Antisense oligodeoxynucleotide to PKC-delta blocks alpha 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

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

A role for protein kinase C (PKC)-delta and -zeta isotypes in alpha 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-delta or -zeta mRNA. Rottlerin, a PKC-delta inhibitor, blocked 72% of basolateral-to-apical, bumetanide-sensitive 36Cl flux in nystatin-permeabilized cell monolayers stimulated with methoxamine, an alpha 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-alpha and -beta 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-delta , but not PKC-zeta . At the subcellular level, methoxamine selectively elevated cytosolic PKC-delta activity and particulate PKC-zeta activity. Pretreatment of cell monolayers with antisense oligodeoxynucleotide to PKC-delta for 48 h reduced the amount of whole cell and cytosolic PKC-delta , diminished whole cell and cytosolic PKC-delta activity, and blocked methoxamine-stimulated Na-K-2Cl cotransport. Sense oligodeoxynucleotide to PKC-delta and antisense oligodeoxynucleotide to PKC-zeta did not alter methoxamine-induced cotransport activity. These results demonstrate the selective activation of Na-K-2Cl cotransport by cytosolic PKC-delta .

bumetanide; immunoprecipitation; tracheal epithelial cells; subcellular fractionation; nystatin permeabilization; transepithelial chloride flux

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha -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 alpha -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 alpha -adrenergic- or PMA-stimulated bumetanide-sensitive cotransport (19). Pretreatment of cells with low concentrations of staurosporine, a PKC inhibitor, also blocks alpha -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 (alpha , beta I, beta II, gamma ) that require Ca2+, DAG, and phosphatidylserine (PtdSer) for activation, novel PKC (nPKC) isotypes (delta , epsilon , eta ) that are Ca2+ independent but require either DAG-PtdSer or PMA-PtdSer for activation, and atypical PKC (aPKC) isotypes (zeta , lambda , µ, phi ) 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-alpha , -beta II, -delta , -epsilon , and -zeta , (20). PKC-alpha and -zeta were localized to a cytosolic fraction, and PKC-beta II and -delta were evenly distributed between cytosolic and particulate fractions. Stimulation with PMA for 30 min induced a transient shift in PKC-delta mass from cytosol to a particulate fraction and increased PKC activity in cytosolic and particulate fractions. More importantly, treatment with the alpha -adrenergic agonist methoxamine for <1 min induced a transient shift in PKC-delta and -zeta 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-delta and -zeta isotypes because prolonged PMA treatment did not deplete PKC isotypes (20). Hence, in the present study, rottlerin, which is an inhibitor of PKC-delta , and antisense oligodeoxynucleotides specific for PKC-delta and PKC-zeta 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-delta and -zeta were selected for these studies. Sequences were complementary to the translation initiation region (nucleotides -6 to 10 for PKC-delta and -6 to 12 for PKC-zeta ) of mouse PKC-delta and human PKC-zeta 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.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials. 36Cl (specific activity 260 MBq/g Cl, 7.5 mCi/g Cl) was purchased from ICN Radiochemical (Irvine, CA), and [gamma -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 Omega  · cm2 (Table 1).

                              
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Table 1.   Integrity of cell cultures after treatment with oligodeoxynucleotide

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-delta (AGGGTGCCATGATGGA) (26) and human PKC-zeta (GCTCCCTTCCATCTTGGG) (16). Sense oligodeoxynucleotides to PKC-delta (TCGATCATGGCACCCT) and PKC-zeta (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 (black-square), 10 µM methoxamine (MOX, black-triangle), or MOX + 10 µM rottlerin (bullet ); 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.

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 Omega  · cm2 (n = 5) to 221 Omega  · 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-delta 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-delta or -zeta 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(beta -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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effect of selective inhibition of PKC-delta on activation of cotransport. Previous studies by this laboratory demonstrated alpha 1-adrenergic stimulation of Na-K-2Cl cotransport that was blocked by the PKC inhibitor staurosporine (19). alpha 1-Adrenergic stimulation also increased the activity of immunoprecipitated PKC-delta and -zeta but had no effect on PKC-alpha and -beta II, indicating selective activation of Ca2+-independent PKC isotypes (20). To determine the contribution of PKC-delta to the regulation of cotransport, we used rottlerin, a PKC inhibitor with a reported 50% inhibitory concentration (IC50) for PKC-delta of 3-6 µM (10). Rottlerin is 5- to 10-fold more potent at blocking PKC-delta than Ca2+-dependent PKC-alpha , -beta , and -gamma and 13- to 33-fold more potent for PKC-delta than for PKC-epsilon , -zeta , and -eta . 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 alpha 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-delta (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-delta . The current study shows that treatment with Go-6976, an inhibitor of PKC-alpha and -beta , or with CGP-53353, an inhibitor of PKC-beta II, did not prevent alpha 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-delta ; this conclusion was confirmed by quantitating kinase activity of immunoprecipitated PKC-delta and -zeta . The data of Fig. 2 show that methoxamine significantly increased PKC-delta and -zeta activities, but pretreatment with rottlerin for 10 min inhibited methoxamine-stimulated PKC-delta activity without altering stimulated PKC-zeta activity. Thus methoxamine activates Ca2+-independent PKC isotypes, with PKC-delta serving as a major contributor to the response.

