Increased expression of gallbladder cholecystokinin: A receptor in prairie dogs fed a high-cholesterol diet and its dissociation with decreased contractility in response to cholecystokinin

Increased expression of gallbladder cholecystokinin: A receptor in prairie dogs fed a high-cholesterol diet and its dissociation with decreased contractility in response to cholecystokinin MASAHITO KANO, JUNICHI SHODA, SUSUMU SATOH, MASAKAZU KOBAYASHI, YASUSHI MATSUZAKI, MASATO ABEI, and NAOMI TANAKA IBARAKI and OSAKA, JAPAN, and EVANSTON, ILLINOIS

A series of our studies have shown that formation of cholesterol-supersaturated bile in patients with cholesterol gallstone disease is causatively related to decreased gallbladder contractility and mucin hypersecretion by the gallbladder. Supersaturated bile may modify the composition of gallbladder membranes so that the transduction of smooth muscle regulatory signals is impaired, and it may enhance the inflammation-induced mucin secretion by the gallbladder. To achieve a better understanding of the mechanism by which supersaturated bile impairs the contractility, we studied changes in the expression levels of gallbladder cholecystokinin (CCK-A) receptor messenger ribonucleic acid (mRNA) in prairie dogs fed a high-cholesterol diet. Levels of pathobiological determinants in arachidonate metabolism which are important for mucin secretion were also measured in their bile. Adult male prairie dogs were randomly assigned to receive either a semisynthetic diet (SSD) or an SSD plus 1.2% cholesterol (a high-cholesterol diet) for 2-, 4-, and 6-week periods. The contractile force in response to CCK-octapeptide (CCK-8) was measured by using gallbladder muscle strips. The mRNA levels of the CCK-A receptor were determined by reverse-transcription polymerase chain reaction (RT-PCR). Parallel to the increase in the cholesterol saturation index, the contractile responses to CCK-8 decreased in the animals fed a high-cholesterol diet for 4 weeks and markedly decreased in the animals with gallstone formation. However, in contrast to the decreased contractility, the steady-state mRNA levels of the gallbladder CCK-A receptor were significantly increased in the animals fed a high-cholesterol diet in comparison with the corresponding control animals. In the bile, a high-cholesterol diet caused an increase in the proportion of arachidonyl-phosphatidylcholine species, where phospholipase A2 activity, prostaglandin E2, and mucin concentrations were increased parallel to the feeding period. Up-regulation of the CCK-A receptor mRNA in the gallbladder of animals fed a high-cholesterol diet associated with decreased contractility may be due to an impairment of CCK signaling related to increased membrane cholesterol contents and its related reaction of biological compensation in order to increase the receptor concentration. The results of the present study suggest that in prairie dogs fed a high-cholesterol diet both a decrease in gallbladder contractility related to impairment of CCK signaling and phospholipase A2 (PLA2)-induced mucosal inflammation in the gallbladder with associated biliary alterations favoring cholesterol crystal formation pathogenetically contribute to the formation of cholesterol gallstones. (J Lab Clin Med 2002;139:285-94) Abbreviations: CCK ⫽ cholecystokinin; DAG ⫽ diacylglycerol; G3PDH ⫽ glyceraldehyde-3-phosphate dehydrogenase; G-protein ⫽ guanine nucleotide-binding protein; IP3 ⫽ inositol-1,4,5-trisphosphate; mRNA ⫽ messenger ribonucleic acid; PGE2 ⫽ prostaglandin E2; PIP2 ⫽ phosphatidylinositol-4,5-bisphosphate; PKC ⫽ phosphokinase C; PLA2 ⫽ phospholipase A2; RT-PCR ⫽ reverse-transcription polymerase chain reaction From the Department of Gastroenterology Surgery, Institute of Clinical Medicine, University of Tsukuba, Ibaraki; Pharmacological Department, Pharmacological Research Laboratories, Fujisawa Pharmaceutical Co, Ltd, Osaka; and Fujisawa Research Institute of America, Inc, Evanston. Supported in part by a grant-in-aid for scientific research from the Ministry of Education (No 05770344), Japan. Parts of this work have been published in abstract form (Gastroenterology 1996;110:A1325).

Submitted for publication August 13, 2001; revision submitted January 7, 2002; accepted January 14, 2002. Reprint requests: Junichi Shoda, MD, PhD, Department of Gastroenterology, Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba-shi, Ibaraki 305– 8575, Japan. Copyright © 2002 by Mosby, Inc. 0022-2143/2002 $35.00 ⫹ 0 5/1/122863 doi:10.1067/mlc.2002.122863

