Impaired cytoprotective function of muscle in human gallbladders with cholesterol stones
Zuo-Liang Xiao,
Joseph Amaral,
Piero Biancani, and
Jose Behar
Department of Medicine, Rhode Island Hospital and Brown University School of Medicine, Providence, Rhode Island
Submitted 15 June 2004
; accepted in final form 6 October 2004
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ABSTRACT
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Acute cholecystitis develops in gallbladders (GB) with excessive bile cholesterol (Ch). Increased membrane Ch content affects membrane function and may affect PGE2 receptors involved in the cytoprotection against acute inflammation. This study was aimed at determining whether the cytoprotective response to PGE2 is affected by lithogenic bile with Ch. Muscle cells from human GB with cholesterol stones (ChS) or pigment stones (PS) were obtained by enzymatic digestion. PGE2 levels were measured by radioimmunoassay, and activities of superoxide dismutase (SOD) and catalase were assayed by spectrophotometry. The contraction in response to H2O2 in muscle cells from PS was 14 ± 0.3%, not different from normal controls, and decreased after the cells were incubated with Ch-rich liposomes (P < 0.05), which increase the Ch content in the plasma membranes. In muscle cells from GB with ChS, H2O2-induced contraction was only 9.2 ± 1.3% and increased to 14 ± 0.2% after Ch-free liposome treatment to remove Ch from the plasma membranes (P < 0.01). H2O2 caused a similar increase in the levels of lipid peroxidation and PGE2 content in muscle cells from GBs with ChS and PS. However, the activities of SOD and catalase were significantly lower in muscle cells from GBs with ChS compared with those with PS. The binding capacity of PGE2 receptors was also significantly lower in muscle cells from GBs with ChS compared with those with PS. In conclusion, the cytoprotective response to reactive oxygen species is reduced in muscle cells from GBs with ChS despite a normal increase in the cellular levels of PGE2. This impaired cytoprotective response may be due to a dysfunction of PGE2 receptors with decreased binding capacity resulting from excessive Ch levels in the plasma membrane.
reactive oxygen species; free-radical scavengers; pigment stones
ACUTE CHOLECYSTITIS IS A FREQUENT complication in patients with supersaturated bile with cholesterol (Ch) as demonstrated by the presence of Ch stones (ChS) and crystals (8, 33, 39). The pathogenesis of acute cholecystitis is unknown (17, 21). It has been postulated that it is due to obstruction of the cystic duct by gallstones. This hypothesis, however, is based on circumstantial evidence. Ligation of the cystic duct does not cause acute cholecystitis unless animals form lithogenic bile with Ch and hydrophobic bile salts are injected into the obstructed gallbladder (GB) (30, 33, 39).
Our previous experimental studies suggest the hypothesis that acute cholecystitis develops in a permissive GB environment that facilitates initiation of this inflammatory process by specific bile constituents. The permissive milieu is characterized by bile stasis caused by the impaired response to CCK-8 and ACh because of greater Ch incorporation in the plasma membranes of GB muscle cells and resulting in increased concentration of bile constituents. These increased Ch levels in the plasma membrane may also impair cytoprotective mechanisms that muscle cells use to protect themselves against injury from concentrated bile and free radicals. This defective cytoprotection may be another factor that contributes to the worsening the permissive environment (40).
Hydrophobic bile salts are potential candidates for initiating this complication, because they can diffuse through the mucosa and affect the smooth muscle layer generating reactive oxygen species (ROS) either by direct action on muscle cells or by increasing the number of inflammatory cells in the lamina propia. ROS are known to damage transmembrane receptors (46). The finding that guinea pigs pretreated with the hydrophilic bile acid ursodeoxycholic acid prevents experimental acute cholecystitis by suppressing the actions of hydrophobic bile acids supports this hypothesis (41).
Therefore, the present studies investigated the effects of Ch on one of the pathways that cells use to protect themselves against injury from free radicals. We examined whether human GB muscle cells have cytoprotective functions and whether their cytoprotective response against the actions of H2O2 is affected by lithogenic bile with Ch.
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MATERIALS AND METHODS
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Patients.
