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BOR - Papers in Press, published online ahead of print January 12, 2005.
Biol Reprod 2005, 10.1095/biolreprod.104.036814
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BIOLOGY OF REPRODUCTION 72, 1125–1133 (2005)
DOI: 10.1095/biolreprod.104.036814
© 2005 by the Society for the Study of Reproduction, Inc.

Functional and Molecular Characterization of Tachykinins and Tachykinin Receptors in the Mouse Uterus1

Eva Patak 4, Francisco M. Pinto 5, Margot E. Story 6, C. Oscar Pintado 5, Anna Fleming 6, Nigel M. Page 7, Jocelyn N. Pennefather 3 8, and M. Luz Candenas 2 5

Department of Anaesthesia,4 Royal Women's Hospital, Carlton, Victoria 3053, Australia Centro de Investigaciones Científicas Isla de La Cartuja,5 Instituto de Investigaciones Químicas, Sevilla 41092, Spain Department of Pharmacology,6 Monash University, Clayton, Victoria 3800, Australia School of Animal and Microbial Sciences,7 University of Reading, Reading RG6 6AJ, United Kingdom Department of Pharmaceutical Biology and Pharmacology,8 Victorian College of Pharmacy, Monash University, Parkville, Victoria 3052, Australia


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The aim of this study was to analyze the function and expression of tachykinins, tachykinin receptors, and neprilysin (NEP) in the mouse uterus. A previous study showed that the uterotonic effects of substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) in estrogen-treated mice were mainly mediated by the tachykinin NK1 receptor. In the present work, further contractility studies were undertaken to determine the nature of the receptors mediating responses to tachykinins in uteri of late pregnant mice. Endpoint and real-time quantitative RT-PCR were used to analyze the expression of the genes that encode the tachykinins SP/NKA, NKB, and hemokinin-1 (HK-1) (Tac1, Tac2, and Tac4); and the genes that encode tachykinin NK1 (Tacr1), NK2 (Tacr2), and NK3 (Tacr3) receptors in uteri from pregnant and nonpregnant mice. The data show that the mRNAs of tachykinins (particularly NKB and HK-1), tachykinin receptors, and NEP are locally expressed in the mouse uterus, and their expression changes during the estrous cycle and during pregnancy. The tachykinin NK1 receptor is the predominant tachykinin receptor in the nonpregnant and early pregnant mouse and may mediate tachykinin-induced uterine contractions in the nonpregnant mouse. The tachykinin NK2 receptor is predominant in the late pregnant mouse and is the main receptor mediating uterotonic responses to tachykinins at late pregnancy. The tachykinin NK3 receptor is expressed in considerable amounts only in uteri from nonpregnant diestrous animals, and its physiological significance remains to be clarified.

female reproductive tract, mouse, neuroendocrinology, pregnancy, signal transduction, tachykinin NK2 receptor, tachykinins, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tachykinins are a family of biologically active peptides that includes substance P (SP), neurokinin A (NKA), neurokinin B (NKB), and the more recently described peptide hemokinin-1 (HK-1) [1–7] and its human orthologs, endokinin A and B (EKA and EKB) [8]. These peptides are widely distributed throughout the periphery within capsaicin-sensitive primary afferent neurons and non-neuronal cells [1, 8, 9] and produce a diverse range of biological effects that are mediated through three receptors, termed NK1, NK2, and NK3 [10, 11].

Accumulating data suggest that tachykinins may be involved in the regulation of reproductive functions [12–14]. In the female reproductive tract, the presence of tachykinin-like immunoreactivity has been demonstrated in numerous species [15–21]. Recent studies have also shown that the genes that encode NKB, HK-1, and EKB are expressed in the uterus [8, 22–24]. We have reported previously that tachykinins produce a direct contractile effect on uterine smooth muscle from rats [22, 25–33], humans [24, 34, 35], and mice [19, 36]. These functional studies together with molecular studies indicate that the NK2 receptor is the predominant receptor involved in mediating tachykinin-induced contractions in uteri from both pregnant and nonpregnant rats and humans [19, 24, 27, 32–35, 37]. In contrast, the NK1 receptor seems predominant in mediating uterine contractility in the nonpregnant, estrogen-primed mouse [36].

The aim of the present study was to examine the contractile effect and the expression of the tachykininergic system in uteri from nonpregnant and pregnant mice. Functional studies were undertaken to characterize the tachykinin receptors involved in mediating myometrial contractility and the influence of peptidase inhibition on tachykinin responses in the mouse uterus. In addition, endpoint and real-time quantitative reverse transcription (RT)-polymerase chain reaction (PCR) were used to analyze the expression of the genes that encode SP/NKA (Tac1), NKB (Tac2), and HK-1 (Tac4), respectively; and the genes encoding the tachykinin NK1 (Tacr1), NK2 (Tacr2), and NK3 (Tacr3) receptors. We also studied the expression of Mme, the gene that encodes neprilysin (NEP), which is the key enzyme involved in tachykinin metabolism in the mammalian uterus.

A preliminary account of some of the findings of this study was communicated to the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists [38, 39].


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Tissue Preparation

Prior ethical approval for these studies was obtained from the Monash University Standing Committee on Ethics in Animal Experimentation (Australia) and the Ethics Committee of Consejo Superior de Investigaciones Científicas (Spain).

Female, nonpregnant (20–25 g) BalbC mice were purchased from Charles River Laboratories (Barcelona, Spain) or bred in the Monash University Animal House. Mice were housed in the corresponding Departmental Animal House in an air conditioned room at 22°C under controlled lighting (12L:12D), with free access to food and water. Female mice were mated with a male overnight to induce pregnancy. Vaginal smears were checked to determine the stage of the estrous cycle in the untreated nonpregnant animals and to confirm pregnancy in mated females. The day on which a vaginal plug was observed was defined as Day 1 of gestation. Mice were killed by decapitation following exposure to CO2, and the uterine horns were rapidly removed and trimmed of surrounding connective tissue. For contractility studies, uteri were obtained from 1) virgin mice treated 24 h before with estradiol cypionate (200 µg/kg, s.c.) and 2) from Day 17 pregnant mice. Uterine horns from nonpregnant animals were opened along the mesometrial border and transected medially to provide four preparations per animal. Uterine horns from Day 17 pregnant mice were similarly opened, and fetuses removed and decapitated before uteri were excised, providing up to eight preparations per mouse. For molecular studies, samples from the middle section of the uterus were obtained from 1) virgin mice in the estrous or diestrous stage of the ovarian cycle, 2) virgin mice treated 24 h before with a single injection of estradiol benzoate (200 µg/kg, s.c.), and 3) pregnant mice on Day 5 or Day 17 of an average 19-day gestation period. Samples of brain cortex used as a positive control of amplification of the target genes were obtained from the same mice. Tissues were quickly frozen in liquid nitrogen and stored at –80°C until use.

