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Am J Physiol Gastrointest Liver Physiol 293: G279-G287, 2007. First published May 10, 2007; doi:10.1152/ajpgi.00488.2006
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HORMONES AND SIGNALING

Phosphatidylinositol 3-kinase is involved in prostaglandin E2-mediated murine duodenal bicarbonate secretion

Bi-Guang Tuo,1,2 Guo-Rong Wen,2 and Ursula Seidler1

1Department of Gastroenterology and Hepatology, Hannover Medical School, Hannover, Germany; and 2Department of Gastroenterology, Affiliated Hospital, Zunyi Medical College, Zunyi, China

Submitted 20 October 2006 ; accepted in final form 12 April 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Prostaglandin E2 (PGE2) plays an important role in the regulation of duodenal bicarbonate (HCO3–) secretion, but its signaling pathway(s) are not fully understood. In the present study, we investigated the signaling pathways involved in PGE2-mediated duodenal HCO3– secretion. Murine duodenal mucosal HCO3– secretion was examined in vitro in Ussing chambers by pH-stat titration in the presence of a variety of signal transduction modulators. Phosphatidylinositol 3-kinase (PI3K) activity was measured by immunoprecipitation of PI3K and ELISA, and Akt phosphorylation was measured by Western analysis with anti-phospho-Akt and anti-Akt antibodies. PGE2-stimulated duodenal HCO3– secretion was reduced by the cAMP-dependent signaling pathway inhibitors MDL-12330A and KT-5720 by 23% and 20%, respectively; the Ca2+-influx inhibitor verapamil by 26%; and the calmodulin antagonist W-13 by 24%; whereas the PI3K inhibitors wortmannin and LY-294002 reduced PGE2-stimulated HCO3– secretion by 51% and 47%, respectively. Neither the MAPK inhibitor PD-98059 nor the tyrosine kinase inhibitor genistein altered PGE2-stimulated HCO3– secretion. PGE2 application caused a rapid and concentration-dependent increase in duodenal mucosal PI3K activity and Akt phosphorylation. These results demonstrated that PGE2 activates PI3K in duodenal mucosa and stimulates duodenal HCO3– secretion via cAMP-, Ca2+-, and PI3K-dependent signaling pathways.

intestine; EP receptor

DUODENAL MUCOSAL BICARBONATE (HCO3–) secretion plays an important role in duodenal mucosal protection against acid-peptic injury. Duodenal ulcer patients have significantly diminished proximal duodenal mucosal HCO3– secretion compared with healthy volunteers (28), suggesting that diminished mucosal HCO3– secretion contributes to the pathogenesis of duodenal ulcer.

Prostaglandins (PGs) are members of a family of lipid mediators derived from cyclooxygenase-mediated metabolism of arachidonic acid and important mediators of normal physiology (13, 50). PGs, especially PGE2, are distributed widely in the gastrointestinal tract, involved in the regulation of a variety of gastrointestinal functions, including blood flow and acid, mucus, and HCO3– secretion, and play an outstanding role in gastroduodenal mucosal protection (14, 61). Although the physiological regulation of duodenal HCO3– secretion involves many neurohumoral factors, PGs are believed to play a crucial role in the regulation of this secretion (30). Indeed, luminal acid, one of the most important physiological stimuli of duodenal HCO3– secretion, causes a mucosal release of PGE2, and the prevention of this PGE2 release by indomethacin inhibits acid-induced duodenal HCO3– secretion (31, 35, 49, 51). PGE2 and its analogs, whether applied luminally or vascularly, stimulate gastroduodenal HCO3– secretion in vivo and in vitro in various species, including humans (27, 61). However, the mechanisms whereby PGE2 mediates duodenal HCO3– secretion are not fully understood. PGE2 stimulates adenylate cyclase activity and elevation of intracellular cAMP levels in duodenal enterocytes (3, 43, 44, 63), but experimental data suggest that PGE2 may stimulate duodenal HCO3– secretion by different signal transduction pathways than an increase in cAMP alone (39, 63). In our recent study in Slc26a6 (a Cl–/HCO3– exchanger) knockout mice (57), the results also demonstrated that a significant effect of PGE2 on duodenal HCO3– secretion must be mediated by a different mechanism than an increase in cAMP levels.

