Departments of Medicine and Surgery, Rhode Island Hospital and Brown University School of Medicine, Providence, Rhode Island 02903
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ABSTRACT |
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Human gallbladders with cholesterol stones (ChS) exhibit
an impaired muscle contraction and relaxation and a lower CCK
receptor-binding capacity compared with those with pigment stones (PS).
This study was designed to determine whether there is an abnormal
receptor-G protein coupling in human gallbladders with ChS using
35S-labeled guanosine
5'-O-(3-thiotriphosphate)
([35S]GTPS) binding,
125I-labeled CCK-8 autoradiography, immunoblotting, and G
protein quantitation. CCK and vasoactive intestinal peptide caused
significant increases in [35S]GTP
S binding
to G
i-3 and Gs
, respectively. The binding
was lower in ChS than in PS (P < 0.01). The reduced
[35S]GTP
S binding in ChS was normalized
after the muscles were treated with cholesterol-free liposomes
(P < 0.01). Autoradiography and immunoblots showed a
decreased optical density (OD) for CCK receptors, an even lower OD
value for receptor-G protein coupling, and a higher OD for uncoupled
receptors or G
i-3 protein in ChS compared with PS
(P < 0.001). G protein quantitation also showed that there were no significant differences in the G
i-3 and
Gs
content in ChS and PS. We conclude that, in addition
to an impaired CCK receptor-binding capacity, there is a defect in
receptor-G protein coupling in muscle cells from gallbladder with ChS.
These changes may be normalized after removal of excess cholesterol
from the plasma membrane.
immunoblotting; 35S-labeled guanosine 5'-O-(3-thiotriphosphate) binding; autoradiography; G protein quantitation; smooth muscle.
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INTRODUCTION |
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MUSCLE STRIPS AND CELLS FROM gallbladders with
cholesterol stones (ChS) exhibit an impaired contraction and relaxation
in response to agonists that act on transmembrane proteins (5, 7, 17,
35). These abnormal responses, however, were normalized when membrane
receptors were bypassed by the G protein activator guanosine
5'-O-(3-thiotriphosphate) (GTPS), second messengers inositol 1,4,5-trisphosphate and
2,3-diphosphate-D-glycerate, the enzyme
calmodulin, or nitric oxide (2, 34, 36). Plasma membranes of muscle
from human gallbladders with ChS and prairie dogs on a high-cholesterol
diet (1.2%) also showed a high cholesterol content and
cholesterol-to-phospholipid ratio (33). These abnormalities were
reversed to normal after these defective muscle cells were incubated
with cholesterol-free liposomes that leach out the excess cholesterol
from the plasma membrane (8, 33). Thus excessive cholesterol
incorporation in plasma membranes may affect the signal-transduction cascade across them, which may account for the muscle dysfunction associated with ChS.
Recent studies in our laboratory showed that the binding capacity of CCK receptors in muscle membranes from human gallbladders with ChS was impaired. These findings suggest that the functions of these membrane proteins are affected by excessive cholesterol incorporation (32). This defect may be located in the extracellular domain (for ligand binding), in the intracellular domain (for G protein coupling) of CCK receptors, or in the G proteins themselves (6, 34). It is known that the interaction of the intracellular domain of receptors and related G proteins can be modulated by the extracellular domain of the receptors (37). Likewise, the binding properties of these receptors can also be influenced by other factors, such as guanine nucleotides (9). G proteins bound to guanine nucleotides decrease receptor-G protein coupling in detergent solutions and in conditions in which the dissociation of receptors from G proteins clearly fails to occur (20).
The mechanisms whereby excess membrane cholesterol affects receptors are not fully understood. The normal distribution of cholesterol in the plasma membrane is asymmetric. Membrane proteins are located in cholesterol rich-domains and cholesterol-poor domains and are even directly associated with cholesterol (27). A normal level of membrane cholesterol is essential for the optimal functional activity of many membrane proteins, such as receptors and ion channels (11). Changes of the cholesterol content in plasma membranes may contribute to the impairment of their functions (12, 19).
CCK acts on CCK-A receptors that activate Gi-3 protein
to cause gallbladder muscle contraction (1, 6). Vasoactive intestinal peptide (VIP) relaxes the gallbladder muscle through receptors that
couple with Gs
(6, 14). However, the functional
integrity of CCK or VIP receptor coupling to G proteins across the
plasma membrane has yet to be demonstrated in human gallbladders with gallstones. Furthermore, there is no direct evidence of how CCK receptors and related G proteins associate in the cell membrane milieu,
nor is it known whether and how excessive membrane cholesterol affects
receptor-G protein coupling.
