Department of Medicine, Rhode Island Hospital and Brown University School of Medicine, Providence, Rhode Island 02903
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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) (GTPS) 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 GTP
S but was normal in response to KCl and
D-myo-inositol 1,4,5-trisphosphate compared with
nonpregnant animals. The stimulation of [35S]GTP
S
binding to G
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 G
q-11 and an increased
Gs
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
G
q-11 protein, which mediates muscle contraction, and
upregulating Gs
protein, which mediates muscle relaxation.
smooth muscle; G protein quantitation; Gs
protein
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
Gi-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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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)
(GTPS) 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]GTP
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.
Gq-11 and Gs
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 G
q-11 and Gs
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
Gi-1, G
i-2,
G
i-3, G
q-11, and Gs
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]GTP
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), GTP
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
|
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 GTPS 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 GTP
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). GTP
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.
|
To further examine the functional integrity of G proteins in antral and
colonic circular smooth muscle during pregnancy,
[35S]GTPS binding was measured after stimulation with
CCK. It has been shown that, in guinea pig ileum, CCK receptors are
coupled with Gq
protein (17, 18). As shown in Fig.
3, CCK-8 at 1 µM caused a significant increase in
[35S]GTP
S binding to G
q-11 but not to
G
i-2, G
i-3, or Gs
in
either antral or colonic circular smooth muscle membranes from control and pregnant guinea pigs. The GTP
S binding to G
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 G
q-11 protein. In pregnant guinea pigs,
however, the stimulation of G
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.
|
To determine whether the expression of Gq-11 protein is
reduced during pregnancy, immunoblot analysis (Western blot) was performed to quantitate the contents of G
q-11 protein in
antral and colonic circular smooth muscle membranes from pregnant and control animals. Figure 4
shows a linear relationship of G
q-11 band densities to
increasing concentrations of G
q-11 protein standards.
The 42-kDa single band of G
q-11 protein was detected in
antral and colonic circular smooth muscle membranes from both pregnant
and nonpregnant guinea pigs. However, the magnitude of G
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.
|
|
To determine whether this reduced expression was limited to G proteins
that mediate contraction, Gs quantitation was performed in antral and colonic circular smooth muscle membranes from pregnant and control animals. Gs
is involved in pathways that
mediate muscle relaxation. Figure 6 shows a linear
relationship of Gs
band densities to increasing
concentrations of Gs
protein standards. The 45-kDa
distinct bands of Gs
protein were evident in antral and
colonic circular smooth muscle membranes from both pregnant and
nonpregnant guinea pigs. However, the magnitude of Gs
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).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 GTPS into the cells after their plasma membranes were permeabilized with saponin. GTP
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 GTP
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]GTPS binding after CCK-8
stimulation. As mentioned previously, activation of G proteins results
in the dissociation of GDP from the
-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
G
q-11 protein, since it caused a significant increase of
[35S]GTP
S binding to G
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
G
q-11 protein to activate phospholipase C
1 and cause
muscle contraction. Our findings indicate that stimulation of
[35S]GTP
S binding to G
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 G
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
Gq-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 G
q-11
proteins and defective muscle contraction since CCK receptors activate
Gq
protein coupled to phospholipase C
, 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,
Gs
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 G
q-11, G
i-2, and G
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 Gs
expression during pregnancy (8). Our
results also showed that Gs
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
Gq-11 protein and possibly to upregulation of
Gs
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
2.
Baron, T. H.,
B. Ramirez,
and
J. Richter.
Gastrointestinal motility disorders during pregnancy.
Ann. Intern. Med.
118:
366-375,
1993
3.
Bolton, T. B.,
and
J. P. Clark.
Effects of histamine, high potassium and carbachol on 42K efflux from the longitudinal muscle of guinea pig intestine.
J. Physiol. (Lond.)
320:
347-361,
1981[Medline].
4.
Bruce, L. A.,
and
F. M. Behsudi.
Progesterone effects on three regional gastrointestinal tissues.
Life Sci.
25:
729-734,
1979[Medline].
5.
Chen, Q.,
V. Chitinavis,
Z. Xiao,
P. Yu,
S. Oh,
P. Biancani,
and
J. Behar.
Impaired G protein function in gallbladder muscle from progesterone-treated guinea pigs.
Am. J. Physiol.
274 (Gastrointest. Liver Physiol. 37):
G283-G289,
1998
6.
Datta, S.,
V. M. Hey,
and
B. J. Pleuvry.
Effects of pregnancy and associated hormones in mouse intestinal in vivo and in vitro.
Pflüegers Arch.
346:
87-95,
1974[Medline].
7.
Elwardy-Merezak, J.,
J. P. Maltier,
J. Cohen-Tannoudji,
J. L. Lecrivain,
V. Vivat,
and
C. Legrand.
Pregnancy-related modifications of rat myometrial Gs proteins: ADP-ribosylation, immunoreactivity and gene expression studies.
