H. Schillers :H. OberleithnerInstitute of Physiology II, University of Muenster,Muenster, Germany

was shown that this cell volume increase was reduced bythe application of amiloride, a specific ENaC blocker [32].In contrast to epithelia, where cell surface appearance ofENaC is well characterized, only little is known aboutexpression and plasma membrane insertion of the channelin vascular endothelia [30]. Therefore, we used HUVEC, awell-established cell model system [33], to study the invitro effects of aldosterone, its receptor antagonist spirono-lactone, and the specific blocker amiloride on α-ENaCprotein levels and cellular localization. We aimed toidentify single ENaC molecules in the plasma membranewith fluorescent quantum-dot (QD)-labeled antibodies, toquantify intracellularly stored ENaC by Western blotting,and to image cell surfaces by using atomic force micros-copy (AFM). We found that (1) aldosterone increased theamount of ENaC in the plasma membrane and inintracellular stores, (2) spironolactone prevented the hor-mone response, and (3) acute application of amiloridedecreased ENaC abundance and the cell surface area. Takentogether, aldosterone is a regulator of ENaC expression andplasma membrane insertion in vascular endothelium. To oursurprise, a functional blockade of ENaC with amiloride ledto a rapid disappearance of the channel from the cell. Thisobserved phenomenon could be due to degradation ofENaC and/or membrane shedding.

Materials and methods

Endothelial cell culture

HUVEC were isolated and cultured as previously described[18, 23] in a way conforming with the principles of theDeclaration of Helsinki. Briefly, cells (passage p0) weregrown in T25 culture flasks coated with 0.5% gelatine(Sigma-Aldrich Chemie, Steinheim, Germany). After reach-ing confluence, cells were split using trypsin and thencultured (passage p1) on eight-well diagnostic microscopeslides (Menzel, Braunschweig, Germany) coated with 0.5%gelatine. For AFM studies, cells were cultured on thin(diameter=15 mm) glass coverslips coated with 0.5%gelatine and cross-linked with 2% glutaraldehyde. Bothslides were placed in Petri dishes filled with culturemedium which was tested negative for aldosterone. Afterreaching either subconfluence (for immunofluorescencestudies) or confluence (for AFM studies), HUVEC wereincubated for 72 h (leads to a 44% decrease in ENaCsurface abundance, a 45% reduction in membrane area: 1)in aldosterone (10 nM, D-aldosterone, Sigma-Aldrich), (2)in 10 nM aldosterone together with 100 nM spironolactone(ICN Biochemicals, Eschwege, Germany), and (3) in 10 nMaldosterone followed by a (before experiment) 1 h incubationin 1 μM amiloride (Sigma-Aldrich). As corresponding

control experiments, HUVEC were incubated in mediumafter addition of the solvent. For immunofluorescenceexperiments, cells were fixed in glutaraldehyde. Weperformed cell fixation directly in the incubator (5% CO2,37°C), a protocol which maintains the cell shape as des-cribed previously [33]. Briefly, glutaraldehyde was gentlyadded to the cell culture medium (final glutaraldehydeconcentration, 0.5%). After a period of 60 min, the fixativewas removed, and cells were stored at 4°C. Althoughendothelial cells are not clearly polarized, we will some-times use the term “apical” for the upper side of the celllayer that faces the culture medium.

Antibody staining

After fixation, HUVEC were gently washed five times inphosphate-buffered saline (PBS, in mM: 140 NaCl, 2 KCl,4 Na2HPO4, 1 KH2PO4, pH 7.4) at room temperature andincubated afterwards for 30 min in 100 mM glycine/PBSsolution. The cells were washed again five times by gentleshaking in PBS and then blocked with 10% normal goatserum (NGS) at room temperature for 1 h. The primarypolyclonal rabbit anti α-ENaC antibody (a kind gift fromDr. M. S. Awayda, Department of Physiology and Bio-physics, Buffalo University School of Medicine, USA) wasdiluted 1:1,000 in 10% NGS and applied to the cells. After1 h of incubation with the primary antibody, the cells werewashed five times in PBS. For QD-labeling, we incubatedthe cells with QD655 goat F(ab′)2 anti-rabbit IgG con-jugates (1:100 and 1:50, respectively) at room temperaturein the dark for 1 h. The QD were purchased at QuantumDot, Hayward, USA. Cells were then washed again fivetimes in PBS, two times in HEPES (in mM: 140 NaCl, 5KCl, 1 MgCl2, 1 CaCl2, 5 glucose, 10 HEPES), and fixedwith 0.5% glutaraldehyde in HEPES buffer for 45 min. Asa negative control, we incubated HUVEC exclusively withthe secondary antibody.