Functional Studies

Uterine preparations from nonpregnant estrogen-treated or Day 17 pregnant mice (10 mm x 5 mm; mean weight = 32.7 ± 2.2 mg, n = 32 preparations) were mounted in 5-ml siliconized organ baths containing a physiological salt solution (PSS) of the following composition (mM): NaCl, 118.0; KCl, 4.7; MgSO4 · 7H20, 1.1; KH2PO4, 1.18; NaHCO3, 25.0; glucose, 11.66; CaCl2 · 2H2O, 1.9; warmed to 37°C. Preparations were bubbled continuously with carbogen gas (95% O2, 5% CO2) to maintain a pH of 7.4. Contractile force produced by the longitudinally oriented smooth muscle fibers was recorded by a Grass FT03 force transducer connected to a MacLab data acquisition system.

Agonist Log Concentration-Response Curves

Uterine preparations were set up under an initial resting force of 1 g (nonpregnant) or 2 g (pregnant) and allowed to equilibrate for 2 h with the PSS replaced every 20 min. Following the equilibration period, discrete concentration-response curves were constructed to SP, NKA, NKB, the NK1 receptor-selective agonist [Sar9Met(O2)11]SP, and the NK2 receptor-selective agonist [Lys5MeLeu9Nle10]NKA(4–10) (0.1 nM to 1 µM). Agonists remained in contact with the tissue for 5 min; the tissue was then washed and a higher concentration of agonist added 15 min later. At the conclusion of the experiment, methacholine (MCh, 10 µM), used as a negative control, was applied for 5 min and washed out, and 15 min later the tissues were exposed to a modified PSS (KPSS) in which 40 mM KCl replaced 40 mM NaCl.

Antagonist Studies

Responses to SP, NKA, [Sar9Met(O2)11]SP, and [Lys5MeLeu9Nle10]NKA(4–10) were compared in the absence and presence of the tachykinin receptor antagonists SR 140333 (NK1 receptor-selective, 10 nM), SR 48968 (NK2 receptor-selective, 10 nM), and SR 142801 (NK3 receptor-selective, 0.3 µM). Antagonists were added to the preparations 1 h before the construction of the concentration-response curve and reapplied following each wash. The peptidase inhibitors thiorphan (3 µM), captopril (10 µM), and bestatin (10 µM) were present throughout construction of log concentration-response curves to SP and NKA.

Peptidase Inhibitor Studies

In a subset of experiments log concentration-response curves to SP and NKA were obtained in the absence and presence of the peptidase inhibitors thiorphan (3 µM), captopril (10 µM), and bestatin (10 µM). Peptidase inhibitors were present throughout the experiment, and were replaced each time the bath was washed out.

Molecular Studies

RNA Extraction and Reverse Transcription Total RNA from approximately 20 mg of mouse uterine tissue or brain cortex was isolated using the method described by Chomczynski and Sacchi [40]. Residual genomic DNA was removed by incubating the RNA samples with RNase-free, fast protein liquid chromatography pure DNase I (Amersham Biosciences, Essex, U.K) and RNasin (Promega Corp., Madison, WI). The quantity of total RNA was determined by spectrophotometric measurement at 260 nM. DNase-treated total RNA (2 µg) was reverse transcribed using a first-strand cDNA synthesis kit (Amersham Biosciences) and the resulting samples were diluted to 400 µl with DNase-free water.

PCR Primers

Specific oligonucleotide primers were used to amplify Tac1, Tac2, Tac4, Tacr1, Tacr2, Tacr3, Mme, and ß-actin in cDNAs from nonpregnant mice in the estrous or diestrous stage of the estrous cycle; nonpregnant, estrogen-treated mice; and Day 5 and Day 17 pregnant mice (five mice per group). The primer pairs were designed with the analysis software Primer 3 [41] and used for both endpoint and real-time PCR. Amplification of the ß-actin gene transcript was used as an internal control of RT-PCR reactions among the samples. The structures of the primers used are shown in Table 1. All primers were synthesized and purified by Sigma Genosys (Cambridge, U.K.).


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  		TABLE 1. Nucleotide sequence of the specific primers used in PCR am plifications.a

Endpoint PCR

An endpoint PCR assay was used to detect the mRNAs of preprotachykinins, tachykinin receptors, and neprilysin and to establish the identity of the amplified products. A 3-µl aliquot of the resulting cDNA was used as a template for PCR amplification using a DNA thermal cycler (MJ Research, Watertown, MA). PCR mixes contained 0.2 µmol primers, 1.5 U Taq polymerase (Amersham Biosciences), the buffer supplied, 2.5 mmol MgCl2, and 200 µmol dNTPs and cDNA in 25 µl. After a hot start (2 min at 94°C), the parameters used for PCR amplification were 10 sec at 94°C; 20 sec at 60°C, and 30 sec at 72°C. These cycles were repeated 35 times for tachykinins, tachykinin receptors, and neprilysin; and 24 times for ß-actin. Serial half dilutions of cDNA were amplified at the indicated number of cycles for each target gene and ß-actin to ensure analysis of products in the linear range of amplification. The PCR products were separated by gel electrophoresis, stained with ethidium bromide, and visualized and photographed under a UV transilluminator (Spectronics Corp., Westbury, NY). Messenger RNA expression for all target genes and ß-actin was analyzed on each tissue, and each PCR experiment was carried out in triplicate. Amplicon sizes were verified by comparison with a DNA mass ladder, and the identity of each PCR product was established by DNA sequence analysis as previously described [32]. 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.

Real-Time PCR

Real-time PCR was used to quantify the expression of Tac2, Tac4, Tacr1, Tacr2, and Mme in the mouse uterus using the iCycler iQ real-time detection system from Bio-Rad Laboratories (Hercules, CA) and SYBR green (Molecular Probes, Leiden, The Netherlands) as previously described [24]. The PCR reaction mixture was identical to that used in the endpoint PCR assay, adding SYBR green I dye (1:75 000 dilution of the 10 000x stock solution). The reactions were performed in 96-well thin-wall PCR plates covered with a sheet of optical-quality sealing film. Thermal cycling conditions were the same as those described for endpoint assays, and fluorescence measurements were recorded during each extension step. For all samples, ß-actin was used as the endogenous control for normalization of initial RNA levels. Control samples without the RT step and with no added RNA were also included with each plate to detect any possible contamination.

