Impaired G protein function in gallbladder muscle from progesterone-treated guinea pigs

Qian Chen, Vikas Chitinavis, Zouliang Xiao, Peirong Yu, Sangik Oh, Piero Biancani, and Jose Behar

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

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

This study was designed to elucidate the mechanism of action of progesterone on gallbladder smooth muscle in guinea pigs. Adult male guinea pigs were treated with either progesterone (2 mg · kg-1 · day-1) or saline for 7 days. Gallbladder muscle cells were isolated by enzymatic digestion with collagenase. Contractile responses to agonists were expressed as percent shortening from control cell length. [35S]guanosine 5'-O-(3-thiotriphosphate) ([35S]GTPgamma S)-binding properties of G proteins were assessed in crude membranes of gallbladder muscle with or without cholecystokinin octapeptide (CCK-8) stimulation. Gallbladder muscle cells from progesterone-treated guinea pigs exhibited an impaired contractile response to CCK-8, GTPgamma S, or aluminum fluoride but a normal response to potassium chloride or D-myo-inositol 1,4,5-trisphosphate compared with controls. Western blot analysis of gallbladder muscle revealed the presence of Gi 1-2, Gi 3, Gq/11, and Gs proteins. The maximal contraction induced by CCK-8 was blocked by pertussis toxin and Gialpha 3-specific antibodies, but not by Gialpha 1-2 or Gq/11alpha antibodies. CCK-8 caused a significant increase in [35S]GTPgamma S binding to Gialpha 3, but not to Gq/11alpha or Gialpha 1-2. The stimulation of Gialpha 3 binding, however, was significantly reduced in gallbladder muscle membranes from progesterone-treated guinea pigs compared with that in control animals. In conclusion, progesterone might cause gallbladder hypomotility by downregulating Gi 3 proteins.

cholecystokinin; smooth muscle

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

PREVIOUS STUDIES HAVE SHOWN a high prevalence of cholelithiasis in pregnant women (2, 8, 33). It has also been demonstrated that pregnancy has a profound effect on the motility of the gallbladder and other segments of the gastrointestinal tract (4, 9, 14). In vitro experiments have shown that progesterone, rather than estrogen, is most likely to cause gallbladder hypomotility (27, 34). Ryan (25, 26) and Ryan and Pellechia (27) have demonstrated that contraction of gallbladder smooth muscle strips in response to cholecystokinin (CCK) and acetylcholine is impaired in pseudopregnant (progesterone-treated) and pregnant guinea pigs. In contrast, the contraction of progesterone-treated gallbladder muscle induced by potassium chloride (KCl) was not different from that of controls. These findings suggest that progesterone affects receptor-dependent, but not receptor-independent, muscle contraction. However, the mechanism by which progesterone causes defective muscle contraction is not fully understood.

The present study was therefore undertaken to investigate the nature of this muscle defect by examining the signal transduction pathways involved in CCK-induced contraction in progesterone- and placebo-treated guinea pigs.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Animals. Male guinea pigs, aged 8-12 wk, were purchased from Charles River Laboratory (Wilmington, MA). Their use was approved by the Animal Welfare Committee of Rhode Island Hospital. Animals were housed in thermoregulated rooms with free access to food and water. Progesterone at a dose of 2 mg/kg body wt (inject vol, 0.1 ml) was injected intramuscularly once daily for 7 days into animals in the experimental group. The same volume of normal saline was given intramuscularly to those in the control group. After an overnight fast, the guinea pigs were anesthetized with an intramuscular injection of ketamine hydrochloride (30 mg/kg) followed by pentobarbital (30 mg/kg ip). The gallbladder was exposed with a midline incision, and the cystic duct was carefully clamped. Bile was removed from the gallbladder, and its cavity was rinsed with ice-cold, oxygenated Krebs solution. The composition of the Krebs solution was as follows (in mM): 116.6 NaCl, 3.4 KCl, 21.9 NaHCO3, 1.2 NaH2PO4, 2.5 CaCl2, 1.2 MgCl2, and 5.4 glucose. The gallbladder was then removed from the liver bed 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 smooth muscle layer was further cleaned by gently removing the remaining connective tissue.

Single cell isolation. Dispersed muscle cells were obtained by a previously reported method (39, 40). The smooth muscle layer was cut into 2 × 2 mm pieces. Tissues from two gallbladders were pooled together to get sufficient muscle cells. Tissue squares were then digested in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered nutrient solution containing 150 U/ml type II collagenase (sp act, 168 U/mg) for 2.5-3 h at 31°C in a shaking water bath. The HEPES buffer consisted of (in mM) 112.5 NaCl, 5.5 KCl, 2.0 KH2PO4, 24 HEPES, 1.9 CaCl2, 0.6 MgCl2, and 10.8 glucose, as well as 0.08 mg/ml soybean trypsin inhibitor and 2% (vol/vol) basal Eagle's medium (50×) amino acids without L-glutamine. The pH of the solution was adjusted to 7.4 at 31°C. The solution was gently gassed with 100% O2 during digestion. At the end of the digestion, the tissue was filtered through a Nitex mesh (Tetko, Elmsford, NY) and rinsed with 30 ml collagenase-free HEPES solution to remove traces of collagenase. The tissue on the filter was collected and incubated in collagenase-free HEPES solution at 31°C for 30 min to allow the cells to disperse freely. The cell suspension was equilibrated at 31°C for 15 min before the experiment was begun.

