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BOR - Papers in Press, published online ahead of print February 19, 2003.
Biol Reprod 2003, 10.1095/biolreprod.102.010785
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BIOLOGY OF REPRODUCTION 68, 2274–2280 (2003)
DOI: 10.1095/biolreprod.102.010785
© 2003 by the Society for the Study of Reproduction, Inc.

Pregnancy

Smoking Enhances Oxytocin-Induced Rhythmic Myometrial Contraction1

Makoto Egawa, Katsuhiko Yasuda2, Tatsuya Nakajima, Hidetaka Okada, Tomoo Yoshimura, Takashi Yuri, Masahiro Yasuhara, Tsuyoshi Nakamoto, Fumie Nagata, and Hideharu Kanzaki

Department of Obstetrics and Gynecology, Kansai Medical University, Moriguchi, Osaka, 570-0074, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although smoking during pregnancy is one of the major risk factors of premature delivery, the underlying mechanism by which smoking causes premature delivery is unknown. In the present study, we examined the effects of smoking on uterine contractility induced by oxytocin and prostaglandin F2{alpha}. Rats inhaled either cigarette smoke or room air from Day 14 to Day 16 of pregnancy through an inhalation apparatus for experimental animals (type "Hamburg II"). After the rats were killed on Day 17 of pregnancy, the uterine contractile sensitivity and activity on exposure to oxytocin or prostaglandin F2{alpha} were investigated. The expression levels of oxytocin-receptor mRNA and prostaglandin F2{alpha} receptor mRNA in the uterus were investigated by reverse transcription-polymerase chain reaction. The contractile activity was assessed as the contractile force and the frequency of rhythmic contractions of myometrial strips that were treated with oxytocin or prostaglandin F2{alpha}. The contractile sensitivity to oxytocin was significantly higher in the smoking group than in the control group (P < 0.01). Although the contractile force of oxytocin-induced contractions did not differ between the smoking and control groups, the frequency of contractions was significantly higher in the smoking group than in the control group (P < 0.01). On the other hand, no significant differences were found in the contractile sensitivity and activity in response to prostaglandin F2{alpha} between the smoking and control groups. The expression of oxytocin-receptor mRNA in the myometrium was significantly increased in the smoking group compared with the control group (P < 0.01). However, no significant difference was found in the level of expression of prostaglandin F2{alpha}-receptor mRNA between the two groups. These results suggest that smoking during pregnancy increases the contractile sensitivity and activity of the myometrium in response to oxytocin by up-regulating the expression of oxytocin-receptor mRNA. The effects of smoking on the contractile sensitivity and activity of the myometrium in response to oxytocin may increase the risk of premature delivery in smokers.

environment, oxytocin, parturition, pregnancy, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It has been established that smoking during pregnancy is one of the most important causes of fetal death and perinatal mortality and morbidity. The risks of low birth weight, spontaneous abortion, and premature delivery were increased among mothers who smoked during pregnancy [1]. Simpson [2] first suggested that smoking was associated with premature delivery in 1957. That author identified a dose-response relationship between cigarette smoking and premature delivery and that the incidence of low birth weight (<2500 g) among smokers was nearly twice that among nonsmokers. Other investigators also reported similar conclusions. Meis et al. [3] analyzed data in the Cardiff Birth Survey, which included more than 50 000 births, and reported that cigarette smoking was associated with a dose-dependent increase in the risk of premature delivery and that the odds ratio was 1.39 (95% confidence interval, 1.15–1.7) among women smoking more than 20 cigarettes daily. Meyer and Tonascia [4] analyzed data from the Ontario Perinatal Mortality Study and reported a 2-fold increase in the incidence of premature delivery (<36 wk) among smokers compared with that among nonsmokers. McDonald et al. [5] analyzed a large data set (n = 40 445) and concluded that smoking was responsible for 11% of all premature deliveries.