At the end of each PCR run, the data were automatically analyzed by the system, and an amplification plot was generated for each DNA sample. From each of these plots, the iCycler software calculates the threshold cycle (CT), defined as the fractional cycle number at which the fluorescence reaches 10x the standard deviation of the baseline. The fold expression or repression of the target gene relative to ß-actin in each sample was then calculated by the formula 2–{Delta}{Delta}CT, where {Delta}CT = CTtarget gene – CTß-actin and {Delta}{Delta}CT = {Delta}CTtest sample – {Delta}CTcontrol.

For each target gene, real-time PCR data from a uterine sample of a virgin mouse in estrus was arbitrarily chosen as control, and this sample was included in all PCR experiments to correct for possible interassay variations. The target gene mRNA:ß-actin mRNA ratio in the control sample was designated as 1. Three serial dilutions of cDNA template were prepared from each tissue, and each dilution was amplified in triplicate. The experimental approach was further validated by the observation that the differences between the CT for the target gene and ß-actin remained essentially constant for each starting DNA amount.

Data Analysis

All values are expressed as the mean ± SEM; except where otherwise stated, n represents the number of mice used.

Contractile responses to each agonist were measured as area under the force-time curve (g·s) for the 5-min period that the agonist remained in contact with the tissue. Spontaneous activity was subtracted from the data when observed at 2 h. Responses were then expressed as a percentage of the corresponding response to KPSS and presented as mean ± SEM. To determine agonist potencies in the absence or presence of antagonists, mean log concentration-response curves were constructed using nonlinear regression analysis in GraphPad PRISM (version 3.0) to determine pD2 values. When pairs of mean regression lines over the linear range of the log concentration-response curve were parallel, a potency ratio with 95% confidence limits was obtained as described previously. Shifts were considered significant when the 95% confidence limits did not include one.

Other statistical procedures used included one-way and two-way analysis of variance (ANOVA) followed by a Tukey or Student-Newman-Keuls pairwise test for multiple comparisons and Student unpaired t-tests to compare the means of two groups. These procedures were undertaken using SigmaStat (version 2.0) or GraphPad PRISM (version 3.0). Statistical significance was accepted when P < 0.05.

Drugs

The drugs used were bestatin (Sigma, St. Louis); captopril (D-3-mercapto-2-methyl propanoyl-L-proline) (Sigma); [Lys5MeLeu9Nle10]NKA(4–10) (RBI, Sigma); methacholine chloride ((2-acetoxypropyl)trimethylammonium chloride) (Sigma); estradiol cypionate and estradiol benzoate (Sigma); neurokinin A (Auspep, Melbourne, Australia); neurokinin B (Auspep); [Sar9Met(O2)11]SP (Auspep); SR 140333 ((1-{2-(3,4-dichlorophenyl)-1-(3-isopropoxyphenylacetyl) piperidin-3-yl]ethyl}-4-phenyl-1-azonia-bicyclo[2.2.2]octane, chloride); SR 48968 ((S)-N-methyl-N[4-acetylamino-4-phenylpiperidino-2-(3,4-dichlorophenyl)butyl]benzamide) and SR 142801 ((S)-(N)-(1-(3-(1-benzoyl-3-(3,4-dichlorophenyl)piperidin-3-yl)propyl)-4-phenylpiperidine-4-yl)-N-methylactamide) (all generous gifts from Sanofi Recherche, Montpellier, France); substance P (Auspep); and DL-thiorphan (Sigma).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Functional Studies

All preparations initially exhibited spontaneous activity, which subsided during the equilibration period. The mean response to KPSS in pregnant mice was 1020.2 ± 67.6 g.s/5 min (n = 32 preparations). The mean response to MCh was 78.0% ± 2.6% of the response to KPSS. No significant differences in the mean responses to either MCh or KPSS were observed in experiments in which the potencies of tachykinin agonists was compared (one-way ANOVA, P > 0.05).

Agonist Studies

All tachykinin peptides tested elicited contractile activity in the pregnant mouse uterus. Figure 1 shows typical contractile responses to increasing concentrations of the NK1 receptor-selective agonist [Sar9Met(O2)11]SP and the NK2 receptor-selective agonist [Lys5MeLeu9Nle10]NKA(4–10), together with responses to MCh (10 µM) and KPSS.



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  		FIG. 1. Trace showing contractile responses of myometrial preparations obtained from a Day 17 pregnant mouse to increasing concentrations of the NK1 receptor-selective agonist [Sar9Met(O2)11]SP (upper) and the NK2 receptor-selective agonist [Lys5MeLeu9Nle10]NKA(4–10) (lower) together with responses to MCh (10 µM) and KPSS

The log concentration-response curves for SP, NKA, NKB, [Sar9Met(O2)11]SP, and [Lys5MeLeu9Nle10]NKA(4– 10) are shown in Figure 2, A and B. The relative order of potency for the mammalian tachykinins based on the positions of the log concentration-response curves was NKA>>SP≥NKB. The tachykinin receptor-selective agonists were approximately equipotent with NKA (one-way ANOVA, P > 0.05) with mean pD2 values of 8.02 ± 0.11, 8.09 ± 0.21, and 7.54 ± 0.11 (n = 6) for [Lys5MeLeu9Nle10]NKA(4–10), [Sar9Met(O2)11]SP, and NKA, respectively. The mean maximal response to NKA was 45.90% of that to KPSS. [Lys5MeLeu9Nle10]NKA(4– 10) produced a maximal response (50.00% ± 3.90% of the response to KPSS, Student unpaired t-test, P > 0.05) similar to that of NKA, which was significantly higher than that of [Sar9Met(O2)11]SP (Fig. 2B, one-way ANOVA, P < 0.05).



