Defect of receptor-G protein coupling in human gallbladder with cholesterol stones

Zuo-Liang Xiao, Qian Chen, Joseph Amaral, Piero Biancani, and Jose Behar

Departments of Medicine and Surgery, Rhode Island Hospital and Brown University School of Medicine, Providence, Rhode Island 02903


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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]GTPgamma S) binding, 125I-labeled CCK-8 autoradiography, immunoblotting, and G protein quantitation. CCK and vasoactive intestinal peptide caused significant increases in [35S]GTPgamma S binding to Galpha i-3 and Gsalpha , respectively. The binding was lower in ChS than in PS (P < 0.01). The reduced [35S]GTPgamma 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 Galpha i-3 protein in ChS compared with PS (P < 0.001). G protein quantitation also showed that there were no significant differences in the Galpha i-3 and Gsalpha 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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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) (GTPgamma S), 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 Galpha i-3 protein to cause gallbladder muscle contraction (1, 6). Vasoactive intestinal peptide (VIP) relaxes the gallbladder muscle through receptors that couple with Gsalpha (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.


    METHODS
TOP
ABSTRACT
INTRODUCTION
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 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 GTPgamma S binding and immunoprecipitation. [35S]GTPgamma 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]GTPgamma 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-Galpha i-1,2, Galpha i-3, Galpha q-11, and Gsalpha 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 Galpha i-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 Galpha i-3 and Gsalpha 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 Galpha i-3 and Gsalpha 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-Galpha i-3 and anti-Gsalpha , 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 Galpha i-3 and Gsalpha were obtained by plotting the band densities against standard G protein concentrations. The contents of Galpha i-3 and Gsalpha 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]GTPgamma S (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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In agreement with previous reports (22), Western blots showed the presence of a full complement of G protein subunits Galpha i-1,2, Galpha q-11, Galpha i-3, and Gsalpha in gallbladder muscle (data not shown). The immunoprecipitation of [35S]GTPgamma S-G protein complex using specific G protein subunit antibodies revealed similar basal levels (without agonist stimulation) of [35S]GTPgamma S activity bound to different G protein subunits in muscle membranes from human gallbladders with ChS and PS (Fig. 1).


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 1.   Basal 35S-labeled guanosine 5'-O-(3-thiotriphosphate) ([35S]GTPgamma S) binding level (without agonist stimulation) of G proteins in muscle membranes from gallbladders with cholesterol stones (ChS) (open bars, n = 4) and pigment stones (PS) (closed bars, n = 3). Membranes from both groups were incubated with [35S]GTPgamma S. Bound components were precipitated by specific G protein subunit (Galpha i-1,2, Galpha q-11, Galpha i-3, and Gsalpha ) antibodies and counted in beta counter. There were no significant differences in [35S]GTPgamma S binding activity between these two groups.

The interaction between receptors and their G proteins was first determined by [35S]GTPgamma S binding. CCK-8 at 1 µM increased the binding of [35S]GTPgamma S to Galpha i-3 up to 77.8 ± 2.7% in muscle membranes from gallbladders with PS. The magnitude of [35S]GTPgamma S bound to Galpha 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 Galpha i-3 because there was no change in [35S]GTPgamma S bound to Galpha i-1,2, Galpha q-11, or Gsalpha . VIP at 1 µM also increased the [35S]GTPgamma S binding to Gsalpha 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.


View larger version (10K):
[in this window]
[in a new window]
 
Fig. 2.   CCK-induced [35S]GTPgamma S binding in gallbladder muscle with ChS (open bars, n = 4) and PS (closed bars, n = 4). Membranes from both groups were incubated with [35S]GTPgamma S and 1 µM CCK-8. Bound [35S]GTPgamma S was precipitated by specific G protein subunit antibodies and counted in beta counter. Data were expressed as percent increase of binding from basal level. CCK receptor coupled with Galpha i-3 and CCK-induced [35S]GTPgamma S binding were significantly lower in human gallbladders with ChS than those with PS. Values are means ± SE; * P < 0.01.



