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Am J Physiol Gastrointest Liver Physiol 292: G1641-G1649, 2007. First published February 15, 2007; doi:10.1152/ajpgi.00495.2006
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HORMONES AND SIGNALING

Role of caveolae in the pathogenesis of cholesterol-induced gallbladder muscle hypomotility

Zuoliang Xiao,1 Frank Schmitz,2 Victor E. Pricolo,1 Piero Biancani,1 and Jose Behar1

1Department of Medicine, Rhode Island Hospital and Brown University School of Medicine, Providence, Rhode Island; and 2Department of Medicine I, St. Josef Hospital, Ruhr-University of Bochum, Bochum, Germany

Submitted 24 October 2006 ; accepted in final form 9 February 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Muscle cells from human gallbladders (GB) with cholesterol stones (ChS) exhibit a defective contraction, excess cholesterol (Ch) in the plasma membrane, and lower binding of CCK-1 receptors. These abnormalities improved after muscle cells were incubated with Ch-free liposomes that remove the excess Ch from the plasma membrane. The present studies were designed to investigate the role of caveolin-3 proteins (Cav-3) in the pathogenesis of these abnormalities. Muscle cells from GB with ChS exhibit higher Ch levels in the plasma membrane that were mostly localized in caveolae and associated with parallel increases in the expression of Cav-3 in the caveolae compared with that in GB with pigment stones (PS). The overall number of CCK-1 receptors in the plasma membrane was not different between muscle cells from GB with ChS and PS, but they were increased in the caveolae in muscle cells from GB with ChS. Treatment of muscle cells from GB with ChS with a G{alpha}i3 protein fragment increased the total binding of CCK-1 receptors (from 8.3 to 11.2%) and muscle contraction induced by CCK-8 (from 11.2 to 17.3% shortening). However, G{alpha}q/11 protein fragment had no such effect. Moreover, neither fragment had any effect on muscle cells from GB with PS. We conclude that the defective contraction of muscle cells with excessive Ch levels in the plasma membrane is due to an increased expression of Cav-3 that results in the sequestration of CCK-1 receptors in the caveolae, probably by inhibiting the functions of G{alpha}i3 proteins.

caveolin-3 proteins; cholecystokinin-1 receptors; G protein fragments

DURING NET FILLING AND STORAGE of bile in the interdigestive phase, the gallbladder (GB) motor function maintains a sustained tonic contraction and intermittent episodes of relaxation. During the digestive phase, most of the stored bile is emptied by strong contractions stimulated by neurohormonal mechanisms that utilize CCK and ACh (13, 24). Lithogenic bile with excessive cholesterol (Ch) impairs the GB contraction induced by CCK and ACh, resulting in bile stasis (1). The mechanism whereby lithogenic bile affects these GB motor functions has not been completely worked out. In vivo and in vitro studies in humans and other animal species have shown that GB muscle cells exposed to lithogenic bile incorporate excessive amounts of Ch in the plasma membrane, which impairs muscle contraction and relaxation in response to G protein-coupled receptors and decreases membrane fluidity and receptor binding of ligands (2, 7, 10, 43). The contraction and relaxation is, however, normal when agonists that bypass receptors stimulate these muscle cells (7, 50). These cellular abnormalities are corrected after the abnormal muscle cells are treated with Ch-free liposomes that remove the excess Ch from the plasma membrane. Conversely, incubation of normal muscle cells with Ch-rich liposomes reproduces all the abnormalities demonstrated in muscle cells from GB with cholesterol stones (ChS) (6, 49).

Recent studies have shown that increased Ch incorporation in the plasma membrane of myometrium decreased the contractility of the uterus in pregnant rats, and depletion of Ch restored the impaired contraction (36). Ch is incorporated by the plasma membrane into the cells through caveolae, specialized structures characterized by invaginations of the membrane clearly seen using electron microscopy (14, 30). Caveolae are microdomains that normally contain high concentrations of Ch, caveolin (Cav) proteins, sphingomyelin, and various preassembled signaling proteins (G proteins, PKC, nitric oxide synthase, among others) (3, 30). These membrane domains are the sites of influx and efflux of free Ch (17). They constitute high-Ch domains that contain caveolin proteins (18), with Cav-3 exclusively present in smooth muscle cells (30). Activated receptors translocate to caveolae, where they couple with G proteins and stimulate preassembled signal molecules (30). Moreover, increased Ch incorporation by the caveolae stimulates the recruitment of higher concentrations of Cav-3, initially from the Golgi network and, later, by stimulating mRNA synthesis (15, 16). The functions of caveolae and Cav-3 are not completely known. Both G protein-coupled receptors and tyrosine kinase receptors are believed to cluster in caveolae and may provide a platform for interactions between the sarcoplasmic reticulum and plasmalemmal ion channels, receptor recycling, and signaling (3, 14). Messengers involved in Ca2+ sensitization of myosin phosphorylation and contraction may depend on caveolae or caveolin proteins (30). Caveolae thus appear to constitute an important signaling domain that plays a role not only in regulation of smooth muscle tone but also in proliferation, such as that seen in neointima formation and atherosclerosis (3). It also has been reported that stretch-induced RhoA and Rac1 activation occurs through caveolae (19).

