CCK receptor dysfunction in muscle membranes from human gallbladders with cholesterol stones

Zuo-Liang Xiao1, Qian Chen1, Joseph Amaral1, Piero Biancani1, Robert T. Jensen2, and Jose Behar1

1 Departments of Medicine and Surgery, Rhode Island Hospital and Brown University School of Medicine, Providence, Rhode Island 02903; and 2 Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
PATIENTS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Human gallbladders with cholesterol stones exhibit impaired muscle contraction induced by agonists that act on transmembrane receptors, increased membrane cholesterol content, and abnormal cholesterol-to-phospholipid ratio compared with those with pigment stones. The present study was designed to investigate the functions of the CCK receptor of gallbladder muscle membranes by radioreceptor assay and cross-linking. 125I-labeled CCK-8 binding was time-dependent, competitive, and specific. Scatchard analysis showed that the maximum specific binding (Bmax) was significantly decreased in cholesterol compared with pigment stone gallbladders (0.18 ± 0.07 vs. 0.38 ± 0.05 pmol/mg protein, P < 0.05). In contrast, the affinity for CCK was higher in cholesterol than pigment stone gallbladders (0.18 ± 0.06 vs. 1.2 ± 0.23 nM). Similar results were observed in binding studies with the CCK-A receptor antagonist [3H]L-364,718. Cross-linking and saturation binding studies also showed significantly less CCK binding in gallbladders with cholesterol stones. These abnormalities were reversible after incubation with cholesterol-free liposomes. The Bmax increased (P < 0.01) and the dissociation constant decreased (P < 0.001) after incubation with cholesterol-free liposomes. In conclusion, human gallbladders with cholesterol stones have impaired CCK receptor binding compared with those with pigment stones. These changes are reversed by removal of the excess membrane cholesterol. These receptor alterations may contribute to the defective contractility of the gallbladder muscle in patients with cholesterol stones.

smooth muscle; cholecystokinin receptors; liposomes


    INTRODUCTION
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ABSTRACT
INTRODUCTION
PATIENTS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE MAIN HUMORAL AGENT affecting gallbladder contraction in the intestinal phase of digestion is CCK (12). We have previously shown that human gallbladders with cholesterol stones exhibit impaired muscle contractility in response to CCK, ACh, and KCl compared with those with pigment stones whose contraction is thought to be normal (2, 5). The magnitude of contraction of these defective muscle cells is normalized when membrane receptors are bypassed by activating directly G proteins with guanosine 5'-O-(3-thiotriphosphate) or by utilizing second messengers such as inositol 1,4,5-trisphosphate and diacylglycerol lipase or by using the enzyme calmodulin (3, 13, 27). This defective muscle contraction is associated with high-cholesterol content and abnormal cholesterol-to-phospholipid ratio in plasma membranes of gallbladder muscle cells from prairie dogs fed a high-cholesterol diet (1.2%). These abnormalities were reversed when muscle cells were subsequently incubated with cholesterol-free liposomes (26). Thus excessive membrane cholesterol content may affect the signal transduction across the cell membrane, especially transmembrane protein functions. Membrane cholesterol distribution is asymmetric, and cholesterol appears to be enriched in the inner leaflet of the membranes (21). Transmembrane proteins are located in cholesterol-rich domains, cholesterol-poor domains, and even specifically associated with cholesterol. An optimum level of membrane cholesterol is essential for the functions of Na+-K+-ATPase, membrane receptors, and ion channels. Thus increased or decreased membrane cholesterol content may induce the impairment of membrane protein functions (9).

CCK is a linear polypeptide hormone of intestinal and neural origin, which exerts a wide variety of effects on multiple target tissues (1, 18). The actions of CCK on the gallbladder have been characterized primarily by measuring the contractile response of gallbladder strips to CCK; differences in the contractile response to CCK in different tissues are partly attributable to the type of muscle cell where CCK receptors are located (2, 10). Based on pharmacological experiments, CCK receptors have been broadly classified as either type A or type B (6). In gallbladder smooth muscles, only CCK-A receptors can be detected (11, 12, 24). Like other members of the G protein-coupled seven transmembrane superfamily, CCK receptors have three main domains: 1) an extramembrane domain for ligand binding, 2) the heptahelical transmembrane domain for signaling, and 3) a cytoplasmic domain for G protein coupling. Usually, the agonist binding site is located in a narrow cleft defined by several transmembrane domains about 15 Å away from the extracellular surface. However, the precise molecular mechanisms of binding and the conformational rearrangement of receptor proteins induced by agonists are still poorly understood (25).

