BIOLOGY OF REPRODUCTION 61, 8–13 (1999)
Endogenous Nitric Oxide Suppresses Rat Myometrial Connexin 43 Gap Junction
Protein Expression during Pregnancy 1
Stephen M. Sladek, Andrea Westerhausen-Larson, and James M. Roberts2
Magee-Womens Research Institute, and the Department of Obstetrics, Gynecology, and Reproductive Sciences,
University of Pittsburgh, Pittsburgh, Pennsylvania 15213
ABSTRACT
Nitric oxide (NO) synthase (NOS) is active in the gravid uter-
us, and its activity decreases prior to the onset of parturition.
We tested the hypothesis that NO helps maintain uterine qui-
escence by suppressing the expression of genes necessary for
parturition. Pregnant rats (18 days gestation) were treated with
inducible NOS (iNOS) inhibitor N-iminoethyl-L-lysine (NIL) or
endothelial NOS inhibitor nitro-L-arginine methyl ester (L-
NAME); 24 h later, uteri were analyzed for myometrial connexin
43 (Cx43) protein by immunoblotting and mRNA by Northern
analysis. Myometrial oxytocin receptors (OTR) were measured
by radioligand binding, and decidual prostaglandin H synthase
(PGHS) protein by immunoblotting. Uterine NOS blockade was
verified by NOS activity assay. We found that NIL, but not L-
NAME, significantly increased myometrial Cx43 protein to par-
turitional levels with treatment at 19 but not 17 days gestation.
Steady state mRNA concentrations were not changed at 24 h.
NOS inhibition did not increase the concentrations of OTR, or
PGHS protein, nor did it decrease maternal serum progesterone.
We conclude that endogenous uterine NO from iNOS suppress-
es myometrial Cx43 gap junction protein expression during rat
pregnancy. Although the exact mechanism is unknown, an in-
crease of uterine wall stretch due to inhibition of relaxation
could account for increased Cx43 gene transcription.
INTRODUCTION
The functions of nitric oxide (NO) produced in the uter-
us during pregnancy have not yet been determined defini-
tively. Because nitric oxide synthase (NOS) is active in the
gravid uterus and its activity decreases at the end of preg-
nancy, the most attractive hypothesis has been that NO
maintains uterine quiescence during pregnancy prior to
term because of its ability to relax smooth muscle [1]. How-
ever, there is no evidence yet that endogenous NO sup-
presses uterine contractions [2–4]. NOS blockade in preg-
nant rats does not induce preterm delivery [5] except when
administered along with anti-progestins [6], which them-
selves are capable of inducing preterm delivery. In addition,
most studies have used nitro-L-arginine methyl ester (L-
NAME), an inhibitor of NOS most potent against vascular
endothelial NOS (eNOS) but also an inhibitor of inducible
NOS (iNOS) and neuronal NOS activity [7–9]. Thus the
results may be nonspecific effects resulting from vasocon-
striction, fetal compromise [10], or endocrine changes [11]
rather than a direct effect of uterine NOS blockade.
Recently we localized iNOS and eNOS to rat uterine
natural killer cells and arterioles of the metrial gland, sug-
gesting immunological and/or vascular functions for uterine
Accepted February 2, 1999.
Received July 21, 1998.
1
This work was supported by NIH Grant (HD-01095) and an NICHD
Clinical Investigator Development Award to S.M.S.
2
Correspondence: James M. Roberts, Magee-Womens Research Insti-
tute, Room 610, 204 Craft Avenue, Pittsburgh, PA 15213-3180. FAX: 412
641 1503; e-mail: jimrob1@pitt.edu
8
NO during pregnancy [12]. Myometrial cells stained posi-
tively for iNOS only if located adjacent to the placental
attachment site. Although it has been suggested that neu-
ronal NOS may mediate relaxation of nonpregnant rat myo-
metrium [13], immunostaining of neuronal NOS (nNOS)
was not seen during pregnancy [12].
