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Effect of cigarette smoking on mRNA and protein levels of oxytocin receptor and on contractile sensitivity of uterine myometrium to oxytocin in pregnant women

European Journal of Obstetrics & Gynecology and Reproductive Biology, pages 142 - 147

Abstract

Objectives

Although smoking is the most important modifiable risk factor associated with preterm delivery, the underlying mechanism by which smoking stimulates premature uterine contractions is still poorly understood. In the present study, we investigated whether cigarette smoking affects the contractile sensitivity of uterine myometrium to oxytocin in pregnant women.

Study design

Cigarette smoking habits of pregnant women were evaluated by direct interviews and by measuring exhaled carbon monoxide (CO). We isolated myometrial strips from pregnant smokers and non-smokers and evaluated uterine contractile sensitivity to oxytocin. Gene expression levels of oxytocin receptors (OTR) were compared between myometrial strips obtained from smokers and non-smokers by real-time PCR. OTR protein levels in the myometrium were evaluated by Western blotting.

Results

The reported number of cigarettes smoked per day by the interviewee significantly correlated with the concentration of exhaled CO. Oxytocin sensitivity increased significantly in smokers (n = 6) compared with non-smokers (n = 11). Real-time PCR analysis did not reveal any significant difference in OTR mRNA expression between smokers and non-smokers. Western blotting revealed that OTR level was significantly increased in smokers compared with non-smokers. Both number of cigarettes smoked per day and the concentration of exhaled CO correlated with oxytocin sensitivity.

Conclusion

Our findings suggest that smoking increases oxytocin sensitivity of pregnant myometrium by increasing OTR levels even though OTR mRNA expression remains unaltered, thereby increasing the risk of preterm delivery in women who smoke during pregnancy. The sensitivity is dependent on number of cigarettes smoked per day.

Keywords: Oxytocin receptor sensitivity, Cigarette smoking, Uterine myometrium, microRNAs.

Introduction

Smoking during pregnancy is related to spontaneous abortion, preterm delivery, intrauterine growth retardation, low birth weight, and fetal congenital anomalies [1] and [2]. It is well known that oxytocin and prostaglandin F2α induce pregnant uterine contractions via the oxytocin receptor (OTR) and prostaglandin F2α receptor (FP), respectively, and that the pathways activated by the binding of oxytocin to OTR and prostaglandin F2α to FP are involved in preterm labor [3] . Although FP mRNA was detected in both pregnant and nonpregnant human myometrium, the level of expression in the pregnant myometrium was lower compared in the nonpregnant myometrium [4] . Contrary, OTR mRNA significantly increased in the human uterus depending on the gestational week [5] . We previously reported that smoking increases the sensitivity of the pregnant myometrium to oxytocin via upregulation of OTR mRNA in a rat model of cigarette smoke inhalation [6] . We also reported that cigarette smoke extract (CSE) directly increases the contractile sensitivity of rat and human preterm myometrium to oxytocin by up regulating the OTR mRNA expression [7] . However, these studies involved experimental animals with acute and compulsive exposure to cigarette smoke or isolated animal and human myometrium incubated with CSE in vitro. Thus, it is hitherto unclear whether smoking stimulates oxytocin sensitivity in women who smoke by preference and those that are chronically exposed to cigarette smoke during pregnancy. In this study, we investigated uterine sensitivity to oxytocin, myometrial levels of OTR mRNA and protein, and a correlation between the sensitivity to oxytocin and the smoking status in smoking and non-smoking pregnant women.

Materials and methods

Chemicals

Oxytocin was purchased from Sigma-Aldrich Corp., USA. TRIzol reagent, phenol red-free Dulbecco's modified Eagle's medium (DMEM), F-12 medium, random primers, DNase I amplification grade and 10× Gene Taq DNA polymerase gene Taq, 10× Gene Taq universal buffer, and RNase inhibitor were purchased from Wako Pure Chemical Co. Ltd, Japan. ReverTra Ace-α-kit(ReverTra Ace reverse transcriptase, dNTP mixture, 5× RT buffer) and anti-Taq high were purchased from Toyobo Co. Ltd, Japan).