In the present study, therefore, we investigated the inhibitory effects of a variety of signal transduction inhibitors on PGE2-mediated duodenal mucosal HCO3– secretion. When we found that two structurally unrelated phosphatidylinositol 3-kinase (PI3K) inhibitors had the strongest inhibitory effects on PGE2-induced mucosal HCO3– secretion, we measured the effect of PGE2 on PI3K activity and the phosphorylation of the downstream effector of PI3K, Akt, in the isolated murine duodenal mucosa and found it to be rapidly activated. Thus, while other signaling pathways are also involved and are likely to interact and modulate PI3K-mediated signaling, we suggest that the activation of the PI3K is of considerable importance in PGE2-stimulated HCO3– secretion.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chemicals and solutions. The reagents PGE2, MDL-12330A, KT-5720, verapamil, W-13, wortmannin, LY-294002, PD-98059, and genistein were purchased from Sigma-Aldrich (Deisenhofen, Germany). These reagents were dissolved in DMSO or water for stock. Anti-PI3K p85 was obtained from Upstate Biotechnology (Lake Placid, NY). All other chemicals in solutions were obtained from Sigma-Aldrich (Deisenhofen, Germany) and Merck (Darmstadt, Germany). The mucosal solution used in Ussing chamber experiments contained (in mmol/l) 140 Na+, 5.4 K+, 1.2 Ca2+, 1.2 Mg2+, 120 Cl–, 25 gluconate, and 10 mannitol. The serosal solution contained (in mmol/l) 140 Na+, 5.4 K+, 1.2 Ca2+, 1.2 Mg2+, 120 Cl–, 25 HCO3–, 2.4 HPO42–, 2.4 H2PO4–, 10 glucose, and 0.001 indomethacin. The osmolalities for both solutions were ~305 mosmol/kgH2O. All reagents were fully soluble in the perfusate, with no precipitation in the concentrations used.

Animal preparation. The protocol was approved by Hannover Medical School, Germany, and the animal welfare committee of Lower Saxony. Experiments were performed in NMRI mice (6–12 wk of age). 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. After brief narcosis with 100% CO2, the mice were killed by cervical dislocation. The abdomen was opened by midline incision, and 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 µmol/l) solution (to suppress trauma-induced PG release). 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 duodenal mucosa was mounted between two chambers with an exposed area of 0.196 cm2 and placed in an Ussing chamber. Parafilm O-ring was used to minimize edge damage to the tissue where it was secured between the chamber halves. The mucosal side was bathed with unbuffered HCO3–-free modified Ringer solution circulated by a gas lift with 100% O2. The serosal side was bathed with modified buffered Ringer solution (pH 7.4) containing 25 mmol/l HCO3– and gassed with 95% O2-5% CO2. Each bath contained 10.0 ml of the respective solution maintained at 37°C by a heated water jacket. Experiments were performed under continuous short-circuited conditions 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 0.5 mmol/l HCl under the automatic control of a pH-stat system (PHM290, pH-Stat Controller, Radiometer, Copenhagen, Denmark). The volume of the titrant infused per unit time was used to quantitate HCO3– secretion. These measurements were recorded at 5-min intervals. The rate of luminal HCO3– secretion is expressed as micromoles per square centimeter per hour. Transepithelial short-circuit current (Isc; reported as µeq·cm–2·h–1) and potential difference (expressed as mV) were measured via an automatic voltage clamp (voltage-current clamp, EVC-4000; World Precision Instruments, Berlin, Germany), and tissue resistance was calculated. After a 30-min measurement of basal parameters, PGE2 (10–6 M) or control vehicle was added to the serosal side of tissue in Ussing chambers. Changes in duodenal HCO3– secretion and Isc during the 40-min period ensuing after the addition of PGE2 were determined.

