Role of PKC
in feedback regulation of Na+
transport in an electrically tight epithelium
Mouhamed S.
Awayda,
Justin D.
Platzer,
Roxanne L.
Reger, and
Abderrahmane
Bengrine
Department of Physiology, Tulane University Health
Sciences Center, New Orleans, Louisiana 70112
 |
ABSTRACT |
It has long been known that
Na+ channels in electrically tight epithelia are regulated
by homeostatic mechanisms that maintain a steady state and allow new
levels of transport to be sustained in hormonally challenged cells.
Little is known about the potential pathways involved in these
processes. In addition to short-term effect, recent evidence also
indicates the involvement of PKC in the long-term regulation of the
epithelial Na+ channel (ENaC) at the protein level
(40). To determine whether stimulation of ENaC involves
feedback regulation of PKC levels, we utilized Western blot analysis to
determine the distribution of PKC isoforms in polarized A6 epithelia.
We found the presence of PKC isoforms in the conventional (
and
), novel (
,
, and
), and atypical (
,
, and
) groups. Steady-state stimulation of Na+ transport with
aldosterone was accompanied by a specific decrease of PKC
protein
levels in both the cytoplasmic and membrane fractions. Similarly,
overnight treatment with an uncharged amiloride analog (CDPC), a
procedure that through feedback regulation causes a stimulation of
Na+ transport, also decreased PKC
levels. These effects
were additive, indicating separate mechanisms that converge at the
level of PKC
. These effects were not accompanied by changes of
PKC
mRNA levels as determined by Northern blot analysis. We propose
that this may represent a novel regulatory feedback mechanism necessary for sustaining an increase of Na+ transport.
Western blotting; aldosterone; protein kinase C
; epithelial
Na+ channel
 |
INTRODUCTION |
IT IS WELL ACCEPTED
that apical membrane Na+ permeability is the rate-limiting
step for Na+ entry and, consequently, transepithelial
Na+ transport in renal, electrically tight epithelia. By
altering apical Na+ permeability, epithelia in the distal
tubule can exhibit a wide range of salt and water transport. It is
therefore not surprising that the activity of the Na+
channel in the apical membrane of these renal epithelia is highly regulated. Besides regulation by a variety of circulating hormones such
as aldosterone and vasopressin, it is also well known that Na+ channels are regulated by feedback mechanisms that
respond to changes of transport by attempting to restore a steady state
(18, 41). Thus it follows that these feedback mechanisms
are modified in hormonally stimulated cells; otherwise, they would tend
to counteract the effects of hormones and attempt to restore baseline rates of Na+ transport. Despite a wealth of information to
the existence of these self- or autoregulatory processes, little is
known about their molecular mechanisms.
The mechanisms of short-term (minutes) feedback regulation of
Na+ transport may be directly related to changes of
intracellular Na+ concentration
([Na+]i) (4). However, the
mechanisms of long-term processes are poorly understood but are likely
to involve the actions of second-messenger protein kinases such as PKC.
PKC is a ubiquitous enzyme with multiple isoforms that fall into three
general categories classified according to their modes of activation
(19). The conventional group (cPKC), which is composed of
isoforms
,
, and
, is activated by diacylglycerol (DAG),
Ca2+, and phosphatidylserine (PS). Members of the second
group (
,
,
, and
) are stimulated by DAG and PS but do not
require Ca2+ for activation and are referred to as novel
PKC isoforms. Members of the third group (
,
, and
) are
stimulated by PS alone and are considered atypical PKC isoforms.
Ample evidence exists supporting isoform-specific roles for PKC in
nonpolarized and polarized low-resistance leaky epithelia (2, 6,
9, 10, 22, 23, 26, 36-38). However, the distribution and
potential functions of these isoforms in electrically tight,
Na+-absorbing epithelia are poorly understood. Preliminary
evidence indicates specific functions for these isoforms. For example, DeCoy et al. (12) have reported that the rabbit cortical
collecting duct (CCD) contains both PKC
and PKC
and that
inhibition of PKC
enhances vasopressin-stimulated Na+
absorption, implicating this isoform in the tissue-specific transient stimulation of Na+ transport by arginine vasopressin.
Wilborn and Schafer (43) found additional evidence for the
presence of PKC
, -
, and -
in the CCD. Interestingly, these
authors also found differences in the expression levels of PKC
and
PKC
between freshly isolated and cultured rabbit CCD cells.
Although the above findings provide strong indirect evidence in support
of the hypothesis of distinct roles for PKC isoforms in the
steady-state or hormonal regulation of the epithelial Na+
channel (ENaC) or of Na+ transport in high-resistance
epithelia, little is know about these specific roles. Indeed, although
a large number of studies have examined ENaC's regulation by PKC in
various preparations (3, 7, 11, 15, 24, 27, 30, 32, 33, 39,
42), only a minor fraction have focused on the potential
regulation by distinct isoforms or a group of isoforms. Moreover, these
studies utilized pharmacological agents believed to be selective for
certain isoforms. For example, Rokaw et al. (33, 34)
utilized rapamycin, which has a higher specificity for cPKC isoforms,
to determine that inhibition of PKC
transiently stimulates ENaC in
A6 epithelia.
Recently, an additional important function for PKC has been elucidated.
Stockand et al. (40) found that long-term stimulation of
PKC results in downregulation of
and
ENaC protein levels, indicating the presence of a chronic interaction between ENaC and PKC.
The PKC isoforms involved in this process are undetermined. It is also
unknown whether a reciprocal or feedback interaction exists between
ENaC or Na+ transport and PKC.
