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BOR - Papers in Press, published online ahead of print May 17, 2006.
Biol Reprod 2006, 10.1095/biolreprod.106.051508
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BIOLOGY OF REPRODUCTION 75, 334–341 (2006)
DOI: 10.1095/biolreprod.106.051508
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Regulation of the Stimulant Actions of Neurokinin A and Human Hemokinin-1 on the Human Uterus: A Comparison with Histamine1

Jocelyn N. Pennefather 2 45 , Eva Patak 4, Sebastian Ziccone 4, Alison Lilley 4, Francisco M. Pinto 6, Nigel M. Page 7, Margot E. Story 8, Sonia Grover 910 , and M. Luz Candenas 3 6

Department of Anaesthesia,4 Royal Women's Hospital, Carlton, Victoria 3053, Australia Department of Pharmaceutical Biology,5 Victorian College of Pharmacy, Monash University, Parkville, Victoria 3052, Australia Instituto de Investigaciones Químicas,6 41092 Sevilla, Spain School of Life Sciences,7 Kingston University London, Kingston-upon-Thames, Surrey KT1 1LQ, United Kingdom Department of Pharmacology,8 Monash University, Clayton, Victoria 3800, Australia Royal Children's Hospital and Mercy Hospital for Women,9 Parkville, Victoria 3052, Australia University of Melbourne Department of Obstetrics and Gynaecology,10 Royal Women's Hospital, Carlton, Victoria 3053, Australia

ABSTRACT

Regulation of the contractile effects of tachykinins and histamine on the human uterus was investigated with biopsy sections of the outer myometrial layer. The effects of neurokinin A (NKA) and human hemokinin-1 (hHK-1) in tissues from pregnant but not from nonpregnant women were enhanced by the inhibition of neprilysin. The effects of NKA and eledoisin were blocked by the NK2 receptor antagonist SR 48968 but not by the NK1 receptor antagonist SR 140333 in tissues from both groups of women. Human HK-1 acted as a partial agonist blocked by SR 48968 and, to a lesser extent, by SR 140333; endokinin D was inactive. In tissues from pregnant women, responses to high potassium-containing Krebs solution were 2–3-fold higher than those from nonpregnant women. Mepyramine-sensitive maximal responses to histamine were similarly enhanced. The absolute maximum responses to NKA and its stable NK2 receptor-selective analogue, [Lys5MeLeu9Nle10]NKA(4–10), were increased in pregnancy, but their efficacies relative to potassium responses were decreased. Tachykinin potencies were lower in tissues from pregnant women than in those from nonpregnant women. These data 1) show for the first time that hHK-1 is a uterine stimulant in the human, 2) confirm that the NK2 receptor is predominant in mediating tachykinin actions on the human myometrium, and 3) indicate that mammalian tachykinin effects are tightly regulated during pregnancy in a manner that would negate an inappropriate uterotonic effect. The potencies of these peptides in tissues from nonpregnant women undergoing hysterectomy are consistent with their possible role in menstrual and menopausal disorders.

eledoisin, endokinin, female reproductive tract, hemokinin, histamine, human myometrium, [Lys5MeLeu9Nle10]NKA(4–10), neprilysin, neurokinin A, neuropeptides, NK1 and NK2 receptors, pregnancy, signal transduction, tachykinins, uterine contractions, uterus

INTRODUCTION

Mammalian tachykinins comprise a family of regulatory peptides characterized by the presence of the common amidated C-terminal sequence Phe-X-Gly-Leu-Met-NH2. In humans, the presently known tachykinins are the products of three different genes [see 1–4 for reviews]. The TAC1 gene gives rise to four different mRNA splicing isoforms that encode substance P (SP), neurokinin A (NKA), and the N-terminally extended forms of NKA named neuropeptide K and neuropeptide {gamma}. The TAC3 gene encodes neurokinin B (NKB). The TAC4 gene can also generate four distinct mRNAs and encodes hemokinin-1 (HK-1) and its N-terminally extended forms named endokinin A (EKA) and endokinin B (EKB). SP, NKA, and NKB are identical in all mammalian species so far investigated, while HK-1 differs structurally in humans and rodents [1–5]. Two additional tachykinin-like peptides derived from TAC4 but having a distinct C-terminus motif (Phe-X-Gly-Leu-Leu-NH2) have also been described and named endokinin C (EKC) and endokinin D (EKD) [5].