Caveolin proteins are known to negatively regulate the function of G proteins by binding to the inactive form of G protein subunit {alpha}-GDP and transiently inhibiting the intrinsic GTPase activity of G proteins (30). In contrast, they do not bind to {alpha}-GTP subunits of activated G proteins, which explains the normal contraction induced by guanosine 5'-O-(3-thiotriphosphate) in muscle cells from GB with ChS (42). Thus Cav-3 modulates G protein dissociation and participates in certain types of heterologous receptor desensitization in muscle cells stimulated by more than one agonist (14, 29). Therefore, they are able to modulate the G protein ability to activate phospholipases and generate second messengers and also may contribute to the recycling of internalized receptors back to the bulk plasma membrane (14, 29). It is therefore conceivable that the increased recruitment of Cav-3 into caveolae by muscle cells from GB with ChS may affect receptor functions through enhanced inhibition of G protein functions (38). The aims of these studies, therefore, were to examine whether excessive Ch incorporation in muscle cells from GB with ChS is located in caveolae, resulting in an increase in the recruitment of Cav-3, and whether they are responsible for the abnormalities of CCK-1 receptors in GB with ChS.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Patients. Human GB were obtained from patients undergoing elective laparoscopic cholecystectomy performed for gallstone disease. None of the patients had a history or clinical evidence of acute cholecystitis. Gallstones were classified as ChS or pigment stones (PS) according to their gross appearance and chemical analysis (6, 10, 31). The GB were kept in ice-cold oxygenated Krebs solution (116.6 mM NaCl, 3.4 mM KCl, 21.9 mM NaHCO3, 1.2 mM NaH2PO4, 2.5 mM CaCl2, 1.2 mM MgCl2, and 5.4 mM 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 cut into strips for further use.

Animals. Male guinea pigs (weight 450–500 g) were purchased from Charles River Laboratory (Wilmington, MA). The Animal Welfare Committee of Rhode Island Hospital approved their use. Animals were housed in thermoregulated rooms with free access to food and water. After an overnight fast, the guinea pigs were anesthetized with an intramuscular injection of ketamine hydrochloride (30 mg/kg) followed by pentobarbital sodium (30 mg/kg ip). The GB was removed, rinsed with ice-cold, oxygenated Krebs solution (8, 9, 11, 44, 45), and placed in a dissecting pan containing the same solution continuously aerated with 95% O2-5% CO2. The mucosa and serosa were carefully peeled off under a dissecting microscope. The GB muscle layer was further cleaned by gently removing the remaining connective tissue.

Preparation of Ch-free/Ch-rich liposomes. Ch-free liposomes were prepared using egg phosphatidylcholine (4, 21). Three milliliters of phosphatidylcholine (20 mg/ml in chloroform) in a glass test tube were 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 8 ml of 0.2% BSA-HEPES buffer (24 mM HEPES, pH 7.4, 112.5 mM NaCl, 5.5 mM KCl, 2.0 mM KH2PO4, 1.9 mM CaCl2, 0.6 mM MgCl2, and 10.8 mM glucose) were mixed to make Ch-free liposomes (~1.2 mg/ml). Ch-rich liposomes were made using Ch-free liposomes plus cholesterol (Ch:phosphatidylcholine ratio = 3:1 mg/mg) (49).

Ch content measurements. Total lipids were extracted from plasma membranes with a mixture of chloroform-methanol (2:1 vol/vol) (49). After centrifugation at 2,000 g for 5 min, the lower organic phase was collected and equally dispensed to six glass test tubes for triplicate measurements of Ch and phospholipids. Samples were dried under a stream of nitrogen. The Ch content was measured using the Ch-oxidase method with a Sigma kit. One milliliter of Ch reagent was added to each dried sample and to 10-µl Ch standards (2 mg/ml), mixed, and incubated at 25°C for 10 min. They were measured at an optical density (OD) of 500 nm in a Beckman DU-7 spectrophotometer (Beckman Instruments, Palo Alto, CA). The Ch content was calculated according to the standards, calibrated by the protein content, and expressed as micrograms per milligram of protein.

