Protein kinase C potentiates cAMP-stimulated mouse duodenal mucosal bicarbonate secretion in vitro

Bi-Guang Tuo, Jimmy Y. C. Chow, Kim E. Barrett, and Jon I. Isenberg{dagger}

Division of Gastroenterology, Department of Medicine, University of California, San Diego, School of Medicine, San Diego, California 92103-8413

Submitted 3 June 2003 ; accepted in final form 22 December 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PKC has been shown to regulate epithelial Cl- secretion in a variety of models. However, the role of PKC in duodenal mucosal bicarbonate secretion is less clear. We aimed to investigate the role of PKC in regulation of duodenal mucosal bicarbonate secretion. Bicarbonate secretion by murine duodenal mucosa was examined in vitro in Ussing chambers using a pH-stat technique. PKC isoform expression and activity were assessed by Western blotting and in vitro kinase assays, respectively. PMA (an activator of PKC) alone had no effect on duodenal bicarbonate secretion or short-circuit current (Isc). When PMA and dibutyryl-cAMP (db-cAMP) were added simultaneously, PMA failed to alter db-cAMP-stimulated duodenal bicarbonate secretion or Isc (P > 0.05). However, a 1-h preincubation with PMA potentiated db-cAMP-stimulated duodenal bicarbonate secretion and Isc in a concentration-dependent manner (from 10-8 to 10-5M) (P < 0.05). PMA preincubation had no effects on carbachol- or heat-stable toxin-stimulated bicarbonate secretion. Western blot analysis revealed that PKC{alpha}, -{gamma}, -{epsilon}, -{theta}, -µ, and -{iota}/{lambda} were expressed in murine duodenal mucosa. Ro 31–8220 (an inhibitor active against PKC{epsilon}, -{alpha}, -{beta}, and -{gamma}), but not Gö 6983 (an inhibitor active against PKC{alpha}, -{gamma}, -{beta}, and -{delta}), reversed the potentiating effect of PMA on db-cAMP-stimulated bicarbonate secretion. PMA also time- and concentration-dependently increased the activity of PKC{epsilon}, an effect that was prevented by Ro 31–8220 but not Gö 6983. These results demonstrate that activation of PKC potentiates cAMP-stimulated duodenal bicarbonate secretion, whereas it does not modify basal secretion. The effect of PKC on cAMP-stimulated bicarbonate secretion is mediated by the PKC{epsilon} isoform.

protein kinase C{epsilon}; bicarbonate secretion; phorbol 12-myristate 13-acetate; adenosine 3',5'-cyclic monophosphate


SECRETION OF BICARBONATE by the epithelium is an important defensive factor in duodenal mucosal protection against acid-peptic damage. Duodenal bicarbonate secretion is regulated by a number of agents including PGE2, cAMP, heat-stable toxin of Escherichia coli (STa), guanylin, and carbachol (12, 16, 26, 37).

PKC consists of a family of phospholipid-dependent serine-threonine kinases, which are widely distributed in mammalian tissues in a cell-specific pattern. At least 11 isoforms of PKC are currently known, and these are usually categorized into three distinct subtypes: conventional PKC (cPKC) isozymes ({alpha}, {beta}, and {gamma}), novel PKC (nPKC) isozymes ({delta}, {epsilon}, {eta}, µ, and {theta}), and atypical PKC (aPKC) isozymes ({zeta} and {iota}/{lambda}) (25, 31). Although PKC isoforms have many structurally similar elements, they vary in their sensitivity to activators and cofactors. cPKCs require Ca2+ for their activation, whereas nPKCs can be activated without Ca2+. Both cPKCs and nPKCs can be activated by phorbol 12-myristate 13-acetate (PMA). aPKCs require only phosphatidylserine for activation and, as a general rule, cannot be activated directly by PMA. PKC plays a key role in the regulation of ion transport, cell growth, and differentiation. It profoundly altered epithelial Cl- secretion (22, 2) and potentiated secretory responses evoked by cAMP in T84 (6), C127 (7), and pancreatic duct cells (36). In the gastrointestinal tract, PKC is also involved in regulation of gastric acid and pepsinogen secretion (21, 30). However, the role of PKC in duodenal mucosal bicarbonate secretion is less clear.

Goals of the present study were to 1) investigate the role of PKC in duodenal bicarbonate secretion, 2) define which PKC isoforms are expressed in the duodenal mucosa, and 3) investigate which PKC isoform(s) participate in the regulation of duodenal bicarbonate secretion.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and solutions. PMA, dibutyryl-cAMP (db-cAMP), carbachol, STa, and myelin basic protein were purchased from Sigma (St. Louis, MO). Ro 31–8220 and Gö 6983 were from Calbiochem (San Diego, CA). Monoclonal antibodies directed against PKC isoforms {alpha}, {beta}, {gamma}, {delta}, {epsilon}, µ, {theta}, and {iota}/{lambda} were from Transduction Laboratories (Lexington, KY). [{gamma}-32P]ATP, with a specific activity of 3,000 Ci/mmol, was purchased from New England Nuclear (Boston, MA). All other chemicals were obtained from Fisher Scientific (Santa Clara, CA). For Ussing chamber studies, the mucosal solution contained the following (in mM): 140 Na+, 5.4 K+, 1.2 Ca2+, 1.2 Mg2+, 120 Cl-, 25 gluconate, and 10 mannitol. The serosal solution contained (in mM): 140 Na+, 5.4 K+, 1.2 Ca2+, 1.2 Mg2+, 120 Cl-, 25 , 2.4 , 2.4 , 10 glucose, and 0.001 indomethacin. The osmolalities for both solutions were ~284 osmol/kg.