Selection of patients and measurement of exhaled carbon monoxide

Pregnant women who smoked at least one cigarette/day (averaged number/day of the pregnancy period) were selected via interviews, and carbon monoxide (CO) levels in exhaled air were measured using CO-oximetry (Micro-Smokerlyzer®; UK). Exhaled CO is a simple and noninvasive method for evaluating smoking status and is often used to verify acute and chronic abstinence from smoking because of the direct relationship between the levels of CO and the number of cigarettes smoked [8] and [9]. Exhaled CO concentration was measured within 6 h after admission. The women were first asked to exhale completely, inhale fully, and then hold their breath for 15 s. Next, they were asked to exhale slowly and fully into the Micro-Smokerlyzer® in order to collect alveolar air. Results were expressed as parts per million (ppm).

Tissue collection

Tissue samples were obtained from patients undergoing elective lower segment cesarean section (ELSCS) for obstetrical indications. Almost all patients underwent ELSCS for previous cesarean section on or after 37 weeks of gestation. Intrauterine growth retardation (IUGR) and IgA nephropathy were observed in one patient in the non-smoking group, and she underwent ELSCS for disease progression of IgA nephropathy at 35 weeks of gestation. IUGR and posttraumatic stress disorder were observed in one patient in the smoking group, and she opted for ELSCS at 35 weeks of gestation. Routine cesarean section was performed under epidural or spinal anesthesia. After delivery of the infant and placenta, a sample of myometrium was incised from the upper margin of the lower uterine segment using tissue forceps and scissors before administering oxytocin. All tissue samples were immediately immersed in normal saline solution followed by ice-cold Viaspan (Belzer UW; Bristol-Myers Squibb, USA) and transported to the laboratory for use within 6 h of dissection. A fraction of each sample was used for assessment of contractile sensitivity, and the remaining sample was used for RNA and protein extraction. This study was approved by the ethics committee of our university and performed according to the Declaration of Helsinki. Written informed consent was obtained from each patient.

Preparation of myometrial strips and contractile sensitivity

Myometrial strips were prepared and the contractile sensitivity was evaluated as described previously [6] and [7]. Briefly, each strip was attached to a holder under 1 g force resting-tension. After equilibration for 60 min in physiological saline solution (PSS), each strip was repeatedly exposed to 72.7 mM KCl solution (high K+ solution) until the response was stabilized. PSS was prepared as described previously [10] . 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 mixture at 37 °C, pH 7.4. After exchanging high K+ solution with PSS, oxytocin was added to the solution to induce rhythmic contractions. Oxytocin sensitivity was defined as the concentration of oxytocin at which the first rhythmic myometrial contraction occurred.

Myometrial contractions were recorded into a personal computer, and the data were analyzed with the Unique Acquisition software package (Unique Medical Co. Ltd., Japan).

RNA extraction and reverse transcription

Total RNA was extracted using TRIzol reagent and dissolved in the appropriate amount of 0.001% diethyl pyrocarbonate water. Reverse transcription (RT) was performed using the RevaTra Ace-α-Kit according to the manufacturer's instructions. Briefly, 1 μg total RNA treated with DNase I was suspended in 20 μL reaction mixture (final concentration: 50 mM Tris–HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol (DTT), 0.1 μM random primer, 1 mM TTP, 0.3 μM (methyl-3H) dTTP, 1 mM dNTP) containing 100U ReverTra Ace reverse transcriptase and 5×RT buffer and incubated at 30 °C for 10 min, 42 °C for 20 min, and 99 °C for 5 min in a TAKARA PCR Thermal Cycler MP (TaKaRa Shuzo, Japan). Successful RT was confirmed for all samples by performing PCR amplification of elongation factor1α (EF1α) as an internal control. PCR product was analyzed by electrophoresis on a 2.0% agarose gel (Invitrogen) and stained with ethidium bromide. The EF-1α oligonucleotide primer sequences were 5′-TCTGGTTGGAATGGTGACAACATGC-3 (forward)′ and 5′-AGAGCTTCACTCAAAGCTTCATGG-3′ (reverse) [11] . The Human OTR oligonucleotide primer sequences were 5′-ATGTGGAGCGTCTGGGATGC-3′ (forward) and 5′-GCTCAGGACAAAGGAGGACG-3′ (reverse) [12] . These sequences amplified a 329-bp (595–923) fragment of EF-1α cDNA and a 237-bp (1508−1744) fragment of human OTR cDNA.