Preparation of permeable smooth muscle cells. Permeable smooth muscle cells were prepared as previously reported (39, 40). At the end of digestion the muscle tissue was rinsed and incubated in "cytosolic buffer," a medium with the following composition (in mM): 20 NaCl, 100 KCl, 25 NaHCO3, 0.96 NaH2PO4, 0.48 CaCl2, 5.0 MgSO4, and 1.0 ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). The medium also contained 2% bovine serum albumin. The pH was adjusted to 7.2 and maintained by equilibrating with 95% O2-5% CO2 at 31°C. After the cells were dispersed, the cell suspension was incubated with saponin (75 µg/ml) and then centrifuged at 200 g for 3 min. Cells were washed once with modified cytosolic buffer by centrifugation and resuspended in modified cytosolic buffer and equilibrated for 15 min before the experiment. The modified cytosolic buffer contained 1.5 mM ATP as an energy source and a regenerating system consisting of 5 mM phosphocreatine and 10 U/ml of phosphocreatine. Antimycin A (10 µM) was also included to prevent substrate oxidation.

Measurements of muscle cell contraction. Contraction was measured in cell suspensions as described previously (39, 40). Briefly, when a sufficient number of cells had dissociated, 0.25-ml aliquots of cell suspension were added to siliconized glass tubes containing appropriate concentrations of agonists. The cells were allowed to react with the agonists for 30 s and were fixed by adding acrolein at a final concentration of 1%. For the measurement of control cell length, the agonists were substituted by an equivalent volume of HEPES buffer. A few drops of the fixed cells were placed on a microscope slide and covered with a coverslip. The coverslip was sealed with nail enamel to prevent evaporation. Thirty consecutive intact cells were measured using a phase-contrast microscope (Carl Zeiss, Oberkochen, Germany) and a television camera (Panasonic CCTV model WV-CD51, Matsushita Communication, Osaka, Japan) connected to a Macintosh IIci computer. The cell lengths were measured by a computer software program (Image 1.33, NIH, Bethesda, MD). Contraction was expressed as the mean of the percent reduction in cell length (%shortening) of 30 individual cells with respect to control (i.e., untreated) cells per experiment. Concentration response curves were constructed using CCK-8, KCl, D-myo-inositol 1,4,5-trisphosphate (IP3), guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S), and aluminum fluoride (AlF4) as agonists. Muscle contraction also was determined with maximally effective concentration of CCK-8 (10-8 M) in permeabilized cells after 1 h preincubation with pertussis toxin (PTX; 1.6 µg/ml) or with various specific G protein antibodies (1:400).

Preparation of gallbladder muscle membranes. Gallbladder muscle squares were homogenized with a Tissue Tearor (Biospec, Racine, WI) for three bursts of 20 s at setting 5 in 20 mM ice-cold HEPES homogenized buffer (pH 7.4) and again with 60 strokes of a Dounce grinder (Wheaton, Millville, NJ). The homogenates were centrifuged at 600 g for 2 min. The supernatant was collected, and the pellet was rehomogenized and filtered through two layers of 200 µm Nitex. The samples were then ultracentrifuged at 40,000 g for 30 min at 4°C. The pellet was resuspended and solubilized for 1 h at 4°C in a buffer containing 20 mM HEPES (pH 7.4), 240 mM NaCl, 2 mM EDTA, 2 mM phenylmethylsulfonyl fluoride, 20 mM leupeptin, 20 mg/ml aprotinin, and 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The solubilized membrane suspension was measured for protein content and was ready for the following binding studies.

Immunoblot analysis of G proteins in gallbladder muscle. The membranes were prepared from gallbladder muscle squares as described above and solubilized on ice for 1 h in 20 mM tris(hydroxymethyl)aminomethane (Tris), 1 mM EDTA, 1 mM dithiothreitol, 100 mM NaCl, and 1% sodium cholate (pH 8.0). The suspension was centrifuged at 13,000 g for 5 min. The supernatant was mixed with sodium dodecyl sulfate (SDS) sample buffer, boiled for 5 min, and centrifuged at 12,000 revolutions/min for 5 min at 4°C. The prestained molecular weight marker was prepared in the same manner. After 50 ml of sample, each lane was loaded and subjected to a 10% SDS-polyacrylamide gel electrophoresis (Mini-PROTEAN II cell, Bio-Rad, Hercules, CA), and the separated proteins were electrically transferred to a nitrocellulose (NC) membrane (Bio-Rad Laboratories, Melville, NY). The NC membranes were blocked with 5% dried milk in phosphate-buffered saline containing 0.2% Tween 20 at room temperature for 1 h, followed by incubation with different G protein-specific antibodies (1:1,000) for 1 h. After three washes, the NC membranes were incubated for 1 h with horseradish peroxidase-conjugated protein A. The G protein bands were identified using enhanced chemiluminescence reagents.