The aim of these studies therefore was to examine receptor-G protein coupling to further investigate whether the functions of CCK and VIP receptors were affected in muscle cells with excessive membrane cholesterol incorporation from human gallbladders with ChS.
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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 evidence of acute cholecystitis. Gallstones were classified as ChS or PS according to their gross appearance and chemical analysis. The gallbladders were kept in ice-cold oxygenated Krebs solution (in mM: 116.6 NaCl, 3.4 KCl, 21.9 NaHCO3, 1.2 NaH2PO4, 2.5 CaCl2, 1.2 MgCl2, and 5.4 glucose). After removal of the serosa and mucosa under a dissecting microscope, the muscle layer was carefully cleaned by further removing the remaining connective tissue and small blood vessels and was cut into strips for further use.
Preparation of cholesterol-free liposomes. Cholesterol-free liposomes were prepared by using egg phosphatidylcholine (3, 16). Phosphatidylcholine (3 ml; 20 mg/ml in chloroform) in a glass test tube was dried under a stream of nitrogen. The dried lipids were suspended in 3 ml of normal saline and sonicated for 30 min with a Branson 2200 sonicator (Branson Ultrasonics, Danbury, CT). The suspension was then centrifuged at 10,000 g for 30 min to sediment the undispersed lipids. Two milliliters of the supernatant and eight milliliters of 0.2% BSA-HEPES buffer (in mM: 24 HEPES, pH 7.4, 112.5 NaCl, 5.5 KCl, 2.0 KH2PO4, 1.9 CaCl2, 0.6 MgCl2, and 10.8 glucose) were mixed to make cholesterol-free liposomes.
Preparation of enriched plasma membranes.
Enriched plasma membranes from muscle strips were prepared and purified
by sucrose gradient centrifugation (28, 32). Muscle strips were
homogenized in sucrose-HEPES buffer using a tissue tearer (Biospec
Products, Racine, WI). This sucrose-HEPES buffer contained 0.25 M
sucrose, 10 mM HEPES, pH 7.4, 0.01% soybean trypsin inhibitor, 0.1 nM
phenylmethylsulfonyl fluoride, 0.1 nM 1,10-phenanthroline, and 1 mM
2-mercaptoethanol. The homogenates were centrifuged at 600 g
for 5 min, and the supernatant was collected in a clean tube and again
centrifuged at 150,000 g for 45 min. The pellet was resuspended
in sucrose-HEPES buffer, layered over a linear 9-60% sucrose
gradient, and centrifuged once more at 90,000 g for 3 h. The
plasma membranes were collected at ~24% sucrose and stored at
70°C.
35S-labeled GTPS binding and
immunoprecipitation.
[35S]GTP
S binding was assayed by
immunoprecipitation (23). Plasma membranes were solubilized for 1 h at
4°C with 1% CHAPS. Solubilized membranes at a concentration of 2.5 mg protein/ml were incubated with 60 nM
[35S]GTP
S in the presence or absence of
agonists (CCK or VIP) at 37°C for 10 min in a solution containing
(in mM) 10 HEPES, pH 7.4, 0.1 EDTA, and 10 MgCl2. For
nonspecific binding, 6 µM GTP was used. The reaction was stopped with
10 volumes of ice-cold Tris buffer (100 mM
Tris · HCl, pH 8.0, 10 mM MgCl2, 100 mM
NaCl, and 20 µM GTP). Aliquots (200 µl) of the reaction mixture
were added to pretreated ELISA wells. The ELISA wells were initially coated with an anti-rabbit immunoglobulin antibody (1:2,000) followed by specific G protein subunit antibodies (1:2,000) at 4°C for 1 h
each. The specific G protein subunit antibodies of
anti-G
i-1,2, G
i-3, G
q-11,
and Gs
were used. After incubation at 4°C for 2 h,
the wells were washed three times with phosphate buffer (in mM: 1 KH2PO4, pH 7.4, 10 Na2HPO4, 137 NaCl, and 2.7 KCl) containing 0.05% Tween-20. The radioactivity of each well was counted by beta
counter. Data were expressed as percent increase over basal levels
(without agonist).
Solubilization of the CCK receptor-G protein complex.
