Effect of altered Na entry on expression of apical and basolateral transport p - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:44 |只看该作者 |倒序浏览 |打印

作者：Jonathan Lebowitz, Bing An, Robert S. Edinger, Mark L. Zeidel, and John P. Johnson作者单位：Renal-Electrolyte Division, Department of Medicine, University ofPittsburgh, Pittsburgh, Pennsylvania 15261 & ^7 m/ [) }" a- C4 ?( d5 |


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      【摘要】7 S8 B0 @3 c! h& a
   In several in vivo settings, prolonged alterations in the rate of apicalNa entry into epithelial cells alter the ability of these cells toreabsorb Na  . We previously modeled this load dependence oftransport in A6 cells by either decreasing Na entry via apicalNa removal or amiloride or enhancing Na entry bychronic short-circuiting (Rokaw MD, Sarac E, Lechman E, West M, Angeski J,Johnson JP, and Zeidel ML. Am J Physiol Cell Physiol 270: C600-C607,1996). Inhibition of Na entry by either method was associated withstriking downregulation of transport rate as measured by short-circuit current( I sc ), which recovered to basal levels of transport over aperiod of hours. Conversely, upregulation of Na entry byshort-circuiting resulted in a sustained increase in transport rate that also returned to basal levels over a period of hours. The current studies wereundertaken to determine whether these conditions were associated withalterations in either the whole cell content or apical membrane distributionof sodium channel (ENaC) subunits or on basolateral expression of either ofthe subunits of the Na -K -ATPase. We compared theseeffects to those achieved by chronic upregulation of Na transportby aldosterone. Whole cell levels of ENaC subunits were measured by immunoblotfollowing 18-h inhibition of Na entry achieved by eithertetramethylammonium replacement of Na or apical amiloride or afteran 18-h increase in Na entry achieved by chronic short-circuiting.None of these maneuvers significantly altered the whole cell content of any ofthe ENaC subunits compared with control cells. We then examined the effects ofthese maneuvers on apical membrane ENaC expression using domain-specificbiotinylation and immunoblot. Inhibition of Na entry by eithermethod was associated with a profound decrease in apical membrane -ENaCwithout significant changes in apical membrane -or -ENaCamounts. Restoration of apical Na and/or removal of amilorideresulted in return of I sc to control levels over 2 h andcoincided with return of apical -ENaC to control levels without changein apical - or -ENaC. Stimulation of Na transport byshort-circuiting, in contrast, did not significantly alter apical membranecomposition of any of the ENaC subunits. Basolateral expression ofNa -K -ATPase was also measured by biotinylation andimmunoblot and was unchanged under all conditions. Aldosterone increasedbasolateral expression of the -subunit ofNa -K -ATPase. These results suggest that chronicdownregulation of transport is mediated, in part, by a selective decrease inapical membrane ENaC expression, consistent with our previous observations ofnoncoordinate regulation of ENaC expression under varying transport conditionsin A6 cells. The chronic increase in the rate of Na entry is not associated with any of the changes in transporter density at either apical orbasolateral membrane seen with aldosterone, suggesting that these twomechanisms of augmenting transport are completely distinct.
      【关键词】 sodium channel amiloride aldosterone Na K ATPase
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IT IS WELL KNOWN that changes in sodium delivery to the tubular epithelium result in changes in sodium handling along the nephron. Variousconditions decrease distal sodium delivery, including volume depletion, lossof upstream glomerular filtration, direct inhibition with diuretics, andobstructive uropathy. Because the sodium delivery is diminished, all theseconditions result in decreased tubular capacity to transport Na ( 8, 15, 26 ). In contrast, increaseddistal Na delivery as occurs in volume expansion and theadministration of loop diuretics lead to hypertrophy of distal convolutedtubule and collecting duct with increased transepithelial Na reabsorption in the distal nephron( 8, 26, 29 ). Additionally, acute orchronic alterations in transporter density at both apical and basolateralmembranes of renal epithelia have been reported with a variety of stimuli, including chronic Na loading, pressure natruiresis, andhypokalemia ( 8, 25, 38 ). We developed a cellculture model to define how chronic changes in the rate of apicalNa entry regulate transepithelial Na transport byinhibiting or stimulating Na entry for 18 h in confluent culturesof A6 cells, grown on filter-bottom supports( 31 ). Inhibition was performedby blockade of Na channels with amiloride or by mole-for-mole replacement of Na in the apical medium with tetramethylammonium (TMA). Chronic stimulation of the Na channel was accomplished by18 h of "flooding" the filter-bottom supports so that the apicalmedia came into direct contact with the basolateral media, effectivelyshort-circuiting the monolayers. With the use of this model, we showed thatchronic inhibition of Na entry by either mechanism decreases bothnet transport rate, measured as amiloride-sensitive short-circuit current( I sc ), and maximal transport capacity, estimated bynystatin-stimulated I sc. Conversely, chronic upregulationof Na entry enhanced both basal I sc andnystatin-stimulated currents. Similarly, ouabain binding was decreased whenapical Na entry was inhibited and ouabain binding was increasedwhen apical Na entry was stimulated. This implies that at leastpart of the effect of altered Na entry on transport rate is due tochanges in Na -K -ATPase activity.