Because of our interest in A6 cells as a model of Na+
transport by electrically tight epithelia, we examined their PKC
isoform content. Three PKC isoforms have been cloned from
Xenopus oocytes: PKI, PKCII (8), and PKC
(Ref. 13 and J. Moscat, M. M. Munico, and M. T. Diaz-Meco, unpublished observations; NCBI accession no. U12588). The
first two, PKCI and PKCII, share the highest homology with mammalian
PKC
and PKC
(13) and are likely to correspond to
these isoforms. Xenopus PKC
shares homology with atypical
PKCs and is likely to correspond to mammalian PKC
(J. Moscat et al.,
unpublished observations). However, it is uncertain which of these and
other unidentified isoforms exist in A6 epithelia. Only PKC
has been
identified at the protein level in A6 cells (1, 33). This
isoform is stimulated by increasing [Ca2+]i,
a procedure known to inhibit Na+ channels in tight
epithelia (18). Given this effect, the data of Rokaw et
al. (33, 34), and our own unpublished observations, it is
likely that PKC
mediates channel inhibition. Thus this isoform would
a priori make a good candidate for its involvement in feedback
regulation of ENaC and Na+ transport.
To determine whether a reciprocal interaction exists between
Na+ transport and these isoforms, and specifically PKC
,
we examined the effects of 1) transport stimulation with the
hormone aldosterone and 2) transport inhibition leading to
feedback regulation, with the uncharged amiloride derivative CDPC. We
report that polarized A6 epithelia contain PKC isoforms in all three
groups. We also found a specific downregulation of PKC
by chronic
aldosterone and CDPC treatment. Furthermore, the extent of
Na+ transport activation with aldosterone or prolonged CDPC
treatment could be enhanced with a specific cell-permeable inhibitor of PKC
, providing further functional evidence for the involvement of
this isoform. We propose that by downregulating an inhibitory kinase,
A6 cells are able to sustain a stimulation of Na+
transport, thereby resetting homeostatic mechanisms to a higher level.
 |
MATERIALS AND METHODS |
A6 epithelia.
Cells were obtained from American Type Culture Collection (ATCC,
Manassas, VA) and were cultured as previously described by Awayda et
al. (3). Cells were either plated on flasks or on permeable polyester membrane inserts (Transwell Clear; Corning-Costar, Acton, MA). Cell polarity was determined from their ability to develop
a transepithelial open-circuit voltage (Voc).
Monolayers which exhibited a Voc > 35 mV
(basolateral with respect to apical) were used for subsequent
experiments. These monolayers exhibited a transepithelial resistance
(RT) in the range of 10 K
· cm2 and an equivalent short-circuit current
(Isc) > 5 µA/cm2. Membrane
voltage and resistance were determined by using a four-electrode system
(EVOM; WPI, Sarasota, FL) designed for measurements of these parameters
in tissue culture inserts without disrupting the cells. The equivalent
Isc was calculated according to Ohm's law.
Confluent filters were treated with CDPC
(6-chloro-3,5-diamino-2-pyrazinecarboxamide) aldosterone (Sigma, St.
Louis, MO), PMA (CalBiochem, San Diego, CA), or a myristoylated PKC
C2 peptide inhibitor
(N-Myr-Ser-Leu-Asn-Pro-Glu-Trp-Asn-Glu-Thr; Biomol, Plymouth
Meeting, PA) as described in RESULTS. All reagents were of
the highest grade available and were dissolved directly into medium or
in stock solution of ethanol (EtOH), DMSO, or medium, respectively.
Cell fractionation and Western blotting.
Confluent monolayers of A6 epithelia were harvested and immunoblotted
as suggested by the manufacturer of the monoclonal antibodies against
each of the PKC isoforms (Transduction Laboratories, San Diego, CA),
with a few modifications as outlined below. Briefly, cells were lysed
in boiling lysis buffer (10 mM Tris, pH 7.5, and 1 mM Na vanadate),
scraped into 15-ml conicals, boiled for 5 min, and passed several times
through a 27-gauge needle. Unbroken cells were pelleted, and an aliquot
of the lysate was removed. This aliquot was the total protein fraction
and was acetone (~6 volumes) precipitated and resuspended in 10 mM
Tris, pH 7.5. The remaining lysate was centrifuged at 100,000 g for 1 h at 4°C. The supernatant contained the
cytoplasmic fraction and was acetone precipitated as above. The pellet
was resuspended in lysis buffer containing 1.0% Triton X-100 and
incubated on ice for 2 h with occasional vortexing. The suspension
was then centrifuged at 14,000 g for 20 min at 4°C, and
the supernatant containing the solubilized membrane fraction was
acetone precipitated as described above. Protein concentrations were
determined with a Bradford-based assay (Bio-Rad, Chicago, IL) by using
BSA as a standard.
