Effect of altered Na+ entry on expression of apical and basolateral transport proteins in A6 epithelia
Jonathan Lebowitz,
Bing An,
Robert S. Edinger,
Mark L. Zeidel, and
John P. Johnson
Renal-Electrolyte Division, Department of Medicine, University of
Pittsburgh, Pittsburgh, Pennsylvania 15261
Submitted 14 December 2001
; accepted in final form 1 May 2003
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ABSTRACT
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In several in vivo settings, prolonged alterations in the rate of apical
Na+ entry into epithelial cells alter the ability of these cells to
reabsorb Na+. We previously modeled this load dependence of
transport in A6 cells by either decreasing Na+ entry via apical
Na+ removal or amiloride or enhancing Na+ entry by
chronic 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 with
striking downregulation of transport rate as measured by short-circuit current
(Isc), which recovered to basal levels of transport over a
period of hours. Conversely, upregulation of Na+ entry by
short-circuiting resulted in a sustained increase in transport rate that also
returned to basal levels over a period of hours. The current studies were
undertaken to determine whether these conditions were associated with
alterations in either the whole cell content or apical membrane distribution
of sodium channel (ENaC) subunits or on basolateral expression of either of
the subunits of the Na+-K+-ATPase. We compared these
effects to those achieved by chronic upregulation of Na+ transport
by aldosterone. Whole cell levels of ENaC subunits were measured by immunoblot
following 18-h inhibition of Na+ entry achieved by either
tetramethylammonium replacement of Na+ or apical amiloride or after
an 18-h increase in Na+ entry achieved by chronic short-circuiting.
None of these maneuvers significantly altered the whole cell content of any of
the ENaC subunits compared with control cells. We then examined the effects of
these maneuvers on apical membrane ENaC expression using domain-specific
biotinylation and immunoblot. Inhibition of Na+ entry by either
method was associated with a profound decrease in apical membrane
-ENaC
without significant changes in apical membrane
-or
-ENaC
amounts. Restoration of apical Na+ and/or removal of amiloride
resulted in return of Isc to control levels over 2 h and
coincided with return of apical
-ENaC to control levels without change
in apical
- or
-ENaC. Stimulation of Na+ transport by
short-circuiting, in contrast, did not significantly alter apical membrane
composition of any of the ENaC subunits. Basolateral expression of
Na+-K+-ATPase was also measured by biotinylation and
immunoblot and was unchanged under all conditions. Aldosterone increased
basolateral expression of the
-subunit of
Na+-K+-ATPase. These results suggest that chronic
downregulation of transport is mediated, in part, by a selective decrease in
apical membrane ENaC expression, consistent with our previous observations of
noncoordinate regulation of ENaC expression under varying transport conditions
in 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 or
basolateral membrane seen with aldosterone, suggesting that these two
mechanisms of augmenting transport are completely distinct.
sodium channel; amiloride; aldosterone; Na+-K+-ATPase
IT IS WELL KNOWN that changes in sodium delivery to the tubular
epithelium result in changes in sodium handling along the nephron. Various
conditions decrease distal sodium delivery, including volume depletion, loss
of upstream glomerular filtration, direct inhibition with diuretics, and
obstructive uropathy. Because the sodium delivery is diminished, all these
conditions result in decreased tubular capacity to transport Na+
(8,
15,
26). In contrast, increased
distal Na+ delivery as occurs in volume expansion and the
administration of loop diuretics lead to hypertrophy of distal convoluted
tubule and collecting duct with increased transepithelial Na+
reabsorption in the distal nephron
(8,
26,
29). Additionally, acute or
chronic alterations in transporter density at both apical and basolateral
membranes of renal epithelia have been reported with a variety of stimuli,
including chronic Na+ loading, pressure natruiresis, and
hypokalemia (8,
25,
38). We developed a cell
culture model to define how chronic changes in the rate of apical
Na+ entry regulate transepithelial Na+ transport by
inhibiting or stimulating Na+ entry for 18 h in confluent cultures
of A6 cells, grown on filter-bottom supports
(31). Inhibition was performed
by 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 by
18 h of "flooding" the filter-bottom supports so that the apical
media came into direct contact with the basolateral media, effectively
short-circuiting the monolayers. With the use of this model, we showed that
chronic inhibition of Na+ entry by either mechanism decreases both
net transport rate, measured as amiloride-sensitive short-circuit current
(Isc), and maximal transport capacity, estimated by
nystatin-stimulated Isc. Conversely, chronic upregulation
of Na+ entry enhanced both basal Isc and
nystatin-stimulated currents. Similarly, ouabain binding was decreased when
apical Na+ entry was inhibited and ouabain binding was increased
when apical Na+ entry was stimulated. This implies that at least
part of the effect of altered Na+ entry on transport rate is due to
changes in Na+-K+-ATPase activity.
