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Regular Article |
a Instituto de Investigaciones Químicas, Centro de Investigaciones Científicas Isla de la Cartuja, 41092 Sevilla, Spain
b Departament de Farmacología, Facultat de Farmàcia, Universitat de València, 46100 Valencia, Spain
c Centre Hospitalier de Versailles et Laboratoire de Pharmacologie, Faculté de Médecine Paris-Ouest, 78257 Versailles, France
ABSTRACT
In the mammalian female reproductive tract, tachykinin neuropeptides, such as substance P (SP), are localized to a population of sensory fibers and their precise physiological role is still unknown. The aim of the present study was to characterize the population of tachykinin receptors in the pregnant rat uterus and to assess their regulation during the course of pregnancy and after delivery. The expression of the tachykinin NK1 receptor (NK1R), the tachykinin NK2 receptor (NK2R), and the tachykinin NK3 receptor (NK3R) in uteri from rats at different stages of pregnancy and on Day 1 postpartum was investigated by using a semiquantitative reverse transcription-polymerase chain reaction. The contractile effect of tachykinin receptor agonists acting selectively on the NK1R, the NK2R, or the NK3R was investigated by conventional organ bath techniques. Serum levels of estrogen and progesterone were measured by RIA. Our data show that the expression and function of NK1R and NK3R varied along the course of pregnancy and at postpartum. Uterine NK2R mRNA levels remain stable during the course of pregnancy and at Day 1 postpartum; and the contractions elicited by activating selectively the NK2 receptor in the presence of the neutral endopeptidase inhibitor phosphoramidon (1 µM) were similar in early, mid, or late pregnancy. These results show that the expression and function of tachykinin receptors within the uterus vary with reproductive state and length of gestation, supporting a role for tachykinins in pregnancy and/or parturition in the rat.
estradiol, female reproductive tract, gene regulation, neuropeptides, pregnancy, progesterone, uterus
INTRODUCTION
The tachykinins represent a family of peptide neurotransmitters including substance P (SP), neurokinin A (NKA) and neurokinin B (NKB). They interact with three distinct types of receptors termed NK1 (NK1R), NK2 (NK2R), and NK3 (NK3R) that are preferentially activated by SP, NKA, and NKB, respectively [1, 2]. The corresponding cDNA coding for these receptors had been cloned by means of molecular biological methods [3–6]. In the rat, NK1R, NK2R, and NK3R consist of 407, 390, and 452 amino acid residues, respectively [3–5, 7], and belong to the family of G protein-coupling cell membrane receptors [6].
Tachykinins are localized in sensory nerves supplying a number of mammalian peripheral tissues [2, 8], and it has been suggested that they may be locally released causing pain and neurogenic inflammation [9–11]. In the female reproductive tract, SP and NKA coexist with calcitonin gene-related peptide in a population of sensory nerves, the presence of which has been demonstrated in virtually all mammalian species examined [12]. In uteri from virgin rats SP, NKA, and NKB induce contraction mainly mediated by activation of NK2R [13–16] and regulated by the hormonal environment [17, 18]. It has also been suggested that NK1R and NK3R may be more important than NK2R in the hormonally dependent fine control of uterine motility [18]. Tachykinin NK1R, NK2R, and NK3R mRNAs are expressed in the nonpregnant rat uterus, and expression levels vary under different hormonal conditions [14, 17, 19]. Moreover, a recent report has shown that the placenta of both human and rats secretes NKB and that an excessive secretion of this tachykinin at late pregnancy causes preeclampsia [20]. These data argue for a role of tachykinins and their receptors in the regulation of uterine functions [13–19]. However, no study has been carried out, to our knowledge, to investigate the role of these neuropeptides in rat myometrial contractility along the course of pregnancy. The aim of the present study was to characterize the population of tachykinin receptors in the rat uterus during pregnancy and after delivery. For this purpose, we studied the expression of tachykinin NK1R, NK2R, and NK3R in uteri from rats at different stages of pregnancy and on Day 1 postpartum. Serum levels of estrogen (E2) and progesterone (P4) were measured on the same animals by RIA. Additionally, functional studies were carried out to investigate the influence of pregnancy on the contractile responses elicited by the tachykinin receptor agonists (Sar9Met[O2]11)-SP, NKA, and (MePhe7)-NKB in the rat uterus. The response to (Sar9Met[O2]11)-SP was studied in the presence of the selective NK2 receptor antagonist SR 48968 (Saredutant) [21] and the selective NK3 receptor antagonist SR 142801 (Osanetant) [22] to act selectively on NK1R. The response to NKA was studied in the presence of the selective NK1 receptor antagonist SR 140333 (Nolpitatium) [23] and SR 142801, to act selectively on NK2R. The response to (MePhe7)-NKB was studied in the presence of SR 140333 and SR 48968 to act selectively on NK3R.