Approximately 7.5 µg of
-mercaptoethanol (
ME)-reduced
protein were separated on 3.9%/7.5% Laemmli gels under denaturing conditions (SDS). Proteins were electrotransferred overnight at 4°C
to nitrocellulose membranes. Blots were blocked in Blotto (10 mM Tris,
pH 7.5, 100 mM NaCl, 0.05% Tween 20, and 5% nonfat dry milk) for
1 h and then incubated for 2 h in primary antiserum diluted
in Blotto at the concentrations recommended by the manufacturer. All
incubations were carried out at room temperature. For monoclonal antibodies, horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antiserum was diluted at 1:100,000 in Blotto, and the blots
were incubated for 45-60 min. Rat anti-rabbit secondary antiserum
was used at 1:160,000 for the polyclonal antibodies. Blots were washed
in 10 mM Tris, pH 7.5, 100 mM NaCl, and 0.05% Tween 20 between
antiserum incubations and in 10 mM Tris, pH 7.5, before being processed
for chemiluminescence (SuperSignal West Dura Extended Duration
Substrate; Pierce, Rockford, IL) according to the manufacturer's
instructions. The blots were then exposed to film and developed in an
automated developer. Films were scanned by using a flatbed scanner,
followed by quantitation of signal intensity using SigmaScan (Jandel
Scientific, San Rafael, CA). A similar procedure was carried out by
using a positive control (rat brain extract) probed with anti-PKC
and anti-GAPDH antibodies to serve as calibration standards.
Equal loading was assessed by using three procedures: 1)
protein concentration determination, 2) Ponceau S staining
of the membranes after electrotransfer, and 3) Western
blotting with a GAPDH-specific antibody (BioDesign International, Saco,
ME). In this case, the bottom half of the blots (<50 kDa) was
separated and probed with the GAPDH antibody.
The antibodies utilized in the present study were raised against highly
conserved protein sequences and allow a high degree of isoform
specificity. This specificity was also verified by testing the
antibodies against known positive controls. These antibodies are also
tested by the manufacturer and are known in many instances to recognize
the same isoform from various species, including human, canine,
rat, mouse, chicken, and frog. Moreover, isoform-specific
anti-mammalian PKC antibodies have been tested by others and are known
to recognize specific Xenopus PKC isoforms (1, 13, 31,
33).
Northern blotting.
Total RNA was isolated from polarized A6 cells by utilizing the Trizol
reagent (Life Technologies, Rockville, MD) following the
manufacturer's instructions modified for a growth area of 4.5 cm2. Briefly, cells in each well were lysed in 500 µl
(initial volume) of Trizol, scraped, and mixed with 0.2× initial
volume chloroform, and the phases were separated by centrifugation at
12,000 g for 15 min at 4°C. The RNA was precipitated from
the aqueous phase with 0.5× initial volume of isopropanol and, after
10 min at room temperature, pelleted by centrifugation at
12,000 g for 10 min at 4°C. The pellet was washed with
2.5× initial volume of 75% EtOH, vortexed, and centrifuged at 7,000 g for 5 min at 4°C. Total RNA was treated with RQ1 DNase
(Promega, Madison, WI), phenol extracted, EtOH precipitated, and
resuspended in RNase-free water. RNA concentrations were determined
spectrophotometrically, and RNA integrity was verified on a 1% agarose
gel by using formaldehyde loading buffer.
To construct the Northern probes, the isolated RNA served as the
template for RT-PCR for the amplification of Xenopus PKCI (also known as PKC
) and GAPDH DNA. Xenopus PKCI primers
were designed to amplify a 303-bp fragment encompassing the V3
region and the adjacent C2 and C3 regions, bases 807-1110, as
determined by Chen et al. (8). The PKCI forward
primer was 5'-gctctttatcttcgggggtgtcagagctgatgaaaatg-3' and the
reverse primer was
5'-ccagtgaattgtaatacgactcactatagggctgccaaccatgac-3'. Primers for Xenopus GAPDH were designed to encompass bases
911-1154 (accession no. GI1136598). The forward primer was
5'-ctcctccatctttgatgctgatgctggaattg-3' and the reverse primer was
5'-ccagtgaattgtaatacgactcactatagggaatgtttcatcatg-3'. The
T7 RNA polymerase promoter was added to the 5' end of each reverse primer (underlined in sequences).
Briefly, 5 µg of control RNA were reverse transcribed (Thermoscript,
Life Technologies) with the gene-specific primer for PKCI or
oligo(dT)20 for GAPDH according to the manufacturer's protocol. The cDNAs (1/20 of each reaction) were then used as the
template for PCR, incorporating the primers described above. Pfu DNA polymerase (Stratagene, La Jolla, CA) was used for
touchdown PCR with the following parameters: initial denaturation at
95°C for 5 min; 15 cycles of 95°C for 30 s; 70°C for 30 s with a 1°C decrease in annealing temperature per cycle, and 74°C
for 1 min, followed by 25 cycles of 95°C for 30 s, 54°C for
30 s, and 74°C for 1 min; and a final extension of 74°C for 10 min. The resulting products were purified on a 3.5% low-melt agarose
gel, phenol extracted, EtOH precipitated, and resuspended in RNase-free
water. Antisense RNA were generated from these products by in vitro
transcription with T7 RNA polymerase (Promega). The RNA was labeled by
using the BrightStar Psoralen-Biotin nonisotopic labeling kit (Ambion, Austin, TX) according to the manufacturer's protocol.
Total RNA (15 µg) from each condition for each of the probes was
separated on a 1% agarose formaldehyde gel. The transfer to the nylon
membrane (MSI) was done in the vacuum blotter according to the
manufacturer's protocols (Bio-Rad Labs, Hercules, CA). After UV
cross-linking, prehybridization (30 min) and hybridization (overnight)
were performed at 55°C in ultrasensitive hybridization buffer
(Ambion). The blots were washed twice at 55°C by using high-stringency wash solution (Ambion). Detection was accomplished by
using the BrightStar BioDetect kit (Ambion) according to the manufacturer's protocol. Hybridization products were detected on Kodak
X-ray film for 2 h.
 |
RESULTS |
Isoform content of polarized A6 cells.
