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Am J Physiol Gastrointest Liver Physiol 291: G672-G680, 2006. First published April 27, 2006; doi:10.1152/ajpgi.00110.2006
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NEUROREGULATION AND MOTILITY

IL-1beta signaling in cat lower esophageal sphincter circular muscle

Weibiao Cao, Ling Cheng, Jose Behar, Piero Biancani, and Karen M. Harnett

Department of Medicine, Rhode Island Hospital and Brown Medical School, Providence, Rhode Island

Submitted 8 March 2006 ; accepted in final form 26 April 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In a cat model of acute experimental esophagitis, resting in vivo lower esophageal sphincter (LES) pressure and in vitro tone are lower than in normal LES, and the LES circular smooth muscle layer contains elevated levels of IL-1beta that decrease the LES tone of normal cats. We now examined the mechanisms of IL-1beta-induced reduction in LES tone. IL-1beta significantly reduced acetylcholine-induced Ca2+ release in Ca2+-free medium, and this effect was partially reversed by catalase, demonstrating a role of H2O2 in these changes. IL-1beta significantly increased the production of H2O2, and the increase was blocked by the p38 MAPK inhibitor SB-203580, by the cytosolic phospholipase A2 (cPLA2) inhibitor AACOCF3, and by the NADPH oxidase inhibitor apocynin, but not by the MEK1 inhibitor PD-98059. IL-1beta significantly increased the phosphorylation of p38 MAPK and cPLA2. IL-1beta-induced cPLA2 phosphorylation was blocked by SB-203580 but not by AACOCF3, suggesting sequential activation of p38 MAPK-phosphorylating cPLA2. The IL-1beta-induced reduction in LES tone was partially reversed by AACOCF3 and by the Ca2+-insensitive PLA2 inhibitor bromoenol lactone (BEL). IL-1beta significantly increased cyclooxygenase (COX)-2 and PGE2 levels. The increase in PGE2 was blocked by SB-203580, AACOCF3, BEL, and the COX-2 inhibitor NS-398 but not by PD-98059 or the COX-1 inhibitor valeryl salicylate. The data suggested that IL-1beta reduces LES tone by producing H2O2, which may affect Ca2+-release mechanisms and increase the synthesis of COX-2 and PGE2. Both H2O2 and PGE2 production depend on sequential activation of p38 MAPK and cPLA2. cPLA2 activates NADPH oxidases, producing H2O2, and may produce arachidonic acid, converted to PGE2 via COX-2.

acetylcholine; smooth muscle

IN VITRO lower esophageal sphincter (LES) tone is thought to be myogenic as it is not affected by neural antagonists, including TTX (10, 28, 37). Myogenic LES tone is associated with the activity of low-molecular-weight secreted group IB phospholipase A2 (PLA2) and the production of arachidonic acid (AA), which is metabolized to prostaglandins and thromboxanes. The AA metabolites PGF2{alpha} and thromboxane A2/B2 act on receptors linked to G proteins to induce the activation of phosphatidylinositol-specific and phosphatidylcholine-specific phospholipases, production of second messengers, activation of PKC, and maintenance of sustained contraction, resulting in the maintenance of LES tone (24).

We have shown that, in a cat model of experimental esophagitis, repeated 45-min esophageal perfusion with 0.1 N hydrochloric acid over 3 days reduces resting in vivo LES pressure and in vitro LES tone (12, 25). In addition, after the induction of experimental esophagitis, the LES circular muscle layer contains elevated levels of inflammatory mediators, such as IL-1beta (16, 26), that increase the production of the reactive oxygen species H2O2 (25). H2O2 increases the production of PGE2, which relaxes the LES, and increases the production of stable prostanoids such as 8-iso-PGF2{alpha}, which, by itself, causes little or no contraction but inhibits the contraction of the LES in response to PGF2{alpha} (25). In addition, H2O2 reduced the amplitude of the acetylcholine (ACh)-induced Ca2+ increase and reproduced the effect of esophagitis on Ca2+ signaling. The esophagitis-induced reduction was partially reversed by the H2O2 scavenger catalase (20).

In the present study, we found that IL-1beta reduced the amplitude of Ca2+ signaling, similarly to H2O2 and esophagitis, and therefore examined in some detail the possible mechanisms of the IL-1beta-induced reduction in LES tone and the signal transduction pathway of IL-1beta-induced H2O2 and PGE2 production.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Tissue dissection. Experimental procedures were approved by the Animal Welfare Committee of Rhode Island Hospital. Adult cats of either sex weighing 3–5 kg were euthanized, and LES smooth muscle squares from the circumferential muscle layer were prepared as previously described (13). The chest and abdomen were opened with a midline incision, exposing the esophagus and stomach. The esophagus and stomach were removed together and pinned on a wax block at their in vivo dimensions and orientation. The esophagus and stomach were opened along the lesser curvature. After opening the esophagus and stomach and identifying the LES, we removed the mucosa and submucosal connective tissue by sharp dissection. The LES was excised, and a 3- to 5-mm-wide strip at the junction of the LES and esophagus was discarded to avoid overlap. The longitudinal muscle layer was also removed under a microscope.

