Antisense oligodeoxynucleotide to PKC-
blocks
1-adrenergic activation of
Na-K-2Cl cotransport
Carole M.
Liedtke and
Thomas
Cole
The Cystic Fibrosis Center and Departments of Pediatrics and of
Physiology and Biophysics, Case Western Reserve University,
Cleveland, Ohio 44106
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ABSTRACT |
A role for protein kinase C (PKC)-
and -
isotypes in
1-adrenergic
regulation of human tracheal epithelial Na-K-2Cl cotransport was
studied with the use of isotype-specific PKC inhibitors and antisense
oligodeoxynucleotides to PKC-
or -
mRNA. Rottlerin, a PKC-
inhibitor, blocked 72% of basolateral-to-apical, bumetanide-sensitive 36Cl flux in
nystatin-permeabilized cell monolayers stimulated with methoxamine, an
1-adrenergic agonist, with a
50% inhibitory concentration of 2.3 µM. Methoxamine increased PKC
activity in cytosol and a particulate fraction; the response was
insensitive to PKC-
and -
II
isotype-specific inhibitors, but was blocked by general PKC inhibitors
and rottlerin. Rottlerin also inhibited methoxamine-induced PKC
activity in immune complexes of PKC-
, but not PKC-
. At the subcellular level, methoxamine selectively elevated cytosolic PKC-
activity and particulate PKC-
activity. Pretreatment of cell
monolayers with antisense oligodeoxynucleotide to PKC-
for 48 h
reduced the amount of whole cell and cytosolic PKC-
, diminished whole cell and cytosolic PKC-
activity, and blocked
methoxamine-stimulated Na-K-2Cl cotransport. Sense oligodeoxynucleotide
to PKC-
and antisense oligodeoxynucleotide to PKC-
did not alter
methoxamine-induced cotransport activity. These results demonstrate the
selective activation of Na-K-2Cl cotransport by cytosolic PKC-
.
bumetanide; immunoprecipitation; tracheal epithelial cells; subcellular fractionation; nystatin permeabilization; transepithelial
chloride flux
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INTRODUCTION |
IN THE LARGE AIRWAYS, epithelial cells regulate fluid
and electrolyte transport in response to hormonal and environmental stimuli. Na-K-2Cl cotransport is a critical electrolyte transporter required for Cl secretion in epithelia, particularly of the airways. Hormone stimulation elicits Cl secretion through the activation of
basolateral Na-K-2Cl cotransport and K channels and apical Cl channels
called cystic fibrosis transmembrane regulators (CFTR). Cotransport is
critical for supplying Cl for secretion and hence has been the focus of
studies in the laboratory. A major signaling mechanism for activation
of Na-K-2Cl cotransport is
-adrenergic-mediated hydrolysis of
phosphatidylinositol bisphosphate, which leads to generation of
inositol trisphosphate and lipid mediators, including diacylglycerol
(DAG) (18, 19). Transient generation of DAG leads to activation of
protein kinase C (PKC) in a time frame coincident with activation of
Na-K-2Cl cotransport (20). Phorbol 12-myristate 13-acetate (PMA), a
tumor-promoting phorbol ester, mimics
-adrenergic activation of
cotransport and also induces secretion in tracheal epithelial cells
that is not as vigorous as adenosine 3',5'-cyclic
monophosphate (cAMP)-stimulated secretion (6, 19). More detailed
studies show that short-term PMA treatment activates
bumetanide-sensitive Cl transport and that long-term treatment with PMA
blunts
-adrenergic- or PMA-stimulated bumetanide-sensitive cotransport (19). Pretreatment of cells with low concentrations of
staurosporine, a PKC inhibitor, also blocks
-adrenergic- or PMA-induced cotransport activation. It is now recognized that PMA
regulates Cl secretion in some epithelia expressing CFTR (3, 6, 13, 28)
by a mechanism that involves PKC-induced phosphorylation (5). How PKC
targets multiple electrolyte transporters required for Cl secretion is
not understood.
Molecular cloning of PKC demonstrates that PKCs comprise a family of
serine-threonine protein kinases that are typically activated by the
second messenger DAG. This multigene family is classified as
conventional PKC (cPKC) isotypes (
,
I,
II,
) that require Ca2+, DAG, and phosphatidylserine
(PtdSer) for activation, novel PKC (nPKC) isotypes (
,
,
) that
are Ca2+ independent but require
either DAG-PtdSer or PMA-PtdSer for activation, and atypical PKC (aPKC)
isotypes (
,
, µ,
) that are dependent on PtdSer
for activation but are not affected by DAG,
Ca2+, or phorbol ester (22, 24).