MATERIALS AND METHODS
Animals and Tissue Preparation
All experiments were conducted in accordance with National Institutes of Health guidelines for the care and use of laboratory animals. Three-month-old virgin female Wistar rats were purchased from Charles River Laboratories (Criffa, Spain). Animals were maintained in an air-conditioned room at 22°C under controlled lighting (12L:12D) and provided with food and water ad libitum. Vaginal smears were checked daily and pregnancy was produced by mating proestrous rats with male rats overnight. The day on which sperm was observed in the vaginal lavage was defined as Day 0 of gestation. Parturition usually occurs on the evening of Day 21 or the morning of Day 22. Uteri were obtained from rats on Days 1, 3, 6, 11, 16, and 21 of pregnancy or from Day 1 postpartum rats, the day of birth being 0 postpartum. Uteri from rats in the estrous stage of the ovarian cycle were used as the nonpregnant myometria. Both pregnant and nonpregnant rats were killed by decapitation at 1000 h. Trunk blood was collected and the uterine horns were rapidly removed, trimmed of surrounding connective tissue, and opened longitudinally. Uteri from pregnant animals were freed of blood, pups, and placenta. The endometrium was carefully scraped with the aid of a binocular microscope. Tissue samples were excised from the longitudinal smooth muscle layer and quickly frozen in liquid nitrogen and stored at -80°C (reverse transcription-polymerase chain reaction [RT-PCR] studies) or used fresh (functional studies).
Reverse Transcription-PCR
The RT-PCR reactions were performed as previously described [14]. Total RNA of approximately 20 mg of rat uterine tissue was isolated according to the acid guanidium isothiocyanate-phenol-chloroform extraction method of Chomczynski and Sacchi [24]. The RNA samples were treated with RNase-free, fast protein liquid chromatographically pure DNase I (Pharmacia Biotech, Uppsala, Sweden) to eliminate contaminating genomic DNA. The integrity of the purified RNA was confirmed by visualization of the 28S and 18S rRNA bands after the electrophoresis of RNA through a 1% agarose-formaldehyde gel. The quantity of total RNA was determined by spectrophotometric measurement at 260 nM. RNA samples (10 µg each) were resuspended in diethylpyrocarbonate-treated water and stored at -80°C until use.
First-strand cDNA was synthesized by using a first-strand cDNA synthesis kit (Pharmacia Biotech, Uppsala, Sweden) in a 15-µl volume reaction containing 5 µg of DNase-treated total RNA. The resulting cDNA samples were amplified by PCR using a DNA thermal cycler (MJ Research, Watertown, MA) and the following specific primer pairs: 1) rat NK1R, forward 5'-ATCTGCTGGCTGCCCTTCC-3' and reverse 5'-CTGTGTCTGGAGGTATCGGG-3', to amplify a PCR product of 261 base pairs (bp); 2) rat NK2R, forward 5'-ATCTGCTGGCTGCCCTACC-3' and reverse 5'-TGTCTTCCTCAGTTGGTGTC-3', giving a PCR product of 223 bp; 3) rat NK3R, forward 5'-GAGAGATCCCAGGAGACA-3' and reverse 5'-TGGGGTCAAACAGCACGG-3', giving a PCR product of 417 bp. Amplification of the rat ß-actin gene transcript was used to control the efficiency of RT-PCR among the samples. Sequences of forward and reverse primers for ß-actin were 5'-CCTAGCACCATGAAGATCAA-3' and 5'-TTTCTGCGCAAGTTAGGTTTT-3', respectively, based on the published sequence of the rat gene [25]. The expected size of the PCR product was 227 bp. PCR mixes contained 0.2 µM primers, 1.5 U of Taq polymerase (Pharmacia), the buffer supplied, 2.5 mM MgCl2, 200 µM dNTPs, and cDNA in 25 µl. After a hot start (2 min at 94°C), the parameters used for PCR amplification were as follows: denaturation, 10 sec at 94°C; annealing, 20 sec at 60°C; extension, 30 sec at 72°C. Cycle numbers were 35 for tachykinin receptors and 25 for ß-actin. The products of the amplification were separated by gel electrophoresis on 1.7% agarose, stained with ethidium bromide, and visualized and photographed under UV transilluminator (Photodyne).