Careful studies of the timing of the decrease of uterine
NOS activity in rats [14] and humans [15] show that the
decline occurs days to weeks before the onset of labor. Dur-
ing this time the uterus is undergoing important prepara-
tions for parturition such as the increased expression of
parturitional proteins: connexin 43 (Cx43) gap junctions,
oxytocin receptors (OTR), and prostaglandin H synthase
(PGHS) [16]. These gene products have invariably been
found in the uterus during labor, whether term or preterm.
In animals, Cx43 and OTR expression are very sensitive to
progesterone withdrawal [16].
Because NO is itself a pluripotent inhibitor of gene ex-
pression [17], we investigated the effect of endogenous NO
on the expression of these parturitional genes. The experi-
ments reported here were designed to test the hypothesis
that NO helps maintains uterine quiescence during preg-
nancy by suppressing the expression of myometrial Cx43
gap junction protein, OTR, and PGHS. In preliminary ex-
periments with pregnant rabbits, the semiselective iNOS in-
hibitor aminoguanidine, but not L-NAME, resulted in the
premature expression of myometrial Cx43 [18]. There was
no premature expression of OTR or increased PGHS en-
zyme activity [19]. Both aminoguanidine and L-NAME in-
hibited rabbit decidual NOS activity ex vivo; hence the NO
specificity of the effect upon Cx43 was not clear. Here we
sought to verify the NO specificity of the effect seen in
rabbits by employing a more selective iNOS inhibitor: N-
iminoethyl-L-lysine (NIL) [20].
MATERIALS AND METHODS
Reagents
Rabbit polyclonal antibody raised against the C-terminal
portion of rat heart Cx43 was a gift of Dr. Dale Laird (Lon-
don, Ontario, Canada) [21]. Cx43 cDNA probe was a gift
of Dr. David Paul (Boston, MA) [22]. Mouse monoclonal
antibodies and standards for PGHS isoform 1 were pur-
chased from Cayman Chemical (Ann Arbor, MI), and for
PGHS 2 from Transduction Laboratories (Lexington, KY).
Molecular weight markers were from Bio-Rad (Hercules,
CA). NOS activity assay components were from the sources
previously described [14]. 125I-Ornithine vasotocin (specific
activity 2200 Ci/mmol) and chemiluminescence reagents
were purchased from New England Nuclear (NEN, Boston,
MA). Serum progesterone RIA kit was purchased from Di-
agnostic Products (Los Angeles, CA). Leupeptin was ob-
tained from Boehringer-Mannheim (Indianapolis, IN), and
all other chemicals from Sigma Chemical Co. (St. Louis,
MO).
 UTERINE NITRIC OXIDE AND MYOMETRIAL CONNEXIN 43
Animals
Time-mated, Sprague-Dawley rats were bred by the ven-
dor (Taconic, Germantown, NY; sperm-positive day 5 Day
1). NIL was tested in doses from 10 to 100 mg/kg. A dose
of 50 mg/kg was chosen because it was the dose that would
inhibit decidual particulate iNOS activity yet not raise mean
arterial pressure (MAP). L-NAME was tested in doses from
50 to 100 mg/kg; 100 mg/kg was chosen because it is
known to completely inhibit eNOS [23] and because it did
not inhibit decidual iNOS in our system (see below). In-
hibitors were administered via oral gavage needle under
metofane anesthesia. In a subset of animals, MAP was re-
corded continuously in conscious, unrestrained animals for
15 min at rest via femoral artery catheters implanted 4 days
previously (method of Conrad [24]). The catheter tips lay
in the abdominal aorta below the renal arteries.
Tissue Preparation
After 24 h, animals were killed on the 17th or 19th day
of gestation via decapitation under metofane anesthesia.
The ‘‘labor’’ group was killed at 1300–1600 h after deliv-
ering one or more pups, but before the delivery of the last
pup. The decidua and metrial gland were scraped from the
myometrium. These tissues as well as placentas and ma-
ternal serum were frozen separately at 2708C until prepa-
ration.