PKC isotypes are differentially expressed in cells, sometimes with a
characteristic subcellular distribution (15, 17). In many cells,
activation by phorbol esters, hormones, or neurotransmitters leads to
differential translocation from cytosol to membrane and/or
activation of specific PKC isotypes (2, 8, 15). Recently, researchers
in this laboratory demonstrated that human tracheal epithelial cells
express five PKC isotypes, PKC-
,
-
II, -
, -
, and -
,
(20). PKC-
and -
were localized to a cytosolic fraction, and
PKC-
II and -
were evenly
distributed between cytosolic and particulate fractions. Stimulation
with PMA for 30 min induced a transient shift in PKC-
mass from
cytosol to a particulate fraction and increased PKC activity in
cytosolic and particulate fractions. More importantly, treatment with
the
-adrenergic agonist methoxamine for <1 min induced a transient shift in PKC-
and -
mass to a particulate fraction; this time frame coincides with activation of bumetanide-sensitive Na-K-2Cl cotransport.
These observations led us to hypothesize that PKC isotype(s) may
differentially regulate initial activation of apical and basolateral
electrolyte transporters required for Cl secretion. Classic paradigms
for the demonstration of a specific in vivo function of a single PKC
isotype have been limited to its translocation and/or phorbol
ester-induced downregulation. These approaches, as applied to tracheal
epithelial cells, provided evidence for PKC involvement in modulation
of cotransport but were not sufficient to allow differentiation of the
role of Ca2+-independent PKC-
and -
isotypes because prolonged PMA treatment did not deplete PKC
isotypes (20). Hence, in the present study, rottlerin, which is an
inhibitor of PKC-
, and antisense oligodeoxynucleotides specific for
PKC-
and PKC-
were used to gain further insight into the role of
these PKC isotypes in the early events leading to cotransport
activation. Oligodeoxynucleotides that hybridize to the
region of the AUG initiation codon for PKC-
and -
were selected
for these studies. Sequences were complementary to the translation
initiation region (nucleotides
6 to 10 for PKC-
and
6
to 12 for PKC-
) of mouse PKC-
and human PKC-
isotypes (16,
26). Sense oligodeoxynucleotides were also used as controls. This
approach allowed investigation of activation of cotransport in cells
deficient in a specific PKC isotype.
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MATERIALS AND METHODS |
Materials.
36Cl (specific activity 260 MBq/g
Cl, 7.5 mCi/g Cl) was purchased from ICN Radiochemical (Irvine, CA),
and [
-32P]ATP
(specific activity 111 TBq/mmol, 3,000 Ci/mol) was purchased from
Amersham (Arlington Heights, IL). Compounds CGP-41251 and CGP-53353
were generously provided by Dr. Doriano Fabbro (Ciba-Geigy, Basel,
Switzerland). Methoxamine HCl was supplied by Burroughs Wellcome
(Research Triangle Park, NC). An enhanced chemiluminescence kit was
purchased from Amersham, and the PKC assay system was purchased from
GIBCO-BRL (Gaithersburg, MD). Calphostin was purchased from Calbiochem
(La Jolla, CA), rottlerin and KN-93 were purchased from Research
Products International (Natick, MA), and nystatin was purchased from
Sigma (St. Louis, MO). Anti-PKC isotype-specific polyclonal antibodies
were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Unconjugated goat anti-rabbit immunoglobin G (IgG) was purchased from
Cappel, and horseradish peroxidase-labeled goat anti-rabbit IgG was
purchased from Bio-Rad (Hercules, CA). Anocell filter inserts were
purchased from Whatman (Fairfield, NJ), and Transwell-Clear Costar
filter inserts from were purchased from Fisher Scientific. Tissue
culture supplies were obtained from GIBCO-BRL. All other chemicals were
reagent grade.
Cell isolation and culture.
Tissue was obtained from human tracheas at the time of autopsy through
the Cystic Fibrosis Center, Case Western Reserve University. Epithelial
cells were isolated and seeded onto
4.52-cm2 filter inserts that were
coated with human placental collagen at a seeding density of 2.5 × 106 cells/filter. Culture
medium was changed at 48-h intervals until confluence was reached.
Confluence was assessed by microscopic examination of the cell
monolayer and by measurement of electrical resistance across the
monolayer
(Rmono).
Rmono was
quantitated with the use of chopstick electrodes and an epithelial
voltohmmeter (EVOM, World Precision Instruments, New Haven, CT). Values
were corrected for background resistance of filter alone bathed in medium. Monolayers were used for experiments between
day 7 and day
9 in culture. Mean
Rmono of
untreated monolayers (n = 21) was
1,328.0 ± 126
· cm2 (Table
1).
Oligodeoxynucleotide treatment of cells.
Phosphodiester oligodeoxynucleotides were purchased from GIBCO-BRL.