Preparation of plasma membranes. Plasma membranes were prepared and purified by sucrose gradient centrifugation as described previously (34, 43). Muscle cells from GB with ChS and PS preincubated with buffer, Ch-free liposomes, or Ch-rich liposomes were homogenized separately using a tissue tearer (Biospec Products, Racine, WI) in 10 volumes by weight of a sucrose-HEPES buffer. The homogenates were centrifuged at 600 g for 5 min. The supernatant was collected in a clean centrifuge tube (Beckman Instruments) and centrifuged at 150,000 g for 45 min. The pellet was resuspended in sucrose-HEPES, layered over a linear 9-to-60% sucrose gradient, and centrifuged at 90,000 g for 3 h. The plasma membranes were collected at ~24% sucrose. They were then diluted and pelleted by centrifugation at 150,000 g for 30 min. The pellet of membranes was stored at –70°C.

Isolation of caveolae. Single muscle cells (~3 x 107 cells from 1 human GB or 3 guinea pig GB) were suspended in 10 ml of buffer A (0.25 M sucrose, 1 mM EDTA, 20 mM tricine, pH 7.8) and homogenized with a Dounce tissue grinder (35, 40). The supernatant was collected, laid on the top of 30% Percoll in buffer A, and centrifuged at 84,000 g for 30 min. The plasma membrane band was collected and sonicated with a Vibra cell sonicator for six sonication bursts. An aliquot of suspension was saved as the total plasma membrane fraction; the remainder was mixed with 1.84 ml of buffer C (0.25 M sucrose, 6 mM EDTA, 120 mM tricine, pH 7.8, and 50% OptiPrep) and 0.16 ml of buffer A (final OptiPrep concentration 23%) in the bottom of a TH641 tube. A linear 20-to-10% OptiPrep gradient was poured on top of the sample and centrifuged at 52,000 g for 90 min. The top 5 ml of the gradient was collected and placed in a fresh TH641 centrifuge tube and mixed with 4 ml of buffer B (0.25 M sucrose, 6 mM EDTA, and 120 mM tricine, pH 7.8). The sample was overlaid with 1 ml of 15% OptiPrep and 0.5 ml of 5% OptiPrep and centrifuge at 52,000 g for 90 min. The band in the 5% interface was collected and designated caveolae membranes.

Autoradiography. Plasma membranes were incubated with radiolabeled ligand (125I-CCK-8) in phosphate-magnesium buffer at room temperature (RT) for 30 min (20, 42). The bound form was obtained by centrifugation at 15,000 rpm for 15 min at 4°C (Microcentrifuge model 235C; Fisher Scientific). The pellet (bound form) was washed and resuspended in the same buffer (250 µl), and DSS (cross-linking agent) up to 5 mM was added. The mixture was centrifuged at 15,000 rpm for 5 min. The pellet (containing cross-linked ligand-receptor and G proteins) was washed twice with HEPES buffer and solubilized with 1% Triton X-100 in HEPES buffer by incubation at 4°C for 30 min. It was then spun at 15,000 rpm for 30 min. The supernatant (containing cross-linked ligand-receptor and solubilized G proteins) was incubated with 5 mM BS3 (cross-linking agent) in HEPES buffer to cross-link ligand-receptor-G proteins. The suspension was diluted with SDS loading buffer and subsequently separated on 9% SDS-PAGE. The radiolabeled proteins were located by autoradiography that was performed by exposing the gels to a film for 1–3 days at –70°C.

Western blot. Plasma membranes and caveolae membranes were separated in 10% SDS-PAGE and transferred to nitrocellulose membranes (22). The membrane was blocked with 5% nonfat dry milk followed by incubation with an anti-Cav-3 and anti-Cav-1 monoclonal antibody (Transduction Laboratories) or anti-CCK-1 receptor antiserum (gift from Dr. F. Schmitz) (32). After three washes, the membrane was incubated with horseradish peroxidase-protein A, and the desired bands were identified with the ECL kit (Amersham International) (42).

125I-CCK-8 binding study. Ligand binding experiments were performed in a final volume of 300 µl (33, 43, 44). The incubation solution consisted of 118 mM NaCl, 4.7 mM KCl, 1 mM EGTA, 5 mM MgCl2, 10 mM 2-(N-morpholino)ethanesulfonic acid, 5 mg/ml BSA, 0.2 mg/ml soybean trypsin inhibitor (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 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-bound 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 unlabeled CCK-8. Radioactivity remaining on the filters was counted in a gamma scintillation counter. Using computer analysis with a ligand-fitting program (25) based on the radio-receptor-assay data, we analyzed the binding results to obtain the maximal specific binding capacity of CCK receptors.

