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1 Departments of Obstetrics and Gynecology and 2 Molecular and Medical Pharmacology, University of California at Los Angeles, Los Angeles 90095; and 3 Department of Pathology, Northridge Hospital, Northridge, California 91238
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES
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In women, during pregnancy, there is decreased motility of the gastrointestinal tract leading to a delay in gastric emptying and an increase in colonic transit time. Whether the rise in estradiol (E2) or progesterone (P4) is responsible for this effect is controversial. As the nitrergic component of the nonadrenergic, noncholinergic (NANC) nerves is responsible for modulating gastrointestinal motility in vivo, the purpose of this study was to evaluate whether the increased release of nitric oxide (NO) from the nitrergic component of the NANC nerves innervating the gastric fundus and colon that occurs during late pregnancy in rats is mediated by E2 or P4. Ovariectomized rats treated with E2 or P4 alone or in combination were used for our studies. We also wanted to assess the cellular and molecular mechanisms involved. The NANC activity was studied by assessing changes in tone after application of electric field stimulation (EFS). The role of NO was determined by observing the effects of EFS in the presence and absence of the NO synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME) and the reversibility of the effects of L-NAME by L-arginine. Our studies indicated that there was increased magnitude of relaxation of isolated strips of rat gastric fundus and rat colon after application of EFS to tissues obtained from animals treated with E2 alone or a combination of E2 + P4 but not from those treated with P4 alone. L-NAME attenuated relaxation responses in E2- and E2 + P4-treated animals. To elucidate whether the increased NO release may be due to an increase in neuronal NOS (nNOS) protein, we used both Western blot analysis and immunohistochemistry. We also used RT-PCR to determine whether there was an increase in nNOS mRNA after treatment with sex steroids. In nonpregnant animals, nNOS was detected by Western blot in the fundus and the colon and was barely detectable in the ileum. In pregnancy, there was an increase in nNOS in both the gastric fundus and the colon. The nNOS protein was also increased in ovariectomized animals treated with either E2 alone or E2 + P4 but not P4 alone when compared with ovariectomized animals receiving vehicle. Our results indicated that there was an increase in nNOS protein that was localized to the neurons of the myenteric plexus in the gastric fundus and colon in E2- and E2 + P4-treated animals, but this increase was not observed in animals treated with P4 alone. This increase in nNOS protein was accompanied by an increase in nNOS mRNA. These results suggest the possibility that E2, rather than P4, may be responsible for the delay in gastric emptying and increase in colonic transit time observed in pregnancy.
nitric oxide; estradiol; progesterone; nonadrenergic, noncholinergic nerves
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES
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IN WOMEN, DURING PREGNANCY, there is decreased motility of the stomach (14, 18) leading to a delay in gastric emptying (10) as well as an increase in colonic transit time (12, 22), but the exact mechanism by which this occurs is not known. Release of nitric oxide (NO) by neuronal NO synthase (nNOS) found in nonadrenergic, noncholinergic (NANC) neurons has been shown to be an important factor controlling gastrointestinal motility and transit time (2-6, 17). We have previously demonstrated an increase in the release of NO in the vascular compartment during late pregnancy (19). We subsequently also demonstrated an increased release of NO from the NANC nerves innervating the gastric fundus and proximal colon obtained from rats in late pregnancy compared with those obtained from nonpregnant animals or animals in midpregnancy. On this basis, we suggested that the delay in gastric emptying and increase in colonic transit time observed during late pregnancy might, in part, be due to increased activity of the nitrergic component of the NANC nerves innervating these organs (23).
During pregnancy, there is an increase both in circulating estradiol (E2) as well as progesterone (P4) concentrations (16). Therefore, we decided to assess whether both of these sex steroids could modulate NANC activity of the gastrointestinal tract and the potential mechanisms involved. Ovariectomized rats were treated with either E2 or P4 or a combination of both, after which the responses to electric field stimulation (EFS) of the isolated gastric fundus and colon were assessed. To elucidate whether the increased NO release may be due to an increase in nNOS protein, we used both Western blot analysis and immunohistochemical techniques. Furthermore, RT-PCR was used to determine whether there was an increase in nNOS mRNA after treatment with the sex steroids. The rat was chosen as the animal model as many of the gastrointestinal changes observed during pregnancy are similar to those observed in women (7, 8). The NANC nerves have also been well characterized in this species, and increased release of NO occurs during EFS of strips of gastric fundus and colon obtained from this species during late pregnancy (23).