Clinical and experimental evidence has indicated that four primary pathogenic mechanisms are involved in premature labor: activation of the hypothalamus-pituitary-adrenal axes of the mother and fetus; inflammation of the amnion, chorion, and decidua; decidual hemorrhage; and pathologic distension of the uterine myometrium. Each mechanism has a distinct epidemiological and clinical profile as well as biochemical and biophysical pathways causing premature delivery; however, they share a common final pathway, resulting in myometrial contraction. Oxytocin and prostaglandin F2{alpha} induce pregnant uterine contractions via the oxytocin receptor (OTR) and prostaglandin F2{alpha} receptor (FP), respectively, and the pathways activated by the binding of oxytocin to the OTR and of prostaglandin F2{alpha} to the FP are involved in premature labor and delivery [6]. Prostaglandin F2{alpha}-receptor gene expression was detected in both pregnant and nonpregnant human myometria, although the level of expression in the pregnant myometrium was 55% of that in the nonpregnant myometrium [7]. Kimura et al. [8] reported that the mRNA expression level of human OTR in the myometrium was increased during pregnancy and was more than 300-fold higher at parturition compared with that in the nonpregnant myometrium. Both oxytocin and prostaglandin F2{alpha} have been clinically used for induction of delivery or termination of pregnancy. Prostaglandin-synthesis inhibitors (e.g., indomethacin) inhibit spontaneous uterine contractions and can be used for the treatment of premature labor and delivery [9]. It was reported that the contractile sensitivity to oxytocin was significantly increased in the premature delivery group (<37 wk) and reduced in the postterm delivery group compared with that in the term delivery group [10]. It was reported that the OTR antagonist, atosiban, reduced uterine contractility in women with threatened premature delivery [11, 12]. These reports suggest that the increased risk of premature labor and delivery in smokers is related to increased contractile sensitivity and activity of the uterine myometrium on exposure to oxytocin and/or prostaglandin F2{alpha} through the respective receptors.

In the present study, we investigated whether inhalation of cigarette smoke increases the contractile sensitivity and activity of the myometrium on exposure to oxytocin and prostaglandin F2{alpha} in the model of pregnant rats. In addition, the expression of OTR mRNA and of FP mRNA in the myometrium of pregnant rats was also evaluated.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals

Commercial cigarettes with filters were purchased from the Japan Tobacco Corporation (Tokyo, Japan). Atonin-0 (oxytocin) was obtained from Teizo (Tokyo, Japan). Prostaglandin F2{alpha} was purchased from Sigma (St. Louis, MO). TRIzol reagent; Superscript II reverse transcriptase; first-strand buffer; dithiothreitol (DTT); deoxy-ATP, -CTP, -GTP, and -TTP mixture (dNTPs); ribonuclease H; and agarose were purchased from Invitrogen (Rockville, MD). Taq DNA polymerase and PCR buffer were supplied by Takara Shuzo (Tokyo, Japan). Glycogen for the molecular biology experiments was purchased from Roche Diagnostics Corporation (Indianapolis, IN).

Animals

Pregnant Wistar rats were obtained from Oriental Bioservice Corporation (Kyoto, Japan). The rats were housed under a controlled condition (12L:12D photoperiod) and were provided with water and rat chow ad libitum. Rats in the smoking group inhaled the smoke of seven cigarettes per day on Day 14 to Day 16 of the pregnancy with an inhalation apparatus for experimental animals (type "Hamburg II"; Heinr. Borgwaldt, Hamburg, Germany). The inhalation was performed in the afternoon (from 1700 to 1800 h), and the duration of inhalation was approximately 6 min (cigarette smoke, 40 sec/1 cigarette; room air [interval], 10 sec). Rats in the control group were treated using the same method, except that room air was used instead of cigarette smoke. All rats were killed under ether anesthesia on Day 17 of the pregnancy (~18 h after the final inhalation). The uterus was removed and used for experiments. The fetuses and placenta were also removed, and the weight was measured to confirm the influence of cigarette smoke on fetal growth. This study was approved by the Animal Committee of Kansai Medical University.