                              
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Table 2.   Effect of PKC inhibitors on methoxamine-stimulated PKC activity


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Fig. 2.   Effect of rottlerin on kinase activity of immunoprecipitated protein kinase C (PKC)-delta (A) and -zeta (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.

Regulation by PKC activity is thought to require translocation of PKC from cytosol to a particulate fraction (2, 8, 15). To determine whether alpha 1-adrenergic stimulation causes translocation of activated PKC isotypes, we measured the activity of PKC-delta and -zeta in subcellular fractions. In contrast to prevailing models, methoxamine differentially elevated cytosolic PKC-delta activity and particulate PKC-zeta activity (Fig. 3). Moreover, as seen above, rottlerin blocked the increase in cytosolic PKC-delta activity but did not affect elevated PKC-zeta activity (Fig. 3). On the other hand, calphostin inhibited elevated PKC-delta and -zeta activities. The striking finding here is a selective activation of cytosolic PKC-delta activity despite an increase in the relative amount of PKC-delta 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-delta (A) or -zeta (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.

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-delta 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-delta . Antisense oligodeoxynucleotide to PKC-delta 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-delta did not significantly affect the stimulatory effect of methoxamine. Cells exposed to sense oligodeoxynucleotide to PKC-delta or to antisense oligodeoxynucleotide to PKC-zeta retained the stimulatory response to methoxamine and, further, displayed rottlerin-sensitive stimulation, indicating that PKC-delta is necessary for cotransport activation (Fig. 4).

                              
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Table 3.   Concentration-dependent decrease in cotransport activation by antisense oligodeoxynucleotide to PKC-delta


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Fig. 4.   Inhibition of MOX-stimulated Na-K-2Cl cotransport by antisense oligodeoxynucleotide to PKC-delta . Monolayer cultures were incubated with sense oligodeoxynucleotide to PKC-delta (B) or antisense oligodeoxynucleotide to PKC-delta (A) or -zeta (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 (black-square), 10 µM MOX (black-triangle), or MOX + 10 µM rottlerin (bullet ). 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.

PKC isotype expression and activity in monolayers treated with oligodeoxynucleotides. Inhibition of cotransport activation by antisense oligodeoxynucleotide to PKC-delta could be the result of a decrease in the amount of PKC-delta , diminished elevation in PKC-delta activity in response to methoxamine, or both. PKC-delta expression was assessed by immunoblot analysis of cytosol and a particulate fraction from cells treated with antisense oligodeoxynucleotide to PKC-delta and, as a control, from Lipofectin-treated cells. Figure 5A illustrates typical results from one of five experiments. Treatment with antisense oligodeoxynucleotide to PKC-delta reduced cytosolic PKC-delta to 20.3 ± 3.1% (n = 3) of control levels and decreased particulate PKC-delta to 38.7 ± 5.4% (n = 3) of control levels. The amount of PKC-alpha , -epsilon , and -zeta were not affected (Fig. 5B). Sense oligodeoxynucleotide to PKC-delta and antisense oligodeoxynucleotide to PKC-zeta did not alter the abundance of PKC-delta (data not shown).


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Fig. 5.   Effect of antisense oligodeoxynucleotide to PKC-delta on amount of PKC isotype. A: PKC-delta expression. Cell monolayers were treated for 48 h with Lipofectin reagent alone (LPF) or with Lipofectin and 1 µM antisense (AS) oligodeoxynucleotide to PKC-delta . 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-delta . Recombinant PKC-delta (STD PKC-delta ) is in lane 1. Molecular mass of PKC-delta is indicated. B: PKC isotype expression. Cells were untreated (UT) or treated with antisense oligodeoxynucleotide to PKC-delta . Detergent lysates of cell monolayers were subjected to immunoblot analysis for the indicated PKC isotypes. Lanes were loaded with 20 µg protein.

To determine whether a decrease in PKC-delta was associated with diminished kinase activity, PKC-delta was immunoprecipitated from cells treated with antisense oligodeoxynucleotide PKC-delta and kinase activity was measured using histone III-S as a substrate. For comparison, kinase activity of immunoprecipitated PKC-zeta was measured in the same cells. The data of Table 4 show that antisense oligodeoxynucleotide to PKC-delta blocked a methoxamine-mediated increase in PKC-delta activity. In contrast, methoxamine increased kinase activity of PKC-zeta . To assess whether treatment of cells with oligodeoxynucleotide could nonspecifically affect PKC-delta activity, the response to methoxamine was measured in cells that were transfected with antisense oligodeoxynucleotide to PKC-zeta . This treatment did not affect alpha -adrenergic stimulation of PKC-delta activity or its sensitivity to rottlerin but did prevent an increase in PKC-zeta activity. The results imply that loss of cytosolic PKC-delta results in loss of PKC-delta activity. This conclusion was tested at the subcellular level in cells treated with antisense oligodeoxynucleotide to PKC-delta followed by stimulation with methoxamine. Antisense treatment significantly reduced methoxamine-stimulated cytosolic PKC-delta activity but did not alter particulate PKC-delta activity (Fig. 6). The results indicate that antisense oligodeoxynucleotide to PKC-delta attenuates activation of Na-K-2Cl by methoxamine by lowering the amount of cytosolic PKC-delta , thus diminishing cytosolic PKC-delta activity.

