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Biology of Reproduction 61, 8-13 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Articles

Endogenous Nitric Oxide Suppresses Rat Myometrial Connexin 43 Gap Junction Protein Expression during Pregnancy1

Stephen M. Sladeka, Andrea Westerhausen-Larsona, and James M. Roberts2,a

a Magee-Womens Research Institute, and the Department of Obstetrics, Gynecology, and Reproductive Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15213


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nitric oxide (NO) synthase (NOS) is active in the gravid uterus, and its activity decreases prior to the onset of parturition. We tested the hypothesis that NO helps maintain uterine quiescence 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 parturitional 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 suppresses myometrial Cx43 gap junction protein expression during rat pregnancy. Although the exact mechanism is unknown, an increase of uterine wall stretch due to inhibition of relaxation could account for increased Cx43 gene transcription.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The functions of nitric oxide (NO) produced in the uterus during pregnancy have not yet been determined definitively. Because nitric oxide synthase (NOS) is active in the gravid uterus and its activity decreases at the end of pregnancy, 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]. However, there is no evidence yet that endogenous NO suppresses uterine contractions [2–4]. NOS blockade in pregnant rats does not induce preterm delivery [5] except when administered along with anti-progestins [6], which themselves 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 vasoconstriction, 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, suggesting immunological and/or vascular functions for uterine NO during pregnancy [12]. Myometrial cells stained positively for iNOS only if located adjacent to the placental attachment site. Although it has been suggested that neuronal NOS may mediate relaxation of nonpregnant rat myometrium [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. During this time the uterus is undergoing important preparations 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 expression [17], we investigated the effect of endogenous NO on the expression of these parturitional genes. The experiments reported here were designed to test the hypothesis that NO helps maintains uterine quiescence during pregnancy by suppressing the expression of myometrial Cx43 gap junction protein, OTR, and PGHS. In preliminary experiments with pregnant rabbits, the semiselective iNOS inhibitor aminoguanidine, but not L-NAME, resulted in the premature expression of myometrial Cx43 [18]. There was no premature expression of OTR or increased PGHS enzyme activity [19]. Both aminoguanidine and L-NAME inhibited 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
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents

Rabbit polyclonal antibody raised against the C-terminal portion of rat heart Cx43 was a gift of Dr. Dale Laird (London, 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 purchased 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 Diagnostic Products (Los Angeles, CA). Leupeptin was obtained from Boehringer-Mannheim (Indianapolis, IN), and all other chemicals from Sigma Chemical Co. (St. Louis, MO).

Animals

Time-mated, Sprague-Dawley rats were bred by the vendor (Taconic, Germantown, NY; sperm-positive day = 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). Inhibitors were administered via oral gavage needle under metofane anesthesia. In a subset of animals, MAP was recorded 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 delivering 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 maternal serum were frozen separately at -70°C until preparation.

NOS Activity Assay

Since the effects of neither L-NAME nor NIL are completely reversible [25, 26], it was possible to determine that these competitors acted selectively in vivo by assessing reduced 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 30°C with [14C]L-arginine (3 µM), CaCl2 (2 mM), calmodulin (500 nM), tetrahydrobiopterin (10 µM), and flavine adenine dinucleotide (2 µM), with and without the essential cofactor nicotinamide adenine dinucleotide phosphate (NADPH; 1 mM). The concentration of arginine substrate used was not saturating 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 calmodulin. The [14C]L-citrulline produced was separated with Dowex 50WX (Dow Corning, Midland, MI) cation exchange resin. NOS activity was defined as NADPH-dependent conversion of arginine to citrulline in NADPH-containing samples minus conversion in samples in which NADPH was absent. Calcium-sensitive activity was calculated 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 4°C by homogenizing 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 µM), and PMSF (1 mM). The homogenate was centrifuged at 1000 x g for 5 min, and the pellet was discarded. The supernatant was recentrifuged at 30 000 x g for 20 min. The resulting pellet was resuspended in 0.6 ml buffer B per gram original tissue, homogenized with a polytetrafluoroethylene pestle, and stored at -70°C until use.