The present study was designed to determine whether there are any alterations in the functions of the CCK receptor that may account for the impairment of muscle contractility in human gallbladders with cholesterol stones and whether membrane cholesterol plays a role.


    PATIENTS AND METHODS
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ABSTRACT
INTRODUCTION
PATIENTS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Patients. Human gallbladders were obtained by elective laparoscopic cholecystectomy performed for gallstone diseases. None of the patients had a history or clinical picture of acute cholecystitis. Nineteen gallbladders were classified as having cholesterol and twelve pigment stones according to their gross appearance and chemical analysis. The gallbladders were kept in ice-cold oxygenated Krebs solution immediately after cholecystectomy and transported to the laboratory for dissection. The Krebs solution consisted of (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 minced to 2 × 2-mm squares that were ready for further use.

Preparation of liposomes. Cholesterol-free liposomes were prepared by using egg phosphatidylcholine as described by others (14). Three milliliters of phosphatidylcholine (20 mg/ml in chloroform) in a glass test tube were dried under a stream of nitrogen and vacuum dried. The dried lipids were suspended in 3 ml of normal saline. They were sonicated for 30 min with a Branson 2200 sonicator (Branson Ultrasonics, Danbury, CT). The suspension was then centrifuged at 13,000 rpm (model J2-21 centrifuge, Beckman Instruments, Fullerton, CA) for 30 min to remove the undispersed lipids. Two milliliters of the supernatant and 8 ml of 0.2% BSA-HEPES buffer were used to make cholesterol-free liposomes. The HEPES buffer contained (in mM) 112.5 NaCl, 5.5 KCl, 2.0 KH2PO4, 24 HEPES, 1.9 CaCl2, 0.6 MgCl2, and 10.8 glucose.

Muscle squares from human gallbladders with cholesterol stones were aliquoted and incubated with either cholesterol-free liposomes in 0.2% BSA-HEPES buffer or 0.2% BSA-HEPES buffer only at 37°C for 4 h. The muscle squares were washed with Krebs solution and kept in liquid nitrogen for further use.

Preparation of enriched plasma membranes. Plasma membranes were prepared and purified by sucrose gradient centrifugation as described by others (22). Muscle squares were homogenized for three bursts of 10 s at a setting 5 by using a tissue tearer (Biospec Products, Racine, WI) in 10 volumes by weight of sucrose-HEPES buffer, which consisted of 0.25 M sucrose, 10 mM HEPES, pH 7.4, 0.01% soybean trypsin inhibitor (STI), 0.1 nM phenylmethylsulfonyl fluoride, 0.1 nM 1,10-phenanthroline, and 1 mM 2-mercaptoethanol and again with 60 strokes in a Dounce grinder (Whenton, Millville, NJ). The homogenates were centrifuged at 600 g for 5 min, the supernatant was collected in a clean centrifuge tube (Beckman Instruments), and the pellet was resuspended in the same buffer, homogenized, and centrifuged again. Supernatants were collected, combined, and centrifuged at 150,000 g for 45 min to make a crude particulate pellet. The pellet was resuspended in sucrose-HEPES medium using 10 strokes of a Dounce grinder with the tight-fitting pestle. This homogenate was then layered over a linear 9-60% sucrose gradient and centrifuged at 90,000 g for 3 h. The plasma membranes were collected at about 24% sucrose. They were then diluted and pelleted by centrifugation at 150,000 g for 30 min. Membranes were stored at -70°C.

Ligand binding studies. Ligand binding experiments were performed in a final volume of 300 µl. The incubation solution consisted of 118 mM NaCl, 4.7 mM KCl, 1 mM EGTA, 5 mM MgCl2, 10 mM MES, 5 mg/ml BSA, 0.2 mg/ml STI, and 0.25 mg/ml bacitracin at pH 6.5 unless otherwise specified. Membranes containing 50 µg of protein were incubated with 50 pM of 125I-CCK-8 for 90 min at 25°C. Three volumes of incubation solution without BSA were added to stop the reaction. Separation of bound from free radioligand was achieved by filtration utilizing a vacuum filtering manifold (Millipore, Bedford, MA) with receptor-binding filter mats (Millipore) and washing the filters with ice-cold incubation medium without BSA. Nonspecific binding was determined in parallel incubations with 0.1 µM of unlabeled CCK-8. Radioactivity remaining on the filters was counted in a gamma scintillation counter.