Both up- and downregulation of transport induced by alterations inNa entry rate were time-dependent phenomena and required 18 h ofaltered Na entry. Short time courses were not associated withsustained changes in transport rate( 31 ). The effects were alsotime dependent in that the recovery to baseline transport took place over 1 toseveral hours. This observation suggested to us the possibility that chronicregulation of transport rate by the rate of Na entry would be dueto alteration in the density of Na transport proteins, eitherepithelial Na channel (ENaC) or Na -K -ATPase, in thewhole cell at the relevant cellular membrane. This possibility wasparticularly interesting as we recently examined the steady-state distributionand effects of a number of hormonal manipulations of transport rate on apical membrane expression of ENaC subunits in A6 cells( 37 ). Our observationssuggested that turnover of apical membrane subunits was somewhat variable,with -ENaC having a shorter half-life after reaching the apical membranethan either -or -ENaC. Interestingly, upregulation of transportby vasopressin or downregulation of transport by brefeldin A (BFA) wasassociated predominantly with changes in apical membrane -ENaC,suggesting the possibility of noncoordinate regulation of ENaC subunitexpression. Our model of chronic regulation of transport rate by rate ofNa entry provides us with another method of alteringNa transport, permitting us to examine the possibility ofnoncoordinate regulation of ENaC expression. Finally, it has been suggestedthat some of the effects of aldosterone might be mediated by chronic upregulation of Na entry. We wished to compare the effects onapical and basolateral transporter density of long-term aldosterone and ourmodel of continuous short-circuiting to determine whether there were directeffects of altered Na entry in the absence of mineralocorticoidhormones.
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METHODS9 _+ k' s" l4 X$ S( ]$ n& o
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Cell lines. A6 cells were maintained in amphibian mediumcontaining 10% FBS (Whittaker Bioproducts, Walkersville, MD) at 28°C inhumidified air with 5% CO 2. Cells were seeded at high density onpermeable supports (Nunc) and used at least 8 days postplating. To ensure thatthe cells were viable, we used an in-hood, short-circuiting device employingsterile KCL-agar salt bridges in apical and basolateral solutions to measure I sc as has been previously described( 18, 31, 37 ).+ X- T. ~, J6 J' i1 s5 g
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Model. A6 cells are seeded onto porous supports (Nunc). Under these conditions, they form a high-resistance, polarized epithelium thatconducts sodium in a vectorial fashion from the apical to the basolateralcompartment. The tonicity of the media is 240 mosmol/kgH 2 O of whichthe primary solute is Na  . As previously described( 31 ), sodium entry could bedecreased by replacing apical Na in the medium with equimolaramounts of TMA for an 18-h period. Additionally, Na entry wasblocked using 10 µM amiloride, a well-described inhibitor of ENaC.Alternatively, Na entry could be chronically stimulated byshort-circuiting, achieved by flooding the porous supports with media so that the apical and basolateral media were brought into contact. Contemporaneouscontrols from the same plating were treated identically except that thecomposition of the apical medium was not altered." Q, g' d" U; q( A4 e, i