NOS Activity Assay
Since the effects of neither L-NAME nor NIL are com-
pletely reversible [25, 26], it was possible to determine that
these competitors acted selectively in vivo by assessing re-
duced activity of the relevant isoform in tissues removed
from the animal. The NOS activity assay method previously
described was employed [14]. Briefly, washed particulate
fraction was incubated for 60 min at 308C with [14C]L-
arginine (3 mM), CaCl2 (2 mM), calmodulin (500 nM), tet-
rahydrobiopterin (10 mM), and flavine adenine dinucleotide
(2 mM), with and without the essential cofactor nicotin-
amide adenine dinucleotide phosphate (NADPH; 1 mM).
The concentration of arginine substrate used was not satu-
rating but was sufficient to determine the effects of the
inhibitors. Calcium-insensitive activity was determined by
adding EGTA (1 mM) in the place of the CaCl2 and cal-
modulin. The [14C]L-citrulline produced was separated with
Dowex 50WX (Dow Corning, Midland, MI) cation ex-
change resin. NOS activity was defined as NADPH-depen-
dent conversion of arginine to citrulline in NADPH-con-
taining samples minus conversion in samples in which
NADPH was absent. Calcium-sensitive activity was cal-
culated as the difference between the total activity minus
the calcium-insensitive activity. Intraassay variation was
5.8%. Samples from the same tissue (decidua or placenta)
were assayed together on the same day.
Protein Concentration Assay
The Bradford method was employed after NaOH (1.5
M) hydrolysis of samples. BSA was used as the standard.
Western Immunoblotting for Cx43
Particulate fraction was prepared at 48C by homogeniz-
ing frozen myometrium for 30 sec in buffer B (5 ml/g)
containing 10 mM potassium phosphate-buffered normal
saline (KPBS, pH 7.40), leupeptin (3 mM), and PMSF (1
9
mM). The homogenate was centrifuged at 1000 3 g for 5
min, and the pellet was discarded. The supernatant was re-
centrifuged at 30 000 3 g for 20 min. The resulting pellet
was resuspended in 0.6 ml buffer B per gram original tis-
sue, homogenized with a polytetrafluoroethylene pestle, and
stored at 2708C until use.
Samples (30 mg protein per lane) were subjected to 12%
PAGE (Bio-Rad Mini-Protean) and electrotransferred (Bio-
Rad Trans-Blot) to nitrocellulose (MSI, Westboro, MA).
Protein was fixed to the membrane by immersing in 1%
glutaraldehyde in 10 mM Tris-buffered normal saline (TBS,
pH 7.40) for 10 min. The membrane was blocked with
Blotto-TX (0.2%; 5% powdered milk in KPBS, pH 7.40,
with 0.2% Triton X-100) for 1 h; then, after rinsing, it was
probed for 1 h with rabbit polyclonal anti-rat heart Cx43
antibody diluted 1:5000 in Blotto-Tween 0.05%. After rins-
ing, a goat anti-rabbit IgG-horseradish peroxidase (HRP)
secondary antibody (Sigma A4914; 1:4000 dilution in
0.05% TBS Tween/1% BSA/2% goat serum) was applied
for 1 h. After rinsing, the membrane was developed for 60
sec with chemiluminescence solution (Renaissance, NEN)
and exposed to x-ray film for 2–5 min. Densitometry was
performed using the Harmony computer program (Videk,
Rochester, NY) on a video camera (Sony CCD, Park Ridge,
NJ) image of the developed x-ray film. Band density was
linear with micrograms protein (5–50 mg) per lane (r2 5
0.97). Samples from the NIL, NAME, and labor groups
were run on separate gels, but each with 3–5 of the same
19-day control samples. Densitometry readings were then
normalized to the average 19-day sample reading to permit
comparisons between the NIL, NAME, and labor groups.