Measurements of in vitro LES tone. Circular muscle strips (2 mm wide) were mounted in separate 1-ml muscle chambers as previously described in detail (12). They were initially stretched to 2.5 g to bring them near conditions of optimum force development and equilibrated for 2 h while perfused continuously with oxygenated physiological salt solution (PSS) at 37°C. During this time, the tension in LES strips increased, attaining a steady level at 2 h. The PSS contained the following (in mmol/l): 116.6 NaCl, 21.9 NaHCO3, 1.2 NaH2PO4, 3.4 KCl, 2.5 CaCl2, 5.4 glucose, and 1.2 MgCl2. The solution was equilibrated with a gas mixture containing 95% O2 and 5% CO2 at 37°C and pH 7.4.

Smooth muscle tension was recorded on a chart recorder (Grass Instruments, Quincy, MA). Passive force was obtained at the end of the experiment by completely relaxing the strips with excess EDTA until no further decrease in resting force was observed. Basal LES tone is defined as the difference between resting and passive force. The percent basal LES tone was calculated by the ratio between the force after the drug exposure and the basal LES tone.

Isolation of smooth muscle cells. LES circular smooth muscle strips were isolated by enzymatic digestion in HEPES-buffered collagenase solution as described previously (17, 77). Briefly, the collagenase solution (pH 7.2) contained 0.5 mg/ml collagenase type F (Sigma, St. Louis, MO), 1 mg/ml papain, 1 mg/ml BSA, 1 mM CaCl2, 0.25 mM EDTA, 10 mM glucose, 10 mM HEPES (sodium salt), 4 mM KCl, 125 mM NaCl, 1 mM MgCl2, and 10 mM taurine. The tissue was kept in enzyme solution at 4°C for about 16 h, warmed up to room temperature for 30 min, and incubated in a water bath at 31°C for about 30 min. At the end of the digestion period, the tissue was poured out over a 200-µm Nitex mesh (Tetko, Elmsford, NY), rinsed in collagenase-free HEPES-buffered solution to remove any trace of collagenase, and incubated in this solution at 31°C and gassed with 100% O2. The collagenase-free HEPES-buffered solution (pH 7.4) contained 112.5 mM NaCl, 3.1 mM KCl, 2.0 mM KH2PO4, 10.8 mM glucose, 24.0 mM HEPES (sodium salt), 1.9 mM CaCl2, 0.6 mM MgCl2, 0.3 mg/ml BME amino acid supplement, and 0.08 mg/ml soybean trypsin inhibitor. Gentle agitation was used to release single cells.

Cytosolic Ca2+ measurements. Freshly isolated cells were loaded with fura 2-AM (1.25 µM) for 40 min and placed in a 5-ml chamber mounted on the stage of an inverted microscope (Carl Zeiss). Cells were allowed to settle onto a coverslip at the bottom of the chamber. The bathing solution was collagenase-free HEPES-buffered solution (normal Ca2+ medium) or HEPES-buffered solution without CaCl2 but with 200 µM BAPTA (Ca2+-free medium). When Ca2+-free medium was used, after cells had settled to the bottom of the chamber, the cells were rinsed twice with Ca2+-free medium before the experiments.

ACh (1 µM) was applied directly to the cells using a pressure ejection micropipette system. Solutions in the pressure ejection micropipettes were identical to the bathing solutions except for the addition of ACh.

The concentrations of agents in the micropipette were considerably higher than those used in cell suspensions. The pipette tip was very small, and it was expected that the solution ejected from the tip would be diluted several times by the buffer surrounding the cells. Thus, the concentration of the agonists reaching the cells was much lower than that present in the micropipette.

Ca2+ measurements were obtained using a modified dual-excitation wavelength imaging system (IonOptix, Milton, MA). The Ca2+ concentrations were measured from the ratios of fluorescence elicited by 340-nm excitation to 380-nm excitation using standard techniques (41). Ratiometric images were masked in the region outside the borders of the cell because low photon counts give unreliable ratios near the edges. We developed a method for generating an adaptive mask that follows the borders of the cell as Ca2+ changes and as the cell contracts. A pseudoisobestic image (i.e., an image insensitive to Ca2+ changes) was formed in computer memory from a weighted sum of images generated by 340-nm excitation and 380-nm excitation. This image was then thresholded, i.e., values below a selected level were considered to be outside the cell and assigned a value of 0. For each ratiometric image, the outline of the cell was determined, and the generated mask was applied to the ratiometric image. This method allows the simultaneous imaging of the changes in Ca2+ and cell length. Our algorithm has been incorporated into IonOptix software. The peak Ca2+ increase was defined as the difference between the peak value and the basal value.

