Introduction

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The tachykinins represent a family of peptide neurotransmitters including substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) (Regoli et al., 1994; Maggi, 1997). They interact with three distinct types of receptors termed NK₁ (NK₁R), NK₂ (NK₂R), and NK₃ (NK₃R), which are preferentially activated by SP, NKA, and NKB, respectively (Sasai and Nakanishi, 1989; Hershey and Krause, 1990; Shigemoto et al., 1990). In the female reproductive tract, SP and NKA are localized in a population of capsaicin-sensitive sensory nerves, the presence of which have been demonstrated in virtually all mammalian species examined (Papka and Shew, 1994). NKB is undetectable in peripheral tissues (Moussaoui et al., 1992), although a recent report has shown that the human and rat placenta secretes NKB during pregnancy (Page et al., 2000). Experimental evidence argues for a role of tachykinins in the modulation of myometrial activity. In uteri from estrogen-treated, virgin rats, SP, NKA, and NKB induce contraction mainly by activation of NK₂R (Pennefather et al., 1993; Magraner et al., 1998). Tachykinin NK₁R, NK₂R, and NK₃R genes are expressed in the nonpregnant rat uterus and their expression and function varied under different hormonal conditions (Barr et al., 1991; Fisher and Pennefather, 1999; Pinto et al., 1999; Hamlin et al., 2000; Patak et al., 2000). However, further studies are needed to characterize the physiological relevance of this sensory innervation in the female reproductive tract.

We have recently found that NK₃R gene expression is strongly down-regulated in uteri from estrogen-treated ovariectomized rats (Pinto et al., 1999) or from late pregnant rats (Candenas et al., 2001). In the present study, we analyzed tachykinin NK₃R mRNA expression in uteri from young (3-month-old) rats at the estrus stage of the estrous cycle and old (30-month-old) nonregularly cycling animals. We also assessed for the first time in the female reproductive tract the expression of preprotachykinin-B (TAC-3), the gene that encodes NKB. In addition, functional studies were carried out to investigate the influence of age on the contractile response elicited by the selective NK₃R agonist [MePhe⁷]-NKB in the isolated rat myometrium.

	Materials and Methods

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Animals and Tissue Preparation. All experiments were conducted in accordance with National Institutes of Health guidelines for the care and use of laboratory animals. 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 (12-h light/12-h dark), with free access to food and water. Vaginal smears were taken and examined microscopically to assess the stage of the estrous cycle. Uteri were obtained from a group of four young (3-month-old) rats at the estrus stage of the ovarian cycle. A group of 10 rats was allowed to reach 30 months of age. Six rats died between 20 and 30 months, giving a final n = 4 for the 30-month-old group. Rats were killed by decapitation after brief exposure to CO₂, and the uterine horns were rapidly removed, trimmed of surrounding connective tissue, and opened longitudinally. Tissue samples were excised from the longitudinal smooth muscle layer, quickly frozen in liquid nitrogen, and stored at 80°C (RT-PCR studies) or used fresh (functional studies).