Measurements of muscle cell contraction. Single muscle cells were obtained by enzymatic digestion (41, 44–47). GB muscle layer was cut into 2-mm-wide strips and digested in HEPES buffer containing 0.5 mg/ml type F collagenase and 2 mg/ml papain (activity of ~13.9 U/mg protein) for 20 min at 35°C in a shaking water bath. The buffer was gently gassed with 100% O2 during digestion. At the end of the digestive process, the tissue was filtered through a Nitex mesh 200 (Tetko, Elmsford, NY) and rinsed with 20 ml of HEPES. The tissue remaining on the filter was collected and incubated in HEPES buffer at 35°C for 15 min to allow the free dispersion of cells. CCK-8-induced muscle contraction was determined in cells from guinea pig GB preincubated with buffer, Ch-free liposomes, or Ch-rich liposomes for 4 h before treatment with G protein fragment for 15 min. Ch-rich liposomes allow excessive Ch to incorporate into the plasma membrane, whereas Ch-free liposomes may cause excess Ch to leach from the plasma membrane (49). Cells were measured in cell suspensions as described previously and fixed by adding acrolein (41, 44–47). Contraction is expressed as the mean of the percent shortening of 30 individual cells with respect to control (i.e., untreated) cells.

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

Drugs and chemicals. Two G protein fragments corresponding to the caveolin-binding domains of G{alpha}q/11, Tyr192-Ala206 (YPFDLQSVIFRMVDA), and G{alpha}i3, Thr187-Val201 (THFTFKELYFKMFDV), were synthesized using the solid phase method and purified by HPLC (Peptidogenic, Livermore, CA). 125I-Bolton-Hunter-labeled CCK-8 (125I-CCK-8; 2,200 Ci/mmol) was obtained from DuPont NEN; CCK-8 and VIP were obtained from Bachem (Torrance, CA); G protein subunit antibodies were purchased from CytoSignal (Irvine, CA); STI was obtained from Worthington Biochemicals (Freehold, NJ); and type F collagenase (specific activity: 2.1 FALGPA units/mg), papain (specific activity: 12.7 units/mg), egg phosphatidylcholine, and other reagents were purchased from Sigma Chemical (St. Louis, MO).

Data analysis. One- and two-factorial repeated-measures ANOVA and unpaired Student's t-test were used for statistical analysis. P < 0.05 was considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Ch content in plasma membranes of muscle cells from human GB with ChS and PS was measured (Fig. 1). In total plasma membranes (Fig. 1A), the Ch content in the control group and in GB with PS was 518.0 ± 6.8 and 528.0 ± 14.7 µg/mg protein, respectively. GB with ChS had a much higher Ch content of 679.0 ± 38.1 µg/mg protein (P < 0.001 by Student's t-test) compared with controls. Pretreatment of the muscle cells from GB with ChS with Ch-free liposomes reduced the Ch content to a level of 515.0 ± 25.1 µg/mg protein, which was not different from controls.


Figure 1
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  		Fig. 1. Cholesterol (Ch) content in plasma membranes (A), caveolae (B), and bulk plasma membrane (BPM; C) of muscle cells from human gallbladders (GB) with cholesterol stones (ChS) and pigment stones (PS). A: the Ch content was higher in muscle cells from GB with ChS (*P < 0.001 by Student's t-test) and was reduced to normal levels after Ch-free liposome treatment. Values are means ± SD; n = 3. B: the higher Ch content of caveolae with ChS (*P < 0.001 by Student's t-test) was reduced to normal levels after Ch-free liposome treatment. Values are means ± SD; n = 3. C: the remnant of the total plasma membranes after extraction of caveolae is the BPM. The Ch content in the BPM was not different among the 4 groups. Values are means ± SD; n = 3.

 
In caveolae membranes isolated from total plasma membranes using the methods of OptiPrep gradient centrifugation (35, 40) (Fig. 1B), the Ch content in the control group and GB with PS was 4,094.0 ± 61.7 and 3,693.0 ± 48.2 µg/mg protein, respectively. The Ch concentration in caveolae was about eight times higher than that in total plasma membranes. The caveolae of muscle cells from GB with ChS showed higher Ch levels, up to 6,692.0 ± 137.6 µg/mg protein (P < 0.001 by Student's t-test), that were reduced to 4,617.0 ± 98.2 µg/mg protein after treatment with Ch-free liposomes.

The remnant of the total plasma membranes after extraction of caveolae is called bulk plasma membranes (40) (Fig. 1C). The Ch content of the remnant was not different among the four groups.

To further confirm these findings, we treated normal muscle cells from guinea pig GB in vitro with Ch-rich liposomes for a period of time to determine the time course of Ch incorporation into plasma membrane (Fig. 2). Increased Ch content was found after 30 min of incubation with Ch-rich liposomes, from 477.0 ± 33.1 µg/mg protein in controls to 555.0 ± 34.2 µg/mg protein, and reached a plateau of 606.0 ± 19.6 µg/mg protein after 1 h of treatment (P < 0.001 by ANOVA).