H2O2 measurement and protein content. LES circular smooth muscle squares (100 mg) were homogenized in PBS buffer to allow the measurement of H2O2 present in the supernatant as well as H2O2 contained inside cells. Homogenization consists of a 20-s burst with a Tissue Tearer (Biospec, Racine, WI) followed by 50 strokes with a Dounce tissue grinder (Wheaton, Melville, NJ). An aliquot of homogenate was taken for protein measurement. The homogenate was centrifuged at 15,000 rpm for 15 min at 4°C in a Beckman J2-21 centrifuge with a fixed-angle JA-20 rotor (Beckman, Palo Alto, CA), and the supernatant was collected.

H2O2 content was measured by BIOXYTECH H2O2-560 Quantitative Hydrogen Peroxide Assay Kit (OXIS, Portland, OR). This assay is based on the oxidation of ferrous ions to ferric ions by H2O2 under acidic conditions. The ferric ion binds with the indicator dye xylenol orange to form a stable colored complex that can be measured at 560 nm.

The amount of protein was determined by colorimetric analysis (Bio-Rad Protein Assay, Bio-Rad Laboratories, Richmond, CA) according to the methods of Bradford (15).

PGE2 measurement. The LES and esophageal circular smooth muscle tissue (100 mg) were homogenized in PGE2 homogenization buffer [0.1 M phosphate buffer (pH 7.4) containing 1 mM EDTA and 20 µg/ml indomethacin] at 4°C. Homogenization consisted of 10- to 20-s bursts with a Tissue Tearer (Biospec) followed by 40–60 strokes with a Dounce tissue grinder (Wheaton). The homogenate was centrifuged at 15,000 rpm at 4°C for 15 min. One hundred microliters of each sample supernatant was used for protein measurement; 2 ml of supernatant was used for PGE2 purification by an Affinity column (Cayman Chemical, Ann Arbor, MI). The resulting extracts were kept at –70°C. The PGE2 concentration was quantified using a PGE2 Competitive Enzyme Immunoassay Kit (Cayman Chemical).

Western blot analysis. LES circular muscle was homogenized in Triton X-100 lysis buffer containing 50 mM Tris·HCl (pH 7.5), 100 mM NaCl, 50 mM NaF, 5 mM EDTA, 1% (vol/vol) Triton X-100, 40 mM beta-glycerolphosphate, 40 mM p-nitrophenylphosphate, 200 µM sodium orthovanadate, 100 µM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, and 1 µg/ml aprotinin. The suspension was centrifuged at 15,000 g for 5 min, and the protein concentration in the supernatant was determined. Western blot analysis was done as previously described (22). Briefly, after the supernatants were subjected to SDS-PAGE, the separated proteins were electrophoretically transferred to a nitrocellulose membrane at 30 V overnight. The nitrocellulose membranes were blocked in 5% nonfat dry milk and then incubated overnight with anti-phosphorylated p38 MAPK antibody (1:1,000), anti-phosphorylated cytosolic PLA2 (cPLA2) antibody (1:1,000), or cyclooxygenase (COX)-2 antibody (1:1,000) followed by 60-min incubation in horseradish peroxydase-conjugated secondary antibodies (Amersham, Arlington Heights, IL). Detection was achieved with an enhanced chemiluminescence agent (Amersham). The cPLA2 antibody recognized phosphorylated Ser505 of cPLA2, and the p38 antibody recognized phosphorylated Thr180 and Tyr182 of p38 MAPK.

After the detection of phosphorylated p38 MAPK or cPLA2, membranes were incubated in stripping buffer (100 mM 2-mercaptoethanol, 2% SDS, and 62.6 mM Tris·HCl; pH 6.7) at 50°C for 30 min, washed three times (10 min each), and then reprobed using anti-p38 MAPK antibody (1:500) or anti-cPLA2 antibody (1:1,000), respectively.

Drugs and chemicals. PD-98059, SB-203580, and AACOCF3 were purchased from Calbiochem; soybean trypsin inhibitor was from Worthington Biochemicals (Freehold, NJ); fura 2-AM and BAPTA were from Molecular Probes; anti-phosphorylated p38 antibody was from New England Biolabs (Beverly, MA); anti-cPLA2 and anti-phosphorylated cPLA2 antibodies were from Cell Signaling Technology (Beverly, MA); anti-p38 MAPK antibody was from Santa Cruz Biotechnology (Santa Cruz, CA); COX-2 antibody, the PGE2 affinity column, and the PGE2 EIA kit were from Cayman Chemical. The H2O2-560 Quantitative Hydrogen Peroxide Assay was from Oxis; human recombinant IL-1beta was from Pierce Endogen (Rockford, IL); and ACh, collagenase type F, papain, catalase, BME amino acid supplement, HEPES sodium, and other reagents were purchased from Sigma.