Antisense oligodeoxynucleotides were complementary to the translation
initiation region of mRNA specific for mouse PKC-
(AGGGTGCCATGATGGA)
(26) and human PKC-
(GCTCCCTTCCATCTTGGG) (16). Sense
oligodeoxynucleotides to PKC-
(TCGATCATGGCACCCT) and PKC-
(CCCAAGATGGAAGGGAGC) were used as controls. Oligodeoxynucleotides were
dissolved in sterile deionized water to a final concentration of 1 mM,
separated into aliquots, and stored at
20°C until ready for
use.
Oligodeoxynucleotides were added to inside wells of confluent cell
cultures in combination with a cationic lipid
N-[1, (2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) and dioleoyl phosphotidylethanolamine (DOPE) (Lipofectin reagent, GIBCO-BRL) as directed by the manufacturer. Preliminary experiments were performed to optimize concentrations of
oligodeoxynucleotide and Lipofectin using bumetanide-sensitive basolateral-to-apical 36Cl flux as
a functional test. Maximal inhibition of radiolabeled electrolyte
fluxes was obtained with 1 µg/ml Lipofectin and 1 µM
oligodeoxynucleotide. This concentration of Lipofectin did not alter
baseline bumetanide-sensitive 36Cl
flux (Fig. 1). Cells were incubated with
oligodeoxynucleotide plus cationic lipid in serum-free and
antibiotic-free culture medium. Oligodeoxynucleotide incubation medium
was replaced every 12 h for 48 h. After this time and just before use
in experiments, Rmono was
measured using an EVOM. Filters were matched by
Rmono and type of
pretreatment for experiments. Mean
Rmono of cultures was not significantly altered by application of Lipofectin or oligodeoxynucleotides (Table 1).

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Fig. 1.
Effect of Lipofectin reagent on basolateral-to-apical
bumetanide-sensitive Cl flux in nystatin-permeabilized tracheal
epithelial cell monolayers. Cells were untreated
(A) or incubated with 1 µg/ml
Lipofectin (B). Medium was replaced
every 10-12 h for 48 h. At end of incubation period, cell
monolayers were permeabilized at apical surface with nystatin and
bumetanide-sensitive 36Cl flux
from basolateral to apical compartments was measured as described in
MATERIALS AND METHODS. Cell monolayers
were pretreated for 10 min with rottlerin before initiation of
36Cl transport. Basal,
unstimulated flux was measured for 10 min, then cells were stimulated
with vehicle ( ), 10 µM methoxamine (MOX, ), or MOX + 10 µM
rottlerin ( ); all additions were to basolateral medium. Samples of
apical medium were taken at 2.5-min intervals for 10 min. Data on
ordinate represent cumulative amount of bumetanide-sensitive 36Cl
recovered from apical compartment up to and including sample taken at
indicated time. Data are means for experiments on 5 different sets of
cell cultures. * P < 0.02 and
** P < 0.005, compared with cells treated with vehicle;
# P < 0.02, compared with cells treated with MOX alone.
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Measurement of Na-K-2Cl cotransport.
After EVOM readings were taken, cell cultures were preincubated for 10 min at 37°C after addition of vehicle or 50 µM bumetanide in the
basolateral solution consisting of
Ringer-N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.5 (11). Monolayers were then preincubated for 10 min
with 175 U/ml nystatin in an apical cytosolic salt solution containing
(in mM) 110 KCl, 20 NaCl, 2 EGTA, 1.0 MgSO4· 7H2O,
and 10 HEPES, at pH 7.5. The concentration of nystatin used was
determined from a dose-response relationship between nystatin concentration and apical 36Cl
efflux from cells preincubated with
36Cl (29). Preliminary studies
demonstrated that a concentration of 175 U/ml nystatin lowered
Rmono by 74%
from baseline of 850
· cm2
(n = 5) to 221
· cm2
(n = 5) and increased apical
36Cl efflux from 0.24 ± 0.06 min
1
(n = 4) to 0.66 ± 0.1 min
1
(n = 4). The rate of apical
36Cl efflux after treatment with
nystatin was equivalent to a forskolin-induced 36Cl basolateral-to-apical flux of
0.62 ± 0.01 min
1
(n = 4). The similarity between the
rate of Cl efflux attained with nystatin and forskolin indicates that
175 U/ml nystatin increased apical Cl permeability, and therefore this
concentration of nystatin was used in subsequent experiments. In some
experiments, immediately after addition of nystatin to the apical
surface, the PKC-
inhibitor rottlerin (10) was added to the
basolateral solution at a 10 µM final concentration.