Both up- and downregulation of transport induced by alterations in
Na+ entry rate were time-dependent phenomena and required 18 h of
altered Na+ entry. Short time courses were not associated with
sustained changes in transport rate
(31). The effects were also
time dependent in that the recovery to baseline transport took place over 1 to
several hours. This observation suggested to us the possibility that chronic
regulation of transport rate by the rate of Na+ entry would be due
to alteration in the density of Na+ transport proteins, either
epithelial Na channel (ENaC) or Na+-K+-ATPase, in the
whole cell at the relevant cellular membrane. This possibility was
particularly interesting as we recently examined the steady-state distribution
and effects of a number of hormonal manipulations of transport rate on apical
membrane expression of ENaC subunits in A6 cells
(37). Our observations
suggested that turnover of apical membrane subunits was somewhat variable,
with
-ENaC having a shorter half-life after reaching the apical membrane
than either
-or
-ENaC. Interestingly, upregulation of transport
by vasopressin or downregulation of transport by brefeldin A (BFA) was
associated predominantly with changes in apical membrane
-ENaC,
suggesting the possibility of noncoordinate regulation of ENaC subunit
expression. Our model of chronic regulation of transport rate by rate of
Na+ entry provides us with another method of altering
Na+ transport, permitting us to examine the possibility of
noncoordinate regulation of ENaC expression. Finally, it has been suggested
that some of the effects of aldosterone might be mediated by chronic
upregulation of Na+ entry. We wished to compare the effects on
apical and basolateral transporter density of long-term aldosterone and our
model of continuous short-circuiting to determine whether there were direct
effects of altered Na+ entry in the absence of mineralocorticoid
hormones.
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METHODS
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Cell lines. A6 cells were maintained in amphibian medium
containing 10% FBS (Whittaker Bioproducts, Walkersville, MD) at 28°C in
humidified air with 5% CO2. Cells were seeded at high density on
permeable supports (Nunc) and used at least 8 days postplating. To ensure that
the cells were viable, we used an in-hood, short-circuiting device employing
sterile KCL-agar salt bridges in apical and basolateral solutions to measure
Isc as has been previously described
(18,
31,
37).
Model. A6 cells are seeded onto porous supports (Nunc). Under
these conditions, they form a high-resistance, polarized epithelium that
conducts sodium in a vectorial fashion from the apical to the basolateral
compartment. The tonicity of the media is 240 mosmol/kgH2O of which
the primary solute is Na+. As previously described
(31), sodium entry could be
decreased by replacing apical Na+ in the medium with equimolar
amounts of TMA for an 18-h period. Additionally, Na+ entry was
blocked using 10 µM amiloride, a well-described inhibitor of ENaC.
Alternatively, Na+ entry could be chronically stimulated by
short-circuiting, achieved by flooding the porous supports with media so that
the apical and basolateral media were brought into contact. Contemporaneous
controls from the same plating were treated identically except that the
composition of the apical medium was not altered.
Antibodies and reagents.
- And
-ENaC antibodies were
generated and affinity purified as described
(37). Anti-
-ENaC
antibodies were obtained from Thomas Kleyman and were described and
characterized by Zuckerman et al.