View larger version (9K):
[in this window]
[in a new window]
 
Fig. 3.   Vasoactive intestinal peptide (VIP)-induced [35S]GTPgamma S binding in human gallbladder muscle with ChS (open bars, n = 4) and PS (closed bars, n = 4). Membranes from both groups were incubated with [35S]GTPgamma S and 1 µM VIP. Bound [35S]GTPgamma S was precipitated by specific G protein subunit antibodies and counted in beta counter. Data were expressed as percent increase of binding from basal level. VIP receptor coupled with Gsalpha and VIP-induced [35S]GTPgamma S binding decreased significantly in human gallbladders with ChS compared with those with PS. Values are means ± SE; * P < 0.01.

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]GTPgamma S bound to Galpha 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]GTPgamma S binding to Gsalpha 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.


View larger version (10K):
[in this window]
[in a new window]
 
Fig. 4.   CCK-induced [35S]GTPgamma S binding in muscle membranes from human gallbladders with ChS pretreated with cholesterol-free liposomes. Muscle squares were incubated with cholesterol-free liposomes (closed bars, n = 5) or HEPES buffer (open bars, n = 5) for 4 h. Then [35S]GTPgamma S binding experiments were performed. CCK-induced [35S]GTPgamma S binding to Galpha i-3 was significantly increased after incubation with cholesterol-free liposomes. Values are means ± SE; * P < 0.01.



View larger version (9K):
[in this window]
[in a new window]
 
Fig. 5.   VIP-induced [35S]GTPgamma S binding in muscle membranes from gallbladders with ChS pretreated with cholesterol-free liposomes. Muscle squares were incubated with cholesterol-free liposomes (closed bars, n = 4) or HEPES buffer (open bars, n = 4) for 4 h. Then [35S]GTPgamma S binding experiments were performed. VIP-induced [35S]GTPgamma S binding to Gsalpha was significantly increased in membranes incubated with cholesterol-free liposomes. Values are means ± SE; * P < 0.01.

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).


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 6.   Identification of Triton X-100-solubilized crosslinked proteins. All muscle membranes were incubated with 125I-labeled CCK-8. Some were pretreated with 1 µM unlabeled CCK-8 (A, lanes 2 and 4) or 100 µM GTPgamma S (B, lane 2). Membranes were exposed to disuccinimidyl suberate (DSS) and extracted in 1% Triton X-100 (A, lanes 1-4). Solubilized supernatants were further incubated with bis(sulfosuccinimidyl) suberate (BS3) (B, lanes 1-3). Desired proteins were analyzed by 9% SDS-PAGE and autoradiography by exposure to Kodak film for 1-3 days at -70°C.

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 GTPgamma S 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 GTPgamma S (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).


View larger version (11K):
[in this window]
[in a new window]
 
Fig. 7.   Optical densities (ODs) of ligand receptor (92 kDa, n = 4) and ligand receptor-G protein coupling (136 kDa, n = 3) in muscle membranes from gallbladders with ChS and PS. A: ligand receptor complexes of 92 kDa (* P < 0.01) are shown. B: ligand receptors (open bars; ** P < 0.001) and ligand receptor-G protein complexes (closed bars; ## P < 0.001) are shown. Pretreatment with GTPgamma S reduced OD of ligand receptor-G protein complex (### P < 0.05).

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 Galpha i-3 alone (42 kDa) was determined both in ChS and PS (Fig. 8A); however, a lower density of the ligand receptor-coupled Galpha i-3 band (136 kDa) and a higher uncoupled Galpha 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 Galpha 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 Galpha 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.


View larger version (30K):
[in this window]
[in a new window]
 
Fig. 8.   Analysis of Triton X-100-solubilized crosslinked proteins by Western blot. Muscle membranes from gallbladders with ChS and PS were solubilized with Triton X-100 only (A) or incubated with 1 µM CCK-8, exposed to DSS, extracted with 1% Triton X-100 again, and further exposed to BS3 (B). Desired proteins were subsequently analyzed by 10% SDS-PAGE and immunoblotted for Galpha i-3 using anti-Galpha i-3 antibody. Arrows indicate the molecular mass of Galpha i-3 (42 kDa) and ligand plus receptor plus Galpha i-3 (136 kDa).



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 9.   Densities of Galpha i-3 (42 kDa, n = 3) and of receptor-Galpha i-3 coupling (136 kDa, n = 3) in muscle membranes from gallbladders with ChS and PS. Immunoblot film was scanned, and OD of each band was obtained. A: only Galpha i-3 is shown. B: Galpha i-3 uncoupled (open bars) with receptors (* P < 0.05 vs. PS) or coupled (closed bars) with receptors (# P < 0.001 vs. PS) are shown.