A semiquantitative RT-PCR assay was used to determine the relative concentrations of tachykinin NK1R, NK2R, and NK3R in uteri from rats at different stages of pregnancy and from nonpregnant and postpartum animals [17, 26]. Equal aliquots of the RT solution for the samples to be compared were serially half diluted and then amplified for a fixed number of cycles, to ensure analysis of products in the exponential range of amplification. The PCR products of each tachykinin receptor and molecular size standard were loaded on the same agarose gel in which ß-actin products and the correspondent molecular size standard were loaded, 30 min ahead of the NK1R, NK2R, or NK3R sample. Messenger RNA levels for the three tachykinin receptors and ß-actin were analyzed on each uterine sample; and experiments in each day of pregnancy, Day 1 postpartum, or nonpregnant uteri were carried out in three to five different animals, with each RT-PCR assay being performed in triplicate. The level of expression of each tachykinin receptor was normalized to ß-actin mRNA levels, and the relative amount of the target sequence in each sample was expressed as a percentage of the value determined in Day 1 pregnant rats. The band intensities were scanned by densitometry using a video documentation system and the image analysis software Intelligent Quantifier (BioImage Systems Corp., Ann Arbor, MI). The identity of each PCR product was established by DNA sequence analysis.
Serum Steroid Levels
Trunk blood was allowed to clot at 4°C. The clotted blood was centrifuged at 2000 x g for 15 min. Sera were collected, frozen, and stored at -80°C until analysis. Serum concentrations of E2 and P4 were measured by using double-antibody RIA kits (DRG Instruments, Marburg, Germany), following instructions by the manufacturer. Both assays used 125I-labeled tracers.
Functional Studies
Strips of longitudinal uterine smooth muscle (8–10 mm in length and 1–2 mm in width) were prepared and mounted in siliconized tissue baths containing 4 ml of physiological salt solution (PSS) of the following composition (mM): NaCl, 118; KCl, 5.6; CaCl2, 1.9; MgSO4, 0.95; NaH2PO4, 1; NaHCO3, 25 and glucose 11. The preparations were bubbled continuously with 95% O2:5% CO2 and warmed to 37°C. Mechanical responses were recorded isometrically by means of force-displacement transducers (Grass FT-03) connected to a Letica amplifier and an ABB Goerz SE 130 multichannel recorder. The tissue was immersed in PSS and equilibrated for 45 min (with changes in bath fluid every 15 min) under a resting tension of 0.5 g. After the equilibration period, the preparation was challenged twice by administration of a maximally effective concentration of acetylcholine (ACh, 1 mM). Uterine strips were then allowed to equilibrate for a further 60-min period before challenge with the tachykinin receptor agonists (Sar9Met[O2]11)-SP (in the presence of the NK2R antagonist SR 48968 and the NK3R antagonist SR 142801), NKA (in the presence of the NK1R antagonist SR 140333 and the NK3R antagonist SR 142801), and (MePhe7)-NKB (in the presence of the NK1R antagonist SR 140333 and the NK2R antagonist SR 48968). Only one agonist was tested on each preparation and only one noncumulative log concentration-response curve to (Sar9Met[O2]11)-SP (1 nM to 1 µM); NKA (1 nM to 1 µM), or (MePhe7)-NKB (1 nM to 1 µM) was constructed on each uterine strip. Each agonist concentration remained in contact with the tissue for 5 min, and the tissue was then washed thoroughly and allowed to rest for 20 (NKA) or 40 min ([Sar9Met(O2)11]-SP and [MePhe7]-NKB) before the addition of the next concentration. The tachykinin receptor antagonists SR 140333 (0.1 µM), SR 48968 (0.1 µM), and/or SR 142801 (0.1 µM) were added to the preparation 60 min before the construction of the noncumulative concentration-response curve to an agonist and reapplied after each concentration of the agonist was washed out. Responses to the agonists were obtained in the absence or in the presence of the neutral endopeptidase (NEP) inhibitor phosphoramidon. A maximally effective concentration of phosphoramidon (1 µM) [14] was added to the bath 20 min before the tachykinin agonist and reapplied 20 min before addition of each agonist concentration. The effects of phosphoramidon and/or antagonist vehicles were assessed in time-matched control strips mounted in parallel and found to have no effect on the responses to the agonists. At the end of the experiment, the preparation was washed repeatedly for 60 min before application of ACh (1 mM), to check the stability of tissue contractility. This last response served as an internal standard for all experiments.