J. Mol. Endocrinol.
13:
23-37,
1994[Abstract].
8.
Europe-Finner, G. N.,
S. Phaneuf,
S. P. Watson,
and
B. A. Lopez.
Identification and expression of G-proteins in human myometrium: upregulation of G alpha s in pregnancy.
Endocrinology
132:
2484-2490,
1993[Abstract].
9.
Fisher, R. S.,
G. S. Robert,
C. J. Grabowski,
and
S. Cohen.
Altered lower esophageal sphincter function during early pregnancy.
Gastroenterology
74:
1233-1237,
1978[Medline].
10.
Joelle, C. T.,
M. Sakina,
E. M. Jamila,
J. L. Lecrivain,
M. T. Robin,
C. Legrand,
and
J. P. Maltier.
Regulation of myometrial Gi2, Gi3, and Gq expression during pregnancy: effect of progesterone and estradiol.
Biol. Reprod.
53:
55-64,
1995[Abstract].
11.
Kiaii, B.,
Q. W. Xu,
and
E. A. Shaffer.
Basis of impaired gallbladder contractility by progesterone in the guinea pig in vitro (Abstract).
Gastroenterology
112:
A1300,
1997.
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
13.
Kubota, Y.,
M. Nomura,
K. N. Kamm,
M. C. Mumby,
and
J. T. Stull.
GTPS-dependent regulation of smooth muscle contractile elements.
Am. J. Physiol.
262 (Cell Physiol. 31):
C405-C410,
1992
14.
Kumar, D.
In vitro inhibitory effect of progesterone on extrauterine human smooth muscle.
Am. J. Obstet. Gynecol.
84:
1300-1304,
1962.
15.
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].
16.
Lazareno, S.,
and
N. J. M. Birdsall.
Pharmacological characterization of acetylcholine-stimulated [35S]GTPS binding mediated by human muscarinic m1-m4 receptors; antagonist studies.
Br. J. Pharmacol.
109:
1120-1127,
1993[Abstract].
17.
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
18.
Murthy, K. S.,
and
G. M. Makhlouf.
Adenosine A1 receptor-mediated activation of phospholipase C-3 in intestinal muscle: dual requirement for
and
subunits of Gi3.
Mol. Pharmacol.
47:
1172-1179,
1995[Abstract].
19.
Okamoto, T.,
T. Ikezu,
Y. Murayama,
E. Ogata,
and
I. Nishimato.
Measurement of GTP gamma S binding to specific G proteins in membranes using G-protein antibodies.
FEBS Lett.
305:
125-128,
1992[Medline].
20.
Ryan, J. P.
Effect of pregnancy on intestinal transit: comparison of results using radioactive and non-radioactive test meals.
Life Sci.
31:
2635-2640,
1982[Medline].
21.
Ryan, J. P.
Effect of pregnancy on gallbladder contractility in the guinea pig.
Gastroenterology
87:
674-678,
1984[Medline].
22.
Ryan, J. P.,
and
A. Bhojwani.
Colonic transit in rats: effect of ovariectomy, sex steroid hormones, and pregnancy.
Am. J. Physiol.
251 (Gastrointest. Liver Physiol. 14):
G46-G50,
1986[Medline].
23.
Ryan, J. P.,
A. Bhojwani,
and
M. B. Wang.
Effect of pregnancy on gastric motility in vivo and vitro in the guinea pig.
Gastroenterology
93:
29-34,
1987[Medline].
24.
Ryan, J. P.,
and
D. Pellechia.
Effect of progesterone pretreatment on guinea pig gallbladder motility in vitro.
Gastroenterology
83:
81-83,
1982[Medline].
25.
Scott, L. D.,
R. Lester,
D. H. Van Thiel,
and
A. Wald.
Pregnancy related changes in small intestinal myoelectric activity in the rat.
Gastroenterology
84:
301-305,
1983[Medline].
26.
Tanfin, Z.,
O. Goureau,
G. Milligan,
and
S. Harbon.
Characterization of G proteins in rat myometrium. A differential modulation of Gi2 and Gi3
during gestation.
Fed. Eur. Biochem. Soc. Lett.
278:
4-8,
1991.
27.
Warsop, H.,
A. Khorija,
D. P. Wichelhaus,
and
C. T. Jones.
Changes in uterine G protein content during pregnancy in the guinea pig.
J. Dev. Physiol. (Eynsham)
19:
91-97,
1993[Medline].
28.
Xiao, Z. L.,
Q. Chen,
P. Biancani,
and
J. Behar.
Mechanisms of pregnancy-induced gallbladder hypomotility in guinea pigs (Abstract).
Gastroenterology
114:
A549,
1998.
29.
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].
30.
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