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  		FIG. 2. Log concentration-response curves to tachykinin peptides on myometrial preparations from Day 17 pregnant mice. Data points are expressed as a percentage of the response to KPSS and represent mean responses, with SEM shown by vertical bars. A) Mean responses to SP, NKA, and NKB. B) Mean responses to [Sar9Met(O2)11]SP and [Lys5MeLeu9Nle10]NKA(4–10) (n = 6–7)

Antagonist Studies

Figure 3 (A and B) shows the log concentration-response curves for NKA and [Lys5MeLeu9Nle10]NKA(4–10) in the absence and presence of SR 140333 (10 nM), SR 48968 (10 nM), or SR 142801 (0.3 µM). The NK2 receptor-selective antagonist SR 48968 (10 nM) produced a significant rightward shift in the position of the log concentration-response curve to NKA (12-fold, 95% CL = 2.5, 150.9; df = 25) and significantly attenuated the maximum response to [Lys5MeLeu9Nle10]NKA(4–10) (two-way ANOVA, P < 0.05). The NK1 receptor-selective antagonist SR 140333 (10 nM) and the NK3 receptor-selective antagonist SR 142801 (0.3 µM) were without significant effects. In contrast, SR 140333 (10 nM) significantly decreased the responses to SP and [Sar9Met(O2)11]SP (two-way ANOVA, P < 0.05; Fig. 3, C and D). Neither SR 48968 (10 nM) nor SR 142801 (0.3 µM) altered responses to SP or [Sar9Met(O2)11]SP (data not shown). None of the antagonists significantly affected the responses to MCh (10 µM) or KPSS (one-way ANOVA, P > 0.05).



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  		FIG. 3. Log concentration-response curves to NKA (A), [Lys5MeLeu9Nle10]NKA(4–10) (B), SP (C), and [Sar9Met(O2)11]SP (D) on myometrial preparations from Day 17 pregnant mice in the absence and presence of the NK1 receptor-selective antagonist SR 140333 (10 nM), the NK2 receptor-selective antagonist SR 48968 (10 nM), and the NK3 receptor-selective antagonist SR 142801 (0.3 µM) in (A) and (B) and SR 140333 (10 nM) in (C) and (D). Data points are expressed as a percentage of the response to KPSS (n = 5–7) and represent mean responses, with SEM shown by vertical bars

Peptidase Inhibitor Studies

In our previous study of the uterotonic effect of tachykinins in the nonpregnant, estrogen-primed mouse uterus [36] we included a cocktail of peptidase inhibitors comprising thiorphan (3 µM), captopril (10 µM), and bestatin (10 µM). In the present study, we compared the effects of this cocktail on responses to SP and NKA in the nonpregnant estrogen-primed and Day 17 pregnant mouse myometrial preparations (Fig. 4). Two-way ANOVAs showed that in tissues from estrogen-primed mice the responses to NKA were significantly larger in the presence of peptidase inhibitors (P < 0.05, df = 144), whereas the responses to SP in the presence of peptidase inhibitors did not differ significantly from control (P > 0.05, df = 128). In the pregnant mouse the responses to NKA (P > 0.05, df = 72) and SP (P > 0.05, df = 80) were not significantly different in the presence of the peptides inhibitor cocktail. The relative potencies of NKA and SP were unchanged from control data.



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  		FIG. 4. Log concentration-response curves to NKA and SP on myometrial preparations from estrogen-primed (A and C, n = 8–10) and Day 17 pregnant mice (B and D, n = 5–7) in the absence and presence of thiorphan (3 µM), captopril (10 µM), and bestatin (10 µM). Data points are expressed as a percentage of the response to KPSS and represent mean responses, with SEM shown by vertical bars

Molecular Studies

By using endpoint RT-PCR, we detected the presence of mRNA transcripts corresponding to the sizes expected for Tac2 (302 base pair; bp), Tac4 (186 bp), Tacr1 (223 bp), Tacr2 (251 bp), Tacr3 (272 bp), and Mme (255 bp). The identity of the amplified fragments was confirmed by DNA sequence analysis. The mRNA of Tac1 (364 bp), the gene that encodes SP and NKA, was absent or, in some uterine samples, present in trace amounts that were detectable only after amplification of higher amounts of cDNA. Figure 5 illustrates an example of an agarose gel showing RT-PCR products obtained by amplification of similar amounts of mouse uterine cDNA, as determined from the previous amplification of the ß-actin sequence. As can be observed, there were marked variations among the levels of expression of the different target genes depending on the stage of the estrus cycle and the stage of gestation.



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  		FIG. 5. Agarose gel showing expression of Tacr1, Tacr2, Tacr3, Tac1, Tac2, Tac4, and Mme mRNAs in uterine samples from nonpregnant mice in estrus or diestrus and from early (Day 5) and late (Day 17) pregnant mice. Equal amounts of each cDNA were amplified by PCR for 24 (ß-actin) or 35 cycles (target genes) with specific primer pairs. The expression of tachykinins, tachykinin receptors, and neprilysin in the mice brain cortex, used as a positive control of amplification of the target genes, is also shown. M, molecular weight standards

Real-time quantitative PCR was therefore used to compare the relative abundance of Tac2, Tac4, Tacr1, Tacr2, and Mme mRNAs among pregnant and nonpregnant mouse uteri. It was not possible to quantify the expression levels of Tac1 and Tacr3 due to the absence of the specific transcripts or, in the case of Tacr3, because quantifiable levels in uterine cDNA were present only in tissue from nonpregnant mice in diestrus.

A low expression of Tac2, the gene that encodes NKB, was detected in two of five nonpregnant mice in diestrous and in all assayed Day 5 and Day 17 pregnant mice (n = 5). There was no detectable expression in uterine cDNA from estrogen-treated (data not shown) animals or from those in estrous (see Fig. 5). The highest mRNA levels were found in Day 5 pregnant mice (Fig. 6A). Tac2 mRNA decreased by 38-fold in Day 17 pregnant mice, compared with those at Day 5 of pregnancy (Fig. 6A).



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  		FIG. 6. Real-time quantitative RT-PCR analysis of expression of (A) neurokinin B precursor (Tac2), (B) hemokinin-1 precursor (Tac4), (C) tachykinin NK1 receptor (Tacr1), and (D) tachykinin NK2 receptor (Tacr2) in cDNA from uteri of nonpregnant (estrous or diestrous) and pregnant (Day 5 or Day 17) mice. Values for Tac2, Tac4, Tacr1, and Tacr2 mRNA levels are shown in arbitrary units, relative to ß-actin mRNA expression. Each bar represents the mean of uterine cDNA samples from five different mice, with SEM shown by vertical lines. aP < 0.05, significant difference versus mRNA levels in uteri from nonpregnant estrous mice; bP < 0.05, significant difference versus mRNA levels in uteri from Day 5 pregnant mice; cP < 0.05, significant difference versus mRNA levels in uteri from Day 17 pregnant mice, one-way ANOVA or Student t-test for unpaired data

The mRNA of Tac4, the gene that encodes HK-1, was present in all uteri assayed. The highest expression was observed in uteri from nonpregnant diestrous mice and from Day 5 pregnant mice (Fig. 6B). Compared with mice in diestrus, Tac4 mRNA decreased by 2.6-fold in uteri from nonpregnant mice in estrus and by 10.2-fold in Day 17 pregnant mice (Fig. 6B).