Drugs
(Sar9Met[O2]11)-SP, NKA, and (MePhe7)-NKB (Bachem, Switzerland). Phosphoramidon (N-[
-L-rhamnopyranosyl-oxyhydroxyphosphinyl]-L-leucyl-L-tryptophan sodium salt), acetylcholine hydrochloride (Sigma, St. Louis, MO). SR 140333 (Nolpitantium, [S]1-[2-[3-(3,4-dichlorophenyl)-1-(3-isopropoxyphenylacetyl)piperidin-3-yl]ethyl]-4-phenyl-1-azoniabicyclo [2.2.2]octane chloride), SR 48968 (Saredutant, [S]-N-methyl-N[4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzamide), and SR 142801 (Osanetant, [R]-[N]-[1-(3-[1-benzoyl-3-(3,4-dichlorophenyl)piperidin-3-yl]propyl)-4-phenylpiperidin-4-yl]-N-methylacetamide) were kindly donated by Dr. X. Emonds-Alt (Sanofi-Synthelabo, Montpellier, France).
Statistical Analysis of Results
Contractile responses were measured as the maximal contractile force or as the area under the force-time curve during the 5-min period that each concentration of an agent was in contact with the preparation. The responses were expressed as a percentage of the maximal contractile force or of the area under the force-time curve measured during a 5-min period for ACh (1 mM). To measure the areas, polygraph tracings were scanned and then processed by using the Sigma-Scan software package (Jandel Scientific Corp., Erkrath, Germany). The pD2 values, defined as the negative log of the concentration of an agonist that produces 50% of the maximal response for that agonist, were calculated by nonlinear regression analysis of individual log concentration-response curves (areas), using Graphpad Prism 3.0 (Graphpad Software, San Diego, CA).
Values are expressed as the mean ± SEM. Unless otherwise indicated, n represents number of different animals. Statistical analysis of differences between two means was assessed by Student t-test. Multiple means were compared by one-way ANOVA followed by Tukey multiple comparison test (GraphPad Prism 3.0). A probability level of P < 0.05 was regarded as significant.
RESULTS
Reverse Transcription-PCR Studies
The RT-PCR assays revealed single bands corresponding to the expected product sizes encoding cDNA for the NK1R (261 bp), the NK2R (223 bp), the NK3R (417 bp) and ß-actin (227 bp), which appeared in uteri from rats at all stages of pregnancy and on Day 1 postpartum (Figs. 1 and 2). The identity of the amplified fragments was confirmed by DNA sequence analysis. This analysis demonstrated that the sequence of the amplified PCR fragments was identical to that previously reported for the rat NK1R (nucleotide positions 787–1047 bp) [7], the rat NK2R (nucleotide positions 1229–1451 bp) [4], and the rat NK3R (nucleotide positions 1017–1433 bp) [5]. No PCR product was detectable when the samples were amplified without the RT step, suggesting that genomic DNA contamination was eliminated by DNase treatment. Similarly, no products were detected when the RT-PCR steps were carried out with no added RNA, indicating that all reagents were free of target sequence contamination.