To initiate transmonolayer flux, 1 µCi
36Cl was added to the basolateral
solution. At 2.5-min intervals, the apical cytosolic medium was
aspirated, transferred to scintillation vials for radioactive counts,
and replaced with an equal volume of medium of the same composition
containing nystatin. Immediately after sampling at 10 min, we added
methoxamine to the basolateral solution to a final concentration of 10 µM, and sampling at 2.5-min intervals continued for 10 min. After the
last sampling, cell monolayers were washed in 1% phosphate-buffered
saline (PBS) and then extracted with 0.5 ml 0.1 N NaOH. Aliquots of the
cell extract were assayed for protein content. The basolateral
perfusate was sampled for radioactive counts to calculate the specific
activity of 36Cl in the
basolateral medium. The accumulation of
36Cl in the apical compartment was
calculated as micromoles Cl per milligrams protein over time.
Immunoblot analysis of PKC isotypes.
Culture medium was replaced with Hanks' balanced salt solution
supplemented with 10 mM HEPES. Cells were treated with vehicle or drugs
of interest at 37°C for times indicated in legends of Figs.
1-4 and 6 and Tables 2 and 3. Cultures were
immediately washed two times with ice-cold PBS and then were harvested
in 1 ml hypotonic buffer containing 1 mM vanadate, 0.1 mM leupeptin, 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF) hydrochloride, and
1 mg/ml aprotinin. Immunoblot analysis of proteins separated on 8%
sodium dodecyl sulfate-polyacrylamide gels with the use of polyclonal
antibodies to PKC-
or -
was performed as previously described
(20). We detected protein bands immunoreactive to PKC isotype-specific
antibodies using enhanced chemiluminescence and analyzed the bands by
laser densiometry in a Sciscan 5000 (United States Biochemical) using
OS-Scan Image Analysis System software package (Oberlin Scientific).
Immunoprecipitation and measurement of immune complex activity of
PKC isotypes.
Primary cell cultures grown on tissue culture plastic were stimulated
with vehicle or the drug of interest at varying time intervals. Cells
were harvested in 1 ml lysis buffer consisting of 100 mM NaCl, 50 mM
NaF, 50 mM tris(hydroxymethyl)aminomethane (Tris) · HCl, pH 7.55, 1% Nonidet P-40,
0.25% sodium deoxycholate, 1 mM EDTA, 1 mM ethylene
glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA), 1 mM sodium vanadate, and the protease inhibitors as
described. Lysates were clarified and incubated with antiserum against
a specific PKC isotype, as previously described (20). In some
experiments, cell monolayers were harvested in hypotonic buffer
consisting of 50 mM Tris · HCl (pH 7.5), 2 mM EGTA, 5 mM MgCl2, 10 µM AEBSF, and 20 µg/ml leupeptin and were sonicated and fractionated by differential
centrifugation. Specific PKC isotypes were immunoprecipitated from
subcellular fractions for assay of immune complex kinase activity.
Kinase activity was measured as previously described (20).
Measurement of PKC activity.
PKC activity was measured with the use of Ac-MBP(4-14)
(AcQKRPSQRSKYL), a substrate peptide that is based after
myosin basic protein sequence and acetylated at the
NH2-terminus, in a PKC assay
system (GIBCO), according to the manufacturer's instructions.
Data analysis.
Protein levels were determined with a Bradford assay kit with the use
of bovine serum albumin as the standard. Data were analyzed by analysis
of variance (ANOVA) followed by Bonferroni multiple-comparison tests or
by Student's t-test for unpaired
samples. Data are means ± SE for the number of cell monolayers
tested.
 |
RESULTS |
Effect of selective inhibition of PKC-
on activation
of cotransport.
Previous studies by this laboratory demonstrated
1-adrenergic stimulation of
Na-K-2Cl cotransport that was blocked by the PKC inhibitor
staurosporine (19).
1-Adrenergic stimulation also
increased the activity of immunoprecipitated PKC-
and -
but had
no effect on PKC-
and -
II,
indicating selective activation of
Ca2+-independent PKC isotypes
(20). To determine the contribution of PKC-
to the regulation of
cotransport, we used rottlerin, a PKC inhibitor with a reported 50%
inhibitory concentration (IC50) for PKC-
of 3-6 µM (10). Rottlerin is 5- to 10-fold more
potent at blocking PKC-
than
Ca2+-dependent PKC-
, -
, and
-
and 13- to 33-fold more potent for PKC-
than for PKC-
, -
,
and -
. Functional studies were performed on cell monolayers grown on
filter inserts and treated at the apical surface with nystatin (11), an
antibiotic that permeabilizes plasma membranes to small monovalent
ions, including Cl. This technique allows study of basolateral Na-K-2Cl
cotransport under conditions in which the rate of cotransport is not
limited by apical Na and Cl permeability.