A6 cells were grown on permeable supports as described in
MATERIALS AND METHODS. Under these conditions, confluent
cells exhibited a spontaneous Voc that
stabilized at 49.5 ± 0.73 mV and an RT of
10.63 ± 0.16 K
· cm2 (n = 116). This resulted in an Isc of 4.71 ± 0.06 µA/cm2. Under these conditions, and consistent with
data previously reported (3), the majority of the
Isc is sensitive to amiloride and to removal of
apical Na+.
Confluent and polarized Na+-transporting A6 cells were
fractionated and probed with isoform-specific PKC antibodies. As
mentioned above, three PKC isoforms have been previously cloned from
Xenopus oocytes. Given their homology with their mammalian
counterparts, it is believed that Xenopus PKCI and PKCII
correspond to PKC
and PKC
, respectively (8).
Therefore, for the purposes of clarity and consistency with other
reports in the literature, we will refer to these Xenopus
isoforms as PKC
and PKC
.
Shown in Fig. 1 is a composite Western
blot of the various immunoreactive species identified in these cells.
Lanes probed with the secondary antibody alone, at the same
concentration as that used with the primary, were blank (not shown).
Specific PKC immunoreactivity was observed in lanes probed with
monoclonal antibodies to PKC
, -
, -
, -
, -
, and -
(Fig.
1A). PKC immunoreactivity was also observed with polyclonal
antibodies to PKC
and -
(Fig. 1B).

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Fig. 1.
PKC isoforms present in polarized A6 epithelia. Composite Western
blot of A6 homogenate was probed with isoform-specific PKC
antibodies. A: A6 proteins were probed with monoclonal
antibodies which recognize unique PKC isoforms. Of the complement of
monoclonal antibodies used, only those directed against PKC and
PKC sequences did not cross-react with A6 protein (data not shown).
B: A6 proteins were probed with polyclonal antibodies
against PKC and PKC . Lanes were separated and individually probed
with a single isoform-specific antibody as described in MATERIALS
AND METHODS.
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|
These commercial isoform-specific antibodies are known to cross-react
with various species, including frog. This is because antigenic regions
chosen to construct these antibodies are conserved among various
species. For example, the PKC
antibody was constructed against a
large 156-amino acid region (amino acids 270-427) of human PKC
,
which is 93% homologous to frog (Xenopus). Thus it is
expected and indeed previously was demonstrated that this antibody will
cross-react with amphibian PKC
. On the other hand, antibodies obtained from other commercial resources made against short regions with little or no homology between Xenopus and human failed
to detect PKC immunoreactivity in A6 cells (data not shown).
In all cases, the Xenopus PKC isoforms recognized by these
antibodies corresponded well with their mammalian counterpart observed in rat brain homogenate (data not shown; also see manufacturer's data). Our finding of PKC
in A6 cells is consistent with previous reports (1, 33), although the identification of other
isoforms represents, to our knowledge, the first published report of
such an attempt.
Summarized in Table 1 is the calculated
mass for the various PKC isoforms in A6 cells. Some isoforms, e.g.,
and
in the novel group and
in the atypical group, migrated as
doublets. In these cases, the masses for each of the immunoreactive
species are summarized in Table 1. With the exception of PKC
, there were no significant differences in the molecular weight of the identified isoform between the cytoplasmic and membrane fractions. On
average there was a small shift in the calculated molecular weight for
PKC
between the two cellular fractions, such that this isoform was
~6 kDa larger in the membrane fraction. It is possible that other
isoforms exhibited a small shift in molecular weight between these two
fractions; however, the differences in PKC
were highlighted once we
focused our attention on this isoform (Fig. 4). The reason for this
small shift in PKC
is unknown but may be attributed to
posttranslational modification, e.g., phosphorylation of the
membrane-associated PKC
(20).
As previously mentioned, conventional PKC isoforms, i.e.,
Ca2+-activated isoforms such as
and
, are thought to
mediate channel inhibition (18, 33, 44). Given these
effects, our long-standing interest in understanding ENaC's inhibition
by PKC (5), and our own unpublished observations with
PKC
, we focused our attention on understanding the roles of these
isoforms in feedback regulation of Na+ channels. Initial
experimentation did not reveal consistent changes of PKC
, and we
therefore focused our attention to examining PKC
protein and mRNA levels.
Role of PKC
in the response to aldosterone.
To examine the effects of sustained stimulation of ENaC, we utilized
the hormone aldosterone. Treatment with 1 µM aldosterone (24 h)
caused a near doubling of Voc from 49.5 ± 0.73 to 96.5 ± 0.66 (n = 36), accompanied by a
nearly fourfold increase of Isc (4.71 ± 0.06 to 15.75 ± 0.27), consistent with the previously reported
effects of this hormone.
The accompanying changes of PKC
protein levels were determined by
using Western blots of membrane and cytoplasmic fractions. As shown in
Fig. 2, PKC
levels were reduced in
both the cytoplasmic and membrane fractions. This decrease of PKC
protein levels was also confirmed by examining the levels of this
isoform in the total cell homogenate. As shown in Table
2, a similar decrease of PKC
was
observed among the cytoplasmic membrane and total fractions, indicating
that this decrease was likely the result of a general downregulation of
protein levels of this isoform.

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Fig. 2.
Downregulation of PKC by aldosterone. Representative
Western blot of the effect of aldosterone on the distribution of PKC
in A6 cell fractions. T, M, and C refer to total, membrane, and
cytoplasmic fractions, respectively. + and refer to 24-h
treatment with 1 µM aldosterone or ethanol vehicle, respectively.