[35S]GTPgamma S binding. [35S]GTPgamma S binding was assayed by the method of Murthy et al. (17) and Okamoto et al. (23). The crude membranes at a concentration of 2.5 mg protein/ml were incubated at 37°C with 30 nM [35S]GTPgamma S in a solution containing 10 mM HEPES (pH 7.4), 0.1 mM EDTA, and 10 mM MgCl2. The stimulation of binding was assayed in the presence or absence of a maximal CCK-8 concentration for tissue squares (10-6 M) with a total volume of 300 µl. The reaction was stopped with 10 vol of ice-cold 100 mM Tris · HCl (pH 8.0) containing 10 mM MgCl2, 100 mM NaCl, and 20 mM GTP. The mixtures of 200 ml each were added to enzyme-linked immunosorbent assay wells that had been coated initially with an anti-rabbit immunoglobulin antibody (1:1,000) and subsequently coated with specific G protein antibodies (1:1,000). After 2 h incubation on ice, the wells were washed three times with phosphate buffer solution containing 0.05% Tween 20. The radioactivity from each well was counted by using a Tri-Carb 1900 CA liquid scintillation analyzer (Packard Instruments, Meriden, CT). Triplicate measurements were carried out for each experiment. Data were expressed as percent stimulation from basal levels.

Protein determination. Protein content in gallbladder muscle membranes was measured according to the method of Bradford using a protein assay kit (Bio-Rad Laboratories). Values were means of triplicate measurements for each sample.

Drugs and chemicals. Type II collagenase and soybean trypsin inhibitor were purchased from Worthington Biochemicals (Freehold, NJ). Polyclone antibodies to Gialpha 1-2, Gialpha 3, and Gq/11alpha and PTX were obtained from Calbiochem (La Jolla, CA). The ability of these G protein antibodies to block activation or inhibition of specific effector enzymes has been demonstrated in recent studies (17, 19, 20). CCK-8 was purchased from Bachem (Torance, CA). [35S]GTPgamma S was purchased from DuPont-NEN (Boston, MA). Horseradish peroxidase-conjugated protein A, enhanced chemiluminescence reagents, and rainbow prestained molecular marker were from Amersham (Arlington Heights, IL). IP3, GTPgamma S, aprotinin, leupeptin, and other reagents were purchased from Sigma Chemical (St. Louis, MO).

Statistics. One and two factorial repeated analysis of variance (ANOVA) and Student's t-test were used for statistical analysis. P < 0.05 was considered to be significant.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The average resting lengths of intact and permeabilized smooth muscle cells isolated from control guinea pig gallbladders were 58.7 ± 1.2 µm (270 cells) and 58.0 ± 0.4 µm (180 cells), respectively. In the progesterone-treated group, they were 56.8 ± 1.0 µm (270 intact cells) and 58.1 ± 1.2 µm (180 permeabilized cells). There was no significant difference in resting cell length between intact and permeabilized or between control and experimental groups (one factor ANOVA).

CCK-8 at concentrations of 10-13 to 10-7 M induced a concentration-dependent contraction of intact gallbladder muscle cells from control and progesterone-treated animals. Maximal contraction of 21.6 ± 1.0% was observed with 10-8 M of CCK-8. In the progesterone-treated group, however, the contraction in response to CCK-8 was significantly lower with a maximal shortening of 11.4 ± 0.8% (Fig. 1).


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Fig. 1.   Dose responses of CCK-8 in intact gallbladder muscle cells from guinea pigs treated with progesterone and control animals. In progesterone-treated guinea pigs, the response to CCK-8 was significantly reduced at all doses, with respect to the control group. Values are means ± SE of 4 experiments. P < 0.01, by ANOVA.

KCl at concentrations of 5 to 25 mM also induced a dose-dependent contraction. Both control and progesterone groups demonstrated comparable contractile responses with maximal contractions of 21.7 ± 0.8% and 20.6 ± 0.7%, respectively (Fig. 2).


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Fig. 2.   Effects of increasing concentrations of potassium chloride (KCl) of intact gallbladder muscle cell contraction in progesterone-treated and control animals. Values are means ± SE of 3 experiments. There was no significant difference between these 2 groups.