For control experiments, we incubated HUVEC with aprimary anti-α-tubulin antibody (1:15,000 and 1:25,000,respectively; Sigma-Aldrich) and Cy3-labeled anti-mousesecondary antibody (1:800; Dianova, Hamburg, Germany).To detect α-tubulin, intracellular cells were permeabilizedby incubating the cells for 10 min in 0.1% Triton X-100/1% sodium dodecyl sulfate (SDS). In another protocol, thestaining procedure was performed without the detergent,and cells remained intact. The staining with the anti-α-tubulin antibody of permeabilized and nonpermeabilizedHUVEC was checked with fluorescence microscopy.

Image analysis

Immunofluorescence images were acquired with aninverted fluorescence microscope (Axiovert 200, Zeiss,

Pflugers Arch - Eur J Physiol

Oberkochen, Germany) equipped with a 100×1.45 oil im-mersion objective. We used the following filters: QD655,420 nm excitation, 655 nm emission (XF302-1 filter,Omega Optical, Brattleboro, VT, USA); Cy3, 546 nm ex-citation, 562 nm emission (filter no. 15, Zeiss). Dataacquisition and analysis were performed with the MetavueSoftware (Visitron, Puchheim, Germany). Therefore, numb-ers of QD-labeled ENaC at the apical surface of the cellswere counted and corrected for QD background levels incell-free areas. As a negative control, we incubatedHUVEC exclusively with the secondary antibody (datanot shown). Surface areas of HUVEC were selectedmanually, and QD-labeled ENaC molecules in the plasmamembrane of the cells were detected by focusing on theupper surface of the cells.

Protein biochemistry

HUVEC were grown and incubated as described above. Forprotein isolation, cells were washed twice with PBS on iceand scraped off with 1 ml of ice-cold lysis buffer (1 mMTris, 15 mM NaCl, 0.2 mM EDTA, 0.125% Triton X-100).Protease inhibitor cocktail was added to this detergentmixture (10 mM Leupeptin, 1 mg/ml Trypsininhibitor,25 μM Pefablock, 100 mM PMSF). The cell lysate wasthen homogenized by repetitive passing through a sterilesyringe (diameter 0.9 mm, Braun, Melsungen, Germany).Afterwards, the homogenized lysate was centrifuged at 4°Cat 4,000×g for 30 min, and the supernatant was transferredinto a new collection tube.

For Western blotting, 40 μg protein was loaded ontoSDS-polyacrylamide gel electrophoresis (7.5% acrylam-ide) and transferred to a polyvinylidene fluoride (PVDF)membrane. Nonspecific binding sites were blocked 4 h by5% nonfat dry milk in Tris-buffered saline/Tween (TBST;10 mM Tris–HCl, pH 7.4; 140 mM NaCl; 0.3% Tween20). The ENaC α-subunit was detected with an anti-ENaCalpha antibody (Dianova) with a concentration of 1:2,500diluted in 5% nonfat dry milk/TBST at 4°C overnight.The immunogen corresponds to the N-terminal amino acidresidues 20–42 from human α-ENaC. After washing inTBST, the membrane was incubated for 1 h at roomtemperature with the goat anti-rabbit IgGs conjugated withalkaline phosphatase (Santa Cruz, California, USA)diluted 1:10,000 in 5% nonfat dry milk/TBST. Themembrane was washed again in TBST, and detectionwas carried out with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate. To determine the proteinamounts of the bands detected by the specific anti α-ENaCantibody in Western blot experiments, we digitized thePVDF membranes and analyzed the blot semiquantita-tively by densitometry using ImageJ analysis software1.36 [1].