To determine if this impaired receptor-G protein interaction was caused by quantitative changes of the G proteins, the contents of Galpha i-3 and Gsalpha proteins were measured. Recombinant Galpha i-3 (43.2 kDa) and Gsalpha (48.5 kDa) His-tagged proteins were used as the standards to calculate the contents of Galpha i-3 and Gsalpha . The immunoblots clearly showed one 42-kDa band for Galpha i-3 (Fig. 10) and two bands of 47-kDa and 45-kDa for Gsalpha (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 Galpha i-3 and Gsalpha were observed in ChS and in PS.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 10.   Quantitation of Galpha i-3 levels in muscle membranes from human gallbladders with PS and ChS. Varying amounts of purified recombinant Galpha i-3 (lane 1, 5 ng; lane 2, 10 ng; lane 3, 20 ng; and lane 4, 40 ng; lane 5 and lane 7 were avoided) were subjected to SDS-PAGE along with membranes from PS (lane 6) and ChS (lane 8). Samples were subsequently immunoblotted for Galpha i-3 using anti-Galpha i-3 antibody. Arrows indicate molecular mass of Galpha i-3 (left: recombinant standard, 43.2 kDa; right: in tissue, 42 kDa).



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 11.   Quantitation of Gsalpha in muscle membranes from human gallbladders with PS and ChS. Purified recombinant Gsalpha (lane 1, 5 ng; lane 2, 10 ng; lane 3, 20 ng; and lane 4, 40 ng; lane 5 and lane 7 were avoided) were subjected to SDS-PAGE along with membranes from PS (lane 6) and ChS (lane 8). Samples were subsequently immunoblotted for Gsalpha using anti-Gsalpha antibody. Arrows indicate molecular mass of Gsalpha (left: recombinant standard, 48.5 kDa; right: in tissue, 47 and 45 kDa).



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 12.   Comparison of G protein levels (A: Galpha i-3; B: Gsalpha ) in muscle membranes from human gallbladders with PS (open bars, n = 3) and ChS (closed bars, n = 3). After immunoblotting, desired blots were scanned to allow construction of standard curves. Such standard curves were generated routinely for quantitation of Galpha i-3 and Gsalpha levels. Data are expressed as nanograms per milligram of protein and presented as means ± SE. There was no significant difference in G protein content between muscle membranes from human gallbladders with ChS and those with PS.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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]GTPgamma S 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]GTPgamma 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 GTPgamma S bound to Gsalpha 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 alpha -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 alpha -subunit and beta gamma -dimer of the G protein, the alpha -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 alpha -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]GTPgamma S 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.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK27389.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Behar, J., K. Y. Lee, W. R. Thompson, and P. Biancani. Gallbladder contraction in patients with pigment and cholesterol stones. Gastroenterology 97: 1479-1484, 1989[ISI][Medline].

2.   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[ISI][Medline].

3.   Bialechi, R. A., and T. N. Tulenko. Excess membrane cholesterol alters calcium channels in arterial smooth muscle. Am. J. Physiol. Cell Physiol. 257: C306-C314, 1989[Abstract/Free Full Text].

4.   Blahos, J., II, S. Mary, J. Perroy, C. De Colle, I. Brabet, J. Bockaert, and J. P. Pin. Extreme C terminus of G protein alpha -subunits contains a site that discriminates between Gi-coupled metabotropic glutamate receptors. J. Biol. Chem. 273: 25765-25769, 1998[Abstract/Free Full Text].

5.   Chen, Q., J. Amaral, S. Oh, P. Biancani, and J. Behar. Gallbladder relaxation in patients with pigment and cholesterol stones. Gastroenterology 113: 930-937, 1997[ISI][Medline].

6.   Chen, Q., V. Chitinavis, Z. L. Xiao, P. Yu, S. Oh, P. Biancani, and J. Behar. Impaired G protein function in gallbladder muscle from progesterone-treated guinea pigs. Am. J. Physiol. Gastrointest. Liver Physiol. 274: G283-G289, 1998[Abstract/Free Full Text].