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MATERIAL AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals
Virgin female and pregnant Sprague-Dawley rats (180-220 g, Harlan, Indianapolis, IN) were housed under conditions of controlled temperature and light cycle and were provided free access to food pellets and water. All animal experiments were performed after ethical approval was obtained from the Animal Research Committee of University of California at Los Angeles. Under anesthesia (pentobarbital sodium, 40 mg/kg ip), a laparotomy was performed, and in animals for certain groups, both the ovaries were removed. The abdominal incision was closed in layers. To assess the effects of E2 and P4, the animals were divided into four groups. After ovariectomy, the animals in group 1 were injected with vehicle alone. Animals in group 2 were ovariectomized and then injected with E2 benzoate dissolved in corn oil at a dose of 100 µg/kg sc for 12 days. Animals in group 3 were ovariectomized and then injected with corn oil alone subcutaneously for 6 days followed by subcutaneous injection of P4 dissolved in corn oil (15 mg/kg) for the following 6 days. After ovariectomy, animals in group 4 received subcutaneous injection of E2 benzoate dissolved in corn oil at a concentration of 100 µg/kg for 12 days, and P4 at a concentration of 15 mg/kg was administered subcutaneously to the animals simultaneously with E2 benzoate for the last 6 days of E2 therapy. The concentrations of E2 and P4 achieved using this protocol in preliminary experiments were 40 ± 7 pg/ml and 190 ± 9 ng/ml, respectively. These values correspond to those observed in rats during late pregnancy (16). These doses of E2 and P4 have also previously been shown to produce a delay in gastric emptying similar to that found in pregnancy (8). The day after the administration of the last dose of steroid, animals were euthanized by intramuscular injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) followed by exsanguination. The abdomen was opened, and the fundus of the stomach, a segment of the ileum, and a segment of the ascending colon just proximal to the cecum were removed.For studies on NANC activity, tissues were placed in Krebs bicarbonate
solution (composition in mM: 118.0 NaCl, 4.7 KCl, 25.0 NaHCO3, 1.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, and 11.5 glucose) gassed with a mixture
of 95% O2 and 5% CO2. For studies on NOS,
i.e., Western blotting and RT-PCR for nNOS, the tissues were frozen in
liquid nitrogen and stored at 
80°C. For studies on
immunohistochemistry for nNOS, tissues were fixed in 10% formalin in PBS.
Chemicals
Acetylcholine chloride, phentolamine, propranolol, atropine, 5-hydroxytryptamine (5-HT), N
-nitro-L-arginine methyl
ester (L-NAME), triethanolamine hydrochloride (TEA-HCl),
L-arginine-HCl, pepstatin, leupeptin, dithiothreitol, sodium acetate, Ponceau S solution, E2 benzoate,
progesterone, and tetrodotoxin were obtained from Sigma Chemical (St.
Louis, MO). Diethylammonium
(Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA-NO) was obtained from Cayman Chemicals (Ann Arbor, MI).
The antibody for nNOS (mouse monoclonal and polyclonal) were obtained from Transduction Laboratories (Louisville, KY). This antibody does not
cross-react with either endothelial NOS or macrophage NOS.
Hybond-polyvinylidene difluoride (PVDF) membrane, horseradish peroxidase-linked secondary antibody, and enhanced chemiluminescence (ECL) Western blotting kit were purchased from Amersham (Arlington Heights, IL). Tri-Reagent was purchased from Molecular Research Center (Cincinnati, OH). Gene AmpRNA PCR kit was obtained from Perkin-Elmer (Norwalk, CT). The primers used for RT-PCR (nNOS, gene
accession number x59949) were obtained from Custom Primers (GIBCO-BRL,
Gaithersburg, MD).