Figure 2
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  		Fig. 2. Ch incorporation by the plasma membranes of normal muscle cells from guinea pig GB treated with Ch-rich liposomes. Ch content increased after 30 min of incubation and reached a plateau after 1 h of treatment (*P < 0.001 by ANOVA). Values are means ± SD; n = 3.

 
Cav-3 in the caveolae is mainly expressed in smooth muscle cells and is essential for the functional integrity of receptors and G proteins. As shown in Fig. 3, both Cav-3 and Cav-1 proteins were expressed in caveolae of muscle cells from human GB with PS. However, the band intensity was much higher in Cav-3 protein compared with Cav-1 protein. Pretreatment of muscle cells with Ch-rich liposomes significantly increased the intensity of the Cav-3 band (i.e., more Cav-3 proteins were detected). These findings suggest that Cav-3 protein is the major caveolin protein in muscle cells from human GB with PS and that changes in Ch content may affect its expression. To further confirm this hypothesis, we determined expression of Cav-3 in the caveolae of normal muscle cells of guinea pig GB treated with Ch-rich liposomes for 90 min (Fig. 4). The preparation of plasma membrane and isolation of caveolae were achieved using the methods mentioned above (35, 40). Pretreatment of muscle cells with Ch-rich liposomes resulted in a biphasic increase in the expression of Cav-3 protein in the caveolae at 30 min and after 2 h (OD values from 174.0 ± 21.2 to 252.0 ± 22.4 and 258.0 ± 20.1, respectively; P < 0.05 vs. control group).


Figure 3
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  		Fig. 3. Expression of caveolin (Cav)-1 and Cav-3 proteins in the caveolae of muscle cells from human GB with PS. Both Cav-3 and Cav-1 proteins were expressed in caveolae. However, the intensity of the Cav-3 protein band was greater than that of Cav-1 protein. Treatment of muscle cells with Ch-rich liposomes significantly increased the intensity of the Cav-3 band. The optical density (OD) value of the Cav-3 band after treatment with Ch-rich liposome was significantly higher than that of controls (*P < 0.01 by Student t-test). Values are means ± SD; n = 3.

 

Figure 4
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  		Fig. 4. Biphasic expression of Cav-3 in the caveolae of normal muscle cells from guinea pig GB treated with Ch-rich liposomes at different time intervals. Treatment with Ch-rich liposomes increased the expression of Cav-3 in the caveolae at 30 min and after 2 h (*P < 0.05 vs. control group). Values are means ± SD; n = 3.

 
To determine the relationships between Ch content and the function of membrane receptors, we performed autoradiography of 125I-CCK-8-bound receptors in total plasma membranes (Fig. 5). Total plasma membranes were incubated with 125I-labeled CCK-8 and a cross-linking agent for 90 min. The ligand-receptor complex was subjected to electrophoresis and detected by autoradiography. GB with ChS showed a small band. The OD value of this band of 632.0 ± 53.5 was lower than that of controls and GB with PS (1,829.0 ± 30.2 and 1,727.0 ± 29.3, respectively; P < 0.001 by Student's t-test). Pretreatment with Ch-free liposomes increased the intensity of the band and the OD value to 1,643.0 ± 60.5, which was not different from those of controls and GB with PS.


Figure 5
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  		Fig. 5. Autoradiography of 125I-labeled CCK-8 bound to its receptors in total plasma membranes of human GB with ChS and PS treated with buffer or Ch-free liposomes. Fewer receptors were detected in ChS but were markedly increased after treatment with Ch-free liposomes. The OD value of this band was lower than that of other groups (*P < 0.001 by Student's t-test). Pretreatment with Ch-free liposomes increased the size of the band and the OD value. Values are means ± SD; n = 3.

 
These findings were further supported by covalenting CCK-1 receptors to their related G proteins (Fig. 6). Solubilized plasma membranes were incubated with CCK-8 and a cross-linking agent for 90 min. Membranes were separated and detected by Western blotting using anti-G{alpha}i3 antibody. As shown in Fig. 6, the intensity of the band in GB with ChS was smaller than that of other groups, and the OD value of 150.0 ± 29.4 in GB with ChS was much lower than that of the control group and GB with PS (414.0 ± 22.3 and 384.0 ± 31.2, respectively; P < 0.001 by Student's t-test). Pretreatment with Ch-free liposomes increased the intensity of the band and the OD value to 300.0 ± 54.8, which was not different from that of the controls and GB with PS. The tendency of the results is similar to that of autoradiography results.


