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Biology of Reproduction 63, 34-41 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Regular Article

Estradiol-17ß Inhibits Nitric Oxide Synthase (NOS)-II and Stimulates NOS-III Gene Expression in the Rat Uterus1

C. Yallampalli2,a, and Y.-L. Donga

a Departments of Obstetrics and Gynecology and Anatomy and Neurosciences, The University of Texas Medical Branch, Galveston, Texas 77555

ABSTRACT

Nitric oxide (NO) is synthesized by NO synthases (NOS) from L-arginine in a variety of tissues, including rat uterus. Progesterone was shown to be required for maintaining elevated NOS II expression in pregnant rat uterus. However, effects of estrogens on uterine NOS II expression remains unclear. In the present study, we examined whether 17ß-estradiol regulates NO production and NOS II expression in the rat uterus during pregnancy and in nonpregnant rats treated with lipopolysaccharide (LPS). Rats on Day 18 of pregnancy received 17ß-estradiol (0.5 or 5 µg/rat). Groups of ovariectomized (ovx) rats received 17ß-estradiol (5 µg/rat) or LPS (1 mg/rat) or a combination of the two or received vehicle only. All rats were sacrificed 24 h after treatments. Nitrite concentrations in uterine cultures were measured by Greiss reaction. Uterine NOS II and NOS III proteins and mRNA levels were determined by Western blotting and reverse transcription polymerase chain reaction, respectively. In the pregnant rat, estradiol administration caused inhibition in total NO production, suppression of both mRNA and protein levels of NOS II enzyme, and increase in NOS III mRNA and protein levels in the uterus in a dose-dependent manner. The data indicate that estradiol inhibits NOS II and total NO generation and stimulates NOS III expression. In ovx rats, LPS stimulated NOS II mRNA and NO production by the uterus. Coadministration of 5 µg estradiol profoundly suppressed NOS II mRNA and NO generation but elevated NOS III mRNA. Thus, estradiol inhibited LPS-induced increases in NOS II mRNA. Estradiol inhibits NO production by NOS II through the inhibition of NOS II expression in the rat uterus. This inhibition of NOS II expression occurs whether NOS II expression is constitutive (pregnancy) or induced (LPS-treated nonpregnant). Estradiol inhibition of NOS II expression occurs in the presence (pregnancy) or absence (ovx) of progesterone. Estradiol may play a role in regulating NOS II expression and NO production and uterine contractility during pregnancy and labor.

estradiol, gene regulation, nitric oxide, parturition, pregnancy, uterus

INTRODUCTION

Myometrial quiescence is a requirement for successful completion of term gestation. Failure to maintain uterine relaxation often results in preterm labor, one of the leading causes of infant mortality (70%) and morbidity [1]. Several studies have implicated a variety of regulatory factors of uterine activity, including steroid hormones [2, 3] and substances that suppress or stimulate myometrial contractility [4, 5]. Myometrial quiescence during pregnancy in the rat coincides with elevated progesterone levels, and increased contractile activity is associated with a decrease in progesterone and an increase in estradiol [2, 6]. The changes in these steroid hormones play a significant role in the regulation of uterine contractility during pregnancy and labor [7].

Recent studies, including ours, suggest a role for nitric oxide (NO) in the regulation of uterine contractility during pregnancy and labor. Nitric oxide is generated by the uterus and inhibits uterine contractility during pregnancy in the rat [8–11], rabbit [12], and human [13]. The NO generation in the uterus in both rats [9–11] and rabbits [12] is elevated during pregnancy and decreased at term. Three isoforms of NO synthase (NOS) have been cloned to date. In the rat uterus, both NOS II and NOS III are expressed during pregnancy [11, 14–16]. Both mRNA and protein for NOS II increase with pregnancy and decrease at term while NOS III expression remains unchanged, and NOS I is not expressed during pregnancy [11, 14–16]. NOS II expression in a variety of tissues is induced primarily by cytokines and bacterial lipopolysaccharides (LPS) [17]. However, in the rat uterus NOS II is expressed during normal pregnancy, but it is absent in the nonpregnant rat uterus [11, 14, 16]. Both NO production and NOS II expression in the pregnant rat uterus are decreased in a time-dependent manner with the administration of RU 486 [11, 14, 16]. In contrast, progesterone reduces the labor-associated fall in uterine NOS II and NO production during pregnancy [11, 16, 18]. However, the role of estradiol on NO production and NOS regulation during pregnancy is not well understood.