AFM measurements

The method of cell imaging by AFM has been described indetail [17, 36]. Briefly, for single cell surface measurements,AFM was performed in electrolyte solution (before and afterapplication of 1 μM amiloride) in living HUVEC at 37°Cusing a Nanoscope III Multimode-AFM (Digital Instruments,Santa Barbara, California, USA) with a J-type scanner(maximal scan area, 100×100 μm). V-shaped oxide sharp-ened DNP-S gold-coated cantilevers with spring constants of0.06 N/m (Digital Instruments) were used. Surface profiles(512×512 pixels) were obtained with scan sizes of10,000 μm2 at a scan rate of 6 Hz. Five to ten images wereobtained from individual samples and analyzed using theNanoscope III software (Digital Instruments). Each imagewas plane-fitted (order 1), and apical endothelial surface ofthe total image (about 7 to 12 cells per image) was analyzed.Single cell surface was calculated by dividing the total imageby the number of cells per image.

Statistics

All results are expressed as means ± SEM (n=number ofobservations). Mean data were tested for significance usingStudent’s t test for paired or unpaired experiments ifapplicable. Differences were considered as statisticallysignificant when p<0.05 or less. All statistical tests wereperformed using Origin version 7.5 for Windows (Origin-Lab, Northampton, MA, USA).

Results

Immunofluorescence

For the immunofluorescence experiments, cells wereincubated for 72 h with solvent (control), aldosterone, oraldosterone plus spironolactone. Furthermore, aldosterone-pretreated HUVEC were exposed to amiloride for 1 hbefore the experiments. Endothelial cells were grown tosubconfluence to identify single cells more easily. ENaCwas detected at the upper surface of the cells with a specificanti-α-ENaC antibody. This polyclonal antibody wasgenerated against a near full-length bovine α-ENaC fusionprotein. Because the predominant immunogen was a 50-kDa α-ENaC protein containing the N-terminus and theextracellular loop, this antibody appeared adequate for themembrane surface detection of ENaC molecules. The spec-ificity and efficacy of this antibody in detecting α-ENaCwas demonstrated by several groups [6, 22]. As a sec-ondary antibody, we used QD-labeled anti-rabbit F(ab′)2conjugates. Experiments were repeated four times, and atleast nine cells for the different conditions were analyzed.


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Figure 1a shows four representative images of ENaCdetected at the surface of endothelial cells. There are visibledifferences in the quantity of ENaC between the differenttreatments of the cells. As summarized in Fig. 1b, we found,in comparison to control cells, a significant increase in ENaC/μm2 at the apical surface of cells incubated in aldosterone.The ENaC surface quantity/μm2 apical surface area wasincreased from 1.27±0.18 ENaC molecules/μm2 apicalsurface (control) to 1.73±0.18 ENaC molecules/μm2 apicalsurface (36%; p<0.01). This effect was effectively preventedby coincubation with the aldosterone receptor antagonistspironolactone (1.29±0.15 ENaC molecules/μm2 apicalsurface). Because amiloride is known to be a specific blockerof ENaC in epithelial cells, we tested whether it modifies theabundance of the sodium channel in the plasma membrane of

HUVEC. Indeed, we found in aldosterone-pretreated cells1 h after amiloride incubation a significant decrease in thenumber of ENaC molecules/μm2 at the cell surface.Amiloride reduced the channel occurrence from 1.73±0.18ENaC molecules/μm2 apical surface (aldosterone) to 1.15±0.1 ENaC molecules/μm2 apical surface as compared toaldosterone-induced ENaC (44% reduction; p<0.01).