Contractile Sensitivity and Activity

After the uterus was removed, myometrial strips were prepared (width, 2–3 mm; length, 10–15 mm). Each strip was attached to a holder under 1 g resting-tension. After equilibration for 60 min in a physiological saline solution, each strip was repeatedly exposed to 72.7 mM KCl solution until the response became stable. In the present study, myometrial strips with spontaneous contraction after preloading of the high K+ solution were excluded, because we would not be able to confirm whether contractions were caused by the agonist. After preloading of the high K+ solution, appropriate concentrations of oxytocin or prostaglandin F2{alpha} were added to evaluate the uterine sensitivity and activity. The physiological saline solution contained the following: NaCl (136.9 mM), KCl (5.4 mM), CaCl2 (1.5 mM), MgCl2 (1.0 mM), NaHCO3 (23.8 mM), glucose (5.5 mM), and EDTA (0.01 mM). The high K+ solution was prepared by replacing NaCl with an equimolar amount of KCl. These solutions were saturated with a 95% O2/5% CO2 (v/v) mixture at 37°C and pH 7.4. Muscle contraction was recorded isometrically with a force-displacement transducer (Model TB611T; Nihon Kohden, Tokyo, Japan) that was connected to a Model 3134 strain amplifier and Model 3056 ink-writing recorder (Yokogawa, Tokyo, Japan). We employed the contraction induced by 72.7 mM K+ solution as a reference response. The amplitude of the high K+-induced muscle contraction was set at 100%, and the amplitude of the oxytocin-induced or prostaglandin F2{alpha}-induced contraction was calculated in reference to the amplitude of the high K+-induced contraction. The number of oxytocin-induced or prostaglandin F2{alpha}-induced contractions were counted over 30 min, and the frequency of contractions was expressed as the mean value per 10 min.

Reverse Transcription-PCR for OTR and FP mRNA

Total RNA extraction and semiquantitative reverse transcription (RT)-PCR were performed using myometrial tissues. The oligonucleotide primers were custom synthesized by Kurabo Industries, Ltd. (Osaka, Japan). The sequences of the oligonucleotide primers used were as follows: OTR, 5'-AATCCGCACGGTGAAGATGACC-3' (forward primer) and 5'-ACGAGCAGAGCAGCAGAAGAAGC-3' (reverse primer); FP, 5'-GAAGTTTAGAAGTCAGCAGC-3' (forward primer) and 5'-ACTCAGAGATAGCAGCAACC-3' (reverse primer). These primers amplified a 241-base pair (bp; 807–1047) fragment of rat OTR cDNA [13] and a 359-bp (690–1048) fragment of rat FP cDNA [14], respectively. The sequences of the oligonucleotide primers of rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were 5'-TTGGCCGCCTGGTCACCAGGGCTGC-3' (forward primer) and 5'-GTTGTCATGGATGACCTTGGCCAGG-3' (reverse primer), and the amplified, 461-bp (35–495) fragment was used as the cDNA loading control.

Total RNA was extracted from myometrial tissues (0.3–0.5 g) by using TRIzol reagent. Four micrograms of total RNA was reverse transcribed with 200 U of Superscript II reverse transcriptase and 25 ng of random hexamer in 20 µl containing 4 µl of 5x first-strand buffer, 10 µM DTT, and 1 mM dNTPs at 42°C for 60 min. For a negative control, the same reaction was performed without reverse transcriptase.

One-fifth of the RT product was used as a template to amplify the cDNA for OTR, and one-tenth of the RT product was used as a template to amplify the cDNA for FP. The PCR was performed in a volume of 100 µl, containing 50 pmol each of forward and reverse primer, 2 µl of the cDNA mixture, 10 µl of 10x PCR buffer, 200 µM dNTPs, and 2.5 U of Taq DNA polymerase. The thermocycling conditions were 95°C for 5 min, followed by 35 cycles of 94°C for 30 sec, 55°C for 30 sec, and 72°C for 60 sec for the amplification of OTR. A final extension was performed at 72°C for 5 min. The thermocycling conditions for amplification of FP were the same, except that the temperature of annealing was 60°C. In the PCR amplification of GAPDH, which was used as an internal control for the PCR of OTR and FP, the temperature of annealing GAPDH primers was 55°C and 60°C, respectively. A linear relationship was observed between the number of cycles and the intensity of the RT-PCR product in the range of amplifications tested (data not shown). The PCR products were electrophoresed on 2% agarose gels containing 0.1% ethidium bromide. We visualized detectable fluorescent bands with an ultraviolet-transilluminator, and we saved the images using FAS III (Toyobo, Tokyo, Japan). The density of the bands for OTR or FP was normalized to that for GAPDH by scanning densitometry.

