Downregulation of Galpha q-11 protein expression in guinea pig antral and colonic circular muscle during pregnancy

Qian Chen, Zuo-Liang Xiao, 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 AND METHODS
RESULTS
DISCUSSION
REFERENCES

Pregnancy has an inhibitory effect on motility of the gastrointestinal tract. The present study was designed to examine the mechanisms responsible for antral and colonic hypomotility in pregnant guinea pigs. Circular smooth muscle cells from the antrum and left colon were isolated by enzymatic digestion with collagenase from pregnant and nonpregnant guinea pigs. Contractile responses to agonists were expressed as percent shortening from resting cell length. The function of G proteins in antral and colonic circular smooth muscle was assessed by [35S]guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S) binding induced by CCK-8 and G protein quantitation. The contraction of antral and colonic circular smooth muscle from pregnant guinea pigs was reduced in response to CCK-8 and to GTPgamma S but was normal in response to KCl and D-myo-inositol 1,4,5-trisphosphate compared with nonpregnant animals. The stimulation of [35S]GTPgamma S binding to Galpha q-11 induced by 1 µM CCK-8 was significantly lower in antral and colonic circular smooth muscle from pregnant guinea pigs than that in controls. Furthermore, Western blot analysis showed a decreased Galpha q-11 and an increased Gsalpha protein content in both tissues during pregnancy. It is concluded that pregnancy appears to impair gastrointestinal circular smooth muscle contractility by downregulating G proteins such as Galpha q-11 protein, which mediates muscle contraction, and upregulating Gsalpha protein, which mediates muscle relaxation.

smooth muscle; G protein quantitation; Gsalpha protein


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

HEARTBURN, DYSPEPSIA, AND constipation are common symptoms in pregnant women. It has been shown that these complaints may be in part related to alterations in gastrointestinal motility (2). Lower esophageal sphincter incompetence (9), delayed gastric emptying (23), and slower small bowel and colonic transit (20, 22) have been reported and are believed to contribute to the gastrointestinal symptoms of pregnancy. Although the mechanisms responsible for the impaired motility remain unclear, there is increasing evidence suggesting that pregnancy is associated with disturbances in the myoelectrical and mechanical properties of the gastrointestinal smooth muscle (6, 25). Progesterone, rather than estradiol and corticosteroids, plays a major role in inhibiting contractile activity of the gastrointestinal smooth muscle during pregnancy (14). Previous studies have shown a significant reduction in contractile activity of esophageal, antral, and colonic smooth muscle from male animals pretreated with progesterone compared with nontreated male animals (4). Other studies (5, 21, 24, 28) have also demonstrated that contraction of gallbladder muscle in response to CCK and ACh is impaired in pseudopregnant (progesterone-treated) and pregnant guinea pigs. In contrast, the contraction of progesterone-treated gallbladder muscle induced by KCl was not different from that of controls. These findings suggested that the defective gallbladder muscle contraction caused by progesterone or pregnancy could involve the G protein-dependent pathways. More recent studies have demonstrated that the impairment of CCK-8-induced gallbladder muscle contraction is associated with decreased GTP binding by and downregulation of Galpha i-3 protein (5). Whether similar mechanisms cause defective muscle contraction in other gastrointestinal segments during pregnancy is not known.

The present study was therefore designed to study the effect of pregnancy on antral and colonic circular smooth muscle by examining the signal transduction pathways mediating CCK-induced muscle contraction in pregnant and nonpregnant female guinea pigs.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Adult nonpregnant female guinea pigs and pregnant guinea pigs that were between 50 and 60 days of gestation were purchased from Elm Hill Breeding Laboratory (Chelmsford, MA). Their use was approved by the Animal Welfare Committee of Rhode Island Hospital. Animals were housed in thermoregulated rooms and had free access to food and water. After an overnight fast, the animals were anesthetized with an intramuscular injection of ketamine hydrochloride (30 mg/kg) followed by pentobarbital sodium (30 mg/kg ip). The antrum and left colon were removed, and their lumen 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 tissue was then 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 circular smooth muscle layer was cleaned further by gently removing the remaining connective tissue.