Nitric oxide generation is elevated in nonpregnant rat uterus during proestrus and in response to estradiol treatment [11]. This increase in uterine NO generation by estradiol is associated with increased NOS III expression [11, 19]. In addition, increased endothelium-dependent vasorelaxation [20, 21], endothelial NO production [22], and expression of NOS III protein content in vessels [23] were reported to be associated with elevated estradiol levels. However, the effect of estradiol on NO production and NOS II regulation in the rat uterus during pregnancy and labor has not been investigated. We previously reported that antiestrogens increased NOS II expression in the pregnant rat uterus [14]. The present studies were designed to investigate whether NO production and mRNA and protein for NOS II in the pregnant rat uterus are regulated by estradiol. Furthermore, we examined whether estradiol inhibits LPS-induced NOS II expression in the nonpregnant rat uterus to determine whether the estradiol effects on NOS II are specific or pregnancy dependent.

MATERIALS AND METHODS

Animals and Treatments

Adult female nonpregnant (180 to 200 g body weight [BW]) and pregnant Sprague-Dawley rats (Harlan Sprague-Dawley, Houston, TX) were maintained on a 12L:12D schedule. Animals received an ad libitum supply of rat chow and water. Two experiments were included in this study. In experiment 1, we examined the changes in NO production and NOS II and NOS III mRNA and protein levels in the uterus during pregnancy upon treatment with various doses of estradiol. In experiment 2, we examined the effects of estradiol on the uterine NO production and NOS expression in the LPS-treated ovariectomized (ovx) nonpregnant rats. All procedures were approved by the Animal Care and Use Committee of the University of Texas Medical Branch.

Experiment 1 Eighteen time-mated rats were randomly distributed into three experimental groups of six animals each. On Day 18 of gestation, these rats received 0.5 or 5.0 µg of estradiol-17ß in 0.2 ml sesame oil or they received sesame oil only. The 5.0-µg dose of estradiol-17ß was chosen to mimic the surge of estradiol that occurs around parturition in rats, and the 0.5-µg dose was used to assess the effects at lower doses. All animals were killed at 24 h following injections using a CO2 inhalation chamber. The 24-h period was chosen based on our previous report using antiestrogen and our unpublished observations. The uteri were removed immediately and cleaned, and the full thickness uterus from the antimesometrial area was utilized for nitrite production assays. For both immunoblotting and reverse transcription polymerase chain reaction (RT-PCR) studies, aliquots of uterine tissues were quickly frozen in liquid nitrogen and stored at -70°C until used.

Experiment 2 Bilateral ovariectomies were performed on nonpregnant rats under general anesthesia using ketamine (Fort Dodge Laboratories, Fort Dodge, IA; 45 mg/kg BW) and xylazine (Burns Veterinary Supply, New York, NY; 5 mg/kg BW). Seven days later, groups of six ovx rats received one of the four different treatments: 1) LPS (Sigma Chemical Co., St. Louis, MO; 1 mg/200 g BW) s.c.; 2) 17ß-estradiol (Sigma; 5 µg/200 g BW) in 0.2 ml sesame oil s.c.; 3) 17ß-estradiol (5 µg/200 g BW) and LPS (1 mg/200 g BW), 17ß-estradiol given 1 h prior to LPS treatment; and 4) sesame oil only (control). Estradiol-17ß was given 1 h prior to LPS to assess whether 17ß-estradiol blocks transcription of NOS II induced by LPS and to compare this effect with the effects of estradiol on already elevated (pregnancy induced) NOS II expression. One milligram of LPS was chosen based upon the doses used for NOS II induction in the uterus [25] and in other tissues [26] in rats. Animals were euthanized 24 h after LPS injection using a CO2 inhalation chamber. Uteri were collected, cleaned, and utilized for nitrite assays and for NOS expression evaluation.