Antibodies and reagents. - And -ENaC antibodies weregenerated and affinity purified as described( 37 ). Anti- -ENaCantibodies were obtained from Thomas Kleyman and were described andcharacterized by Zuckerman et al.( 39 ). Antibodies to -and -subunits of Na -K -ATPase were obtained fromUpstate Biotechnology (Lake Placid, NY). All other reagents were purchasedfrom Sigma unless otherwise noted.

Quantification of whole cell ENaC. A6 cells were grown on six-well filter inserts and subjected to control, TMA, amiloride, short-circuit, oraldosterone conditions for 18 h. Cells were washed with ice-cold PBS x 5to remove FBS and harvested by scraping. Cells were sonicated at 7.5 for 7 s x 2 and protein assays were performed. For whole cell measurements, 50µg of cell lysate were placed in sample buffer( 37 ), and proteins separatedon SDS-PAGE gels and Western blots were performed with the antibodies to ENaCsubunits. Control and experimental samples for each observation were runtogether, transferred to nitrocellulose, and visualized by enhancedchemiluminescence as previously described( 37 ). Samples were analyzedtogether to control for both sample loading and exposure time. As wepreviously described, the anti- -ENaC antibody recognizes a doublet of150, 180 kDa in Western blots from A6 cells, with typically more of the180-kDa form in apical membrane-biotinylated samples. This antibodyspecifically recognizes -ENaC expressed in vitro( 39 ) or in HeLa cells at 75kDa ( 37 ) and recognizes newlysynthesized -ENaC by immunoprecipitation from radiolabeled cells alsoat 75 kDa in A6 cells ( 37 ).Western blot analysis of cell lysates from A6 consistently give the highermolecular weight bands described above, which are competed away bypreincubation with immunizing peptides( 21, 37, 39 ). We feel that this highermolecular weight protein represents the fully mature form of -ENaCexpressed at the apical membrane in A6( 21, 37, 39 ). Identicalhigh-molecular-weight bands are seen in A6 lysate using an antibody raisedagainst a COOH-terminal epitope of -ENaC( 39 ) and using the -rENaC antibody generated by Knepper and colleagues( 24 ) and kindly provided bythose investigators (not shown). Other investigators described -ENaC asmigrating at different molecular weights in A6 cells. With the use ofantibodies prepared against different epitopes of the protein, Alverez De LaRosa et al. ( 1 ) described -ENaC as two bands appearing at 85 and 65 kDa, and Stockand et al. ( 34 ) described a proteinmigrating at 85-90 kDa. The antibodies to - and -ENaC weregenerated in our laboratory and recognize bands at 97 kDa in A6 cells( 37 ). These bands werequantitated by densitometry, and all results are expressed as a percentage ofthe mean values of simultaneously measured control levels.
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Quantification of apical ENaC and basolateralNa -K -ATPase. A6 cells weregrown on six-well filter inserts and subjected to control, TMA, amiloride,short-circuit, or aldosterone conditions for 18 h. Cells were washed withice-cold PBS with agitation at 28°C x 5 to remove FBS-containingmedium. The cells were then biotinylated in borate buffer on the apicalsurface (for quantification of membrane-bound ENaC subunits) or on thebasolateral surface (for quantification of membrane-boundNa -K -ATPase). The nonbiotinylated side of themonolayer was bathed in medium containing FBS to prevent biotinylation. After20 min, basolateral and apical sides were aspirated and FBS-containing mediumwas placed on the cells to quench the signal. Monolayers were then washed x 5 with ice-cold PBS with agitation at 28°C, and the cells wereharvested. Cell homogenate was obtained by sonication at 7.5 for 7 s x 2in a cooling block and then centrifuged on a tabletop device x 5 min at5,000 rpm. Cell homogenate was then assayed for protein, and 300 µg wereplaced on 150 µl of avidin beads( 37 ). Samples from avidinbeads were collected in 2 x sample buffer and heated to 100°C for 8 min to ensure complete collection. Proteins were separated by SDS-PAGE asdescribed ( 37 ), and sampleswere transferred to nitrocellulose membranes and subjected to Western blotanalysis with the appropriate antibodies and visualized with enhanced chemiluminescence (PerkinElmer Life Science). Simultaneous controls (untreatedcells) were always separated on the same gels with experimental samples.Multiple time exposures were carried out for each blot to ensure that signalswere quantified when they were increasing in the linear range. Antibodies tothe three ENaC subunits were visualized at molecular weights described above.The -subunit of the Na -K -ATPase was visualizedat 110 kDa and the -subunit at 50 kDa. The results werequantified by densitometry.5 E) G# M# H7 M' y  |3 \