Figure 1 shows that 10 µM rottlerin strongly inhibited activation of
cotransport by methoxamine, an
1-adrenergic agonist. The
IC50 value for rottlerin
inhibition of cotransport was 2.28 ± 0.26 (n = 3) µM, a value comparable to
the reported inhibition constant for rottlerin inhibition of PKC-
(10). As seen in Table 2, methoxamine also
increased PKC activity in cytosolic and particulate fractions, as
previously reported (20). To ascertain the PKC isotype contributing to
this response, a panel of PKC inhibitors was used to block all PKC
isotypes, cPKC isotypes, or PKC-
. The current study shows that
treatment with Go-6976, an inhibitor of PKC-
and -
, or with
CGP-53353, an inhibitor of
PKC-
II, did not prevent
1-adrenergic-induced
increase in PKC activity in cytosolic and particulate
fractions (Table 2). In contrast, staurosporine, calphostin, CGP-41251
(a compound derived from staurosporine), and rottlerin blocked the
response to methoxamine. Inhibition by rottlerin suggests that
methoxamine activated PKC-
; this conclusion was confirmed by
quantitating kinase activity of immunoprecipitated PKC-
and -
.
The data of Fig. 2 show that methoxamine
significantly increased PKC-
and -
activities, but pretreatment
with rottlerin for 10 min inhibited methoxamine-stimulated PKC-
activity without altering stimulated PKC-
activity. Thus methoxamine
activates Ca2+-independent PKC
isotypes, with PKC-
serving as a major contributor to the response.

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Fig. 2.
Effect of rottlerin on kinase activity of immunoprecipitated protein
kinase C (PKC)- (A) and -
(B). Cells were incubated with
vehicle or 10 µM MOX or pretreated with 10 µM rottlerin for 10 min
before stimulation with MOX. After 40 s incubation, cells were lysed
with detergent buffer. PKC isotypes were immunoprecipitated and
immediately assayed for kinase activity as described in
MATERIALS AND METHODS. Data are means ± SE for individual experiments with cells from 6 different cell
preparations. * P < 0.02, compared with cells treated with vehicle;
# P < 0.01, compared with cells treated with MOX alone.
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Regulation by PKC activity is thought to require translocation of PKC
from cytosol to a particulate fraction (2, 8, 15). To determine whether
1-adrenergic stimulation causes
translocation of activated PKC isotypes, we measured the activity of
PKC-
and -
in subcellular fractions. In contrast to prevailing
models, methoxamine differentially elevated cytosolic PKC-
activity
and particulate PKC-
activity (Fig. 3).
Moreover, as seen above, rottlerin blocked the increase in cytosolic
PKC-
activity but did not affect elevated PKC-
activity (Fig. 3).
On the other hand, calphostin inhibited elevated PKC-
and -
activities. The striking finding here is a selective activation of
cytosolic PKC-
activity despite an increase in the relative amount
of PKC-
in a particulate fraction.

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Fig. 3.
MOX differentially activates PKC isotypes at subcellular level. Where
indicated, cell monolayers were pretreated for 10 min with 50 µM
calphostin or 10 µM rottlerin before stimulation with MOX. Cells were
lysed in a hypotonic buffer and subjected to differential centrifugation to recover subcellular fractions, as previously described (20). PKC- (A) or -
(B) was isolated as immune
complexes, and kinase activity of immunoprecipitated enzyme was
measured on same day as cell fractionation procedures. Data are
calculated as PKC-isotype activity in MOX-treated cells divided by
PKC-isotype activity in cells treated with vehicle and are reported as
means ± SE for individual experiments with 4-6 different cell
preparations. * P < 0.01 and
** P < 0.001 compared with
vehicle (ratio of 1.0); # P < 0.04 and
## P < 0.01 compared with MOX alone.
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Effect of sense and antisense oligodeoxynucleotides on activation of
cotransport.
Another approach often used to investigate the role of PKC in a
physiological function is to downregulate PKC by prolonged incubation
with phorbol ester. One caveat with this method is that phorbol esters
bind to receptors in addition to PKC. In our hands, long-term treatment
with the phorbol ester PMA did not deplete PKC isotypes. Hence we used
antisense RNA technology to downregulate PKC isotypes. Tracheal
epithelial cells were cultured in the presence of antisense
oligodeoxynucleotide to PKC-
for 48 h. Cationic lipids have been
shown to increase the potency of antisense oligodeoxynucleotide and
therefore were used in this study to reduce the concentration of
oligodeoxynucleotide necessary to achieve a maximal effect. Preliminary
experiments demonstrated that Lipofectin at concentrations up to 1 µg/ml did not affect baseline bumetanide-sensitive
36Cl transmonolayer flux (Fig. 1).