Samples (30 µg 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 rinsing, 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 µg) per lane (r2 = 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 micrograms 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 65°C for 5 h. The membrane was then hybridized with a 32P-labeled (1.4 µCi/ng, MultiPrime; Amersham Pharmacia Biotech, Piscataway, NJ) Cx43 cDNA (rat heart, 900 bp, a gift of Dr. David Paul [22]) for 22 h at 65°C in Church buffer without EDTA. The membrane was then washed four times in 100 ml fresh Church buffer at 60°C for 20 min. The 32P signal was quantified overnight in a PhosphorImager (Bio-Rad). The membrane was subsequently stripped (100°C in 2% SDS, 5 min), rehybridized with a 32P-labeled (1.0 µCi/ng) 18S ribosomal RNA cDNA (human, 400 bp, a gift of Dr. Phil Rauk), washed, and quantified 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 30°C with 125I-ornithine vasotocin (2200 Ci/mmol), 24 000 dpm/tube = 0.05 nM, and 0.1–20 nM nonradioactive ornithine vasotocin. Bound ligand was collected on glass filters [27]. Binding curves were analyzed with an iterative, nonlinear curve-fitting computer program (Wavemetrics, Oswego, OR). Kd was 0.38 ± 0.07 nM at 19 days gestation and decreased to 0.18 ± 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 polyacrylamide 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 secondary antibody (Sigma A8924) was employed. Band density was linear with micrograms protein (10–50 µg) per lane (r2 = 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).

Statistical Analysis

Tests were performed with StatView 4.5 or SuperANOVA software (Abacus Concepts, Berkeley, CA). The Cx43 Western immunoblot band densities (normalized to 19-day control samples) from any one animal were averaged 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 values were compared via repeated measures ANOVA; p values less than 0.05 were considered significant. Data are presented throughout as mean ± SEM.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Figure 1 shows that selective NOS inhibition was achieved in vivo. Twenty-four hours after treatment with the iNOS inhibitor, NIL decreased calcium-insensitive decidual iNOS activity by 70% (p = 0.04, one-tailed test), while calcium-sensitive placental eNOS activity was not affected (p = 0.45). Conversely, treatment with the eNOS inhibitor L-NAME completely blocked placental eNOS activity (p = 0.01) and did not decrease decidual iNOS activity. Rather, L-NAME more than doubled decidual iNOS activity 24 h after administration (p = 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 ± 4, 115 ± 5, and 107 ± 12 mm Hg before versus 5 and 24 h after treatment, respectively (mean ± SEM, n = 3, p > 0.20). In contrast, consistent with previous findings [5, 23], the vasoconstricting eNOS inhibitor L-NAME did significantly increase MAP: 105 ± 0, 142 ± 8, and 140 ± 5 mm Hg before versus 5 and 24 h after treatment, respectively (mean ± SD, n = 2, p < 0.02).



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  		FIG. 1. Effect of in vivo NOS inhibitors on ex vivo gravid rat uterine NOS activity (particulate fraction NADPH-sensitive L-arginine to L-citrulline activity). Mean ± SEM, n as indicated. Treatments were administered by gavage at 18 days gestation, uteri harvested 24 h later (19 days). Control, water; NIL, iNOS inhibitor (50 mg/kg); L-NAME, eNOS inhibitor (100 mg/kg). *p < 0.04 one-tailed test and {ddagger}p < 0.01 versus control by ANOVA and Fisher post hoc test.

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 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 = 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 labor was observed.



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  		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 µg protein loaded per lane; MW, molecular weight markers (x 10-3); Control, rat heart (5 µg protein).



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  		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 ± SEM, n as indicated. *p < 0.05 versus control by ANOVA and Fisher post hoc test.



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  		FIG. 4. Northern blot of rat myometrial Cx43 mRNA. Twenty micrograms 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.



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  		FIG. 5. Effect of in vivo NOS inhibitors on steady state concentrations of rat myometrial Cx43 mRNA. Northern blotting with band density normalized to 18S rRNA bands. Mean ± SEM, n as indicated. *p < 0.05 versus control by ANOVA and Fisher post hoc test.

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 + 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 increases in OTR [27], PGHS 1, and PGHS 2 [28]. Maternal serum progesterone concentrations did not prematurely decrease: 88 ± 3, 91 ± 6, 109 ± 10, and 7.4 ± 1.5 ng/ml for control 19 days, NIL 19 days, L-NAME 19 days, and labor 22 days, respectively (mean ± SEM, n as in Fig. 5).