RESULTS0 A7 n7 ~5 }0 h( Y" U

We first examined the effect of chronic regulation of apical Na entry on I sc and apical membrane expression of the three ENaC subunits. As shown in Fig.1, chronic alterations in the rate of Na entry alteredtransport rates similar to our previous description( 31 ). Chronic short-circuitingresulted in a significant increase in transport rate compared withsimultaneous controls, whereas chronic inhibition of Na entry byeither substitution of apical Na by TMA or by chronic exposure toapical amiloride significantly inhibited transport rate when A6 cells wereinitially reexposed to apical Na  . Downregulation of Na by either 18-h replacement of apical Na with TMA or 18 h in 10µM amiloride followed by replacement with normal apical media wasassociated with a marked change in the apical expression of ENaC. As shown in Fig. 2, replacement of apicalNa with TMA resulted in no change in the surface expression ofeither - or -ENaC but caused a large and significant decreaseselectively in apical membrane -ENaC expression. Treatment of A6 cellsfor 18 h with 10 µM amiloride ( Fig.3 ) also caused a significant decrease in apical -ENaCexpression compared with control cells. In this case, there was also somedecrease in the apical expression of - and -subunits, althoughthese decreases did not reach statistical significance. Both downregulationconditions therefore were associated with selective decreases in apicalmembrane -ENaC expression, similar to the effect previously noted by uswhen the transport rate was downregulated by BFA( 37 ).

Fig. 1. Effect of altered conditions of apical Na entry onshort-circuit current ( I sc ) in A6 epithelia. A6 cellsgrown on filter supports were either short-circuited (SC) for 18 h by floodingfilters so that apical and basolateral media were in contact, exposed to 10µM amiloride on the apical surface for 18 h, or had total replacement ofapical Na by tetramethylammonium (TMA) for 18 h. After eachtreatment, cells were washed 3 times in regular media, I sc was measured within 5-10 min and compared with simultaneous controls. Allconditions are significantly different from control, P 2 s5 D0 E( T* F! w+ |

Fig. 2. Effect of inhibition of apical Na entry by TMA substitution onapical membrane expression of epithelial Na channel (ENaC) subunits.Na entry was blocked by 18-h substitution of TMA forNa  . After 18 h under these conditions, cells were returned tocontrol conditions and apical membrane proteins were isolated bydomain-specific biotinylation as described. Results are the mean of 6observations each. TMA resulted in a significant decrease in apical membrane -ENaC alone. Results are expressed as percentage of control mean ENaCdensitometry. Top : representative immunoblots for each subunit.Molecular weights are given in METHODS. * Significantlydifferent from control, P 7 T" x/ s) x- ~* t6 {