7.   Chen, Q., G. D. Petris, P. Yu, J. Amaral, P. Biancani, and J. Behar. Different pathways mediate cholecystokinin actions in cholelithiasis. Am. J. Physiol. Gastrointest. Liver Physiol. 272: G838-G844, 1997[Abstract/Free Full Text].

8.   Chen, Q., J. Amaral, P. Biancani, and J. Behar. Excessive membrane cholesterol alters human gallbladder muscle contractility and membrane fluidity. Gastroenterology 116: 678-685, 1999[ISI][Medline].

9.   Chidiac, P. Rethinking receptor-G protein-effector interactions. Biochem. Pharmacol. 55: 549-556, 1998[ISI][Medline].

10.   Fong, T. M., and M. G. Mcnamee. Stabilization of acetylcholine receptor secondary structure by cholesterol and negatively charged lipids. Biochemistry 26: 3871-3880, 1987[ISI][Medline].

11.   Gimpl, G., K. Burger, and F. Fahrenholz. Cholesterol as modulator of receptor function. Biochemistry 36: 10959-10974, 1997[ISI][Medline].

12.   Gleason, M. M., M. S. Medow, and T. N. Tulenko. Excess membrane cholesterol alters calcium movements, cytosolic calcium levels, and membrane fluidity in arterial smooth muscle cells. Circ. Res. 69: 216-227, 1991[Abstract].

13.   Harmar, A. J., A. Arimura, I. Gozes, L. Journot, M. Laburthe, J. R. Pisegna, S. R. Rawlings, P. Robberecht, S. I. Said, S. P. Sreedharan, S. A. Wank, and J. A. Waschek. International Union of Pharmacology. XVIII. Nomenclature of receptors for vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide. Pharmacol. Rev. 50: 265-270, 1998[Abstract/Free Full Text].

14.   Kermode, J. C., A. W. DeLuca, A. Zilberman, J. Valliere, and S. M. Shreeve. Evidence for the formation of a functional complex between vasoactive intestinal peptide, its receptor, and Gs in lung membranes. J. Biol. Chem. 267: 3382-3388, 1992[Abstract/Free Full Text].

15.   Kim, G. D., I. C. Carr, L. A. Anderson, J. Zabavnik, K. A. Eidne, and G. Miligan. The long isoform of the rat thyrotropin-releasing hormone receptor down-regulates Gq proteins. J. Biol. Chem. 269: 19933-19940, 1994[Abstract/Free Full Text].

16.   Klein, I., L. Moore, and I. Pastan. Effect of liposomes containing cholesterol on adenylate cyclase activity of cultured mammalian fibroblasts. Biochim. Biophys. Acta 506: 42-53, 1978[ISI][Medline].

17.   Lamorte, W. W., E. A. Brotsschi, T. E. Scott, and L. F. Williams. Pigment gallstone formation in the cholesterol-fed guinea pig. Hepatology 5: 21-27, 1985[ISI][Medline].

18.   Li, Y. F., N. W. Weisbrodt, and F. G. Moody. Effect of bile diversion and sphincterotomy on gallbladder muscle contractility and gallstone formation. Am. J. Surg. 162: 31-35, 1991[ISI][Medline].

19.   Liscum, L., and K. W. Underwood. Intracellular cholesterol transport and compartmentation. J. Biol. Chem. 270: 15443-15446, 1995[Free Full Text].

20.   Matesic, D. F., D. R. Manning, B. B. Wolfe, and G. R. Luthin. Pharmacological and biochemical characterization of complexes of muscarinic acetylcholine receptor and guanine nucleotide-binding protein. J. Biol. Chem. 264: 21638-21645, 1989[Abstract/Free Full Text].

21.   Mitchell, P. B., H. K. Manji, G. Chen, L. Jolkovsky, E. Smith-Jackson, K. Denicoff, M. Schmidt, and W. Z. Potter. High levels of Galpha s in platelets of euthymic patients with bipolar affective disorder. Am. J. Psychiatry 154: 218-223, 1997[Abstract].

22.   Murthy, K. S., D. H. Coy, and G. M. Makhloup. Somatostatin receptor-mediated signaling in smooth muscle: activation of phospholipase C-beta 3 by Gbeta gamma and inhibition of adenylyl cyclase by Galpha i1 and Galpha 0. J. Biol. Chem. 271: 23458-23463, 1996[Abstract/Free Full Text].