Animal preparation. Experiments were performed on White Swiss mice (6–10 wk of age). All studies were approved by the University of California, San Diego, Committee on Investigations Involving Animals. The mice were housed in a standard animal care room with a 12:12-h light-dark cycle and were allowed free access to food and water. Before experiments, the mice were deprived of food and water for <=1 h. After anesthesia with a cocktail of hypnorm and midazolam (10 ml/kg ip), the abdomen was opened by midline incision. The proximal duodenum (a portion stretching approximately from 2 mm distal to the pylorus to the common bile duct ampulla) was removed and immediately placed in ice-cold isosmolar mannitol and indomethacin (1 µM) solution (to suppress trauma-induced prostaglandin release). The anesthetized mice were then killed by cervical dislocation. The duodenum was opened along the mesenteric border and stripped of external serosal and muscle layers by sharp dissection in the above mentioned ice-cold isosmolar mannitol and indomethacin solution.

Ussing chamber experiments. The mucosa was mounted between two Lucite half chambers with an exposed area of 0.1 cm2 and placed in an Ussing chamber. The duodenal tissue from each animal was randomly divided among three chambers for experiments. The mucosal side was bathed with unbuffered bicarbonate-free modified Ringers solution circulated by a gas lift with 100% O2. The serosal side was bathed with modified buffered Ringers solution (pH 7.4) containing 25 mM and gassed with 95% O2-5% CO2. Each bath contained 3.0 ml of the respective solution maintained at 37°C by a heated water jacket. Experiments were performed under continuous short-circuited conditions (Voltage-Current Clamp, model VCC 600; Physiological Instruments, San Diego, CA) to maintain the electrical potential difference at zero, except for a brief period (<2 s) at each time point when the open-circuit potential difference was measured. Luminal pH was maintained at 7.40 by the continuous infusion of 5 mM HCl under the automatic control of a pH-stat system (model ETS 822; Radiometer America, Westlake, OH). The volume of the titrant infused per unit time was used to quantitate bicarbonate secretion. These measurements were recorded at 5-min intervals, and mean values for consecutive 10-min periods were calculated. The rate of luminal bicarbonate secretion is expressed as micromoles per square centimeter per hour. Short-circuit current (Isc) was measured in microamperes and converted into microequivalents per square centimeter per hour, and potential difference was measured in millivolts.

To investigate the effect of PMA on basal duodenal mucosal bicarbonate secretion and Isc, a different dose of PMA (10-8 to 10-5 M) or control vehicle was added to the serosal side of individual chambers for 2 h after 30 min of measurement of basal parameters. To investigate the effect of PMA on cAMP-stimulated duodenal bicarbonate secretion and Isc, db-cAMP was added to the serosal side simultaneously, or after preincubation with different doses of PMA (10-8 to 10-5 M) on the serosal side for 60 min.

To investigate the effect of PKC inhibitors on the ability of PMA to regulate duodenal bicarbonate secretion, Ro 31–8220 or Gö 6983 were added to the serosal side 30 min before the administration of PMA.

Western blot analysis. Segments of duodenal tissue (~25 mg) were stripped of seromuscular layers as described above for Ussing chamber experiments. Tissue samples were frozen immediately in liquid nitrogen. Protein was extracted by homogenization on ice in 500 µl lysis buffer containing (in mM) 20 Tris·HCl (pH 7.5), 150 NaCl, 1 disodium EDTA, 1 EGTA, 2.5 sodium pyrophosphate, 1 {beta}-glycerophosphate, 1 sodium orthovanadate, and 1% Triton X-100, and complete protease inhibitor cocktail (Sigma, St. Louis, MO). Equal amounts of protein, as determined by Lowry assay (Dc assay; BioRad, Hercules, CA), were combined with 2x Laemmli sample buffer and boiled for 5 min. Proteins were separated by electrophoresis on 7.5% SDS-PAGE gels and transblotted to nitrocellulose membranes. The protein-bound nitrocellulose sheets were first incubated overnight at 4°C in blocking buffer containing 5% nonfat dry milk in distilled water. Nitrocellulose sheets were then incubated with monoclonal antibodies to different PKC isoforms diluted in blocking buffer (1:1,000) for 1 h at room temperature and rinsed for 1 h with a wash buffer containing 20 mM Tris, pH 7.5, 500 mM NaCl, and 1% Tween 20. The membranes were then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG antibody for 30 min at room temperature and washed for 1 h with agitation, changing the wash buffer every 15 min. PKC bands were visualized with ECL Plus detection reagents (Amersham, Pharmacia, Piscataway, NJ).