Fig. 3. Effect of inhibition of apical Na entry by amiloride on apicalmembrane expression of ENaC subunits. Na entry was blocked by 18-hexposure to 10 µM amiloride. Cells were returned to control conditions andapical membrane proteins were isolated by domain-specific biotinylation asdescribed. Results are the mean of 7 observations each. Amiloride resulted ina significant decrease in apical membrane -ENaC alone compared withsimultaneous controls. Results are expressed as percentage of control ENaCsubunit densitometry. Top : representative immunoblots for eachcondition. * Significantly different from control, P
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If the decrease in apical -ENaC was of significance in the decline in I sc, then a minimum expectation would be that a return of I sc to control levels following removal of amiloride orrestoration of apical Na would be accompanied by a return ofapical -ENaC amounts to control levels. We therefore examined the effect of recovery from inhibition of apical Na entry on the apical expression of -, -, and -ENaC. Cells were exposed to 18-hdownregulation of Na entry by either apical amiloride orreplacement of apical Na as described above. After restoration ofnormal apical media, I sc was monitored for return of ENaCfunction compared with control, untreated monolayers. The transport raterecovered 70% in 1 h and 100% in 2 h, consistent with our initial observations using this model ( 31 ). At thetime of full recovery, control and both amiloride/recovery and TMA/recoverysamples were subjected to apical membrane biotinylation as described tomeasure apical expression of each ENaC subunit. As shown in Fig. 4, recovery of I sc to control levels following removal of inhibition of apical Na entry was associated with return of apical membrane -ENaC to control levels.. M1 Y: Z/ N  K, R

Fig. 4. Effect of recovery from inhibition of apical Na entry on apicalmembrane expression of ENaC subunits. Na entry was blocked by 18-hexposure to 10 µM amiloride or by replacement of TMA for Na inapical media. After 18 h, filters were washed and normal apical Na was restored. I sc was measured and returned to controllevels 2 h following restoration of apical Na  . At that point,apical membrane proteins were isolated by biotinylation from both recoverysamples and simultaneous untreated, control A6 cells. Results are the means of4 observations each and are expressed as percentage of control mean ENaCsubunit densitometry.

In contrast to the remarkable effects of downregulation of transport onapical membrane ENaC expression, upregulation of transport by 18 h ofshort-circuiting had no substantial effect on the apical membrane expressionof any of the three ENaC subunits ( Fig.5 ). There was a slight tendency toward an increase in apical -ENaC expression, but this did not reach significance.

Fig. 5. Effect of upregulation of apical Na entry by chronicshort-circuiting on apical membrane expression of ENaC subunits. A6 cellsgrown on filter supports were chronically short-circuited as described in METHODS. After 18 h, cells were returned to control conditions andapical membrane proteins were isolated by biotinylation. Results are a mean of5 observations each. There are no differences between control and SCmeasurements. Top : representative immunoblots for each condition.! [# {! I/ I2 M/ Q0 X' C
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Because apical membrane ENaC expression had been altered by chronicdownregulation of Na transport, we next sought to determinewhether this was a reflection of altered whole cell amounts of ENaC subunitsor alternatively whether it might represent a redistribution of ENaC subunitsin response to the altered Na entry. Whole cell ENaC subunitdensities under the two conditions are shown in Fig. 6. Replacement of apical Na with TMA selectively reduced apical membrane -ENaC but only reduced -ENaC in whole cells. Blockade of apical Na entry with 18 h of 10 µM amiloride significantly and markedly reducedapical membrane-associated -ENaC with slight but not significant changesin the other subunits but had no significant effect on the whole cell amountsof either - or -ENaC.6 {  b' c+ w- O6 X/ I
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Fig. 6. Effect of chronic downregulation of apical Na on whole celllevels of ENaC subunits. A6 cells underwent either 18-h substitution of TMAfor apical Na or 18 h in 10 µM amiloride. Cells were then lysedand proteins were separated by SDS-PAGE as described. Lysates were probed withsubunit-specific antibodies and results were quantitated by densitometry.Treatment with amiloride had no effect on subunit abundance. TMA replacementresulted in a significant decrease in -ENaC expression alone. Top : representative immunoblots; n = 5-6. * Significantly different from control, P # b( ^( s! y- [, v; P
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Because alterations in the rate of Na entry were associated with significant changes in the activity ofNa -K -ATPase activity that persisted after return tonormal growth conditions in our initial study( 31 ), we also examined theeffect of these maneuvers on the basolateral membrane expression of -and -subunits of the ATPase. As shown in Fig. 7, neither chronicupregulation of transport rate by short-circuiting nor chronic downregulation of transport by TMA substitution for apical Na had any effect onthe amount of either - or -subunit of theNa -K -ATPase present in the basolateral membrane.8 M6 ^6 Q1 r( g+ u( v. @' r