Statistical analysis. Data are expressed as means ± SE. Statistical differences between two groups were determined by Student's t-test. Differences between multiple groups were tested using ANOVA and checked for significance using Fisher's protected least-significant difference test.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Possible mechanisms of the IL-1beta-induced reduction in LES tone. We have previously shown that IL-1beta dose dependently decreased the tone of in vitro LES strips and that the reduction in tone was partially reversed by catalase, suggesting a role of H2O2 in the reduction. We have shown that IL-1beta significantly increased H2O2 production and that H2O2 dose dependently reduced LES tone (25), suggesting that IL-1beta reduces tone by producing H2O2. H2O2, in turn, contributes to the reduction by affecting the Ca2+ release from intracellular stores (23) and by inducing the formation of PGE2 and products of lipid peroxidation (25).

To directly show that IL-1beta affects intracellular Ca2+ stores through the production of H2O2, we incubated normal LES cells in Ca2+-free medium with IL-1beta (100 U/ml for 2 h). After the IL-1beta treatment, the ACh-induced peak Ca2+ increase was significantly decreased (Fig. 1) compared with untreated cells in Ca2+-free medium, suggesting that IL-1beta may affect intracellular Ca2+ stores or Ca2+-release mechanisms from stores. In addition, the IL-1beta-induced reduction in the Ca2+ release was in part restored by catalase, indicating that the formation of H2O2 may contribute to the IL-1beta-induced reduction in Ca2+ release.


Figure 1
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  		Fig. 1. Freshly isolated cells were loaded with fura 2-AM (1.25 µM) for 40 min. The bathing solution was collagenase-free HEPES-buffered solution without CaCl2 but with 200 µM BAPTA (Ca2+-free medium). Cells were incubated in Ca2+-free medium to selectively examine the effect of IL-1beta on the release of Ca2+ from intracellular stores. Left: decrease in Ca2+ release after cells were incubated in IL-1beta. Right: partial restoration of the Ca2+ signal when cells were exposed simultaneously to IL-1beta and catalase (78 U/ml, 2 h), indicating that the reduction in Ca2+ release is mediated by the production of H2O2 in response to IL-1beta. Data are shown as means ± SE; n = 8 cells for control, IL-1beta, and IL-1beta + catalase.

 
Signaling in IL-1beta-induced H2O2 production. To confirm a role of H2O2 in the IL-1beta-induced reduction in tone, we measured the production of H2O2 in normal LES circular muscles in response to IL-1beta (200 U/ml, 2 h; Fig. 2). Consistently with previous findings (25), IL-1beta significantly increased the production of H2O2. The IL-1beta-induced production of H2O2 was significantly reduced by the p38 MAPK inhibitor SB-203580 (10–5 M) (29), the cPLA2 inhibitor AACOCF3 (10–5 M, P < 0.01 by ANOVA) (79), and the NADPH oxidase inhibitor apocynin, but not by the MEK inhibitor PD-98059 (10–5 M) (1), suggesting that IL-1beta-induced H2O2 production is mediated by the activation of p38 MAPK, cPLA2, and NADPH oxidase. To confirm the roles of p38 MAPK and cPLA2, we examined the phosphorylation of p38 MAPK and cPLA2 after IL-1beta treatment. Because a 2-h treatment with IL-1beta produced a significant inhibition of muscle contractility and significant stimulation of H2O2 production, we examined the phosphorylation of p38 MAPK and cPLA2 at 2 h after IL-1beta treatment. IL-1beta significantly increased the phosphorylation of p38 MAPK and cPLA2 (Fig. 3), confirming the IL-1beta-induced activation of p38 MAPK and cPLA2. The IL-1beta-induced p38 MAPK phosphorylation was not blocked by the cPLA2 inhibitor AACOCF3 (Fig. 3), indicating that p38 MAPK phosphorylation does not depend on the activation of cPLA2. The IL-1beta-induced cPLA2 phosphorylation, however, was blocked by the p38 MAPK inhibitor SB-203580, indicating that the IL-1beta-induced cPLA2 phosphorylation depends on the activation of p38 MAPK (Fig. 3).


Figure 2
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  		Fig. 2. Production of H2O2 in normal lower esophageal sphincter (LES) circular muscles in response to IL-1beta (200 U/ml, 2 h). IL-1beta significantly increased the production of H2O2 [*statistically significant difference (P < 0.05) with respect to the control]. The production of H2O2 was significantly reduced by the p38 MAPK inhibitor SB-203580 (10–5 M), by the cytosolic phospholipase A2 (cPLA2) inhibitor AACOCF3 (10–5 M, P < 0.01 by ANOVA), and by the NADPH oxidase inhibitor apocynin (10–4 M) [#statistically significant difference (P < 0.05) with respect to IL-1beta]. The production of H2O2 was not significantly reduced by the MEK inhibitor PD-98059 (10–5 M). The data indicate that IL-1beta-induced H2O2 production is mediated by the activation of p38 MAPK, cPLA2, and NADPH oxidase. IL-1beta was added after the muscle strips were preincubated with Krebs solution or inhibitors for 30 min. Data are shown as means ± SE of 3 experiments.