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The multifactorial nature of cholesterol gallstone formation requires hepatic secretion of cholesterol-supersaturated bile, nucleation of cholesterol monohydrate crystals in the gallbladder, and subsequent growth and agglomeration of these crystals to form gallstones.1 In these etiological processes, evidence is mounting that gallbladder stasis due to a gallbladder motility defect plays an important role in the pathogenesis of cholesterol gallstones.2-7 Once bile becomes supersaturated with cholesterol, stasis provides the time necessary for cholesterol nucleation and then retention of the precipitated microcrystals, allowing the crystals to agglomerate into stones.3-5 Cholesterol gallstone disease is often associated with gallbladder hypomotility as a potential etiology, possibly due to either impaired cholecystokinin (CCK) release in response to a meal or the absence of CCK stimulation through a dysfunction of CCKreceptor.8,9 CCK represents the key hormonal stimulant of gallbladder contraction via CCK-A receptor (one of the two major subtypes of CCK receptors) on smooth muscle cells.10 Dysfunction of CCK signaling has been suggested as a potential cause of gallbladder inertia with associated gallstone disease. A previous series of studies has focused attention on the level of the CCK receptor or its coupling to its G-protein as the site of a critical defect in the pathogenesis of cholesterol gallstone disease.11-14 Furthermore, it has been reported that the cellular site of the contractile defect does not reside in the intracellular signal transduction pathways or in the contractile apparatus but, instead, involves the sarcolemmal membrane of gallbladder smooth muscle.15 Therefore, it is of great interest to study the key factors underlying the dysfunction of CCK signaling, such as a defect of the CCK-A receptor itself14,16 or the altered plasmalemmal environment affecting CCK-A receptor signaling.15 Prairie dogs fed a high-cholesterol diet are a wellestablished animal model for studying early events in cholesterol gallstone formation. In animals, changes in gallbladder contractility and mucin secretion by the gallbladder reportedly antedate the formation of gallstones as the biliary cholesterol saturation increases.17,18 In the present study, to gain a better understanding of the molecular mechanism by which cholesterol-supersaturated bile impairs gallbladder motility, we studied alterations in gallbladder contractility in response to CCK-octapeptide (CCK-8) and expression levels of the CCK-A receptor in the gallbladders of prairie dogs fed a high-cholesterol diet. Furthermore, we examined the effects of a high-cholesterol diet on sequential events of biochemical changes in biliary composition that lead to mucin hypersecretion by the gallbladder, ie, increases in phospholipase A2 activity, arachidonyl-phosphatidylcholine species, prostaglandin E2, and mucin glyco-

protein levels, in the animals fed a high-cholesterol diet. METHODS Experimental animals. Fifty-four male prairie dogs (Cynomys ludovicians, trapped in the wild), weighing between 0.8 kg and 1.2 kg, were purchased from Otto Martin Locke (Braunfels, Tex). The animals were caged in rooms maintained at 23°C with alternating 12-hour periods of light and dark. They were allowed continuous access to food and water. After a 2-week equilibration period, the animals were weighed and randomly assigned into six groups (Table 1). Animals in groups A (n ⫽ 8), B (n ⫽ 9), and C (n ⫽ 4) were maintained on a semisynthetic diet (SSD, Teklad, Madison, Wis) containing a trace amount of cholesterol ad libitum for 2, 4, and 6 weeks, respectively, and animals in groups D (n ⫽ 11), E (n ⫽ 13), and F (n ⫽ 9) were maintained on a high-cholesterol (lithogenic) diet that consisted of an SSD plus 1.2% cholesterol ad libitum for 2, 4, and 6 weeks, respectively, as described previously.19 The diet consisted of sucrose, 56.5%; cornstarch, 13.9%; soy protein, 20.2%; corn oil, 1.2%; cellulose, 2.6%; and a mineral and vitamin mix. Cholesterol (1.2%) was incorporated into the diet as egg yolk (0.6%) and crystalline cholesterol (0.6%). At the end of the feeding period, the animals were anesthetized with sodium pentobarbital (Fort Dodge Laboratories Inc, Fort Dodge, Iowa) (30 mg/kg, injected intraperitoneally), and the abdomen was opened. Blood was collected from the abdominal aorta for determination of plasma cholesterol concentration. The cystic duct was then ligated and the gallbladder removed. Bile was withdrawn from the gallbladder by fine needle aspiration, and one drop was examined for cholesterol crystals under a polarizing microscope. The gallbladder was then opened and examined along with the bile for the presence of cholesterol stones, defined as macroscopically visible sediment. The liver was removed, and aliquots were obtained for determination of liver cholesterol level and preparation of microsomes. Four prairie dogs in group A, 4 in group B, 4 in group C, 6 in group D, 5 in group E, and 9 in group F were randomly selected and used for the determination of steady-state mRNA levels of the gallbladder CCK-A receptor (Table I). In the other 4 animals in group A, 5 in group B, 5 in group D, and 8 in group E, a longitudinal strip cut from each gallbladder was used for measuring smooth muscle contractility in response to CCK-8. The average initial weights and final weights of the animals in groups A through F were not statistically different (Table I). During the 24-hour period before sacrifice, the animals were starved to assure adequate quantities of gallbladder bile for biliary lipids and mucins. Chemicals. All solvents were of analytical grade and were redistilled. Colorimetric enzymatic assay kits (Cholesterol E-Test Wako, Phospholipid C-Test Wako, and Total Bile Acids-Test Wako) were obtained from Wako Pure Chemical Industries, Ltd (Osaka, Japan). Cholesterol concentration in plasma. Plasma cholesterol concentrations were determined by enzymatic methods using Cholesterol E-Test Wako.20