In vitro kinase assay. After incubation with PMA, duodenal mucosal samples were frozen immediately in liquid nitrogen. When PKC inhibitors were used, the tissue was preincubated with the PKC inhibitor for 30 min before PMA. Protein was extracted by homogenization on ice in 500 µl lysis buffer containing (in mM) 20 Tris·HCl (pH 7.5), 150 NaCl, 1 disodium EDTA, 1 EGTA, 2.5 sodium pyrophosphate, 1 mM {beta}-glycerophosphate, 1 sodium orthovanadate, and 1% Triton X-100, and complete protease inhibitor cocktail. Monoclonal antibody against nPKC{epsilon} (1:1,000) was added to each sample for overnight rotation at 4°C. After incubation, immune complexes were precipitated with protein A agarose beads, washed, resuspended in 20 µl kinase buffer containing (in mM) 25 Tris·HCl (pH 7.5), 5 {beta}-glycerophosphate, 2 DTT, 0.1 sodium orthovanadate, 10 magnesium chloride, and 10 µCi [{gamma}-32P]ATP (New England Nuclear), and then incubated with 10 µg myelin basic protein as a substrate at 30°C for 30 min. After incubation, the reaction was terminated by adding 2x Laemmli sample buffer to the samples, and the samples were boiled for 5 min. Supernatants were subjected to SDS-PAGE (12% gels; Bio-Rad), and the gel was dried and subjected to autoradiography.

Statistical analysis. All results are expressed as means ± SE. Net 1-h bicarbonate secretion refers to stimulated responses minus mean basal values. Data were analyzed by one-way ANOVA followed by Student-Newman-Keuls post hoc test or, when appropriate, by Student's t-tests using GraphPad Prism 2.0 (San Diego, CA). P < 0.05 was considered statistically significant.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effect of PMA on basal duodenal mucosal bicarbonate secretion. After measuring basal duodenal bicarbonate secretion, different concentrations of PMA (10-8 to 10-5 M) were added serosally, and the incubation was continued for up to 2 h. Fig. 1, A and B shows a time course of the effect of PMA (10-5 M) on basal duodenal bicarbonate secretion and Isc. Basal duodenal bicarbonate secretion was 0.94 ± 0.18 µmol·cm-2·h-1. After incubation with PMA on the serosal side for 2 h, duodenal bicarbonate secretion was 0.96 ± 0.19 µmol·cm-2·h-1, not different from basal bicarbonate secretion. Lower doses of PMA were similarly ineffective in changing bicarbonate secretion and Isc (data not shown).



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Fig. 1. Time course of the effect of PMA on basal duodenal mucosal bicarbonate secretion (A) and short-circuit current (Isc) (B). Mouse duodenal mucosae were mounted in Ussing chambers, and PMA (10-5 M) was added to the serosal side at the time indicated by the arrow. PMA failed to alter basal bicarbonate secretion or Isc. Data are shown as means ± SE for 5 experiments.

 

Effect of PMA on cAMP-stimulated duodenal bicarbonate secretion. cAMP is an important intracellular messenger regulating duodenal bicarbonate secretion (9). Some studies have shown that PMA can potentiate chloride secretory responses evoked by cAMP in T84 (6), C127 (7), and pancreatic duct cells (36). We therefore examined whether PMA had any effect on cAMP-stimulated duodenal bicarbonate secretion. When PMA (10-5 M) and db-cAMP (10-3 M) were added simultaneously to the serosal side of mouse duodenum, duodenal bicarbonate secretion and Isc were no different from those produced by db-cAMP in tissues treated only with the vehicle (Fig. 2, A and B) (P > 0.05). However, after a 1-h preincubation with PMA (10-5 M), db-cAMP-stimulated duodenal bicarbonate secretion and Isc responses were significantly potentiated, compared with the control group (Fig. 3, A and B) (P < 0.01). Moreover, the effect of PMA on db-cAMP-stimulated duodenal bicarbonate secretion was concentration-dependent (10-8 to 10-5M, P < 0.05) (Fig. 4). At the highest concentration of PMA, the effect of db-cAMP on bicarbonate secretion was increased by ~50%. For comparison, we assessed any effects of PMA on carbachol- and STa-induced duodenal bicarbonate secretion. These agents act via calcium and cGMP, respectively. Moreover, there is evidence that at least part of their effect on bicarbonate secretion may be independent of the CFTR chloride channel. The results demonstrated that preincubation with PMA (10-5 M) for 1 h had no effect on either carbachol- or STa-induced duodenal bicarbonate secretion (Fig. 5).



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Fig. 2. Effect of coincubation of PMA and dibutyryl-cAMP (db-cAMP) on cAMP-stimulated duodenal mucosal bicarbonate secretion (A) and Isc (B). Mouse duodenal mucosae were mounted in Ussing chambers and PMA (10-5M) or control vehicle and db-cAMP (10-3 M) were added serosally to the tissue at the same time as indicated by the arrow. PMA had no effect on cAMP-stimulated duodenal bicarbonate secretion or Isc (P > 0.05). Data are shown as means ± SE for 10 experiments.

 


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Fig. 3. Effect of PMA pretreatment for 1 h on cAMP-stimulated duodenal mucosal bicarbonate secretion (A) and Isc (B). Mouse duodenal mucosae were mounted in Ussing chambers and PMA (10-5 M) or control vehicle was added to serosal side of the tissue. db-cAMP (10-3 M) was added serosally 60 min later. PMA markedly potentiated cAMP-stimulated duodenal bicarbonate and Isc (P < 0.01) by 1-way ANOVA. Data are shown as means ± SE for 10 experiments.

 


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Fig. 4. Concentration-dependent effect of PMA on cAMP-stimulated duodenal mucosal bicarbonate secretion. This figure shows results from a series of experiments similar to those in Fig 3. Mouse duodenal mucosae were mounted in Ussing chambers and different doses of PMA or control vehicle were added serosally. db-cAMP (10-3 M) was added serosally 60 min later. Each concentration of PMA was tested independently in a separate tissue. PMA potentiated cAMP-stimulated duodenal bicarbonate secretion in a concentration-dependent manner by one-way ANOVA with Student-Newman-Keuls post hoc test. Data are shown as means ± SE for at least 9 experiments at each PMA concentration. *P < 0.05, **P < 0.01 (compared with control).