Tachykinin peptides are present within capsaicin-sensitive sensory nerves supplying the female reproductive tract (SP and NKA) and/or in nonneuronal cells and the placenta (NKB, SP, and HK-1) [1, 2, 6, 7]. This distribution suggests an important role for tachykinins in intercellular communication within both the pregnant and nonpregnant uterus. These peptides may play roles in inflammatory uterine events, e.g., in stress-induced abortion, menstrual disorders, and preterm labor [1, 7, 8]. In particular, the close association of different tachykinin-expressing cells with uterine smooth muscle indicates that tachykinins released influence uterine contractility. In previous studies, it has been shown that tachykinins are potent contractile agents in the mammalian myometrium and that their effects are regulated by pregnancy and steroidal hormones [1, 6, 7, 9–15]. Compared with SP and NKB, NKA is a potent uterotonic agent in both the late pregnant and nonpregnant human myometrium [14, 15].

The biological actions of tachykinins are mediated through three receptors belonging to the family of G protein-coupled receptors, denoted TACR1 (NK1), TACR2 (NK2), and TACR3 (NK3), which have the highest affinity for SP and HK-1, NKA, and NKB, respectively [4, 7, 16, 17]. In the myometrium from late pregnant women, the order of potency of the mammalian tachykinins (NKA > SP {approx} NKB) and the effects of tachykinin receptor-selective agonists indicated that the NK2 receptor mediates the uterotonic effects of the tachykinins. Furthermore, the tachykinin NK2 receptor-selective antagonist SR 48968 [18] was a potent antagonist of the NK2 receptor-selective agonist [Lys5MeLeu9Nle10]NKA(4–10), confirming the importance of NK2 receptors in mediating myometrial contraction. Molecular studies of the human uterus indicated that these receptors as well as the tachykinins are expressed in both the pregnant and nonpregnant myometrium [2, 15].

MME, the gene encoding neprilysin, the major cell surface peptidase that degrades tachykinins [19–21], is also present in the human uterus and is more strongly expressed in the pregnant than in the nonpregnant uterus [15]. Inhibitors of this enzyme markedly potentiate the uterotonic effects of substrate tachykinins in the rat [6, 9, 10] but are less effective in the mouse [13]. We have previously reported that responses to NKA in myometrial preparations from women undergoing hysterectomies were unaffected by the neprilysin inhibitor thiorphan, alone or in combination with the aminopeptidase inhibitor bestatin [15]. While the uterotonic effects of SP on the pregnant human uterus are potentiated by thiorphan in combination with bestatin [14], the role of neprilysin, if any, in regulating the effects of NKA and human (h)HK-1 in pregnancy is not known.

The aims of the present study with human myometrial preparations were to 1) examine further the uterotonic actions of tachykinins, particularly of hHK-1 and NKA, 2) characterize the receptors mediating their effects, and 3) examine the effects of neprilysin inhibitors on responses to tachykinins. In addition, we compared the effects of gestational status on responses to tachykinins with those of the nonpeptide, uterotonic, and inflammatory mediator named histamine. This monoamine is one of the key constitutive components of human uterine mast cells and may contribute to uterine contractility in both normal and premature labor [22–26]. For this purpose, we analyzed the contractile effects of tachykinins and histamine on the myometrium from nonpregnant women undergoing hysterectomies as well as from late pregnant women.

MATERIALS AND METHODS

Prior ethical approval for this study was obtained from the Ethics Committee of the Royal Women's Hospital (Victoria, Australia). All women had given written informed consent.

Tissue Preparation

Human myometrium was obtained from 1) 35 women (median age = 33.5 yr) undergoing elective lower uterine segment cesarean section (LUSCS) at 37–40-wk gestation and 2) 28 nonpregnant women (median age = 44 yr) undergoing hysterectomies for benign uterine disease. The difference between the mean ages of the women was statistically significant (Student unpaired t-test, P < 0.001; data not shown). The pregnant women were not in labor.

All women undergoing an LUSCS received ranitidine as a preanesthetic medication. This was followed by either spinal or epidural anesthesia induced by a combination of bupivicaine and fentanyl or marcain and fentanyl. Specimens were excised from the upper edge of the incision.