Isolation, permeabilization of single muscle cells. Circular smooth muscle cells were obtained and permeabilized by the methods that have been reported previously (5, 17, 29, 30). Briefly, the circular smooth muscle layer from the antrum and colon was separately cut into pieces and then digested with type II collagenase (specific activity: 168 U/mg) in HEPES buffer for 2.5-3 h at 31°C in a shaking water bath. The cells were collected by filtering through a Nitex mesh and equilibrated at 31°C for 20 min before beginning the experiment. For the permeabilized cells, the cells were washed with cytosolic buffer and exposed briefly to saponin to permeabilize the cells. Cell contraction was measured after agonist stimulation for 30 s and was fixed by acrolein. Thirty consecutive intact cells were measured using a phase contrast microscope (Carl Zeiss, Oberkochen, Germany) and a TV 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; National Institutes of Health (NIH), Bethesda, MD]. Contraction per experiment was expressed as the mean of the percent reduction in cell length (%shortening) with respect to control (i.e., untreated) cells.

Preparation of muscle membranes. Circular smooth muscle squares from the antrum and colon were separately homogenized with a tissue tearer (Biospec, Racine, WI) with 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 (PMSF), 20 mM leupeptin, 20 mg/ml aprotinin, and 1% 3-[(3-chloramidopropyl)dimethylammonio]-l-propanesulfonate. The solubilized membrane suspension was measured for protein content and was ready for the following binding studies.

[35S]guanosine 5'-O-(3-thiotriphosphate) binding. [35S]guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S) binding was assayed by the method of Okamoto et al. (19) as described previously (5). The solubilized 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 CCK-8 (1 µM) 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 µM GTP. The mixtures of 200 µl each were added to ELISA 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 of each well was counted by using a Tri-Carb 1900 CA Liquid Scintillation Analyzer (Packard Instrument, Meriden, CT). Triplicate measurements were carried out for each experiment. Data are expressed as percent increase from basal levels (without stimulation).

Quantitation of G proteins in antral and colonic circular smooth muscle. Galpha q-11 and Gsalpha protein contents of antral and colonic circular smooth muscle were assayed by a G protein quantitation kit (CytoSignal, Irvine, CA). Immunoblot analysis (Western blot) was performed as described previously (5). Briefly, the crude membranes were prepared from antral and colonic circular smooth muscle squares as described above and were solubilized on ice for 30 min in 20 mM HEPES (pH 7.4), 240 mM NaCl, 2 mM EDTA, 2 mM PMSF, 20 µM leupeptin, 20 µg/ml aprotinin, and 1% sodium choleate. The suspension was centrifuged at 13,000 g for 5 min. The supernatant was mixed with SDS sample buffer (50 mM Tris · HCl, 2% SDS, 0.2 M 2-mercaptoethanol, 10% glycerol, and 0.005% bromphenol blue, pH 6.8), boiled for 5 min, and kept on ice for 10 min. Pure Galpha q-11 and Gsalpha protein subunit standards (5, 10, 20, and 40 ng/lane) were prepared in the same manner. After 50 µl/lane of sample or G protein standard were loaded and subjected to a 10% SDS-PAGE (Mini-PROTEAN II cell; Bio-Rad, Hercules, CA), the separated proteins were electrically transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Melville, NY). The nitrocellulose membrane was blocked with 5% nonfat dried milk in PBS [consisting of (in mM) 80 Na2HPO4, pH 7.5, 20 NaH2PO4, and 100 NaCl) containing 0.1% Tween 20 at room temperature for 1 h, followed by incubation with specific G protein subunit antibodies (1:2,000) for 1 h at room temperature. After three washes (10 min each time), the nitrocellulose membrane was incubated for 1 h with horseradish peroxidase-conjugated protein A (1:2,000) at room temperature. The membrane was washed three times (10 min each), and the desired G protein bands were identified with enhanced chemiluminescence reagent (ECL kit, Amersham International) autography. Quantitation of the immunoblots was performed by densitometric scanning of the bands by means of an image analysis system (NIH, version 1.44). G protein subunit standard curves were plotted with standard G protein subunit contents and correlative band densities. The G protein subunit contents in the samples were calculated from the band density by using the standard curve and are expressed as nanograms per milligram membrane protein.