 


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Fig. 5. Effect of PMA pretreatment for 1 h on carbachol- and heat-stable toxin of Escherichia coli (STa)-stimulated duodenal mucosal bicarbonate secretion. Mouse duodenal mucosae were mounted in Ussing chambers and PMA (10-5M) or control vehicle was added to serosal side of the tissue. Carbachol (10-4M) or STa (10-7 M) were added serosally 60 min later. PMA had no effects on carbachol- or STa-stimulated duodenal bicarbonate secretion. Data are shown as means ± SE for at least 9 experiments.

 

Effects of isozyme-selective PKC inhibitors on the ability of PMA to potentiate transport responses to cAMP. To demonstrate which PKC isoform(s) are involved in the regulation of duodenal bicarbonate secretion, we first examined the effect of two PKC inhibitors, Gö 6983 and Ro 31–8220, which have been shown to inhibit differentially cPKC and nPKC isoforms. Neither drug altered bicarbonate secretion induced by db-cAMP by themselves (Fig. 6). Likewise, serosal addition of Gö 6983 (10-5 M), an inhibitor of PKC{alpha}, -{gamma}, -{beta}, and -{delta}, for 30 min before PMA, failed to reverse the potentiation of cAMP-stimulated duodenal bicarbonate secretion induced by PMA. In contrast, the addition of Ro 31–8220 (5 x 10-6 M), an inhibitor of PKC{epsilon}, -{alpha}, -{beta}, and -{gamma}, completely reversed the potentiation of cAMP-stimulated duodenal bicarbonate secretion induced by PMA (Fig. 6).



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Fig. 6. Effect of PKC inhibitors on the ability of PMA to potentiate cAMP-stimulated bicarbonate secretion. Mouse duodenal mucosae were incubated sequentially with Ro 31–8220 (5 x 10-6 M), Gö 6983 (10-5 M), or the vehicle control (DMSO), followed 30 min later by PMA (10-5 M) or vehicle, followed 1 h later by db-cAMP. Ro 31–8220 reversed the potentiating effect of PMA on cAMP-stimulated duodenal bicarbonate secretion, whereas Gö 6983 did not. Data are expressed as the net bicarbonate output stimulated by db-cAMP and are means ± SE for at least 9 experiments. **Significantly different from control, P < 0.01; ++significantly different from tissues treated with PMA alone or PMA + Gö 6983, P < 0.01 by 1-way ANOVA with Student-Newman-Keuls post hoc test.

 

Expression of PKC isoforms in murine duodenal mucosa. At least 11 isoforms of PKC have been identified. The presence of PKC{alpha}, -{beta}, -{gamma}, -{delta}, -{epsilon}, -µ, -{theta}, and -{iota}/{lambda} isoforms in murine duodenal mucosa was investigated by Western blot analysis. With the use of specific antibodies against these isoforms, PKC{alpha} and -{gamma} in the conventional family, PKC{epsilon}, -µ, and -{theta} in the novel family, and PKC{iota}/{lambda} in the atypical family were detected in murine duodenal mucosa. PKC{beta} and -{delta} were not reproducibly detected in these tissues (Fig. 7).



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Fig. 7. Western blot analysis of conventional PKC (cPKC), atypical PKC (aPKC), and novel PKC (nPKC) isoforms in murine duodenal mucosa. Tissue lysates were subjected to electrophoresis through 7.5% SDS-PAGE and transferred to PVDF membranes. Membranes were probed with monoclonal antibodies specific for various PKC isoforms ({alpha}, {beta}, {gamma}, {epsilon}, {delta}, µ, {theta}, {lambda}, and {iota}). Expression in murine duodenal mucosa (S) was assessed by comparison with molecular mass standards and comigration with positive control preparations of rabbit brain (P). PKC isoforms were detected through the use of an enhanced chemiluminescent methodology as described in MATERIALS AND METHODS. These Western blots are representative of 3 similar experiments.

 

Effects of PMA and PKC inhibitors on activity of PKC{epsilon}. The pharmacological studies described above were consistent with the hypothesis that PKC{epsilon} is responsible for the ability of PMA to potentiate cAMP-stimulated duodenal bicarbonate secretion. We therefore made a further investigation of the effects of PMA and PKC inhibitors (Gö 6983 and Ro 31–8220) on the activity of this specific isozyme, shown to be present in the tissues (Fig. 7). PMA concentration- and time-dependently increased the activity of PKC{epsilon}. This was demonstrated by a steady increase in the intensity of the 19-kDa MBP band detected on autoradiograms of samples derived from in vitro kinase assays, reflective of PKC{epsilon} activity (Figs. 8 and 9). PKC{epsilon} kinase activity was increased significantly at a PMA concentration of 10-7 M and maximally at 10-5 M (Fig. 8). In time course studies, PKC{epsilon} kinase activity was first notably increased after 30 min of exposure to PMA (10-5 M) and reached a peak at 60 min (Fig. 9). Pretreatment with Ro 31–8220 (5 x 10-6 M) for 30 min before the addition of PMA completely inhibited PMA-stimulated PKC{epsilon} activity, whereas Gö 6983 (10-5 M) did not inhibit PMA-induced activation of PKC{epsilon} (Fig. 10).