Uterine Tissue Cultures and Nitrite Assay

Nitrite production by the uterus was measured as previously described [8, 10]. Full-thickness uterine tissues were cut into 2-mm strips, weighed, placed in 0.5 ml of minimum essential medium containing 1% penicillin and 1% streptomycin, and incubated in a humidified chamber with 5% carbon dioxide/95% oxygen at 37°C for an initial 1-h equilibration period. The medium was then replaced with 0.5 ml of fresh medium. After a 24-h incubation period, the medium was collected to measure nitrites (metabolic byproduct of NO).

Total nitrite concentration in the tissue culture medium was measured in triplicate by the microplate assay with the Griess reagent as previously described [27]. The Griess reagent (0.5% sulfanilamide and 0.05% naphthalene diamine dihydrochloride in 2.5% orthophosphoric acid, 100 µl) was added to 100-µl aliquots of medium, and optical densities were measured at 550 nm in a microplate reader after a 10-min incubation at room temperature. Nitrite values were determined using sodium nitrite as the standard. Background nitrite values of medium without tissues were subtracted from values of medium with tissues, and values were expressed as nanomoles per gram of wet uterine tissue. The sensitivity of nitrite assay was 1 µmol/L, and the coefficient of variation was less than 10% for both intra- and interassay variation [28, 29].

Relative Levels of NOS II and NOS III mRNA Measured by RT-PCR

Total cellular RNA was extracted from the rat uterus by a single-step guanidine thiocyanate method [30] using the reagent Trizol (Biotex, Friendswood, TX). First strand cDNA synthesis was primed with oligodeoxythymidine using 2 µg of total extranuclear RNA with 10 units of reverse transcriptase and oligodeoxythymidine as primer in a total volume of 20 µl at 42°C for 40 min as described previously [14, 18]. Ten percent of the cDNA made from 2 µg of total RNA was used for amplification by PCR with 35 cycles, for both NOS enzymes and ß-actin, which are in the linear region of the amplification, as described previously [18]. Polymerase chain reaction primers used in the current study were derived from the sequences of murine NOS II cDNA [31] and rat kidney NOS III cDNA [32]. The primers used for amplification of the housekeeping gene ß-actin were derived from the published rat ß-actin cDNA sequence [33]. The specific primers used for amplification were as follows: 1) NOS II: forward primer 5'-ATGGCTTGCCCCTGGAAGTTTCTC-3', reverse primer 5'-GCCGACCTGATGTTGCCACTGTT-3' (with an expected amplified length of 718 bp); 2) NOS III: forward primer 5'-GGACTTCATCAACCAGTAC-3', reverse primer 5'-GATGTAGGTGAACATTTCC-3' (with an expected amplified length of 250 bp); 3) ß-actin: forward primer 5'-GTCGACAACGGCTCCGGCA-3', reverse primer 5'-GTCAGGTCCCGGCCAGCCA-3' (with an expected length of 530 bp). The PCR was performed in 100 µl of buffer containing 5 µl cDNA, 2 mM magnesium chloride, 200 µM dNTP mixture, 4 µM primer, and 2 units of Taq DNA polymerase. The reactions were denatured at 94°C for 30 sec, annealed at 50°C for 30 sec, and elongated at 72°C for 2 min. An aliquot of the mixture from each reaction was then electrophoresed on 1% agarose, and the reaction products were detected by ethidium bromide staining of the agarose gels. With these primers, the PCR products were identical to those of the published sequences of NOS II and NOS III [13, 34] as confirmed by direct double-strand sequencing (Fmol DNA Sequencing Kit, Promega, Madison, WI; data not shown). The relative concentrations of NOS mRNA were determined by densitometric analysis of the photographs of the ethidium bromide-stained reaction products. The results were expressed as the ratio of densitometric readings for NOS to ß-actin; therefore, values indicate relative changes in NOS mRNA levels.