Studies After Application of EFS to Strips of Fundus, Ileum, and Colon
Studies with fundus. For preparation of the rat fundal strips (25), the fundal portion of the stomach was dissected free, cut along the antimesenteric surface, and rinsed of any residual contents with Krebs buffer. Longitudinal strips (5 × 10 mm) were cut and placed in a dish of clean Krebs. One side of a Tungsten wire triangle was woven through either narrow end of the tissue, and the tissues were suspended in a 25-ml, water-jacketed (37°C) organ bath containing Krebs bicarbonate solution gassed continuously with 95% O2 and 5% CO2. The tissues were gradually stretched to a resting tension of 1 g (recorded with a Grass FTO3 force-displacement transducer attached either to a Soltec chart or recorder) and allowed to equilibrate for 30 min. The appropriate resting tension for fundal strips and segments of colon were determined in initial experiments as described by us previously (23). Strips were placed under progressive increments of tension, and contractile responses to potassium chloride (120 mM in Krebs bicarbonate solution) were measured under the various resting tension conditions. Optimal length-tension relationships were achieved with resting tensions of 1 g for the fundal strips. Therefore, a resting tension of 1 g was applied to the tissues, and changes in tension were recorded with a Grass FTO3 force-displacement transducer attached to a Soltec chart recorder. Tissues were incubated with atropine (10 µM), phentolamine (10 µM), and propranolol (10 µM) for 30 min to ensure blockade of adrenergic and cholinergic receptors. To observe relaxations, tone was raised 60-80% of maximal contraction produced by 120 mM KCl by addition of 5-HT to a final concentration of ~30 µM. After maintenance of active tone for 30 min, EFS was performed with two parallel platinum electrodes connected to a current amplifier and a stimulator (Grass Instruments, SD9) at parameters of 0.5-ms pulse width and supramaximal voltage (40 V) during each 10-s train of stimulation throughout the experiment. On the basis of preliminary experiments, these parameters were selected after construction of frequency-response curves and at various voltages and then observing the absence of relaxation at the parameters used when EFS was applied in the presence of tetrodotoxin (1 µM), which blocks nerve conductance by blocking the Na+ channel. Stimulation was delivered at varying frequencies (1, 2, 5, 10, 20, and 40 Hz), and the resulting changes in response were measured. The same protocol was repeated after the addition of either L-NAME (100 µM) alone or in combination with L-arginine (1 mM).
Studies with colon. The colon was rinsed intralumenally with Krebs bicarbonate solution to remove any remaining contents. One side of a Tungsten triangle was inserted through the antimesenteric side of the colon at either end of the longitudinal segment and then suspended in a 25-ml, water-jacketed organ bath. The tissue was gradually stretched to a resting tension of 2 g recorded by a force-displacement transducer (as above) and allowed to equilibrate for 30 min. Tissues were incubated with atropine (10 µM), phentolamine (10 µM), and propranolol (10 µM) for 30 min to block the adrenergic and cholinergic receptors. Two parallel platinum wire electrodes were positioned on either side of the tissue, and EFS was applied using the same parameters as stated above. It was difficult to maintain a consistent active tone of segments of colon with any available pharmacological agents except carbachol. In these tissues, application of EFS resulted in a quiescence of the intrinsic activity of the tissue. EFS was applied at varying frequencies (1, 5, 10, 15, 30, and 45 Hz), and the responses were measured as a function of the length of time of quiescence (23). This protocol was repeated in a similar manner after preincubation of the tissues with either L-NAME (100 µM) alone or in combination with L- or D-arginine (1 mM). The sensitivity to NO of the fundus and colon was assessed using the NO donor DEA-NO as has been previously described (23).
Western Blot Analysis for nNOS
Fundal, ileal, and colonic tissues were excised from virgin female rats and from rats from various groups of animals, i.e., ovariectomized rats, animals in late pregnancy (18-20 days), and/or ovariectomized rats treated with E2 alone, P4 alone, or with a combination of E2 and P4.Fundal, ileal, and colonic tissues were excised from female rats. The
tissues obtained from four animals in each of the various groups were
homogenized in buffer A [20% (wt/vol); 50 mM TEA, pH 7.4, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM 1,4-dithiothreitol, 1.0 µM pepstatin
A, 2.0 µM leupeptin, and 1.0 mM phenylmethylsulfonyl fluoride] on
ice and then centrifuged at 20,000 g at 4°C for 1 h,
and the supernatant of each sample was removed and analyzed for protein
content using the Bio-Rad protein assay kit. The remaining samples were
then diluted with Laemmli's loading buffer (50 mM Tris · HCl,
pH 6.8, 14.3 mM 
-mercaptoethanol, 2% SDS, 0.1% bromophenol blue)
and boiled for 3 min. High molecular weight standard positive controls
(150 µg) and a volume of sample containing 150 µg of protein were
electrophoresed onto a 7.5% SDS-polyacrylamide gel and transferred to
PVDF membrane using a semidry transfer apparatus. This concentration of
protein was selected based on preliminary experiments wherein we loaded
our positive controls and samples at different protein concentrations
for fixed exposure times. This protein concentration gave a linear
relationship between protein mass and optical densities. The PVDF
membrane was incubated in blocking buffer [5% nonfat dry milk in
PBS-Tween 20, 0.1% (vol/vol)] overnight at 4°C. The membrane was
then washed three times for 10 min each in PBS-Tween 20 and incubated
at room temperature with diluted (1:2,500) monoclonal nNOS antibody for
60 min. The membrane was washed again in PBS-Tween 20 for three times,
10 min each, and then incubated in diluted (1:1,000) secondary antibody for 60 min at room temperature. The membrane was washed three times for
15 min each in PBS-Tween 20. The presence of bound antibody on the
membrane was determined using ECL-Western blot detection kit by
exposing the membrane to autoradiography film (Hyperfilm-ECL, Amersham). The relative intensities were quantified by densitometric analysis.