Hysterectomies were performed under general anesthetic induced by propofol and maintained with isoflurane, sevoflurane, or nitrous oxide either alone or in combination. Tissue obtained after hysterectomy was from an area corresponding to that taken at LUSCS.

Tissue samples were immediately placed in a Petri dish containing a modified Krebs-Henseleit solution (in millimoles: NaCl, 118.0; KCl, 4.7; MgSO4 7H2O, 1.1; KH2PO4, 1.18; NaHCO3, 25.0; glucose, 11.66; and CaCl2 2H2O, 1.9), and the inner and outer myometrial layers were identified. Four to eight preparations (3 x 3 x 10 mm) were obtained from the outer layer and attached to tissue holders to measure the contractile force produced by the longitudinally oriented smooth muscle fibers. The use of four to eight myometrial strips permitted an investigation of the effects of peptidase inhibitors, antagonists, or both on the response to an agonist in the uterus of each woman. Preparations were then placed in siliconized 5-ml organ baths containing the modified Krebs-Henseleit solution maintained at 37°C and continuously bubbled with carbogen (5% CO2 in O2; pH = 7.4). Each preparation was attached to a Grass FT03 force transducer that was connected to a MACLAB data acquisition system.

Agonist Log Concentration-Response Curves

Each tissue was allowed to equilibrate for 60–90 min under an initial force of 15–20 g before agonists were added. Discrete log concentration-response curves, usually with a progression ratio of one-half log unit over 5.5 orders of magnitude, were then constructed for NKA, [Lys5MeLeu9Nle10]NKA(4–10), eledoisin, EKD, hHK-1, and histamine. Each agonist concentration remained in contact with the tissue for 5 min; the tissue was then washed with two to three times the bath volume, and a higher concentration of agonist was added 15 min later. Only one concentration-response curve was constructed on each preparation. At the conclusion of the experiment, each tissue was exposed to a modified Krebs physiological saline solution (KPSS), in which 40 mM KCl replaced 40 mM NaCl. The reproducibility of the protocol has been established in our earlier studies [6, 14, 15]. Tissues were weighed at the end of each experiment.

Effects of Peptidase Inhibitors

The effects of the neprilysin inhibitors thiorphan and phosphoramidon, alone or in combination with the angiotensin-converting enzyme inhibitor captopril and/or the aminopeptidase inhibitor bestatin, on log concentration-response curves to NKA, [Lys5MeLeu9Nle10]NKA(4–10), hHK-1, and eledoisin were examined in tissues from pregnant women. The effects of inhibitors on responses to NKA and hHK-1 in tissues from nonpregnant women were also investigated. The inhibitors were added 20 min before the first agonist addition and were replaced after each wash.

Effects of Antagonists

A series of experiments examining the effects of the tachykinin NK2 receptor-selective antagonist SR 48968 (1 nM) [18] on responses to NKA, eledoisin, and hHK-1 were conducted. The effects of the NK1 receptor-selective antagonist SR 140333 (1 nM) [27] and the NK3 receptor-selective antagonist SR 142801 (0.3 µM) [28] on responses to NKA and [Lys5MeLeu9Nle10]NKA(4–10) were examined in tissues from nonpregnant women. We examined the effects of SR 140333 on responses to eledoisin and hHK-1 in tissues from pregnant women only. The antagonists were added at the beginning of the equilibration period and replaced after each wash. Captopril (10 µM), phosphoramidon (10 µM), or both were present in experiments with preparations from pregnant women, except in the subset of experiments with eledoisin, in which the peptidase inhibitors present were thiorphan (3 µM), captopril (10 µM), and bestatin (10 µM).

In a further set of experiments on tissues from five pregnant and five nonpregnant women, we examined the effects of the histamine H1, H2, and H3 antagonists, mepyramine (0.3 µM), ranitidine (10 µM), and thioperamide (1 µM), respectively, on responses to NKA and histamine.