Human GBs were obtained by elective laparoscopic cholecystectomy performed for Ch and pigment gallstones. None of the patients had a history or clinical evidence of acute cholecystitis. Gallstones were classified as Ch and pigment according to their gross appearance and chemical analysis (810, 30). Muscle cells from human GBs with pigment stones were considered control muscle cells, because they contract normally in response to an intravenous infusion of CCK-8 in vivo (2) and the percentage of shortening in response to agonists that are G protein-coupled receptor dependent and the levels of Ch in the plasma membrane are not different from those of muscle cells from GBs of normal prairie dogs and guinea pigs (8, 10, 49). The GBs were kept in ice-cold oxygenated Krebs solution (in mM: 116.6 NaCl, 3.4 KCl, 21.9 NaHCO3, 1.2 NaH2PO4, 2.5 CaCl2, 1.2 MgCl2, and 5.4 glucose). After removal of the serosa and mucosa under a dissecting microscope, the muscle layer was carefully cleaned by removing the remaining connective tissue and small blood vessels and then cut into strips for further use.
Preparation of Ch-free liposomes.
Ch-free liposomes were prepared by using egg phosphatidylcholine (5, 19). Three milliliters of phosphatidylcholine (20 mg/ml in chloroform) in a glass test tube were dried under a stream of nitrogen. The dried lipids were suspended in 3 ml of normal saline and sonicated for 30 min with a Branson 2200 sonicator (Branson Ultrasonics, Danbury, CT). The suspension was then centrifuged at 10,000 g for 30 min to sediment the undispersed lipids. Two milliliters of the supernatant and 8 ml of 0.2% BSA-HEPES buffer [in mM: 24 HEPES (pH 7.4), 112.5 NaCl, 5.5 KCl, 2.0 KH2PO4, 1.9 CaCl2, 0.6 MgCl2, and 10.8 glucose] were mixed to make Ch-free liposomes.
Isolation and permeablization of muscle cells.
Single muscle cells were obtained by enzymatic digestion (4446, 5052). GB muscle layer was cut into 2-mm-wide strip and digested in HEPES buffer containing 0.5 mg/ml type F collagenase and 2 mg/ml papain (activity of
13.9 U/mg protein) for 20 min at 35°C in a shaking water bath. The buffer was gently gassed with 100% O2 during digestion. At the end of the digestive process, the tissue was filtered through a Nitex mesh 200 (Tetko, Elmsford, NY) and rinsed with 20 ml HEPES. The tissue remaining on the filter was collected and incubated in HEPES buffer at 35°C for 15 min to allow the free dispersion of cells.
Muscle cells were permeabilized with saponin. The partially digested muscle layer was rinsed with "cytosolic buffer" (5052). After cells were dispersed, the cell suspension was briefly treated with saponin (75 µg/ml) during centrifugation at 200 g for 3 min. Cells were washed and resuspended in modified cytosolic buffer for further use.
Studies on muscle cell contraction.
Muscle contraction was determined in cells from GBs with ChS and pigment stones (PS) preincubated with buffer, Ch-free liposomes, or Ch-rich liposomes for 4 h before H2O2 (70 µM) for 15 min. Ch-rich liposome treatment allows excessive Ch to incorporate into the plasma membrane, whereas Ch-free liposomes may leach out excess Ch from it (49). Cells were measured in cell suspensions as described previously and were fixed by adding acrolein (43, 45, 46). Contraction was expressed as the mean of the percent shortening of 30 individual cells with respect to control (i.e., untreated) cells.
Preparation of plasma membranes.
Plasma membranes were prepared and purified by sucrose gradient centrifugation as previously described (32, 42). Muscle cells from GBs with ChS and PS preincubated with buffer, Ch-free liposomes, or Ch-rich liposomes were homogenized separately by using a tissue tearer (Biospec Products, Racine, WI) in 10 vol by weight of a sucrose-HEPES buffer. The homogenates were centrifuged at 600 g for 5 min. The supernatant was collected in a clean centrifuge tube (Beckman Instruments, Fullerton, CA) and centrifuged at 150,000 g for 45 min. The pellet was resuspended in sucrose-HEPES buffer, layered over a linear 960% sucrose gradient, and centrifuged at 90,000 g for 3 h. The plasma membranes were collected at
24% sucrose. They were then diluted and pelleted by centrifugation at 150,000 g for 30 min. The pellet of membranes was stored at 70°C.
Radioreceptor assay.