Membrane (1 mg; 200 µl) was incubated with 280 pM
125I-labeled CCK-8 in phosphate-magnesium buffer (1 mM
MgSO4 and 50 mM Na3PO4, pH 7.4) at
room temperature for 30 min (total volume 500 µl) (14, 26). The bound
complex was obtained by addition of 0.5 ml of phosphate-magnesium
buffer and centrifuged at 15,000 rpm (Microcentrifuge, model 235C,
Fisher Scientific) for 15 min at 4°C. The pellet was washed with
the same buffer and resuspended (250 µl). The crosslinking agent
disuccinimidyl suberate (DSS) in DMSO was added (final concentration 5 mM) and incubated at room temperature for 30 min. Then the reaction was
quenched by adding 1 M Tris (pH 7.5, final concentration 20 mM) and incubating at room temperature for 10 min. After centrifugation at 15,000 rpm for 5 min, the pellet was washed twice with HEPES buffer
and solubilized with 1% Triton X-100 at 4°C for 30 min and again
spun at 15,000 rpm for 30 min. The resulting supernatant was incubated
with 5 mM bis(sulfosuccinimidyl) suberate (BS3)
(crosslinking agent) in HEPES buffer at room temperature for 1 h. The
mixture was diluted with SDS-loading buffer (2% SDS, 62.5 mM Tris, pH
6.8, 1% 2-mercaptoethanol, 10% glycerol, and 0.01% bromphenol blue)
and separated on 9% SDS-PAGE (Mini-Protean II cell, Bio-Rad, Hercules,
CA). Autoradiography was performed to locate the radiolabeled proteins
by exposing the gels to a Kodak film for 1-3 days at
70°C. The desired protein bands were determined by
densitometric scanning using the NIH Image analysis system (National
Institutes of Health, version 1.44).
Immunoblotting analysis of receptor-G protein coupling.
Immunoblotting analysis was performed by a slight modification of a
method reported by Kermode et al. (14). Membranes were incubated with 1 µM CCK-8 at room temperature for 30 min and centrifuged at 15,000 rpm
(Microcentrifuge, model 235C) for 15 min at 4°C. After the pellet
was washed and resuspended (250 µl), the crosslinking agent DSS was
added (final concentration 5 mM) and this was incubated at room
temperature for 30 min. The reaction was quenched by adding 1 M Tris
(pH 7.5, final concentration 20 mM) and incubating at room temperature
for 10 min. The mixture was centrifuged at 15,000 rpm for 5 min; the
pellet was washed twice and solubilized with 1% Triton X-100,
incubated at 4°C for 30 min, and again spun at 15,000 rpm for 30 min. The supernatant was incubated with 5 mM BS3 at room
temperature for 1 h. The reaction mixture was diluted with SDS-loading
buffer and separated on 10% SDS-PAGE (Mini-Protean II cell).
Immunoblotting was performed to locate the desired proteins using a
specific antibody against Gi-3. The G protein bands were identified by using enhanced chemiluminescence reagents (ECL kit; Amersham International, Amersham, UK) and quantitated by densitometric scanning using NIH Image 1.44.
G protein quantitation.
The contents of Gi-3 and Gs
were
determined by using a G protein quantitation kit (CytoSignal, Irvine,
CA) (15, 21). Membranes were solubilized on ice for 30 min with 1%
sodium choleate. The suspension was centrifuged at 13,000 g for
5 min and the supernatant was mixed with SDS-loading buffer (100 µg
membrane protein/lane). Pure G
i-3 and Gs
subunit standards (5, 10, 20, and 40 ng/lane) were also prepared in the
same manner. The samples were boiled and subjected to 10% SDS-PAGE
(Mini-Protean II cell). The separated proteins were electrically
transferred to nitrocellulose membrane (Bio-Rad). The membrane was
blocked with blocking solution, which consists of 5% nonfat dried
milk, 0.01% antiform A, 0.02% sodium azide, and 0.02% Tween-20 in
PBS (in mM: 80 Na2HPO4, pH 7.5, 20 NaH2PO4, and 100 NaCl) at room temperature for
1 h. Then the membranes were incubated with anti-G protein subunit
antibodies (anti-G
i-3 and anti-Gs
,
separately, 1:2,000 dilution) in a blocking solution at room
temperature for another hour. The membranes were washed three times
with a blocking solution without nonfat dried milk and incubated with
horseradish peroxidase-conjugated protein A (1:2,000 dilution) in
blocking solution at room temperature for 1 h. The G protein bands were
identified by using the ECL kit. Quantitation of the immunoblots was
performed by densitometric scanning of the bands by means of NIH Image
1.44. The standard curves of G proteins for G
i-3 and
Gs
were obtained by plotting the band densities against
standard G protein concentrations. The contents of G
i-3
and Gs
in muscle membranes were then calculated from the
band densities of the standard curves and expressed as nanograms per
milligram of membrane protein.