RT-PCR
Tissues were harvested from animals in various treatment groups and frozen in liquid nitrogen. Total RNA (pooled from 4 animals in each of the treatment groups) from the frozen tissues was extracted using Tri-Reagent as described by the manufacturer's protocol (Molecular Research Center). Reverse transcription was performed with 4 µg of total RNA, 50 units of Moloney murine leukemia RT, and 100 pmol of oligo(dT) and running the reaction at 42°C for 20 min as recommended by the manufacturer's protocol of the RT-PCR kit (Gene Amp RNA-PCR core kit from Perkin-Elmer). The resulting cDNA samples were PCR-amplified using the Gene Amp RNA-PCR kit and 100 ng each of sense and antisense primers. Five-hundred nanograms of RNA were used for GAPDH amplification. GAPDH was used as the housekeeping gene for comparison as this is not regulated by either E2 or P4 (20). The following amplification cycle was used: initial denaturation at 94°C for 1 min, primer annealing at 60°C for 1 min, and extension at 72°C for 1.5 min for a total of 35 cycles. The following sets of primers were used: nNOS sense, 5'-GAATACCAGCCTGATCCATGGAA-3'; antisense, 5'-TCCTCCAGGAGGGTGTCCACCGCATC-3'; GAPDH sense, 5'-GTGAAGGTCGGTGTCAACGGATTT-3'; antisense, 5'-CACAGTCTTCTGAGTGGCAGTGAT-3' (24). The resulting PCR products were analyzed on a 1% agarose gel using appropriate molecular weight markers to verify the required size of the final PCR product. The identity of PCR products was confirmed by DNA sequencing (data not shown). The relative intensities of the bands were quantified by densitometric analysis (Personal Densitometer SI, Molecular Dynamics). The nNOS band was normalized to the GAPDH band, and the values were expressed as percent change from the control group (ovariectomized and vehicle only).Immunohistochemistry
After animals were euthanized, tissue from the various groups was collected and fixed in 10% formalin in PBS. Whole tissues were embedded in paraffin, and sections of 3-5 µm were cut and mounted on slides coated with poly-L-lysine. Slides were dried at 70°C for 10-15 min and rehydrated in distilled water. They were then placed in citrate buffer (10 mM, pH 6.0) and heated in an 800-W microwave oven at high power for 5 min (×2), allowed to cool for at least 20 min in citrate buffer, and then washed in distilled water. Slides were placed in 1% H2O2/methanol for 20 min and washed in running tap water for 2-3 min and then placed in PBS (pH 7.4). Either polyclonal nNOS antibody (dilution 1:20) was placed in normal goat serum for 20 min or monoclonal nNOS antibody (dilution 1:1,600) was placed in normal horse serum for 20 min. The horse or goat serum was decanted, and sections were covered in primary antibody, diluted in PBS, and incubated overnight at room temperature in a humidity chamber then washed with PBS. For polyclonal and monoclonal nNOS, slides were incubated in biotinylated anti-rabbit IgG for 30 min and biotinylated anti-mouse IgG for 45 min, respectively. The slides were incubated with antibody conjugated for 45 min followed by avidin-biotin complex for 30 min. The slides then were incubated in diaminobenzidine for 2-3 min, washed in running tap water for 2 min, stained with hematoxylin, washed in tap water, dehydrated, and mounted on slides as described previously (24).In each section, 10 fields were counted for negative, weakly positive, and strongly positive by an individual blinded to the treatment groups, and the mean obtained from at least three animals in each group was taken and displayed as percent positive neuronal cells.