Western Blotting of NOS Enzymes

Immunoblotting methods were similar to those previously reported [11, 19]. Equal amounts of protein (30 µg) were size fractionated with 7.5% (w/v) SDS-PAGE and transferred onto a polyvinylidene difluoride membrane. The blots were allowed to air dry and were placed in blocking buffer (1% [w/v] BSA in 10 mM Tris buffer with 100 mM NaCl, 0.1% [v/v] Tween-20, pH 7.5) for 1 h at room temperature. All primary antibodies were monoclonal (Transduction Laboratories, Lexington, KY) and were used at different final dilutions (NOS II, 1:1000; NOS III, 1:500 [v/v]) in the blocking buffer. The blots were washed three times for 30 min each with wash buffer (10 mM Tris, 100 mM NaCl, 0.1% [v/v] Tween-20, pH 7.5) and then incubated with horseradish peroxidase–conjugated goat anti-mouse immunoglobin antibody (Transduction Laboratories) diluted in 5% (w/v) nonfat milk in a wash buffer. The membranes were washed with wash buffer three times for 30 min each, and the enhanced chemiluminescence reagent (ECL Kit; Amersham, Arlington Heights, IL) was added and incubated for 1 min at room temperature. The blots were exposed to autoradiographic film, and the intensity of specific immunoreactive bands was quantified using densitometric scanning. Densitometric units of specific protein bands were expressed relative to the values from nonpregnant animals. Both the elimination of primary antibodies and the use of a non-NOS-related monoclonal antibody indicated the specificity of the NOS protein bands at appropriate molecular sizes. In each blot, at least one lane was loaded with protein from an appropriate positive control, i.e., protein from rat uterus on Day 19 of pregnancy.

Statistics

Results are expressed as means ± SEM for each group. Differences between treatment groups were analyzed using a one-way ANOVA followed by Bonferroni t-test. Differences were considered significant at P < 0.05.

RESULTS

Effects of 17ß-Estradiol on NO Production and the Expression of NOS II and NOS III Enzymes in the Rat Uterus During Pregnancy

Nitrite production We examined whether administration of 17ß-estradiol to pregnant rats during mid-late pregnancy would inhibit NO production by the uterus. Figure 1 shows the total nitrite generation by the uterus on Day 19 of gestation in rats 24 h after receiving various doses of 17ß-estradiol. Estradiol-17ß inhibited total nitrite production by the uterus at both 0.5 (P < 0.05) and 5.0 (P < 0.01) µg/200 g BW. This finding indicates that estradiol is inhibitory to uterine NO generation in the pregnant rat uterus and that these inhibitory effects are dose dependent, with more profound inhibition in the rats receiving 5.0 µg estradiol (P < 0.01).



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  		FIG. 1. Nitric oxide production by uterine tissues from pregnant rat treated with 17ß-estradiol. Uterine tissues were obtained from rats euthanized 24 h after injection of sesame oil (control) or 17ß-estradiol (E2, 0.5 or 5.0 µg/rat, s.c.) given on Day 18 of gestation. Rat uteri were incubated in minimum essential medium for 24 h, and nitrite levels in culture medium were determined by Griess reaction. Results are mean ± SEM (n = 6). Bars with different letters on the top differ significantly (P < 0.05, ANOVA)

NOS II protein and mRNA expression To determine whether the estradiol-induced decreases in NO production by the pregnant rat uterus were due to the inhibition of NOS II protein, we measured NOS II protein in these tissues. As shown in Figure 2, a single band was seen with the predicted size of 130 kDa for NOS II. Injection of estradiol at both 0.5 and 5.0 µg/200 g BW caused a significant (P < 0.01) reduction in the NOS II protein content in the uterus during pregnancy, indicating estradiol inhibits NOS II protein in the rat uterus. To further assess whether this inhibitory effect of estradiol is at the level of mRNA expression for NOS II, we measured NOS II mRNA in the tissues from pregnant rats treated with estradiol. Figure 3 shows that the relative expression of mRNA for NOS II in the uterus was significantly (P < 0.01) inhibited by estradiol at both 0.5 and 5.0 µg and that these effects were dose dependent. Estradiol administration to pregnant rats caused a decrease in the NOS II expression at the transcription level.



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  		FIG. 2. Western blot analysis of NOS II protein in the rat uterus. Uterine tissues were obtained from rats euthanized 24 h after a single injection of sesame oil (control) or 17ß-estradiol (E2, 0.5 or 5.0 µg/rat, s.c.) given on Day 18 of gestation. A) Protein bands from two rats representing each treatment group. B) Densitometric measurements of 130-kDa band in Western blots of rat uterus. Each bar graph represents mean ± SEM of samples from three rats. Bars with different letters on the top differ significantly (P < 0.01, ANOVA). M{Phi}: mouse macrophage