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  		FIG. 6. Effect of in vivo NOS inhibitors on rat myometrial OTR concentration (fmol/mg protein 125I-ornithine vasotocin binding). Mean ± SEM, n as indicated. Different letters indicate p < 0.05 by ANOVA and Fisher post hoc test.



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  		FIG. 7. Western immunoblot of rat decidual PGHS 1 and PGHS 2. Thirty micrograms of protein loaded per lane. MW: molecular weight markers (x 10-3); PGHS 1 control, ram seminal vesicle; PGHS 2 control, lysate from mouse macrophages stimulated with interferon-{gamma} and lipopolysaccharide.



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  		FIG. 8. Effect of in vivo NOS inhibitors on rat decidual PGHS 1 and PGHS 2 expression. Western immunoblotting with band density normalized to at least 3 control 19-day (19d) samples repeated on each blot. Mean ± SEM, n as indicated. *p < 0.05 versus control by ANOVA and Fisher post hoc test.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Blocking all forms of NOS activity (iNOS, eNOS, nNOS) in pregnant animals may be too blunt a tool to separate out the functions of endogenous uterine NO. For example, specific effects of blockade of decidual or myometrial NOS must be distinguished from nonspecific effects mediated by vasoconstriction (due to eNOS blockade). NOS inhibitors are semiselective at best, with multiple isoforms inhibited by any one compound if high enough doses are administered [8]. In this study we demonstrated selective inhibition of iNOS and eNOS (Fig. 1 and MAP measurements). Tissue preparation and washing of the particulate 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 activity 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 treatment. 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 prior to term (Fig. 3). Further, it is NO produced by iNOS and not eNOS that seems responsible for suppressing Cx43, because premature increase in expression occurred with the iNOS inhibitor NIL and not the eNOS inhibitor L-NAME. Interestingly, the significant increase in decidual iNOS activity 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 significance (p = 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 inhibition 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 concentration 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 expression with iNOS inhibition might be an increase in uterine wall tension after eliminating a relaxing effect of endogenous 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 protein 1 (AP-1) transcription factor is involved in the activation of Cx43 gene transcription [32], and NO has recently been shown to decrease the activity of the AP-1 [33]. However we did not find an increase in steady state Cx43 mRNA concentrations 24 h after treatment with iNOS inhibitor (Fig. 5). Whether Cx43 mRNA concentrations had peaked earlier than 24 h, or whether there are posttranslational enhancements of Cx43 gene expression with iNOS blockade remains to be determined. A caveat regarding increased 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 inhibition 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 concentrations 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 inhibition 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 amnion, 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 expression 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 expression 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.


   FOOTNOTES
 
1 This work was supported by NIH Grant (HD-01095) and an NICHD Clinical Investigator Development Award to S.M.S. Back

2 Correspondence: James M. Roberts, Magee-Womens Research Institute, Room 610, 204 Craft Avenue, Pittsburgh, PA 15213-3180. FAX: 412 641 1503; jimrob+{at}pitt.edu Back

Accepted: February 2, 1999.