Statistical Analysis

The Mann-Whitney U test was used for statistical analysis of uterine sensitivity. The results were expressed as the mean ± SEM, and the nonpaired Student t-test was used for statistical analysis of uterine activity and receptor expression. A value of P < 0.05 was considered to be significant in the present study.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of Cigarette Smoking on Fetal and Placental Growth

Inhalation of the smoke of seven cigarettes per day on Day 14 to Day 16 of the pregnancy influenced the growth of the fetuses and placenta of the rats. The weight of the fetus on Day 17 of pregnancy was 796 ± 7 mg (n = 189) in the smoking group and 839 ± 8 mg (n = 188) in the control group. The weight of the fetus was significantly lower in the smoking group than in the control group (P < 0.0001). The weight of the placenta in the smoking group was also significantly lower than that in the control group (322 ± 5 mg for n = 189 vs. 368 ± 6 mg for n = 188, P < 0.0001).

Contractile Sensitivity to Oxytocin and Prostaglandin F2{alpha}

In the smoking group, spontaneous and rhythmic contractions of the myometrial strips occasionally occurred before stimulation by oxytocin or prostaglandin F2{alpha}. However, only a few spontaneous contractions occurred in the control group. Strips with spontaneous contraction were excluded from analysis in the present study. However, this finding indicates that the irritability was much greater in the smoking group than in the control group. As shown in Table 1, 8 (33%) of 24 myometrial strips in the smoking group responded to 25 µU/ml of oxytocin (final concentration). However, of the 24 myometrial strips in the control group, only 2 (8.3%) responded to the same dose of oxytocin. Although 20 strips (83%) in the smoking group were sensitive to oxytocin at 50 µU/ml or less (8 strips responded to 25 µU/ml and 12 strips to 50 µU/ml); 13 (54%) in the control group were sensitive to the same concentrations of oxytocin (2 strips responded to 25 µU/ml and 11 strips to 50 µU/ml). In addition, all myometrial strips in the smoking group responded to oxytocin at 100 µU/ml or less, although two strips in the control group did not contract on treatment with 100 µU/ml of oxytocin. Statistical analysis revealed that the contractile sensitivity to oxytocin was significantly increased in the smoking group compared with that in the control group (P < 0.01). The contractile sensitivity to prostaglandin F2{alpha} was slightly higher in the smoking group than in the control group, although the difference was not significant.


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  		TABLE 1. Contractile sensitivity of myometrial strips to oxytocin and prostaglandin F2{alpha} in the smoking and control groups.a

Contractile Activity (Force and Frequency)

In each group, the contractile force and frequency were evaluated in myometrial strips that had the same sensitivity to oxytocin or prostaglandin F2{alpha}. As shown in Figure 1, 50 µU/ml of oxytocin induced rhythmic contractions of myometrial strips in both the smoking and control groups. The contractile force was expressed relative to the amplitude of the high K+-stimulated contraction. Twelve myometrial strips in the smoking group responded to oxytocin at a concentration of 50 µU/ml, and the relative amplitude was 110.4 ± 6.3%. Eleven myometrial strips in the control group responded to 50 µU/ml of oxytocin, and the relative amplitude was 98.4 ± 4.7%. No significant difference was found in the contractile force between the two groups. In addition, the relative amplitude of the contractions of myometrial strips with the same sensitivity to prostaglandin F2{alpha} (10-7 M) in the smoking and control groups was 102.3 ± 15.5% (n = 7) and 104 ± 23.6% (n = 7), respectively. No significant difference was found between the two groups.