Tachykinin NK1 receptor mRNA levels were similar in uteri from nonpregnant estrous, diestrous (Fig. 6C), and estrogen-treated (data not shown) mice. Compared with its expression in estrous and diestrous mice, Tacr1 mRNA increased by approximately 2-fold on Day 5 of pregnancy and decreased by approximately 11-fold on Day 17 of pregnancy (Fig. 6C).

The mRNA levels of the tachykinin NK2 receptor in uteri from either nonpregnant or pregnant mice were low (see Fig. 5) and it was necessary to use a higher number of cycles (42 cycles) for real-time PCR detection. Tacr2 expression was slightly higher in pregnant compared with nonpregnant animals (Fig. 6D). However, there were no significant differences between the mRNA levels in the different conditions assayed (P > 0.05; n = 5 mice per group, one-way ANOVA; Fig. 6D).

The mRNA of neprilysin was detected in all uteri assayed both from pregnant and nonpregnant mice. Among the genes assayed, the expression of NEP varied most among the different uterine samples. Thus, compared with nonpregnant mice in diestrus (n = 5), Mme mRNA levels decreased by 14.5-fold in nonpregnant estrous mice (P < 0.01; n = 5), by 4-fold in Day 5 pregnant mice (P < 0.05; n = 5), and by 9-fold at Day 17 of pregnancy (P < 0.01; n = 5), (Fig. 7). The expression of neprilysin was similar in estrous mice and in Day 17 pregnant animals (P > 0.05, one-way ANOVA; Fig. 7).



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  		FIG. 7. Real-time quantitative RT-PCR analysis of expression of neprilysin (Mme) in cDNA from uteri of nonpregnant (estrous or diestrous) and pregnant (Day 5 or Day 17) mice. Values for Mme mRNA levels are shown in arbitrary units, relative to ß-actin mRNA expression. Each bar represents the mean of uterine cDNA samples from five different mice, with SEM shown by vertical lines. aP < 0.05, significant difference versus mRNA levels in uteri from nonpregnant estrous mice. bP < 0.05, significant difference versus mRNA levels in uteri from Day 5 pregnant mice. cP < 0.05, significant difference versus mRNA levels in uteri from Day 17 pregnant mice, one-way ANOVA


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The main findings of the present study demonstrate that pregnancy induced a change in the predominant tachykinin receptor type that mediates the contractile effects of tachykinins in the mouse uterus. This shift appears due in part to down-regulation of the expression of the tachykinin NK1 receptor during late pregnancy. Furthermore, the mRNAs of the tachykinins NKB and HK-1, and the tachykinin receptors, are shown to be locally expressed in the mouse uterus with their expression levels changing during the estrous cycle and during pregnancy.

Several recent findings at both central [42, 43] and peripheral [12–14, 24, 33, 35, 44] levels argue for a role for tachykinins in the regulation of female reproductive function. Studies carried out with uteri from humans and rats have shown the presence of all mammalian tachykinins, their receptors, and the tachykinin-metabolizing enzyme neprilysin [8, 19, 22–24, 32–36, 45]. In late pregnant and nonpregnant women and rats, tachykinins induced myometrial contractions that were mainly mediated by the tachykinin NK2 receptor, with minor participation by the NK1 receptor [22, 24, 27–35, 37, 46, 47]. In contrast, in nonpregnant estrogen-treated BalbC mice, tachykinin-induced uterine contractions were predominantly mediated through NK1 receptor stimulation, with NK2 receptors playing a less important role in the response induced by NKA or NKB [36].

The present functional studies in contrast demonstrate that contractions induced by tachykinins in longitudinally oriented uterine smooth muscle from late pregnant BalbC mice were predominantly mediated by activation of tachykinin NK2 receptors, with a relatively minor role for NK1 receptors. Thus, in uteri from late pregnant animals, 1) SP, NKA, and NKB produced concentration-related contractions of uterine preparations with an order of potency of NKA>>SP≥NKB; 2) the maximum response to the NK2 receptor-selective agonist [Lys5MeLeu9Nle10]NKA(4–10) was approximately 20 times greater than that previously reported [36] for preparations from nonpregnant estrogen primed mice; 3) the tachykinin NK2 receptor-selective antagonist SR 48968 (10 nM) shifted the log concentration-response curve to NKA to the right and reduced the effect of [Lys5MeLeu9Nle10]NKA(4–10); 4) the tachykinin NK1 receptor-selective antagonist SR 140333 (10 nM) reduced the effects of SP and [Sar9Met(O2)11]SP but did not affect responses to NKA and [Lys5MeLeu9Nle10]NKA(4–10); and 5) the tachykinin NK3 receptor-selective antagonist SR 142801 (0.3 µM) did not affect responses to any of the agonists tested. Taken together, these data indicate that the NK2 receptor predominates in uteri from late pregnant mice.

The data above indicate that physiological changes in the hormonal environment are capable of modifying the hierarchy of tachykinin receptor types mediating uterine contractions in the mouse. The present molecular studies show that Tacr1 mRNA levels decreased by about 11-fold at Day 17 of pregnancy compared with uteri from nonpregnant estrous, or estrogen-treated mice. These findings demonstrate, in good correlation with functional data, that the tachykinin NK1 receptor was the predominant receptor expressed in uteri from the nonpregnant mouse and that its expression decreased markedly at late pregnancy. Tachykinin NK2 receptor mRNA levels were slightly higher in pregnant animals, an observation that could explain the enhanced effectiveness of the NK2 receptor-selective agonist [Lys5MeLeu9Nle10]NKA(4–10), but remained essentially constant in the different hormonal stages assayed, a result identical to that previously found in the rat uterus [33]. The shift observed in functional responses in uteri from late pregnant animals appears therefore to be caused by 1) the decrease in tachykinin NK1 receptor protein levels, 2) a slight increase of the tachykinin NK2 receptor, and 3) a possible increase in the Ca2+ sensitivity of the contractile machinery [37, 48, 49]. This is in line with the two described primary ways to modulate signal transduction through G protein-coupled receptors at the receptor level: first by structural modification of the various interactions between the receptor and other molecules that determine signal amplification and termination; and second, by a variation in the effective number of receptors available to the ligand. A third additional possibility is that there could be changes in the populations of receptor isoforms for each of the tachykinin receptor types. To this end in human nonpregnant uteri, one of us (unpublished observations) has confirmed the presence of the short NK1 receptor, previously described by Fong et al. [50], which is missing its 96 amino acid C-terminal tail. This isoform occurs in a ratio of 1 to 2.3 for the short NK1 receptor to the long NK1 receptor isoform. It is not yet known whether such a phenomenon occurs in the mouse, but these two NK1 receptor isoforms have been shown to have altered effector systems [50], and hence differential isoforms could provide another dimension to controlling uterine contractility.