[3H]PGE2 binding studies were performed in a final volume of 300 µl (31, 43). Membranes containing 50 µg of protein were incubated with 100 pM of [3H]PGE2 and unlabeled PGE2 (1011 to 104 M) for 60 min at 25°C. Separation of bound from free radioligand was achieved by filtration using a vacuum filtering manifold (Millipore, Billerica, MA) with receptor-binding filter mats (Millipore), and the filters were washed with ice-cold incubation medium without BSA. Radioactivity remaining on the filters was counted in a
-scintillation counter. The results are expressed as specific binding (%) achieved by subtracting nonspecific binding (assumed as the binding in the presence of 104 M unlabeled PGE2) from total binding.
Analysis of data.
We used computer analysis with a ligand-fitting program (25) based on the radioreceptor-assay data, which analyzes the binding results to obtain the maximal specific binding capacity of CCK and PGE2 receptors.
Assessment of lipid peroxidation.
Purified plasma membranes were resuspended with 1.15% KCl (29, 43, 45, 46) and mixed with 0.2 ml of 8.1% SDS, 1.5 ml of 20% acetic acid solution (pH 3.5), and 1.5 ml of 0.8% aqueous solution of 2-thiobarbituric acid. This mixture was added to a volume of 4 ml with distilled water. The sample was heated at 95°C for 60 min. After being cooled, 1 ml of distilled water and 5 ml of the mixture of n-butanol and pyridine [15:1 (vol/vol)] were added and shaken vigorously, The organic layer was taken after it was spun at 2,000 g for 10 min, and its absorbance (red pigment) was measured at 532 nm. 1,1,3,3-Tetramethoxypropane was used as an external standard. The level of lipid peroxides was expressed as nanomoles of malonaldehyde (MDA; a secondary product of lipid peroxidation)/100 mg protein.
Measurements of PGE2 levels.
The content of PGE2 was measured by using a radioimmunoassay kit from New England Nuclear (Life Science Products, Boston, MA) (15, 43, 45). Muscle cells were homogenized in HEPES buffer containing EDTA/indomethacin to inhibit the metabolism of arachidonic acid to prostaglandins. The suspension was centrifuged at 10,000 g for 15 min. PGE2 was extracted from the supernatant by the methods of Kelly et al. (18). Extracted PGE2 was measured by converting this prostaglandin into its methyl oximate derivative using the methyl oximation reagent. The determination of PGE2 content was achieved by following the kit's protocol. The content of PGE2 was expressed as picograms per milligram of protein.
Measurement of catalase activity.
The catalase activity was determined by using the method reported by others (11, 28, 35, 43, 46). Muscle cells were homogenized in HEPES buffer, and the suspension was centrifuged at 10,000 g for 15 min. Duplicate 25-µl aliquots of each sample of the supernatant and 75 µl of 10 mM phosphate buffer, pH 7.4, were placed into 3-ml cuvettes. Duplicate blanks (B) of a solutions containing 90 µl phosphate buffer, 10 µl of 1% sodium azide, 200 µl 6 N sulfuric acid, and 1 ml of 6 mM H2O2 were also prepared. The enzymatic reactions were initiated sequentially at fixed intervals by adding 1 ml of 6 mM H2O2 to the samples. After 3 min, the reactions were stopped sequentially at the same fixed intervals by rapidly adding and mixing 200 µl 6 N H2SO4. Finally, 1.4 ml of 0.01 N KMnO4 were added to each cuvette and mixed thoroughly. A spectrophotometric standard (St) was prepared by adding 7.0 ml 0.01 N KMnO4 to a mixture of 5.5 ml phosphate buffer and 1.0 ml 6 N H2SO4. The absorbency of the solution was read at 480 nm within 3060 s. One unit of catalase activity (k) was defined as the amount of enzyme that consumed 1 µmol H2O2·mg protein1·min1. Calculation of the catalase activity was achieved according to the equation: k = log (S0/SE) x 2.3/t. (S0 was obtained by subtracting the absorbency of B from the St; SE was obtained by subtracting the absorbency of the samples from St; t = 3 min in this experiment). Data were expressed as units per milligram of protein.
Measurement of superoxide dismutase activity.