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Tachykinin NK1R mRNA levels were lower in early pregnant rats (Days 1–6), increased by 2.3- and 2.8-fold on pregnancy Days 16 and 21, respectively, and declined on Day 1 postpartum, with a return to levels similar to those at early pregnancy (Fig. 2). Tachykinin NK1R mRNA levels were not significantly different in uteri from nonpregnant estrous rats, compared to Day 1 of pregnancy (140.8 ± 15.7%, P > 0.05, n = 4).
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No significant alterations in NK2R mRNA expression were observed in uteri from rats at any stage of pregnancy or on Day 1 postpartum (Fig. 2). Tachykinin NK2R mRNA expression was also similar in uteri from nonpregnant estrous rats (115.3 ± 21.2% of mRNA levels on Day 1 of pregnancy, P > 0.05, n = 5).
Compared to Day 1 of pregnancy, NK3R mRNA levels decreased by 3.5-fold in uteri from Day 16 pregnant rats and by 18.8-fold on Day 21 pregnant rats (Fig. 2). On Day 1 postpartum, there was an increase in tachykinin NK3R mRNA, with a return to levels similar to those observed at early pregnancy (Fig. 2). In uteri from nonpregnant estrous rats, NK3R mRNA levels were 31.4 ± 3.7% of those observed on Day 1 of pregnancy (P < 0.001, n = 5).
Effect of Tachykinin Receptor Agonists on the Mechanical Activity of Rat Uterus
The log concentration-response curves obtained for the NK1R selective agonist (Sar9Met[O2]11)-SP (in the presence of 0.1 µM SR 48968 and 0.1 µM SR 142801) at different stages of pregnancy are shown in Figure 3. Both in the absence and presence of phosphoramidon (1 µM), (Sar9Met[O2]11)-SP (1 nM to 1 µM) elicited contractions of a small area, compared to the control response to ACh (1 mM) (Fig. 3). In the presence of phosphoramidon, the maximal amplitude of the contraction ranged between 22.7 ± 8.8% (pregnancy Day 3) and 54.6 ± 9.2% (pregnancy Day 11) of the maximal response to ACh. The contractions were highly variable between preparations and were observed to be more consistent when obtained in the presence of the NEP inhibitor. Their area was of higher magnitude at mid and late pregnancy (Days 11–21) than on early pregnant rats (Days 1–6) (Fig. 3). The response to (Sar9Met[O2]11)-SP (1 nM to 1 µM) declined after labor where contractions were similar to those observed on pregnancy Day 1 or in nonpregnant, estrous animals (not shown).
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Figure 4 shows the log concentration-response curves obtained for NKA (in the presence of 0.1 µM SR 140333 and 0.1 µM SR 142801) in the rat uterus during the course of pregnancy. NKA (1 nM to 1 µM) induced concentration-dependent contractile responses that were markedly increased in the presence of phosphoramidon (1 µM). In the absence of the peptidase inhibitor, the log concentration-response curves in early pregnancy (Days 1 and 6) were displaced to the right, compared to curves on late pregnancy (Days 16–21). In the presence of phosphoramidon, the log concentration-response curves to NKA were virtually superimposed in uteri from rats at different stages of pregnancy (Fig. 4), at Day 1 postpartum or at the estrous stage of the ovarian cycle (not shown). The pD2 values calculated for this agonist from the areas under the force-time curve in the presence of phosphoramidon were 7.9 ± 0.1 (n = 4), 7.7 ± 0.1 (n = 4), 8.0 ± 0.2 (n = 5), 7.9 ± 0.1 (n = 5), 7.9 ± 0.2 (n = 4), 7.8 ± 0.2 (n = 5), and 7.7 ± 0.3 (n = 4) at pregnancy Days 1, 6, 11, 16, and 21, Day 1 postpartum, and nonpregnant estrous rats, respectively. Due to the limited availability of the peptide, only partial log concentration-response curves were obtained in the absence of phosphoramidon and the pD2 values could not be calculated. In the presence of the NEP inhibitor, NKA showed the higher Emax (both in terms of area and of maximal contractile force) among the tachykinin agonists tested. The maximal increase in force produced by NKA in the presence of phosphoramidon ranged between 86.1 ± 6.3% (pregnancy Day 11) and 101.7 ± 8.0% (pregnancy Day 6) of the maximal contraction to ACh.