Table 3 illustrates results of 48-h
exposure to a combination of Lipofectin and various concentrations of
antisense oligodeoxynucleotide to PKC-
. Antisense
oligodeoxynucleotide to PKC-
caused a concentration-dependent
inhibition of methoxamine-stimulated cotransport with maximal
inhibition at 1 µM. At this concentration, rottlerin-sensitive
cotransport was also abolished (Fig. 4,
left). Lower concentrations of
antisense oligodeoxynucleotide to PKC-
did not significantly affect
the stimulatory effect of methoxamine. Cells exposed to sense
oligodeoxynucleotide to PKC-
or to antisense oligodeoxynucleotide to
PKC-
retained the stimulatory response to methoxamine and, further,
displayed rottlerin-sensitive stimulation, indicating that PKC-
is
necessary for cotransport activation (Fig. 4).

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Fig. 4.
Inhibition of MOX-stimulated Na-K-2Cl cotransport by antisense
oligodeoxynucleotide to PKC- . Monolayer cultures were incubated with
sense oligodeoxynucleotide to PKC-
(B) or antisense
oligodeoxynucleotide to PKC- (A)
or - (C) at 1 µM final
concentration + 1 µg/ml Lipofectin for 48 h. Basolateral-to-apical
bumetanide-sensitive 36Cl flux was
measured as described in Fig. 1 and MATERIALS AND METHODS. Cells were stimulated with vehicle ( ), 10 µM MOX ( ), or MOX + 10 µM rottlerin ( ). Data are reported as
means for 4-6 different cell cultures.
* P < 0.001 compared with
cells treated with vehicle;
# P < 0.003 compared with cells treated with methoxamine alone.
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PKC isotype expression and activity in monolayers treated with
oligodeoxynucleotides.
Inhibition of cotransport activation by antisense oligodeoxynucleotide
to PKC-
could be the result of a decrease in the amount of PKC-
,
diminished elevation in PKC-
activity in response to methoxamine, or
both. PKC-
expression was assessed by immunoblot analysis of cytosol
and a particulate fraction from cells treated with antisense
oligodeoxynucleotide to PKC-
and, as a control, from
Lipofectin-treated cells. Figure
5A
illustrates typical results from one of five experiments. Treatment
with antisense oligodeoxynucleotide to PKC-
reduced cytosolic
PKC-
to 20.3 ± 3.1% (n = 3) of
control levels and decreased particulate PKC-
to 38.7 ± 5.4%
(n = 3) of control levels. The amount
of PKC-
, -
, and -
were not affected (Fig.
5B). Sense oligodeoxynucleotide to
PKC-
and antisense oligodeoxynucleotide to PKC-
did not alter the
abundance of PKC-
(data not shown).

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Fig. 5.
Effect of antisense oligodeoxynucleotide to PKC- on amount of PKC
isotype. A: PKC- expression. Cell
monolayers were treated for 48 h with Lipofectin reagent alone (LPF) or
with Lipofectin and 1 µM antisense (AS) oligodeoxynucleotide to
PKC- . Cell monolayers were harvested in hypotonic buffer and
fractionated into cytosol (C) and a particulate (P) fraction, as
previously described (20). Lanes were loaded with 20 µg protein.
Immunoblots were probed with polyclonal antibody to PKC- .
Recombinant PKC- (STD PKC- ) is in lane
1. Molecular mass of PKC- is indicated.
B: PKC isotype expression. Cells were
untreated (UT) or treated with antisense oligodeoxynucleotide to
PKC- . Detergent lysates of cell monolayers were subjected to
immunoblot analysis for the indicated PKC isotypes. Lanes were loaded
with 20 µg protein.
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To determine whether a decrease in PKC-
was associated with
diminished kinase activity, PKC-
was immunoprecipitated from cells
treated with antisense oligodeoxynucleotide PKC-
and kinase activity
was measured using histone III-S as a substrate. For comparison, kinase
activity of immunoprecipitated PKC-
was measured in the same cells.
The data of Table 4 show that antisense
oligodeoxynucleotide to PKC-
blocked a methoxamine-mediated increase
in PKC-
activity. In contrast, methoxamine increased kinase activity
of PKC-
. To assess whether treatment of cells with
oligodeoxynucleotide could nonspecifically affect PKC-
activity, the
response to methoxamine was measured in cells that were transfected
with antisense oligodeoxynucleotide to PKC-
. This treatment did not
affect
-adrenergic stimulation of PKC-
activity or its
sensitivity to rottlerin but did prevent an increase in PKC-
activity. The results imply that loss of cytosolic PKC-
results in
loss of PKC-
activity. This conclusion was tested at the subcellular
level in cells treated with antisense oligodeoxynucleotide to PKC-
followed by stimulation with methoxamine. Antisense treatment
significantly reduced methoxamine-stimulated cytosolic PKC-
activity
but did not alter particulate PKC-
activity (Fig.