(39). Antibodies to
-
and
-subunits of Na+-K+-ATPase were obtained from
Upstate Biotechnology (Lake Placid, NY). All other reagents were purchased
from 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, or
aldosterone conditions for 18 h. Cells were washed with ice-cold PBS x5
to remove FBS and harvested by scraping. Cells were sonicated at 7.5 for 7 s
x2 and protein assays were performed. For whole cell measurements, 50
µg of cell lysate were placed in sample buffer
(37), and proteins separated
on SDS-PAGE gels and Western blots were performed with the antibodies to ENaC
subunits. Control and experimental samples for each observation were run
together, transferred to nitrocellulose, and visualized by enhanced
chemiluminescence as previously described
(37). Samples were analyzed
together to control for both sample loading and exposure time. As we
previously described, the anti-
-ENaC antibody recognizes a doublet of
150, 180 kDa in Western blots from A6 cells, with typically more of the
180-kDa form in apical membrane-biotinylated samples. This antibody
specifically recognizes
-ENaC expressed in vitro
(39) or in HeLa cells at 75
kDa (37) and recognizes newly
synthesized
-ENaC by immunoprecipitation from radiolabeled cells also
at 75 kDa in A6 cells (37).
Western blot analysis of cell lysates from A6 consistently give the higher
molecular weight bands described above, which are competed away by
preincubation with immunizing peptides
(21,
37,
39). We feel that this higher
molecular weight protein represents the fully mature form of
-ENaC
expressed at the apical membrane in A6
(21,
37,
39). Identical
high-molecular-weight bands are seen in A6 lysate using an antibody raised
against a COOH-terminal epitope of
-ENaC
(39) and using the
-rENaC antibody generated by Knepper and colleagues
(24) and kindly provided by
those investigators (not shown). Other investigators described
-ENaC as
migrating at different molecular weights in A6 cells. With the use of
antibodies prepared against different epitopes of the protein, Alverez De La
Rosa et al. (1) described
-ENaC as two bands appearing at 85 and 65 kDa, and Stockand et al.
(34) described a protein
migrating at 85-90 kDa. The antibodies to
- and
-ENaC were
generated in our laboratory and recognize bands at 97 kDa in A6 cells
(37). These bands were
quantitated by densitometry, and all results are expressed as a percentage of
the mean values of simultaneously measured control levels.
Quantification of apical ENaC and basolateral
Na+-K+-ATPase. A6 cells were
grown on six-well filter inserts and subjected to control, TMA, amiloride,
short-circuit, or aldosterone conditions for 18 h. Cells were washed with
ice-cold PBS with agitation at 28°C x5 to remove FBS-containing
medium. The cells were then biotinylated in borate buffer on the apical
surface (for quantification of membrane-bound ENaC subunits) or on the
basolateral surface (for quantification of membrane-bound
Na+-K+-ATPase). The nonbiotinylated side of the
monolayer was bathed in medium containing FBS to prevent biotinylation. After
20 min, basolateral and apical sides were aspirated and FBS-containing medium
was placed on the cells to quench the signal. Monolayers were then washed
x5 with ice-cold PBS with agitation at 28°C, and the cells were
harvested. Cell homogenate was obtained by sonication at 7.5 for 7 s x2
in a cooling block and then centrifuged on a tabletop device x5 min at
5,000 rpm. Cell homogenate was then assayed for protein, and 300 µg were
placed on 150 µl of avidin beads
(37). Samples from avidin
beads were collected in 2x sample buffer and heated to 100°C for 8
min to ensure complete collection. Proteins were separated by SDS-PAGE as
described (37), and samples
were transferred to nitrocellulose membranes and subjected to Western blot
analysis with the appropriate antibodies and visualized with enhanced
chemiluminescence (PerkinElmer Life Science). Simultaneous controls (untreated
cells) were always separated on the same gels with experimental samples.
Multiple time exposures were carried out for each blot to ensure that signals
were quantified when they were increasing in the linear range. Antibodies to
the three ENaC subunits were visualized at molecular weights described above.
The
-subunit of the Na+-K+-ATPase was visualized
at
110 kDa and the
-subunit at
50 kDa. The results were
quantified by densitometry.
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RESULTS
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We first examined the effect of chronic regulation of apical Na+
entry on Isc and apical membrane expression of the three
ENaC subunits. As shown in Fig.