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Biliary lipids and mucin glycoproteins. Cholesterol and phospholipid concentrations were determined by enzymatic methods using Cholesterol E-Test Wako20 and Phospholipid C-Test Wako,21 respectively, after eliminating the bilirubin using a Bond-Elut NH2 (aminopropyl) cartridge.22 Total bile acid concentration was determined by an enzymatic method using Total Bile Acids-Test Wako.23 The cholesterol saturation of bile was calculated according to the critical tables for cholesterol saturation based on the total lipid concentration.24 The fatty acid pattern of phospholipids in bile was analyzed by gas-liquid chromatography as described previously.25 The phospholipids extracted on a Bond-Elut NH2 cartridge were methylated by a reflux for 1 hour in 3N HCl at 75°C. The resulting fatty acid methyl esters were analyzed on a fusedsilica capillary column bonded with methylsilicon DB-1HT (J&W Scientific, Folsom, Calif). Mucin concentration in bile was quantitated according to the method described by Miquel et al.26 A specimen was diluted 1:1 (vol/vol) with 0.1 mol/L Tris-HCl buffer (pH 7.5). The diluted specimen was gently shaken for 24 hours at 4°C and then centrifuged at 12,000g for 10 minutes. The supernatant was subsequently fractionated using Sepharose 4B-Cl gel chromatography (35 ⫻ 1 cm column; Pharmacia, Uppsala, Sweden). Mucin was eluted in the void volume. Mucin concentration was determined using a fluorometric assay.27 Enzyme activity of phospholipase A2 in bile. PLA2 enzyme activity was determined as described previously.28 In brief, the assay was performed using 0.8 mmol/L 1-palmitoyl2-oleoyl-sn-glycero-3-phosphoglycerol as a substrate in the presence of 5 mmol/L cholate. Fatty acids released by PLA2 were labeled with 9-anthryldiazomethane, and the derivatized fatty acids were separated by high-performance liquid chromatography. The oleic acid was quantitated using margaric acid as an internal standard. Prostaglandin E2 assay. Prostaglandin E2 (PGE2) levels in bile were determined by a highly specific radioimmunoassay (anti-PGE2 antibody, Amersham, Roosendaal, The Netherlands) according to the method described previously.29 The final results are expressed as picograms of PGE2 per milliliter. Preparation of liver microsomes. One hundred to 150 mg of liver tissue was placed in 9 vol (wt/vol) of ice-cold 50 mmol/L Tris-HCl buffer (pH 7.4) containing 0.3 mol/L sucrose, 10 mmol/L DTT, 10 mmol/L EDTA, and 50 mmol/L NaF, and the microsomal fraction was prepared as described previously.30 The resulting microsomal fraction was suspended in 50 mmol/L Tris-HCl buffer (pH 7.4) containing 0.3 mol/L sucrose, 5 mmol/L DTT, and 1 mmol/L EDTA. Microsomal protein contents were determined by the method described by Bradford.31 Assay of HMG-CoA reductase and cholesterol 7␣hydroxylase activities in liver microsomes. Microsomal

fractions (50-200 ␮g of protein) were preincubated for 10 minutes at 37°C in a total volume of 225 ␮l containing 50 mmol/L Tris-HCl buffer (pH 7.4), 0.3 mol/L sucrose, 5 mmol/L DTT, 1 mmol/L of EDTA, 12 mmol/L glucose-6phosphate, and 0.1 mmol/L deuterated HMG-CoA. Assays were then initiated by the addition of 1 unit of glucose-6phosphate dehydrogenase and 750 nmol of NADPH dis-