Northern Analysis for Cx43 mRNA
RNA was isolated from frozen myometrium by the acid
phenol-guanidine isothiocyanate method. Twenty micro-
grams of total RNA in formamide was loaded per lane onto
a 1% agarose/20% formaldehyde gel and electrophoresed
at 215 V for 16 h. The RNA was transferred to a nylon
membrane (Magna, MSI) by capillary action with 20-
strength saline-sodium citrate (3 M NaCl, 0.3 M sodium
citrate) for 22 h. The membrane was dried in a vacuum
oven and UV cross-linked (120 mJ/cm2; Fisher BioTech,
Pittsburgh, PA) before prehybridization in 20 ml Church
buffer (0.25 M sodium phosphate pH 7.2, 7% SDS, 0.1 M
EDTA) at 658C for 5 h. The membrane was then hybridized
with a 32P-labeled (1.4 mCi/ng, MultiPrime; Amersham
Pharmacia Biotech, Piscataway, NJ) Cx43 cDNA (rat heart,
900 bp, a gift of Dr. David Paul [22]) for 22 h at 658C in
Church buffer without EDTA. The membrane was then
washed four times in 100 ml fresh Church buffer at 608C
for 20 min. The 32P signal was quantified overnight in a
PhosphorImager (Bio-Rad). The membrane was subse-
quently stripped (1008C in 2% SDS, 5 min), rehybridized
with a 32P-labeled (1.0 mCi/ng) 18S ribosomal RNA cDNA
(human, 400 bp, a gift of Dr. Phil Rauk), washed, and quan-
tified in the PhosphorImager. To correct for RNA loading
per lane, the Cx43 mRNA signal was expressed as the ratio
of Cx43 to 18S 32P counts.
OTR Assay
Microsomal preparations in Tris (50 mM, pH 7.40),
MgCl2 (4 mM), BSA (0.5%) were incubated for 45 min at
308C with 125I-ornithine vasotocin (2200 Ci/mmol), 24 000
dpm/tube 5 0.05 nM, and 0.1–20 nM nonradioactive or-
nithine vasotocin. Bound ligand was collected on glass fil-
10 SLADEK ET AL.
FIG. 1. Effect of in vivo NOS inhibitors on ex vivo gravid rat uterine
NOS activity (particulate fraction NADPH-sensitive L-arginine to L-citrul-
line activity). Mean 6 SEM, n as indicated. Treatments were administered
by gavage at 18 days gestation, uteri harvested 24 h later (19 days). Con-
trol, water; NIL, iNOS inhibitor (50 mg/kg); L-NAME, eNOS inhibitor (100
mg/kg). *p , 0.04 one-tailed test and ‡p , 0.01 versus control by AN-
OVA and Fisher post hoc test.
ters [27]. Binding curves were analyzed with an iterative,
nonlinear curve-fitting computer program (Wavemetrics,
Oswego, OR). Kd was 0.38 6 0.07 nM at 19 days gestation
and decreased to 0.18 6 0.05 nM with term labor. Intra-
and interassay variation was 15.2% and 37%, respectively.
Western Immunoblotting for PGHS 1 and 2
PGHS 1 and 2 were examined in decidua. Particulate
was prepared as for the NOS assay, and Western blotting
was performed as with Cx43 except that the polyacryl-
amide gel was 10%; Cayman mouse monoclonal anti-
PGHS 1 antibody (#160110) and Transduction Laboratories
mouse monoclonal anti-PGHS 2 antibody (#C22420) were
used at 1:300 dilution; and goat anti-mouse IgG-HRP sec-
ondary antibody (Sigma A8924) was employed. Band den-
sity was linear with micrograms protein (10–50 mg) per
lane (r2 5 0.94 and 0.89 for PGHS 1 and 2, respectively).
The PGHS 1 antibody reacted with PGHS 1 and not PGHS
2 standard. In contrast to previous findings [28], newer lots
of the PGHS 2 antibody also reacted somewhat with PGHS
1 standard.
Serum Progesterone
A commercial RIA kit (Diagnostic Products) was used
according to the manufacturer’s instructions (sensitivity 0.1
ng/ml, intra- and interassay coefficients of variation, 5.9%
and 15.5%, respectively).
FIG. 2. Western immunoblot of rat myometrial Cx43 protein. Rats were
treated at 16 or 18 days gestation, and tissue was harvested 24 h later
(17d and 19d). Labor: 22 days gestation, untreated; 30 mg protein loaded
per lane; MW, molecular weight markers (3 1023); Control, rat heart (5
mg protein).