The total superoxide dismutase (SOD) activity was measured by using a spectrophotometric assay kit (R&D systems, Minneapolis, MN) (27, 43, 46). Muscle cells were homogenized in HEPES buffer. The supernatant was obtained after the suspension was centrifuged at 8,500 g for 10 min at 4°C. Ice-cold extraction reagent [400 µl; ethanol/chloroform, 62.5/37.5 (vol/vol)] was added to 250 µl of the supernatant, vortex for at least 30 s and centrifuged at 3,000 g for 10 min at 4°C. Then, the aqueous upper layer was collected for assay of the SOD activity. The determination of SOD activity was achieved by following the kit's protocol. Data were expressed as units per milligram of protein.
Protein determination.
The protein content of the muscle membranes was measured using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Melville, NY). Values for each sample were means of triplicate measurements.
Drugs and chemicals.
H2O2 was obtained from Fisher. [5H]PGE2 and PGE2 radioimmunoassay kit were obtained from New England Nuclear Life Science Products. Type F collagenase, papain, and other reagents were purchased from Sigma (St. Louis, MO).
Data analysis.
One- and two-factorial repeated ANOVA and unpaired Student's t-test were used for statistical analysis. P < 0.05 was considered to be statistically significant.
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RESULTS
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Single muscle cells from human GBs with ChS or PS were obtained by enzymatic digestion. The average resting lengths of intact cells was 59.8 ± 1.5 and 59.1 ± 1.2 µm, respectively. The resting length of muscle cells after incubating with Ch-free or Ch-rich liposomes for 4 h was 59.6 ± 0.7 and 59.3 ± 0.9 µm, respectively. No significant differences in resting cell length were observed among them (1-factor ANOVA).
Our previous studies have shown that muscle cells from GBs with PS have normal concentrations of plasma membrane Ch and that their functions that are not different from normal muscle cells of human and guinea pig GBs (8, 43, 44). We have also shown that treatment with Ch-rich liposomes increases the Ch content in the plasma membranes and reproduces all the abnormalities observed in muscle cells from GBs with Ch stones. Conversely, treatment of muscle cells from GBs with ChS with Ch-free liposomes removes the excess Ch from the plasma membranes and normalizes their functions (49). In a previous study, we had also shown that H2O2-induced muscle contraction was 14.3 ± 0.4% in normal muscle cells (45) but only 9.2 ± 1.3% in muscle cells from GBs with ChS preincubated with buffer (Fig. 1A). Pretreatment of these muscle cells with Ch-free liposomes increased H2O2 (70 µM)-induced muscle cell contraction from 9.2 ± 1.3 to 14.4 ± 0.6% (*P < 0.05) that was not different from that of controls. Ch-rich liposomes treatment of muscle cells from GBs with ChS before H2O2 had no additional effect on H2O2-induced contraction. Ch-free or Ch-rich liposomes by themselves did not induce muscle contraction.

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Fig. 1. Contraction induced by H2O2 (70 µM) in muscle cells from gallbladder (GB) with cholesterol (Ch) stones (ChS; A) and pigment stones (PS; B) preincubated with buffer, Ch-free liposomes (Ch-free L), or Ch-rich liposomes (Ch-rich L) for 4 h. A: neither Ch-free L nor Ch-rich L caused muscle contraction by themselves. Pretreatment with Ch-free L increased the H2O2 induced contraction when compared with those pretreated with buffer (*P < 0.05). Ch-rich L treatment had no effect on H2O2-induced contraction. B: preincubation with Ch-free L had no effect on H2O2-induced contraction. However, the contraction induced by H2O2 was significantly reduced after Ch-rich L preincubation (*P < 0.05). Values are means ± SE of 3 experiments.
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In muscle cells from GBs with PS preincubated with buffer, H2O2 (70 µM)-induced contraction was 14.1 ± 0.2% (Fig. 1B) and was not different from that of controls (45). Preincubation with Ch-free liposomes had no effect on H2O2-induced contraction. However, the contraction induced by H2O2 was significantly reduced from 14.1 ± 0.2 to 10.1 ± 0.2% after treatment with Ch-rich liposomes (*P < 0.05) and was not different from the contraction observed in muscle cells from GBs with ChS pretreated with buffer. These data suggest that excessive membrane Ch content impairs H2O2-induced muscle cell contraction.