To test the integrity of intracellular pathways, muscle cells were permeabilized by brief exposure to saponin to allow the second messenger IP3 and the G protein activator GTPgamma S to diffuse across the plasma membrane. IP3 (10-10 to 10-5 M) caused concentration-dependent contraction of muscle cells from both progesterone-treated and control animals with maximal contractions of 20.9 ± 0.8% and 21.3 ± 0.6%, respectively (Fig. 3). The magnitude of contraction caused by IP3 in both groups was not different from that induced by CCK-8 in intact cells from control gallbladders. GTPgamma S contracted muscle cells from control gallbladders in a dose-dependent manner, with a maximal contraction of 21.1 ± 0.6% at 10-5 M. However, the response to GTPgamma S in gallbladder muscle cells from progesterone-treated animals was significantly weaker, with a maximal contraction of only 10.9 ± 1.0% (Fig. 4).


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Fig. 3.   Dose responses of D-myo-inositol 1,4,5-trisphosphate (IP3) of gallbladder muscle cell contraction in progesterone-treated and control animals. Cells were permeabilized with saponin to allow diffusion of IP3 into the cytosol. Values are means ± SE of 3 experiments. There was no significant difference between progesterone-treated and control animals.


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Fig. 4.   Effects of increasing concentrations of [35S]guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S) on permeabilized gallbladder muscle cells from guinea pigs treated with progesterone (n = 3) and control animals (n = 3). The dose-response relationships between the 2 groups were significantly different. P < 0.01, by ANOVA.

These results suggest that contraction involving receptor-G protein activation is affected by progesterone. In contrast, contraction induced by KCl was not affected by progesterone probably because it is G protein independent and mediated by calcium influx. To further support this hypothesis, we tested the contraction evoked by G protein-dependent calcium influx in progesterone-treated gallbladder muscle with another G protein activator, AlF4. Intact muscle cells were stimulated with AlF4 in a calcium-free 4 mM strontium medium. Strontium influx can replace the role of extracellular calcium and cause muscle contraction (16), but it cannot substitute for the functions of stored calcium. Strontium is incorporated in the endoplasmic reticulum but cannot be released, therefore blocking the pathways requiring intracellular calcium release (5, 30, 35). AlF4 was prepared by mixing 20 µM aluminum chloride with 10-7 to 10-3 M sodium fluoride. Unlike GTPgamma S, AlF4 can diffuse through the plasma membranes of intact cells. AlF4 caused a full contraction of gallbladder muscle cells from the control animals in a dose-dependent fashion with a maximal contraction of 20.1 ± 1.0%. In animals treated with progesterone, however, maximal contraction was only 13.3 ± 0.6% (Fig. 5). Similar results were also observed after gallbladder muscle cells from the progesterone-treated guinea pigs were pretreated with 10-6 M thapsigargin. Thapsigargin has been shown to deplete the intracellular calcium stores by releasing calcium and blocking calcium accumulation within IP3-sensitive pools (3, 10). After thapsigargin pretreatment, the maximal contraction induced by AlF4 was significantly reduced in progesterone-treated guinea pigs. In contrast, the maximal contraction caused by KCl was similar to that induced in control guinea pigs (Fig. 6). These data further indicate that G protein-mediated calcium influx is impaired in the gallbladder muscle from the progesterone-treated guinea pigs.


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Fig. 5.   Effect of aluminum fluoride (AlF4) on intact gallbladder muscle cells from progesterone-treated animals (n = 3) and controls (n = 3) in 4 mM strontium medium. P < 0.01, by ANOVA.


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Fig. 6.   Maximal contraction induced by KCl and AlF4 in gallbladder muscle cells from progesterone-treated guinea pigs after 10-6 M thapsigargin pretreatment. Pretreatment with thapsigargin depletes intracellular calcium stores. Maximal contraction induced by 10-4 M AlF4 was significantly less than that induced by 20 mM KCl. Values are means ± SE of 3 experiments. * P < 0.01, by t-test.

The presence of G protein subtypes in gallbladder muscle was determined by immunoblot analysis (Western blot), and their role in mediating CCK-induced muscle contraction was demonstrated by using a panel of specific G protein antibodies. Figure 7 shows the presence of a full complement of G proteins in gallbladder muscle; distinct bands were evident with antibodies to Gialpha 1-2, Gialpha 3, Gq/11alpha , and Gsalpha . The maximal contraction induced by 10-8 M CCK-8 in permeabilized gallbladder muscle cells was significantly inhibited by pretreatment of muscle cells with PTX and Gialpha 3 specific antibody, but not by pretreatment with Gialpha 1-2 or Gq/11alpha specific antibodies (Figs. 8 and 9A). In contrast, in guinea pig ileal circular muscle cells, CCK-induced contraction was selectively blocked by Gq/11alpha specific antibody but was unaffected by PTX and by Gialpha 1-2 or Gialpha 3 antibodies (Figs. 8 and 9B).