RT-PCR Studies. These reactions were performed as previously described (Pinto et al., 1999). Total RNA of approximately 20 mg of rat uterine tissue was isolated according to the method of Chomczynski and Sacchi (1987). Residual genomic DNA was removed by incubating the RNA samples with RNase-free, fast-pressure liquid chromatography pure DNase I (Amersham Pharmacia Biotech, Uppsala, Sweden). First strand cDNA was synthesized using Moloney murine leukemia virus reverse transcriptase and random hexamers according to the manufacturer's instructions (First-strand cDNA synthesis kit; Amersham Pharmacia Biotech). The resulting cDNA samples were amplified by PCR using a DNA thermal cycler (MJ Research, Watertown, MA) and the following specific primer pairs: a) rat TAC-3, forward 5'-TGATCTCTCTCTGCTACCTCCAC-3' and reverse 5'-CCCTGTCTTTATGATGCAG TCC-3' to amplify a PCR product of 300 base pairs (bp), based on the published rat cDNA sequence (Bonner et al., 1987); and b) rat NK₃ receptor, forward 5'-GAGAGATCCCAGGAGACA-3' and reverse 5'-TGGGGTCAAACAGCACGG-3' giving a PCR product of 417 bp (Shigemoto et al., 1990). Amplification of the rat -actin gene transcript was used to control the efficiency of RT-PCR among the samples. Sequences of forward and reverse primers for -actin were 5'-CCTAGCACCATGAAGATCAA-3' and 5'-TTTCTGCGCAAGTTAGGTTTT-3', respectively, based on the published sequence of the rat gene (Nudel et al., 1983). The expected size of the PCR product was 227 bp. We also analyzed the expression of glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and protein phosphatase type 1 -isoform (PP1-), two of the most widely employed housekeeping genes. Sequences of forward and reverse primers for GAPDH were 5'-CTACCCACGGCAAGTTCAAT-3' and 5'-CTTCTGAGTGGCAGTGATGG-3', respectively, based on the published rat cDNA sequence (Tso et al., 1985), giving a PCR product of 407 bp. Sequences of forward and reverse primers for PP1- were 5'-AACCATGAGTGTGCTAGCATCA-3' and 5'-CACCAGCATTGTCAAACTCGCC-3', based on the published cDNA sequence of rat PP1- (Sasaki et al., 1990), and were designed to amplify a PCR product of 472 bp. All primers were synthesized and purified by Amersham Pharmacia Biotech. 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: 15 s at 94°C, 20 s at 60°C, and 30 s at 72°C. Cycle numbers were 36 for TAC-3 and NK₃R, 25 for -actin, and 28 for GAPDH and PP1-. Serial half-dilutions of cDNA were amplified at the indicated number of cycles for each of the amplification products to ensure analysis in the linear range of amplification (Pinto et al., 1999). The PCR products were separated by gel electrophoresis on 1.7% agarose, stained with ethidium bromide, and visualized and photographed under UV transilluminator (Spectronics Corp., Rochester, NY). 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). mRNA levels for TAC-3, NK₃R, GAPDH, PP1-, and -actin were determined on each uterine sample, and the band densities determined for all cDNA dilutions within the linear amplification range were analyzed by linear regression analysis using Microsoft Excel (Microsoft, Redmond, WA). The level of expression of each PCR product was normalized to the -actin mRNA level, and the relative amount of the target sequence in young rats was expressed as a percentage of the value determined in old animals. The identity of each PCR product was established by DNA sequence analysis.

Functional Studies. The experiments were carried out essentially as previously described (Magraner et al., 1998). 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 of the following composition (in 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, warmed to 37°C, and equilibrated under a resting tension of 0.5 g. Mechanical responses were recorded isometrically (FT-03 transducers; Grass Instruments, Quincy, MA). Uterine strips were challenged twice at 30-min intervals by a supramaximal effective concentration of acetylcholine (ACh, 1 mM; Sigma, St. Louis, MO) and allowed to equilibrate for a further 60-min period before challenge with the selective tachykinin NK₃R agonist [MePhe⁷]-NKB (Bachem, Bubendorf, Switzerland). One noncumulative log concentration-response curve to [MePhe⁷]-NKB (0.1 nM-0.1 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 40 min before the addition of the next concentration. Responses to [MePhe⁷]-NKB were obtained in the absence or in the presence of the neutral endopeptidase inhibitor phosphoramidon [N-(-L-rhamnopyranosyl-oxyhydroxyphosphinyl)-L-leucyl-L-tryptophan sodium salt, Sigma] at a maximal effective concentration (1 µM; Magraner et al., 1998). The effect of phosphoramidon vehicle was assessed in uterine strips mounted in parallel and found to have no effect on the responses to the agonist. At the end of the experiment, the preparation was challenged again with ACh (1 mM), to check the stability of tissue contractility. This last response served as an internal standard for all experiments. Contractions were measured as the peak increase in 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 (Magraner et al., 1998). Responses were expressed as a percentage of the peak increase in 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).

Statistical Analysis. All values are expressed as the mean ± S.E.M.; n represents number of different experiments in n different animals. In RT-PCR assays, each experiment with the cDNA from each animal was carried out in triplicate. Statistical significance of differences between two means was assessed by Student's paired t test. Multiple means were compared by one-way analysis of variance followed by Tukey's multiple comparison test (Prism 3.0; GraphPad Software, San Diego, CA). A probability level of P < 0.05 was regarded as significant.