Protein determination. Protein content in plasma membranes was measured by using the Bio-Rad protein assay kit. Values are means of triplicate measurements of each sample.
Materials.
Bolton-Hunter-labeled 125I-CCK-8 (2,200 Ci/mmol) and
[35S]GTPS (1,250 Ci/mmol) were from DuPont
NEN; CCK-8 and VIP were from Bachem (Torrance, CA); DSS and
BS3 were from Pierce (Rockford, IL); G protein subunit
antibodies and G protein quantitation kit were purchased from
CytoSignal (Irvine, CA); horseradish peroxidase-conjugated protein A
and ECL kit were from Amersham; soybean trypsin inhibitor was from Worthington Biochemicals (Freehold, NJ); egg phosphatidylcholine and
other reagents were purchased from Sigma Chemical (St. Louis, MO).
Statistical analysis. Results are expressed as means ± SE. Statistical significance was evaluated by using Student's t-test for unpaired and paired values. P < 0.05 was considered significantly different.
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RESULTS |
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In agreement with previous reports (22), Western blots showed the
presence of a full complement of G protein subunits
Gi-1,2, G
q-11, G
i-3, and
Gs
in gallbladder muscle (data not shown). The
immunoprecipitation of [35S]GTP
S-G protein
complex using specific G protein subunit antibodies revealed similar
basal levels (without agonist stimulation) of [35S]GTP
S activity bound to different G
protein subunits in muscle membranes from human gallbladders with ChS
and PS (Fig. 1).
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The interaction between receptors and their G proteins was first
determined by [35S]GTPS binding. CCK-8 at 1 µM increased the binding of [35S]GTP
S to
G
i-3 up to 77.8 ± 2.7% in muscle membranes from
gallbladders with PS. The magnitude of
[35S]GTP
S bound to G
i-3 was
significantly lower in gallbladders with ChS (32.8 ± 8.5%) than in
those with PS (Fig. 2, P < 0.01). The CCK receptor only coupled to G
i-3
because there was no change in [35S]GTP
S
bound to G
i-1,2, G
q-11, or
Gs
. VIP at 1 µM also increased the
[35S]GTP
S binding to Gs
in
gallbladders with PS by 88.2 ± 9.0%, compared with 36.5 ± 9.4% in
gallbladders with ChS (Fig. 3, P < 0.01). These findings suggest that receptor-G protein activation stimulated by agonists is impaired in human gallbladders with ChS.
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As shown in our previous studies (8), muscle membranes from human
gallbladders with ChS exhibit excessive cholesterol content and
abnormal cholesterol-to-phospholipid ratio. To determine whether the
defective receptor-G protein activation was related to excessive membrane cholesterol content, muscle strips from human gallbladders with ChS were pretreated with cholesterol-free liposomes. CCK-induced [35S]GTPS bound to G
i-3 was
significantly increased, from 30.5 ± 5.7% (untreated) to 54.9 ± 5.4% after treatment with cholesterol-free liposomes (Fig.
4, P < 0.01). Similarly, the
[35S]GTP
S binding to Gs
induced by VIP was also increased (from 43.2 ± 2.3% to 66.3 ± 1.2%) (Fig. 5, P < 0.01). These
findings support our hypothesis that the impaired receptor-G protein
coupling induced by agonists in human gallbladders with ChS is due to
excessive membrane cholesterol content.
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To examine the nature of this impairment, covalent crosslinking of
125I-CCK-8 to its receptors was performed using the
crosslinking agent DSS and followed by electrophoretic analysis and
autoradiography (Fig.
6A). A single radioactive band
of 92 kDa for CCK receptors was found in human gallbladders with PS and
ChS. The density of the band from gallbladder with ChS was lower than
that from PS (lanes 1 and 3). These bands were
eliminated completely by the addition of 1 µM unlabeled CCK-8
(lanes 2 and 4).
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The interaction of CCK receptors with their related G proteins was
examined by adding the crosslinking reagent BS3 after the
membranes were solubilized with Triton X-100 (G proteins are insoluble
in regular buffer) (Fig. 6B). Two bands of 92 kDa for CCK
receptors and 136 kDa for the ligand receptor-G protein complex were
detected in each lane (lanes 1-3). A higher density of the
136-kDa band and a lower density of the 92-kDa band (lane 1)
were detected in PS compared with those in ChS (lane 3). The addition of GTPS caused a significant decrease in the density of the
136-kDa band in PS (lane 2). These results are in complete agreement with previous studies (9).