1, chronic alterations in the rate of Na+ entry altered
transport rates similar to our previous description
(31). Chronic short-circuiting
resulted in a significant increase in transport rate compared with
simultaneous controls, whereas chronic inhibition of Na+ entry by
either substitution of apical Na+ by TMA or by chronic exposure to
apical amiloride significantly inhibited transport rate when A6 cells were
initially reexposed to apical Na+. Downregulation of Na+
by either 18-h replacement of apical Na+ with TMA or 18 h in 10
µM amiloride followed by replacement with normal apical media was
associated with a marked change in the apical expression of ENaC. As shown in
Fig. 2, replacement of apical
Na+ with TMA resulted in no change in the surface expression of
either
- or
-ENaC but caused a large and significant decrease
selectively in apical membrane
-ENaC expression. Treatment of A6 cells
for 18 h with 10 µM amiloride (Fig.
3) also caused a significant decrease in apical
-ENaC
expression compared with control cells. In this case, there was also some
decrease in the apical expression of
- and
-subunits, although
these decreases did not reach statistical significance. Both downregulation
conditions therefore were associated with selective decreases in apical
membrane
-ENaC expression, similar to the effect previously noted by us
when the transport rate was downregulated by BFA
(37).

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Fig. 1. Effect of altered conditions of apical Na+ entry on
short-circuit current (Isc) in A6 epithelia. A6 cells
grown on filter supports were either short-circuited (SC) for 18 h by flooding
filters so that apical and basolateral media were in contact, exposed to 10
µM amiloride on the apical surface for 18 h, or had total replacement of
apical Na+ by tetramethylammonium (TMA) for 18 h. After each
treatment, cells were washed 3 times in regular media, Isc
was measured within 5-10 min and compared with simultaneous controls. All
conditions are significantly different from control, P < 0.01;
n = 8-12.
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Fig. 2. Effect of inhibition of apical Na+ entry by TMA substitution on
apical membrane expression of epithelial Na channel (ENaC) subunits.
Na+ entry was blocked by 18-h substitution of TMA for
Na+. After 18 h under these conditions, cells were returned to
control conditions and apical membrane proteins were isolated by
domain-specific biotinylation as described. Results are the mean of 6
observations each. TMA resulted in a significant decrease in apical membrane
-ENaC alone. Results are expressed as percentage of control mean ENaC
densitometry. Top: representative immunoblots for each subunit.
Molecular weights are given in METHODS. *Significantly
different from control, P < 0.05.
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Fig. 3. Effect of inhibition of apical Na+ entry by amiloride on apical
membrane expression of ENaC subunits. Na+ entry was blocked by 18-h
exposure to 10 µM amiloride. Cells were returned to control conditions and
apical membrane proteins were isolated by domain-specific biotinylation as
described. Results are the mean of 7 observations each. Amiloride resulted in
a significant decrease in apical membrane -ENaC alone compared with
simultaneous controls. Results are expressed as percentage of control ENaC
subunit densitometry. Top: representative immunoblots for each
condition. *Significantly different from control, P <
0.05.
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If the decrease in apical
-ENaC was of significance in the decline in
Isc, then a minimum expectation would be that a return of
Isc to control levels following removal of amiloride or
restoration of apical Na+ would be accompanied by a return of
apical
-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-h
downregulation of Na+ entry by either apical amiloride or
replacement of apical Na+ as described above. After restoration of
normal apical media, Isc was monitored for return of ENaC
function compared with control, untreated monolayers. The transport rate
recovered 70% in 1 h and 100% in 2 h, consistent with our initial observations
using this model (31). At the
time of full recovery, control and both amiloride/recovery and TMA/recovery
samples were subjected to apical membrane biotinylation as described to
measure apical expression of each ENaC subunit. As shown in
Fig. 4, recovery of
Isc to control levels following removal of inhibition of
apical Na+ entry was associated with return of apical membrane
-ENaC to control levels.