To determine whether the excess Ch incorporation into plasma membrane affects membrane lipid peroxidation, the level of lipid peroxidation was measured in muscle cells from GBs with ChS preincubated with buffer, Ch-free liposomes, or Ch-rich liposomes for 4 h before exposure to H2O2 (70 µM; Fig. 2A). Ch-free or Ch-rich liposomes by themselves had no effect on the level of lipid peroxidation. H2O2 exposure for 15 min increased the level of lipid peroxidation from a basal level of 179 ± 19 to 300 ± 11 nmol MDA/100 mg protein (*P < 0.01) in the buffer-treated group. Preincubation with Ch-free liposomes or Ch-rich liposomes before H2O2 exposure had no effect on the increase in the levels of lipid peroxidation induced by H2O2.

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Fig. 2. Lipid peroxidation induced by H2O2 (70 µM) in muscle cells from GB with ChS (A) and PS (B) preincubated with buffer, Ch-free L, or Ch-rich L for 4 h. A: H2O2 exposure for 15 min caused an increase in the level of lipid peroxidation (*P < 0.01) in buffer-treated group. Preincubation with Ch-free L or Ch-rich L had no effect on the elevated level of lipid peroxidation induced by H2O2. B: H2O2 exposure for 15 min caused an increase in the level of lipid peroxidation (*P < 0.01) in the buffer-treated group. Preincubation with Ch-free L or Ch-rich L had no effect on the elevated level of lipid peroxidation induced by H2O2. Values are means ± SE of 3 experiments.
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Similarly, the level of lipid peroxidation induced by H2O2 (70 µM) was also measured in muscle cells from GBs with PS preincubated with buffer, Ch-free liposomes, or Ch-rich liposomes for 4 h (Fig. 2B) before H2O2 exposure. Ch-free or Ch-rich liposomes by themselves had no effect on the level of lipid peroxidation. H2O2 exposure for 15 min increased the level of lipid peroxidation from 210 ± 11 nmol MDA/100 mg protein of control to 328 ± 17 nmol MDA/100 mg protein (*P < 0.01) in the buffer-treated group. Preincubation with Ch-free liposomes or Ch-rich liposomes before H2O2 had no effect on the expected increase in the levels of lipid peroxidation induced by H2O2, suggesting that these changes are not influenced by increases in the content of membrane Ch and do not contribute to the impaired muscle response to H2O2.
H2O2 generates PGE2 in muscle cells (45). Therefore, PGE2 generation was examined to determine whether it was affected by excessive incorporation of Ch in the plasma membrane. PGE2 levels were measured in muscle cells from GBs with ChS preincubated with buffer, Ch-free liposomes, or Ch-rich liposomes for 4 h before treatment with H2O2 (Fig. 3A). Ch-free or Ch-rich liposomes by themselves had no effect on PGE2 generation. A 15-min exposure with H2O2 (70 µM) increased PGE2 production maximally from a basal level of 6.3 ± 2.4 to 14.1 ± 0.6 ng/mg protein (*P < 0.05) in the buffer-treated group. Preincubation with Ch-free liposomes or Ch-rich liposomes before H2O2 had no effect on the increases in PGE2 content induced by H2O2.

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Fig. 3. H2O2-induced PGE2 generation in muscle cells from GB with ChS (A) and PS (B) preincubated with buffer, Ch-free L, or Ch-rich L for 4 h. A: H2O2 exposure for 15 min caused a significant increase in PGE2 production (*P < 0.05). Preincubation with Ch-free L or Ch-rich L had no effect on the increased PGE2 content caused by H2O2. B: H2O2 exposure for 15 min caused a significant increase in PGE2 production (*P < 0.05). Preincubation with Ch-free L or Ch-rich L had no effect on the increased PGE2 content caused by H2O2. Values are means ± SE of 3 experiments.
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H2O2 (70 µM)-induced PGE2 generation was also measured in muscle cells from GBs with PS preincubated with buffer, Ch-free liposomes, or Ch-rich liposomes for 4 h (Fig. 3B). H2O2 exposure for 15 min increased significantly the PGE2 production from 7.4 ± 2.1 ng/mg protein of control to 15.7 ± 1.6 ng/mg protein (*P < 0.05) in the buffer-treated group. Preincubation with Ch-free liposomes or Ch-rich liposomes before H2O2 had no effect on the increased PGE2 content caused by H2O2.