Protein determination. Protein content in muscle membranes was measured according to the method of Bradford using the Bio-Rad protein assay kit (Bio-Rad Laboratories). Values are 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 Galpha i-1, Galpha i-2, Galpha i-3, Galpha q-11, and Gsalpha were obtained from Calbiochem (La Jolla, CA). The ability of these G protein antibodies to block activation of specific effector enzymes has been demonstrated in recent studies (5, 18). The G protein quantitation kit was purchased from CytoSignal. CCK-8 was purchased from Bachem (Torance, CA). [35S]GTPgamma S was purchased from DuPont New England Nuclear (Boston, MA). Horseradish peroxidase-conjugated protein A, ECL kit, and rainbow prestained molecular weight marker were from Amersham (Arlington Heights, IL); D-myo-inositol 1,4,5,-trisphosphate (IP3), GTPgamma S, aprotinin, leupeptin, and other reagents were purchased from Sigma Chemical (St. Louis, MO).

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


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The average resting lengths of intact circular smooth muscle cells isolated from control guinea pig antrum and colon were 91.76 ± 3.1 and 85.6 ± 2.6 µm (mean ± SE, n = 6), respectively. In pregnant guinea pigs, they were 92.8 ± 2.3 and 88.1 ± 2.0 µm (mean ± SE, n = 6), respectively. There were no significant differences in resting cell length between control and pregnant groups.

CCK-8 at concentrations of 0.1 pM to 0.1 µM induced a concentration-dependent contraction of both antral and colonic circular smooth muscle cells from control guinea pigs with a maximal contraction of 22.1 ± 1.1 and 21.7 ± 0.9% shortening at 10 nM, respectively. Contraction in response to CCK-8, however, was significantly less in both antral and colonic circular smooth muscle cells from pregnant animals, with a maximal shortening of only 15.2 ± 0.2 and 14.5 ± 0.7%, respectively (Fig. 1, P < 0.01 by ANOVA). KCl, which is a receptor-G protein-independent agonist, at concentrations of 5-25 mM, also induced a dose-dependent contraction of both antral and colonic circular smooth muscle cells. However, the contractile responses of antrum and colon from pregnant guinea pigs were not significantly different from those in controls (data not shown). These data suggest that receptor-G protein-independent but not receptor-G protein-dependent contractile pathways are intact during pregnancy.


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Fig. 1.   Dose-response studies with CCK-8 in intact antral (A) and colonic (B) circular smooth muscle cells from pregnant and control guinea pigs. Contractile responses to CCK-8 were significantly reduced at all doses in both antrum and colon from pregnant guinea pigs compared with those in the control groups. Values are means ± SE of 3 experiments. *P < 0.01 by ANOVA.

To determine the defective site of the intracellular pathway that mediates CCK-induced muscle contraction, 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 at concentrations of 10 nM to 10 µM caused concentration-dependent contraction of antral circular smooth muscle cells from both pregnant and control animals. The magnitude of contraction caused by IP3 was not different from that induced by CCK-8 in intact cells from control guinea pigs (Fig. 2A). Contraction in response to direct G protein activation with GTPgamma S, however, was significantly less in antral circular smooth muscle cells from pregnant guinea pigs than that in controls (Fig. 2B, P < 0.01 by ANOVA). GTPgamma S at 10 µM caused a maximal shortening of only 11.9 ± 0.8% in antral circular smooth muscle cells from pregnant animals as opposed to 20.1 ± 0.4% shortening in controls. Similar results were observed in colonic smooth muscle cells from pregnant animals (data not shown). These results suggest that, during pregnancy, contraction involving receptor-G protein activation is affected, whereas that induced by IP3, which directly releases Ca2+ from intracellular stores, was not affected.