Binding of [3H]L-364,718 was measured as described previously (11). Membrane protein (50 µg) and [3H]L-364,718 (300 pM) were incubated in polypropylene tubes for 90 min at 25°C. After the incubation, duplicate 100-µl samples were added to 1 ml of ice-cold incubation solution without BSA and filtered through a vacuum filtering manifold (Millipore) with receptor-binding filter mats (Millipore). Membranes retained on the filters were washed three times with 1 ml of the same solution. Filters were placed in scintillation vials containing liquid scintillation fluid (Ecolume, ICN) and assayed for radioactivity with the Tri-Carb 1900CA liquid scintillation analyzer (Packard Instrument, Meriden, CT). Nonspecific binding was achieved by parallel incubations with 1 µM of nonradioactive L-364,718.

Saturation binding experiments were performed using a method modified from Zhang et al. (28). Membranes containing 30 µg of protein were incubated with increasing concentrations of 125I-CCK-8 (range from 10 to 10,000 pM) at 25°C for 90 min. Nonspecific binding was determined by parallel incubations with 0.1 µM of unlabeled CCK-8. Separation of bound from free radioligand was achieved by filtration utilizing a vacuum filtering manifold (Millipore) with receptor-binding filter mats (Millipore) and washing the filters with ice-cold incubation medium without BSA.

Scatchard analysis. We used computer analysis of the ligand-fitting program (15), based on the displacement curve and saturation binding data, to analyze the binding results and to obtain the maximum specific binding capacity (Bmax) and affinity (Kd-1, the dissociation constant) of CCK receptors.

Ligand-receptor cross-linking. Radioligand binding experiments were carried out as previously described (20) except that 100 µg of membrane protein and 280 pM of 125I-CCK-8 were used. The bound form was obtained by addition of 0.3 ml of incubation solution without BSA and centrifuged at 15,000 rpm for 15 min at 4°C (microcentrifuge, model 235C, Fisher Scientific). The pellet was washed with HEPES buffer and resuspended in the same buffer; the bifunctional cross-linking reagent disuccinimidyl suberate (DSS) was then added to a final concentration of 5 µM and incubated at 4°C for 5 min. The cross-linking reaction was quenched with Tris buffer (final concentration 20 mM) for 5 min. The reaction mixture was diluted and solubilized with SDS-loading buffer (2% SDS, 62.5 mM Tris, pH 6.8, 1% 2-mercaptoethanol, 10% glycerol, 0.01% bromphenol blue). The solubilized membrane proteins were subsequently separated on 9% SDS-PAGE. The gels were stained with Coomassie brilliant blue and dried. To locate the radiolabeled proteins, autoradiography was performed by exposing the gels to Kodak film for 1-5 days at -70°C.

Protein determination. Protein content in plasma membrane was measured by using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Melville, NY). Values are means of triplicate measurements for each sample.

Materials. 125I-Bolton-Hunter-labeled-CCK-8 (125I-CCK-8, 2,200 Ci/mmol) and [3H]L-364,718 (73.9 Ci/mmol) were from DuPont NEN. CCK-(26---33) (CCK-8) was from Bachem (Torrance, CA). STI was from Worthington Biochemicals (Freehold, NJ). 1,10-Phenanthroline was from ICN Biomedicals (Aurora, OH). DSS was from Pierce (Rockford, IL). Phosphatidylcholine, MES, BSA (fraction V), bacitracin, and other reagents were purchased from Sigma Chemical (St. Louis, MO). L-364,718 was a gift from Dr. Paul Andersen, Merck Sharp and Dohme (West Point, PA).

Statistical analysis. Results are expressed as means ± SE. Statistical significance was evaluated using Student's t-test for unpaired and paired values. P < 0.05 were considered to be significantly different.