Quantitative real-time PCR (qPCR)

qPCR was performed in a volume of 25 μL, comprising 2.5 μL cDNA template, 1 μL each of forward and reverse primers (final concentration 375 nM) and 12.5 μL THUNDERBIRD SYBR qPCR Mix (TOYOBO). Samples were processed on a Rotor-Gene G (Qiagen) at the following conditions: 1 min, 95 °C (1 cycle); 40 cycles of 15 s at 95 °C, 15 s at 60 °C, and 45 s at 72 °C. During PCR, fluorescence accumulation resulting from DNA amplification was recorded, and cycle threshold (Ct) values were obtained from the exponential phase of PCR amplification. The Ct values for each gene were normalized against Ct values for EF1α as follows: ΔCt = specific gene Ct −EF1α Ct. Relative expression was then calculated according as 2−ΔCt.

Western blotting

For Western blotting of OTR protein levels, samples of myometrium were homogenized for 1 min on ice in radioimmunoprecipitation assay (RIPA) buffer (Wako, Japan) and protease inhibitor cocktail set III (Calbiochemas®, Germany). Samples were centrifuged at 10,000 × g for 10 min at 4 °C. Supernatants were collected, and protein concentrations were determined by Pierce® BCA™ Protein Assay Kit (TAKARA BIO INC). Twenty μg of protein was mixed with 5× sample buffer boiled for 5 min, electrophoresed on a 7.5% sodium dodecyl sulphate–polyacrylamide gel (200 V, 60 min) and transferred to Immune-Blot® polyvinylidene difluoride membrane (Bio-Rad, Laboratories, USA) (15 V, 30 min). Non-specific binding sites were blocked with 10% skimmed milk powder in Tris-buffered saline for 30 min. Blots were then incubated for 1 h at room temperature with the goat monoclonal oxytocin receptor (N-19) antibody (1:200; Santa Cruz Biotechnology, USA) or mouse monoclonal β-actin antibody (1:5000; Sigma-Aldrich Corp., USA). Membranes were then incubated with anti-goat IgG peroxidase-labeled secondary antibody (1:5000; Santa Cruz, USA) or anti-mouse IgG peroxidase-labeled secondary antibody (1:10000; GE, USA). Immune complexes were visualized by enhanced chemiluminescence (ECL) plus Western Blotting Detection Reagents (GE, NYSE, USA). OTR levels were analyzed using image J software, and relative protein intensity was calculated by normalizing the OTR expression values to those of β-actin.

Statistical analysis

Data are expressed as mean ± SEM. Results were analyzed with a statistical software package, StatView II version 4.0 (Abacus Concepts, CA). Differences in the measured parameters across the different groups were statistically assessed using ANOVA with repeated measurements followed by t-test. A level of P < 0.05 was considered statistically significant.

Results

Characteristics of non-smoking and smoking groups

We interviewed 6 smokers (smoking group) and 11 non-smokers (non-smoking group) about smoking status. Patients in the non-smoking group neither smoked before pregnancy nor did they have family members who smoked. The characteristics of the 2 groups are shown in Table 1 . No significant differences in gestational age, birth weight, placenta weight, and occurrence rate of pregnancy complications between the 2 groups were observed. Maternal age in the smoking group was significantly lower than that in the non-smoking group. The concentration of exhaled CO in the smoking group was significantly greater than in the non-smoking group. Additionally, there was a significant correlation between the concentration of exhaled CO and the number of cigarettes smoked per day (Y = 0.8X + 1.035, R2 = 0.545, P < 0.0001).

Table 1 Characteristics of the non-smoking and smoking groups.

  Non-smoking group (n = 11) Smoking group (n = 6)
Smoking status (no. of cigarettes/day) 0 (0, 0) 11.3 (3.9, 2–20)
Exhaled CO (ppm) 0.46 (0.25, 0–2) 11.17 ** (4.7, 0–26)
Age (years old) 36.5 (1.0, 30–41) 28.3 *** (1.8, 24–35)
Gestation period 37W3D (1.9D, 35W0D–38 W3D) 37W2D (2.2D, 35W0D–38 W0D)
Baby weight (g) 2968.2 (175.2, 1490–3450) 2596.0 (63.6, 2470–2900)
Placenta weight (g) 560.9 (38.2, 300–790) 564.2 (10.5, 535–600)
Pregnancy complications GDM (n = 1), PD (n = 1), IUGR (n = 1) PROM (n = 1), PD (n = 1), IUGR (n = 2), NRFS (n = 1)
Maternal complications IgA nephropathy (n = 1) PTSD (n = 1)

** Shows significant difference P < 0.01.

*** Shows significant difference P < 0.001.

Data are represented as mean (SE, range). GDM, gestational diabetes mellitus; IUGR, intrauterine growth retardation; PROM, premature rupture of the membrane; PD, preterm delivery; NRFS, non-reassuring fetal status; PTSD, posttraumatic stress disorder. One smoker showed three complications, IUGR, PROM, and NRFS.