As mentioned previously, muscle cells protect themselves against free radicals by producing scavengers (43, 45). The activity of the free-radical scavenger SOD was examined in muscle cells from GBs with ChS preincubated with buffer, Ch-free liposomes, or Ch-rich liposomes for 4 h before H2O2 exposure (Fig. 4A). Ch-free or Ch-rich liposomes by themselves had no effect on SOD activity. H2O2 (70 µM) exposure for 15 min increased significantly the SOD activity from 2.8 ± 0.6 in the buffer-treated group (controls) to 10.3 ± 0.9 U/mg protein (*P < 0.001). The higher SOD activity induced by H2O2 was further increased from 10.3 ± 0.9 to 14.4 ± 0.4 U/mg protein by preincubating these muscle cells with Ch-free liposomes (**P < 0.01). Ch-rich liposomes pretreatment before H2O2 exposure had no effect on the SOD activity of these muscle cells.

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Fig. 4. Superoxide dismutase (SOD) activity in muscle cells from GB with ChS (A) and PS (B) preincubated with buffer, Ch-free L, or Ch-rich L for 4 h. A: H2O2 exposure for 15 min significantly increased the SOD activity (*P < 0.001). The magnitude of SOD activity induced by H2O2 was further elevated by Ch-free L preincubation (**P < 0.01). Ch-rich L pretreatment before H2O2 had no effect on H2O2-induced SOD activity. B: H2O2 exposure for 15 min significantly increased the SOD activity (*P < 0.001). The increased SOD activity induced by H2O2 was not affected by Ch-free L pretreatment. However, preincubation with Ch-rich L reduced the elevated SOD activity induced by H2O2 (**P < 0.01). Values are means ± SE of 3 experiments.
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SOD activity was also measured in muscle cells from GBs with PS preincubated with buffer, Ch-free liposomes, or Ch-rich liposomes for 4 h before H2O2 exposure (Fig. 4B). H2O2 (70 µM) significantly increased the SOD activity from 2.2 ± 0.3 in the buffer (control)-treated group to 14.3 ± 0.5 U/mg protein (*P < 0.001). Preincubation with Ch-rich liposomes reduced the increase in SOD activity caused by H2O2 from 14.3 ± 0.5 to 8.6 ± 1.4 U/mg protein (**P < 0.01) and was not different from the activity levels found in muscle cells from GBs with ChS treated with buffer. These data suggest that excessive membrane Ch content reduces the activity of the free-radical scavenger SOD.
Catalase activity was also measured in muscle cells from GBs with ChS preincubated with buffer, Ch-free liposomes, or Ch-rich liposomes for 4 h (Fig. 5A). H2O2 (70 µM) exposure for 15 min significantly increased the catalase activity to 15.1 ± 1.2 U/mg protein (*P < 0.05). The magnitude of catalase activity induced by H2O2 was further increased by preincubation of these muscle cells with Ch-free liposomes from 15.1 ± 1.2 to 23.6 ± 1.2 U/mg protein (**P < 0.01). Ch-rich liposomes pretreatment before H2O2 had no effect on catalase activity induced by H2O2.

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Fig. 5. Catalase activity in muscle cells from GB with ChS (A) and PS (B) preincubated with buffer, Ch-free L, or Ch-rich L for 4 h. Ch-free L or Ch-rich L had no effect on catalase activity by themselves. A: H2O2 exposure for 15 min significantly increased the catalase activity (*P < 0.05). The magnitude of catalase activity induced by H2O2 was further elevated by Ch-free L preincubation (**P < 0.01). Ch-rich L pretreatment before H2O2 had no effect on H2O2-induced catalase activity. B: H2O2 exposure for 15 min significantly increased the catalase activity (*P < 0.01). The increased catalase activity induced by H2O2 was not affected by Ch-free L pretreatment. However, preincubation with Ch-rich L reduced the elevated catalase activity induced by H2O2 (**P < 0.01). Values are means ± SE of 3 experiments.
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In muscle cells from GBs with PS preincubated with buffer, H2O2 exposure for 15 min significantly increased the catalase activity to 25.1 ± 2.2 U/mg protein (*P < 0.01) (Fig. 5B). The increased catalase activity was not affected by Ch-free liposomes pretreatment but significantly reduced by preincubation with Ch-rich liposomes (**P < 0.01).