Data Analysis
Values are expressed as mean ± SE obtained from five to seven animals in each group except in experiments involving Western blotting and RT-PCR, in which four animals from each group were pooled for each experiment, and three such experiments were performed. For studies involving immunohistochemistry, three animals were used in each group. Values between groups were compared using two-way analysis of variance with Duncan's t-range or Student's t-test where appropriate.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Study 1
Studies following application of EFS to strips of fundus and colon.
studies with fundal strip.
Application of EFS resulted in a transient decrease in tone, and the
magnitude of relaxation was frequency dependent. The relaxant response
started at the time of application of EFS. The time taken for the tone
to return to baseline correlated directly with the intensity and
frequency of the stimulus. Figure 1 shows representative traces obtained following application of EFS to a fundal
strip obtained from an ovariectomized animal and an animal treated with
both E2 and P4. Relaxant responses in tissues
obtained from animals treated with E2 + P4
or E2 alone were of a similar magnitude and were
significantly greater than those obtained from ovariectomized
animals. The relaxant responses in
tissues obtained from P4 (alone)-treated animals were not
significantly different from those obtained from ovariectomized animals
(Fig. 2). The role of NO was assessed by observing the magnitude of
relaxation after application of EFS in the absence and presence of
L-NAME alone (after incubation of tissue with
L-NAME for at least 15 min) or in the presence of
L-NAME and either L- or D-arginine. In the presence of L-NAME, there was attenuation of the
relaxant responses, and this was significantly greater at lower
frequencies in tissues obtained from animals treated with
E2 alone (data not shown) or E2 and
P4 (Fig. 3), compared with
ovariectomized controls. There was no significant difference observed
in tissues obtained from P4-treated animals compared with
controls (data not shown). L- but not
D-arginine significantly reversed the effects of
L-NAME (data not shown). Pretreatment with tetrodotoxin to
the tissue bath for 30 min abolished all responses to EFS (data not
shown).
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Study 2
Studies assessing magnitude of relaxant responses of strips of
fundus and colon to DEA-NO, an NO donor.
In these experiments, the strips of fundus and colon were set up in
isolated organ baths, and phentolamine (10 µM), propronolol (10 µM), and L-NAME were added to the oxygenated Krebs
bicarbonate solution. Active tone was induced by carbachol (10 µM).
Concentration-response curves were generated using DEA-NO
(10
8 to 104 M). The EC50 values
for the fundal strips and segment of colon obtained from
non-hormone-treated animals were 60 ± 15 and 45 ± 8 µM,
respectively. The corresponding EC50 values for the fundal strip and colon obtained from E2-treated animals were
68 ± 11 and 53 ± 9 µM, respectively. Comparison of the
EC50 values to DEA-NO showed no significant difference
between tissues obtained from E2- and vehicle-treated animals.
Study 3
Effect of pregnancy on nNOS protein in the
gastrointestinal tract or treatment of ovariectomized animals with
E2 alone, P4
alone, or E2 + P4
on nNOS protein in rat colon using Western blot
analysis.
The Western blot analyses of nNOS of the gastric fundus, ileum, and
colon from nonpregnant and pregnant animals are shown in Fig.
6. In nonpregnant animals, nNOS was
detected in the fundus and colon and was barely detectable in the
ileum. In pregnancy, there was an increase of nNOS both in gastric
fundus and in the colon. Western blot analyses of nNOS of colonic
tissues from the various groups of animals are shown in Fig.
7. Densitometric analysis showed that
tissues from ovariectomized animals had significantly lower nNOS
protein compared with those obtained from late-pregnant animals. There
was also a greater increase in nNOS immunoreactivity in tissues
obtained from E2- and E2 + P4-treated animals, whereas there was no change in nNOS in
tissues obtained from animals treated with P4 alone
compared with those obtained from ovariectomized animals. Results from
fundal tissue showed a similar trend (data not shown).
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Study 4
Effect of pregnancy or treatment of ovariectomized animals with
E2 + P4 or
E2 or P4 alone on
immunohistochemical localization of nNOS in rat fundus
and colon.
nNOS-positive staining was observed in the myenteric plexus in
the fundus and colon. This immunostaining was localized to the neurons
and nerve fibers from these cells that formed a network in the circular
and longitudinal smooth muscle layer. The surrounding Schwannian cells
were not stained. Comparison of the fundus (Fig. 8) as well as the colon (Fig.