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solved in 225 ␮l of the preincubated buffer. Incubations were carried out at 37°C for 15 minutes and were terminated by the addition of 50 ␮l of 1 mol/L NaOH. Purification of reaction products and their analysis by gas—liquid chromatography—mass spectrometry (GC-MS) were carried out as described previously.30 Cholesterol concentration in liver. Cholesterol concentrations in liver specimens were determined using the method described by Schaffer et al.32 Deuterated cholesterol and chloroform-methanol 2:1 (vol/vol) were added to 10 ␮L of the liver homogenates. The chloroform phase was then evaporated, and the residue was hydrolyzed with 0.5 mol/L KOH, extracted with n-hexane, converted into dimethylethylsilyl ether, and analyzed by GC-MS. Measurements of gallbladder muscle contractility. The measurement of gallbladder contractility was performed according to the technique described previously.33 In brief, muscle strips were mounted in organ baths filled with KrebsRinger’s solution, and muscle optimal length for active force development was determined by stimulating with bethanechol (10⫺5 mol/L; Sigma Chemicals, St Louis, Mo). Isometric contractile force in response to CCK-8 (1.5 ⫻ 10⫺11 to 1.5 ⫻ 10⫺7 mol/L; Sigma Chemicals) was recorded. RNA isolation and complementary DNA synthesis. Total RNA was isolated from the gallbladder specimens as described previously.34 First-strand cDNAs were synthesized by the random primer method using a cDNA synthesis kit (Boehringer Mannheim Yamanouchi, Tokyo, Japan). Northern hybridization. Northern blot analysis was performed to determine steady-state mRNA levels of the gallbladder CCK-A receptor in prairie dogs. One ␮g of polyadenylated RNA was subjected to electrophoresis on 1.0% agarose gels in the presence of formaldehyde and then transferred onto charged nylon membranes (Hybond-N⫹, Amersham Pharmacia Biotech, Uppsala, Sweden). Hybridization was performed at 60°C for 19 hours in Church buffer (0.5 mol/L sodium phosphate, 1% BSA, 7% SDS, 1 mmol/L EDTA) with 32P-labeled CCK-A receptor cDNA probe 200 ng, which was amplified by PCR with the primers designed for this study as follows: sense 5⬘-AACTTGGTGCCTTTTAC-3⬘, antisense 5⬘-GGAAGAAGAGGACCAC-3⬘. These primers were designed from cDNA sequences of the rat CCK-A receptor.35 The sequences of this probe were analyzed, and it was confirmed that this probe has ⬎80% homology with rat,35 guinea pig,36 and human CCK-A receptors.37 After hybridization, the membranes were washed once with washing buffer (0.5% BSA, 5% SDS, 40 mmol/L sodium phosphate, 1 mmol/L EDTA) for 20 minutes at 55°C and then twice with the washing buffer for 10 minutes at 55°C. Exposure to x-ray film was performed at ⫺80°C using an intensifying screen. Reverse transcription-polymerase chain reaction. Reverse-transcription polymerase chain reaction (RT-PCR) was performed as described previously,38 using DNA Thermal Cycler (model PJ 2000; Applied Biosystems, Inc, Foster City, Calif). PCR-assisted amplification was performed using 50 pmol of each primer, 20 ␮L of cDNA template, 1.25 units of Taq polymerase, 0.2 mmol/L dNTPs, and 1⫻ reaction buffer

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Table I. Basal data and incidence of formation of cholesterol gallstones and biliary cholesterol crystals in prairie dogs

A B C D E F

Average food intake (g/day)

Liver

Diet

No.

SSD, 2 weeks SSD, 4 weeks SSD, 6 weeks SSD ⫹ 1.2% cholesterol, 2 weeks SSD ⫹ 1.2% cholesterol, 4 weeks SSD ⫹ 1.2% cholesterol, 6 weeks

8 9 4 11

914 ⫾ 38 996 ⫾ 42 886 ⫾ 34 1052 ⫾ 39 909 ⫾ 25 1137 ⫾ 30 901 ⫾ 31 1001 ⫾ 34

26 ⫾ 2 28 ⫾ 3 32 ⫾ 5 28 ⫾ 2

3.8 ⫾ 0.1 7.4 ⫾ 0.4 4.3 ⫾ 0.3 7.7 ⫾ 0.5 3.5 ⫾ 0.1 7.6 ⫾ 0.6 7.7 ⫾ 0.5† 11.9 ⫾ 1.7†

8.9 ⫾ 0.8 8.7 ⫾ 0.5 9.5 ⫾ 1.2 2.1 ⫾ 0.3†

2.3 ⫾ 0.3 3.2 ⫾ 0.4 3.3 ⫾ 0.4 9.8 ⫾ 0.7†

4 4 4 6

13

948 ⫾ 28 1097 ⫾ 32

29 ⫾ 3

12.2 ⫾ 1.3† 13.0 ⫾ 1.9†

1.6 ⫾ 0.4†

10.9 ⫾ 0.9†

5

9

923 ⫾ 26 1182 ⫾ 32

31 ⫾ 3

11.0 ⫾ 1.4† 12.4 ⫾ 2.0†

1.3 ⫾ 0.2†

10.2 ⫾ 0.7†

9

Group

Average final weight (g)

Plasma

Average initial weight (g)

Cholesterol (mmol/L)

Cholesterol (␮mol/g)

HMG-CoA R (pmol/min/mg)

Ch-7case proteins

No.

Values are expressed as mean ⫾ SEM. CCK, Cholecystokinin; Ch, cholesterol; Ch-7␣ase, cholesterol 7␣-hydroxylase; HMG-CoA R, 3-hydroxy5-methylglutanyl enzyme A reductase, SSD, semisynthetic diet. *P ⬍ .05. †P ⬍ .01, significantly different from corresponding control group.