                              
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Table 4.   Kinase activity of immunoprecipitated PKC isotypes


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Fig. 6.   Effect of antisense oligodeoxynucleotides to PKC-delta on PKC-delta activity. Cell monolayers were treated for 48 h with Lipofectin reagent alone or with Lipofectin and 1 µM antisense oligodeoxynucleotide to PKC-delta . Kinase activity of immunoprecipitated PKC-delta was quantitated, as described in Fig. 3. Relative kinase activity is calculated as PKC-delta activity in methoxamine-treated cells divided by PKC-delta activity in cells treated with vehicle and is reported as mean ± SE for 4 experiments. * P < 0.02 compared with Lipofectin-treated cells.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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-delta and -zeta , by methoxamine, an alpha 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-delta and -zeta 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-delta 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 alpha 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-delta . 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. alpha 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-alpha and -beta 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-delta inhibitor, demonstrates that rottlerin blocks an alpha 1-adrenergic-mediated increase in Ca2+-independent PKC isotypes, most likely PKC-delta . Measurement of kinase activity of immunoprecipitated PKC-delta and -zeta supported this conclusion (Figs. 2 and 3). Pretreatment of cells with rottlerin blocked increases in PKC-delta activity but did not prevent increases in PKC-zeta activity. Similar results were observed at the subcellular level, where rottlerin blocked an increase in cytosolic PKC-delta activity but did not alter elevated particulate PKC-zeta activity. These results demonstrate the selectivity of rottlerin for PKC-delta in tracheal epithelial cells. More importantly, this study demonstrates, for the first time, that alpha 1-adrenergic stimulation specifically activates PKC-delta and -zeta 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-delta , 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-delta and -zeta 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-delta or -zeta 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-delta for 48 h potently blocked methoxamine-induced Na-K-2Cl cotransport activation (Fig. 4). Sense oligodeoxynucleotide to PKC-delta and antisense oligodeoxynucleotide to PKC-zeta did not alter the response to methoxamine (Fig. 4).

Antisense oligodeoxynucleotide to PKC-delta also reduced cytosolic PKC-delta by 80% and particulate PKC-delta by 62%, as detected by immunoblot analysis (Fig. 5A). Downregulation of the amount of PKC-delta is in agreement with the half-life of ~24 h for PKC in vitro. The specificity of antisense oligodeoxynucleotide to PKC-delta for PKC-delta was assessed from immunoblot analysis for other PKC isotypes. PKC-alpha , -epsilon , and -zeta were apparently unaltered in cells treated with antisense oligodeoxynucleotide to PKC-delta (Fig. 6). Downregulation of PKC-delta is also indicated by diminished alpha 1-adrenergic-stimulated whole cell (Table 4) and cytosolic PKC-delta 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-beta I (27). In the current study, treatment with antisense oligodeoxynucleotide to PKC-delta did not affect PKC-zeta activity (Table 4). Likewise, treatment with antisense oligodeoxynucleotide to PKC-zeta did not affect PKC-delta activity. Thus aptameric inhibition of PKC-delta or -zeta 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 alpha 1-adrenergic stimulation of human tracheal epithelial cotransport. For example, vascular smooth muscle cells deficient in PKC-delta 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-alpha was found necessary for the regulation of phospholipase D (1) and phorbol ester-stimulated release of arachidonate and its metabolites (9). alpha 1-Adrenergic stimulation of tracheal epithelial cells, in comparison, fails to activate PKC-alpha (20) and does not involve phospholipase D activation (14), suggesting a target for PKC-delta downstream of phospholipases. In another study, PKC-epsilon was found necessary for inhibition of vasopressin-stimulated Na+ transport in rabbit cortical collecting duct cells, suggesting cAMP-mediated activation of PKC-epsilon (7). In human tracheal epithelial cells, alpha -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-delta in tracheal epithelial cells.

In summary, the current studies show, for the first time, a requirement for increased PKC-delta activity during alpha 1-adrenergic-stimulated Na-K-2Cl cotransport. Hormone stimulation is sensitive to rottlerin, a PKC-delta inhibitor, and is blocked in cells made deficient in PKC-delta . The results indicate a critical role for PKC-delta 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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Cell Physiol 273(5):C1632-C1640
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