 

Figure 3
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  		Fig. 3. Top left: Western blot analysis of phosphorylated (p-p38) and nonphosphorylated p38 MAPK in normal (control) and IL-1beta-treated (200 U/ml, 2 h) LES circular muscles. IL-1beta significantly increased the phosphorylation of p38 MAPK. IL-1beta-induced p38 MAPK phosphorylation was not blocked by the cPLA2 inhibitor AACOCF3 (10–5 M), indicating that p38 MAPK phosphorylation does not depend on the activation of cPLA2. Bottom left: bar graph showing means ± SE of 3 experiments. *Statistically significant difference (P < 0.05) with respect to the control. Top right: Western blot analysis of phosphorylated (p-cPLA2) and nonphosphorylated cPLA2 in normal (control) and IL-1beta-treated (200 U/ml, 2 h) LES circular muscles. IL-1beta significantly increased the phosphorylation of cPLA2. IL-1beta-induced cPLA2 phosphorylation was blocked by the p38 MAP kinase inhibitor SB-203580 (10–5 M), indicating that cPLA2 phosphorylation depends on the activation of p38 MAPK. IL-1beta was added after the muscle strips were preincubated with Krebs solution or inhibitors for 30 min. Bottom right: bar graph showing means ± SE of 3 experiments. *Statistically significant difference (P < 0.05) with respect to the control; #statistically significant difference (P < 0.05) with respect to IL-1beta.

 
These results confirm that IL-1beta causes the production of H2O2 and demonstrate that H2O2 production depends on the sequential activation of p38 MAPK to phosphorylate cPLA2, presumably resulting in the production of AA.

AA production within muscle or other cells may derive from cPLA2 as well as from Ca2+-insensitive PLA2 (iPLA2) (4, 5, 30–32, 57, 84). The cPLA2 inhibitor AACOCF3 and the iPLA2 inhibitor bromoenol lactone (BEL) (46) partially reversed the IL-1beta-induced reduction in LES tone (P < 0.05 by ANOVA) when used individually (Fig. 4) and abolished it when used together (Fig. 5), suggesting that the production of AA (or relaxant AA metabolites, e.g., PGE2) may be required for the IL-1beta-induced reduction in LES tone. In addition, because nitric oxide (NO) relaxes LES circular muscle, we examined a possible role of NO in the IL-1beta-induced reduction of LES tone. The IL-1beta-induced reduction in LES basal tone was not affected by the NO synthase inhibitor N-nitro-L-arginine, indicating that the IL-1beta-induced reduction of LES tone does not involve the production of NO.


Figure 4
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  		Fig. 4. Basal LES tone was dose dependently reduced by IL-1beta, as shown in the bottom curves (both left and right). Each dose of IL-1beta was applied for 1 h. The cPLA2 inhibitor AACOCF3 (10–5 M; left) partially reversed the IL-1beta-induced reduction in LES tone (P < 0.05 by ANOVA). The Ca2+-insensitive PLA2 (iPLA2) inhibitor bromoenol lactone (BEL; 10–6 M; right) partially reversed the IL-1beta-induced reduction in LES tone (P < 0.05 by ANOVA). IL-1beta was added after the muscle strips were preincubated with Krebs solution, AACOCF3, or BEL for 30 min. Data are shown as means ± SE of 3 experiments

 

Figure 5
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  		Fig. 5. Basal LES tone (control) was significantly reduced by IL-1beta (200 U/ml, 2 h, *P < 0.05 with respect to the control). The decreased tone was partly restored by the iPLA2 inhibitor BEL (10–6 M) and by the cPLA2 inhibitor AACOCF3 (10–5 M). Tone was completely restored by BEL and AACOF3 when used in combination (#statistical significance with respect to IL-1beta). IL-1beta was added after the muscle strips were preincubated with Krebs solution or inhibitors for 30 min. Data are shown as means ± SE of 3 experiments.

 
Signaling in IL-1beta-induced PGE2 production. We have previously shown that one of the components of the IL-1beta/H2O2-induced reduction of LES tone depends on the formation of PGE2 (25). To confirm a role of PGE2 in the IL-1beta-induced reduction in tone, we measured the production of PGE2 in normal LES circular muscles incubated with IL-1beta (200 U/ml, 2 h). IL-1beta significantly increased PGE2 production (Fig. 6), and the production of PGE2 was abolished by the NADPH oxidase inhibitor apocynin, indicating that the formation of H2O2 is a required step in the IL-1beta-induced production of PGE2.


Figure 6
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  		Fig. 6. Production of PGE2 in normal LES circular muscles in response to IL-1beta (200 U/ml, 2 h). IL-1beta significantly increased the production of H2O2 [*statistically significant difference (P < 0.05) with respect to the control]. Production of H2O2 was significantly reduced by the NADPH oxidase inhibitor apocynin (10–4 M) [#statistically significant difference (P < 0.05) with respect to IL-1beta]. There were no statistically significant differences between IL-1beta + apocynin and control PGE2 levels. IL-1beta was added after the muscle strips were preincubated with Krebs solution or apocynin for 30 min. Data are shown as means ± SE of 3 experiments.