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Fig. 7.   Immunoblot analysis of G proteins in guinea pig gallbladder muscle. Solubilized membranes from gallbladder muscle were loaded into an SDS-polyacrylamide gel electrophoresis system, electrophoretically transferred to a nitrocellulose membrane, and then probed with G protein-specific antibodies and horseradish peroxidase-conjugated protein A. G protein bands were identified by enhanced chemiluminescence reagents.


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Fig. 8.   Inhibition of maximal CCK-induced contraction in guinea pig gallbladder muscle and ileal circular muscle cells by pertussis toxin (PTX). Permeabilized muscle cells were preincubated with PTX (1.6 mg/ml) for 1 h. Values are means ± SE of 3 experiments. * P < 0.01, by Student's t-test.


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Fig. 9.   Inhibition of maximal CCK-induced contraction in guinea pig gallbladder muscle (A) and ileal circular muscle (B) cells by antibodies to G protein subunits. Permeabilized muscle cells were preincubated with various G protein-specific antibodies (1:400) for 1 h. Values are means ± SE of 3 experiments. * P < 0.01, by Student's t-test.

To further determine the functional integrity of the G proteins that mediate CCK-induced muscle contraction in progesterone-treated gallbladder muscle, the magnitude of [35S]GTPgamma S binding was measured after CCK stimulation. The time course of binding was first determined in gallbladder muscle membranes from control animals in the presence or absence of 10-6 M CCK-8. These experiments revealed that maximal binding induced by CCK-8 was reached at 10 min. The subsequent experiments were carried out with an incubation time of 10 min. To demonstrate the specificity of the GTP binding to G proteins, displacement curves were performed with unlabeled GTPgamma S and GDP. Both GTPgamma S and GDP dose dependently inhibited [35S]GTPgamma S binding, suggesting that [35S]GTPgamma S binding is specific for G proteins. We then identified the subtypes of G proteins that couple with CCK receptors. We also compared CCK-induced stimulation of the [35S]GTPgamma S binding with different G protein subtypes in progesterone-treated and control (saline-treated) animals using specific G protein antibodies to immunoprecipitate the activated specific G proteins. CCK-8 (10-6 M) caused a significant increase in [35S]GTPgamma S binding to Gialpha 3, but not to Gialpha 1-2 and Gq/11alpha in control animals with a stimulation of 54.7 ± 6.2% over basal level. In progesterone-treated guinea pigs, however, the stimulation of Gialpha 3 binding induced by CCK-8 was significantly reduced with a maximal stimulation of only 27.2 ± 6.2% (Fig. 10).


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Fig. 10.   Stimulation of [35S]GTPgamma S binding with 10-6 M of CCK-8 in gallbladder muscle membranes in progesterone-treated guinea pigs (n = 4) and control animals (n = 4). Data are expressed as %increase of binding from basal level (without CCK-8). * P < 0.05, by Student's t-test.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

CCK is involved in the physiological regulation of gallbladder contraction and emptying of bile. CCK may cause gallbladder contraction by stimulating cholinergic neurons and the smooth muscle (31, 38). Recent studies from our laboratory (39, 40) have shown that CCK contracts the gallbladder muscle via the activation of phospholipase C (PLC), which hydrolyzes phosphatidylinositol 4,5-bisphosphate to IP3 and diacylglycerol (DAG). IP3 releases intracellular calcium, which at low concentrations potentiates DAG to activate protein kinase C. Higher CCK and IP3 concentrations may release larger amounts of calcium that activate calmodulin and myosin light chain kinase.

The present study shows that gallbladder muscle cells from progesterone-treated guinea pigs contract poorly in response to CCK-8 but normally in response to extracellular KCl compared with control animals. These results are in complete agreement with previous findings in muscle strips (27). It has been previously shown that CCK-8 contracts gallbladder smooth muscle by binding to specific receptors that in turn activate G proteins. On the other hand, the contraction evoked by KCl is receptor and G protein independent. KCl depolarizes the plasma membrane and causes calcium influx directly. The finding that KCl-induced contraction of gallbladder muscle cells from the progesterone-treated group is normal suggests that calcium influx through calcium channels is not affected by this hormone.

To localize the defect in the contractile signal transduction pathways, exogenous IP3 and GTPgamma S were introduced into the cells after permeabilizing plasma membranes with saponin. GTPgamma S is a GTP analog that binds to G proteins but cannot be hydrolyzed. GTPgamma S therefore turns the G proteins into a constant "on" state (12, 13, 19). It has been widely used as a G protein stimulator in a variety of cells (12, 13, 19). In progesterone-treated gallbladder muscle cells, the contraction evoked by GTPgamma S was reduced, whereas that induced by IP3 was not affected. These findings suggest that the intracellular calcium stores and the contractile apparatus are functionally intact, whereas either the G proteins and/or the phospholipases and phosphoinositol precursors are affected by progesterone.