Data Analysis

Responses to all agonists were measured as the area under the force-time curve (AUC) for the 5-min period that the agonist remained in contact with the tissue. In most tissues, spontaneous activity ceased during the equilibration period. In the cases in which some spontaneous activity remained, a measurement of AUC for a 5-min period was made from the period immediately prior to the agonist addition and subtracted from AUC in the presence of the agonist. In this way, the possibility that spontaneous contractions could be misconstrued as an agonist-induced response was avoided. Responses were then expressed as a percentage of the corresponding response to 40 mM KPSS and presented as the mean ± SEM; n values refer to the number of subjects.

Estimates of the mean maxima and negative log EC50 values (negative log concentrations producing 50% of maximal responses) were determined from log concentration-response curves constructed by nonlinear regression analysis with fixed slope in the GraphPad PRISM (version 4.0) program, and the lowest response was set to zero. Analysis in PRISM as described by Motulsky and Christopoulos [29] with an F-test allowed a determination of whether the negative log EC50 values and maxima determined were significantly different. When log concentration-response curves to an agonist in the presence of an antagonist did not reach a clear plateau, the mean log concentrations producing 50% of the maximum response to that agonist in the absence of the antagonist were determined. Other statistical procedures used included one- and two-way analyses of variance, which were followed by the Student-Neuman-Keuls pairwise test for multiple comparisons and the Student unpaired t-test with or without the Welsh correction to compare the means of two groups. These procedures were undertaken with PRISM (4.0). Statistical significance was accepted when P < 0.05.

Drugs and Solutions

The drugs used were bestatin HCl (N-[2S,3R)-3-amino-2-hydroxy-4-phenylbutyryl]- L-leucine hydrochloride; Sigma, Castle Hill, Australia); captopril (D-3-mercapto-2-methyl propanoyl-L-proline; Sigma); [Lys5MeLeu9Nle10]NKA(4–10) (Sigma); eledoisin (AUSPEP, Melbourne, Australia); EKD (Department of Biochemistry, University of Bristol, Bristol, U.K.); histamine acid phosphate (Sigma); hHK-1 (Department of Biochemistry, University of Bristol); NKA (AUSPEP); phosphoramidon (Sigma); 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-methylacetamide) (all generous gifts from Sanofi Recherche, Montpellier, France); and DL-thiorphan (Sigma). SR 140333, SR 48968, and SR 142801 were dissolved in absolute ethanol. All remaining compounds were dissolved in dilute hydrochloric acid (0.01 M) or distilled water. Stock solutions of bestatin (10 mM), captopril (10 mM), and SR 140333, SR 48968, and SR 142801 (1 mM) were stored at 4°C. Standard solutions (1 mM) of all peptides, thiorphan, and phosphoramidon were aliquoted into Eppendorf tubes and stored at –20°C.

RESULTS

Responses to High Potassium Krebs Solution

Only tissues that contracted in response to KPSS, indicating their viability, were included in this study. The mean response to KPSS was 36.95 ± 3.98 g.s/5 min mg1 tissue in control preparations from the pregnant women (n =34; mean weight, 84.33 ± 5.83 mg) and 18.22 ± 2.56.g.s/5 min mg–1 tissue in those from nonpregnant women (n = 27; mean weight, 69.27 ± 5.77 mg). The approximately 2-fold difference in mean KPSS responses was statistically significant (Student unpaired t-test, P < 0.01), while the mean tissue weights were not. There was no correlation between subject age and response to KPSS in either group of tissues. The presence of antagonists or peptidase inhibitors did not affect mean responses to KPSS in tissues from either pregnant or nonpregnant women (one-way ANOVAs, P > 0.05), nor did responses to KPSS in each group differ depending on agonists used. Table 1 shows mean responses to KPSS per milligram of tissue weight.


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  		TABLE 1. Effects of agonists and KPSS on isolated human myometrium

Responses to Peptide Agonists in the Absence and Presence of Peptidase Inhibitors

As shown in our previous studies [14, 15], the protocol used allowed the construction of reproducible concentration-response curves to agonists in myometrial preparations from both groups of women (see Fig. 1 for a representative trace showing the response to NKA in the uterus of a pregnant woman). Figure 2 shows log concentration-response curves to the stable NK2 receptor-selective agonist [Lys5MeLeu9Nle10]NKA(4–10) (Fig. 2A) and to NKA (Fig. 2B) in tissues from pregnant women. In these preparations, the effects of NKA were potentiated by thiorphan (3 and 10 µM), and phosphoramidon (10 µM), while those of [Lys5MeLeu9Nle10]NKA(4–10) were unaffected. The responses to NKA in tissues from nonpregnant women were also unaffected by the neprilysin inhibitors (Fig. 2C).