Figure 6
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  		Fig. 6. Covalent binding of CCK-1 receptor-G protein (R-G) in total plasma membranes of human GB with ChS and PS treated with buffer or Ch-free liposomes. Solubilized plasma membranes were incubated with CCK-8 and a cross-linking agent for 90 min. Membranes were separated, and G{alpha}i3 proteins were detected by Western blotting using anti-G{alpha}i3 antibody. GB with ChS had a smaller band and lower OD value than those of other groups (*P < 0.001 by Student's t-test), consistent with those determined using autoradiography. Values are means ± SD; n = 3.

 
The distribution of CCK-1 receptors in plasma membranes, bulk plasma membranes, and caveolae was investigated directly by Western blotting using a specific antibody against CCK-1 receptors (Fig. 7). In GB with ChS, a higher expression of CCK-1 receptors in caveolae (OD value of 890.0 ± 32.3; P < 0.001 by Student's t-test) and a lower expression in the bulk plasma membranes (OD value of 516.0 ± 11.3; P < 0.001 by Student's t-test) were found compared with GB with PS (OD value of 539.0 ± 21.1 and 912.0 ± 22.7, respectively). There was no difference in CCK-1 receptor expression in total plasma membrane between GB with ChS (OD value of 750.0 ± 55.3) and GB with PS (OD value of 723.0 ± 39.4).


Figure 7
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  		Fig. 7. Expression of CCK-1 receptors in different membrane components of muscle cells from human GB with ChS and PS. Expression of CCK-1 receptors was higher in the caveolae of ChS (*P < 0.001 by Student's t-test) and lower in the BPM (**P < 0.001 by Student's t-test) compared with GB with PS. No difference in CCK-1 receptor expression was found in plasma membrane (PM) from GB with ChS and PS. Values are means ± SD; n = 3.

 
To further confirm these findings, we performed autoradiography of CCK-1 receptors in caveolae membranes. As shown in Fig. 8, GB with ChS had a bigger and darker band compared with other groups. The OD value of the band of 1,994.0 ± 146.9 was higher than that of control groups and GB with PS (1,556.0 ± 44.5 and 1,637.0 ± 78.3, respectively; P < 0.05 by Student t-test). Pretreatment with Ch-free liposomes reduced the intensity of the band and the OD value to 1,524.0 ± 43.3, which was not different from that of controls and GB with PS. These data suggest that an excess of Ch in the plasma membrane may cause the sequestration of receptors in caveolae of muscle cells from human GB with ChS.


Figure 8
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  		Fig. 8. Autoradiography of 125I-CCK-8 bound to its receptors in caveolae of human GB with ChS and PS treated with buffer or Ch-free liposomes. A higher number of receptors was detected in ChS and was reduced after treatment with Ch-free liposomes. The OD value of the band from GB with ChS was higher than that of other groups (*P < 0.05 by Student's t-test). Values are means ± SD; n = 3.

 
To determine a possible mechanism whereby the excess of Ch and Cav-3 in caveolae causes the sequestration of receptors in these domains, we treated normal muscle cells of guinea pig GB with CCK-1 receptor antagonist L364,718 (Fig. 9). It is known that receptor antagonists freeze the receptor in the bulk plasma membrane and therefore block receptor redistribution (38). Pretreatment with L364,718 significantly reduced the density of CCK-1 receptors in caveolae in normal muscle cells (OD value from 198.3 ± 16.5 to 120.0 ± 6.2; P < 0.001 by Student's t-test). Treatment with Ch-rich liposomes increased the distribution of CCK-1 receptors in caveolae (from 198.3 ± 16.5 to 283.3 ± 24.5; P < 0.001 by Student's t-test). Pretreatment with L364,718 also inhibited the expected increase of CCK-1 receptors in caveolae caused by treatment with Ch-rich liposomes (from 283.3 ± 24.5 to 214.3 ± 9.6; P < 0.001 by Student's t-test). These data suggest that higher expression of Cav-3 proteins may increase the sequestration of receptors in caveolae.


Figure 9
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  		Fig. 9. Effect of CCK-1 receptor antagonist L364,718 on the distribution of CCK-1 receptors in normal muscle cells from guinea pig GB. L364,718 pretreatment inhibited the translocation of CCK receptors from BPM to caveolae (C) (*P < 0.001 by Student's t-test). Treatment with Ch-rich liposomes increased the density of CCK-1 receptors in caveolae (**P < 0.001 by Student's t-test) and was blocked by pretreatment with L364,718 before incubation with Ch-rich liposomes (***P < 0.001 by Student's t-test). Values are means ± SD; n = 3. The OD values represent the band densities of CCK-1 receptors present in caveolae.