 
In addition, PGE2 production (Fig. 7) was significantly reduced by SB-203580 (10–5 M) and by the COX-2 inhibitor NS-398 (8, 49) but not by PD-98059 (10–5 M) or the COX-1 inhibitor valeryl salicylate (11, 49), suggesting that the IL-1beta-induced PGE2 production is mediated by p38 MAPK and COX-2. We therefore examined COX-2 levels in LES circular muscles before and after treatment with IL-1beta.


Figure 7
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  		Fig. 7. Production of PGE2 in normal LES circular muscles in response to IL-1beta (200 U/ml, 2 h). IL-1beta significantly increased the production of PGE2 [*statistically significant difference (P < 0.05) with respect to the control]. Production of PGE2 was significantly reduced by the p38 MAPK inhibitor SB-203580 (10–5 M) and by the cyclooxygenase (COX)-2 inhibitor NS-398 (10–4 M, P < 0.01 by ANOVA) [#statistically significant difference (P < 0.05) with respect to IL-1beta]. Production of H2O2 was not significantly reduced by the MEK inhibitor PD-98059 (10–5 M) or by the COX-1 inhibitor valeryl salicylate (10–4 M). The data indicate that IL-1beta-induced PGE2 production is mediated by the activation of p38 MAPK and COX-2. IL-1beta was added after the muscle strips were preincubated with Krebs solution or inhibitors for 30 min. Data are shown as means ± SE of 3 experiments.

 
Figure 8 shows the Western blot analysis of COX-2 in LES muscle before and after IL-1beta treatment. The data indicate that IL-1beta caused a statistically significant increase in COX-2.


Figure 8
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  		Fig. 8. COX-2 levels in normal (control) and after IL-1beta-treated (200 U/ml, 2 h) LES circular muscles were examined by Western blot analysis (top). IL-1beta significantly increased COX-2 levels. Bottom: bar graph showing means ± SE of 3 experiments. The increase in COX-2 in response to IL-1beta was statistically significant (*P < 0.05).

 
Because the IL-1beta-induced reduction in LES tone depends on the activation of both cPLA2 and iPLA2 (Figs. 4 and 5), we examined whether the production of PGE2 depends on the activation of the same phospholipases. Figure 9 shows that the cPLA2 inhibitor AACOCF3 and the iPLA2 inhibitor BEL reduced the IL-1beta-induced production of PGE2 (P < 0.05 by ANOVA) when used individually and abolished it when used together, suggesting that AA production by the two phospholipases may be required for the production of PGE2.


Figure 9
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  		Fig. 9. Production of PGE2 in normal LES circular muscles in response to IL-1beta (200 U/ml, 2 h). Production of PGE2 (control) was significantly increased by IL-1beta (*P < 0.05 with respect to the control). The increase in PGE2 was partly reduced by the iPLA2 inhibitor BEL (10–6 M) and by the cPLA2 inhibitor AACOCF3 (10–5 M). The increase in PGE2 was abolished by BEL and AACOF3 when used in combination (#statistical significance with respect to IL-1beta). IL-1beta was added after the muscle strips were preincubated with Krebs solution or inhibitors for 30 min. Data are shown as means ± SE of 3 experiments.

 
Because the production of PGE2 was blocked by the same antagonists that blocked the production of H2O2, including the NADPH oxidase inhibitor apocynin, these data are consistent with H2O2 being a required step in the IL-1beta-induced production of PGE2. The production of both H2O2 and PGE2 requires the activation of p38 MAPK and the production of AA through cPLA2 and iPLA2 to activate NADPH oxidase.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
IL-1beta-induced reduction of LES tone through the formation of AA and H2O2. We have previously shown in an in vivo model of esophagitis, induced by repeated esophageal acid perfusion (25), that esophageal inflammation is associated with increased levels of IL-1beta in circular muscle, that IL-1beta causes a dose-dependent reduction of LES tone, and that the reduction is reversed by catalase, because it is, in part, mediated by H2O2.

H2O2 contributes to the reduction of tone through several mechanisms (52). Reactive oxygen species have been shown to consistently depress the Ca2+-ATPase responsible for the uptake of Ca2+ into the endoplasmic reticulum (38–40, 58, 73). The inhibition of Ca2+-ATPase shifts the balance between Ca2+ uptake and Ca2+ release, resulting in a net Ca2+ release from intracellular stores through both ryanodine- and inositol (1,4,5)-trisphosphate-sensitive Ca2+ channels (52), eventually depleting releasable Ca2+ stores (20).

We now show that, similarly to esophagitis (20), IL-1beta causes a reduction in ACh-induced Ca2+ release that is reversed in part by the H2O2 scavenger catalase, indicating that the effect of IL-1beta on Ca2+ release is mediated in part through H2O2.