Figure 1
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  		FIG. 1. Representative trace showing contractile responses elicited by increasing concentrations of NKA and by 40 mM KPSS in a myometrial preparation obtained from a 38-wk pregnant woman. Thiorphan (3 µM), captopril (10 µM), and bestatin (10 µM) were present throughout. Black circle indicates washing


Figure 2
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  		FIG. 2. Log concentration-response curves to tachykinin peptides on myometrial preparations from pregnant and nonpregnant women. Data points are means ± SEM and are expressed as a percentage of the response to KPSS. A) Mean responses to [Lys5MeLeu9Nle10]NKA(4–10) in the absence and in the presence of the peptidase inhibitors thiorphan (3 and 10 µM) or phosphoramidon (10 µM) in tissues from pregnant women (n = 8–9). B) Mean responses to NKA in the absence and presence of the peptidase inhibitors as in A (n = 8–9). C) Mean responses to NKA in tissues from nonpregnant women in the absence and presence of thiorphan (10 µM) or phosphoramidon (10 µM) (n = 8–9)

In tissues from pregnant women, the log concentration-response curve to the nonmammalian tachykinin eledoisin (0.1 nM–3 µM), was shifted approximately 3-fold to the left in the presence of thiorphan (3 µM), captopril (10 µM), and bestatin (10 µM) (data not shown). The negative log EC50 value for eledoisin in the presence of inhibitors was 6.81 ± 0.09 (n = 6). A negative log EC50 value could not be calculated for eledoisin in the absence of peptidase inhibitors, as the curve did not asymptote.

Figure 3 shows the effects of hHK-1 in the absence and presence of phosphoramidon and captopril. In tissues from pregnant women, its effects were potentiated in the presence of the peptidase inhibitors. In contrast, the actions of hHK-1 in tissues from nonpregnant women were unaffected by them. Figure 3 illustrates that the potency of hHK-1 was higher in tissues from nonpregnant women.


Figure 3
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  		FIG. 3. Log concentration-response curves to hHK-1 on myometrial preparations from pregnant and nonpregnant women (n = 6). Data points are means ± SEM and are expressed as a percentage of the response to KPSS. Responses to hHK-1 in tissues from pregnant women were potentiated by a combination of captopril (10 µM) and phosphoramidon (10 µM), but those in tissues from nonpregnant women were not

In three experiments with preparations from both pregnant and nonpregnant women, EKD (1 nM–10 µM) was completely devoid of uterotonic activity, irrespective of the presence or absence of peptidase inhibitors.

Antagonist Studies

SR 48968 (1 nM) produced significant concentration-related rightward shifts in the position of the log concentration-response curves to NKA in tissues from both pregnant and nonpregnant women (Fig. 4). The effects of eledoisin examined in tissues from pregnant women and of hHK-1 in tissues from nonpregnant women were also reduced by SR 48968 (Fig. 5, A and B).


Figure 4
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  		FIG. 4. Log concentration-response curves to NKA on myometrial preparations from (A) pregnant (n = 5) and (B) nonpregnant (n = 4) women in the absence and presence of SR 48968 (1 nM). The peptidase inhibitors captopril (10 µM) and phosphoramidon (10 µM) were present in experiments with tissues from pregnant women. Data points are means ± SEM and are expressed as a percentage of the response to KPSS


Figure 5
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  		FIG. 5. A) Log concentration-response curves to eledoisin on myometrial preparations from pregnant women (n = 5–6) in the absence and presence of SR 140333 (1 nM) and SR 48968 (1 nM). Data points are means ± SEM and are expressed as a percentage of the response to KPSS. The peptidase inhibitors bestatin (10 µM), captopril (10 µM), and thiorphan (3 µM) were present. B) Log concentration-response curves to hHK-1 (with 10 µM captopril and 10 µM phosphoramidon) in tissues from nonpregnant women in the absence and presence of SR 140333 (1 nM) and SR 48968 (1 nM)

The NK1 receptor-selective antagonist SR 140333 (1 nM) [27] was without significant effect on the positions or maxima of the log concentration-response curves to any of the peptides, except for hHK-1, in tissues from either group of women (Figs. 5, A and B, and 6). Figure 6 illustrates that the NK3 receptor-selective antagonist SR 142801 (0.1 µM) [28] produced small but significant shifts in the positions of log concentration-response curves to [Lys5MeLeu9Nle10]NKA(4–10).