Until recently, peripherally located tachykinins were regarded as being primarily of neuronal origin [7, 51]. The major tachykinins so expressed, including SP and NKA, are the products of the Tac1 gene and affect visceral function, including that mediated by inflammatory and immune cells; by endocrine and exocrine glands; and by vascular and visceral smooth muscle [6, 7, 9], including uterine smooth muscle from rats, mice, and humans [19]. More recently, NKB, produced by the Tac2 (human TAC3) gene, has also been reported to be present in the periphery [22, 52], and this peptide, and new members of the tachykinin family, including mouse HK-1 [1], and their human orthologs [5, 8] derived from the Tac4 (human TAC4) gene are now known to be expressed in cells of non-neuronal origin, including those within the rat and human uterus, and the placenta [8, 19, 23, 52, 53]. In the present molecular studies the differential distributions and levels of expression of the three tachykinin-encoding genes are of particular interest in that they suggest 1) different cellular or regional locations (or both) of Tac1 on the one hand, and Tac2 and Tac4 on the other hand; and 2) differential modulation of these genes by hormonal and gestational status.

SP- and NKA-like immunoreactivity has been localized within uterine nerves in different species, including the mouse [15–19]. To our knowledge, the presence of NKB and HK-1 in these nerves has not been demonstrated. The present molecular studies show that the Tac4 gene, encoding for HK-1, was consistently expressed in both estrous and diestrous mice and on Days 5 and 17 of pregnancy. The Tac2 gene was also expressed in some, but not all, diestrous uteri, and also on Days 5 and 17 of pregnancy. Most notably, there was marked down-regulation of both Tac2 and Tac4 between early and late pregnancy. In contrast, although SP-like immunoreactivity can be observed in nonpregnant mouse uterus [19], Tac1 transcripts were not detected in the uterus either during the estrous cycle or pregnancy. This finding is reminiscent of our recent findings with the human myometrium [24], and may be similarly explained, because the cell bodies of the sensory neurons in which SP and NKA are synthesized before their transport to the peripheral terminals are not present in the myometrium of either species. In contrast, the products of the Tac2 and Tac4 genes in the mouse are locally produced and most likely expressed in the variety of non-neuronal cells in, or associated with, the uterine endometrium and myometrium, as reported previously for both genes in different peripheral tissues [1, 8, 22, 23, 53].

As yet, the presence of NKB and HK-1 peptides in the uterus has not been described; however, our real-time PCR data suggest that they might be expected to be present in highest levels in diestrus and early pregnancy. Moreover, treatment of mice in vivo with the translation inhibitor cycloheximide caused a significant increase in Tac2 and Tac4 expression levels in uterine cDNA, suggesting that, under physiological conditions, these mRNAs are being translated (unpublished observations). These data strongly suggest that NKB and HK-1 peptides are locally synthesized and may play an endocrine or paracrine role (or both) in the mammalian uteri.

The tachykinin NK3 receptor is expressed in uteri from all mammalian species so far assayed, and its expression exhibits strong variation depending on the hormonal conditions [22, 24, 32, 45]. Little is known, however, about its physiological role in the female genital tract. Our data in humans [19, 24], rats [22, 30, 33], and mice (present study) indicate that the NK3 receptor may be primarily involved in functions other than mediating uterine contraction.

The present study shows that expression of the Mme gene in the mouse uteri varied markedly during the estrous cycle and during pregnancy, as also occurs in the rat and human [24, 32, 54, 55]. In rat uterus (see [19] for references) inhibitors of the enzyme neprilysin cause a marked potentiation of the uterotonic actions of tachykinins, indicating that this enzyme plays a major role in limiting these actions. In contrast, our present functional experiments did not indicate an unequivocal role for this enzyme in limiting tachykinin-induced contractions. This peptidase degrades a number of other biologically active peptides [56], and, accordingly, may have other roles in the mouse uterus.

In conclusion, the present data show that mRNAs for Tac2 and Tac4, the genes that encode NKB and HK-1, respectively, and for the three tachykinin receptors, are expressed in the mouse uteri and their expression levels varied during the estrous cycle and throughout pregnancy. At least two of these receptors, NK1 and NK2, are involved in uterine contractility, with a switch from a receptor type to another depending on the hormonal environment. The distribution of these peptides and their receptors in tissues of the female mouse reproductive tract, together with their modulation by hormonal and gestational status, as has been shown previously for rat and human, indicates a role in mammalian reproductive function.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. X. Emonds-Alt for his generous gift of tachykinin receptor antagonists SR 140333, SR 48968, and SR 142801.


   FOOTNOTES
 
1 Supported by grants from the National Health and Medical Research Council of Australia; the Royal Women's Hospital, Carlton, Victoria, Australia; the Medical Research Council, United Kingdom; and Ministerio de Educación y Ciencia (SAF2002-04080-CO2-01), Spain. E.P and F.M.P. contributed equally to this work. Back

2 Correspondence: M. Luz Candenas, Centro de Investigaciones Científicas Isla de La Cartuja, Instituto de Investigaciones Químicas, Avda. Americo Vespucio s/n, 41092 Sevilla, Spain. FAX: 34 95 446 0565; luzcandenas{at}iiq.csic.es Back

3 Correspondence: Jocelyn N Pennefather, Department of Pharmaceutical Biology and Pharmacology, Victorian College of Pharmacy, Monash University, Parkville, Victoria 3052, Australia. FAX: 61 39 833 3754; jocelyn.oneil{at}vcp.monash.edu.au Back

Received: 4 October 2004.

First decision: 24 October 2004.