Effects of signaling pathway inhibitors on PGE2-stimulated duodenal mucosal HCO3– secretion and Isc. To explore the signaling pathways involved in the action of PGE2, after a 30-min measurement of basal parameters, cAMP-dependent signaling pathway inhibitor MDL-12330A (10–5 M) or KT-5720 (10–5 M), Ca2+-dependent signaling pathway inhibitor verapamil (5 x 10–5 M) or W-13 (5x10–5 M), PI3K inhibitor wortmannin (10–7 M) or LY-294002 (2x10–5 M), MAPK inhibitor PD-98059 (10–5 M), or tyrosine kinase inhibitor genistein (10–5 M) was added into the serosal side or to both sides (only genistein). Thirty minutes later, PGE2 was added into the serosal side. Duodenal HCO3– secretion and Isc during the 40-min period after addition of PGE2 were then determined.

To assess tissue viability, glucose (25 mM) was added to the mucosal reservoir of all tissues at the end of the experiment. As expected, stimulation of Na+-glucose transport by glucose addition increased Isc and abolished HCO3– secretion, indicating that tissues were still viable after the end of experiments (48).

Immunoprecipitation of PI3K and ELISA for detection of PI3K activity. The mice were prepared as described above. Segments of duodenum (~50 mg) were placed in buffered serosal solution containing 10–6 M indomethacin at 37°C, gassed with 95% O2/5% CO2 for incubation. After stabilization for 20 min, different concentration of PGE2 or control was added to the bathing solution and incubated for different time points. At the end of incubation, the tissue was immediately washed three times with ice-cold buffer A [137 mM NaCl, 20 mM Tris·HCl (pH 7.4), 1 mM CaCl2, 1 mM MgCl2, and 1 mM sodium orthovanadate]. The mucosa was then scraped and placed into 1 ml lysis buffer [buffer A plus 1% (vol/vol) Nonidet P-40, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyfluoride] for homogenization at 4°C. After centrifugation at 10,000 g for 10 min at 4°C, 50 µl of the supernatant was for protein assay (Bradford), and the other (~5 mg protein extract) was immunoprecipitated with anti-PI3K p85 antibody for PI3K assay as described previously in the study by Hutchinson and Bengtsson (25). PI3K activity was measured in vitro using a competitive ELISA format (Echelon Biosciences, Salt Lake City, UT) according to the manufacturer's instructions. Briefly, the bead-bound immunoprecipitated enzyme was incubated with phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] substrate (100 pmol) in kinase reaction buffer [4 mm MgCl2, 20 mm Tris (pH 7.4), 10 mm NaCl, and 25 µm ATP] for 2 h at room temperature with shaking. The supernatant was then incubated with a phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P3] detector protein for 1 h at room temperature, and the reaction mixes were transferred to PI(3,4,5)P3-coated detection plates for 1 h at room temperature. After washing in wash buffer [150 mm NaCl, 10 mm Tris (pH 7.5), and 0.05% (vol/vol) Tween 20], secondary detection reagent (supplied with the kit) was added, plates were washed again, developing solution (supplied with the kit) was added, and PI(3,4,5)P3 detector protein binding to the plate was determined by measuring the absorbance at 450 nm. Enzyme activity was estimated by comparing the values from samples containing enzymatic reaction products to the values in the standard curve. The result was expressed as percentage of control.

Western blot analysis for measurement of Akt-phosphorylation. Segments of duodenum (~50 mg) were placed in buffered serosal solution at 37°C gassed with 95% O2-5% CO2 for incubation. After stabilization for 20 min, PGE2 (10–6 M) or control was added to the bathing solution and incubated for different times. In some cases, tissues were pretreated with 100 nM wortmannin for 30 min. At the end of incubation, the tissue was processed as described in Immunoprecipitation of PI3K and ELISA for detection of PI3K activity. Aliquots of the supernatants containing ~50 µg of protein were applied to each plane on to 10% SDS-PAGE gels followed by electrotransferring onto the polyvinylidene difluoride membranes. Blots were blocked for 1 h at room temperature with 5% nonfat milk and incubated at 4°C overnight with anti-phospho-Akt S473 or anti-Akt (diluted 1:1,000, Cell Signaling) as primary antibody. The membranes were washed in Tris-buffered saline containing 0.1% Tween-20 and were incubated with a second antibody (1:5,000, Pierce). After additional washing, they were developed with enhanced chemiluminescence reagents (Amersham Life Science) and exposed to films in a dark room.