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  		FIG. 3. mRNA expression for NOS II in the rat uterus. Uterine tissues were obtained from rats euthanized 24 h after a single injection of sesame oil (control) or estradiol-17ß (E2, 0.5 or 5.0 µg/rat, s.c.) given on Day 18 of gestation. A) NOS II and ß-actin PCR products from two rats representing each treatment group. Molecular weight (bp) ladder is shown on the left. B) Relative levels of NOS II mRNA. Densitometric readings of NOS II are presented as ratio of readings of corresponding ß-actin samples. Each bar graph represents mean ± SEM of samples from three rats. Bars with different letters on the top differ significantly (P < 0.01, ANOVA). M{Phi}: mouse macrophage

NOS III protein and mRNA expression Because estradiol is known for its stimulatory effects on NOS III expression in other tissues [35], we measured the protein and mRNA content for NOS III in the pregnant rat uterus treated with estradiol. Figure 4 shows a single band on a Western blot with the predicted size of 140 kDa for NOS III. Figure 5 shows a single PCR product of 250 bp for NOS III. Significant increases (P < 0.05) were noted in both protein and mRNA levels of NOS III in the uterus upon administration of various doses of estradiol. These increases in NOS III expression occurred at the mRNA level and at the time when NOS II expression and total nitrite production were decreasing. These increases are dose dependent, with more substantial increases in rats receiving 5.0 µg estradiol (P < 0.01).



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  		FIG. 4. Expression of NOS III protein in the rat uterus using Western blot analysis. Uterine tissue was obtained from rats euthanized 24 h after a single injection of sesame oil (control) or 17ß-estradiol (E2, 0.5 or 5.0 µg/rat, s.c.) given on Day 18 of gestation. A) Protein bands from two rats representing each treatment group. B) Densitometric measurements of 140-kDa band in Western blots of rat uterus. Each bar graph represents mean ± SEM of samples from three rats. Bars with different letters on top differ significantly (P < 0.01, ANOVA). HE, Human endothelial cells



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  		FIG. 5. mRNA expression for NOS III in rat uterus. Uterine tissue was obtained from rats euthanized 24 h after a single injection of sesame oil (control) or 17ß-estradiol (E2, 0.5 or 5.0 µg/rat, s.c.) given on Day 18 of gestation. A) NOS III and ß-actin PCR products from two rats representing each treatment group. Molecular weight (bp) ladder is shown on the left. B) Relative levels of NOS III mRNA. Densitometric readings of NOS III are presented as ratio of readings of corresponding ß-actin samples. Each bar graph represents mean ± SEM of samples from three rats. Bars with different letters on the top differ significantly (P < 0.01, ANOVA). BPAE: bovine pulmonary artery endothelial cells

Effects of 17ß-Estradiol on NO Production and the Expression of NOS II and NOS III Enzymes in the LPS-Treated Nonpregnant Rats

Because 17ß-estradiol inhibited NOS II expression and NO production in the pregnant rat uterus, we investigated whether 17ß-estradiol suppresses the LPS-induced NOS II expression and NO production by the nonpregnant uterus.

Nitrite production Figure 6 shows that injection of LPS to ovx rats caused a significant (P < 0.01) increase in nitrite production by the uterus. Similarly, 17ß-estradiol administration also increased nitrite production by the uterus (P < 0.05). However, coadministration of LPS and 17ß-estradiol significantly (P < 0.05) attenuated the LPS-induced increases in nitrite generation to the levels similar to the group receiving 17ß-estradiol alone. These data indicate that 17ß-estradiol suppresses inducible nitrite production in the rat uterus.



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  		FIG. 6. Nitric oxide production by uterine tissues from ovx rats. Uterine tissue was obtained from ovx rats euthanized 24 h after s.c. injection of sesame oil (control), 17ß-estradiol (E2, 5.0 µg/rat), LPS (1 mg/rat), or a combination of E2 and LPS. Rat uteri were incubated in minimum essential medium for 24 h, and nitrite levels in culture medium were determined by Griess reaction. Results are mean ± SEM (n =6). Bars with different letters on the top differ significantly (P < 0.01 and P < 0.05, ANOVA)

NOS II mRNA expression Because 17ß-estradiol inhibited NOS II expression at the mRNA level, we examined NOS II mRNA levels in the ovx rats treated with LPS and 17ß-estradiol. Figure 7 shows that NOS II mRNA levels were minimal in ovx rats and were not induced by 17ß-estradiol. Treatment with LPS caused a dramatic (P < 0.01) elevation in the mRNA for NOS II in the ovx rat uterus. However, this LPS-induced increase in NOS II mRNA was abolished (P < 0.01) by 17ß-estradiol, indicating 17ß-estradiol suppresses the expression of NOS II.