    RESULTS
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ABSTRACT
INTRODUCTION
PATIENTS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Studies were performed at different temperatures and duration of incubation times to determine the optimal conditions for binding 125I-CCK-8 to plasma membranes of gallbladder muscles. Binding of 125I-CCK-8 was time and temperature dependent. Maximum binding occurred at 25°C (data not shown). Binding at 25°C was one-half maximal at 15 min, and steady-state binding was reached at 90 min and remained at a plateau for up to 240 min (Fig. 1). Membranes from gallbladders with pigment stones exhibit higher 125I-CCK-8 binding than that from gallbladders with cholesterol stones with nonspecific binding being almost the same for both. After a steady state was reached, addition of 0.1 µM unlabeled CCK-8 significantly reduced the binding of 125I-CCK-8 (Fig. 1). The dissociating process is faster in gallbladders with pigment stones than that in gallbladders with cholesterol stones (P < 0.05, determined by the unpaired Student's t-test).


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Fig. 1.   Time course of 125I-CCK-8 binding to and dissociation from human gallbladder smooth muscle membranes with cholesterol (CS; triangle  and black-triangle) and pigment (PS; open circle  and ) stones. Muscularis membranes were incubated at membrane protein concentration of 150 µg/ml for 90 min in absence (total binding; triangle  and open circle ) and presence (nonspecific binding;  and ) of 0.1 µM of unlabeled CCK-8. To determine reversibility of 125I-CCK binding, 0.1 µM unlabeled CCK-8 was added after 120-min incubation and continued for another 120 min (black-triangle and ). Values shown are means ± SE. In each experiment each value was measured in triplicate, and results given are means from 3 separate experiments. B/T, bound/total radioactivity.

Displacement studies using increasing concentrations of unlabeled CCK-8 in gallbladders with cholesterol and pigment stones reveal a shift to the left of the dose-response relationships for those with cholesterol stones compared with those with pigment stones (Fig. 2A). Computer analysis of this competitive inhibition of 125I-CCK-8 binding by unlabeled CCK-8 was also used to determine the binding capacity and affinity of these receptors. The displacement curves were best fitted by a single class of binding site for CCK-8. The binding capacities were significantly decreased in gallbladders with cholesterol stones of 0.18 ± 0.07 pmol/mg protein compared with 0.38 ± 0.05 pmol/mg protein for gallbladders with pigment stones (P < 0.05). A lower Kd of 0.18 ± 0.06 nM was also observed in muscle cells from cholesterol stone compared with 1.2 ± 0.23 nM in muscle cells from pigment stone gallbladders (P < 0.01). Expressed as percentage, the binding by cholesterol stone gallbladders was only about 60% of the binding of radiolabeled ligand by pigment stone gallbladders measured as 100% (Fig. 2B).


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Fig. 2.   CCK-8 induced inhibition of binding of 125I-CCK-8 to human gallbladder smooth muscle membranes with CS and PS. Membranes were incubated for 90 min at 25°C with 50 pM 125I-CCK-8 plus indicated concentrations of unlabeled CCK-8. A: results are expressed as percentage of value measured with radiolabeled ligand alone (i.e., percent control). B: results are expressed as percentage of value for PS gallbladders measured with radiolabeled ligand alone, which was considered as 100%. In each experiment each value was measured in triplicate, and results given are means from 4 separate experiments.

Similar displacement curves were obtained with the specific CCK-A receptor antagonist L-364,718 to inhibit binding of [3H]L-364,718 (Fig. 3A). The Bmax (0.35 ± 0.04 pmol/mg protein) for gallbladders with cholesterol stones was lower than that for gallbladders with pigment stones (1.60 ± 0.39 pmol/mg protein; P < 0.001). The Kd also decreased from 6.05 ± 1.06 nM for gallbladders with pigment stones to 0.62 ± 0.12 nM for gallbladders with cholesterol stones (P < 0.0001). The percent value for gallbladders with cholesterol stones was only 80% of that for pigment stone gallbladders measured with the radiolabeled ligand alone (Fig. 3B).


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Fig. 3.   CCK-A receptor antagonist L-364,718 induced inhibition of binding of [3H]L-364,718 to human gallbladder smooth muscle membranes with CS and PS. Membranes were incubated for 90 min at 25°C with 300 pM [3H]L-364,718 plus indicated concentrations of nonradioactive L-364,718. A: results are expressed as percentage of value measured with radiolabeled ligand alone (i.e., percent control). B: results are expressed as percentage of value for gallbladders with PS measured with radiolabeled ligand alone, which was considered as 100%. In each experiment each value was measured in triplicate, and results given are means from 3 separate experiments.