Statistics. All results are expressed as means ± SE. {Delta}HCO3– and {Delta}Isc refer to stimulated peak responses minus basal levels. Basal values for HCO3– and Isc refer to an average taken over the 20-min period before stimulation of PGE2. Data were analyzed by one-way ANOVA followed by Newman-Keuls post hoc test or, when appropriate, by Student's t-tests. P < 0.05 was considered statistically significant; N is the number of individual experiments from individual mice.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of cAMP- and Ca2+-dependent signaling pathway inhibitors on PGE2-stimulated duodenal mucosal HCO3– secretion and Isc. As in previous studies (57, 63), PGE2 markedly stimulated duodenal mucosal HCO3– secretion and Isc (P < 0.0001) (Fig. 1, A and B). Adenylate cyclase inhibitor MDL-12330A (10–5 M) and cAMP-dependent protein kinase (PKA) inhibitor KT-5720 (10–5 M) significantly inhibited PGE2-stimulated duodenal HCO3– secretion and Isc (P < 0.05 and P < 0.01) (Fig. 2, A and B). MDL-12330A and KT-5720 reduced PGE2-stimulated {Delta}HCO3– secretion by 23% and 20%, respectively and {Delta}Isc by 28% and 24%. The Ca2+ channel blocker verapamil (5 x 10–5 M) and calmodulin antagonist W-13 (5 x 10–5 M) also significantly inhibited PGE2-stimulated duodenal {Delta}HCO3– secretion and {Delta}Isc by 26% and 24% and by 44% and 49%, respectively (P < 0.01 and P < 0.0001, Fig. 3, A and B). As shown in Figs. 1–3, all inhibitors had no effects on basal duodenal bicarbonate secretion and Isc. They had no effect on transepithelial resistance of duodenal mucosa either (data not shown).


Figure 1
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  		Fig. 1. Time course of change for PGE2-stimulated duodenal HCO3– secretion (A) and transepithelial short-circuit current (Isc; B). PGE2 (10–6 M) or control vehicle was added serosally at the time indicated by the arrow. Values are expressed as means ± SE; N = 8 in each series. PGE2 markedly stimulated duodenal HCO3– secretion and Isc (P < 0.0001 by 1-way ANOVA with Student-Newman-Keuls post hoc test).

 

Figure 2
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  		Fig. 2. Effects of cAMP-dependent signaling pathway inhibitor on PGE2-stimulated duodenal HCO3– secretion [time course (A1) and {Delta}HCO3– (A2)] and Isc [time course (B1) and {Delta}Isc (B2)] in murine duodenum, where {Delta}HCO3– and {Delta}Isc refer to stimulated peak responses minus basal levels. Adenylyl cyclase inhibitor MDL-12330A (10–5 M), PKA inhibitor KT-5720 (10–5 M), or control vehicle was added into the serosal side 30 min before PGE2 (10–6 M). Values are expressed as means ± SE; N = 8 in each series. MDL-12330A and KT-5720, respectively, reduced PGE2-stimulated {Delta}HCO3– secretion by 23% and 20%, and {Delta}Isc by 28% and 24%. *P < 0.05, **P < 0.01 compared with control by 1-way ANOVA with Student-Newman-Keuls post hoc test.

 

Figure 3
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  		Fig. 3. Effects of Ca2+-dependent signaling pathway inhibitor on PGE2-stimulated duodenal HCO3– secretion [time course (A1) and {Delta}HCO3– (A2)] and Isc [time course (B1) and {Delta}Isc (B2)] in murine duodenum. The Ca2+ channel blocker verapamil (5 x 10–5 M), calmodulin antagonist W-13 (5 x 10–5 M), or control vehicle was added into the serosal side 30 min before PGE2 (10–6 M). Values are expressed as means ± SE; N = 9 in each series. Verapamil and W-13, respectively, reduced PGE2-stimulated {Delta}HCO3– secretion by 26% and 24%, and {Delta}Isc by 44% and 49%. **P < 0.01, ****P < 0.0001 compared with control by 1-way ANOVA with Student-Newman-Keuls post hoc test.