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Fig. 4. Effect of recovery from inhibition of apical Na+ entry on apical
membrane expression of ENaC subunits. Na+ entry was blocked by 18-h
exposure to 10 µM amiloride or by replacement of TMA for Na+ in
apical media. After 18 h, filters were washed and normal apical Na+
was restored. Isc was measured and returned to control
levels 2 h following restoration of apical Na+. At that point,
apical membrane proteins were isolated by biotinylation from both recovery
samples and simultaneous untreated, control A6 cells. Results are the means of
4 observations each and are expressed as percentage of control mean ENaC
subunit densitometry.
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In contrast to the remarkable effects of downregulation of transport on
apical membrane ENaC expression, upregulation of transport by 18 h of
short-circuiting had no substantial effect on the apical membrane expression
of 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.

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Fig. 5. Effect of upregulation of apical Na+ entry by chronic
short-circuiting on apical membrane expression of ENaC subunits. A6 cells
grown on filter supports were chronically short-circuited as described in
METHODS. After 18 h, cells were returned to control conditions and
apical membrane proteins were isolated by biotinylation. Results are a mean of
5 observations each. There are no differences between control and SC
measurements. Top: representative immunoblots for each condition.
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Because apical membrane ENaC expression had been altered by chronic
downregulation of Na+ transport, we next sought to determine
whether this was a reflection of altered whole cell amounts of ENaC subunits
or alternatively whether it might represent a redistribution of ENaC subunits
in response to the altered Na+ entry. Whole cell ENaC subunit
densities 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 reduced
apical membrane-associated
-ENaC with slight but not significant changes
in the other subunits but had no significant effect on the whole cell amounts
of either
- or
-ENaC.

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Fig. 6. Effect of chronic downregulation of apical Na+ on whole cell
levels of ENaC subunits. A6 cells underwent either 18-h substitution of TMA
for apical Na+ or 18 h in 10 µM amiloride. Cells were then lysed
and proteins were separated by SDS-PAGE as described. Lysates were probed with
subunit-specific antibodies and results were quantitated by densitometry.
Treatment with amiloride had no effect on subunit abundance. TMA replacement
resulted in a significant decrease in -ENaC expression alone.
Top: representative immunoblots; n = 5-6.
*Significantly different from control, P < 0.05.
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Because alterations in the rate of Na+ entry were associated
with significant changes in the activity of
Na+-K+-ATPase activity that persisted after return to
normal growth conditions in our initial study
(31), we also examined the
effect of these maneuvers on the basolateral membrane expression of
-
and
-subunits of the ATPase. As shown in
Fig. 7, neither chronic
upregulation of transport rate by short-circuiting nor chronic downregulation
of transport by TMA substitution for apical Na+ had any effect on
the amount of either
- or
-subunit of the
Na+-K+-ATPase present in the basolateral membrane.

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Fig. 7. Effect of 18-h altering apical Na+ entry and aldosterone on
basolateral membrane expression of Na+-K+-ATPase
subunits. Cells were treated with aldosterone (1 µM) or underwent chronic
SC or replacement of apical Na+ with TMA for 18 h, and basolateral
membrane proteins were isolated by domain-specific biotinylation and probed
for individual subunits of the ATPase. Aldosterone induced a significant
increase in -subunit of the ATPase. No other changes are significant.
Top: representative immunoblots. Molecular weights of the subunits
are given in METHODS; n = 5.
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Although we were unable to demonstrate any effect of chronic upregulation
of Na+ entry on the expression of apical or basolateral transport
proteins, we previously demonstrated that long-term (18 h) but not short-term
(3 h) exposure of A6 cells to 10-6 M aldosterone results
in a selective increase in apical membrane expression of
-ENaC
(37). This occurred without an
effect 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
-ENaC
expression without a change in
- or
-ENaC has been described by
Stockand and colleagues (34)
following long-term aldosterone exposure. Although we were unable to
demonstrate changes in basolateral Na+-K+-ATPase amounts
following alterations in Na+ entry, the effects of aldosterone on
both transcription and translation of both subunits of this enzyme are well
described (2,
36). Additionally, we
previously demonstrated upregulation of enzyme activity in A6 cells, which is
unaffected 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 in
basolateral membrane without affecting basolateral amount of
-subunit.