Data are representative of 7 experiments. See MATERIALS AND
METHODS for more details.
|
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To determine whether this effect is attributed to a decrease of PKC
protein synthesis, a question especially important given that
aldosterone is a steroid hormone with multiple genomic and nongenomic
effects, we utilized Northern blotting with riboprobes constructed from
Xenopus PKC
as described in MATERIALS AND
METHODS. A similar signal was observed in RNA from control and
aldosterone-treated cells (Fig. 3),
indicating that the decrease of PKC
protein levels was not the
result of a direct genomic downregulation of the PKC
gene by
aldosterone. However, we were uncertain whether these effects were
specifically attributed to the actions of aldosterone or generally
related to prolonged stimulation of Na+ transport, leading
to potential secondary changes of [Na+]i. To
address these issues, we carried out experiments in which Na+ channels were stimulated by aldosterone but in which
the effects on transport were restored to near baseline by overnight
(24 h) incubation with a specific ENaC blocker.

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Fig. 3.
Lack of direct genomic effects of aldosterone on PKC .
A: Northern blotting with PKC -specific probes.
B: parallel Northern blot with a GAPDH-specific probe was
carried out to assure equality of loading. The intensity of
ethidium-stained ribosomal RNA was also examined (data not shown).
Conditions were as described in MATERIALS AND METHODS,
except that 10 µg of the total RNA were used and washes were carried
out at 65°C. This may have resulted in decreased signal intensity
(see Fig. 6 for comparison). The intensities of control cells
(lanes 1 and 3) were similar to those observed
from aldosterone-treated cells (lanes 2 and 4).
Note the presence of 2 products in both control and aldosterone-treated
cells. These may represent different forms of PKC and correspond to
sizes of ~3.6 and 6.5 kB, respectively. Data are representative of 3 experiments.
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Effects of CDPC: feedback regulation.
We utilized the electroneutral amiloride analog CDPC to reduce
Na+ transport in aldosterone-treated tissues to control
prestimulated levels. We specifically chose this blocker because it is
electroneutral and is therefore not susceptible to changes of
intracellular voltage leading to changes of its blocking efficacy. We
initially chose a concentration of 50-100 µM CDPC, which is in
the range of the half-maximal block for ENaC by this compound
(3). However, because of the distribution of channels from
the closed to open and blocked states, this concentration was not
sufficient in providing an appreciable inhibition of the
Isc, even under short-term conditions. We
increased this concentration and found that 300-400 µM CDPC acutely decreased the Isc in aldosterone-treated
cells to values similar to those in the untreated ones.
The effects of separate and combined CDPC and aldosterone treatment are
shown in Fig. 4. It is clear that the
effects of aldosterone on PKC
were not mediated by a simple increase
of Na+ transport, because the protein levels of this
isoform also decreased in epithelia treated with aldosterone and CDPC.
However, and surprisingly, tissues treated with both CDPC and
aldosterone exhibited the largest decrease of PKC
. Again, this was
observed in the cytoplasmic and membrane fractions and was a reflection
of decreased PKC
protein levels. Consistent with this observation
was the finding that CDPC alone was also capable of decreasing PKC
levels. Although these findings with aldosterone and CDPC may appear
puzzling given the opposite effects of these agents on Na+
transport, these effects may be reconciled by the ability of both CDPC
and aldosterone to cause prolonged stimulation of transport (see
below).

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Fig. 4.
Additive downregulation of PKC protein levels by aldosterone and
uncharged amiloride analog (CDPC). A: representative Western
blot demonstrating that PKC is downregulated by the separate and
combined treatments with aldosterone and CDPC. Note that the largest
decrease was observed in cells treated with both aldosterone and CDPC.
0, A, C, and C + A refer to control (vehicle), 1 µM aldosterone,
400 µM CDPC, and CDPC and aldosterone, respectively. B:
corresponding signal intensities relative to those observed in the
control ethanol-treated cells (lanes 1 and 2).
Data are representative of 4 experiments with 400 µM CDPC and 5 additional experiments with 200-300 µM CDPC. Aldosterone and
CDPC were added for 24 and 21 h, respectively.
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Shown in Fig. 5 are the effects of
aldosterone and CDPC on the Na+ transport properties of A6
cells. As seen above, aldosterone caused a threefold increase of the
Isc. Short-term treatment with CDPC (10 min)
caused a large decrease of the Isc in both
control and aldosterone-treated cells. However, at 21 h, the
Isc in cells treated with CDPC and aldosterone
exhibited a complete recovery to values even higher than those found in
tissues treated with aldosterone alone. A similar recovery to values
above control was also observed in tissues treated with CDPC alone.
These processes are likely a continuation of the short-term feedback or
autoregulation described in many Na+-transporting
epithelia, where the Isc gradually recovers
toward control values after inhibition of Na+ transport,
manifested by the ability of cells to stimulate the apical
Na+ channels (16, 18, 41). Thus, in both the
CDPC- and aldosterone-treated cells, a downregulation of PKC
was
associated with conditions that require a sustained increase of
Na+ transport. As with the effects of aldosterone alone,
the decrease of PKC
protein levels was not accompanied by a decrease
of mRNA levels (Fig. 6). These findings
indicate that these effects were not the result of direct genomic
modulation of the PKC
gene.

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Fig. 5.