	Results

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RT-PCR Studies. RT-PCR assays revealed single bands corresponding to the expected product sizes encoding cDNA for TAC-3 (300 bp); NK₃R (417 bp); PP1- (472 bp); GAPDH (407 bp); and -actin (227 bp), which appeared in uteri from young and old animals (Fig. 1). The identity of the amplified fragments was confirmed by DNA sequence analysis. 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.

View larger version (36K): [in this window] [in a new window]   Fig. 1.   Agarose gels showing RT-PCR products for uterine cDNA from young (3-month-old) and old (30-month-old) rats. After normalization to -actin mRNA levels, serial half-dilutions of cDNA were amplified for 25 (-actin), 28 (GAPDH and PP1-), or 36 (NK3R and NKB) cycles with specific oligonucleotide primers. Lane 1 represents the more diluted samples in each series. The observation of a steadily declining yield of product at each dilution step confirmed that the comparison of the two samples was made in the exponential portion of the amplification curve. The amount of NK3R, NKB, GAPDH, and PP1- mRNA was then assessed relative to the amount of the coamplified -actin fragment. m, molecular size standards. Data are representative of typical results in four young and four old animals.

Functional Studies. Figure 2 shows the log concentration-response curves obtained for [MePhe⁷]-NKB on myometrial contractility in young and old rats. Whatever the age of the animal, the contractile response to the selective NK₃R agonist in the presence of phosphoramidon was similar in amplitude and time course to that obtained in the absence of the neutral endopeptidase inhibitor (Fig. 2). In uteri from 3-month-old estrus animals, [MePhe⁷]-NKB (0.1 nM-0.1 µM) elicited contractions of small area, compared with the control response to 1 mM ACh (n = 4; Figs. 2A and 3A). The contraction consisted of one, two, or three rhythmic contractions with a rapid return to the basal tone (Fig. 3A). The peak increase in force reached 63.5 ± 8.1% of the maximal response to ACh (mean value in the presence of phosphoramidon; Fig. 2B). The maximal effect was reached at 10 nM, and higher concentrations (30 nM-0.1 µM) elicited contractile responses of similar amplitude and area. The contractile response was significantly higher in old animals (Figs. 2 and 3B). [MePhe⁷]-NKB (0.1 nM-0.1 µM) elicited uterine contractions characterized by bell-shaped log concentration-response curves (Fig. 2). The NK₃R agonist induced a tonic contraction with superimposed rhythmic oscillations (Fig. 3B). The maximal effect was reached at a concentration of 3 nM, and higher concentrations elicited contractions of decreasing amplitude and area (Fig. 2). In the presence of phosphoramidon, the maximal response to [MePhe⁷]-NKB was 83.2 ± 8.4% of the control response to ACh in terms of area and 93.4 ± 10.2% of the control response to ACh in terms of peak increase in force (P > 0.05 versus ACh in both cases, n = 4)

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Noncumulative log concentration-response curves for the selective NK3R agonist [MePhe7]-NKB in longitudinally arranged uterine smooth muscle from young (3-month-old, , ) and old (30-month-old, , ) rats. Experiments were performed in the absence (, ) and in the presence (, ) of the neutral endopeptidase inhibitor phosphoramidon (1 µM). Data points represent areas under the force-time curve (A) or peak increases in contractile force (B) expressed as a percentage of the area (A) or the peak increase in force (B) of the control response to ACh (1 mM). Each value is the mean ± S.E.M. of four experiments in four different animals. , P < 0.05, significant differences from [MePhe7]-NKB responses in old rats in the presence of phosphoramidon, one-way analysis of variance.

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Representative tracings showing the contractile response induced by [MePhe7]-NKB (Me-NKB) in uteri from young (A, 3-month-old) and old (B, 30-month-old) rats. ACh, reference contraction produced by acetylcholine (1 mM). W, washing. Traces are representative of typical results of four experiments per age group.

	Discussion

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This study demonstrates that NKB and NK₃R are expressed in the rat uterus and that the expression and function of the tachykinin NK₃R are strongly increased in old animals.

Recent advances in our knowledge of the physiological role played by tachykinins and their receptors have focused on NK₁R and NK₂R. Studies on NK₁R knockout mice have proved a role for this receptor in nociception, stress and neurogenic inflammation (De Felipe et al., 1998). The NK₂R appears to be the most important tachykinin receptor involved in smooth muscle contraction (Pennefather et al., 1993; Maggi, 1997; Advenier et al., 1999). Conversely, little is known about the physiological role played by the NK₃R. This tachykinin receptor is mainly found in the central nervous system being absent or present in small amounts in peripheral tissues (Tsuchida et al., 1990).