6). The results indicate that antisense
oligodeoxynucleotide to PKC-
attenuates activation of Na-K-2Cl by
methoxamine by lowering the amount of cytosolic PKC-
, thus
diminishing cytosolic PKC-
activity.

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Fig. 6.
Effect of antisense oligodeoxynucleotides to PKC- on PKC-
activity. Cell monolayers were treated for 48 h with Lipofectin reagent
alone or with Lipofectin and 1 µM antisense oligodeoxynucleotide to
PKC- . Kinase activity of immunoprecipitated PKC- was quantitated, as described in Fig. 3. Relative kinase activity is calculated as
PKC- activity in methoxamine-treated cells divided by PKC- activity in cells treated with vehicle and is reported as mean ± SE
for 4 experiments. * P < 0.02 compared with Lipofectin-treated cells.
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 |
DISCUSSION |
PKC has been implicated in activation of Na-K-2Cl cotransport and in
the regulation of CFTR, an apical Cl channel that is defective in the
disease cystic fibrosis, in tracheal and nasal polyp epithelial cells
(3, 6, 19). Researchers in this laboratory recently reported the
expression of a panel of PKC isotypes in human tracheal epithelial
cells and the differential activation of two PKC isotypes, PKC-
and
-
, by methoxamine, an
1-adrenergic agonist that also
activates Na-K-2Cl cotransport in these cells (19, 20). Because
long-term treatment with the phorbol ester PMA for 18 h did not deplete
PKC isotype activity or abundance, downregulation of PKC with the use
of PMA cannot be used to investigate functional consequences of
increased PKC-
and -
activities. Hence, in the current study, we
took advantage of new isotype-specific PKC inhibitors and, more
importantly, of antisense technology, to induce a deficiency in one of
the PKC isotypes. The novel finding of a requirement for PKC-
for activation of Na-K-2Cl cotransport is the first demonstration of
differential regulation of epithelial cotransport by a specific PKC
isotype.
In permeabilized monolayers, the mallotoxin rottlerin blocked 71.8% of
1-adrenergic-mediated
bumetanide-sensitive Cl transport (Fig. 1). Rottlerin has been reported
to also inhibit
Ca2+-calmodulin-protein kinase
(10); hence it was important to verify that rottlerin mediated its
effects through blockage of PKC-
. For this reason, PKC activity in
whole cell fractions and immune complexes was determined using buffers
that were supplemented with KN-93, a water-soluble
Ca2+-calmodulin-protein kinase II
inhibitor.
1-Adrenergic
stimulation elevated cytosolic and particulate PKC activity 2.0- and
2.9-fold, respectively (Table 2). The PKC inhibitors Go-6976 and
CGP-53353 failed to block this response, indicating that cPKC isotypes
PKC-
and -
II were not
activated by methoxamine. This finding, together with inhibition by the
general PKC inhibitors staurosporine, calphostin, and CGP-41251 and
rottlerin, a PKC-
inhibitor, demonstrates that rottlerin blocks an
1-adrenergic-mediated increase
in Ca2+-independent PKC isotypes,
most likely PKC-
. Measurement of kinase activity of
immunoprecipitated PKC-
and -
supported this conclusion (Figs. 2
and 3). Pretreatment of cells with rottlerin blocked increases in
PKC-
activity but did not prevent increases in PKC-
activity.
Similar results were observed at the subcellular level, where rottlerin
blocked an increase in cytosolic PKC-
activity but did not alter
elevated particulate PKC-
activity. These results demonstrate the
selectivity of rottlerin for PKC-
in tracheal epithelial cells. More
importantly, this study demonstrates, for the first time, that
1-adrenergic stimulation
specifically activates PKC-
and -
activities associated with
different subcellular fractions even though both PKC isotypes shift
from cytosol to a particulate fraction (20). These results suggest that
the two PKC isotypes target substrates in different subcellular
fractions for phosphorylation or, in the case of PKC-
, might
interact with an intermediary protein to induce activation of Na-K-2Cl
cotransport.
In a second approach, antisense techniques were used to investigate a
role for PKC-
and -
in methoxamine-induced activation of Na-K-2Cl
cotransport. The proposed mechanisms by which antisense oligodeoxynucleotides produce a specific effect include stimulation of
mRNA degradation by ribonuclease H, inhibition of new protein synthesis
by translational arrest, prevention of mRNA maturation and transport
out of the nucleus, and inhibition of gene transcription by formation
of a triple helix with DNA (30). The specific site of mRNA to which
antisense oligodeoxynucleotides hybridize and the chemical
characteristics of the oligodeoxynucleotides are also thought to be
contributing factors (26). In this study, antisense
oligodeoxynucleotides to the translation initiation region of mRNA for
PKC-
or -
were selected because this region is single stranded
and because an oligodeoxynucleotide complementary to this region is
particularly effective in blocking mRNA processing, transport, or
translation (12, 23). Treatment of cell monolayers with antisense
oligodeoxynucleotide to PKC-
for 48 h potently blocked
methoxamine-induced Na-K-2Cl cotransport activation (Fig. 4). Sense
oligodeoxynucleotide to PKC-
and antisense oligodeoxynucleotide to
PKC-
did not alter the response to methoxamine (Fig. 4).