Accepted: 20 December 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Zhang Y, Lu L, Furlonger C, Wu GE, Paige CJ. Hemokinin is a hematopoietic-specific tachykinin that regulates B lymphopoiesis. Nat Immunol 2000 1:392-397[CrossRef][Medline]
  2. Morteau O, Lu B, Gerard C, Gerard NP. Hemokinin 1 is a full agonist at the substance P receptor. Nat Immunol 2001 2:1088[CrossRef][Medline]
  3. Bellucci F, Carini F, Catalani C, Cucchi P, Lecci A, Meini S, Patacchini R, Quartara L, Ricci R, Tramontana M, Giuliani S, Maggi CA. Pharmacological profile of the novel mammalian tachykinin, hemokinin 1. Br J Pharmacol 2002 135:266-274[CrossRef][Medline]
  4. Camarda V, Rizzi A, Calo G, Guerrini R, Salvadori S, Regoli D. Pharmacological profile of hemokinin 1: a novel member of the tachykinin family. Life Sci 2002 71:363-370[CrossRef][Medline]
  5. Kurtz MM, Wang R, Clements M, Cascieri M, Austin C, Cunningham B, Chicchi G, Liu Q. Identification, localization and receptor characterization of novel mammalian substance P-like peptides. Gene 2002 296:205-212[CrossRef][Medline]
  6. Patacchini R, Lecci A, Holzer P, Maggi CA. Newly discovered tachykinins raise new questions about their peripheral roles and the tachykinin nomenclature. Trends Pharmacol Sci 2004 25:1-3[CrossRef][Medline]
  7. Pennefather JN, Lecci A, Candenas ML, Patak E, Pinto FM, Maggi CA. Tachykinins and tachykinin receptors: a growing family. Life Sci 2004 74:1445-1463[CrossRef][Medline]
  8. Page NM, Bell NJ, Gardiner SM, Manyonda IT, Brayley KJ, Strange PG, Lowry PJ. Characterization of the endokinins: human tachykinins with cardiovascular activity. Proc Natl Acad Sci U S A 2003 100:6245-6250[Abstract/Free Full Text]
  9. Maggi CA. The effects of tachykinins on inflammatory and immune cells. Regul Pept 1997 70:75-90[CrossRef][Medline]
  10. Henry JL. Discussions of nomenclature for tachykinins and tachykinin receptors. In: Henry JL, Couture R, Cuello AC, Pelletier G, Quirion R, Regoli D (eds.), Substance P and Neurokinins. New York: Springer-Verlag; 1986:xvii–xviii
  11. Regoli D, Nguyen QT, Jukic D. Neurokinin receptor subtypes characterized by biological assays. Life Sci 1994 54:2035-2047[CrossRef][Medline]
  12. Page NM, Woods RJ, Lowry PJ. A regulatory role for neurokinin B in placental physiology and pre-eclampsia. Regul Pept 2001 98:97-104[CrossRef][Medline]
  13. Loffler S, Schulz A, Hunt SP, Spanel-Borowski K. Increased formation of corpora lutea in neurokinin 1-receptor deficient mice. Mol Reprod Dev 2004 68:408-414[CrossRef][Medline]
  14. Pintado CO, Pinto FM, Pennefather JN, Hidalgo A, Baamonde A, Sanchez T, Candenas ML. A role for tachykinins in female mouse and rat reproductive function. Biol Reprod 2003 69:940-946[Abstract/Free Full Text]
  15. Alm P, Alumets J, Brodin E, Hakanson R, Nilsson G, Sjoberg NO, Sundler F. Peptidergic (substance P) nerves in the genito-urinary tract. Neuroscience 1978 3:419-425[CrossRef][Medline]
  16. Fried G, Meister B, Radestad A. Peptide-containing nerves in the human pregnant uterine cervix: an immunohistochemical study exploring the effect of RU 486 (mifepristone). Hum Reprod 1990 5:870-876[Abstract/Free Full Text]
  17. Heinrich D, Reinecke M, Forssmann WG. Peptidergic innervation of the human and guinea pig uterus. Arch Gynecol 1986 237:213-219[CrossRef][Medline]
  18. Huang WM, Gu J, Blank MA, Allen JM, Bloom SR, Polak JM. Peptide-immunoreactive nerves in the mammalian female genital tract. Histochem J 1984 16:1297-1310[CrossRef][Medline]
  19. Pennefather JN, Patak E, Pinto FM, Candenas ML. Mammalian tachykinins and uterine smooth muscle: the challenge escalates. Eur J Pharmacol 2004 500:15-26[CrossRef][Medline]
  20. Reinecke M, Gauwerky JF, Schneider K. Peptidergic (NPY, NT, VIP, SP, CGRP) innervation of the functional systems of the uterus and fallopian tube in the human. Arch Gynecol Obstet 1989 245:399-401[CrossRef][Medline]
  21. Shew RL, Papka RE, McNeill DL. Substance P and calcitonin gene-related peptide immunoreactivity in nerves of the rat uterus: localization, colocalization and effects on uterine contractility. Peptides 1991 12:593-600[CrossRef][Medline]
  22. Cintado CG, Pinto FM, Devillier P, Merida A, Candenas ML. Increase in neurokinin B expression and in tachykinin NK(3) receptor-mediated response and expression in the rat uterus with age. J Pharmacol Exp Ther 2001 299:934-938[Abstract/Free Full Text]
  23. Pinto FM, Cintado CG, Devillier P, Candenas ML. Expression of preprotachykinin-B, the gene that encodes neurokinin B, in the rat uterus. Eur J Pharmacol 2001 425:R1-R2[CrossRef][Medline]
  24. Patak E, Candenas ML, Pennefather JN, Ziccone S, Lilley A, Martín JD, Flores C, Mantecón AG, Pinto FM. Tachykinins and tachykinin receptors in human uterus. Br J Pharmacol 2003 139:523-532[CrossRef][Medline]
  25. Fisher L, Pennefather JN. Potencies of agonists acting at tachykinin receptors in the oestrogen-primed rat uterus: effects of peptidase inhibitors. Eur J Pharmacol 1997 335:221-226[CrossRef][Medline]
  26. Fisher L, Pennefather JN. Structure-activity studies of analogues of neurokinin A mediating contractions of rat uterus. Neuropeptides 1998 32:405-410[CrossRef][Medline]
  27. Fisher L, Pennefather JN. Tachykinin receptors mediating contractions of oestrogen-primed rat uterus: classification using non-peptide antagonists. Clin Exp Pharmacol Physiol 1999 26:729-735[CrossRef][Medline]
  28. Fisher L, Pennefather JN, Hall S. Tachykinin receptors in the rat isolated uterus. Regul Pept 1993 46:396-398[CrossRef][Medline]
  29. Pennefather JN, Zeng XP, Gould D, Hall S, Burcher E. Mammalian tachykinins stimulate rat uterus by activating NK-2 receptors. Peptides 1993 14:169-174[CrossRef][Medline]