 
Effects of PI3K inhibitors on PGE2-stimulated duodenal mucosal HCO3– secretion and Isc. Both cAMP- and Ca2+-dependent signaling pathway inhibitors only partially inhibited PGE2-stimulated duodenal HCO3– secretion, indicating that there may be other mechanism(s) involving the regulation of duodenal HCO3– secretion. The involvement of PI3K-dependent pathway in PGE2 signaling in other systems has been recently shown (19, 20, 21). We therefore attempted to determine whether the PI3K-dependent pathway is involved in the regulation of PGE2-mediated duodenal HCO3– secretion. As shown in Fig. 4, A and B, both wortmannin and LY-294002, PI3K inhibitors, significantly inhibited PGE2-stimulated duodenal HCO3– secretion and Isc (P < 0.0001). Wortmannin and LY-294002 reduced PGE2-stimulated {Delta}HCO3– secretion by 51% and 47% and {Delta}Isc by 41% and 38%, respectively. Likewise, both wortmannin and LY-294002 had no effects on basal duodenal HCO3– secretion and Isc as shown in Fig. 4. They had no effect on transepithelial resistance of duodenal mucosa either (Data not shown).


Figure 4
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  		Fig. 4. Effects of phosphatidylinositol 3-kinase (PI3K) inhibitor on PGE2-stimulated duodenal HCO3– secretion [time course (A1) {Delta}HCO3– (A2)] and Isc [time course (B1) and {Delta}Isc (B2)] in murine duodenum. PI3K inhibitors, wortmannin (10–7 M) or LY-294002 (2 x 10–5 M), or control vehicle was added into the serosal side 30 min before PGE2 (10–6 M). Values are expressed as means ± SE; N = 11 in each series. Wortmannin and LY-294002, respectively, reduced PGE2-stimulated {Delta}HCO3– secretion by 51% and 47%, and {Delta}Isc by 41% and 38%. ****P < 0.0001 compared with control by 1-way ANOVA with Student-Newman-Keuls post hoc test.

 
Effect of MAPK and tyrosine kinase inhibitors on PGE2-stimulated duodenal mucosal HCO3– secretion and Isc. Some studies have shown that MAPK- and tyrosine kinase-dependent pathways are involved in the action of PGE2 (8, 54). Therefore, we investigated whether these pathways are also involved in the regulation of PGE2-mediated duodenal HCO3– secretion. As shown in Fig. 5, A and B, the MAPK inhibitor PD-98059 (10–5 M), which can effectively inhibit MAPK at that concentration (2), had no effect on PGE2-stimulated duodenal HCO3– secretion or Isc. Likewise, the tyrosine kinase inhibitor genistein (10–5 M), which can effectively inhibit tyrosine kinase at that concentration (1), had no effect on PGE2-stimulated duodenal HCO3– secretion or Isc either. The additions of PD-98059 and genistein had no effect on transepithelial resistant of duodenal mucosa (data not shown).


Figure 5
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  		Fig. 5. Effects of MAPK and tyrosine kinase inhibitor on PGE2-stimulated duodenal HCO3– secretion (A) and Isc (B) in murine duodenum. The MAPK inhibitor PD-98059 (10–5 M), the tyrosine kinase inhibitor genistein (10–5 M), or control vehicle was added into the serosal side or both sides (genistein) 30 min before PGE2 (10–6 M). Values are expressed as means ± SE; N = 8 in each series. Neither PD-98059 nor genistein altered PGE2-stimulated duodenal HCO3– secretion. #P > 0.05 compared with control by 1-way ANOVA with Student-Newman-Keuls post hoc test.

 
Effect of PGE2 on PI3K activity in duodenal mucosa. The pharmacological studies described above indicated that PI3K-dependent pathway is involved in PGE2-mediated duodenal mucosal HCO3– secretion and Isc. To confirm the involvement of PI3K in the action of PGE2, we further examined the effect of PGE2 on duodenal mucosal PI3K activity. The results showed that PGE2 increased duodenal mucosal PI3K activity in a concentration-dependent manner (10–9 M to 10–5 M) (P < 0.0001) (Fig. 6A). PGE2 stimulated PI3K activity in a dose-dependent manner with a maximal effect at 10–6 M. PGE2 markedly induced the activation of PI3K as early as 1 min after stimulation. The activity of PI3K was maximal within 5 min (Fig. 6B).