Received: July 21, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Sladek SM, Magness RR, Conrad KP. Nitric oxide and pregnancy. Am J Physiol 1997; 272:R441–463.
  2. Jones GD, Poston L. The role of endogenous nitric oxide synthesis in contractility of term or preterm human myometrium. Br J Obstet Gynaecol 1997; 104:241–245.[Medline]
  3. Mirabile CP, Figueroa JP. The role of nitric oxide in the maintenance of uterine quiescence in the nonpregnant and pregnant ewe in vivo. J Soc Gynecol Invest 1998; 5:64A.
  4. Chaud MA, Franchi AM, Beron de Astrada M, Gimeno MF. Role of nitric oxide on oxytocin-evoked contractions and prostaglandin synthesis in isolated pregnant rat uterus. Prostaglandins Leukot Essent Fatty Acids 1997; 57:323–329.[CrossRef][Medline]
  5. Molnar M, Hertelendy F. N-Omega-nitro-L-arginine, an inhibitor of nitric oxide synthesis, increases blood pressure in rats and reverses the pregnancy-induced refractoriness to vasopressor agents. Am J Obstet Gynecol 1992; 166:1560–1567.[Medline]
  6. Yallampalli C, Buhimschi I, Chwalisz K, Garfield RE, Dong YL. Preterm birth in rats produced by the synergistic action of a nitric oxide inhibitor (NG-nitro-L-arginine methyl ester) and an antiprogestin (onapristone). Am J Obstet Gynecol 1996; 175:207–212.[CrossRef][Medline]
  7. Gross SS, Jaffe EA, Levi R, Kilbourn RG. Cytokine-activated endothelial cells express an isotype of nitric oxide synthase which is tetrahydrobiopterin-dependent, calmodulin-independent and inhibited by arginine analogs with a rank-order of potency characteristic of activated macrophages. Biochem Biophys Res Commun 1991; 178:823–829.[CrossRef][Medline]
  8. Joly GA, Ayres M, Chelly F, Kilbourn RG. Effects of NG-methyl-L-arginine, NG-nitro-L-arginine, and aminoguanidine on constitutive and inducible nitric oxide synthase in the rat aorta. Biochem Biophys Res Commun 1994; 199:147–154.[CrossRef][Medline]
  9. Lambert LE, Whitten JP. Nitric oxide synthesis in the CNS, endothelium, and macrophages differs in its sensitivity to inhibition by arginine analogs. Life Sci 1991; 48:69–75.[CrossRef][Medline]
  10. Miller MJ, Voelker CA, Olister S. Fetal growth retardation in rats may result from apoptosis: role of peroxynitrite. Free Radical Biol Med 1996; 21:619–629.[CrossRef][Medline]
  11. Giordano M, Vermeulen M, Trevani AS, Dran G, Andonegui G, Geffner JR. Nitric oxide synthase inhibitors enhance plasma levels of corticosterone and ACTH. Acta Physiol Scand 1996; 157:259–264.[CrossRef][Medline]
  12. Sladek SM, Kanbour-Shakir A, Watkins S, Berghorn KA, Hoffman GE, Roberts JM. Granulated metrial gland cells contain nitric oxide synthases during pregnancy in the rat. Placenta 1998; 19:55–65.[CrossRef][Medline]
  13. Shew RL, Papka RE, McNeill DL, Yee JA. NADPH-diaphorase-positive nerves and the role of nitric oxide in CGRP relaxation of uterine contraction. Peptides 1993; 14:637–641.[CrossRef][Medline]
  14. Sladek SM, Roberts JM. Nitric oxide synthase activity in the gravid rat uterus decreases a day before the onset of parturition. Am J Obstet Gynecol 1996; 175:1661–1667.[CrossRef][Medline]
  15. Sladek SM, Stranko CP, Roberts JM. Human decidual nitric oxide synthase activity decreases by the last week of gestation but not with labor. In: Soc Gynecol Invest 41st annual meeting; 1994; Chicago, IL. P422 (abstract).
  16. Challis JRG. Characteristics of parturition. In: Creasy RK, Resnik R (eds.), Maternal Fetal Medicine. Philadelphia: W.B. Saunders; 1994.
  17. Stamler JS. Redox signaling: nitrosylation and related target interactions of nitric oxide. Cell 1994; 78:931–936.[CrossRef][Medline]
  18. Sladek SM, Roberts JM. Connexin 43 is prematurely expressed in pregnant rabbit myometrium in response to aminoguanidine, a nitric oxide synthase inhibitor. J Soc Gynecol Invest 1997; 4:165a (abstract).
  19. Sladek SM, Roberts JM. Blockade of nitric oxide synthase in pregnant rabbits does not hasten uterine preparations for parturition. J Soc Gynecol Invest 1996; 3:332A (abstract).[CrossRef]