9) demonstrated an increase in the intensity of immunoreactivity for nNOS as well as an increase in the
number of nNOS-positive cells in the myenteric plexus in animals in
late pregnancy and those treated with either E2 (Figs. 8
and 9) or E2 + P4 but not in animals
treated with P4 alone (data not shown) when compared with
nonpregnant ovariectomized animals. The percentage of cells in the
myenteric plexus that were negative, weakly positive, or strongly
positive for nNOS in the rat colon from the various groups is indicated
in Fig. 10. The results in the fundus
showed a similar trend to that observed in the colon (data not shown).
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Study 5
Analysis of nNOS gene expression in the rat colon
after exposure to sex steroids.
In these studies, we assessed whether the increase in nNOS mRNA was
responsible for the increase in nNOS protein during late pregnancy in
the colon and whether this increase was modulated by E2
and/or P4 or both. Because levels of total RNA obtained were not sufficient to perform Northern blot analysis, RT-PCR was
employed. Results indicated that there was an increase in nNOS mRNA in
tissues obtained from animals in late pregnancy (42 ± 8%) and
those treated with either E2 alone (35 ± 7%) or
E2 + P4 (51 ± 12%), but not
P4 alone (decrease of 12 ± 8%), when compared with
ovariectomized animals treated with vehicle alone (Fig.
11).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The precise mechanism(s) responsible for the changes in gastrointestinal motility that occur during pregnancy is not known. It appears that sex steroids play an important role in modulating these effects as postmenopausal women being treated with sex hormone-replacement therapy had a decreased rate of gastric emptying of solids compared with men. In contrast, postmenopausal women without hormone replacement had rates of solid emptying similar to those of men (13). Similarly, rats in diestrus had slower gastric emptying than ovariectomized rats (7).
The precise sex steroid that is responsible for these changes is controversial. It has been suggested that P4 may be responsible for this effect, because high concentrations of P4, when added to the tissue bath, could completely inhibit the spontaneous contractile activity of colonic smooth muscles in vitro (12, 15, 21, 22). This inhibitory effect appeared to be specific for P4, because E2 and corticosterone, when added to the tissue bath, were without effect. However, the concentration of P4 required to produce this effect (µM range) is much higher than the circulating concentration of P4 (nM range) observed during pregnancy. On the other hand, other investigators have reported that parenteral administration of E2 and P4 simultaneously or E2 alone to rats inhibited gastric emptying, whereas P4 alone enhanced it (7). In another study, parenteral administration of relatively low doses of E2 to male rats significantly delayed gastric emptying, whereas only very high doses of P4 were required to produce this effect (8). It therefore appears that the effects of P4 in inhibiting colonic motility in vitro were only observed when P4 was added to the tissue bath in supraphysiological concentrations, whereas E2 and not P4 inhibited gastrointestinal motility when administered systemically.
Irrespective of whether E2 or P4 is responsible for this effect, the mechanism by which they cause slowing of gastrointestinal motility is not known. The release of NO by NANC nerves has been shown to be an important factor controlling gut motility in vitro (2-5, 17) as well as in vivo (6), and this release is increased during pregnancy (23). The primary objective of this study, therefore, was to assess whether E2 and/or P4 during pregnancy was responsible for the increased activity of the nitrergic component of the NANC nerves innervating the gastrointestinal tract. We also wanted to evaluate the cellular and molecular mechanisms that may be involved. The rat gastric fundus and rat colon were chosen for most of the studies as our previous studies indicated that there was an increase in the release of NO from the nitrergic component of the NANC nerves innervating the gastric fundus and colon of pregnant rats but not that of the ileum (23).
Our results indicate that after EFS, there was increased magnitude of relaxation of isolated strips of gastric fundus and colon of rats treated with either E2 alone or E2 and P4 and was of a similar magnitude but not from those treated with P4 alone. The relaxation after application of EFS was due to stimulation of the NANC nerves as the effects were observed in the presence of adrenergic and cholinergic receptor blockers, and the responses were abolished in the presence of tetrodotoxin. Similar to our previous studies (23) and those reported by others (2, 3, 5), we observed that application of EFS at low frequencies (1-20 Hz) to precontracted strips of gastric fundus relaxed the strips only during the period of transmural stimulation, and the tone returned to initial levels immediately after cessation of EFS, indicating the release of a mediator with a very short half-life such as NO. In contrast, EFS at higher frequencies (20 Hz and greater) induced relaxation that persisted for some time even after cessation of the stimulus. The relaxant responses at higher frequencies, because of the slow recovery, suggest that in addition to NO, another long-acting mediator such as vasoactive intestinal peptide (VIP) (3) may also be involved. L-NAME, an inhibitor of NO synthesis, decreased the magnitude of relaxation, and this decrease was greater in tissues obtained from animals treated with either E2 alone or E2 and P4 compared with control or P4-treated animals. As there was no difference in the sensitivity of the gastric fundus obtained from the different groups to DEA-NO, one can conclude that the effects observed in rats treated with either E2 or a combination of E2 and P4 were most likely due to increased synthesis and release of NO from the NANC nerves.