Fig. 7. Effect of 18-h altering apical Na entry and aldosterone onbasolateral membrane expression of Na -K -ATPasesubunits. Cells were treated with aldosterone (1 µM) or underwent chronicSC or replacement of apical Na with TMA for 18 h, and basolateralmembrane proteins were isolated by domain-specific biotinylation and probedfor individual subunits of the ATPase. Aldosterone induced a significantincrease in -subunit of the ATPase. No other changes are significant. Top : representative immunoblots. Molecular weights of the subunitsare given in METHODS; n = 5.

Although we were unable to demonstrate any effect of chronic upregulationof Na entry on the expression of apical or basolateral transportproteins, we previously demonstrated that long-term (18 h) but not short-term(3 h) exposure of A6 cells to 10 - 6 M aldosterone resultsin a selective increase in apical membrane expression of -ENaC( 37 ). This occurred without aneffect on the apical membrane expression of either - or -ENaC,the mirror image of what we described with downregulation of Na entry by TMA substitution. A similar change in whole cell amounts -ENaCexpression without a change in - or -ENaC has been described byStockand and colleagues ( 34 )following long-term aldosterone exposure. Although we were unable todemonstrate changes in basolateral Na -K -ATPase amountsfollowing alterations in Na entry, the effects of aldosterone onboth transcription and translation of both subunits of this enzyme are welldescribed ( 2, 36 ). Additionally, wepreviously demonstrated upregulation of enzyme activity in A6 cells, which isunaffected by blockade of Na entry with amiloride( 18 ). Figure 7 demonstrates the effect of 18-h aldosterone treatment on the basolateral membrane amounts of - and -subunits of Na -K -ATPase.Aldosterone significantly upregulates the amount of the -subunit inbasolateral membrane without affecting basolateral amount of -subunit.* k- S: k  _4 B' q: Z  H0 ^& R

DISCUSSION* y$ ~  {5 G) ^+ g0 b7 U
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It has been shown in a number of mammalian models that chronic alterationsin delivery or chronic blockade of specific Na transporters mayinduce adaptive changes in transport rates that appear to be dependent on load( 8, 15, 27, 29, 38 ). In in vitro systems,acute stimulation of Na entry results in reduction of ENaCactivity through a process of feedback inhibition( 16, 19, 30 ), whereas acute inhibitionof Na entry is associated with increased ENaC activity( 11, 30 ). Because chronic increasesin Na load or delivery in animals seem to be associated withtransporter upregulation ( 8, 27 ), whereas chronicinhibition of transport seems to be associated with downregulation oftransporters ( 15 ), we examinedthe long-term effects of these maneuvers in vitro. We established a model ofchronic regulation of Na transport by the rate of apicalNa entry in the cultured A6 cell line, where Na transport is primarily mediated by apical membrane ENaC and the basolateralNa -K -ATPase( 31 ). In this model, chronic upregulation of Na entry by continuous short-circuiting of theepithelia for 18 h resulted in upregulation of transport rate when normalpolarity was restored. Transport rates declined toward basal levels over aperiod of hours. In contrast, chronic downregulation of apical Na entry by either removal of apical Na or ENaC blockade withamiloride for 18 h resulted in a decreased rate of transport. When normalapical Na entry was restored, I sc returned tocontrol levels within 2 h. In contrast to our results, studies in primarycultures derived from the rabbit kidney collecting system showed little effectof chronic short-circuiting and chronic inhibition of Na entrywith benzamil-enhanced transport rates( 6 ). In these experiments,chronic short-circuiting, which had little effect on either basal I sc or -ENaC levels, blocked the effect ofaldosterone and this inhibition was overcome by incubation with benzamil( 6 ). Noting the differencebetween their results and our initial studies( 31 ), the authors speculated that intracellular Na concentration or an alteration in rates ofNa entry might have dual effects, depending on the absence orpresence of aldosterone. The current experiments were designed to reevaluateour model of chronic regulation and test the possibility that chronicalterations in Na entry altered channel subunit expression orbasolateral enzyme expression under conditions of chronic up- ordownregulation. Additionally, we sought to compare the effects of chronicupregulation by increased Na entry to those seen with chronicupregulation of transport by aldosterone.