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DISCUSSION
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It has been shown in a number of mammalian models that chronic alterations
in delivery or chronic blockade of specific Na+ transporters may
induce 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 ENaC
activity through a process of feedback inhibition
(16,
19,
30), whereas acute inhibition
of Na+ entry is associated with increased ENaC activity
(11,
30). Because chronic increases
in Na+ load or delivery in animals seem to be associated with
transporter upregulation (8,
27), whereas chronic
inhibition of transport seems to be associated with downregulation of
transporters (15), we examined
the long-term effects of these maneuvers in vitro. We established a model of
chronic regulation of Na+ transport by the rate of apical
Na+ entry in the cultured A6 cell line, where Na+
transport is primarily mediated by apical membrane ENaC and the basolateral
Na+-K+-ATPase
(31). In this model, chronic
upregulation of Na+ entry by continuous short-circuiting of the
epithelia for 18 h resulted in upregulation of transport rate when normal
polarity was restored. Transport rates declined toward basal levels over a
period of hours. In contrast, chronic downregulation of apical Na+
entry by either removal of apical Na+ or ENaC blockade with
amiloride for 18 h resulted in a decreased rate of transport. When normal
apical Na+ entry was restored, Isc returned to
control levels within 2 h. In contrast to our results, studies in primary
cultures derived from the rabbit kidney collecting system showed little effect
of chronic short-circuiting and chronic inhibition of Na+ entry
with benzamil-enhanced transport rates
(6). In these experiments,
chronic short-circuiting, which had little effect on either basal
Isc or
-ENaC levels, blocked the effect of
aldosterone and this inhibition was overcome by incubation with benzamil
(6). Noting the difference
between their results and our initial studies
(31), the authors speculated
that intracellular Na+ concentration or an alteration in rates of
Na+ entry might have dual effects, depending on the absence or
presence of aldosterone. The current experiments were designed to reevaluate
our model of chronic regulation and test the possibility that chronic
alterations in Na+ entry altered channel subunit expression or
basolateral enzyme expression under conditions of chronic up- or
downregulation. Additionally, we sought to compare the effects of chronic
upregulation by increased Na+ entry to those seen with chronic
upregulation of transport by aldosterone.
Our results confirmed some but not all of our expectations. Chronic
downregulation of Na+ entry by either mechanism was associated with
decreased expression of ENaC subunits at the apical membrane without
significant changes in whole cell subunit content. Of particular interest,
only one subunit,
-ENaC, was decreased in apical expression. This
observation is similar to what we described with downregulation of transport
by the agent BFA (37). BFA
disrupts delivery of proteins to post-Golgi targets and markedly reduced
Isc in A6 cells but resulted in a decrease in apical
expression only of
-ENaC with no significant change in
- or
-expression. This occurred over a period of 1- to 3-h incubation with
BFA, similar to the time course of BFA inhibition of apical membrane channel
activity measured by noise analysis
(9). This surprising result was
explained to some degree when we examined the half-life of ENaC subunits that
reach 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 this
phenomenon of apparent differential turnover of apical membrane ENaC subunits
as noncoordinate regulation. It appears that downregulation of Na+
entry in A6 cells may be another example of this.