Feedback regulation of Na+ transport after
prolonged inhibition with the electroneutral blocker CDPC. The initial
effects of CDPC were measured 10 min after its addition to control or
aldosterone-prestimulated cells (3-h treatment). Overnight changes were
reported at 24 h after addition of aldosterone and 21 h after
addition of CDPC. Lanes 1 and 2 refer to control
and 3-h aldosterone-treated cells. Lanes 3 and 4 refer to the same tissues 10 min after the addition of CDPC.
Lanes 5, 6, and 7 refer to filters
chronically treated (21 or 24 h) with CDPC, aldosterone, and
CDPC + aldosterone, respectively. As expected, CDPC caused a large
initial inhibition of transport. However, at 24 h, cells were able
to compensate and restore their transport rates to values slightly
higher than those in the absence of blocker. Note the changes of
transepithelial resistance (RT) observed with
aldosterone and CDPC treatments. These changes indicate that, under our
culture conditions, RT is predominantly a
reflection of the cellular resistance and that the observation of an
RT > 4 K · cm2
under all experimental conditions indicates the absence of adverse
cellular effects.
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Fig. 6.
Lack of direct genomic effects of CDPC and combined
CDPC/aldosterone treatments on PKC . Blots were conducted as
described in MATERIALS AND METHODS. Lanes 1 through 4 correspond to RNA from control, aldosterone-,
CDPC-, and CDPC + aldosterone-treated cells, respectively. The
top panel was probed with a PKC -specific probe, while the
bottom panel was probed with a GAPDH-specific probe. Note
that lanes 1 and 2 have similar intensities,
whereas lanes 3 and 4 are slightly higher than
1 and 2. Thus the decrease of PKC protein
levels cannot be accounted for by its observed levels of RNA. As with
Fig. 3, 2 bands are observed for PKC . Data are representative of 3 additional experiments.
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Given the importance of these findings, we further characterized
Xenopus PKC
. Specifically, we tested whether this isoform exhibits cytoplasm-to-membrane translocation subsequent to its activation. As shown in Fig.
7A, 1-h treatment with 100 nM
PMA was capable of translocating the majority of cytoplasmic PKC
to
the plasma membrane. This indicates that the mechanism of activation of
Xenopus PKC
is similar to that described in other
systems. As expected, the addition of PMA caused a large decrease of
Isc and Voc (Fig.
7B). This was accompanied by an increase of
RT due to inhibition of the apically located
ENaC.

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|
Fig. 7.
PMA causes translocation of PKC to the membrane,
subsequent to its acute activation, and inhibition of Na+
transport. A: Western blot of the effect of 1-h treatment
with 100 nM PMA on the distribution of PKC . This provides further
evidence for the specificity of the PKC antibody in recognizing
Xenopus PKCI. B: transport properties of these
cells were severely inhibited by PMA, consistent with a previous report
by Els et al. (15). The increase of
RT with PMA's inhibition of
Isc provides further evidence for the integrity
of the cells under our experimental conditions.
|
|
Role of PKC
in feedback stimulation.
The above findings indicate that two different conditions that require
a sustained stimulation of Na+ transport are also
accompanied by a decrease of PKC
activity subsequent to a massive
decrease of its protein levels. Thus it follows that specific
inhibition of this kinase may result in sustaining higher rates of
transport. This hypothesis was tested as shown in Fig.
8. In these sets of experiments, we
examined the time course of the changes of Na+ transport
and the effects of specific PKC
inhibition with a cell-permeable
peptide inhibitor. The sequence of this inhibitor is derived from the
C2 (Ca2+-binding) region of PKCs (35). Because
levels are not detectable, and because
does not appear to
participate in the response to aldosterone or CPDC, the effects of this
peptide can be interpreted in terms of inhibition of PKC
membrane
localization.

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Fig. 8.
Additional sustained stimulation of Na+
transport after prolonged inhibition of PKC . Experiments for
control, CDPC-, and aldosterone-treated cells were carried out as
previously described. Experiments with the cell-permeable PKC
inhibitor (C2-4 peptide) were carried out by adding 10 µM
peptide to the medium at time 0. The transient stimulation
observed in untreated cells was purely due to the solution exchange.
Data were normalized to the value of current immediately before the
solution exchange. n = 9, 11, and 11 for A,
B, and C, respectively. See text for additional
details.
|
|
As shown in the Fig. 8A, addition of this peptide did not
significantly affect Na+ transport over a 24-h period such
that the equivalent current was indistinguishable from that observed in
control untreated cells at 4, 18, and 24 h. On the other hand, the
recovery of current after prolonged addition of CDPC was enhanced with
the PKC
inhibitor such that the values at 2.5, 4, 18, and 24 h
were significantly higher than those observed with CDPC alone (Fig.
8B). As shown in Fig. 8C, a similar trend was
also observed with aldosterone treatment, except that the differences
were only marginally significant at 18 and 24 h (P < 0.07).
 |
DISCUSSION |
Numerous studies have examined the regulation of epithelial
Na+ channels. Although it is clear that many hormonal and
humoral factors regulate this channel, it is also accepted that other partially defined processes act on these channels to maintain homeostasis and attempt to restore activity in response to various experimental stimuli. It is a priori expected that such mechanisms would also affect the response of the channel to hormones and circulating factors, and, thus, a better understanding of these feedback homeostatic processes is essential to our understanding of
channel regulation in tight epithelia. We put forth the hypothesis that
such processes are likely to involve modulation of channel regulation
by kinases. We began by defining the PKC isoforms present in our model
system. Second, we tested the above hypothesis by determining the
effects of channel stimulation on an inhibitory PKC isoform, PKC
. We
report that sustained stimulation of Na+ channels by
hormonal and nonhormonal mechanisms is accompanied by specific, and
potentially nongenomic, downregulation of this kinase. Moreover, both
processes were additive in their downregulation of PKC
, indicating
distinct signaling cascades that converge at the level of this kinase.