Figure 6
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  		Fig. 6. Effect of PGE2 on duodenal mucosal PI3K activity. Murine duodenal mucosa was treated for 5 min with various concentrations of PGE2 (A) or for various periods of time with PGE2 (10–6 M) (B). Mucosal extract was immunoprecipitated with anti-PI3K P85 antibody in vitro. PI3K activity was measured as described in MATERIALS AND METHODS and expressed as % of control. Values are means ± SE of 4 independent experiments. PGE2 increased PI3K activity concentration dependently. PGE2 induced activation of PI3K in as early as 1 min and reached maximal value within 5 min. #P > 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with control by 1-way ANOVA with Student-Newman-Keuls post hoc test.

 
Effect of PGE2 on Akt phosphorylation. The phosphorylation of Akt, a downstream effector of PI3K, by PGE2 was then studied in intact murine duodenum. The experimental protocol for incubation of the tissue was similar to that for measuring PI3K activity. PGE2 at 10–6 M caused a rapid phosphorylation of Akt, and the time course of Akt phosphorylation was similar to that for activation of PI3K (Fig. 7). Wortmannin, a specific PI3K inhibitor, significantly inhibited PGE2-stimulated phosphorylation of Akt. Thus the results indicate that PGE2 activates PI3K and its downstream effector in murine duodenal mucosa.


Figure 7
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  		Fig. 7. Time course of PGE2-stimulated phosphorylation of Akt and effect of PI3K inhibitor wortmannin on PGE2-stimulated phosphorylation of Akt. A: duodenal mucosae were incubated with 10–6 M PGE2 for the indicated times and were subjected to Western blot analysis as described in MATERIALS AND METHODS. PGE2 stimulated phosphorylation of Akt and reached a peak at 5 min. The blot shown is a representative of 3 independent experiments with each antibody and indicates the relative density of the band compared with the control. B: duodenal mucosae were pretreated for 30 min with 100 nM wortmannin before incubating with PGE2 for 5 min. The blot shown is a representative of 3 independent experiments with each antibody and indicates the relative density compared with control. pAkt, phospho-Akt; C, control without administration of PGE2; W + P, wortmannin was administrated before PGE2; P, administration of PGE2 without pretreatment with wortmannin. Wortmannin markedly inhibited PGE2-stimulated phosphorylation of Akt. *P < 0.01 compared with both C and P.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
PGE2 plays an important role in the regulation of duodenal HCO3– secretion and is the most important intramucosal mediator of stimulation of duodenal HCO3– secretion by luminal acid and other noxious agents (31, 51, 52). However, the intracellular signal transduction pathways underlying the PGE2-induced duodenal secretory response have not been completely understood. In this study, our results demonstrated that multiple signaling pathways, cAMP, Ca2+, and PI3K dependent, are involved in PGE2-mediated duodenal HCO3– secretion and that the PI3K-dependent pathway is an important part of the signaling pathway in PGE2-mediated duodenal HCO3– secretion.

As described in the introduction, early studies implicated cAMP as the intracellular modulator of duodenal mucosal HCO3– secretion (18, 30), but several recent studies suggested that PGE2 may stimulate duodenal HCO3– secretion by additional mechanisms (39, 57, 63). We found in this study that both MDL-12330A, an adenylate cyclase inhibitor, and KT-5720, a specific PKA inhibitor, significantly reduced PGE2-stimulated duodenal HCO3– secretion, but only by ~20%.

Ca2+ also functions as an intracellular modulator in duodenal HCO3– secretion (24, 56). We previously observed some similarities between PGE2 and carbachol-induced secretory response (57). In addition, experiments in rats in vivo found that the Ca2+ channel blocker verapamil markedly mitigated PGE receptor subtype 3 (EP3) agonist (sulprostone)-stimulated duodenal HCO3– secretion (53). In this study, our results also showed that both verapamil, a Ca2+ channel blocker, and W-13, a calmodulin antagonist, reduced PGE2-stimulated duodenal HCO3– secretion in vitro, but only by ~25%. In addition, studies by us (unpublished observation) and by others (11) failed to see the classic Ca2+ peaks in isolated murine and human duodenocytes, as seen with carbachol, when PGE2 was used as the stimulus.