FIG. 3. Effect of in vivo NOS inhibitors on rat myometrial Cx43 protein
expression. Western immunoblotting, with band density normalized to at
least 3 control 19-day (19d) samples that were repeated on each blot.
Mean 6 SEM, n as indicated. *p , 0.05 versus control by ANOVA and
Fisher post hoc test.
Statistical Analysis
Tests were performed with StatView 4.5 or Super-
ANOVA software (Abacus Concepts, Berkeley, CA). The
Cx43 Western immunoblot band densities (normalized to
19-day control samples) from any one animal were aver-
aged from multiple replicates. These values for each animal
were then averaged for each experimental group. The group
means were compared by t-tests with Bonferroni correction
for multiple comparisons. Group means for NOS activity,
Cx43 mRNA, OTR, PGHS 1, and PGHS 2 concentrations
were compared via one-way factorial ANOVA and the
Fisher protected least-significant difference test. MAP val-
ues were compared via repeated measures ANOVA; p val-
ues less than 0.05 were considered significant. Data are
presented throughout as mean 6 SEM.
RESULTS
Figure 1 shows that selective NOS inhibition was
achieved in vivo. Twenty-four hours after treatment with
the iNOS inhibitor, NIL decreased calcium-insensitive de-
cidual iNOS activity by 70% (p 5 0.04, one-tailed test),
while calcium-sensitive placental eNOS activity was not
affected (p 5 0.45). Conversely, treatment with the eNOS
inhibitor L-NAME completely blocked placental eNOS ac-
tivity (p 5 0.01) and did not decrease decidual iNOS ac-
tivity. Rather, L-NAME more than doubled decidual iNOS
activity 24 h after administration (p 5 0.001). Changes in
maternal MAP also indicated the specificity of blockade.
The iNOS inhibitor NIL did not inhibit eNOS enough to
significantly increase MAP: 102 6 4, 115 6 5, and 107 6
12 mm Hg before versus 5 and 24 h after treatment, re-
spectively (mean 6 SEM, n 5 3, p . 0.20). In contrast,
consistent with previous findings [5, 23], the vasoconstrict-
ing eNOS inhibitor L-NAME did significantly increase
MAP: 105 6 0, 142 6 8, and 140 6 5 mm Hg before
versus 5 and 24 h after treatment, respectively (mean 6
SD, n 5 2, p , 0.02).
In vivo inhibition of iNOS with NIL, but not eNOS with
L-NAME, resulted in significantly increased expression of
rat myometrial Cx43 protein at 19 days gestation (Figs. 2
and 3). This did not occur at 17 days gestation. The increase
 UTERINE NITRIC OXIDE AND MYOMETRIAL CONNEXIN 43
FIG. 4. Northern blot of rat myometrial Cx43 mRNA. Twenty micro-
grams total RNA was loaded per lane and reprobed for 18S rRNA to
control for lane loading. Treatments were administered to pregnant rats
by gavage at 18 days gestation and uteri harvested 24 h later.
in Cx43 protein at 19 days reached the levels present during
spontaneous labor at term. With L-NAME treatment, which
increased decidual iNOS activity (Fig. 1), there was a trend
toward a decrease in Cx43 protein (Fig. 3, p 5 0.15).
Northern analysis revealed that 24 h after administration of
NOS inhibitors, the steady state concentration of mRNA
was not significantly changed (Figs. 4 and 5). The expected
increase of Cx43 transcripts in myometrial mRNA with la-
bor was observed.
In contrast, NOS inhibition did not result in a premature
increase of OTR (Fig. 6) or PGHS 1 or 2 protein (Figs. 7
and 8). (Even if the PGHS 2 antibody is showing total
[PGHS 1 1 2] enzyme mass, one can infer that since PGHS
1 did not increase, and the total did not increase, then
PGHS 2 enzyme mass must not have increased significantly
either.) Untreated laboring animals showed the expected in-
FIG. 5. Effect of in vivo NOS inhibitors on steady state concentrations
of rat myometrial Cx43 mRNA. Northern blotting with band density nor-
malized to 18S rRNA bands. Mean 6 SEM, n as indicated. *p , 0.05
versus control by ANOVA and Fisher post hoc test.