To further confirm these findings, maximal binding was performed by increasing the concentration of radiolabeled CCK up to 10 nM (Fig. 4). The binding became saturated after the concentration of 125I-CCK-8 increased to 3 nM. Computer analysis (15) using a nonlinear least-squares curve-fitting program (LIGAND) demonstrated that the data were best fit by a single binding site model. The maximum binding capacity was 0.38 ± 0.02 pmol/mg protein for gallbladders with pigment stones and 0.18 ± 0.02 pmol/mg protein for gallbladders with cholesterol stones. These results are similar to the Bmax values obtained from displacement curves and were significantly different between gallbladders with pigment and cholesterol stones (P < 0.01).


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Fig. 4.   Saturation binding of 125I-CCK-8 to CCK receptors in human gallbladder smooth muscle membranes with CS and PS. Increasing concentrations of 125I-CCK-8 (10-10,000 pM) were incubated with the membranes as described in experimental procedures. Specific bindings (open circle  and ) were defined as arithmetic difference between total binding and nonspecific binding (NSB; ) observed in presence of 0.1 µM unlabeled CCK-8. Data points represent means of triplicate determinations in single experiment. Similar data were obtained in 2 additional experiments.

After the muscle tissue from cholesterol stone gallbladders was incubated with cholesterol-free liposomes for 4 h, the displacement curve of CCK receptors shifted to the right compared with that incubated with HEPES buffer (Fig. 5A). The binding capacity increased significantly from 0.13 ± 0.03 pmol/mg protein in HEPES buffer to 0.31 ± 0.06 pmol/mg protein after incubation with cholesterol-free liposomes (P < 0.01), which was not significantly different from the binding capacity of gallbladders with pigment stones. Likewise, the Kd increased from 0.13 ± 0.03 in HEPES to 0.61 ± 0.05 nM after incubation with cholesterol-free liposomes (P < 0.001). The percent increased in the Bmax for gallbladders with cholesterol stones after incubation with cholesterol-free liposomes was 186% compared with that after incubation with HEPES buffer, which was considered as 100%. This finding indicates that the impaired CCK receptor binding capacity and affinity in membranes from gallbladders with cholesterol stones were reversible (Fig. 5B).


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Fig. 5.   Effects of cholesterol-free liposomes incubation on CCK receptor binding properties in human gallbladder smooth muscle membranes with cholesterol stones. Aliquots of smooth muscle squares were first incubated with either cholesterol-free liposomes in HEPES buffer () or HEPES buffer only (open circle ), and membranes from each group were then incubated for 90 min at 25°C with 50 pM 125I-CCK-8 plus indicated concentrations of unlabeled CCK-8. A: results are expressed as percentage of value measured with radiolabeled ligand alone (i.e., percent control). B: results are expressed as percentage of value for HEPES buffer-treated group measured with radiolabeled ligand alone, which was considered as 100%. In each experiment each value was measured in triplicate, and results given are means from 3 separate experiments.

Covalent cross-linking of 125I-CCK-8 to membrane CCK receptors using DSS followed by electrophoretic analysis in 9% SDS-PAGE and autoradiography revealed a single radioactive band of molecular mass equal to 92 kDa (Fig. 6) in each lane. The inclusion of nonradioactive CCK-8 markedly reduced the extent of 125I-CCK-8 incorporation into this band. Density scan was performed (Fig. 7) using an optical density (OD) to represent the specific binding of CCK to its receptors. It was calculated by using total binding minus nonspecific binding. The results showed a much lower OD in gallbladders with cholesterol stones than in gallbladders with pigment stones, indicating a lower CCK receptor binding in the former specimens (P < 0.01).


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Fig. 6.   Comparison of membrane-bound CCK receptors in human gallbladder smooth muscle membranes with CS and PS using cross-linking agents and analysis by SDS-PAGE. Membranes were incubated for 30 min at 25°C with 280 pM 125I-CCK-8 in the absence (lanes 1 and 3) and presence (lanes 2 and 4) of 0.1 µM nonlabeled CCK-8. Membranes were cross-linked with 5 mM disuccinimidyl suberate and analyzed in 9% gels. Gels were dried and subjected to autoradiography. Lanes 1 and 2, gallbladders with pigment stones; lanes 3 and 4, gallbladders with cholesterol stones. Arrow indicates molecular mass of specifically labeled proteins.