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(MePhe7)-NKB (1 nM to 1 µM, in the presence of SR 140333 and SR 48968, both at 0.1 µM) elicited uterine contractions characterized by bell-shaped log concentration-response curves (Fig. 5). For the same day of pregnancy, the contractile response to the NK3R selective agonist in the presence of phosphoramidon was similar in amplitude and time-course to that obtained in the absence of the peptidase inhibitor. The maximal increase in contractile force ranged between 2.8 ± 1.9% (pregnancy Day 16) and 76.6 ± 18.3% (pregnancy Day 3) of the maximal response to ACh. The maximal effect was reached at a concentration of 10–30 nM, and higher concentrations elicited contractile responses of decreasing amplitude and area (Fig. 5). The contractile responses to (MePhe7)-NKB were of higher magnitude in uteri from early or mid pregnant rats (Days 1–11) and were almost absent in uteri from late pregnant rats (Days 16–21) or from nonpregnant, estrous animals (not shown).
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Serum Steroid Levels
Circulating levels of ovarian steroids and the ratio of E2:P4 serum levels in pregnant rats during the course of pregnancy, at Day 1 postpartum and in nonpregnant rats at the estrous stage of the estrous cycle are shown in Table 1. Serum E2 concentrations were lower during early and mid pregnancy and increased throughout late pregnancy. Serum P4 levels were higher during early and mid pregnancy and declined at the end of gestation. A complex correlation exists between contractile activity derived from activation of tachykinin NK1R, its mRNA levels and the ratio of E2:P4 serum levels. Tachykinin NK3R responsiveness and mRNA levels correlate inversely with the ratio of E2:P4 serum levels.
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DISCUSSION
The present study demonstrates that NK1R, NK2R, and NK3R mRNAs are present in uteri from pregnant rats, and their expression shows differential variations along the course of pregnancy. Our results also show that changes in the amount of tachykinin receptor mRNAs correlate with changes in contractile responses to agonists acting selectively at NK1R, NK2R, or NK3R.
The three known tachykinin receptors are present in the nonpregnant rat uterus and their expression and responsiveness varied depending upon the hormonal conditions [14–19]. This suggests that tachykinins, acting at any of their receptors, could play a role in regulating reproductive functions. However, the physiological relevance of tachykinins in the female reproductive tract remains unknown, and to our knowledge, no previous studies have been carried out to investigate the expression and role of tachykinin receptors on uterine contractility along the course of pregnancy. Our results show that (Sar9Met[O2]11)-SP, NKA, and (MePhe7)-NKB elicited contractions in the pregnant rat uterus, demonstrating the coexistence of functionally active NK1R, NK2R, and NK3R. Among the agonists tested, NKA (in the presence of phosphoramidon and the selective antagonists SR 140333 and SR 142801, to block activation of NK1R and NK3R) showed the higher efficacy (Emax) in uteri from rats at all stages of pregnancy and at postpartum. Therefore, the NK2R appears to be the most important mediator of contractile responses to tachykinins during pregnancy and after labor, as occurred in the nonpregnant rat uterus [13, 14] and the pregnant human uterus at term [27]. The present results also demonstrate that NK2R mRNA levels remains stable during pregnancy, after labor or in nonpregnant estrous rats. This is in agreement with previous data in ovariectomized rats, where uterine NK2R mRNA expression did not change significantly between animals treated with E2, P4, or E2 + P4 [17].
The contractile response elicited by NKA was markedly increased in the presence of phosphoramidon. This indicates that metabolism of endogenous tachykinins by NEP may be a crucial step in the regulation of uterine responses to these neuropeptides during pregnancy. We previously found that NEP mRNA expression is downregulated in uteri from ovariectomized rats under conditions of estrogen dominance [17]. The present study shows that, in the absence of the peptidase inhibitor, the log concentration-response curves to NKA in early pregnancy were displaced to the right, compared to curves on late pregnancy. In the presence of phosphoramidon, the log concentration-response curves to NKA were virtually superimposed in uteri from rats at all stages of pregnancy. These findings suggest that the activity of NEP could vary during the course of pregnancy, being higher at early than at late pregnancy.