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  		FIG. 7. mRNA expression for NOS II in rat uterus. Uterine tissue was obtained from ovx rats euthanized 24 h after s.c. injection of sesame oil (control), 17ß-estradiol (E2, 5.0 µg/rat), LPS (1 mg/rat), or a combination of E2 and LPS. A) PCR products from two rats representing each treatment group. Molecular weight (bp) ladder is shown on the left. B) Relative levels of NOS II mRNA. Densitometric readings of NOS II are represented as ratio of readings of corresponding ß-actin samples. Each bar graph represents mean ± SEM of samples from three rats. Bars with different letters on the top differ significantly (P < 0.01, ANOVA). M{Phi}: mouse macrophage

NOS III mRNA expression In contrast to the NOS II mRNA levels, NOS III mRNA in the uterus was substantially (P < 0.01) elevated with 17ß-estradiol treatment, as compared with levels in ovx animals (Fig. 8). Furthermore, LPS has no significant effect on uterus NOS III mRNA expression in ovx rats or in ovx rats treated with 17ß-estradiol, indicating that LPS does not regulate NOS III expression in the rat uterus.



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  		FIG. 8. mRNA expresson for NOS III in rat uterus. Uterine tissue was obtained from ovx rats euthanized 24 h after s.c. injection of sesame oil (control), 17ß-estradiol (E2, 5.0 µg/rat), LPS (1 mg/rat), or a combination of E2 and LPS. A) PCR products from two rats representing each treatment group. Molecular weight (bp) ladder is shown on the left. B) Relative levels of NOS III mRNA. Densitometric readings of NOS III are presented as ratio of readings of corresponding ß-actin samples. Each bar graph represents mean ± SEM of samples from three rats. Bars with different letters on the top differ significantly (P < 0.01, ANOVA). BPAE: bovine pulmonary artery endothelial cells

DISCUSSION

In the present study, we examined whether 17ß-estradiol affects the NO production and NOS II expression in the rat uterus during pregnancy, a physiological state when NOS II expression is elevated without exogenous administration of cytokines. Results show that 17ß-estradiol when administered to pregnant rats on Day 18 of gestation inhibited NO production by the uterus, suppressed both mRNA and protein of NOS II in the uterus, and increased the levels of mRNA and protein for NOS III in the uterus. These results demonstrate that 17ß-estradiol inhibits NOS II and stimulates NOS III expression, and the total NO production by the uterus is reduced. These results of estradiol administration together with our previous findings [14] in which antiestrogen, ICI 164 384, time-dependently stimulated the expression of mRNA for NOS II in the pregnant rat uterus provide strong evidence that estradiol during pregnancy is inhibitory to NOS II expression in the uterus. Thus, we suggest that decreased NO production at term through the reduction in NOS II expression in the uterus may be due to increased estradiol at term. These effects of estradiol may be even more pronounced with the concomitant decreases in progesterone levels that also occur at term, because progesterone is stimulatory to NOS II expression [11, 16, 18].

To further validate these findings in the pregnant rat uterus, we examined the effects of 17ß-estradiol on LPS-induced NOS II expression in the nonpregnant ovx rat. We also tried to separate the effects of estradiol from those of progesterone on NOS II expression; progesterone has been shown to be required for maintaining NOS II levels in the rat uterus during pregnancy [11, 16, 18]. Because NOS II was not detectable in the nonpregnant rats [11, 14, 16], we administered LPS to induce expression of NOS II mRNA in the uterus. Similar to reports for other tissues [36, 37], NOS II was induced in the uterus and thus mimicked the elevated NOS II expression condition without the pregnancy. Nakaya et al. [25] showed that injections of LPS (15 mg/kg) induced a significant increase in mRNA levels of iNOS in the rat uterus. These data are consistent with our results. This observation is critical for assessing whether estradiol effects on NOS II expression are related to pregnancy. Our findings show that 17ß-estradiol profoundly inhibited the LPS-induced NOS II expression in the ovx rats. This effect was seen in the absence of progesterone (ovx) or in the presence of progesterone (pregnancy), indicating that estradiol effects on NOS II are specific. These results indicate that estradiol inhibits NOS II, whether NOS II is expressed constitutively as in the case of pregnancy or is induced with LPS in the nonpregnant rat. In addition, we previously reported that nitrite production by the uterus from prepubertal rats was also inhibited by estradiol in both the presence and the absence of progesterone treatment. The molecular mechanism(s) by which estrogen inhibits the expression of NOS II remains to be elucidated.