G protein activation may cause muscle contraction by directly increasing calcium influx as well as by stimulating second messenger generation (11, 36, 37). Because calcium influx itself appears to be normal in the progesterone-treated group as demonstrated by the effects of KCl, the function of G proteins on calcium influx can be directly assessed if a G protein stimulator is used in conjunction with substances such as strontium or thapsigargin that block or deplete intracellular calcium release. To preserve the process of calcium influx across the plasma membranes, AlF4 was used since it can activate G proteins in intact cells. AlF4 is a compound that can be formed by aluminum chloride and sodium fluoride, has a similar structure of PO<SUP>−3</SUP><SUB>4</SUB>, and is able to interact with GDP on the alpha -subunits in intact cells, resulting in activation of G proteins by mimicking GTP (21, 24, 41). To block intracellular calcium release, we incubated muscle cells in a medium in which calcium was replaced by 4 mM strontium. Strontium is incorporated in the endoplasmic reticulum, displacing calcium from high-affinity binding sites, and is not readily released (30, 35). However, strontium can replace the role of extracellular calcium since the gallbladder muscle contraction induced by KCl is similar in strontium as well as in normal calcium medium (16). Indeed, the L-type calcium channel has a close conductance and permeability to strontium and calcium (29, 32). AlF4 failed to maximally contract the gallbladder muscle cells of progesterone-treated animals in strontium medium, suggesting that the G proteins that mediate gallbladder muscle contraction are likely to be abnormal. This finding was further supported by the results obtained with AlF4 after thapsigargin treatment.

To further examine the hypothesis that G proteins are affected by progesterone, we determined the GTP binding induced by CCK in control and progesterone-treated muscle cells. Activation of G proteins results in the dissociation of GDP from the alpha -subunit and subsequent binding of GTP. Hence, it is possible to assess the function of G proteins by analyzing their GTP-binding properties (15, 23). In the present study, specific [35S]GTPgamma S binding was demonstrated by its inhibition with the nonlabeled GTPgamma S and, more importantly, by GDP. The reduced [35S]GTPgamma S binding induced by CCK-8 in gallbladder muscle membranes from progesterone-treated guinea pigs suggests that the G proteins might be qualitatively or quantitatively abnormal. In the myometrium, progesterone is thought to uncouple alpha -receptors from inhibition of adenylate cyclase (7, 22), and during pregnancy there is also a suppressed G protein coupling to phosphoinositide hydrolysis, which may contribute to the relaxation of the uterus (1).

Most studies have shown that CCK activates Gq/11 proteins. In the circular muscle of the guinea pig ileum, CCK-induced contraction is mediated by activation of PTX-insensitive Gq/11 alpha -subunits that results in stimulation of PLC-beta 1 (18). However, it has also been shown that CCK can activate Gi and Gs proteins (28). Our findings suggest that CCK causes gallbladder muscle contraction by activating PTX-sensitive Gi 3 proteins since it was blocked by PTX and Gialpha 3 specific antibodies. In addition, CCK caused a significant increase of [35S]GTPgamma S binding to Gialpha 3 subunits in gallbladder muscle but not to other G protein subunits. We have also shown that CCK-induced contraction is mediated by the activation of PLC with subsequent generation of IP3 and DAG in gallbladder muscle from cats and humans (39, 40). Further studies need to be performed to determine the PLC isoform activated by Gi 3 proteins.

In conclusion, these findings suggest that defective muscle contraction induced by CCK in progesterone-treated guinea pig gallbladders may be explained by an impairment of G protein function due to downregulation of Gialpha 3 or change in its affinity for GTP binding.

    ACKNOWLEDGEMENTS

This work was supported by National Institutes of Health Grant R01-DK27389.

    FOOTNOTES

Portions of this work were presented at the Annual Meeting of the American Gastroenterological Association in San Diego, CA, in May 1995, and have been previously published in abstract form (Gastroenterology 108: A583, 1995).

Address for reprint requests: J. Behar, Division of Gastroenterology, Ambulatory Patient Center 421, 593 Eddy St., Providence, RI 02903.

Received 7 May 1997; accepted in final form 29 October 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Arkinstall, S. J., and C. T. Jones. Pregnancy suppresses G protein coupling to phosphoinositide hydrolysis in guinea pig myometrium. Am. J. Physiol. 259 (Endocrinol. Metab. 22): E57-E65, 1990[Abstract/Free Full Text].

2.   Barbara, L., C. Sama, A. Maria, M. Labate, F. Taroni, A. G. Rusticali, D. Festi, C. Sapio, E. Roda, C. Banterle, A. Puci, F. Formentini, S. Colasanti, and F. Nardin. A population study on the prevalence of gallstone disease: the Sirmione study. Hepatology 7: 913-917, 1987[Medline].

3.   Bian, J., T. K. Ghosh, J. Wang, and D. L. Gill. Identification of intracellular calcium pools. J. Biol. Chem. 266: 8801-8806, 1991[Abstract/Free Full Text].