The mRNA encoding NK1R is expressed in the nonpregnant rat uterus, and this receptor type plays a role in mediating uterine contractile responses to tachykinins [14–18]. In pregnant rat uteri, the NK1R selective agonist (Sar9Met[O2]11)-SP elicited contractions at all stages of pregnancy that reached a maximum at a low concentration. The contractile responses were obtained in the presence of the selective NK2R antagonist SR 48968 and the selective NK3R antagonist SR 142801, providing evidence that they were specifically mediated by activation of NK1R. Both in the absence and presence of phosphoramidon, the log concentration-response curves to (Sar9Met[O2]11)-SP in uteri from late pregnant rats were displaced to the left, compared to the log concentration-response curves in early pregnant animals. A direct correlation was observed between the magnitude of the contractile response to (Sar9Met[O2]11)-SP and the level of NK1R mRNA, suggesting that changes in steady-state mRNA levels are accompanied by changes in the amount of functional protein produced. Previous studies from our laboratory have shown that uterine NK1R gene expression decreased after treatment of ovariectomized rats with P4 and increased after treatment with E2 [17]. This and the complex relation observed between tachykinin NK1R mRNA levels and the ratio of E2:P4 serum levels suggest that both E2 and P4 regulate the expression of the NK1R during the course of pregnancy.
The tachykinin NK3R is widely distributed in the central nervous system but is present in smaller amounts or absent in peripheral tissues, including the uterus from nonpregnant estrogen-primed or estrous rats [5, 14, 28]. The present data show that the NK3R selective agonist (MePhe7)-NKB (in the presence of the selective NK1R antagonist SR 140333 and the selective NK2R antagonist SR 48968) elicited contractile responses in early and mid pregnancy but failed to contract the uterus of late pregnant rats. Our data also demonstrate that the expression of the NK3R gene in the rat uterus was strongly altered during the course of pregnancy, being almost undetectable at late pregnancy and being present in a 20-fold higher amount at early pregnancy. Moreover, NK3R mRNA levels and function correlated inversely with the ratio of E2:P4 serum levels. These results and those obtained in ovariectomized rats [17] suggest that E2 has a marked negative effect in NK3R gene expression. Previous studies by Hamlin et al. [18] and by us [17] demonstrated that the contractile response elicited by NK3R activation in the nonpregnant rat uterus is highly dependent upon the hormonal environment. These data and the strong repression of the NK3R gene under conditions of estrogen dominance reinforce the suggestion that the NK3R may play an important role in the regulation of uterine functions.
In conclusion, the present results demonstrate that functionally active tachykinin NK1R, NK2R, and NK3R are present in uteri from pregnant rats and that functional responses and mRNA expression levels for the three tachykinin receptor types varied selectively and differentially during the course of pregnancy and at postpartum. These data strongly argue for a role of tachykinins in the regulation of reproductive functions in mammals.
ACKNOWLEDGMENTS
The authors are very grateful to Dr. M. Paya for his invaluable help with RIA and to Dr. X. Emonds-Alt for the generous gift of SR 140333, SR 48968, and SR 142801.
FOOTNOTES
First decision: 18 January 2001.
1 This work was supported by grants from the Ministry of Science and Technology (PB 97-1123) and Fundación Ramón Areces (Spain). 
2 Correspondence: Francisco M. Pinto, Instituto de Investigaciones Químicas,
Avenida Americo Vespucio s/n, Isla de la Cartuja, 41092 Sevilla,
Spain. FAX: 34 95 4460565; mluz{at}cica.es
Accepted: April 3, 2001.
Received: December 13, 2000.
REFERENCES
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L. H. Crane, M. J. Williams, A. J. Nimmo, and G. P. Hamlin Estrogen-Dependent Regulation of Neurokinin 3 Receptor-Mediated Uterine Contractility in the Rat Biol Reprod, November 1, 2002; 67(5): 1480 - 1487. [Abstract] [Full Text] [PDF]
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P < 0.05, significant difference from mRNA levels in pregnancy Day 3; 
P < 0.05, significant difference from mRNA levels in pregnancy Day 6; 
P < 0.05, significant difference from mRNA levels in pregnancy Day 21, one-way ANOVA