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  		FIG. 1. Oxytocin-induced contractions of myometrial strips of the smoking and control groups. Treatment of pregnant rats and preparation of myometrial strips were performed as described in the Materials and Methods. The uterine activity in both groups was evaluated by the relative amplitude and frequency of contractions. The two samples presented here had the same sensitivity to oxytocin (50 µU/ml)

The frequency of oxytocin (50 µU/ml)-induced contractions was 12.2 ± 1.6 per 10 min (range, 3–20) in the smoking group and 6.4 ± 1.0 per 10 min (range, 2–12) in the control group, showing a significant difference (P < 0.01) (Fig. 2A). However, no significant difference was found in the frequency of prostaglandin F2{alpha} (10-7 M)-induced contractions between the smoking group (12.0 ± 4.9 per 10 min, n = 7) and control group (12.9 ± 4.7 per 10 min, n = 7) (Fig. 2B).



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  		FIG. 2. Comparison of the frequency of oxytocin- and prostaglandin F2{alpha}-induced contractions in the smoking and control groups. After the contractions started, the frequency of oxytocin-induced or prostaglandin F2{alpha}-induced contractions was measured for 30 min and expressed as the mean value per 10 min. Values are expressed as the mean ± SEM. A) Myometrial strips of the smoking group (n = 12) and control group (n = 11) that responded to oxytocin at a concentration of as low as 50 µU/ml. B) Myometrial strips of the smoking group (n = 7) and control group (n = 7) that responded to prostaglandin F2{alpha} at a concentration of as low as 10-7 M. NS, Not significant

OTR and FP mRNA Expression

Because the level of OTR mRNA in the myometrium was very low, we compared the level of OTR mRNA in the myometrium of the smoking and control groups using the RT-PCR method, although this methodology was semiquantitative at best. We employed five myometrial strips in each group. In the smoking group, four of the five strips were sensitive to 25 µU/ml (final concentration) of oxytocin, and one strip was sensitive to 50 µU/ml of oxytocin. In the control group, all five strips were sensitive to 100 µU/ml of oxytocin. The RT-PCR revealed a single band of 241 bp, which was the position expected by the OTR primers (Fig. 3A). As shown in Figure 3B, the relative intensity of OTR mRNA to GAPDH mRNA was significantly greater in the smoking group than in the control group (0.34 ± 0.03 vs. 0.09 ± 0.03, P < 0.01).



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  		FIG. 3. RT-PCR analysis of mRNA expression of OTR in the smoking and control groups. A) RT-PCR products of OTR (top) and GAPDH mRNA (bottom). The PCR product of GAPDH was used as an internal control. Four micrograms of total RNA were reverse transcribed. One-fifth of the RT product was used as a template to amplify the cDNA for OTR and GAPDH. The thermocycling conditions were 95°C for 5 min, followed by 35 cycles of 94°C for 30 sec, 55°C for 30 sec, and 72°C for 60 sec. The arrow indicates the 241-bp fragment of rat OTR cDNA. Lanes 1–5 show samples in the smoking group. These samples responded to 25 µU/ml of oxytocin, except for the sample in lane 2, which responded to 50 µU/ml of oxytocin. Lanes 6–10 show samples in the control group. All samples in the control group responded to 100 µU/ml of oxytocin. Lane 11 shows a negative control. B) Comparison of the relative level of mRNA expression of OTR in the smoking and control groups. The level of OTR mRNA in each sample was expressed relative to the level of GAPDH mRNA in that sample. The data were analyzed by the nonpaired Student t-test. #Values were significantly different (P < 0.01)

We also performed RT-PCR for FP mRNA using the same myometrial tissues that had been used for evaluating the OTR mRNA. In the smoking group, one strip was sensitive to 5 x 10-8 M, two strips to 10-7 M, and two strips to 4 x 10-7 M of prostaglandin F2{alpha}. In the control group, three strips were sensitive to 10-7 M and two strips to 2 x 10-7 M of prostaglandin F2{alpha}. A single band of 359 bp was observed in the position expected by the FP primers (Fig. 4A). The relative intensity of FP mRNA to GAPDH mRNA in the smoking and control groups was 0.61 ± 0.09 and 0.69 ± 0.09, respectively (Fig. 4B). No significant difference was found in the level of FP mRNA expression between the smoking and control groups.



