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
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ABSTRACT |
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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
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INTRODUCTION |
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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.
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PATIENTS AND METHODS |
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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
(Kd1,
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-(2633) (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.
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RESULTS |
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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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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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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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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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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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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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DISCUSSION |
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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 -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.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-27389.
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FOOTNOTES |
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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.
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