Our results confirmed some but not all of our expectations. Chronicdownregulation of Na entry by either mechanism was associated withdecreased expression of ENaC subunits at the apical membrane withoutsignificant changes in whole cell subunit content. Of particular interest,only one subunit, -ENaC, was decreased in apical expression. Thisobservation is similar to what we described with downregulation of transportby the agent BFA ( 37 ). BFAdisrupts delivery of proteins to post-Golgi targets and markedly reduced I sc in A6 cells but resulted in a decrease in apicalexpression only of -ENaC with no significant change in - or -expression. This occurred over a period of 1- to 3-h incubation withBFA, similar to the time course of BFA inhibition of apical membrane channelactivity measured by noise analysis( 9 ). This surprising result wasexplained to some degree when we examined the half-life of ENaC subunits thatreach the apical membrane in A6( 37 ). -ENaC turns over with a half-life of several hours, whereas - and -ENaC that reach the apical membrane appear to be considerably more long lived( 21, 37 ). We referred to thisphenomenon of apparent differential turnover of apical membrane ENaC subunitsas noncoordinate regulation. It appears that downregulation of Na entry in A6 cells may be another example of this." g) z9 b/ W$ a: e
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Because there is no reason to believe that physiologically significant ENaCfunction is mediated by anything other than fully formed heterotrimericchannels, the observation of noncoordinate regulation implies that channelsmay be assembling or disassembling at some point beyond the endoplasmicreticulum (ER). As Rotin and colleagues( 32 ) recently pointed out,there is no evidence to support such a concept that emerges from the manystudies of ENaC assembly or function in oocytes or heterologous expression systems, which clearly demonstrate that ENaC assembles into completetetrameric channels in ER soon after biosynthesis as do most multimericproteins. It is interesting to note, however, that multimeric proteins may beprocessed differently in endogenously expressing cells than in overexpressingcells. The T cell antigen receptor complex (TCR-CD3) of T cell hybridomas hasserved as an established model of ER assembly of a multimeric membraneprotein, but when examined in normal T cells, the putative limiting -subunit appears to exhibit more rapid turnover from membrane-boundcomplexes than do the other subunits( 28 ). Similarly, the threesubunits of the interleukin-2 receptor in T lymphocytes appear to have varyingsurface half-lives and endocytic fates( 14 ). In cells or tissuesendogenously expressing ENaC, there are multiple examples of apparentnoncoordinate regulation of individual subunits. In rat distal nephron,aldosterone induces expression of -ENaC with an apparent shift ofcytosolic - and -subunits to the apical membrane with a shortcourse in early distal nephron and a more prolonged course in collecting duct( 10, 23, 24 ). Physiologicalmanipulations to rats have been described that result in either selectiveregulation of - and -ENaC( 7, 13, 20 ) or of alone( 3 ). Selective upregulation of -and -ENaC has also been described to alter the biophysical properties of ENaC expressed in alveolar cells( 22 ), and long-term PKCstimulation inhibits the transport rate in A6 epithelia in association with aselective decrease in - and -ENaC without changes in -ENaC( 34 ). Overexpression of -ENaC in A6 cells results in enhanced transport rates( 4 ), but surface subunit amounts were not measured in this study. The precise mechanism by whichnoncoordinate regulation of ENaC subunit expression results in changes in theapical density of fully active channels is unclear, but in each case,regulation of the transport rate appears to correspond with altered expressionof some, but not all, ENaC subunits( 3, 7, 13, 20, 22 - 24, 34, 37 ). A recent report byAlvarez De La Rosa et al. ( 1 ),which describes fully coordinate expression and turnover of cellular andapical membrane ENaC subunits in response to aldosterone in A6 cells, clearlycontradicts the notion of noncoordinate regulation. The reasons for thedifferences in results between this study and our own previous findings aswell as those of other groups ( 10, 29, 32 ) are not clear, becausemany of the same methods were employed. Alvarez De La Rosa et al.( 1 ) describe an extremely shorthalf-life (12-17 min) for ENaC subunits that reach apical membrane, a periodin marked contrast to the longer half-life we( 37 ) and others( 21 ) described in A6 cells anddifficult to reconcile with the time course of BFA inhibition of ENaC surfacedensity measured by noise analysis( 9 ).