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  		FIG. 4. RT-PCR analysis of mRNA expression of FP in the smoking and control groups. A) RT-PCR products of FP (top) and GAPDH mRNA (bottom). Samples are the same as those presented in Figure 3. One-tenth of the RT product was used as a template for amplification of FP or GAPDH. The thermocycling conditions were 95°C for 5 min, followed by 35 cycles of 94°C for 30 sec, 60°C for 30 sec, and 72°C for 60 sec. The arrow indicates the 359-bp fragment of rat FP cDNA. Lanes 1–5 show samples in the smoking group. The samples in lanes 1 and 4 reacted to 4 x 10-7 M of prostaglandin F2{alpha}, those in lanes 2 and 3 to 10-7 M of prostaglandin F2{alpha}, and that in lane 5 to 5 x 10-8 M of prostaglandin F2{alpha}. Lanes 6–10 show samples in the control group. The samples in lanes 6 and 7 reacted to 2 x 10-7 M of prostaglandin F2{alpha} and the samples in lanes 8–10 to 10-7 M of prostaglandin F2{alpha}. Lane 11 shows a negative control. B) Comparison of the relative level of mRNA expression of FP in the smoking and control groups. The level of FP mRNA in a sample was expressed relative to the level of GAPDH mRNA in that sample


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Many authors have reported that cigarette smoking not only causes intrauterine growth retardation (IUGR) but also premature delivery. These studies revealed that the risk of premature delivery among smokers was 1.2- to 2.6-fold higher than that among nonsmokers [15–21]. However, the mechanism through which cigarette smoke causes premature labor and premature delivery has not been known. Oxytocin and prostaglandin F2{alpha} induce pregnant uterine contractions and are involved in premature delivery [22]. In 1980, Takahashi et al. [10] performed the oxytocin challenge test (n = 268) and reported no significant difference in uterine contractility among the premature, term, and postterm delivery groups; however, uterine sensitivity to oxytocin was significantly increased in the premature delivery group (<37 wk) and was reduced in the postterm delivery group in comparison with that in the term group [10]. In 1984, Fuchs et al. [23] measured the human OTR concentration in pregnant uterine muscle using a binding assay and reported that the OTR concentration was low at 13–17 wk but had increased approximately 12-fold by 37–41 wk. Furthermore, the OTR concentration in the pregnant uterine muscle of patients in premature labor (28–36 wk) who underwent delivery by cesarean section was significantly higher than that of patients in normal term pregnancy before labor [23]. Prostaglandin-synthesis inhibitors inhibit premature labor. Additionally, it has been reported that the oxytocin-antagonist atosiban inhibited premature labor and was comparable in clinical effectiveness to conventional ritodrine therapy [11, 12]. These reports suggested to us that smoking during pregnancy influences oxytocin-induced and/or prostaglandin-induced contractions.