Figure 6
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  		FIG. 6. Log concentration-response curves to tachykinin peptides on myometrial preparations from pregnant and nonpregnant women. Data points are means ± SEM and are expressed as a percentage of the response to KPSS. A) Mean responses to NKA in tissues from nonpregnant women in the absence and presence of SR 140333 (1 nM) and SR 142801 (0.3 µM) (n = 8). The peptidase inhibitors captopril (10 µM) and phosphoramidon (10 µM) were present throughout. B, C) Mean responses to [Lys5MeLeu9Nle10]NKA(4–10) in myometrial preparations from nonpregnant (B) and pregnant (C) women in the absence and presence of SR 140333 (1 nM) and SR 142801 (0.3 µM) (n = 8)

Effects of Histamine Antagonists on Responses to NKA and Histamine

Histamine (10 nM–100 µM) produced concentration-related contractions in preparations from five pregnant and five nonpregnant women, with a decrease in its maximal response relative to KPSS in the latter group (Table 1 and Fig. 7, A and B). The H1 receptor-selective antagonist mepyramine (0.1 µM) was the only one of the three investigated that antagonized responses to histamine (Fig. 7B). In myometrial strips mounted in parallel, none of the histamine antagonists affected the responses to NKA (data not shown).


Figure 7
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  		FIG. 7. Log concentration-response curves to histamine on myometrial preparations from pregnant and nonpregnant women. Data points are means ± SEM and are expressed as a percentage of the response to KPSS. Mean responses to histamine in tissues from pregnant (A) and nonpregnant (B) women in the absence and presence of mepyramine (0.1 µM), ranitidine (10 µM), and thioperamide (1 µM) (n = 5)

Effect of Pregnancy on Log Concentration-Response Curves to Agonists

Previous studies [14, 15], as well as the results shown in Figure 3 for hHK-1, indicated that pregnancy led to decreases in tachykinin potencies and in their efficacies relative to KPSS. It was for this reason that we compared their effects with those of another inflammatory mediator, namely histamine.

Table 1 shows the responses to 40 mM KPSS, the maximal responses to the agonists, and the positive EC50 estimates for the agonists. Inspection of the mean responses to KPSS and the estimates of the responses to the agonists expressed as a percentage of those to KPSS shows that the absolute maximum responses to all agonists and KPSS were higher in tissues from pregnant than from nonpregnant women. However, the contractions induced by NKA and [Lys5MeLeu9Nle10]NKA(4–10) increased to a lesser extent than did the responses to KPSS and histamine in these tissues. Both tachykinins were significantly more potent in tissues from nonpregnant women (Student unpaired t-test, P < 0.05; Table 1). The same occurred for hHK-1 within the range of concentrations assayed (Table 1). The log concentration-response curves for NKA, [Lys5MeLeu9Nle10]NKA(4–10), and hHK-1 in tissues from pregnant women lay markedly to the right of those obtained in tissues from nonpregnant women (see Figs. 3, 4, and 6).

In contrast to the tachykinins, the mean maximum effect of histamine relative to that of KPSS was significantly enhanced in tissues from pregnant women (Table 1), and the concentration-response curves lay to the left of those obtained for this agonist in tissues from nonpregnant women (Fig. 7).

DISCUSSION

The main findings of the present study are that 1) the effects of the mammalian tachykinins NKA and hHK-1 and of the nonmammalian tachykinin eledoisin are enhanced by the inhibition of neprilysin in myometrial tissues from pregnant women, 2) the inhibitors of neprilysin are without effect on responses to NKA and hHK-1 in tissues from nonpregnant women, 3) the NK2 receptor is the predominant tachykinin receptor mediating the contractile responses to tachykinins in the myometrium from both pregnant and nonpregnant women, 4) the NK1 receptor may participate to a minor extent in the responses to hHK-1 but not to the other peptides, while [Lys5MeLeu9Nle10]NKA(4–10) may activate the NK3 receptor to a small extent, and 5) pregnancy upregulates the maximum responses to high potassium-containing solution, to histamine, and, to a lesser extent, to tachykinins. However, the potency of hHK-1, like that of NKA and of [Lys5MeLeu9Nle10]NKA(4–10), is less in tissues from pregnant than from nonpregnant women, even when inhibitors are present to reduce inactivation by peptidases.