Similarly, in studies with the proximal colon, the magnitude of relaxant effects after EFS and its inhibition after L-NAME was significantly greater in tissues obtained from E2- or E2 and P4-treated animals but not those obtained from P4 (only)-treated animals compared with controls. These studies, therefore, indicate that E2 rather than P4 was responsible for the increase in nitrergic activity.
We next assessed the cellular and molecular mechanisms by which E2 increased the nitrergic activity similar to that observed in pregnancy. Our results indicated that the nNOS protein, as assessed by Western blot analysis, was increased in the gastric fundus and colon but not in the ileum in late pregnancy compared with nonpregnant controls. This finding correlated well with our previous study (23) in which we observed an increase in nitrergic activity during late pregnancy only in the gastric fundus and colon but not in the ileum. We also observed an increase in nNOS protein in the fundus and colon of animals treated with either E2 alone or a combination of both E2 and P4 but not in animals treated with P4 alone when compared with nonpregnant controls. This again correlated very well with the finding of other investigators (26, 27), who showed an increase of nNOS with E2, and with our studies showing increased nitrergic activity in these groups of animals.
The increase in nNOS protein was mainly localized to the neurons in the myenteric plexus and the nerve axons coursing in parallel with the inner circular muscle. There were more nNOS-positive neurons in the colon compared with the fundus, and similar regional differences have also been reported by other investigators (1, 9, 11). Analysis by immunohistochemistry revealed that parallel with changes in the intensity of nNOS staining, the number of detectable nNOS-positive cells was also increased during late pregnancy and after treatment with a combination of E2 and P4 or E2 alone but not with P4 alone. These data suggest that in addition to the possibility that E2 stimulates individual neurons to produce more nNOS, they are consistent with the interpretation that E2 alone, or a combination of E2 and P4, causes the recruitment of additional neurons to the active nNOS pool.
This increase in nNOS protein may have been due to increased gene expression or to an increase in mRNA stability mediated by E2. The increase in nNOS protein by E2 may also have been due to increased translation or posttranslational stability, although these aspects were not assessed in this study. Our results are similar to those of other investigators who also observed an increase in the expression of nNOS in the gastrointestinal tract during pregnancy and after E2 (26, 27). There is no estrogen-responsive element in the promoter region for nNOS gene and the mechanism by which E2 increased nNOS gene expression or the type of estrogen receptor involved was not assessed in this study, and this is the subject for future studies.
Although our studies specifically focused on the role of E2 and P4 in modulating the nitrergic component of the NANC nerves, we have not ruled out the possibility that increased release of other inhibitory mediators like VIP from the NANC nerves may also modulate gastrointestinal activity during pregnancy. Our studies, therefore, support the concept that increased nitrergic activity of the NANC nerves may, at least in part, be responsible for the decrease in motility of the gastric fundus and colon during pregnancy. Our studies also suggest the possibility that this effect is most likely mediated by E2 and not by P4. Further studies are needed to assess whether physiological concentrations of P4 also cause a decrease in gastrointestinal motility by other mechanisms.
Perspectives
Increased release of NO from NANC nerves innervating the gastric fundus is modulated by E2. This may be a potential explanation for many of the gastrointestinal changes such as esophageal reflux, delayed gastric emptying, and constipation experienced by women during pregnancy.| |
ACKNOWLEDGEMENTS |
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This work was supported in part by National Institutes of Health Grants HD-35991 and HL-46843.
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FOOTNOTES |
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Address for reprint requests and other correspondence: G. Chaudhuri, Dept. of Obstetrics and Gynecology, UCLA School of Medicine, 10833 Le Conte, Los Angeles, CA 90095-1740.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 30 May 2000; accepted in final form 8 January 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
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