Antisense oligodeoxynucleotide to PKC-
also reduced cytosolic
PKC-
by 80% and particulate PKC-
by 62%, as detected by
immunoblot analysis (Fig. 5A).
Downregulation of the amount of PKC-
is in agreement with the
half-life of ~24 h for PKC in vitro. The specificity of antisense
oligodeoxynucleotide to PKC-
for PKC-
was assessed from
immunoblot analysis for other PKC isotypes. PKC-
, -
, and -
were apparently unaltered in cells treated with antisense
oligodeoxynucleotide to PKC-
(Fig. 6). Downregulation of PKC-
is
also indicated by diminished
1-adrenergic-stimulated whole
cell (Table 4) and cytosolic PKC-
activity (Fig. 6).
Oligodeoxynucleotides are reported to also act as aptamers that bind to
proteins in a partially or totally sequence-independent manner (27).
One recent study showed that phosphodiester or phosphothioate
oligodeoxynucleotide inhibited purified
PKC-
I (27). In the current
study, treatment with antisense oligodeoxynucleotide to PKC-
did not
affect PKC-
activity (Table 4). Likewise, treatment with antisense
oligodeoxynucleotide to PKC-
did not affect PKC-
activity. Thus
aptameric inhibition of PKC-
or -
by oligodeoxynucleotide is not
likely involved in mediating the effects of the antisense treatment.
Application of antisense techniques to other cells has been used to
investigate the relation of PKC isotypes to intracellular signaling
mechanisms and to functional events that occur within a rapid time
frame similar to that of
1-adrenergic stimulation of
human tracheal epithelial cotransport. For example,
vascular smooth muscle cells deficient in PKC-
failed to display
phorbol ester-induced activation of mitogen-activated protein kinase, indicating a role for this PKC isotype in the regulation of this superfamily of protein kinases (4). In Madin-Darby canine kidney cells, PKC-
was found necessary for the regulation of
phospholipase D (1) and phorbol ester-stimulated release of
arachidonate and its metabolites (9).
1-Adrenergic stimulation of
tracheal epithelial cells, in comparison, fails to activate PKC-
(20) and does not involve phospholipase D activation (14), suggesting a
target for PKC-
downstream of phospholipases. In another study, PKC-
was found necessary for inhibition of vasopressin-stimulated Na+ transport in rabbit cortical
collecting duct cells, suggesting cAMP-mediated activation of PKC-
(7). In human tracheal epithelial cells,
-adrenergic stimulation
rapidly activates Na-K-2Cl cotransport but is not sufficient to induce
Cl secretion (21). However, elevated cAMP levels are sufficient to
produce a sustained Cl secretion with a vigorous bumetanide-sensitive
Na-K-2Cl cotransport activity (25), suggesting cross talk between
cAMP-dependent protein kinase isoforms and PKC isotypes. Further
studies are necessary to discern whether cAMP regulates PKC-
in
tracheal epithelial cells.
In summary, the current studies show, for the first time, a requirement
for increased PKC-
activity during
1-adrenergic-stimulated Na-K-2Cl cotransport. Hormone stimulation is sensitive to rottlerin, a
PKC-
inhibitor, and is blocked in cells made deficient in PKC-
. The results indicate a critical role for PKC-
during hormonal regulation of Na-K-2Cl cotransport, most likely through
phosphorylation. The deduced primary structure of mammalian NKCC1, a
secretory isoform of Na-K-2Cl cotransport found in epithelia (25), with multiple putative PKC phosphorylation sites suggests a key role for PKC
in the regulation of cotransport activity. In addition, the results
point to a critical role for PKC isotypes in the regulation of a Cl
secretory response in mammalian tracheal epithelium.
 |
ACKNOWLEDGEMENTS |
This research was supported by grants HL-43907 and HL-50160 from
the National Heart, Lung, and Blood Institute.
 |
FOOTNOTES |
Address for reprint requests: C. M. Liedtke, Pediatric Pulmonology,
Case Western Reserve Univ., Biomedical Research Bldg., Rm. 827, 2109 Adelbert Rd., Cleveland, OH 44106-4948.
Received 12 March 1997; accepted in final form 15 July 1997.
 |
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