The tachykinin peptides appear to play a role in the regulation of reproductive function. In some invertebrates, tachykinins and the ancestral tachykinin receptor are expressed in the gonads [30, 31]. In mammals, SP, NKA, NKB, and the tachykinin receptors are present at all main levels throughout the reproductive axis: the hypothalamus, the anterior pituitary, and the reproductive tract of both males and females [7, 32]. Human HK-1, the most recently discovered member of the tachykinin family in humans, is mainly expressed in nonneuronal cells at the peripheral level [5]. In one comparison of 25 different human tissues, the uterus and the placenta were found to be the tissues with the highest expression of the predominant splice isoform {alpha}TAC4 v2 that encodes hHK-1 [2]. However, the physiological role of this tachykinin, particularly in the female genital tract, remains unknown.

While mammalian tachykinins can act at all three tachykinin receptor subtypes, the present data show that the NK2 receptor is the predominant tachykinin receptor mediating the contractile responses to tachykinins in the myometrium from both pregnant and nonpregnant women. This conclusion is supported by the following observations: 1) NKA and [Lys5MeLeu9Nle10]NKA(4–10) showed the highest potency and efficacy among a number of mammalian and nonmammalian tachykinins and tachykinin analogues assayed, 2) the potency of the nonmammalian tachykinin eledoisin is consistent with it acting on the NK2 receptor [33], and 3) the tachykinin NK2 receptor-selective antagonist SR 48968 is a potent antagonist of uterine contractions elicited by all tachykinin receptor agonists [14, 15, the present study]. By quantitative real-time RT-PCR, we observed that the mRNA levels of the tachykinin NK2 receptor were 95-fold lower in the myometrium from pregnant than from nonpregnant women [15]. This is consistent with the finding that the potency, as well as the efficacy relative to that of high potassium, of tachykinins is less in tissues from pregnant than from nonpregnant women [14, 15, the present study] and adds further support to the notion that the NK2 receptor is the main or the sole tachykinin receptor involved in contractile responses to tachykinins in human uteri [1, 7].

Human HK-1 and its mouse/rat orthologue, mHK-1, possess a remarkable selectivity for the tachykinin NK1 receptor [5, 34, 35]. In addition, mHK-1 behaves as a full agonist at the tachykinin NK2 and NK3 receptors [34, 35]. In our study, hHK-1 caused a modest contractile effect in the uteri from nonpregnant women and an even more minor and less potent response in the pregnant uteri. In the nonpregnant myometrium, the contraction evoked by hHK-1 was inhibited by SR 48968 but also by the tachykinin NK1 receptor-selective antagonist SR 140333. The TACR1 gene that encodes the NK1 receptor is expressed in the myometrium from both pregnant and nonpregnant women [15] and may participate to a minor extent in the contractile responses to hHK-1, although it is not involved in the uterine contraction induced by tachykinins other than hHK-1. Human HK-1 may, therefore, uncover the presence of a minor population of functional tachykinin NK1 receptors in the human nonpregnant myometrium. This adds support to the suggestion that this tachykinin is the most important nonneuronal activator of the tachykinin NK1 receptor at the peripheral level [5]. Moreover, in the presence of SR 140333, the concentration-response curve for hHK-1 showed a low maximum response, suggesting that, at least in the human uterus, hHK-1 acts as a partial agonist at the tachykinin NK2 receptor.

We also observed a small but significant inhibition of the contractile response to [Lys5MeLeu9Nle10]NKA(4–10) in the presence of the tachykinin NK3 receptor-selective antagonist SR 142801. Further studies with other tachykinin receptor antagonists are needed to identify whether [Lys5MeLeu9Nle10]NKA(4–10) is able to activate the tachykinin NK3 receptor or whether the effects of SR 142801 are due to the blockade of the tachykinin NK2 receptor.