 
To further assess whether the interaction between G protein and Cav-3 modulates G protein-dependent receptors, we performed CCK receptor binding studies in muscle cells treated with G protein fragments that block the specific binding site for G proteins in Cav-3 (Fig. 10). Previous studies in our laboratory have shown that maximum binding occurs after 90 min of incubation (43) and that CCK-1 receptors couple with G{alpha}i3 proteins in GB muscle cells (42). Specific G protein fragment preincubation can block the interaction of intact G protein with Cav-3. Treatment with G{alpha}i3 protein fragment increased the total binding of 125I-CCK to its receptors in both plasma membranes and bulk plasma membranes in muscle cells from human GB with ChS (from 8.10 ± 0.28 to 11.10 ± 0.14% and from 5.75 ± 0.21 to 7.75 ± 0.07%, respectively; P < 0.01 and P < 0.05 by Student's t-test; i.e., the specific binding of 125I-CCK to its receptors increased from 0.25 ± 0.03 to 0.35 ± 0.07 pmol/mg protein and from 0.15 ± 0.06 to 0.23 ± 0.02 pmol/mg protein, respectively) (Fig. 10A). G{alpha}q/11 protein fragment had no effect on the binding capacity of CCK-1 receptors in both plasma membranes and bulk plasma membranes. The nonspecific binding is shown in Fig. 10B, and there is no significant difference among all groups.


Figure 10
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  		Fig. 10. CCK-1 receptor binding studies performed in muscle cells from human GB with ChS and PS treated with G protein fragments. A: in cells from GB with ChS, treatment with G{alpha}i3 protein fragment increased the total binding of 125I-CCK to its receptors in both PM (*P < 0.01 by Student's t-test) and BPM (membranes without caveolae) (**P < 0.05 by Student's t-test). Values are means ± SD; n = 3. In contrast, treatment with G{alpha}q fragment had no effect on 125I-CCK-8 binding. B: there was no significant difference in the nonspecific binding among all groups.

 
These findings were further supported by the studies of muscle cell contraction using the same protocol (Fig. 11). Incubation of normal muscle cells from guinea pig GB with Ch-rich liposomes reduced CCK-8 induced contraction (from 21.2 ± 1.3 to 11.2 ± 1.4%). Pretreatment with G{alpha}i3 protein fragment in the presence of Ch-rich liposomes increased the CCK-induced muscle cell contraction (from 11.2 ± 1.4 to 17.3 ± 0.5%; P < 0.001 by Student's t-test). G{alpha}q/11 protein fragment had no effect on CCK-8-induced muscle cell contraction.


Figure 11
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  		Fig. 11. Effect of G protein fragments on CCK-8-induced muscle cell contraction of guinea pig GB pretreated with Ch-free or Ch-rich liposomes. Treatment with G{alpha}i3 protein fragment increased the contraction induced by CCK-8 in muscle cells pretreated with Ch-rich liposomes (*P < 0.001 by Student's t-test). Values are means ± SD; n = 3. In contrast, treatment with G{alpha}q fragment had no effect.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present studies indicate that the excessive incorporation of Ch takes place mainly in the caveolae in muscle cells from GB with ChS. Normal muscle cells exposed to Ch-rich liposomes start to incorporate Ch as early as 30 min, and this levels off after 60 min. Higher Ch incorporation increases the expression of Cav-3 in the caveolae. These results are in agreement with the findings that increased Ch influx into the plasma membrane of fibroblasts increases the transfer of caveolin proteins from the Golgi to the caveolae and stimulates the mRNA expression of caveolin proteins, whereas inhibition of Ch transport to the cell surface by oxysterols downregulates the mRNA expression of caveolin proteins (15, 16). Moreover, as suggested by previous studies in our laboratory (48), the total number of CCK-1 receptors in the whole plasma membrane as determined by Western blotting using a specific CCK-1 receptor antibody is not different between muscle cells from GB with ChS and PS. There is, however, an increased number of receptors in the caveolae and a reduced number of receptors in the bulk plasma membrane. These findings suggest that a lower number of receptors return to the bulk plasma membrane, where they bind ligands, in muscle cells from GB with ChS and in normal muscle cells treated with Ch-rich liposomes. These observations may explain the decreased binding of agonists and antagonists found in muscle cells from GB with ChS (43). These findings are further supported by measurements of functionally active receptors in the bulk plasma membrane and caveolae obtained by autoradiography and cross-linking studies of receptor-G protein coupling. These studies revealed decreased binding of CCK-8 in the bulk plasma membrane and coupling to G proteins as well as increased binding by these receptors present in the isolated caveolae. These findings suggest that receptors may undergo spontaneous recycling, that is, in the absence of stimulation by their specific ligands. The findings are in agreement with previous studies using fluorescence labeling techniques to track receptor recycling showing CCK-1 receptors being internalized and recycled spontaneously without being activated by agonists (38). These studies also showed that treatment with a CCK-1 receptor antagonist freezes the receptor in the bulk plasma membrane, preventing its spontaneous internalization (38). These findings were confirmed in our studies of the CCK-1 receptor antagonist L364,718 in normal muscle cells before incubation with either buffer or Ch-rich liposomes. The antagonist significantly decreased receptor translocation to the caveolae in normal muscle cells incubated with buffer and prevented the expected increase in receptor sequestration in the caveolae after incubation with Ch-rich liposomes. Thus these results suggest that increased Ch and Cav-3 in the caveolae cause sequestration and reduced recycling of CCK-1 receptors similar to that observed in other cell types and receptors (5, 12).