(10 mmol/L Tris-HCl, pH 8.3, containing 50 mmol/L KCl, 1.5 mmol/L MgCl2, and 0.01% gelatin) in a 50-␮L reaction volume. The reactions were subject to each cycle as follows: CCK-A receptor, 25; glyceraldehyde-3-phosphate dehydrogenase (G3PDH), 19 at 94°C for 1 minute, 60°C for 2 minutes, and 72°C for 2 minutes. Aliquots of the reaction mixture were electrophoresed on a 2% agarose gel. In experiments involving quantitative assessment, the amounts of fluorescence intensity were measured by FluorImager (Molecular Dynamics, Sunnyvale, Calif). The densitometrically measured abundance of mRNA was corrected by the recovery of G3PDH mRNA, and the data were expressed relative to the amounts of G3PDH mRNA present in each specimen. Since the cDNA sequence of the prairie dog CCK-A receptor has not yet been cloned, PCR primers were designed according to the cDNA sequences of the CCK-A receptor of rat,35 guinea pig,36 and human,37 and then synthesized by an Applied Biosystems DNA synthesizer (model 392; Applied Biosystems, Inc, Foster City, Calif) as follows: G3PDH, sense 5⬘-GAACGGGAAGCT CACTGGCATGGC-3⬘, antisense 5⬘-TGAGGTCCACCACCCTGTTGCTG-3⬘; CCK-A receptor, sense 5⬘-ACCATGAAGACCCTCCTACTG-3⬘, antisense 5⬘-GAAGAGGGGACTCAGCAACG-3⬘. Statistics. Values are given as means ⫾ SEM. The statistical significance of intergroup differences in values was evaluated using the Mann-Whitney test (two-tailed test). A P value of ⬍ .05 was defined as statistically significant. RESULTS Animal weights, food intake, and incidence of formation of cholesterol crystals and gallstones. The effects of

a high-cholesterol (lithogenic) diet on animal weights,

food intake, and the incidence of formation of cholesterol crystals and gallstones are summarized in Table 1. No crystals and gallstones were found in the gallbladder of any prairie dogs fed a control diet (an SSD including a trace amount of cholesterol). In contrast, cholesterol crystals appeared in all prairie dogs fed on a high-cholesterol diet (an SSD plus 1.2% cholesterol). Furthermore, cholesterol gallstones were found in 1 of 11 animals (9%) at 2 weeks, 7 of 13 animals (54%) at 4 weeks, and 5 of 9 animals (56%) at 6 weeks. Cholesterol concentration in plasma. Plasma cholesterol concentrations were determined at 2, 4, and 6 weeks after a high-cholesterol diet was started (Table I). Consistent with the previous observation,41 the prairie dogs fed a high-cholesterol diet had significantly elevated plasma cholesterol concentrations, averaging 7.7 mmol/L (control animals: 3.8 mmol/L) at 2 weeks, 12.2 (4.3) at 4 weeks, and 11.0 (3.5) at 6 weeks. Cholesterol concentrations in liver and enzyme activities of HMG-CoA reductase and cholesterol 7␣-hydroxy-

Cholesterol concentrations in liver were determined at 2, 4, and 6 weeks after a high-cholesterol diet was started (Table I). Consistent with the previous observation,41 the prairie dogs fed a high-cholesterol diet had significantly elevated hepatic cholesterol concentrations, averaging 11.9 ␮g/g (control animals: 7.4 ␮g/g) at 2 weeks, 13.0 (7.7) at 4 weeks, and 12.4 (7.6) at 6 weeks. To study the effects of a high-cholesterol diet on hepatic cholesterogenesis and bile acid synthesis in the lase in liver microsomes.

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Gallbladder contractility in response to CCK-8

CCK-A receptor assay Ch gallstones

Ch crystals

mRNA % G3PDH

No.

Ch gallstones

Ch crystals

0 0 0 0

0 0 0 6

58 ⫾ 12 55 ⫾ 13 56 ⫾ 11 131 ⫾ 9*

4 5 — 5

0 0 — 1

0 0 — 5

2

5

139 ⫾ 17†

8

5

8

5

9

187 ⫾ 28†

—

—

—

animals, the catalytic activity of hepatic HMG-CoA reductase, the rate-limiting enzyme of cholesterol synthesis, and that of cholesterol 7␣-hydroxylase, the ratelimiting enzyme of bile acid synthesis, were determined using the method of isotope-dilution mass spectrometry.30 Reflecting the depressed hepatic cholesterol de novo synthesis due to the high-cholesterol diet, the HMG-CoA reductase activities in the animals fed a high-cholesterol diet were significantly lower than those in the control animals, averaging 2.1 pmol/ min/mg protein (control animals: 8.9) at 2 weeks, 1.6 (8.7) at 4 weeks, and 1.3 (9.5) at 6 weeks. On the other hand, in relation to the increased substrate amount for bile acid synthesis due to the high-cholesterol diet, the cholesterol 7␣-hydroxylase activities in the animals fed a high-cholesterol diet were significantly higher than those in the control animals, averaging 9.8 pmol/ min/mg protein (control animals: 2.3) at 2 weeks, 10.9 (3.2) at 4 weeks, and 10.2 (3.3) at 6 weeks. The changes in enzyme activities of the reductase and the hydroxylase were analogous to those observed in the previous studies.39,40 Biliary lipid composition. Biliary lipid compositions in the six groups of prairie dogs are summarized in Table II. In the gallbladder bile, the molar percentages of cholesterol and phospholipids were both significantly higher in the animals fed a high-cholesterol diet than in the corresponding control animals, whereas the percentage of bile acids was significantly lower in the animals fed a high-cholesterol diet than in the corre-