Contractile sensitivity to oxytocin

A myometrial strip from a non-smoker was sensitive to oxytocin at 50 μU/ml, whereas that from a smoker was sensitive at as low as 5 μU/ml ( Fig. 1 ).

gr1

Fig. 1 Oxytocin-induced rhythmic contractions of myometrial strips from a non-smoker and smoker.

The contractile sensitivity was 40.46 ± 4.01 μU/ml (20−50 μU/ml) and 16.67 ± 7.38 μU/ml (5−50 μU/ml) in the non-smoking and smoking groups, respectively, i.e. significantly greater than that in the non-smoking group (P < 0.01) ( Fig. 2 ). A significant correlation between contractile sensitivity and the smoking status (Y = −1.674X + 38.754. R2 = 0.475, P < 0.01) ( Fig. 3 A) and contractile sensitivity and the concentration of exhaled CO was observed (Y = −1.487X + 38.357. R2 = 0.44, P < 0.01) ( Fig. 3 B).

gr2

Fig. 2 Sensitivity of myometrial strips to oxytocin in the non-smoking (n = 11) and smoking (n = 6) groups. O indicates non-smoker. △ indicates smoker. ** (P < 0.01).

gr3

Fig. 3 (A) Relationship between oxytocin sensitivity and smoking status. (B) Relationship between oxytocin sensitivity and concentration of exhaled CO. O indicates non-smoker. △ indicates smoker.

Expression of OTR mRNA in myometrium

RT-PCR analysis revealed single bands of 237 bp and 329 bp corresponding to OTR and EF-1α cDNAs, respectively ( Fig. 4 A). OTR mRNA intensity appeared to be lower among smokers compared to non-smokers, whereas the intensity of EF-1α was comparable.

gr4

Fig. 4 (A) Agarose gel electrophoresis with ethidium bromide staining of RT-PCR products amplified with specific primers for OTR and EF-1α. RT-PCR of OTR and EF-1α mRNA was performed with 27 cycles and 25 cycles, respectively. (B) Comparison of OTR mRNA expression in the non-smoking and smoking groups.

The relative OTR mRNA level was 43.36 ± 14.44 × 10−3 in the non-smoking and 8.94 ± 1.07 × 10−3 in the smoking group. Although OTR mRNA level was lower in the smoking group compared to the non-smoking group, the difference was not significant ( Fig. 4 B).

Expression of OTR protein in myometrium

Western blotting revealed a band of 66 kDa corresponding to OTR protein [12] ( Fig. 5 A). β-Actin (internal standard) revealed a 45 kDa band [13] . The relative OTR protein level was 0.484 ± 0.046 in the non-smoking group and 0.914 ± 0.082 in the smoking group. The relative OTR protein level was significantly greater in the smoking group than in the non-smoking group (P < 0.001) ( Fig. 5 B). A significant correlation between oxytocin sensitivity and the OTR protein level was observed (Y = −37.274X + 56.359, R2 = 0.268, P < 0.05).

gr5

Fig. 5 (A) Western blot analysis of OTR in pregnant myometrium from non-smokers and smokers. β-Actin was used as internal standard. (B) Comparison of OTR protein level in the non-smoking and smoking groups. *** (P < 0.001).

Comments

We evaluated the relationship between the exhaled CO concentration and interviewee's smoking status during pregnancy and showed a significant correlation between these two factors. The results support the credibility of our interview and the benefit of measuring exhaled CO for evaluating the smoking status during pregnancy. The smoking group included younger women compared to the non-smoking group, thereby suggesting that younger pregnant women have poor self-control and are unable to quit smoking during pregnancy.

We compared the sensitivity to oxytocin between smokers and non-smokers. As expected, the sensitivity to oxytocin is significantly greater in the smokers than in the non-smokers. Additionally, a significant correlation between the sensitivity to oxytocin and the smoking status or exhaled CO was obtained. In the present study, maternal age was significantly lower in the smoking group than in the non-smoking group. Therefore, maternal age might affect contractile sensitivity to oxytocin in pregnant myometrium. However, our preliminary experiment did not yield significant differences in the sensitivity to oxytocin between non-smokers in their 20s and non-smokers in their 30s in term pregnancy (data not shown). These findings suggest that smoking increases the sensitivity of pregnant myometrium to oxytocin, and the sensitivity is dependent on smoking status and exhaled CO. In the present study, we found that the rate of pregnancy complications in the non-smoking vs. smoking group was equivalent. This result may contradict previously published data that indicate smoking increases pregnancy complications. The smoking group was probably too small for such smoking-related complications of pregnancy to be visible, although it was sufficient for detecting the differences in oxytocin sensitivity.