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Fig. 8. Concentration-dependent effect of PMA on PKC{epsilon} activity in murine duodenal tissues. Mouse duodenal mucosae were stimulated with different concentrations of PMA (10-8 M-10-5 M) as shown. The tissues were then lysed. PKC{epsilon} activities in these lysates were determined as described in MATERIALS AND METHODS. Top: data are from a single experiment representative of 6 similar experiments. Bottom: means ± SE for densitometric assessments of the intensity of the MBP band expressed in arbitrary units (A.U.). *P < 0.05, **P < 0.01, significantly different from control by one-way ANOVA with Student-Newman-Keuls post hoc test.

 


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Fig. 9. Time course of PKC{epsilon} activity in murine duodenal tissues after the addition of PMA. Mouse duodenal mucosae were stimulated for various times as shown with PMA (10-5 M). The tissues were then lysed. PKC{epsilon} activities in these lysates were determined as described in MATERIALS AND METHODS. Top: data are from a single experiment representative of 6 similar experiments. Bottom: means ± SE for densitometric assessments of the intensity of the MBP band expressed as a %value obtained in unstimulated tissue. **P < 0.01, ***P < 0.001, significantly different from control by one-way ANOVA with Student-Newman-Keuls post hoc test.

 


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Fig. 10. Effects of Gö 6983 and Ro 31–8220 on PKC{epsilon} activity in murine duodenal tissues. Isolated tissues were pretreated with Ro 31–8220 (5 x 10-6 M) or Gö 6983 (10-5 M) for 30 min before PMA (10-5M). Tissues were then lysed. PKC{epsilon} activities in these lysates were determined as described in MATERIALS AND METHODS. Top: data from a single experiment representative of 6 similar experiments. Bottom: means ± SE for densitometric assessments of the intensity of the MBP band expressed in A.U. ***Significantly different from control, P < 0.001; +++Significantly different from tissues treated with PMA or PMA + Gö 6983, P < 0.001 by 1-way ANOVA with Student-Newman-Keuls post hoc test.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Duodenal bicarbonate secretion is currently accepted as a critical defense mechanism against acid-peptic injury (9, 13, 17). This transport process is regulated via the activation of different signaling pathways. cAMP, Ca2+, and cGMP are three important positive regulators of duodenal bicarbonate secretion (8, 9). PKC is a Ca2+- and phospholipid-dependent protein kinase and plays an important role in signal transduction in many systems, including the regulation of ion transport, cell growth, and differentiation. Immunohistochemistry has confirmed the presence of some PKC isoforms in the rat duodenum (20). However, the role of PKC in duodenal bicarbonate secretion has not been fully elucidated.

In this study, we found that PMA, an activator of PKC, had no significant effect on basal duodenal bicarbonate secretion in mice. Previous studies have shown that PKC potentiated the chloride secretory response induced by cAMP in T84 (6), C127 (7), and pancreatic duct cells (36). We therefore examined the effect of PMA on cAMP-stimulated duodenal bicarbonate secretion. We found that when PMA and db-cAMP were added simultaneously to the serosal side of mouse duodenum, PMA failed to alter db-cAMP-stimulated duodenal bicarbonate secretion. However, serosal pretreatment of the tissue with PMA for 1 h significantly potentiated db-cAMP-stimulated bicarbonate secretion. Moreover, the ability of PMA to potentiate secretion was concentration-dependent and consistent with the concentration-dependency for the effect of PMA on PKC activation in other intact tissues (24, 32). Odes et al. (27) reported that the intravenous infusion of PMA into guinea pigs resulted in a marked stimulation of active duodenal bicarbonate secretion. This was accompanied by a time-dependent translocation of cytosolic, lipid-dependent PKC activity into the particulate fraction. Furukawa et al. (10) likewise found that luminal perfusion of PMA in rats increased basal duodenal bicarbonate secretion, and that staurosporine, an inhibitor of PKC, inhibited acid-induced duodenal bicarbonate secretion. These prior results indicated that PKC is involved in the regulation of duodenal bicarbonate secretion. However, in the present study, we found that PMA had no significant effect on basal duodenal bicarbonate secretion in mice as studied in vitro. This difference between our studies and the prior work of others may be accounted for by the presence in vivo of cAMP-dependent secretagogues that set a considerable basal tone for bicarbonate secretion, which could then be increased further by PMA.

The PKC family consists of at least 11 isoforms. Our Western blot analysis demonstrated that seven PKC isoforms, PKC{alpha}, -{gamma}, -{epsilon}, -µ, -{theta}, and -{iota}/{lambda}, are expressed in the duodenal mucosa of Swiss mice, representative of three major classes of PKC isoforms. PKC{beta} and -{delta} were not detected in duodenal mucosa in mice. The study of Balogh et al. (1), however, showed that PKC{alpha}, -{beta}, and -{delta}, but not PKC{epsilon} and -{theta} were expressed in rat duodenal mucosa. This discrepancy may be related to species or to methodological differences. To understand which isoforms of PKC participate in the regulation of duodenal bicarbonate secretion, we first took a pharmacological approach. This showed that Ro 31–8220 (an inhibitor active against PKC{epsilon}, -{alpha}, -{beta}, and –{gamma}), but not Gö 6983 (an inhibitor active against PKC{alpha}, -{beta}, -{gamma} and -{delta}), completely reversed the potentiating action of PMA on cAMP-stimulated duodenal bicarbonate secretion. These data implied that PKC{epsilon} was a candidate to mediate the effect of PMA on duodenal bicarbonate secretion. However, these pharmacological studies are complicated by the fact that no isoform-specific pharmacological inhibitors of PKC are currently available. Thus in addition to inhibiting PKC{epsilon}, Ro 31–8220 is also an inhibitor of PKC{alpha}, -{beta}, and -{gamma} (19, 38). On the other hand, Gö 6983 was also shown to inhibit PKC{alpha}, -{beta}, and -{gamma} but has no inhibitory effect on PKC{epsilon} (3, 11). In addition, there is apparently no expression of PKC{beta} in the duodenal mucosa of mice, at least in our hands. Similarly, a PKC{iota}/{lambda} is not directly activated by PMA. Therefore, by a process of elimination, we identified PKC{epsilon} as the most likely isoform to mediate the potentiating effect of PMA on duodenal bicarbonate secretion.