We propose that this mechanism represents a novel regulatory pathway
invoked when cells require sustained increase of Na+ transport.
Effects of aldosterone.
It is well established that the actions of aldosterone are to cause
prolonged and sustained increases of Na+ transport. This is
in contrast to the effects of other renal hormones, e.g., antidiuretic
hormone (ADH), where the response is transient in nature. This
sustained increase is observed despite the presence of homeostatic
mechanisms that tend to inactivate Na+ channels. For
example, channel activation by aldosterone would tend to increase
[Na+]i, which in turn could inactivate the
apical membrane resident ENaC via various feedback and self-inhibitory
mechanisms (4, 18, 41). However, the response to
aldosterone is clearly not a transient one, and one such possibility is
the subsequent downregulation of kinases responsible for maintaining
feedback inhibition, such as PKC
.
PKC
has been previously implicated in the regulation of ENaC and the
response to aldosterone utilizing a pharmacological approach. Rokaw et
al. (33, 34) found that the immunosuppressive agent
rapamycin causes a transient (peak 1 h) stimulation of the Isc in A6 cells. This was later confirmed by Yue
and colleagues (44) to be an effect on ENaC activity.
Short-term treatment with this agent was found to inhibit PKC activity,
despite what appears to be an increase in the membrane localization of
this isoform (see Fig. 4 in Ref. 33). Long-term treatment
(>18 h) with rapamycin was effective in nearly abolishing the
stimulation of Na+ transport observed with aldosterone.
Thus it is possible, and indeed likely, that the long-term effects of
rapamycin on aldosterone are related to the increased translocation of
PKC
in the membrane which, consistent with our hypothesis, would
tend to counteract the stimulatory effects of aldosterone.
The precise link between PKC
and aldosterone is unknown. Doolan and
Harvey (14) previously attributed some of the nongenomic and rapid (minutes) effects of aldosterone to stimulation of PKC. Until
recently, the isoforms involved in this response were undetermined. In
a recent followup (21), it was concluded that aldosterone directly stimulates PKC
activity in vitro. This effect was specific to PKC
and was observed in a cell-free system containing purified enzyme. In these experiments, addition of 1 µM aldosterone caused an
~30% increase of PKC
activity. These experiments provide
additional evidence in support of interaction between the
aldosterone-signaling pathway and the
-isoform of PKC. However, it
is important to recognize that the experiments of Doolan and Harvey
represent a different time scale than those in the current study
(minutes vs. 24 h) and that the effects on PKC
are opposite in
direction. Nonetheless, it is possible that the early stimulatory
effects of aldosterone on PKC
lead to prolonged inhibition. In this
case, the long-term effects would be more physiologically relevant in understanding the sustained increase of Na+ transport,
especially because PKC
is likely to inhibit ENaC (see below and
Refs. 33, 34, and 44).
A second possibility that may explain the link between aldosterone and
PKC
may involve changes of transport leading to potential changes of
[Na+]i. This possibility is further explored
with the addition of CDPC (see below). In either case, it is important
to recognize that the findings from the Northern blot experiments are
consistent with no direct effects of aldosterone on the PKC
gene.
This does not rule out the possibility of indirect genomic effects on a PKC
protease or other upstream regulators of PKC
protein levels. Unfortunately, these issues could not be directly assessed, because experiments utilizing actinomycin D resulted in complete dissolution of
polarized A6 cells at concentrations (5 µM) routinely used to inhibit
de novo protein synthesis in other epithelial preparations (data not
shown). Nonetheless, the similarity of these effects to those observed
with CDPC argue against direct genomic effects.
Effects of CDPC: feedback regulation.
As mentioned in RESULTS, CDPC was used to decrease
Na+ transport in the presence of aldosterone to test the
role of [Na+]i. These experiments were chosen
in favor of those with reduced external apical [Na+],
because custom formulation of Na+-free medium is
prohibitively costly and, moreover, appreciable basolateral
Na+ back-leak would drastically alter the
[Na+] in the external vicinity of the channel. We found
that CDPC also decreased PKC
protein levels and that this decrease
was additive with aldosterone. These changes coincided with the ability of the cells to counteract the inhibitory effects of this passive blocker and restore Na+ transport to control levels either
in the absence or presence of aldosterone. Because the
Isc returned to pre-CDPC levels, it is likely
that the steady-state [Na+]i was similar to
that in the absence of CDPC. In this case, it is unlikely that
prolonged changes in [Na+]i are the simple
explanation that ties in the effects of aldosterone and CDPC. However,
it remains possible that the time course of changes of
[Na+]i is important in setting in motion a
prolonged signaling cascade that may over the course of 24 h
affect PKC
protein levels. This hypothesis awaits further
experimental verification.
The stimulation observed with CDPC is similar to that observed with a
variety of other maneuvers that alter Na+ transport and has
been historically termed feedback regulation. This is a homeostatic
mechanism that represents the first line of cellular regulation of
Na+ transport. This process is thought to involve potential
direct effects of the Na+ ions, a process termed
self-inhibition, in addition to delayed cell signaling-mediated effects
termed feedback inhibition. Two previous reports from the Palmer and
Eaton groups (17, 25) indicate that the latter phenomenon
involves the activation of PKC leading to inhibition of apical ENaCs.