  30. Moodley N, Lau WA, Pennefather JN, Story ME, Fisher L. NK2 receptors mediate tachykinin-induced contractions of rat uterus during the oestrous cycle. Eur J Pharmacol 1999 376:53-60[CrossRef][Medline]
  31. Magraner J, Pinto FM, Anselmi E, Hernandez M, Perez R, Martín JD, Advenier C, Candenas ML. Characterization of tachykinin receptors in the uterus of the oestrogen-primed rat. Br J Pharmacol 1998 123:259-268[CrossRef][Medline]
  32. Pinto FM, Armesto CP. Magraner J, Trujillo M. Martín JD, Candenas ML. Tachykinin receptor and neutral endopeptidase gene expression in the rat uterus: characterization and regulation in response to ovarian steroid treatment. Endocrinology 1999 140:2526-2532[Abstract/Free Full Text]
  33. Candenas ML, Magraner J, Armesto CP, Anselmi E, Nieto PM, Martin JD, Advenier C, Pinto FM. Changes in the expression of tachykinin receptors in the rat uterus during the course of pregnancy. Biol Reprod 2001 65:538-543[Abstract/Free Full Text]
  34. Patak EN, Pennefather JN, Story ME. Effects of tachykinins on uterine smooth muscle. Clin Exp Pharmacol Physiol 2000 27:922-927[CrossRef][Medline]
  35. Patak EN, Ziccone S, Story ME, Fleming AJ, Lilley A, Pennefather JN. Activation of neurokinin NK(2) receptors by tachykinin peptides causes contraction of uterus in pregnant women near term. Mol Hum Reprod 2000 6:549-554[Abstract/Free Full Text]
  36. Patak E, Pennefather JN, Fleming A, Story ME. Functional characterization of tachykinin NK(1) receptors in the mouse uterus. Br J Pharmacol 2002 137:1247-1254[CrossRef][Medline]
  37. Shintani Y, Nishimura J, Niiro N, Hirano K, Nakano H, Kanaide H. Mechanisms underlying the neurokinin A-induced contraction of the pregnant rat myometrium. Br J Pharmacol 2000 130:1165-1173[CrossRef][Medline]
  38. Patak E, Fleming A, Story ME, Candenas ML, Pennefather JN. Pregnancy-induced switch in receptors mediating the uterotonic effects of tachykinins in the mouse. Proc Aust Soc Clin Exp Pharmacol Toxicol 2003 11:A125
  39. Candenas L, Patak E, Ventura S, Story M, Pinto F, Pennefather JN. Expression of tachykinins and tachykinin receptors in the mouse uterus. Proc Aust Soc Clin Exp Pharmacol Toxicol 2003 11:A202
  40. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987 162:156-159[Medline]
  41. Rozen S, Skaletsky H. Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 2000 132:365-386[Medline]
  42. Debeljuk L, Lasaga M. Modulation of the hypothalamo-pituitary-gonadal axis and the pineal gland by neurokinin A, neuropeptide K and neuropeptide gamma. Peptides 1999 20:285-299[CrossRef][Medline]
  43. Goubillon ML, Forsdike RA, Robinson JE, Ciofi P, Caraty A, Herbison AE. Identification of neurokinin B-expressing neurons as a highly estrogen-receptive, sexually dimorphic cell group in the ovine arcuate nucleus. Endocrinology 2000 141:4218-4225[Abstract/Free Full Text]
  44. Debeljuk L. Tachykinins in the normal and gonadotropin-stimulated ovary of the mouse. Peptides 2003 24:1445-1448[CrossRef][Medline]
  45. Pinto FM, Magraner J, Ausina P, Anselmi E, Martín JD, Candenas ML. Regulation by oestrogens of tachykinin NK3 receptor expression in the rat uterus. Eur J Pharmacol 1997 324:125-127[CrossRef][Medline]
  46. Hamlin GP, Williams MJ, Nimmo A, Crane LH. Hormonal variation of rat uterine contractile responsiveness to selective neurokinin receptor agonists. Biol Reprod 2000 62:1661-1666[Abstract/Free Full Text]
  47. Williams MJ, Hamlin GP, Nimmo AJ, Crane LH. Circular versus longitudinal myometrial contractile activity to selective tachykinin receptor agonists in the rat. Reprod Fertil Dev 2003 15:311-316[CrossRef][Medline]
  48. Trujillo M, Candenas L, Cintado CG, Magraner J, Fernandez J, Martin JD, Pinto FM. Hormonal regulation of the contractile response induced by okadaic acid in the rat uterus. J Pharmacol Exp Ther 2001 296:841-848[Abstract/Free Full Text]
  49. Cario-Toumaniantz C, Reillaudoux G, Sauzeau V, Heutte F, Vaillant N, Finet M, Chardin P, Loirand G, Pacaud P. Modulation of RhoA-Rho kinase-mediated Ca2+ sensitization of rabbit myometrium during pregnancy—role of Rnd3. J Physiol 2003 552:403-413[Abstract/Free Full Text]
  50. Fong TM, Anderson SA, Yu H, Huang RR, Strader CD. Differential activation of intracellular effector by two isoforms of human neurokinin-1 receptor. Mol Pharmacol 1992 41:24-30[Abstract]
  51. Maggi CA, Meli A. The sensory-efferent function of capsaicin-sensitive sensory neurons. Gen Pharmacol 1988 19:1-43[Medline]
  52. Page NM, Woods RJ, Gardiner SM, Lomthaisong K, Gladwell RT, Butlin DJ, Manyonda IT, Lowry PJ. Excessive placental secretion of neurokinin B during the third trimester causes pre-eclampsia. Nature 2000 405:797-800[CrossRef][Medline]
  53. Pinto FM, Almeida TA, Hernandez M, Devillier P, Advenier C, Candenas ML. mRNA expression of tachykinins and tachykinin receptors in different human tissues. Eur J Pharmacol 2004 494:233-239[CrossRef][Medline]
  54. Casey ML, Smith JW, Nagai K, Hersh LB, MacDonald PC. Progesterone-regulated cyclic modulation of membrane metalloendopeptidase (enkephalinase) in human endometrium. J Biol Chem 1991 266:23041-23047[Abstract/Free Full Text]
  55. Ottlecz A, Walker S, Conrad M, Starcher B. Neutral metalloendopeptidase associated with the smooth muscle cells of pregnant rat uterus. J Cell Biochem 1991 45:401-411[CrossRef][Medline]
  56. Roques BP, Noble F, Dauge V, Fournie-Zaluski MC, Beaumont A. Neutral endopeptidase 24.11: structure, inhibition, and experimental and clinical pharmacology. Pharmacol Rev 1993 45:87-146[Medline]



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