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sponding control animals. The cholesterol saturation index (CSI%) in the animals fed a high-cholesterol diet was significantly higher than that in the corresponding control animals, averaging 105% (control animals: 54%) at 2 weeks, 122% (65%) at 4 weeks, and 126% (68%) at 6 weeks. When the fatty acid composition of phosphatidylcholine was studied in the cholesterol-supersaturated bile, the relative amount of arachidonyl-phosphatidylcholine species was significantly increased in the animals fed a high-cholesterol diet compared with the amount in the corresponding control animals, averaging 13.4% (control animals: 4.2%) at 2 weeks, 13.7% (4.5%) at 4 weeks, and 13.9% (6.7%) at 6 weeks (Table 2). In addition, the enzyme activity of PLA2 in gallbladder bile was significantly increased in the animals fed a high-cholesterol diet compared with the activity in the corresponding control animals, averaging 1.8 nmol/ min/mL (control animals: 1.2 nmol/min/mL) at 2 weeks, 2.9 (1.5) at 4 weeks, and 2.9 (1.1) at 6 weeks (Table II). The increased PLA2 in the bile may hydrolyze the increased arachidonyl-phosphatidylcholine and yield free arachidonate, the substrate for PGE2 synthesis. In coordination with the increased amount of arachidonyl-phosphatidylcholine species and the increased PLA2 activity, the PGE2 concentrations were increased in the bile of animals fed a high-cholesterol diet compared to the concentrations in the corresponding control animals, averaging 2531 pg/mL (control animals: 1455 pg/mL) at 2 weeks, 2893 (1476) at 4 weeks, and 3550 (1600) at 6 weeks (Table II). Reflecting a mucin secretagogue action of PGE2, the total mucin concentrations in the bile were significantly increased in the animals fed a high-cholesterol diet compared with the concentrations in the corresponding control animals, averaging 239 ␮g/mL (control animals: 155 ␮g/mL) at 2 weeks, 337 (135) at 4 weeks, and 369 (134) at 6 weeks (Table II). Gallbladder muscle contractility in response to chole-

CCK-8 at concentrations ranging from 10⫺10 to 10⫺6 mol/L was observed to produce concentration-dependent contractions in the gallbladder muscle strip preparations (Fig 1). At the end of 2 weeks, the animals fed a high-cholesterol diet showed a slight decrease in the tension generated in response to CCK-8 at concentrations of 10⫺7 to 10⫺6 mol/L as shown in the left part of Fig 1. In contrast, at the end of 4 weeks, the animals fed a high-cholesterol diet, particularly those with cholesterol gallstone formation, showed a marked decrease in the tension as shown in the right part of Fig 1. The decrease in the animals fed a high-cholesterol diet at each concentration was stacystokinin-octapeptide.

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Table II. Effect of high cholesterol on biliary lipid composition in prairie dogs

Group

A B C D E F

Diet

SSD, 2 weeks SSD, 4 weeks SSD, 6 weeks SSD ⫹ 1.2% cholesterol, 2 weeks SSD ⫹ 1.2% cholesterol, 4 weeks SSD ⫹ 1.2% cholesterol, 6 weeks

Molar percentage (%)

No.

Total lipid conc. (g/dL)

Cholesterol

Phospholipids

8 9 4 11

8.4 ⫾ 0.6 8.6 ⫾ 0.9 8.2 ⫾ 1.1 8.6 ⫾ 1.1

1.9 ⫾ 0.2 1.9 ⫾ 0.1 2.1 ⫾ 0.2 3.9 ⫾ 0.4†

8.0 ⫾ 0.5 6.7 ⫾ 0.2 7.2 ⫾ 1.0 14.2 ⫾ 0.9†

90.0 ⫾ 0.6 91.6 ⫾ 0.3 90.7 ⫾ 1.3 81.8 ⫾ 1.2†

54 ⫾ 4 65 ⫾ 3 68 ⫾ 2 105 ⫾ 7†

13

9.2 ⫾ 1.2

5.0 ⫾ 0.4†

13.4 ⫾ 0.8†

81.6 ⫾ 1.0†

122 ⫾ 8†

9

8.7 ⫾ 1.4

5.5 ⫾ 0.4†

13.3 ⫾ 0.5†

81.2 ⫾ 0.8†

126 ⫾ 7†

Bile acids

Cholesterol saturation index (%)

Values are expressed as mean ⫾ SEM Mucin, Mucin glycoprotein; PC, phosphidatidy choline; PGE2 , prostaglandin E2; PLA2 , phospholipidase A2; SSD, semisynthetic diet. *P ⬍ .05. †P ⬍ .01, significantly different from corresponding control group.

tistically significant compared with the tension generated in the control animals. Northern hybridization of cholecystokinin-A receptor in

Northern hybridization of gallbladder poly (A)⫹ RNA prepared from the gallbladder tissues of prairie dogs was carried out with a 32P-labeled CCK-A receptor cDNA probe that was amplified by PCR (Fig 2). As shown in the figure, the expression levels of the CCK-A receptor mRNA were significantly increased in the gallbladders of the animals fed a highcholesterol diet for 2 weeks, in those fed a high-cholesterol diet for 4 weeks, and in those fed a highcholesterol diet for 6 weeks, compared with the levels in the gallbladders of the corresponding control animals. the gallbladder.