23.   Okamoto, T., T. Ikezu, Y. Murayama, E. Ogata, and I. Nishimato. Measurement of GTPgamma S binding to specific G proteins in membranes using G-protein antibodies. FEBS Lett. 305: 125-128, 1992[ISI][Medline].

24.   Pellegrini, C. A., T. Ryan, W. Broderick, and L. W. Way. Gallbladder filling and emptying during cholesterol gallstone formation in the prairie dog. Gastroenterology 90: 143-149, 1986[ISI][Medline].

25.   Raber, J., and A. Bast. Changes in receptor response by the effect of disease on membrane fluidity. Med. Hypotheses 28: 169-171, 1989[ISI][Medline].

26.   Schjoldager, B., X. Molero, and L. J. Miller. Functional and biochemical characterization of the human gallbladder muscularis cholecystokinin receptor. Gastroenterology 96: 1119-1125, 1989[ISI][Medline].

27.   Schroeder, F., J. R. Jefferson, A. B. Kier, J. Knittel, T. J. Scallen, W. G. Wood, and I. Hapala. Membrane cholesterol dynamics: cholesterol domains and kinetic pools. Proc. Soc. Exp. Biol. Med. 196: 235-252, 1991[Abstract].

28.   Shaw, M. J., E. M. Hadac, and L. J. Miller. Preparation of enriched plasma membrane from bovine gallbladder muscularis for characterization of cholecystokinin receptors. J. Biol. Chem. 29: 14313-14318, 1987.

29.   Slotte, J. P., and B. Lundberg. Transfer of (3H) cholesterol between lipid vesicles and rat arterial smooth muscle cells in vitro. Biochim. Biophys. Acta 750: 434-439, 1983[ISI][Medline].

30.   Wank, S. A. G protein-coupled receptors in gastrointestinal physiology. I. CCK receptors: an exemplary family. Am. J. Physiol. Gastrointest. Liver Physiol. 274: G607-G613, 1998[Abstract/Free Full Text].

31.   Wu, V., M. Yang, J. A. McRoberts, J. Ren, R. Seensalu, N. Zeng, M. Dagrag, M. Birnbaumer, and J. H. Walsh. First intracellular loop of the human cholecystokinin-A receptor is essential for cyclic AMP signaling in transfected HEK-293 cells. J. Biol. Chem. 272: 9037-9042, 1997[Abstract/Free Full Text].

32.   Xiao, Z. L., Q. Chen, J. Amaral, P. Biancani, R. T. Jensen, and J. Behar. CCK receptor dysfunction in muscle membranes from human gallbladders with cholesterol stones. Am. J. Physiol. Gastrointest. Liver Physiol. 276: G1401-G1407, 1999[Abstract/Free Full Text].

33.   Yu, P., Q. Chen, P. Biancani, and J. Behar. Membrane cholesterol alters gallbladder muscle contractility in prairie dogs. Am. J. Physiol. Gastrointest. Liver Physiol. 271: G56-G61, 1996[Abstract/Free Full Text].

34.   Yu, P., Q. Chen, K. M. Harnett, J. Amaral, P. Biancani, and J. Behar. Direct G protein activation reverses impaired CCK signaling in human gallbladders with cholesterol stones. Am. J. Physiol. Gastrointest. Liver Physiol. 269: G659-G665, 1995[Abstract/Free Full Text].

35.   Yu, P., G. De Petris, P. Biancani, and J. Behar. Cholecystokinin-coupled intracellular signaling in human gallbladder muscle. Gastroenterology 106: 763-770, 1994[ISI][Medline].

36.   Yu, P., K. M. Harnett, P. Biancani, G. De Petris, and J. Behar. Interaction between signal transduction pathways contributing to gallbladder tonic contraction. Am. J. Physiol. Gastrointest. Liver Physiol. 265: G1082-G1089, 1993[Abstract/Free Full Text].

37.   Zhao, M. M., R. J. Gaivin, and D. M. Perez. The third extracellular loop of the beta 2-adrenergic receptor can modulate receptor/G protein affinity. Mol. Pharmacol. 53: 524-529, 1998[Abstract/Free Full Text].


Am J Physiol Gastroint Liver Physiol 278(2):G251-G258
0193-1857/00 $5.00 Copyright © 2000 the American Physiological Society