In contrast to our expected finding of ENaC downregulation by the rate ofNa entry, we could find no evidence of a change in apical membraneENaC expression with chronic short-circuiting and no evidence of a change inbasolateral Na -K -ATPase by either chronic adaptation.Clearly, altered enzyme activity in chronic adaptation is not a function of agreater or lesser number of pumps expressed in the membrane. Although we could measure no direct effects of upregulation of Na entry byshort-circuiting on apical or basolateral transporter density, chronicupregulation of transport by aldosterone clearly affected both. Once again, the characteristic of this regulation was noncoordinate both with respect toENaC and ATPase. Selective upregulation of apical -ENaC by aldosteronein A6 has previously been noted ( 37 ), and data from Stockandet al. ( 34 ) suggest this isdue to increased expression of this subunit at the whole cell level. Bothtranscriptional and translational regulation of both subunits ofNa -K -ATPase by aldosterone have been well documented previously ( 2, 17, 29, 36 ). A recent observation incultured cortical collecting duct cells demonstrates a roughly similar increase in basolateral -subunit to that which we describe here, but nodata on -subunit expression at basolateral membrane are available( 35 ). Previous studies suggestthat the two subunits may have slightly different time courses of membrane insertion ( 5, 12 ), and relative differencesin fold-changes in the two subunits expression following regulatory stimuli have been described ( 25, 38 ). These observationssuggest that the enzyme may not traffic exclusively as -heterodimers. Moreover, it has been shown that increases in asingle subunit of the Na -K -ATPase not only occur buthave also been implicated in increasing pump activity( 33 ). Our observations wouldsuggest that enzyme function may be increased by alteration in basolateral expression of -subunit alone, which implies that there may be an excessof -subunit already present in or near the membrane. Whatever themechanism, aldosterone clearly induces alterations in apical ENaC andbasolateral Na -K -ATPase, which are not seen withchronic upregulation of Na entry alone. Our data are in agreementwith the conclusion by Summa and colleagues ( 35 ) that the effect ofaldosterone on basolateral transport proteins is not mediated by apicalNa entry.

DISCLOSURES4 I6 q+ v' V/ }/ c1 G% N8 T
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This work was supported by National Institute of Diabetes and Digestive andKidney Diseases Grants 57718 (to J. P. Johnson) and 43955 and 48217 (to M. L.Zeidel) and by funds from Dialysis Clinics.
      【参考文献】
Alvarez De La Rosa D, Hui L, and Canessa CM. Effects of aldosteroneon biosynthesis, traffic and functional expression of epithelial sodiumchannels in A6 cells. J Gen Physiol 119: 427-442,2002.
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