To verify that we detected ENaC exclusively at the cellsurface (and not intracellularly), we used anti-α-tubulinantibodies for control experiments. They were carried outwith and without permeabilization of the cell membrane.Figure 2 shows that no α-tubulin could be detected whenthe standard antibody staining protocol without permeabi-lization was applied. This indicates that only the ENaClocated in the plasma membrane was visualized.

Protein biochemistry

In addition to ENaC located in the cell surface, we analyzedthe total cellular amount of the sodium channel protein andperformed semiquantitative Western blot experiments witha specific anti-α-ENaC subunit antibody, which is highlysuitable for immunoblotting approaches [11]. Cells wereexposed for 72 h to the solvent (control) or aldosterone. Inaddition, aldosterone-pretreated HUVEC were exposed toamiloride for 1 h. As displayed in Fig. 3a, the anti-α-ENaCantibody recognizes a specific protein band with anapparent molecular mass of about 95 kDa, which mostlikely represents a glycosylated form of the α-ENaCsubunit. Additionally, another band was detected in therange of 55 kDa, which could represent an endogenousproteolytically cleaved form of the α-ENaC protein [20,21]. The specificity of this antibody was also tested in

Fig. 1 a Detection of ENaC at the cell surface of HUVEC(representative images). Single ENaC molecules were detected withthe combination of specific polyclonal anti-α-ENaC antibodies andQD-labeled secondary antibodies. The background staining of thecells is due to fixation with glutaraldehyde, which allows theidentification of cell borders. Control HUVEC incubated with solvent,aldo HUVEC incubated with 10 nM aldosterone for 72 h, aldo+spirocoincubation with 10 nM aldosterone and 100 nM spironolactonefor 72 h, aldo+ami aldosterone-pretreated cells were incubated with1 μM amiloride for 1 h. b Quantification of single ENaC molecules atthe cell surface. The abundance of QD-labeled ENaC molecules/μm2

apical surface of HUVEC was counted for each cell (numbers ofanalyzed cells are given in the bars). In this bar, diagram mean valuesare given. Control HUVEC incubated with solvent, aldo HUVECincubated with 10 nM aldosterone for 72 h, aldo+spiro coincubationwith 10 nM aldosterone and 100 nM spironolactone for 72 h, aldo+ami aldosterone-pretreated cells were incubated with 1 μM amiloridefor 1 h. The asterisk indicates the significant difference in comparisonwith control (p<0.01)

nonpermeabilized permeabilized

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Fig. 2 Detection of α-tubulin inpermeabilized and nonpermea-bilized HUVEC (representativeimages). Cytosolic α-tubulinwas detected with an α-tubulinantibody and Cy3 as secondaryantibody in permeabilizedHUVEC (right side). In non-permeabilized cells (left side),no tubulin could be detected inthe cytosol. This experimentserves as a control for the stain-ing of proteins at the cell surface

R


Western blot experiments with proteins isolated fromENaC-expressing oocytes used as a positive control (pc),where it recognized one distinct ENaC band in the range of95 kDa (Fig. 3a). As a negative control, we used non-

injected oocytes where we found no indication (i.e., nobands) for any protein binding (not shown). These experi-ments were repeated five times, and the mean values arepresented in Fig. 3b. In aldosterone-treated cells, totalENaC protein was significantly increased (by 91%) ascompared with control (aldosterone-deficient) cells. To oursurprise, the functional blockade by amiloride (1 h beforethe experiments) led to a dramatic decrease by 84% in theamount of the sodium channel protein.

Atomic force microscopy

In another series of experiments, we tested whetheramiloride modifies the apically exposed cell surface ofaldosterone-pretreated HUVEC. In paired experimentsapplying AFM in living HUVEC, we found within 3 to6 min a significant decrease in apical cell surface area(mean value, −45±5.6%; n=10; p<0.01). A representativeexperiment in aldosterone-pretreated cells is shown inFig. 4. In contrast, HUVEC, which were not aldosterone-pretreated, did not respond to amiloride (mean change incell surface, −1±3.8%; n=10). The substantial reduction inthe cell surface in response to the acute application ofamiloride in the aldosterone-treated cells correlates with thedecrease in the ENaC molecules in the plasma membrane asdescribed above.