Ligand-bound receptors couple to their respective G proteins, are phosphorylated by activated kinases, and then translocate to caveolae. Caveolin proteins bind inactive G proteins, since they preferentially interact with high affinity with GDP-G{alpha} subunits (39). This binding domain of caveolin proteins is specific for each type of G protein, since any mutational change prevents the binding and cofractionation with caveolin proteins (23, 39). The specific caveolae binding domain is located within a 41-amino acid region of the cytoplasmic NH2 terminus. The Cav-G protein interaction regulates the GTPase activity of inactive heterotrimeric G proteins (GDP-G{alpha} subunits) allowing the displacement of GDP by GTP and dissociation of G proteins into {alpha}-GTP and beta{gamma} subunits to stimulate signaling molecules preassembled in caveolae (37). A polypeptide derived from the G protein-binding domain of caveolin proteins (residues 61–101) effectively suppresses the basal activity of purified G proteins in vitro by inhibiting the GDP/GTP exchange (23). The sequence of this region is homologous to that of the Rab GDP dissociation inhibitor, a known inhibitor of the GDP/GTP exchange for Rab proteins (23). Thus, under normal physiological conditions, the dissociation of G protein is negatively regulated by nanomolar concentrations of Cav-3 that behave like a GDP dissociation inhibitor. Caveolin proteins also may act as chaperones of receptors facilitating their trafficking, since they have been located in noncaveolae pools of recycling endosomes (28). Whether caveolin proteins bind receptors directly or regulate their functions through G proteins, however, is unclear (26).

The mechanism whereby higher levels of Cav-3 in the caveolae increase the sequestration of these receptors was examined by treating muscle cells with a G protein fragment. G protein fragment can bind to a specific site of the Cav-3 proteins and prevent the subsequent binding of a specific endogenous inactive {alpha}-subunit of G proteins to Cav-3. Since receptor redistribution depends on G protein activation/inactivation that interacts with Cav-3 in the caveolae, inhibition of G protein binding to Cav-3 by G protein fragment may affect the distribution of receptors (23, 27, 29). In contrast to other smooth muscle cells of the gastrointestinal tract, CCK-1 receptors couple to G{alpha}i3 proteins in the GB muscle cells (42). Treatment with a G{alpha}i3 fragment after muscle cells were incubated with Ch-rich liposomes significantly increased the binding of 125I-CCK-8 and muscle contraction induced by CCK-8. In contrast, fragments of G{alpha}q/11 had no effect in cells treated with buffer or Ch-rich liposomes. The improvement in binding and contraction in muscle cells treated with a G{alpha}i3 fragment occurred despite the persistent high Ch and Cav-3 levels in the caveolae. These findings suggest that Cav-3 play a key role in the pathogenesis of the defective muscle contraction associated with lithogenic bile with Ch and greater incorporation of Ch levels by the plasma membrane. The findings also suggest that Cav-3 proteins modulate receptor recycling, most likely indirectly through their inhibition of G protein dissociation and activation.

In summary, the present studies indicate that the excess of Ch incorporated by the plasma membrane of muscle cells from GB with ChS localizes mainly in caveolae, resulting in a biphasic increase in Cav-3. This increase in Cav-3 causes greater sequestration of CCK-1 receptors in the caveolae and decreased recycling to the bulk plasma membrane, where they bind to their respective ligands. The blockade of binding sites of Cav-3 to specific G proteins using their binding fragments increases the binding capacity of receptors and muscle contraction induced by CCK-8 despite the high Ch and Cav-3 levels in the caveolae. These findings may explain the abnormalities of CCK-1 receptors in muscle cells from GB with ChS.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01 DK-27389.


   ACKNOWLEDGMENTS
 
These data were presented in part at the Annual Meeting of the American Gastroenterological Association, San Francisco, CA, in May 2002.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Z. Xiao, Division of Gastroenterology, APC 406, Rhode Island Hospital/Brown Univ. Medical School, 593 Eddy St., Providence, RI 02903 (e-mail: zuoliangxiao2000{at}yahoo.com)

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. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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