Semiquantification of mRNA of cholecystokinin-A re-

The steady-state levels of the CCK-A receptor mRNA in the gallbladders of prairie dogs were determined by RT-PCR. The results are shown in Fig 3 and expressed as CCK-A receptor mRNA/G3PDH mRNA ratios (percent). The expression levels of the CCK-A receptor mRNA were increased in the animals fed a high-cholesterol diet compared with the levels in the corresponding control animals, averaging 131% (control animals: 58%) at 2 weeks, 139% (55%) at 4 weeks, and 187% (56%) at 6 weeks. The difference between each group of animals fed a high-cholesterol diet and the corresponding group of control animals was statistically significant (Table 1). Importantly, the magnitude of the up-regulation was significantly greater in the animals with gallstone formation (180% of G3PDH mRNA at the end of 4 weeks, P ⬍ .01, and 251% at the end of 6 weeks, P ⬍ .01), ceptor in gallbladders.

despite the profoundly decreased contractility in response to CCK-8, than in those without the formation (112% at the end of 4 weeks and 108% at the end of 6 weeks). DISCUSSION

Gallbladder stasis due to contractility defects is a well-known key feature of cholesterol stone formation in humans2,3 and in animal models.4-7 However, the cellular mechanisms responsible for the evolution of gallbladder hypomotility remain obscure. In the present study, changes in gallbladder contractility were associated with an increase in biliary cholesterol-supersaturation. Parallel to the period of high-cholesterol diet, gallbladder muscle strip prepared from the prairie dogs fed a high-cholesterol diet, particularly those with cholesterol stone formation, showed decreases in isometric tensions generated in response to CCK-8. CCK is a physiologic regulator of gallbladder muscle contraction during the postprandial state.41 A number of factors are involved in regulation of the smooth muscle contractility. CCK influences the contractility via the phosphoinositide pathway,14 and the signal transduction involves several membrane-associated elements, including CCK-A receptor, G-proteins, phospholipase C, diacylglycerol (DAG), and phosphokinase C (PKC). CCK-induced contraction via CCK-A receptor is reportedly mediated by activation of G-proteins and subsequent hydrolysis of phosphatidylinositol-4,5bisphosphate (PIP2) to inositol-1,4,5-trisphosphate (IP3) and DAG.13 IP3 releases calcium from the endoplasmic reticulum, forming a calcium-calmodulin complex that causes smooth muscle contraction through an

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Arachidonyl– PC (%)

PLA2 activity (nmol/min/mL)

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PGE2 conc. (pg/mL)

Mucin conc. (␮g/mL)

4.2 ⫾ 0.2 4.5 ⫾ 0.6 6.7 ⫾ 1.0 13.4 ⫾ 0.3†

1.2 ⫾ 0.1 1.5 ⫾ 0.1 1.1 ⫾ 0.1 1.8 ⫾ 0.1

1455 ⫾ 174 1476 ⫾ 159 1600 ⫾ 99 2521 ⫾ 188†

155 ⫾ 11 135 ⫾ 9 134 ⫾ 27 239 ⫾ 19*

13.7 ⫾ 0.9†

2.9 ⫾ 0.3†

2893 ⫾ 220†

337 ⫾ 22†

13.9 ⫾ 0.7†

2.9 ⫾ 0.4†

3550 ⫾ 271†

369 ⫾ 45†

activation of myosin light chain kinase.42-44 DAG activates PKC that may influence the phosphorylation of a different subset of cellular proteins involved in smooth muscle contraction.45,46 In the present study, our interest should be focused on the possibility that changes in expression levels of the gallbladder CCK-A receptor are caused by alterations in the biochemical properties of bile, that is, cholesterol-supersaturated bile. The results of Northern hybridization showed that the amount of the CCK-A receptor mRNA was increased in the gallbladders of prairie dogs fed a high-cholesterol diet for 2, 4, and 6 weeks compared with the levels in the corresponding control animals. Consistent with the results of the Northern hybridization, the results of semiquantitative RT-PCR showed that the expression levels of the gallbladder CCK-A receptor mRNA in the animals fed a high-cholesterol diet were approximately 2.3-fold higher at the end of 2 weeks, approximately 2.5-fold higher at the end of 4 weeks, and approximately 3.3fold higher at the end of 6 weeks than the levels of the corresponding control animals. However, in contrast to the up-regulation of the CCK-A receptor mRNA, the contractile responses to CCK-8 were decreased in the gallbladders of the animals fed a high-cholesterol diet at the end of 4 weeks and markedly decreased in the animals with cholesterol stone formation. A previous series of studies focused on the level of the CCK receptor or its coupling to its G protein as the site of a critical defect in the pathogenesis of cholesterol gallstone disease.10-12 The results of the present experiments include only the data on expression levels of CCK-A receptor mRNA in prairie dogs fed a high-