Previous studies have demonstrated the presence of a functionally active heterogeneous population of NK₁R, NK₂R, and NK₃R in the rat uterus and the selective and differential regulation of each tachykinin receptor by ovarian steroids (Pinto et al., 1999; Patak et al., 2000; Hamlin et al., 2000). Tachykinin NK₃R gene expression is strongly down-regulated under conditions of estrogen dominance (Pinto et al., 1999; Candenas et al., 2001). To our knowledge, this is the only described mechanism of regulation of the NK₃R. The present data show that NK₃R mRNA expression is dramatically increased in the uterus of old rats. The increase in the expression level was associated with a clear augmentation of the potency and magnitude of contractile responses to the selective NK₃R agonist [MePhe⁷]-NKB. Moreover, the nature of the contractile response to [MePhe⁷]-NKB was markedly different in old rats, compared with young, estrus animals (see Figs. 2 and 3). The reason for this different contractile pattern remains unclear. Hamlin et al. (2000) observed that the selective NK₃R agonist senktide induced a sustained contraction in uteri from nonestrogen-dominated states and a short-lived transient contraction in estrogen-dominated uteri. These authors suggested that estrogen and progesterone might have a direct regulatory effect on NK₃R-mediated uterine responsiveness. An alternative explanation is that ovarian steroids and/or other age-related factors regulate not only NK₃R mRNA expression but also the cellular localization of this tachykinin receptor within the uterus. In this case, the nature of the contractile response would depend on the type of cell expressing predominantly NK₃R. Further studies are needed to investigate NK₃R localization and whether the observed changes in functional responses are a consequence of aging, differences in the hormonal environment, or a combination of both factors.

Although each endogenous tachykinin is not selective enough and can bind to the three tachykinin receptors, NKB is the most potent endogenous agonist for the NK₃R. The expression of this neurokinin is restricted to certain areas of the central nervous system, but it has recently been shown that the human and rat placenta secretes NKB during pregnancy (Page et al., 2000). The present study shows that TAC-3, the gene that encodes NKB, is expressed in the rat uterus and its expression is increased in old animals. Our data do not permit us to establish the precise cellular localization of NKB within the rat uterus. However, it must be taken into account that neuropeptides are synthesized in the cell soma and transported through the axon to sites of release in the nerve terminals (Maggi, 1997). Tachykinins are localized in sensory fibers innervating the uterus, and these primary afferent neurons have their cell bodies in the dorsal root ganglia (Papka and Shew, 1994; Maggi, 1997). This suggests that NKB may be synthesized and released from uterine non-neuronal cells, as reported for the placenta (Page et al., 2000). The present data suggest that the uterus is not only a site of NKB action but also a site of NKB production. The result in uteri is in line with the identification of a sexually dimorphic and estrogen-receptive population of neurons expressing NKB in ovine females (Goubillon et al., 2000) and the increase in expression of NKB in the hypothalami of postmenopausal women (Rance and Young, 1991).

Several recent findings in certain hypothalamic neurons (Goubillon et al., 2000), the placenta (Page et al., 2001), or the uterus (Candenas et al., 2001) argue for a role of tachykinins in regulating reproductive functions. The present study shows: a) NK₃R mRNA levels increased by approximately 45-fold in 30-month-old compared with 3-month-old rats, b) TAC-3 mRNA levels increased by about 2.5-fold in old animals, and c) [MePhe⁷]-NKB induced small contractions in uteri from young rats; in old animals, low concentrations (1-3 nM) of this selective NK₃R agonist elicited contractions similar to those produced by a maximal effective concentration of ACh (1 mM). From these data, it can tentatively be hypothesized that the NK₃R/NKB receptor-ligand pair could be involved or, at least, be an indicator of estrogen-related pathophysiologies. In this context, it has recently been shown that an excessive placental secretion of NKB causes pre-eclampsia (Page et al., 2000), a disease that is associated with low circulating levels of estrogens (Innes and Byers, 1999).

In summary, the rat uterus represents a peripheral tissue that expresses NKB and, under certain physiological conditions, is highly sensitive to NK₃R activation.