Because there is no reason to believe that physiologically significant ENaC
function is mediated by anything other than fully formed heterotrimeric
channels, the observation of noncoordinate regulation implies that channels
may be assembling or disassembling at some point beyond the endoplasmic
reticulum (ER). As Rotin and colleagues
(32) recently pointed out,
there is no evidence to support such a concept that emerges from the many
studies of ENaC assembly or function in oocytes or heterologous expression
systems, which clearly demonstrate that ENaC assembles into complete
tetrameric channels in ER soon after biosynthesis as do most multimeric
proteins. It is interesting to note, however, that multimeric proteins may be
processed differently in endogenously expressing cells than in overexpressing
cells. The T cell antigen receptor complex (TCR-CD3) of T cell hybridomas has
served as an established model of ER assembly of a multimeric membrane
protein, but when examined in normal T cells, the putative limiting
-subunit appears to exhibit more rapid turnover from membrane-bound
complexes than do the other subunits
(28). Similarly, the three
subunits of the interleukin-2 receptor in T lymphocytes appear to have varying
surface half-lives and endocytic fates
(14). In cells or tissues
endogenously expressing ENaC, there are multiple examples of apparent
noncoordinate regulation of individual subunits. In rat distal nephron,
aldosterone induces expression of
-ENaC with an apparent shift of
cytosolic
- and
-subunits to the apical membrane with a short
course in early distal nephron and a more prolonged course in collecting duct
(10,
23,
24). Physiological
manipulations to rats have been described that result in either selective
regulation 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 PKC
stimulation inhibits the transport rate in A6 epithelia in association with a
selective 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 which
noncoordinate regulation of ENaC subunit expression results in changes in the
apical density of fully active channels is unclear, but in each case,
regulation of the transport rate appears to correspond with altered expression
of some, but not all, ENaC subunits
(3,
7,
13,
20,
22-24,
34,
37). A recent report by
Alvarez De La Rosa et al. (1),
which describes fully coordinate expression and turnover of cellular and
apical membrane ENaC subunits in response to aldosterone in A6 cells, clearly
contradicts the notion of noncoordinate regulation. The reasons for the
differences in results between this study and our own previous findings as
well as those of other groups
(10,
29,
32) are not clear, because
many of the same methods were employed. Alvarez De La Rosa et al.
(1) describe an extremely short
half-life (12-17 min) for ENaC subunits that reach apical membrane, a period
in marked contrast to the longer half-life we
(37) and others
(21) described in A6 cells and
difficult to reconcile with the time course of BFA inhibition of ENaC surface
density measured by noise analysis
(9).
In contrast to our expected finding of ENaC downregulation by the rate of
Na+ entry, we could find no evidence of a change in apical membrane
ENaC expression with chronic short-circuiting and no evidence of a change in
basolateral Na+-K+-ATPase by either chronic adaptation.
Clearly, altered enzyme activity in chronic adaptation is not a function of a
greater or lesser number of pumps expressed in the membrane. Although we could
measure no direct effects of upregulation of Na+ entry by
short-circuiting on apical or basolateral transporter density, chronic
upregulation of transport by aldosterone clearly affected both. Once again,
the characteristic of this regulation was noncoordinate both with respect to
ENaC and ATPase. Selective upregulation of apical
-ENaC by aldosterone
in A6 has previously been noted
(37), and data from Stockand
et al. (34) suggest this is
due to increased expression of this subunit at the whole cell level. Both
transcriptional and translational regulation of both subunits of
Na+-K+-ATPase by aldosterone have been well documented
previously (2,
17,
29,
36). A recent observation in
cultured cortical collecting duct cells demonstrates a roughly similar
increase in basolateral
-subunit to that which we describe here, but no
data on
-subunit expression at basolateral membrane are available
(35). Previous studies suggest
that the two subunits may have slightly different time courses of membrane
insertion (5,
12), and relative differences
in fold-changes in the two subunits expression following regulatory stimuli
have been described (25,
38). These observations
suggest that the enzyme may not traffic exclusively as

-heterodimers. Moreover, it has been shown that increases in a
single subunit of the Na+-K+-ATPase not only occur but
have also been implicated in increasing pump activity
(33). Our observations would
suggest that enzyme function may be increased by alteration in basolateral
expression of
-subunit alone, which implies that there may be an excess
of
-subunit already present in or near the membrane. Whatever the
mechanism, aldosterone clearly induces alterations in apical ENaC and
basolateral Na+-K+-ATPase, which are not seen with
chronic upregulation of Na+ entry alone. Our data are in agreement
with the conclusion by Summa and colleagues
(35) that the effect of
aldosterone on basolateral transport proteins is not mediated by apical
Na+ entry.
 |
DISCLOSURES
|
---|
This work was supported by National Institute of Diabetes and Digestive and
Kidney Diseases Grants 57718 (to J. P. Johnson) and 43955 and 48217 (to M. L.
Zeidel) and by funds from Dialysis Clinics.
 |
FOOTNOTES
|
---|
Address for reprint requests and other correspondence: J. P. Johnson, 935
Scaife Hall, 3550 Terrace St., Pittsburgh, PA 15213 (E-mail:
Johnson{at}msx.dept-med.pitt.edu).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
 |
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