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Fig. 2.   Dose-response studies with D-myo-inositol 1,4,5-trisphosphate (IP3; A) and guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S; B) in antral circular smooth muscle cells from pregnant and control guinea pigs. Cells were permeabilized with saponin to allow diffusion of IP3 and GTPgamma S into the cytosol. There was no significant difference in cell contraction induced by IP3 between the pregnant and the control animals. However, the contractile responses to GTPgamma S were significantly reduced at all doses in antrum from the pregnant animals compared with the control groups. Values are means ± SE of 3 experiments. *P < 0.01 by ANOVA.

To further examine the functional integrity of G proteins in antral and colonic circular smooth muscle during pregnancy, [35S]GTPgamma S binding was measured after stimulation with CCK. It has been shown that, in guinea pig ileum, CCK receptors are coupled with Gqalpha protein (17, 18). As shown in Fig. 3, CCK-8 at 1 µM caused a significant increase in [35S]GTPgamma S binding to Galpha q-11 but not to Galpha i-2, Galpha i-3, or Gsalpha in either antral or colonic circular smooth muscle membranes from control and pregnant guinea pigs. The GTPgamma S binding to Galpha q-11 increased over basal levels (without CCK-8) by 84.7 ± 6.2 and 78.9 ± 5.7%. These data are in agreement with previous studies in antral and colonic circular smooth muscle membranes showing that CCK receptors are coupled with Galpha q-11 protein. In pregnant guinea pigs, however, the stimulation of Galpha q-11 binding induced by 1 µM CCK-8 was significantly reduced with an increase of only 47.2 ± 4.2 and 50.2 ± 3.2% in pregnant guinea pigs.


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Fig. 3.   Stimulation of [35S]GTPgamma S binding with 1 µM CCK-8 of antral (A) and colonic (B) circular smooth muscle membranes from pregnant and control guinea pigs. CCK-8 caused a significant increase in [35S]GTPgamma S binding to Galpha q-11, but not to Galpha i-1, Galpha i-2 (Galpha i1,2), Galpha i-3, or Gsalpha . Stimulation of GTPgamma S binding to Galpha q-11 induced by CCK-8 was significantly reduced in both antral and colonic circular smooth muscle membranes from pregnant guinea pigs. Data are expressed as percent increase of binding from basal level (without stimulation). Values are means ± SE of 3 experiments. *P < 0.05 by Student t-test.

To determine whether the expression of Galpha q-11 protein is reduced during pregnancy, immunoblot analysis (Western blot) was performed to quantitate the contents of Galpha q-11 protein in antral and colonic circular smooth muscle membranes from pregnant and control animals. Figure 4 shows a linear relationship of Galpha q-11 band densities to increasing concentrations of Galpha q-11 protein standards. The 42-kDa single band of Galpha q-11 protein was detected in antral and colonic circular smooth muscle membranes from both pregnant and nonpregnant guinea pigs. However, the magnitude of Galpha q-11 protein bands was significantly less in both antral and colonic circular smooth muscle membranes from pregnant guinea pigs than in controls (Figs. 4 and 5, P < 0.05 by Student's t-test). These data further support the hypothesis that a reduction in contractile G protein expression contributes to the impaired G protein activation and defective muscle contraction during pregnancy.


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Fig. 4.   Quantitative analysis of Galpha q-11 by immunoblot analysis of antral and colonic circular smooth muscle membranes from control (C) and pregnant (P) guinea pigs. Crude membranes from antral and colonic circular smooth muscle were solubilized with 1% sodium choleate in HEPES buffer. Galpha q-11 protein standards (44.9 kDa) and the solubilized membranes were loaded in an SDS-PAGE system, electrophoretically transferred to a nitrocellulose membrane, and then probed with Galpha q-11 protein specific antibody and horseradish peroxidase-conjugated protein A. The 42-kDa Galpha q-11 bands were detected in both antral and colonic circular smooth muscle membranes by an enhanced chemiluminescence (ECL) kit.