To determine whether the decreased production of free-radical scavengers in GBs with ChS or in Ch-rich liposome-treated muscle cells is related to altered function of PGE2 receptors, receptor binding studies were performed in plasma membranes of muscle from GBs with ChS preincubated with buffer, Ch-free liposomes, or Ch-rich liposomes for 4 h (Fig. 6A). The maximal specific binding of [3H]PGE2 to its receptors was 2.8 ± 0.2% in the buffer-treated group. Preincubation with Ch-free liposomes increased significantly the magnitude of the specific binding of [3H]PGE2 to its receptors from 2.8 ± 0.2 to 5.4 ± 0.1% (P < 0.001 by ANOVA). In muscle cells from GBs with ChS, incubation with Ch-rich liposomes had no effect on the specific binding of [3H]PGE2 to its receptors.

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Fig. 6. Specific binding of [3H]PGE2 in plasma membranes of muscle from GB with ChS (A) and PS (B) preincubated with buffer, Ch-free L, or Ch-rich L for 4 h. A: preincubation with Ch-free L significantly increased the magnitude of specific binding of [3H]PGE2 to its receptors (P < 0.001 by ANOVA). Ch-rich L preincubation had no effect on the specific binding of [3H]PGE2 to its receptors. B: Ch-free L preincubation had no effect on the specific binding of 3H-PGE2 to its receptors. Preincubation with Ch-rich L significantly reduced the magnitude of specific binding of [3H]PGE2 to its receptors (P < 0.001 by ANOVA). Values are means ± SE of 3 experiments.
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The binding studies were also performed in plasma membranes of muscle from GBs with PS preincubated with buffer, Ch-free liposomes, or Ch-rich liposomes for 4 h (Fig. 6B). The maximal specific binding of [3H]PGE2 to its receptors was 6.4 ± 0.1% in the buffer-treated group. Preincubation with Ch-free liposomes had no effect on the specific binding of [3H]PGE2 to its receptors. In contrast, preincubation with Ch-rich liposomes significantly reduced the magnitude of specific binding of [3H]PGE2 to its receptors and was not different from those in muscle cells from GB with ChS treated with buffer (Fig. 6; P < 0.001 by ANOVA).
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DISCUSSION
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The present studies show that increased Ch levels in the plasma membrane of muscle cells from human GBs with ChS impair the contraction and decrease the levels of scavengers of free radicals in response to stress induced by exogenous ROS. In contrast, muscle cells from GBs with PS have normal Ch levels in the plasma membrane and a normal response to ROS that is not different from that of normal guinea pig GB muscle cells (8, 43, 44). This reduced cytoprotective response to oxidative stress occurs despite normal increases in the levels of lipid peroxidation that are likely to include PAF-like lipids and of PGE2 (40). The data also showed that the impaired muscle responses are associated with a decrease in the binding capacity of PGE2 receptors. The defective muscle cells, however, seem to have a normal number of receptors, because the removal of the excess Ch from the plasma membrane by treatment with Ch-free liposomes for 4 h restores receptor binding capacity and muscle responses. Our recent studies suggest that abnormal incorporation of Ch in the plasma membrane localizes in caveolae that are Ch-rich domains resulting in an increase in the recruitment and concentrations of caveolin proteins. The increase in caveolin proteins appears to increase their inhibition on the functions of G proteins and slow receptor return to the bulk plasma membrane where they bind their respective ligands (26, 47).
During inflammatory processes, activated polymorphonuclear cells are the main source of ROS (13, 38) that damage membranes of epithelial and smooth muscle cells (3, 4, 14). It has been postulated that PMN cells release NH2Cl, which is converted by SOD to H2O2 and then inactivated by catalase (20, 22). H2O2 can directly act on plasma membranes or readily cross the membranes and give rise to more radical species inside the cells where they affect several sites (20). ROS have been shown to disrupt the intestinal motility, causing an initial contraction followed by a slow relaxation as well as blocking the contraction induced by methacholine (37). ROS also inhibit GB muscle contraction induced by CCK-8, ACh, and KCl in muscle strips and dissociated muscle cells (16, 23, 24, 45). GB muscle cells generate ROS in response to hydrophobic bile acids that are also capable of damaging constituents of cell membranes. These bile acids have been suggested as possible bile components that may initiate an acute inflammatory process in the presence of a permissive environment (41, 46).