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cholesterol diet. Because of the lack of data on the protein content of CCK-A receptor or the receptor activity, the possibility of decreased levels of CCK-A receptor, due to a decrease in translocation of mRNA, posttranslational processing or an increase in receptor turnover, cannot be excluded. Nevertheless, since inositol triphosphate reportedly restores normal gallbladder contractility to CCK-8,47 the cellular site of the contractile defect may not reside in the intracellular signal transduction pathways or in the contractile apparatus but, instead, may involve the sarcolemmal membrane of gallbladder smooth muscle to influence receptor function or interactions between the receptor and G-proteins.13 Up-regulation of the CCK-A receptor mRNA in the gallbladder of animals fed a high-cholesterol diet with decreased contractility may be due to an impairment of CCK signaling and its related reaction of biological compensation in order to increase the receptor concentration. The formation of cholesterol-supersaturated bile may be a key factor underlying the dysfunction of CCK signaling through alterations in the plasmalemmal environment, ie, a stiffening of sacroplasmal membranes caused by increased membrane cholesterol contents.48 Besides a gallbladder contractility defect, mucosal inflammation of the gallbladder may be of pathogenetic importance since inflammation-induced mucin is a potent promoter of cholesterol nucleation as well as crystal growth in vitro.49-51 In our previous studies,52,53 induced group IIA phospholipase A2 (PLA2), an isoform of the secretory-type PLA2 family, was found to play a critical role in etiological process of cholesterol stone formation, probably through stimulating arachidonate metabolism and thereby potentiating gallbladder mucosal inflammation. The coordinate increases in levels of biliary cholesterol supersaturation, free arachidonate, PGE2, and mucin glycoproteins in obese patients during rapid weight loss54,55 and in postmenopausal women56 suggest that inflammatory responses mediated by the cascade of arachidonate may be activated and then proceed in the presence of supersaturated bile. Supporting this speculation, the animal experiments done in the previous studies7,57-59 and the present study have shown that a high-cholesterol diet causes increases in biliary PLA2 activity57 and levels of arachidonylphosphatidylcholine species,58 PGE2,7,58,59 and mucins.58,59 In summary, the results of the present experiments have confirmed that in prairie dogs fed a high-cholesterol diet, changes in gallbladder contractility and mucin secretion by the gallbladder are associated with an increase in cholesterol-supersaturation of bile but antedate the formation of cholesterol stones. The results further suggest that in the cholesterol-fed animals both

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a decrease in gallbladder contractility related to impairment of CCK signaling and PLA2-induced inflammation in gallbladder mucosa with associated biliary alterations favoring cholesterol crystal formation may pathogenetically contribute to the stone formation.

REFERENCES

Fig 1. Gallbladder contractility in response to CCK-8 showing active stress developing in response to CCK-8 by gallbladder muscle strips from prairie dogs fed a control diet including a trace amount of cholesterol (open circle) and those from prairie dogs fed a 1.2% cholesterol diet (a high-cholesterol diet) (closed circle). Values are means ⫾ SEM. The response in the animals fed a high-cholesterol diet for 4 weeks (closed circle), especially in the animals with gallstone formation (closed diamond), was significantly lower than that in the corresponding animals fed a control diet (open circle). *P ⬍ .05, significantly different from the animals fed a control diet. **P ⬍ .01, significantly different from the animals fed a control diet.

Fig 2. Northern hybridization of the CCK-A receptor in the gallbladders of prairie dogs fed a control diet including a trace amount of cholesterol and in those of prairie dogs fed a 1.2% cholesterol diet (a high-cholesterol diet). SSD, A semisynthetic diet including a trace amount of cholesterol; ⫹ Ch, an SSD plus 1.2% cholesterol. The steady-state mRNA levels of the gallbladder CCK-A receptor were found to be higher in the animals fed a high-cholesterol diet for 2, 4, and 6 weeks.

Fig 3. RT-PCR-assisted amplifications of the CCK-A receptor mRNA in the gallbladders of prairie dogs fed a control diet including a trace amount of cholesterol and in those of prairie dogs fed a 1.2% cholesterol diet (a high-cholesterol diet). SSD, A semisynthetic diet including a trace amount of cholesterol; ⫹ Ch, an SSD plus 1.2% cholesterol; GS, gallstones. In experiments involving quantitative assessment, the densitometrically measured abundance of the CCK-A receptor mRNA was corrected by the recovery of G3PDH mRNA, and the data were expressed relative to the amounts of G3PDH mRNA present in each specimen. The PCR products were 260 bp in size for the CCK-A receptor and 311 bp in size for G3PDH. Consistent with the results of Northern hybridization, the steady-state mRNA levels of the gallbladder CCK-A receptor were found to be higher in the animals fed a high-cholesterol diet for 2, 4, and 6 weeks.

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