We therefore examined the possible mechanisms mediating H2O2 production in response to IL-1beta. The IL-1beta-induced production of H2O2 was not significantly reduced by the ERK1/2 inhibitor PD-98059 but was reduced by the p38 MAPK inhibitor SB-203580, indicating the involvement of p38 MAPK in the formation of H2O2. In addition, the production of H2O2 was reduced by the cPLA2 inhibitor AACOCF3 and by the NADPH oxidase inhibitor apocynin, suggesting a role of AA and NADPH oxidase in the formation of H2O2.

The link between p38 MAPK and cPLA2 is consistent with recent data on the ACh-induced contraction of rabbit intestinal smooth muscle (85) and in agreement with the IL-1beta-induced activation of p38 MAPK (33, 44, 66) and cPLA2 (9, 47) in a variety of cells. Our data show that IL-1beta significantly increased the phosphorylation of p38 MAPK and cPLA2 and that the IL-1beta-induced cPLA2 phosphorylation was blocked by a p38 MAPK inhibitor (SB-203580), whereas the inhibition of cPLA2 by AACOCF3 had no effect on p38 phosphorylation. The data thus demonstrate that the IL-1beta-induced production of H2O2 depends on the sequential activation of p38 MAPK phosphorylating cPLA2 to produce AA, in agreement with data from several laboratories on other cell species (42, 67, 82, 85, 86).

AA is released in response to a large number of bioactive molecules and is involved in the mediation of several important biological functions, including the maintenance of LES tone (18, 19, 21), vascular contraction/relaxation, cell proliferation/differentiation, and cell survival/apoptosis (59, 68, 70, 72, 84). PLA2 is the major rate-limiting enzyme in the release of AA in many cell types (5, 57, 59, 84). Among the growing number of PLA2 enzymes that have been isolated and characterized thus far, cPLA2 and iPLA2 have been shown to play important roles in AA release in response to a number of stimulants (4, 5, 46, 57, 84). Studies from several laboratories have reported that cPLA2 activity is regulated by phosphorylation (62, 72, 84). The mechanism of regulation of iPLA2 activity has not been completely elucidated, but iPLA2 also appears to be important in AA release in response to various agonists. For instance, iPLA2 plays a predominant role in thrombin-induced AA release and DNA synthesis in vascular smooth muscle cells, and these responses are mediated by p38 MAPK (84).

In LES circular muscle, we find that the cPLA2 inhibitor AACOCF3 and the iPLA2 inhibitor BEL partially reversed the IL-1beta-induced reduction in LES tone and PGE2 production when used individually and completely prevented them when used together, suggesting that the formation of AA may be a necessary step in the IL-1beta-induced reduction in LES tone and production of PGE2 and that p38 MAPK-mediated phosphorylation may be the mechanism responsible for the activation of PLA2 and production of AA.

The data are consistent with IL-1beta-induced production of AA by the activation of cPLA2 and iPLA2, with activation of cPLA2 [and possibly of iPLA2 (84)] depending on phosphorylation of the enzyme through a p38-dependent mechanism.

AA-induced activation of NADPH oxidase. AA, in turn, may activate NADPH oxidase (14, 50, 75), resulting in the production of H2O2. The mechanisms of NADPH oxidase-mediated H2O2 production in phagocytic cells has been elucidated to a reasonable extent (55). NADPH oxidase consists of transmembrane and cytosolic subunits with monomeric G proteins (2). Upon stimulation, the cytosolic subunits translocate to the plasma membrane and associate with the transmembrane subunits to form a complete enzyme.

According to the current topology of NADPH oxidases, superoxide is formed outside of the cells and reacts with itself or is degraded by superoxide dismutase to form H2O2 (55). This may explain why catalase, which does not cross the cell membrane, neutralizes the H2O2 produced in response to IL-1beta.

Our data show that the IL-1beta-induced production of H2O2 is significantly inhibited by the NADPH oxidase inhibitor apocynin, a potent and selective inhibitor of NADPH oxidase (43) that inhibits the assembly of NADPH oxidase by preventing the translocation of cytosolic components to the membrane (78) to associate with the catalytic subunit to form the active enzyme (55). Apocynin is extensively used to identify the production of H2O2 by NADPH oxidase, as opposed to other possible sources of reactive oxygen species, in cells and in experimental models of inflammation (6).

Our data are consistent with cPLA2-dependent activation of NADPH oxidase. It is known that cPLA2 translocates from the cytosol to the nuclear membrane and to the endoplasmic reticulum in response to Ca2+ ionophores or agonists such as histamine or IgE/antigen, which increase cytoplasmic Ca2+ in a variety of cells (36, 69, 74, 76). The perinuclear translocation of cPLA2 is in agreement with its role in prostaglandin formation and not consistent with translocation to the plasma membrane to activate NADPH oxidase. The activation of NADPH oxidase in monocytes, however, depends on cPLA2-generated AA (3, 61), with cPLA2 transiently recruited to the plasma membrane (75). This mechanism is consistent with the existence of alternative pathways that induce the translocation of cPLA2 not involving elevation of cytoplasmic Ca2+, for instance, PMA-induced activation and translocation of cPLA2 (45, 71). The colocalization of both enzymes (the assembled NADPH oxidase and cPLA2) in the same compartment and their direct binding during the onset of superoxide production provides a means by which AA released by cPLA2 activates the assembled NADPH oxidase. Several studies have suggested that AA induces structural changes in NADPH oxidase components that may promote interactions between the different oxidase subunits (75), enabling full oxidase activation.