To further delineate the action of PKC{epsilon} in duodenal mucosa, we performed an in vitro kinase activity assay for PKC{epsilon}. PMA concentration- and time-dependently increased the activity of PKC{epsilon}. The activity of PKC{epsilon} was markedly increased at 30 min after exposure to PMA, and was maximal at 60 min. Ro 31–8220, but not Gö 6983, completely inhibited the activation of PKC{epsilon} by PMA. When PMA and db-cAMP were added simultaneously to duodenal tissue, PMA failed to alter db-cAMP-stimulated duodenal bicarbonate secretion, whereas when it was added 1 h before db-cAMP, PMA potentiated db-cAMP-stimulated duodenal bicarbonate secretion. This is therefore consistent with the kinetics of PMA-induced PKC{epsilon} kinase activity in the tissue. We conclude that events downstream of PKC{epsilon} activation must exist in the tissues at the time of db-cAMP addition for augmented secretion to occur. Such events are presumably delayed in time after PKC{epsilon} activation, but could persist once initiated. This could account for seeming discrepancies in the details of the time courses for PKC activation and augmented secretion. In any event, these results further substantiate the role of PKC{epsilon} in the regulation of duodenal bicarbonate secretion. In the long term, molecular techniques will be needed to verify definitively the role of PKC{epsilon}.

The precise mechanisms whereby PMA potentiates cAMP-stimulated duodenal bicarbonate secretion also require additional study, but some speculations are possible. PKC is known to regulate CFTR. Several recent studies have shown that CFTR channel also plays an important role in regulating duodenal bicarbonate secretion (5, 14, 15, 29, 33), and cAMP predominately regulates epithelial Cl- and secretion in epithelia by activating CFTR. CFTR is a 1,480-amino acid protein with a unique structure characterized by three cytoplasmic domains, two nucleotide binding folds, and a regulatory domain that contains consensus sequences for phosphorylation by PKA and PKC (28). In membrane patches excised from cells expressing CFTR, the addition of exogenous PKC resulted in a modest increase in CFTR channel activity and enhanced the rate and magnitude of subsequent PKA stimulation of open probability (35). Jia et al. (18) found that PKC-mediated phosphorylation not only potentiated CFTR opening in response to cAMP, but, in fact, was essential for acute PKA activation of CFTR channels. Thus one mechanism whereby PMA could potentiate cAMP-stimulated duodenal bicarbonate secretion would be by regulating CFTR channels. In addition, we found that PMA had no effect on carbachol- or STa-induced duodenal bicarbonate secretion. Duodenal bicarbonate secretion induced by these agents involves pathways that are independent of CFTR (29). Our results demonstrate that PKC likely has no effect on Ca2+- and cGMP-dependent duodenal bicarbonate secretion.

Considering PKC{epsilon} specifically, Liedtke and Cole (23) showed that this isoform regulated the function of CFTR in Calu-3 cells. On the other hand, Chow et al. (4) showed that PKC{epsilon} was activated in T84 cells in response to epidermal growth factor, and participated in the negative regulation of Ca2+-dependent Cl- secretion. Similarly, Song et al. (34) showed that PMA could inhibit cAMP-stimulated Cl- secretion in T84 cells by activating PKC{epsilon}. This latter effect was apparently due to an increased rate of basolateral endocytosis in PMA-treated cells, resulting in the retrieval of transport proteins needed for the overall chloride secretory mechanism from the plasma membrane. In the present study, however, PKC{epsilon} was associated with an increase rather than a decrease in epithelial ion transport. This might suggest a different balance of positive (on CFTR) and negative (on basolateral transportors) effects of PKC{epsilon} on the machinery needed for bicarbonate vs. chloride secretion, or that specific phosphorylation events on CFTR might modulate its substrate specificity, or some combination of these and/or additional mechanisms.

In summary, the present study demonstrates that PKC [or at least PMA-sensitive isoform(s)] likely does not participate in basal duodenal bicarbonate secretion in mice, but potentiates cAMP-stimulated duodenal bicarbonate secretion. Western blot analysis showed the expression of PKC{alpha}, -{gamma}, -{epsilon}, -µ, -{theta}, and -{iota}/{lambda} in duodenal mucosa in mice. Activation of PKC{epsilon} specifically regulates duodenal bicarbonate secretion. Our findings contribute to the understanding of the cellular mechanisms that underlie duodenal bicarbonate secretion, and additionally underscore the complexities and specificity of regulatory mechanisms for anion secretion in the mammalian intestine.