The generation of NO by the uterus in response to estradiol administration was significantly reduced in both the pregnant and the LPS-treated ovx rats, and this reduction appears to be due to the suppression of NOS II expression. The reduction in NO generation by the uterus in both of these models of study occurred despite increases in the NOS III mRNA and protein. These results also suggest that the contribution from NOS II activity is greater than NOS III activity for the total NO production by the uterus in both of these models. These results also confirm those of previous studies [11, 14, 16] demonstrating that NOS II is the primary isoform that contributes to the majority of changes in NO production that occur during pregnancy and labor. These observations are consistent with the published reports that NOS II generates large amounts of NO, as compared with NOS III [17].

17ß-Estradiol inhibits basal nitrite production by unactivated hepatocytes in culture [38]. In addition, estradiol also reduces nitrite production by the stimulated mouse macrophages [39]. Furthermore, estradiol reduces the interleukin-1ß-induced excessive NO production in isolated rat thoracic aortic rings [40] and the LPS-induced elevation in plasma nitrite levels in ovx rats [41]. These reports indicate that estradiol inhibits NO production via reducing NOS II expression, and the present findings in the rat uterus are consistent with the results in these reports.

Both NO production and NOS II expression in the uterus were substantially reduced when 17ß-estradiol was exogenously administered to the pregnant rat. Estradiol levels during pregnancy are significantly elevated, and therefore the exogenously administered estradiol could be considered excessive and could result in inhibition of NOS II expression. This does not appear to be the case because NOS II expression was inhibited at both 0.5-µg and 5.0-µg doses of 17ß-estradiol and, more importantly, an antiestrogen, ICI 164 384, when administered to pregnant rats, increased NOS II expression significantly [16]. The results in this latter report [16] together with those of the current study demonstrate that during pregnancy estradiol is inhibitory to NOS II expression and that the well-documented precipitous elevation in serum estrogens [2] at term could suppress NOS II expression and thus NO production, facilitating labor. These inhibitory effects of estradiol on NOS II could also be amplified with the increase in the estrogen:progesterone ratio as a result of the fall in progesterone at term [2].

17ß-Estradiol up-regulated the NOS III expression in the uterus of nonpregnant ovx rats, confirming our previous findings [11, 19]. These observations are consistent with the reports of estrogen effects on the uterus of sheep [42] and on vascular tissues [43, 44]. Activity and expression of constitutive NOS have been reported to increase with estradiol administration and with pregnancy in several other tissues [43, 44]. Moreover, the 17ß-estradiol also increased NOS III expression in the uterus of pregnant rats in a dose-dependent manner, indicating the specificity of NOS III regulation by estradiol [11].

Our findings indicate that 17ß-estradiol inhibits NO production through the inhibition of NOS II expression in the rat uterus. This inhibition of NOS II expression by 17ß-estradiol occurs in both pregnant and nonpregnant ovx rats given LPS. The effects of 17ß-estradiol on NOS II expression appears to be at the transcriptional level. Therefore, 17ß-estradiol may play a role in regulating uterine NOS II expression and NO generation and thus uterine activity during pregnancy and labor.

FOOTNOTES

First decision: 24 September 1999.

1 This work was supported in part by NIH grants HD 30273 and HL 58144 to C.Y. Back

2 Correspondence: Chandra Yallampalli, Department of Obstetrics and Gynecology, The University of Texas Medical Branch, 301 University Boulevard, Route 1062, Galveston, TX 77555. FAX: 409 747 0475; chyallam{at}utmb.edu Back

Accepted: February 4, 2000.

Received: August 11, 1999.

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