4.   Braverman, D. Z., M. L. Johnson, and F. Kern, Jr. Effects of pregnancy and contraceptive steroids on gallbladder function. N. Engl. J. Med. 302: 362-364, 1980[Abstract].

5.   Brown, A. M., and L. Birnbaumer. Direct G protein gating of ion channels. Am. J. Physiol. 254 (Heart Circ. Physiol. 23): H401-H410, 1988[Abstract/Free Full Text].

6.   Chen, Q., S. Oh, P. Yu, Z. L. Xiao, P. Biancani, and J. Behar. Pertussis toxin-sensitive Gi 3 mediates CCK induced contraction in gallbladder smooth muscle (Abstract). Gastroenterology 112: A711, 1997.

7.   Europe-Finner, G. N., S. Phaneuf, S. P. Watson, and B. A. Lopez. Identification and expression of G proteins in human myometrium: up-regulation of Galpha s in pregnancy. Endocrinology 132: 2484-2490, 1993[Abstract].

8.   Everson, G. T. Pregnancy and gallstones. Hepatology 17: 159-161, 1993[Medline].

9.   Everson, G. T., C. McKinley, M. Lawson, M. Johnson, and F. Kern, Jr. Gallbladder function in the human female: effect of the ovulatory cycle, pregnancy and contraceptive steroids. Gastroenterology 82: 711-719, 1982[Medline].

10.   Foskett, J. K., C. M. Roifman, and D. Wong. Activation of calcium oscillations by thapsigargin in parotid acinar cells. J. Biol. Chem. 266: 2778-2782, 1991[Abstract/Free Full Text].

11.   Hamilton, S. L., J. Codina, M. J. Hawkes, A. Yatani, T. Sawada, F. M. Strickland, S. C. Froehner, A. M. Spiegel, L. Toro, E. Stefani, L. Birnbaumer, and A. M. Brown. Evidence for direct interaction of Gsalpha with the Ca2+ channel of skeletal muscle. J. Biol. Chem. 266: 19528-19535, 1991[Abstract/Free Full Text].

12.   Kitazawa, T., S. Kobayashi, K. Horiuti, A. V. Somlyo, and A. P. Somlyo. Receptor-coupled, permeabilized smooth muscle. Role of the phosphatidylinositol cascade, G proteins, and modulation of the contractile response to Ca2+. J. Biol. Chem. 264: 5339-5342, 1989[Abstract/Free Full Text].

13.   Kubota, Y., M. Nomura, K. N. Kamm, M. C. Mumby, and J. T. Stull. GTPgamma S-dependent regulation of smooth muscle contractile elements. Am. J. Physiol. 262 (Cell Physiol. 31): C405-C410, 1992[Abstract/Free Full Text].

14.   Lawson, M., F. Kern, Jr., and G. T. Everson. Gastrointestinal transit time in human pregnancy: prolongation in the second and third trimesters followed by postpartum normalization. Gastroenterology 89: 996-999, 1985[Medline].

15.   Lazareno, S., and N. J. M. Birdsall. Pharmacological characterization of acetylcholine-stimulated [35S]GTPgamma S binding mediated by human muscarinic m1-m4 receptors: antagonist studies. Br. J. Pharmacol. 109: 1120-1127, 1993[Abstract].

16.   Lee, K. Y., P. Biancani, and J. Behar. Calcium sources utilized by cholecystokinin and acetylcholine in the cat gallbladder muscle. Am. J. Physiol. 256 (Gastrointest. Liver Physiol. 19): G785-G788, 1989[Abstract/Free Full Text].

17.   Murthy, K. S., D. H. Coy, and G. M. Maklouf. Somatostatin receptor-mediated signaling in smooth muscle: activation of phospholipase C-beta 3 by Gbeta gamma and inhibition of adenylyl cyclase by Galpha i 1 and Galpha o. J. Biol. Chem. 271: 23458-23463, 1996[Abstract/Free Full Text].

18.   Murthy, K. S., J. R. Grider, and G. M. Makhlouf. InsP3-dependent Ca2+-mobilization in circular but not longitudinal muscle cells of intestine. Am. J. Physiol. 261 (Gastrointest. Liver Physiol. 24): G937-G944, 1991[Abstract/Free Full Text].

19.   Murthy, K. S., J. R. Grider, and G. M. Makhlouf. Receptor-coupled G proteins mediate contraction and Ca2+ mobilization in isolated intestinal smooth muscle cells. J. Pharmacol. Exp. Ther. 260: 90-97, 1992[Abstract].

20.   Murthy, K. S., and G. M. Makhlouf. Adenosine A1 receptor-mediated activation of phospholipase C-beta 3 in intestinal muscle: dual requirement for alpha  and beta gamma subunits of Gi 3. Mol. Pharmacol. 47: 1172-1179, 1995[Abstract].