    ACKNOWLEDGMENTS
 
GRANTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-33491 (to J. I. Isenberg) and DK-28305 (to K. E. Barrett) and by a Fellowship Grant from the Crohn's and Colitis Foundation of America (to J. Y. C. Chow).


    FOOTNOTES
 

Address for reprint requests and other correspondence: K. E. Barrett, Univ. of California, San Diego Medical Center, Div. of Gastroenterology, 8414, 200 W. Arbor Dr., San Diego, CA 92103-8413 (E-mail: kbarrett{at}ucsd.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.

{dagger} Deceased October 10, 2003. Back


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Balogh G, Boland R, and Boland AR. 1,25 (OH)2-vitamin D3 affects the subcellular distribution of protein kinase C isozymes in rat duodenum. Influence of aging. J Cell Biochem 79: 686-694, 2000.[CrossRef][ISI][Medline]
  2. Barthelson RA, Jawby DB, and Widdicombe JH. Regulation of chloride secretion in dog tracheal epithelium by protein kinase C. Am J Physiol Cell Physiol 253: C802-C808, 1987.[Abstract/Free Full Text]
  3. Chang LC and Wang JP. Examination of the signal transduction pathways leading to activation of extracellular signal-regulated kinase by formyl-methionyl-leucyl-phenylalanine in rat neutrophils. FEBS Lett 454: 165-168, 1999.[CrossRef][ISI][Medline]
  4. Chow JYC, Uribe JM, and Barrett KE. A role for protein kinase C{epsilon} in the inhibitory effect of epidermal growth factor on calcium-stimulated chloride secretion in human colonic epithelial cells. J Biol Chem 275: 21169-21176, 2000.[Abstract/Free Full Text]
  5. Clarke LL and Harline M. Dual role of CFTR in cAMP-stimulated secretion across murine duodenum. Am J Physiol Gastrointest Liver Physiol 274: G718-G726, 1998.[Abstract/Free Full Text]
  6. Decheccehi MC, Rolfini R, Tamanini A, Camberi C, Berton G, and Cabrini G. Effect of modulation of protein kinase C on the cAMP-dependent chloride conductance in T84 cells. FEBS Lett 311: 25-28, 1992.[CrossRef][ISI][Medline]
  7. Dechecchi MC, Tamanini A, Berton G, and Cabrini G. Protein kinase C activates chloride conductance in C127 cells stably expressing the cystic fibrosis gene. J Biol Chem 268: 11321-11325, 1993.[Abstract/Free Full Text]
  8. Flemstrom G. Gastric and duodenal mucosal secretion of bicarbonate. In: Physiology of the Gastrointestinal Tract (3rd ed.), edited by Johnson LR. New York: Raven, 1994, p. 1285-1309.
  9. Flemstrom G and Isenberg JI. Gastroduodenal mucosal alkaline secretion and mucosal protection. News Physiol Sci 16: 23-28, 2001.[Abstract/Free Full Text]
  10. Furukawa O, Hirokawa M, Guth PH, Engle E, and Kaunitz JD. Role of protein kinases on acid-induced duodenal bicarbonate secretion in rats. Pharmacology 67: 99-105, 2003.[CrossRef][ISI][Medline]
  11. Gschwendt M, Dieterich S, Rennecke J, Kittstein W, Mueller HJ, and Johannes FJ. Inhibition of protein kinase C mu by various inhibitors. Differentiation from protein kinase C isoenzymes. FEBS Lett 392: 77-80, 1996.[CrossRef][ISI][Medline]
  12. Guba M, Kuhn M, Forssmann WG, Classen M, Gregor M, and Seidler U. Guanylin strongly stimulates rat duodenal secretion: proposed mechanism and comparison with other secretagogues. Gastroenterology 111: 1558-1568, 1996.[ISI][Medline]
  13. Hogan DL, Ainsworth MA, and Isenberg JI. Gastroduodenal bicarbonate secretion. Aliment Pharmacol Ther 8: 475-488, 1994.[ISI][Medline]
  14. Hogan DL, Crombie DL, Isenberg JI, Svendsen P, Schaffalitzky de Muckadell OB, and Ainsworth MA. CFTR mediates cAMP- and Ca2+-activated duodenal epithelial secretion. Am J Physiol Gastrointest Liver Physiol 272: G872-G878, 1997.[Abstract/Free Full Text]
  15. Hogan DL, Crombie DL, Isenberg JI, Svendsen P, Schaffalitzky De Muckadell OB, and Ainsworth MA. Acid-stimulated duodenal bicarbonate secretion involves a CFTR-mediated transport pathway in mice. Gastroenterology 113: 533-541, 1997.[ISI][Medline]
  16. Hogan DL, Yao BG, and Isenberg JI. Modulation of bicarbonate secretion in rabbit duodenum: the role of calcium. Dig Dis Sci 43: 120-125, 1998.[CrossRef][ISI][Medline]
  17. Hojgaard L, Mertz-Neilson A, and Rune SJ. Peptic ulcer pathophysiology: acid, bicarbonate, and mucosal function. Scand J Gastroenterol Suppl 216: 10-15, 1996.[Medline]
  18. Jia Y, Mathews CJ, and Hanrahan JW. Phosphorylation by protein kinase C is required for acute activation of cystic fibrosis transmembrane conductance regulator by protein kinase A. J Biol Chem 272: 4978-4984, 1997.[Abstract/Free Full Text]