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Fig. 5.   Galpha q-11 protein contents in antral (A) and colonic (B) circular smooth muscle membranes from pregnant and control guinea pigs. The Galpha q-11 protein contents in the samples were calculated from the band optimal densities (OD) of samples according to the standard curve and are expressed as ng/mg membrane protein. Values are expressed as means ± SE of 3 experiments. *P < 0.05 by Student's t-test.

To determine whether this reduced expression was limited to G proteins that mediate contraction, Gsalpha quantitation was performed in antral and colonic circular smooth muscle membranes from pregnant and control animals. Gsalpha is involved in pathways that mediate muscle relaxation. Figure 6 shows a linear relationship of Gsalpha band densities to increasing concentrations of Gsalpha protein standards. The 45-kDa distinct bands of Gsalpha protein were evident in antral and colonic circular smooth muscle membranes from both pregnant and nonpregnant guinea pigs. However, the magnitude of Gsalpha protein bands was significantly higher in both antral and colonic circular smooth muscle membranes from pregnant guinea pigs than in controls (Figs. 6 and 7, P < 0.05 by Student's t-test).


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Fig. 6.   Quantitation of Gsalpha levels by Western blot in antral (A) and colonic (B) circular smooth muscle membranes from control (C) and pregnant (P) guinea pigs. Crude membranes from antral and colonic circular smooth muscle were solubilized with 1% sodium choleate in HEPES buffer. Gsalpha protein standards (48.5 kDa) and the solubilized membranes were loaded into an SDS-PAGE system, electrophoretically transferred to a nitrocellulose membrane, and then probed with Gsalpha protein specific antibody and horseradish peroxidase-conjugated protein A. Mainly 45-kDa Gsalpha bands were detected in both antral and colonic circular smooth muscle membranes by ECL kit.


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Fig. 7.   Gsalpha protein contents in antral (A) and colonic (B) circular smooth muscle membranes from pregnant and control guinea pigs. Gsalpha protein contents in the samples were calculated from the band OD of samples according to the standard curve and are expressed as ng/mg membrane protein. Values are expressed as means ± SE of 3 experiments. *P < 0.05 by Student's t-test.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study shows that both antral and colonic circular smooth muscle cells from pregnant guinea pigs exhibit a defective contraction in response to CCK-8 compared with those from control animals. These results are in complete agreement with previous in vivo and in vitro findings that pregnancy or progesterone decreases gastric emptying and colonic transit (6, 15, 20, 22, 23), suggesting that pregnancy affects the contractility of the smooth muscle throughout the gastrointestinal tract.

It has been shown that CCK-8 contracts antral and colonic smooth muscle by binding to specific receptors that, in turn, activate G proteins (17). In contrast, the contraction evoked by KCl is receptor and G protein independent. KCl contracts the smooth muscle by depolarizing plasma membranes and causing Ca2+ influx (3). In the present study, KCl-induced contraction of antral and colonic circular smooth muscle cells from pregnant animals is similar to that from controls. These data are in agreement with previous findings in gallbladder muscle (5, 11) that Ca2+ influx is not affected during pregnancy or by pretreatment with progesterone.

To define the site of the muscle defect, the signal transduction pathway activated by CCK was investigated by introducing exogenous IP3 and GTPgamma S into the cells after their plasma membranes were permeabilized with saponin. GTPgamma S is a GTP analog that binds to G proteins but can not be hydrolyzed and, therefore, turns G proteins into a constant "on" state. It has been used widely as a G protein stimulator in a variety of cells (12, 13). IP3 is a product of phosphatidylinositol 4,5-bisphosphate hydrolysis and functions as an intracellular second messenger that mediates the actions of CCK (17, 29). IP3 acts on the endoplasmic reticulum, releasing Ca2+ from intracellular stores that activates calmodulin-dependent pathways, leading to muscle contraction. In both antral and colonic circular smooth muscle cells from pregnant guinea pigs, the contraction evoked by GTPgamma S was reduced significantly, whereas that induced by IP3 was not affected when compared with those from controls. These findings suggest that G protein activation is affected during pregnancy, whereas the intracellular Ca2+ stores and the contractile apparatus are functionally intact.