The optical density (OD) of these bands is shown in Fig.
7. The OD of the 92-kDa band for the ligand
receptor complex was 20.3 ± 4.3 for ChS, which was lower than that
for PS (45.7 ± 3.2, P < 0.01) (Fig. 7A). After the
membranes were treated with the crosslinking agent BS3
(Fig. 7B), the OD of the 136-kDa band (ligand receptor-G
protein complex) was 3.0 ± 2.4 for ChS (lane 3), which is
lower than that for PS (lane 1) (15.4 ± 0.2, P < 0.001). In contrast, the OD of the 92-kDa band (uncoupled with G
proteins) was 12.4 ± 1.4 for ChS, which is higher than that for PS
(3.4 ± 1.1, P < 0.001). These findings suggest lower
receptor-G protein coupling in muscle membranes from gallbladders with
ChS. Pretreatment of plasma membranes with GTPS (lane 2)
also affected the receptor-G protein interaction in PS (OD of 136-kDa
band decreased from 15.4 ± 0.2 to 10.7 ± 1.4, P < 0.05).
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Moreover, the assumption that less receptor-G protein coupling occured
in ChS was further supported by immunoblotting analysis (Fig. 8). Similar density of the band for
Gi-3 alone (42 kDa) was determined both in ChS and PS
(Fig. 8A); however, a lower density of the ligand
receptor-coupled G
i-3 band (136 kDa) and a higher
uncoupled G
i-3 band (42 kDa) were observed in muscle membranes from gallbladders with ChS compared with those in muscle membranes from gallbladders with PS (Fig. 8B). A density scan (Fig. 9) showed a similar OD value of the
G
i-3 band in ChS and PS (Fig. 9A) but a much
lower OD value of 3.2 ± 1.4 for ligand receptor-G protein complex and
a higher OD of 8.4 ± 2.4 for uncoupled G
i-3 in ChS
compared with that in PS (14.4 ± 3.2 and 2.4 ± 1.1; P < 0.001 and P < 0.05 vs PS, respectively) (Fig. 9B).
These results indicate that the receptor-G protein interaction after
agonist stimulation is impaired in muscle membranes from human
gallbladders with ChS.
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To determine if this impaired receptor-G protein interaction was caused
by quantitative changes of the G proteins, the contents of
Gi-3 and Gs
proteins were measured.
Recombinant G
i-3 (43.2 kDa) and Gs
(48.5 kDa) His-tagged proteins were used as the standards to calculate the
contents of G
i-3 and Gs
. The
immunoblots clearly showed one 42-kDa band for G
i-3
(Fig. 10) and two bands of 47-kDa and
45-kDa for Gs
(Fig. 11).
These immunoblots also showed a dose-dependent increase in the band
density, with increased concentrations of G protein standards. The G
protein contents were calculated from standard curve (OD/mg protein)
and OD value of the desired G protein band (Fig.
12). No significant differences in the
contents of G
i-3 and Gs
were observed in
ChS and in PS.
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DISCUSSION |
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Although previous studies have shown an impaired muscle contraction and relaxation in human and animal gallbladders with ChS (1, 5, 18, 24), the mechanisms responsible for this defect are still not fully understood. We have shown that muscle cells from gallbladders with ChS have an abnormal CCK receptor-binding capacity (32) and a normal signal-transduction cascade distal to the activation of G proteins (34, 36). This abnormal CCK receptor-binding capacity may be due to the presence of an excessive cholesterol content, high cholesterol-to-phospholipid ratio, and decreased membrane fluidity (8), which may directly or indirectly affect the functions of transmembrane proteins, such as receptor binding and receptor-G protein coupling. These assumptions are supported by the finding that they are normalized in vitro by removing the excessive membrane cholesterol from the defective muscle using cholesterol-free liposomes (6, 8, 32, 33).