  19. Johnson MS, MacEwan DJ, Simpson J, and Mitchell R. Characterization of protein kinase C isoforms and enzymic activity from the alpha T3–1 gonadotroph-derived cell line. FEBS Lett 333: 67-72, 1993.[CrossRef][ISI][Medline]
  20. Kawakita N, Nagahata Y, Saitoh Y, and Ide C. Protein kinase C {alpha}-, {beta}-, and {gamma}-subspecies in basal granulated cells of rat duodenal mucosa. Anat Embryol (Berl) 191: 329-336, 1995.[ISI][Medline]
  21. Kopp R and Pfeiffer A. Effect of phorbol ester treatment on dibutyryl cyclic adenosine-5' monophosphate- and carbachol-stimulated aminopyrine accumulation in isolated rat parietal cells. Scand J Gastroenterol 35: 686-693, 2000.[CrossRef][ISI][Medline]
  22. Li M, McCann JD, Anderson MP, Clancy JP, Liedtke CM, Nairn AC, Greengard P, and Welch MJ. Regulation of chloride channels by protein kinase C in normal and cystic fibrosis airway epithelia. Science 244: 1353-1356, 1989.[ISI][Medline]
  23. Liedtke CM and Cole TS. Antisense oligonucleotide to PKC{epsilon} alters cAMP-dependent stimulation of CFTR in Calu-3 cells. Am J Physiol Cell Physiol 275: C1357-C1364, 1998.[Abstract/Free Full Text]
  24. Mauduit P, Zoukhri D, and Rossignol B. Direct activation of a protein kinase activity from rat lacrimal gland by PMA in a phospholipid-free system. FEBS Lett 252: 5-11, 1989.[CrossRef][ISI][Medline]
  25. Mochly-Rosen D and Gordon AS. Anchoring proteins for protein kinase C: a means for isozyme selectivity. FASEB J 12: 35-42, 1998.[Abstract/Free Full Text]
  26. Odes HS, Muallem R, Reimer R, Beil W, Schwenk M, and Sewing KF. Cholinergic regulation of guinea pig duodenal bicarbonate secretion. Am J Physiol Gastrointest Liver Physiol 265: G270-G276, 1993.[Abstract/Free Full Text]
  27. Odes HS, Reimer R, Muallem R, Schwenk M, Beil W, and Sewing KF. Role of protein kinase C in duodenal mucosal bicarbonate secretion in the guinea pig. Pharmacology 53: 60-65, 1996.[ISI][Medline]
  28. Picciotto MR, Cohn JA, Bertuzzi G, Greengard P, and Nairn AC. Phosphorylation of the cystic fibrosis transmembrane conductance regulator. J Biol Chem 267: 12742-12752, 1992.[Abstract/Free Full Text]
  29. Pratha VS, Hogan DL, Martensson BA, Bernard J, Zhou R, and Isenberg JI. Identification of transport abnormalities in duodenal mucosa and duodenal enterocytes from patients with cystic fibrosis. Gastroenterology 118: 1051-1060, 2000.[ISI][Medline]
  30. Raufman JP. Gastric chief cells: receptors and signal transduction mechanisms. Gastroenterology 102: 699-710, 1992.[ISI][Medline]
  31. Row D and Kazanietz MG. New insights into the regulation of protein kinase C and novel phorbol ester receptors. FASEB J 13: 1658-1676, 1999.[Abstract/Free Full Text]
  32. Sakai Y, Inazu M, Shamoto A, Zhu B, and Homma I. Contractile hyperreactivity and alteration of PKC activity in gastric fundus smooth muscle of diabetic rats. Pharmacol Biochem Behav 49: 669-674, 1994.[CrossRef][ISI][Medline]
  33. Seidler U, Blamenstein I, Kretz A, Viellard-Baron D, Rossmann H, Colledge WH, Evans DF, Ratcliff R, and Gregor M. A functional CFTR protein is required for mouse intestinal cAMP-, cGMP-, and Ca2+-dependent secretion. J Physiol 505: 411-423, 1997.[Abstract]
  34. Song JC, Hanson CM, Tsai V, Farokhzad OC, Lotz M, and Matthews JB. Regulation of epithelial transport and barrier function by distinct protein kinase C isoforms. Am J Physiol Cell Physiol 281: C649-C661, 2001.[Abstract/Free Full Text]
  35. Tabcharani JA, Chang XB, Riordan JR, and Hanrahan JW. Phosphorylation-regulated Cl- channel in CHO cells stably expressing the cystic fibrosis gene. Nature 352: 628-631, 1991.[CrossRef][ISI][Medline]
  36. Winpenny JP, McAlory HL, Gray MA, and Argent BE. Protein kinase C regulates the magnitude and stability of CFTR currents in pancreatic duct cells. Am J Physiol Cell Physiol 268: C823-C828, 1995.[Abstract/Free Full Text]
  37. Yao BG, Hogan DL, Bukhave K, Koss MA, and Isenberg JI. Bicarbonate transport by rabbit duodenum in vitro: effect of vasoactive intestinal polypeptide, prostaglandin E2, and cyclic adenosine monophosphate. Gastroenterology 104: 732-740, 1993.[ISI][Medline]
  38. Zhang Y and Akhtar RA. Epidermal growth factor stimulates phospholipase D independent of phospholipase C, protein kinase C or phosphatidylinositol-3 kinase activation in immortalized rabbit corneal epithelial cells. Curr Eye Res 17: 294-300, 1998.[CrossRef][ISI][Medline]