  20. Connor JR, Manning PT, Settle SL. Suppression of adjuvant-induced arthritis by selective inhibition of inducible nitric oxide synthase. Eur J Pharmacol 1995; 273:15–24.[CrossRef][Medline]
  21. Laird DW, Revel JP. Biochemical and immunochemical analysis of the arrangement of connexin 43 in rat heart gap junction membranes. J Cell Sci 1990; 97:109–117.[Abstract/Free Full Text]
  22. Beyer EC, Paul DL, Goodenough DA. Connexin43: a protein from rat heart homologous to a gap junction protein from liver. J Cell Biol 1987; 105:2621–2629.[Abstract/Free Full Text]
  23. Lubarsky SL, Ahokas RA, Friedman SA, Sibai BM. The effect of chronic nitric oxide synthase inhibition on blood pressure and angiotensin II responsiveness in the pregnant rat. Am J Obstet Gynecol 1997; 176:1069–1076.[CrossRef][Medline]
  24. Conrad KP, Colpoys MC. Evidence against the hypothesis that prostaglandins are the vasodepressor agents of pregnancy. J Clin Invest 1986; 77:236–245.
  25. Olken NM, Rusche KM, Richards MK, Marletta MA. Inactivation of macrophage nitric oxide synthase activity by NG-methyl-L-arginine. Biochem Biophys Res Commun 1991; 177:828–833.[CrossRef][Medline]
  26. Bryk R, Wolff DJ. Mechanism of inducible nitric oxide synthase inactivation by aminoguanidine and L-N6-(1-iminoethyl)lysine. Biochemistry 1998; 37:4844–4852.[CrossRef][Medline]
  27. Riemer RK, Goldfien AC, Goldfien A, Roberts JM. Rabbit uterine oxytocin receptors and in vitro contractile response: abrupt changes at term and the role of eicosanoids. Endocrinology 1986; 119:699–709.[Abstract]
  28. Dong YL, Gangula PR, Fang L, Yallampalli C. Differential expression of cyclooxygenase-1 and-2 proteins in rat uterus and cervix during the estrous cycle, pregnancy, labor and in myometrial cells. Prostaglandins 1996; 52:13–34.[CrossRef][Medline]
  29. Salter M, Duffy C, Hazelwood R. Determination of brain nitric oxide synthase inhibition in vivo: ex vivo assays of nitric oxide synthase can give incorrect results. Neuropharmacology 1995; 34:327–334.[CrossRef][Medline]
  30. Lye SJ, Nicholson BJ, Mascarenhas M, MacKenzie L, Petrocelli T. Increased expression of connexin-43 in the rat myometrium during labor is associated with an increase in the plasma estrogen:progesterone ratio. Endocrinology 1993; 132:2380–2386.[Abstract]
  31. Ou CW, Orsino A, Lye SJ. Expression of connexin-43 and connexin-26 in the rat myometrium during pregnancy and labor is differentially regulated by mechanical and hormonal signals. Endocrinology 1997; 138:5398–5407.[Abstract/Free Full Text]
  32. Piersanti M, Lye SJ. Increase in messenger ribonucleic acid encoding the myometrial gap junction protein, connexin-43, requires protein synthesis and is associated with increased expression of the activator protein-1, c-fos. Endocrinology 1995; 136:3571–3578.[Abstract]
  33. Nikitovic D, Holmgren A, Spyrou G. Inhibition of AP-1 DNA binding by nitric oxide involving conserved cysteine residues in Jun and Fos. Biochem Biophys Res Commun 1998; 242:109–112.[CrossRef][Medline]
  34. Garfield RE, Blennerhassett MG, Miller SM. Control of myometrial contractility: role and regulation of gap junctions. Oxf Rev Reprod Biol 1988; 10:436–490.[Medline]
  35. Swierkosz TA, Mitchell JA, Warner TD, Botting RM, Vane JR. Co-induction of nitric oxide synthase and cyclo-oxygenase: interactions between nitric oxide and prostanoids. Br J Pharmacol 1995; 114:1335–1342.[Medline]
  36. Dong YL, Yallampalli C. Interaction between nitric oxide and prostaglandin E2 pathways in pregnant rat uteri. Am J Physiol 1996; 270:E471–476.
  37. Teixeira FJ, Zakar T, Hirst JJ. Prostaglandin endoperoxide-H synthase (PGHS) activity and immunoreactive PGHS-1 and PGHS-2 levels in human amnion throughout gestation, at term, and during labor. J Clin Endocrinol Metab 1994; 78:1396–1402.[Abstract]



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