Our findings extend those observations to specifically implicate PKC
and to provide additional evidence supporting the notion that
downregulation of this isoform is accompanied by feedback stimulation
of Na+ transport. Our findings from Western blotting are
also functionally validated, as further stimulation of this process is
observed by the addition of a specific PKC
inhibitor.
Isform-specific regulation by PKC
.
Our findings of isoform-specific regulation of a physiological process
in tight epithelia is consistent with the emerging hypothesis of
distinct roles of PKC isoforms (see Introduction). In the present
study, we find that specific inhibition of PKC
with a cell-permeable
peptide inhibitor did not alter baseline rates of transport. After the
initial transient stimulation observed with cell feeding and solution
exchange, the Isc returned to values not
different from control. This indicates little or no regulation of
baseline rates of transport by PKC
(Fig. 8). These data are in
contrast to the observations from other laboratories examining the
baseline regulation by PKC. An important difference between the
majority of previously published observations and our present findings
is that we are dealing with inhibition of a specific PKC isoform.
Because it is our overall hypothesis that the regulation of ENaC by PKC
is isoform specific, it is nearly impossible to compare our present
findings with those using pharmacological agents that affect multiple
isoforms in all three PKC subtypes.
However, two previously published reports indicate that rapamycin
inhibits Na+ transport at the macroscopic (33)
and microscopic (44) levels. Because rapamycin has been
elegantly demonstrated by Rokaw et al. (33) to affect
purified PKC
in vitro, and because the effects of rapamycin could be
mimicked by inhibiting conventional PKC with a myristoylated
pseudosubstrate peptide, these data would appear to contradict our
conclusion of no baseline regulation by this isoform. A major
difference between those studies and ours is that our inhibitor binds
to the C2 domain (Ca2+ binding) of PKCs and inhibits their
translocation to the plasma membrane (35), whereas the
peptide used by Rokaw et al. acts as a pseudosubstrate for cPKCs. Thus,
in our case, we expect reduced membrane distribution of PKC
with
little or no effects on the activity of cytoplasmic PKC, whereas in the
experiments of Rokaw et al., we expect reduced activity throughout the
cell. A second possibility is differences in the levels of PKC
or
the presence of PKC
between cells grown under our conditions and
those of Rokaw et al. (33) and Yue et al.
(44). Because PKC
levels are not detectable under our
conditions, effects of these inhibitors on this isoform can be ignored.
A third potential explanation is that the previously published
experiments with rapamycin are short term, whereas ours extend to the
time scale of many hours (24 h). Thus it is possible that the
short-term stimulatory effects of rapamycin are masked in our
experiments by the transient effects observed from the medium exchange
that result in current stimulation by itself. On the other hand, the
prolonged effects of PKC
inhibition, which are not complicated by
the feeding transient, also indicate a lack of transport inhibition.
However, these data may be consistent with the experiments with
rapamycin and the cPKC pseudosubstrate, because prolonged incubation
with these agents may eventually lead to the return of transport to
baseline levels. This indeed is the trend observed after 60 min from
the data of Rokaw et al. (see Fig. 3 in Ref. 33).
Resolution of these issues awaits the development of additional
isoform-specific agonists/antagonists.
Our conclusions are different with CDPC and aldosterone, where the
Isc was sustained at 25 and 45% higher levels
(24 h), respectively. Because PKC
levels are already downregulated
by aldosterone and CDPC, it is not expected that further
pharmacological inhibition will result in dramatic changes in the
sustained level of stimulation. Thus the observed small additional
stimulation is entirely consistent with this explanation. Moreover,
in both cases (CDPC and aldosterone treatment), the changes of
transport were apparent at longer time points, consistent with the
hypothesis that PKC
is downregulated under conditions where
long-term stimulation of Na+ transport is required.
In conclusion, we have described the PKC isoforms present in A6
epithelia. We have also identified a novel mechanism for the regulation
of Na+ transport in these epithelia involving a specific
isoform, PKC
. Two different conditions that require sustained
Na+ transport resulted in the downregulation of this
isoform. These effects are not mediated via steady-state changes of
[Na+]i, because aldosterone and CDPC are
expected to have opposite effects on this parameter. This can also be
ruled out because the Isc and presumably
[Na+]i are essentially the same at 24 h
between untreated and CDPC-treated tissues. The decrease of PKC
protein levels is unlikely to be a direct genomic effect, because
PKC
mRNA levels are unchanged and, moreover, it is unexpected that a
passive channel blocker would exhibit genomic effects. The simplest
explanation for our findings is that stimulation of ENaC is sustained
by downregulation of PKC
through novel signaling mechanisms yet to
be defined. It is noteworthy that the transient stimulation of
Na+ transport observed in various epithelia in response to
ADH has been linked to secondary activation of PKC and, more
specifically, Ca2+-dependent cPKC isoforms
(1). Thus it seems plausible that PKC
may be under
precise cellular control by various feedback and feedforward mechanisms
that impinge on long-term regulation of Na+ transport.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-55626.
 |
FOOTNOTES |
Address for reprint requests and other correspondence:
M. S. Awayda, Dept. of Physiology, SL 39, Tulane University
Health Sciences Center, New Orleans, LA 70112 (E-Mail:
mawayda{at}tulane.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.
May 29, 2002;10.1152/ajpcell.00142.2002
Received 29 March 2002; accepted in final form 23 May 2002.
 |
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