The translocation of cPLA2 to the cell periphery does not occur in the absence of functional NADPH oxidase, and it can be induced with the PKC activator PMA, which does not cause an elevation of cytoplasmic Ca2+ (75).

Thus, the assembled NADPH oxidase may be the major determinant in directing cPLA2 to the plasma membrane, although the interaction sites among cPLA2, NADPH oxidase, and the plasma membrane have not yet been defined. Thus, the mechanism that permits the participation of cPLA2 in two different processes in the same cell (the regulation of NADPH oxidase and eicosanoid production) may be controlled by localization of the enzyme in different subcellular compartments (75).

Similarly to phagocytic NADPH oxidases, NADPH oxidase of LES circular muscle may be a downstream target of AA, inducing the production of H2O2 and resulting in the reduction of LES tone, as previously demonstrated (25, 27).

IL-1beta/H2O2 and PGE2. A second mechanism for the H2O2-induced reduction of LES tone involves the production of PGE2, which relaxes LES circular muscle (25). We therefore examined the possible mechanisms for the IL-1beta/H2O2-induced production of PGE2. As expected, similarly to the IL-1beta-induced production of H2O2, the IL-1beta-induced PGE2 production was abolished by inhibition of p38 MAPK, cPLA2, and iPLA2 and by the NADPH inhibitor apocynin, indicating that the p38 MAPK-mediated production of AA and H2O2 is required for the synthesis of PGE2. IL-1beta-induced PGE2 production was also inhibited by the COX-2 inhibitor NS-398 (8, 49) but not by the COX-1 inhibitor valeryl salicylate (11, 49), indicating that IL-1beta-induced PGE2 production is through the activation of COX-2.

H2O2 is known to induce the expression and activation of COX-2 in numerous experimental preparations (6, 7, 34, 48, 51, 60, 64, 81, 83). In these preparations, the NADPH oxidase-induced production of H2O2 causes the translocation of NF-{kappa}B to the nucleus, causing upregulation, increased protein expression, and activation of COX-2. In LES smooth muscle, COX-2 is present even in the absence of IL-1beta, a relatively short (2 h) exposure to IL-1beta is sufficient to increase COX-2 protein levels (Fig. 8), and IL-1beta-induced PGE2 synthesis is mediated through the activation of COX-2 (Fig. 7). It is known that cells regulate COX-2 by intracellular compartimentation, accessory proteins, arachidonate levels, and the availability of H2O2 activators (53, 65) and that the availability of O2 in most tissues is sufficient to sustain a high rate of prostaglandin biosynthesis (56). Thus, the regulation of COX-2 activity depends to a large extent on the production of AA by PLA2. Both COX-1 and COX-2 have similar primary amino acid sequences, crystal structures, and catalytic mechanisms, and the constitutive regulation of COX-1 and COX-2 catalysis is similar with only a few differences. For instance, catalysis by the COX-2 isoenzyme can proceed at H2O2 levels 10-fold lower than those needed to promote catalysis by COX-1. Investigators have speculated that this may be a mechanism to generate prostaglandins preferentially by COX-2 when both isoforms occur in the same intracellular compartment (54, 63), and this difference in regulatory mechanisms may be sufficient to explain the preferential activation of COX-2 in LES circular muscle to produce PGE2 (35). Because in LES circular muscle the activation of NADPH oxidase with the production of superoxide/H2O2 is required for the synthesis of PGE2, it is possible that the presence of reactive oxygen species and AA may preferentially activate COX-2, perhaps because of the proximity of COX-2 and PGE2 synthase (80).

Whether iPLA2, either directly or through the production of AA/H2O2, may contribute to the activation of NADPH oxidase has not yet been demonstrated.

We conclude that IL-1beta reduces LES tone by producing H2O2, which may affect Ca2+-release mechanisms and increase synthesis of PGE2. Both H2O2 and PGE2 production depend on the sequential activation of p38 MAPK and cPLA2. cPLA2 may produce AA, activating NADPH oxidases and producing H2O2, which may facilitate the conversion of AA to PGE2 via COX-2.


   GRANTS
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 ABSTRACT
 METHODS
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1-DK-57030.


   ACKNOWLEDGMENTS
 
These data were presented in part at the 101st annual meeting of the American Gastroenterological Association in San Diego, CA, in May 2000.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. M Harnett, Dept. of Medicine, Brown Medical School and Rhode Island Hospital, 55 Claverick St., Rm. 336, Providence, RI 02903 (e-mail: Karen_Harnett{at}brown.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.


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