To further examine whether the defective muscle contraction observed in pregnancy was due to impaired G protein activation, their function was studied by determining [35S]GTPgamma S binding after CCK-8 stimulation. As mentioned previously, activation of G proteins results in the dissociation of GDP from the alpha -subunit and subsequent binding of GTP. Therefore, it is possible to assess the function of G proteins by analyzing their GTP-binding properties (5, 16, 19). G proteins were activated by CCK in control and pregnant guinea pigs. CCK causes antral and colonic circular smooth muscle contraction by activating Galpha q-11 protein, since it caused a significant increase of [35S]GTPgamma S binding to Galpha q-11, but not to other G protein subunits. These data are consistent with previous studies (17, 18) showing that CCK receptors are coupled to Galpha q-11 protein to activate phospholipase Cbeta 1 and cause muscle contraction. Our findings indicate that stimulation of [35S]GTPgamma S binding to Galpha q-11 induced by CCK-8 in both antral and colonic circular smooth muscle membranes from pregnant guinea pigs was significantly lower than that in controls. These results further support the hypothesis that the activation of G proteins that mediate muscle contraction such as Galpha q-11 is impaired during pregnancy (28).

Because the impairment of G protein functions could result from qualitative or quantitative abnormalities, G protein measurements were performed using Western blot. There is increasing evidence showing that steroid hormones may alter G protein content in several target tissues (7, 8, 26, 27). These changes in specific G protein expression may be one of the molecular mechanisms by which steroids modulate the efficiency of transmembrane signaling pathways and, consequently, cell responsiveness. Our findings show that the expression of Galpha q-11 proteins was significantly reduced in both antral and colonic circular smooth muscle from pregnant guinea pigs. These results could explain the reduced GTP binding by Galpha q-11 proteins and defective muscle contraction since CCK receptors activate Gqalpha protein coupled to phospholipase Cbeta , leading to muscle contraction in gastrointestinal circular muscle (17, 18). Furthermore, the quantitative reduction of G proteins appears to be selective and confined to those that mediate contraction. In contrast, Gsalpha proteins are upregulated, suggesting that muscle cells may be more sensitive to neurotransmitters that induce muscle relaxation such as vasoactive intestinal peptide. Previous studies in pregnant rats have shown that myometrial Galpha q-11, Galpha i-2, and Galpha i-3 are regulated by progesterone (10). These abnormalities could also explain the reduced phospholipase C activity that has been reported during pregnancy (1). Studies in rat and human myometrium have also shown that there is an increase in Gsalpha expression during pregnancy (8). Our results also showed that Gsalpha protein expression is upregulated in the guinea pig antral and colonic smooth muscle, which may contribute to the hypomotility of the gastrointestinal tract during pregnancy.

In conclusion, both antral and colonic circular smooth muscle from pregnant guinea pigs show an impaired muscle contraction in response to CCK. The muscle defect associated with pregnancy is, at least in part, due to downregulation of G proteins that mediate contraction such as Galpha q-11 protein and possibly to upregulation of Gsalpha proteins, which mediate muscle relaxation.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-27389.


    FOOTNOTES

These data were presented at the Annual Meeting of the American Gastroenterological Association in May 1998, New Orleans, LA, and were published as an abstract (Gastroenterology 114: A733, 1998).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: J. Behar, Div. of Gastroenterology, APC 421, 593 Eddy St., Providence, RI 02903. (E-mail: jose_behar{at}brown.edu).

Received 17 September 1998; accepted in final form 20 December 1998.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Gastroint Liver Physiol 276(4):G895-G900
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