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Fig. 7.   Optical density (OD) of autoradiographs of 125I-CCK-8 cross-linked to human gallbladder muscle membranes with cholesterol and pigment stones. Films obtained from autoradiography were scanned to measure OD. OD, which represents specific bound CCK receptors, was obtained by using OD value for total binding minus OD value for nonspecific binding. Results given are means ± SE from 3 gallbladders.


    DISCUSSION
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ABSTRACT
INTRODUCTION
PATIENTS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Although there is a large body of evidence showing an impaired muscle contractility in gallbladders with cholesterol stones in human and animal models (2, 17), the mechanisms responsible for this defective contraction have not been fully elucidated. We have shown that smooth muscle cells from gallbladders with cholesterol stones have normal signal transduction pathways distal to the activation of G proteins (27), suggesting that the defect resides in the plasma membrane. Furthermore, these membranes contain excessive amounts of cholesterol, abnormal cholesterol-to-phospholipid ratio, and decreased membrane fluidity (26). These membrane lipid alterations might directly or indirectly affect the functions of transmembrane proteins, such as G protein-coupled receptors, which could contribute to the defective muscle contraction.

Therefore, the purpose of the present study was to determine whether CCK receptor numbers or affinity was altered in muscle membranes from gallbladders with cholesterol stones compared with gallbladders with pigment stones. Our results support the conclusion that CCK receptor binding is altered in muscle membranes from gallbladders with cholesterol stones. First, the maximal binding capacity of muscle membranes from gallbladders with cholesterol stones was significantly reduced when assessed by studies with radiolabeled CCK or directly by cross-linking studies. The decreased number in available binding sites was supported by the finding that the binding of the CCK-A receptor antagonist [3H]L-364,718 was also reduced and the high-affinity sites do not depend on G protein activation (4). Second, the affinity for both radiolabeled agonist CCK and antagonist L-364,718 was significantly higher in muscle membranes from gallbladders with cholesterol stones than those from gallbladders with pigment stones. Third, these receptor-binding abnormalities were related to changes caused by the presence of excessive cholesterol in the plasma membrane inasmuch as they were all reversed after incubations with cholesterol-free liposomes. The CCK receptor itself appears to be unaffected, since Nardone et al. (16) found no evidence of any sequence mutation or polymorphism in the CCK receptor gene of muscle membranes from specimens with either cholesterol or pigment stones.

The source of cholesterol that is excessively incorporated by the plasma membranes of the gallbladder muscle layer has not been determined. Bile cholesterol diffuses normally into the gallbladder mucosa (19). It is therefore conceivable that when gallbladder bile is supersaturated cholesterol may diffuse at higher rates, reaching the muscle layer where it is readily incorporated by the plasma membranes, resulting in an increased cholesterol content. The serum is an unlikely source of cholesterol inasmuch as gallbladder muscle cells are not more susceptible than all other gastrointestinal muscle cells that remain unaffected. Ileal muscle cells from prairie dogs exhibit a normal contraction after a high-cholesterol diet. However, like normal gallbladder muscle cells, they become defective after they are incubated with cholesterol-rich liposomes (26).

The mechanisms by which cholesterol-lipid changes might cause these alterations in the muscle membrane are not completely clear. However, there are a number of findings that provide some important insights. Incorporated free cholesterol can move freely in the plasma membrane (23). It is inserted in the bilayer with its long axis perpendicular to the plasma membrane, physically associated with sphingomyelin. It may also interact directly with transmembrane receptors by fitting between the grooves of the alpha -helices and restrict the conformational changes that are necessary for optimal binding of ligands, coupling with G proteins and activation of effector enzymes (7). Excessive incorporation of cholesterol by the membrane also changes the membrane fluidity, which by itself could affect membrane protein functions (9). A previous study failed to show any differences in CCK receptor binding between muscles from "controls" and gallbladders with gallstones (20). This study, however, included a heterogeneous group of gallbladders that did not take into account the nature of the stone. Our results were obtained in two different gallbladder populations whose muscle cells have been extensively characterized. We have shown that the muscle cells from gallbladders with cholesterol stones have an abnormal contraction and relaxation as well as specific biochemical abnormalities localized in the plasma membrane (2, 3, 5, 26, 27).