Discussion

Previous investigations indicated that aldosterone increasesthe surface area of endothelial cells [19, 33]. Thisaldosterone-induced cell expansion was explained by apicalsodium entry via ENaC into the cells. In the present study,we examined the aldosterone response with regard to thequantity of ENaC molecules located in the cell membraneof vascular endothelium. In addition, the functional role ofENaC was tested by the application of the specific blockeramiloride.

Aldosterone activates ENaC expression and plasmamembrane insertion

The kidney is the major target for aldosterone. A typicalcellular response to the hormone is the expression ofENaC and its insertion into the apical surface of renalcollecting duct cells that allows filtered sodium to beretained [35]. Although endothelial cells are not known asthe classical targets for mineralocorticoids, it was realizedrecently that they express aldosterone receptors and sodiumchannels similar as renal epithelial tissue [9, 16, 32].

To detect and quantify the ENaC α-subunit located inthe apical plasma membrane, we used QD, which allow

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Fig. 3 a Detection of the cellular ENaC amount. Representativeimmunoblot of proteins prepared from HUVEC. Before proteinisolation, cells were exposed to solvent (control), 10 nM aldosterone(aldo), and 1 μM amiloride after aldosterone pretreatment (aldo+ami). 40 microgram protein was loaded on each lane and separated ona 7.5% SDS gel. Proteins were blotted onto PVDF membranes, andWestern blots were carried out with a specific anti-α-ENaC antibodyand an alkaline phosphatase-labeled secondary antibody. A distinctprotein band in the range of 95 kDa was detected, which is typical forthe α-ENaC subunit. The second protein band in the range of 55 kDamost likely represents an endogenous proteolytically cleaved form ofthe α-ENaC protein. As a positive control (pc), rat ENaC proteinexpressed in oocytes was incubated with the specific anti-α-ENaCantibody. There are obvious differences in the protein amount of α-ENaC between control, aldosterone, and aldosterone+amiloride:Aldosterone treatment increases the protein amount, while incubationwith amiloride reduces the abundance of α-ENaC in comparison withthe aldo-induced ENaC amount. b Densitometric analysis of theWestern blots. All blots were digitized, and densitometric analyseswere carried out by using the ImageJ software. Aldo HUVECincubated with 10 nM aldosterone for 72 h, aldo+ami aldosterone-pretreated cells were incubated with 1 μM amiloride for 1 h. Given isthe relative expression of α-ENaC in HUVEC in comparison tocontrol (normalized to 1=100%). Mean values of five different blotsare shown


the identification of proteins at the single molecule level[29]. With this experimental approach, we could show thataldosterone elevates the abundance of ENaC in theapically exposed cell membrane by 36% related to1 μm2 of the apical cell surface. As recently described,aldosterone, in addition, substantially enlarges the apicalcell surface by 64% [33]. This indicates that the totalincrease in ENaC molecules per cell (found on the cellsurface) in response to aldosterone treatment is muchhigher than 36%.

Western blotting experiments reveal a significant aldo-sterone-mediated increase in the total amount of the cellularENaC α-subunit by even 91%. These data suggest thataldosterone primarily increases cellular ENaC, but inaddition, promotes the insertion of ENaC molecules in theplasma membrane. As already mentioned, the mineralocor-ticoid hormone aldosterone is known to be a major ENaCregulator activating the sodium channel at the genomic and/or the nongenomic level. Because spironolactone preventsthe response of HUVEC to aldosterone, we conclude thatENaC activation by aldosterone in the endothelium occursvia the genomic pathway which however does not excludea nongenomic early action of this steroid [4]. Recently,besides sgk1, another early aldosterone-induced gene wasidentified (Usp2-45) which deubiquitylates and thereforeactivates ENaC [12].