Although the use of inhibitor molecules to elements of intracellular signaling cascades in intact duodenal mucosa is always fraught with uncertainties, we and others have found that these molecules have a much greater effect in the same tissue when used to inhibit HCO3– secretion induced by other agonists. For example, verapamil completely blocked the carbachol-induced HCO3– secretory response both in vivo (40) and in vitro (24). Likewise, the cAMP-signaling cascade inhibitors KT-5720 was shown to effectively block PKA-mediated events in whole rat ileum in vitro (26, 32), and MDL-12330A has been shown to inhibit HCO3– secretory responses to other agonists much more effectively in the same murine duodenal preparation (56). Therefore, our results demonstrated that both cAMP- and Ca2+-dependent signaling pathways do not fully explain signaling in PGE2-mediated duodenal HCO3– secretion.

PI3K is a lipid kinase, which is responsible for the phosphorylation of the 3 position of the inositol ring of PI(4,5)P2 to generate PI(3,4,5)P3. It has been shown that PI3K plays an important role in the control of metabolism; cell growth, proliferation, survival, and migration; and membrane transport and secretion (9, 15, 41, 62). We therefore attempted to determine whether PI3K is involved in PGE2-mediated duodenal HCO3– secretion. Our results showed that wortmannin (100 nM), a PI3K inhibitor, markedly reduced PGE2-stimulated duodenal HCO3– by 51%. LY-294002 (20 µM), another structurally and mechanistically distinct PI3K inhibitor, exerted similar effects on PGE2-stimulated duodenal HCO3– secretion. Both LY-294002 and wortmannin, at these concentrations, have been shown to target PI3K activity (42, 60). Either of the two also has other, but different, targets, and the fact that both agents have a very similar inhibitory effect on PGE2-induced HCO3– secretion strongly suggested that the PI3K-dependent pathway may also be involved in PGE2-stimulated duodenal HCO3– secretion. We further measured PI3K activity in murine duodenal mucosa and found that PGE2 increased duodenal mucosal PI3K activity concentration dependently, the maximal response reached fivefold of the control, and that activation of PI3K was observed as early as 1 min and reached a peak within 5 min. The downstream effector of PI3K, Akt, was also rapidly phosphorylated by incubation of the duodenal mucosa with PGE2, and wortmannin significantly inhibited PGE2-stimulated phosphorylation of Akt. Taken together, multiple signaling pathways mediated PGE2-stimulated duodenal HCO3– secretion, but the strong and similarly potent inhibition of structurally unrelated PI3K inhibitors suggests that the activation of PI3K, which we indeed found to occur rapidly on PGE2 application in the duodenal mucosa, is an important mechanism in PGE2-stimulated duodenal HCO3– secretion.

PGE2 mediates its physiological effects by interactions with subtype of PGE2 receptors (EP). Four pharmacologically classified EP receptors (EP1, EP2, EP3, and EP4) have been cloned, which are members of the G protein-coupled family of receptors. Morimoto et al. (38) investigated the regional and cellular distribution of mRNAs for EP-receptor subtypes in the mouse gastrointestinal tract by in situ hybridization. They found that the expression of EP4 receptor mRNA was particularly strong in the epithelial layer of the duodenum. The high PGE2 concentrations that stimulate duodenal mucosal HCO3– secretion in vitro are consistent with the concept that the major receptor for PGE2-stimulated HCO3– secretion in isolated duodenal mucosa is the EP4 receptor (4). In addition, a recent study, using a specific EP4-receptor antagonist, has implicated the EP4 receptor to mediate the PGE2-induced stimulation of duodenal HCO3– secretion in human biopsies in vitro (33). Despite the linkage of EP4 receptor activation to cAMP generation, we felt that, for reasons mentioned above, cAMP-dependent signaling cannot be the only pathway in PGE2-mediated duodenal HCO3– secretion.