It has been shown in the G protein-coupled superfamily of receptors that ligand binding occurs in a pocket formed by the ring-like arrangement of the seven transmembrane domains and appears to be initiated by ion-ion interaction between a positively charged amino head group and a conserved aspartic acid residue. Usually, the agonist binding site is located in a narrow cleft defined by several transmembrane domains of the receptors about 15 Å away from the extracellular surface (25). Because of excessive membrane cholesterol incorporation, it is conceivable that the distance between transmembrane domains of the CCK receptors may increase, interfering with the ion-ion interaction. Alternatively, excessive cholesterol in the plasma membrane, directly or indirectly by decreasing the membrane fluidity, may restrict the conformational changes of the transmembrane domains, so the formation of the active binding center may be less accessible to the ligand. This may result in dysfunction of some CCK receptors reducing the maximal binding capacity.

Cell membranes are dynamic structures with mechanisms capable of continuous cholesterol incorporation and transfer from the membrane to the cytoplasm by esterification. It is conceivable that both processes may be limited because the cholesterol content and cholesterol-to-phospholipid ratio of plasma membranes in prairie dog gallbladder muscle fed with a high-cholesterol diet for 4 wk is not different from that of human gallbladders with cholesterol stones that are exposed to supersaturated bile with cholesterol presumably for a prolonged period of time (26).

The present studies also showed that the reduced numbers of specific CCK binding sites can be reversed when the muscle layers were incubated with cholesterol-free liposomes for 4 h. Cholesterol leaches out from cell membranes after incubation with cholesterol-free liposomes, resulting in normal contractility and membrane fluidity (26). These findings therefore suggest that CCK receptors are quantitatively normal but a percentage of them are functionally defective because of the excessive amount of cholesterol or because of increased membrane stiffness. They also suggest that the reversibility of gallbladder muscle contractility induced by CCK may be mainly due to the recovery of its receptor functions. Thus impairments of receptor functions by excessive cholesterol incorporation may be the key factor for the defect of muscle contractility in human gallbladders with cholesterol stones. Our findings, however, cannot explain the discrepancies in the Bmax and the Kd of the muscle between gallbladders with cholesterol and pigment stones. It is possible that receptor affinity is not affected, but their number able to bind ligands may be reduced by the excessive incorporation of cholesterol. Under these conditions, labeled CCK may be displaced from fewer binding sites by lower concentrations of nonradioactive CCK given the artificial impression of a higher affinity (lower Kd) in specimens with cholesterol stones than in those with pigment stones. Muscarinic receptors exhibit similar discrepancies between the Bmax and Kd in cortical neurons asso- ciated with changes in plasma membrane fluidity (8).

In conclusion, our study shows that the binding characteristics of CCK receptors in human gallbladders with cholesterol stones are affected compared with those with pigment stones. Changes in CCK receptor binding capacity and affinity in human gallbladders with cholesterol stones may be caused by excessive membrane cholesterol incorporation. Defective CCK receptor functions might be the leading cause for the impaired muscle contractility of human gallbladders with cholesterol stone diseases. Removal of excessive membrane cholesterol by cholesterol-free liposomes can reverse the defects of CCK receptor functions and muscle contractility. Further studies are needed to investigate the receptor-G protein interaction in human gallbladders with cholesterol stones.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-27389.


    FOOTNOTES

These data were accepted and presented at the annual meeting of the American Gastroenterological Association, New Orleans, LA, in May 1998, and an abstract was printed in Gastroenterology 114: A861, 1998.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: J. Behar, Div. of Gastroenterology, APC 421, 593 Eddy St., Providence, RI 02903 (E-mail: Jose_Behar{at}brown.edu).

Received 15 December 1998; accepted in final form 15 February 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
PATIENTS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Behar, J., and P. Biancani. Pharmacologic characterization of excitatory and inhibitory cholecystokinin receptors of the cat gallbladder and sphincter of Oddi. Gastroenterology 92: 764-770, 1987[Medline].

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3.   Behar, J., B. Y. Rhim, W. Thompson, and P. Biancani. Inositol triphosphate restores impaired human gallbladder motility associated with cholesterol stones. Gastroenterology 104: 563-568, 1993[Medline].

4.   Blevins, G. T., E. M. van de Westerlo, C. D. Logsdon, P. M. Blevins, and J. A. Williams. Nucleotides regulate the binding affinity of the recombinant type A cholecystokinin receptor in CHO K1 cells. Regul. Pept. 61: 87-93, 1996[Medline].

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Am J Physiol Gastroint Liver Physiol 276(6):G1401-G1407
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