21.   Nadakavukaren, J. J., D. K. Welsh, and S. M. Reppert. Aluminum fluoride reveals a phosphoinositide system within the suprachiasmatic region of rat hypothalamus. Brain Res. 507: 181-188, 1990[Medline].

22.   Nimmo, A. J., E. M. Whitaker, and J. F. Morrison. Progesterone promotes the coupling of beta  adrenoceptors to G proteins in rat myometrium (Abstract). Biochem. Soc. Trans. 19: 181S, 1991[Medline].

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[Medline].

24.   Ratz, P. H., and P. F. Blackmore. Differential activation of rabbit femoral arteries by aluminum fluoride and sodium fluoride. J. Pharmacol. Exp. Ther. 254: 514-520, 1990[Abstract].

25.   Ryan, J. P. Effect of pregnancy on gallbladder contractility in the guinea pig. Gastroenterology 87: 674-678, 1984[Medline].

26.   Ryan, J. P. Calcium and gallbladder smooth muscle contraction in guinea pig: effect of pregnancy. Gastroenterology 89: 1279-1285, 1985[Medline].

27.   Ryan, J. P., and D. Pellechia. Effect of progesterone pretreatment on guinea pig gallbladder motility in vitro. Gastroenterology 83: 81-83, 1982[Medline].

28.   Schnefel, S., A. Profrock, K. Hinsch, and I. Schulz. Cholecystokinin activates Gi 1-, Gi 2-, Gi 3- and several Gs-proteins in rat pancreatic acinar cells. Biochem. J. 269: 483-488, 1990[Medline].

29.   Shimada, T. Voltage-dependent calcium channel current in isolated gallbladder smooth muscle cells of guinea pig. Am. J. Physiol. 264 (Gastrointest. Liver Physiol. 27): G1066-G1076, 1993[Abstract/Free Full Text].

30.   Somlyo, A. V., and A. P. Somlyo. Strontium accumulation by sarcoplasmic reticulum and mitochondria in vascular smooth muscle. Science 174: 955-958, 1971[Medline].

31.   Takahashi, T., D. May, and C. Owyang. Cholinergic dependence of gallbladder response to cholecystokinin in the guinea pig in vivo. Am. J. Physiol. 261 (Gastrointest. Liver Physiol. 24): G565-G569, 1991[Abstract/Free Full Text].

32.   Tsien, R. W., P. Hess, E. W. McCleskey, and R. L. Rosenberg. Calcium channels: mechanisms of selectivity, permeation and block. Annu. Rev. Biophys. Biophys. Chem. 16: 265-290, 1987[Medline].

33.   Valdivieso, V., C. Covarrubias, F. Siegel, and F. Cruz. Pregnancy and cholelithiasis: pathogenesis and natural course of gallstones diagnosed in early puerperium. Hepatology 17: 1-4, 1993[Medline].

34.   Xu, Q. W., R. B. Scott, and E. A. Shaffer. Adverse effect of female sex hormones on biliary lipid composition in the ground squirrel: different influences on the liver versus gallbladder and intestinal motility (Abstract). Gastroenterology 110: A1362, 1996.

35.   Yasuda, N., and Y. Sakai. A possible explanation for effects of Sr2+ on contraction-relaxation cycle in canine stomach. Comp. Biochem. Physiol. A Physiol. 78: 35-41, 1984.

36.   Yatani, A., J. Codina, Y. Imoto, J. P. Reeves, L. Birnbaumer, and A. M. Brown. A G protein directly regulates mammalian cardiac calcium channels. Science 238: 1288-1292, 1987[Medline].

37.   Yatani, A., Y. Imoto, J. Codina, S. L. Hamilton, A. M. Brown, and L. Birnbaumer. The stimulatory G protein of adenylyl cyclase, Gs, also stimulates dihydropyridine-sensitive Ca2+ channels. J. Biol. Chem. 263: 9887-9895, 1988[Abstract/Free Full Text].

38.   Yau, W. M., G. M. Makhlouf, L. E. Edwards, and T. Farrar. Mode of action of cholecystokinin and related peptides on gallbladder muscle. Gastroenterology 65: 451-456, 1973[Medline].

39.   Yu, P., G. DePetris, P. Biancani, J. Amaral, and J. Behar. Cholecystokinin-coupled intracellular signaling in human gallbladder muscle. Gastroenterology 106: 763-770, 1994[Medline].

40.   Yu, P., K. M. Harnett, P. Biancani, G. DePetris, and J. Behar. Interaction between signal transduction pathways contributing to tonic gallbladder contraction. Am. J. Physiol. 265 (Gastrointest. Liver Physiol. 28): G1082-G1089, 1993[Abstract/Free Full Text].

41.   Zeng, Y. Y., C. G. Benishin, and P. K. T. Pang. Guanine nucleotide binding proteins may modulate gating of calcium channels in vascular smooth muscle. I. Studies with fluoride. J. Pharmacol. Exp. Ther. 250: 343-351, 1989[Abstract].


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