11
FIG. 6. Effect of in vivo NOS inhibitors on rat myometrial OTR concen-
tration (fmol/mg protein 125I-ornithine vasotocin binding). Mean 6 SEM,
n as indicated. Different letters indicate p , 0.05 by ANOVA and Fisher
post hoc test.
creases in OTR [27], PGHS 1, and PGHS 2 [28]. Maternal
serum progesterone concentrations did not prematurely de-
crease: 88 6 3, 91 6 6, 109 6 10, and 7.4 6 1.5 ng/ml
for control 19 days, NIL 19 days, L-NAME 19 days, and
labor 22 days, respectively (mean 6 SEM, n as in Fig. 5).
DISCUSSION
Blocking all forms of NOS activity (iNOS, eNOS,
nNOS) in pregnant animals may be too blunt a tool to sep-
arate out the functions of endogenous uterine NO. For ex-
ample, specific effects of blockade of decidual or myome-
trial NOS must be distinguished from nonspecific effects
mediated by vasoconstriction (due to eNOS blockade).
NOS inhibitors are semiselective at best, with multiple iso-
forms inhibited by any one compound if high enough doses
are administered [8]. In this study we demonstrated selec-
tive inhibition of iNOS and eNOS (Fig. 1 and MAP mea-
surements). Tissue preparation and washing of the partic-
ulate fraction should serve to decrease the concentration of
inhibitor during ex vivo assay [29]. This may explain why
a dose of NIL (50 mg/kg) known to inhibit iNOS in other
animal systems [20] decreased ex vivo decidual iNOS ac-
FIG. 7. Western immunoblot of rat decidual PGHS 1 and PGHS 2. Thirty
micrograms of protein loaded per lane. MW: molecular weight markers
(3 1023); PGHS 1 control, ram seminal vesicle; PGHS 2 control, lysate
from mouse macrophages stimulated with interferon-g and lipopolysac-
charide.
12 SLADEK ET AL.
FIG. 8. Effect of in vivo NOS inhibitors on rat decidual PGHS 1 and
PGHS 2 expression. Western immunoblotting with band density normal-
ized to at least 3 control 19-day (19d) samples repeated on each blot.
Mean 6 SEM, n as indicated. *p , 0.05 versus control by ANOVA and
Fisher post hoc test.
tivity by 70% and not 100%. Placental eNOS activity was
not reduced 24 h after administration of the iNOS inhibitor
NIL. Conversely, eNOS inhibition was achieved with L-
NAME; and rather than a lack of effect on decidual iNOS
activity, there was an increase 24 h after L-NAME treat-
ment. This effect has been reported previously, although
the mechanism is unknown [10]. The major finding of this
study is that endogenous NO suppresses rat myometrial
Cx43 gap junction protein expression during pregnancy pri-
or to term (Fig. 3). Further, it is NO produced by iNOS and
not eNOS that seems responsible for suppressing Cx43, be-
cause premature increase in expression occurred with the
iNOS inhibitor NIL and not the eNOS inhibitor L-NAME.
Interestingly, the significant increase in decidual iNOS ac-
tivity with L-NAME (Fig. 1) resulted in a trend toward
decreased myometrial Cx43 protein (Fig. 3), although the
decrease did not achieve our chosen level of statistical sig-
nificance (p 5 0.16 rather than 0.05). Future studies will
be necessary to determine whether exogenous NO donors
can suppress myometrial Cx43 expression.
Although the direct mechanism by which iNOS inhibi-
tion increases myometrial Cx43 protein is not yet known,
at least two possibilities seem unlikely: vasoconstriction or
luteolysis [30]. Vasoconstriction is an unlikely mechanism
because the increase with NIL treatment occurred without
an increase in maternal blood pressure. Luteolysis is also
an unlikely mechanism because serum progesterone con-
centration did not decrease with iNOS inhibitor treatment.