The normal GB protects itself from the deleterious actions of these hydrophobic bile acids and other bile constituents (1) by periodic emptying of bile during the interdigestive and postprandial periods and by cytoprotective mechanisms of the epithelial and muscle cells. GB emptying in the interdigestive period occurs mostly during the antral phase III of the migrating motor complex, and in the digestive period, it is stimulated by the vagus nerve and by CCK. Both stimuli are mediated by cholinergic mechanisms (1). The muscle response to hormones and neurotransmitters is affected by lithogenic bile with excess Ch that impairs GB emptying (2). Acute inflammatory processes may be initiated by the deleterious effects of concentrated bile constituents and ROS formation worsening the GB stasis caused by lithogenic bile with an excess Ch. They cause additional inhibition of the CCK- and ACh-induced contraction in vivo (43, 46). GB stasis may therefore be one of the factors that facilitates the development of acute cholecystitis (34).
There are other factors, however, that may also protect the GB from the actions of bile constituents such as hydrophobic bile salts and against ROS. GB muscle cells appear to have cytoprotective mechanisms in which PGE2 may play an important role. The increase in PGE2 levels in muscle cells in response to oxidative stress protects its receptors and associated signal transduction, preserving the ability of this prostaglandin to stimulate the activity of scavengers of free radicals and thus moderate the deleterious actions of ROS (40, 45). The mechanisms whereby PGE2 activates SOD and catalase are not known. It has been reported that both SOD and catalase are activated by rapid phosphorylation in response to oxidative stress. SOD is phosphorylated by phosphoproteins (12), and catalase is phosphorylated by nonreceptor tyrosine kinases c-Abl and Arg or protein kinases A and C (6, 7, 48).
In human GBs with ChS and lithogenic bile, the increase in the levels of lipid peroxidation and PGE2 induced by ROS was not different from those generated in human GBs with PS or controls. However, PGE2-induced contraction and stimulation of the activity of SOD and catalase were significantly reduced. The lower increase in scavengers of free radicals may be explained by the finding that the functional integrity of PGE2 receptors is impaired in GBs with lithogenic bile with excess Ch. The data suggest that this cytoprotective pathway is partially disrupted by the defective PGE2 receptors. The lower receptor binding by EP receptors (a PGE2 receptor) could be an additional factor making these GBs more susceptible to damage from bile constituents and ROS released by the increased number of inflammatory cells in the lamina propia or produced within the muscle cells. Unlike our previous report on normal GB muscle cells, where increases in endogenous PGE2 protects its receptors from damage by ROS and taurochenodeoxycholate (40), similar levels of endogenous PGE2 in human GB with ChS may be unable to protect its already defective receptors from the harmful effects of ROS because of the excess Ch in the plasma membrane that impairs PGE2 receptor binding.
Thus the excess Ch content in plasma membrane seems to create a permissive environment by causing GB stasis and by impairing the cytoprotective responses that may also affect epithelial cells. This permissive environment created by the lithogenic bile with excess Ch may explain the relatively higher incidence of acute cholecystitis in human GB with ChS (33). Moreover, the findings that the permissive environment created by the lithogenic bile with excess Ch is reversible by removal of abnormal Ch levels in plasma membranes suggest possible therapeutic approaches in high surgical risk patients with symptomatic GB with Ch stones.
In summary, the present studies show that excessive incorporation of Ch by the plasma membrane of GB muscle cells impair one of their cytoprotection mechanisms by causing a dysfunction of PGE2 receptors and their signal transduction that would otherwise contribute to GB contraction and generation of free-radical scavengers that minimize the inflammatory processes in which ROS may be involved. The defective response to PGE2 in muscle cells from human GB with ChS is due to impaired receptor function. These data may explain the high incidence of acute cholecystitis in GB with symptomatic Ch stones (36).
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GRANTS
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This work is supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-27389.
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ACKNOWLEDGMENTS
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These data were partially presented at the Annual Meeting of the American Gastroenterological Association in May, 2000, San Diego, CA.
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FOOTNOTES
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Address for reprint requests and other correspondence: J. Behar, Division of Gastroenterology, APC 421, 593 Eddy St., Providence, RI 02903 (E-mail address: Jose_Behar{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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