The present studies show that the impaired muscle response to agonists
that act on transmembrane receptors may be due to a defect in both
ligand receptor binding and receptor-G protein coupling in muscle
membranes from human gallbladders with ChS. First,
[35S]GTPS binding to specific G
protein-induced by CCK-8 or VIP was significantly reduced in muscle
membranes from gallbladders with ChS compared with those from
gallbladders with PS. The decrease in CCK-8-induced
[35S]GTP
S binding cannot be completely
explained by the reduced receptor-binding capacity because the
magnitude of the receptor-G protein coupling was even lower than that
of ligand receptor binding in ChS compared with those in PS. Second,
the decreased receptor-G protein coupling is probably due to a
functional defect of the intracellular domain of the receptors where G
protein couples, since there was no significant change in G protein
content of gallbladder muscle with ChS. These abnormalities are not
confined to the receptor-G protein cascade that mediates muscle
contraction, since VIP-induced GTP
S bound to Gs
that
mediates muscle relaxation was also affected. These abnormal receptor-G
protein couplings appear to be related to the presence of excessive
cholesterol incorporation in the plasma membrane because they were
normalized after incubation with cholesterol-free liposomes.
Like other G protein-coupled receptors, the actions of CCK and VIP are
mediated through their receptors and related G proteins. The peptide
sequences of these receptors have seven putative transmembrane domains
that are largely constituted of hydrophobic amino acids and three
hydrophilic loops. The COOH-terminal portion of the third cytoplasmic
loop (Ci-3) of these receptors contains a stretch of
charged residues that are thought to form an amphipathic -helical extension of the sixth transmembrane domain in a critical orientation for G protein activation (13, 30). Moreover, the first intracellular loop of these receptors may also play a role in receptor-G protein coupling (31). Although receptors may contact with both the
-subunit
and
-dimer of the G protein, the
-subunit of G protein was
shown to play a critical role in determining the specificity of
receptor-G protein coupling by interacting with certain motifs within a
cavity formed by the third intracellular loop of the receptors (4).
The mechanisms whereby cholesterol-lipid or cholesterol-protein
interactions might cause these alterations in the muscle membrane are
not completely clear. However, there are a number of observations that
provide insights that may be relevant to our findings. Incorporated free cholesterol is inserted in the bilayer of the plasma membrane and
can move freely in the plasma membrane or be physically associated with
sphingomyelin (29). Gimpl et al. (11) reported that ligand binding of
CCK receptors was strongly dependent on the level of the membrane
fluidity. Excessive cholesterol incorporation of plasma membrane
affects not only the membrane fluidity but also a variety of other
properties of the membrane bilayer (thickness, curvature, dipole
potential, and so forth) as well as membrane protein functions (25). It
is conceivable that excessive cholesterol incorporation may interact
directly with transmembrane proteins by fitting between the grooves of
the -helices and may restrict the conformational changes or increase
the distance between transmembrane domains of the receptor (10).
Therefore, the signal-transduction cascade through receptor and G
protein could become more difficult. These may explain the decreased
CCK receptor-G protein coupling in muscle membranes from human
gallbladders with ChS. These results are in complete agreement with our
previous findings in gallbladder muscle from specimens with ChS and PS
(1, 2, 7, 8, 33, 35, 36).
The present studies also showed that the impairment of agonist-induced
[35S]GTPS binding could be reversed to
normal when the muscle strips were incubated with cholesterol-free
liposomes. Our previous studies demonstrated that cholesterol-free
liposomes may leach out excess cholesterol from plasma membranes,
resulting in normal cholesterol content, cholesterol-to-phospholipid
ratio, and membrane fluidity as well as agonist-induced muscle cell
contraction and relaxation (8, 33). These findings, therefore, suggest
that, although the membrane receptors are quantitatively normal, a
percentage of them are functionally defective because of the excessive
amount of cholesterol or because of increased membrane stiffness. The findings that the gallbladder muscle contraction and relaxation are
reversible may be due to the recovery of receptor functions. Thus
impairments of membrane receptor functions by excessive cholesterol incorporation may be the key factor for the defective muscle
contraction and relaxation in human gallbladders with ChS.
In conclusion, our study showed that the interaction of membrane receptor and related G protein was impaired in human gallbladders with ChS compared with those with PS. These changes may be due to membrane receptor dysfunction caused by excessive membrane cholesterol incorporation since the G protein content is not affected. Defective membrane receptor functions might be the leading cause of the impaired muscle contraction and relaxation of human gallbladders with ChS. Removal of excessive membrane cholesterol by cholesterol-free liposomes normalized the impaired functions of receptor-G protein coupling and muscle contraction and relaxation.
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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-DK27389.
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FOOTNOTES |
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These data were presented at the Annual Meeting of the American Gastroenterological Association, New Orleans, LA, May 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, Division of Gastroenterology, APC 421, 593 Eddy St., Providence, RI 02903 (E-mail: jose_behar{at}brown.edu).
Received 23 August 1999; accepted in final form 9 October 1999.
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