Luteolysis would also have induced an increase in OTR
[27], but this was not seen.
A third possibility for the increase in Cx43 protein ex-
pression with iNOS inhibition might be an increase in uter-
ine wall tension after eliminating a relaxing effect of en-
dogenous NO. Stretch of the uterine wall has been shown
to be necessary for the increased expression of Cx43 at the
end of pregnancy [31]. The data presented here could be
novel, indirect evidence that endogenous uterine NO does
indeed affect myometrial contractility in vivo. At 17 days
gestation this mechanism may not operate because fetal
growth may not yet be sufficient to stretch the uterine wall
to the threshold necessary to stimulate Cx43 expression
when NO is removed.
A fourth possible mechanism is a direct effect of NO or
the lack thereof on the transcription of the Cx43 gene.
There are several precedents for suppressive effects of NO
on gene transcription [17]. In particular, the activator pro-
tein 1 (AP-1) transcription factor is involved in the acti-
vation of Cx43 gene transcription [32], and NO has recently
been shown to decrease the activity of the AP-1 [33]. How-
ever we did not find an increase in steady state Cx43
mRNA concentrations 24 h after treatment with iNOS in-
hibitor (Fig. 5). Whether Cx43 mRNA concentrations had
peaked earlier than 24 h, or whether there are posttransla-
tional enhancements of Cx43 gene expression with iNOS
blockade remains to be determined. A caveat regarding in-
creased Cx43 protein is that the presence of the protein
does not necessarily lead to increased cell-to-cell coupling.
The Cx43 gap junction proteins must be brought from the
endoplasmic reticulum to the plasma membrane, assembled
into hexamers, linked to a hexamer on an adjoining cell,
and avoid inactivation by phosphorylation in order to form
a gap junction capable of transmitting a depolarization [34].
Whether the increase in Cx43 protein seen with iNOS in-
hibition results in increased cell-to-cell coupling remains to
be determined.
The interaction of uterine NO and prostaglandin (PG)
synthesis during pregnancy has increasingly been a subject
of study. Previously, studies of other organ systems had
shown low concentrations of NO increase, and high con-
centrations decrease, PG synthesis [35]. In vitro, myocytes
cultured from pregnant rat uteri responded to NO donors
with increased PG synthesis [36], whereas myometrial
strips from 21-day-gestation rats increased PG synthesis in
response to oxytocin only after iNOS had been inhibited
[4]. In the experiments reported here, iNOS and eNOS in-
hibition at 18–19 days gestation did not change the mass
of PGHS 1 or 2. The expected increase in PGHS 1 and 2
mass with labor [28] was evident in our studies (Fig. 8). A
failure to increase PGHS mass does not necessarily exclude
an effect on PG synthesis, because at least in human am-
nion, PGHS activity but not mass increased with labor [37].
Nonetheless our findings do not support the hypothesis that
NO maintains a generalized inhibitory effect upon the ex-
pression of parturitional proteins.
In summary, a role for uterine NO during rat pregnancy
is demonstrated by in vivo experiments with pharmacologic
inhibitors of NOS. Of genes that are important in preparing
the uterus for parturition, Cx43 gap junction protein was
prematurely expressed at 19 days gestation as a result of
inhibiting NO production. This effect was specific to iNOS
and not eNOS inhibition and was independent of luteolysis
and vasoconstriction. Myometrial OTR and decidual PGHS
1 and 2 concentrations were unaffected by NOS blockade.
The increase in Cx43 occurred when the uterine wall was
stretched further at 18–19 days gestation, not at 16–17 days
gestation. The mechanism of NO’s effect on Cx43 expres-
sion remains to be determined.
ACKNOWLEDGMENTS
We gratefully acknowledge Dr. Dale Laird of University of Western
Ontario University for the gift of the Cx43 antibody; Dr. David L. Paul
of Harvard University for the gift of the Cx43 cDNA probe; and Francine
Yao, Camilo Borrero, Kirk Conrad, and Marcia Gallaher for technical
assistance.
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