In the present study, we found that inhalation of cigarette smoke for 3 days (seven cigarettes/day) by pregnant Wistar rats significantly reduced both the fetal and placental weight and could confirm that smoking during pregnancy is an important cause of low birth weight, which has been reported in the literature. We also found that the sensitivity of the pregnant rat uterus to oxytocin was increased in the smoking group compared with the control group. Additionally, the frequency of rhythmic contractions induced by 50 µU/ml of oxytocin was significantly higher in the smoking group than in the control group, although the amplitude of the contractions was similar in the two groups. Thus, inhalation of cigarette smoke during pregnancy increased not only the contractile sensitivity but also the contractile activity in response to oxytocin. However, although inhalation of cigarette smoke significantly increased the contractile sensitivity and activity in response to oxytocin, it did not significantly change the contractile sensitivity and activity in response to prostaglandin F2{alpha}. Both the sensitivity and activity on exposure to 10-7 M prostaglandin F2{alpha} were similar in the smoking and control groups. These results suggest that the number of OTRs in the uterine myometrium was higher in the smoking group than in the control group and that the number of FPs were similar in the two groups. As expected, RT-PCR revealed that the level of OTR mRNA expression in the myometrium of the smoking group was significantly higher than that in the control group, whereas the level of FP mRNA expression in the myometrium was similar in the two groups. This result indicates that cigarette smoke increased, either directly or indirectly, the number of OTRs in the pregnant myometrium.

Many factors influence the OTR level in the pregnant uterus. Steroid hormones (e.g., estrogen and progesterone), inflammatory cytokines (e.g., interleukin-1ß and interleukin-6), oxytocin, and lactation-suckling influence the expression of OTR at the mRNA or protein level [24]. Progesterone decreased the levels of uterine OTR mRNA and protein in both nonpregnant and pregnant rats [25], and estrogen increased the level of OTR in the rat uterus and in human cultured myometrial cells [26, 27]. Cigarette smoke contains thousands of chemical compounds that cross the placenta after direct or passive exposure to cigarette smoke. These compounds include nicotine, cotinine, carbon monoxide, polyaromatic hydrocarbons, thiocyanate, and many metal ions, including cadmium, lead, chromium, aluminum, and copper. Cigarette-smoke alkaloids (nicotine, cotinine, and anabasine) inhibited the production of progesterone in MA-10 Leydig tumor cells [28], and cigarette-smoke cadmium inhibited the production of progesterone in cultured human trophoblast [29]. However, pregnant women who smoked had lower estrogen levels than nonsmokers [30, 31]. Nicotine, cotinine, and anabasine inhibited aromatase in human granulosa cells and choriocarcinoma cells [32, 33], whereas cadmium inhibited estrogen production in whole-ovary cultures from nonpregnant and pregnant rats [34]. It is not known which inhibitory effect of smoking on steroid hormone production is dominant in the regulation of the OTR level in the myometrium. The stress involved in breathing acrid cigarette smoke may increase the levels of glucocorticoids (e.g., cortisol and corticosterone) in the maternal and/or fetal plasma, and the increased levels of glucocorticoids may influence OTR expression in the myometrium. However, the effect of glucocorticoids on OTR expression was varied in previous studies. Glucocorticoids increased the expression of OTR mRNA and the number of oxytocin-binding sites in the pregnant sheep uterus and cultured rabbit amnion cells [35, 36], although they reduced the number of oxytocin-binding sites induced by estrogen in cultured human uterine myometrial cells [37]. Thus far, no report, to our knowledge, on whether glucocorticoids up-regulate the expression of OTR mRNA in rat and human uterine myometria has appeared. Glucocorticoids also influence the levels of steroid hormones. Administration of glucocorticoids stimulated the conversion of pregnenolone to estrogen in sheep placenta by inducing placental 17{alpha}-hydroxylase [38] and resulted in an increased estrogen:progesterone ratio that had been observed at normal term in sheep. However, the human placenta lacks the enzyme 17{alpha}-hydroxylase, and the conversion of pregnenolone to estrogen does not take place. Therefore, estrogen synthesis in the placenta depends on the supply of its precursor, dehydroepiandrosterone sulfate, from the adrenal gland. In the human adrenal gland, however, glucocorticoids inhibit 17{alpha}-hydroxylase activity, which is involved in dehydroepiandrosterone sulfate production [39]. Interleukin-1ß (inflammatory cytokine) and oxytocin itself each reduced the OTR mRNA level in human myometrial cells and tissues [40–44]. Interleukin-6 had various effects on the expression of OTR mRNA. Schmid et al. [41] and Fang et al. [45] reported that interleukin-6 decreased the expression of OTR mRNA in human myometrial cells but increased its expression in the pregnant rat whole uterus (endometrium and myometrium) and did not influence its expression in the nonpregnant rat whole uterus. However, no report, to our knowledge, on whether smoking during pregnancy influences the levels of any cytokines in the pregnant uterus has previously appeared.