Recent work by Fujino et al. provided evidence for another signaling pathway coupled to EP4 receptors. They demonstrated that the maximal level of PGE2-stimulated cAMP formation in EP4 receptor-transfected cells was only 14% that achieved in EP2 receptor-transfected cells, even though both receptors were expressed to nearly the same extent (19). In addition, the group provided evidence for PI3K-mediated phosphorylation of Akt after EP4 but not EP2 receptor stimulation. They demonstrated that PGE2 stimulation of EP4 receptors, but not EP2 receptors, leads to phosphorylation of the extracellular signal-regulated kinases (ERKs) through a PI3K-dependent mechanism (20, 21). A further study gave evidence for the coupling of EP4 receptors to a pertussis toxin-sensitive inhibitory G protein that can inhibit cAMP-dependent signaling and activate PI3K signaling (22). EP4 agonists and antagonists have been developed, and it would be interesting to see whether they are able to elicit, and antagonize, PI3K activation in the duodenal mucosa. However, they are not available to use at the present time.

In an intestinal cell line, CFTR activation by forskolin was found not to involve PI3K activation (16). Later, the group of Barrett et al. (6) showed that in T84 cells, transactivation of the EGF receptor is required for the full expression of cAMP-dependent Cl– secretory responses, through a mechanism that likely involves PI3K. PI3K activation of other apical and basolateral solute absorbing mechanisms is amply described in the literature (7, 17, 23, 34, 47). PGE2-mediated HCO3– secretion likely involves both CFTR-mediated conductive HCO3– secretion (48) as well as CFTR-dependent Cl–/HCO3– exchange (Walker NM, Simpson JE, Schweinfest CW, Clarke LL, personal communication) and CFTR-independent, Slc26a6 mediated Cl–/HCO3– exchange (57). In addition, HCO3– uptake and generation mechanisms are differentially expressed along the crypt-villus axis. Since the different time course of the changes in Isc and HCO3– secretion has been observed previously (37, 55) and is believed to be due to the fact that a change in ionic movement is immediately reflected in Isc, but titration of increased HCO3– is delayed owing to slow diffusion through mucus and other unstirred layers, Isc minus HCO3– secretory rates do not accurately reflect Cl– secretion either. Thus it is presently impossible to exactly delineate which HCO3– secretory mechanisms—Cl–/HCO3– exchangers or anion conductive pathways—may be exclusively or more strongly affected by PI3K inhibition. Heterologous expressions systems may be useful to answer this question.

The finding of PI3K activation by PGE2 in the gastrointestinal epithelium also sheds some new light on the old puzzle of the "cytoprotective" effect of PGE2 against mucosal injury inflicted by a large variety of substances (10, 12, 45). While many scientists believed at the time that this effect could be fully contributed to the effects of PGs on mucosal blood flow, inhibition of acid, and/or stimulation of mucus and HCO3– secretion (29, 14), these cytoprotective effects were also seen in isolated gastric cells (46) and, as later noticed, also in other cell types like hepatic (36), pancreatic (5), and immune cells (59). It was also noticed that PGE2 prolonged epithelial cell life (58). The central role of PI3Ks in mediating antiapoptotic signals (41, 62) may explain part of these cytoprotective effects. Thus PI3K activation by PGE2 may be involved in mediating both secretory and antiapoptotic events in the gastrointestinal mucosa.

In summary, our findings provide a novel signaling mechanism for the regulation of duodenal HCO3– secretion. It shows that in the native duodenum, PI3K signaling is an important pathway in PGE2-mediated duodenal HCO3– secretion in addition to cAMP- and Ca2+-dependent pathways. These data may help to solve the puzzle why the characteristics of the PGE2-elicited secretory response are distinctly different from that elicited by cAMP-, cGMP-, or Ca2+-dependent stimuli.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by grants from the Deutsche Forschungsgemeinschaft (Se 460/13-1/2) and Affiliated Hospital of Zunyi Medical College, Zunyi, China, to B.-G. Tuo.


   ACKNOWLEDGMENTS
 
We thank Anja Krabbenhöft and Regina Engelhardt for technical skill.


   FOOTNOTES
 

Address for reprint requests and other correspondence: U. Seidler, Dept. of Gastroenterology, Hepatology, and Endocrinology, Hannover Medical School, Carl-Neuberg Str. 1, 30625 Hannover, Germany (e-mail: Seidler.Ursula{at}mh-hannover.de)

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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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