The increased sensitivity to oxytocin suggests an increased OTR mRNA expression in the smokers compared with the non-smokers. Surprisingly, real-time PCR revealed that the OTR mRNA expression was lower in smokers than in non-smokers although the difference was not significant. This result is contradictory to previous studies using a rat model of forced inhalation of cigarette smoke and in vitro experiments concerning direct effect of CSE on OTR mRNA expression in rat and human myometrium [6] and [7].

We therefore evaluated the OTR protein level using Western blotting, and found that OTR protein level was significantly higher in the smokers than in the non-smokers and significantly correlated with the sensitivity to oxytocin. Many factors affect OTR levels in pregnant uterus. Steroid hormones, inflammatory cytokines, oxytocin, and lactation affect the expression of OTR at mRNA or protein level. Estrogen up-regulates OTR mRNA in rat uterus and OTR protein level increases concomitantly in the rat uterus; however, progesterone inhibits the effect of estrogen [14] . Therefore, estrogen/progesterone ratio may be important to evaluate the effect of steroid hormones on OTR mRNA expression and protein levels. Smoking is known to have adverse effects on placental growth and function and to influence the production and metabolism of steroid hormones in human placenta [15], [16], and [17]. Therefore, production and metabolism of estrogen and/or progesterone in placenta are probably altered in pregnant smokers. Some reports indicate that smoking decreases the level of estrogen in pregnant women [16] and [17]. Whether smoking increases estrogen/progesterone ratio in humans, however, remains obscure. Both OTR mRNA and protein significantly increase in the human uterus depending on the gestational week [5] . Thus, the protein level of OTR is thought to depend on OTR mRNA expression in the uterus. However, our results are not consistent with the earlier reports. The changes in OTR mRNA expression did not correlate with the changes in OTR protein level.

Recent reports show that protein levels are regulated by specific microRNAs (miRNAs) [18] and [19]. miRNA is known to inhibit protein translation and promote mRNA degradation. The miRNA-200 family modulates OTR mRNA and connexin-43 leading to uterine contraction in pregnant myometrium [20] . In addition, changes in miRNA expression are seen in smoking-related diseases, such as cancer, chronic obstructive pulmonary disease, and cardiovascular diseases [21] .We therefore speculate that smoking inhibits the miRNA that inhibits the translation of OTR protein from OTR mRNA in the myometrium of pregnant women. OTR protein levels increase and oxytocin sensitivity subsequently increases in the myometrium of pregnant smokers. Even though oxytocin sensitivity in the myometrium was significantly greater in the smoking group than in the non-smoking group, no significant differences were observed in terms of incidence of preterm delivery between the groups probably because of the small sample size of the smoking group. Moreover, both OTR and oxytocin are important in inducing myometrial contractions. Very low concentrations of oxytocin may not induce myometrial contractions even though pregnant uterus may have significant levels of OTR. Therefore, the concentration of oxytocin in the smoking group was probably not enough to induce myometrial contractions, but was sufficient to elicit oxytocin sensitivity via the increased number of OTR. In such a case, preterm delivery may not always occur even in the smoking group. No reports exist concerning the correlation between OTR mRNA, miRNA, and OTR protein in the pregnant myometrium. Further studies are required to elucidate the mechanism by which miRNAs regulate OTR protein expression.

Condensation

Smoking increases oxytocin sensitivity of uterine myometrium in pregnant women by increasing OTR protein levels, but not OTR mRNA expression, thereby increasing preterm delivery risk.

Acknowledgments

We thank Ms. Miyuki Imai for her excellent technical assistance and Ms. Khono for her secretarial assistance. This work was supported by grant from the Japan Smoking Research Foundation.

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Footnotes

a Department of Obstetrics and Gynecology, Affiliated Takii Hospital, Kansai Medical University, Osaka, Japan

b Division of Cardiology, Department of Medicine II, Kansai Medical University, Osaka, Japan

c Department of Obstetrics and Gynecology, Kansai Medical University, Osaka, Japan

lowast Corresponding author. Tel.: +81 6 6992 1001; fax: +81 6 6992 8779.