Previous reports have shown that EKC and EKD are inactive at the human tachykinin NK1 and NK2 receptors in Ca2+ mobilization assays [5, 36]. EKD did not induce any functional response in the human uterus, adding further support to the suggestion that this tachykinin-like peptide does not activate any of the known mammalian tachykinin receptors.

The results of earlier studies [14, 15] and of the present study suggest that neprilysin is the most important enzyme involved in tachykinin metabolism in the human myometrium. MME mRNA levels were significantly lower in the nonpregnant myometrium than in the myometrium from pregnant women [15]. The present data extend these observations and show that the effects of the mammalian tachykinins NKA and mHK-1 and of the nonmammalian tachykinin eledoisin were enhanced by the inhibition of neprilysin in the tissues from pregnant women but not from nonpregnant women.

Pregnancy is associated with important changes in the electric and metabolic properties of uterine smooth muscle [37–39]. In the pregnant rat uterus, L-type Ca2+ currents increase during the course of pregnancy and are maximal at term, just before labor [38]. It has also been shown that the Ca2+ sensitivity of the contractile machinery is greatly increased at term [39]. These findings could explain the higher contractile response to KCl in the myometrium from pregnant women when compared with tissues from nonpregnant women. There are also important changes in the expression of different membrane receptors, including a marked increase in oxytocin receptors [40, 41]. In the present study, we observed a pregnancy-related increase in the maximal myometrial response to histamine. Its effects on the human myometrium have been investigated previously [22, 23]; however, to our knowledge, its potency and efficacy in tissues from pregnant women have not been directly compared with those in tissues from nonpregnant women. It has been proposed that this monoamine, present in uterine mast cells in close proximity to myometrial cells [26, 42], has important effects on the latter in late pregnancy [24]. However, although its relative efficacy was increased, its potency remained unchanged in pregnancy; indeed, it was approximately 100 times less potent than NKA. This low potency, together with the finding that mepyramine did not antagonize the effects of NKA, indicates that, under the present experimental conditions, NKA does not exert an uterotonic action through its release. These findings are consistent with an earlier report showing that SP does not release histamine from human uterine mast cells [25]. Nevertheless, the present data do not negate the possibility that, under allergic and other pathological conditions, histamine plays a role in premature labor and delivery, as has been demonstrated recently [26].

In contrast to the enhancement of oxytocin action and as shown in this study of the efficacy of histamine action in late pregnancy, the present data show that the potencies of hHK-1, like those of NKA and [Lys5MeLeu9Nle10]NKA(4–10), are lower in tissues from pregnant than from nonpregnant women, even when inhibitors are present to reduce inactivation by peptidases. Moreover, the expression and activity of neprilysin appear to be more important at late pregnancy, at least in comparison with the nonpregnant tissues used in the present study. The potent effects of tachykinins in nonpregnant women, especially compared with those of the inflammatory mediator histamine, raise the possibility that they play a role in the inflammation and pain associated with menstrual disorders. In addition, the data indicate regulatory mechanisms that might preclude inappropriate tachykinin-induced uterine activity in the late stages of normal pregnancy. This tight regulation could suggest that an augmentation in tachykinin activity contributes to pregnancy disorders by increasing uterine contractility and cervical ripening. Any roles for these peptides in the induction of normal or preterm labor remain to be determined.

ACKNOWLEDGMENTS

We are grateful to Dr. Emonds-Alt for gifts of SR 140333, SR 48968, and SR 142801.

FOOTNOTES

1 Supported by grants from the National Health and Medical Research Council of Australia to J.N.P. and the Ministerio de Educación y Ciencia (BFU2005-04495-C02-01/BFI), Spain. J.N.P. and E.P. contributed equally to this work. Back

2 Correspondence. FAX: 61 39 833 3754; jocelyn.oneil{at}vcp.monash.edu.au Back

3 Correspondence: M. Luz Candenas, Centro de Investigaciones Científicas Isla de La Cartuja, Instituto de Investigacones Químicas, Avda. Americo Vespucio 49, 41092 Sevilla, Spain. FAX: 34 95 446 0565; e-mail: luzcandenas{at}iiq.csic.es Back

Received: 7 February 2006.

First decision: 27 February 2006.

Accepted: 15 May 2006.

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