ENaC abundance is prevented when aldosterone receptorsare blocked

Recently, it was shown that endothelial cell volumeexpansion due to aldosterone could be prevented by thetreatment with intracellular aldosterone receptor antagonists[19, 33]. Because the expansion of cell volume is mostlikely due to apical sodium entry via ENaC, the effect of

spironolactone, a specific aldosterone receptor antagonist,on ENaC abundance was investigated. Detection of singleENaC molecules in the cell membrane by using QD-labeledantibodies revealed that spironolactone prevents the aldo-sterone-induced increase in the number of sodium channels.This strongly indicates that ENaC activation due toaldosterone action occurs at the genomic level and iscomparable to that in epithelial cells [2].

Amiloride application leads to the reduction in cellularENaC

The third finding of our study may open a new perspectivein terms of ENaC regulation. While we tested thefunctionality of ENaC with the channel blocker amiloride,we observed in the aldosterone-pretreated cells that thesodium channel abundance was reduced upon acuteamiloride application. Unexpectedly, both total cellularENaC and cell membrane surface ENaC were reduced by84 and 44%, respectively. This means that aldosterone-induced augmentation of ENaC protein expression can bereversed by amiloride. In flanking AFM experiments, weadditionally showed that the apical surface area of the cellsshrinks significantly by 45% when amiloride is applied.Quantitative differences between changes in membrane areaand ENaC expression could be due to further unknowneffects of amiloride. This means that amiloride couldnot only abolish the aldosterone effect but may also in-fluence any aldosterone-independent mechanisms of ENaCexpression.

It is known that amiloride binds ENaC reversibly in theouter pore and blocks the sodium channel [37, 25].However, until now, it has not been reported that thesodium channel blocker is able to reduce the quantity ofcellular ENaC. In previous AFM measurements, amiloride-

Fig. 4 AFM imaging of livingHUVEC. Cells were maintainedfor 72 h in aldosterone-containingmedium. The paired experiment,performed in buffered electrolytesolution at 37°C, shows individ-ual endothelial cells beforeamiloride application (left image)and 5 min after amiloride expo-sure (right image). Over thisshort period of time, cells de-crease in apical surface by 45%


induced cell shrinkage in aldosterone-pretreated cells wasdetected [32]. Obviously, sodium influx across the apicalplasma membrane is inhibited while sodium extrusioncontinues through the Na+/K+-ATPase. This volume changeis closely linked to a decrease in apical surface area asshown in the present study. It has been reported that ENaCheterologously expressed in oocytes is sensitive to osmoticpressure [24]. Cell shrinkage is most likely paralleled byendocytosis because cell volume and transmembranepressure are crucial regulators of endo- and exocytoticprocesses [5, 26, 27]. Furthermore, cell shrinkage couldserve as a signal for degradation or ubiquitination of ENaC.In this context, it was also reported that low doses ofamiloride effectively prevent cell swelling [32]. Ubiquiti-nation and degradation of ENaC probably occurs viaNedd4-2, a ubiquitin ligase, which binds to a PY motif inthe cytoplasmic tail of the channel [38, 39, 41].

In conclusion, ENaC is expressed in vascular endothe-lium and regulated by aldosterone. The steroid induces theexpression of ENaC and its insertion into the plasmamembrane. In contrast to the activating role of aldosterone,the sodium channel blocker amiloride downregulates theabundance of ENaC molecules in the cell. The observedeffects of aldosterone and amiloride apply only to the ENaCα-subunit. It remains open whether the β- and the +-subunits of the ENaC were also affected.

Our findings open a new perspective in the regulation ofENaC in the vascular endothelium: The functional blockadeof the sodium channel not only inhibits sodium entry intothe cell but also reduces its cellular availability. This noveleffect of ENaC inhibition by amiloride could have apotential impact for cardiovascular and renal pathologiesas pointed out recently [40].

Acknowledgment We are grateful to Prof. Dr. W.-M. Weber(Institute of Animal Physiology, University of Muenster, Germany)for support and discussion and to Dr. Peter Hanley for criticallyreading the manuscript. The project was supported by grants of theDeutsche Forschungsgemeinschaft (Re1284/2-1 and Ob 63/16-1) andEU grant Tips4cells.

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