As mentioned above, the major compounds in cigarette smoke are nicotine and carbon monoxide. These compounds may directly influence OTR mRNA expression in the uterine myometrium. However, carbon monoxide itself does not have the ability to induce smooth muscle contraction, and thus far, no report, to our knowledge, regarding the relationship between carbon monoxide and OTR mRNA expression has appeared. Because carbon monoxide causes hypoxia, it might be involved in increasing the sensitivity and activity in response to oxytocin, and it may affect the expression of OTR mRNA in the myometrium. However, hypoxia reduced the myometrial contractile response to oxytocin as well as the number of oxytocin-binding sites in the pregnant rat uterus [46–48]. Additionally, it was reported that hypoxia did not influence the expression of OTR mRNA in cultured human uterine smooth muscle cells [42]. Nicotine has the ability to induce arterial smooth muscle contraction, tracheal smooth muscle contraction, and gastrointestinal smooth muscle contraction, which in turn may increase the uterine smooth muscle contractile response to oxytocin. Nicotine is rapidly and extensively metabolized to many different compounds, the most notable being cotinine, which has a much longer half-life than nicotine, in the liver [49]. The half-life of nicotine following i.v. administration was reported to range from 0.9 to 1.1 h in rats [50, 51] and from 1 to 3 h in humans (men) [49]. On the other hand, the half-life of cotinine was reported to range from 4.8 to 5.3 h in rats [50] and from 10 to 17 h in humans (men) [49, 52–54]. In addition, it was reported that the nicotine concentration in the maternal plasma decreased after overnight cigarette abstinence, although the cotinine concentration remained high and was more than 10-fold higher than the nicotine concentration [55]. Therefore, not only nicotine but also cotinine and other nicotine metabolites with a long half-life might cause the increases in the contractile sensitivity and activity of the myometrium in response to oxytocin through a mechanism involving stimulation of OTRs. However, no report, to our knowledge, on whether nicotine and cotinine increase the OTR level in the pregnant uterus has appeared. Further studies are needed to examine the mechanism through which smoking up-regulates OTR mRNA expression in the pregnant myometrium.

In summary, the present results in an animal model indicate that smoking during pregnancy affects fetal and placental growth and increases the contractile sensitivity and activity of the myometrium in response to oxytocin through a mechanism involving the stimulation of OTR mRNA expression. These findings suggest that smoking during pregnancy increases not only the risk of IUGR but also the risk of premature labor and delivery in humans by increasing the level of myometrial OTRs. Further studies on compounds that up-regulate the level of OTR mRNA in the myometrium may yield valuable information regarding the physiology and pathophysiology in parturition.


   ACKNOWLEDGMENTS
 
The authors thank Dr. Michiaki Mishima (Department of Respiratory Medicine, Kyoto University, Japan) who kindly offered the inhalation apparatus for experimental animals (type "Hamburg II") used in the present study. The authors also thank Drs. Hiroshi Ozaki and Masatoshi Hori (Department of Veterinary Pharmacology, University of Tokyo, Japan) for their advice. The secretarial assistance of Ms. Miyuki Imai, Ms. Noriko Sugie, Ms. Wakako Okamoto, and Ms. Ayuko Morikawa is also greatly appreciated.


   FOOTNOTES
 
1 Supported in part by grants from the Japan Smoking Research Foundation. Back

2 Correspondence: Katsuhiko Yasuda, Department of Obstetrics and Gynecology, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi, Osaka, 570-0074, Japan. FAX: 81 6 6992 3438; yasuda{at}takii.kmu.ac.jp Back

Received: 6 October 2002.

First decision: 24 October 2002.

Accepted: 24 January 2003.


   REFERENCES
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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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