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Gene regulation of neurokinin B and its receptor NK3 in late
pregnancy and pre-eclampsia
N.M.Page1,3, J.Dakour2 and D.W.Morrish2
1School of Life Sciences, Kingston University London, Penrhyn Road, Kingston-upon-Thames, Surrey, UK and 2Department
of Medicine, The University of Alberta, Edmonton, Canada
3To whom correspondence should be addressed at: School of Life Sciences, Kingston University London, Penrhyn Road,
Kingston-upon-Thames, Surrey KT1 2EE, UK. E-mail: firstname.lastname@example.org
Elevated circulating levels of the tachykinin, neurokinin B (NKB), have been observed in women with pre-eclampsia during the
third trimester of pregnancy. Currently, the molecular mechanisms responsible for these increased levels remain unknown. To
understand the molecular regulation, we have compared the differences in gene expression of the tachykinins and their receptors
in control and pre-eclamptic placentae and the responses of the TAC3 gene encoding NKB to proposed physiological triggers of
pre-eclampsia including hypoxia and oxidative stress using real-time quantitative PCR. We have determined the placenta to be
the main site of TAC3 expression with levels 2.6-fold higher than the brain. TAC3 expression was found to be significantly higher
in pre-eclamptic placenta (1.7-fold, P < 0.05) than in normal controls. No evidence was found that hypoxia and oxidative stress
were responsible for increases in TAC3 expression. In rat placenta, a longitudinal study in normal late pregnancy was associated
with a significant down-regulation of the NKB/NK3 ligand–receptor pair (P < 0.05). The present data suggest that the increased
placental expression of TAC3 is part of the mechanism leading to the increased circulating levels of NKB in pre-eclampsia.
Key words: neurokinin B/pre-eclampsia/pregnancy
The tachykinins are a family of peptides that comprise substance
P (SP), neurokinin A (NKA), neurokinin B (NKB) and the species-
divergent endokinins including endokinin B (EKB) in humans
(reviewed by Page, 2004, 2005). These tachykinins are encoded on
three different genes, preprotachykinin 1 (TAC1) encoding SP and
NKA, TAC3 encoding NKB and TAC4 encoding EKB (reviewed by
Page, 2004, 2005). Three tachykinin receptors have been identified,
which interact with these tachykinins: NK1, NK2 and NK3, whereby
SP and EKB show the greatest potency for NK1, NKA for NK2 and
NKB for NK3 (Page et al., 2003).
Traditionally, these peptides have been classified as neurotransmit-
ters being found in discrete neurons and immune cells (reviewed by
Page, 2004, 2005). Recently, this conceived dogma was challenged
when the placenta, a tissue devoid of nerves, was found to be a source
of TAC3 gene expression (Page et al., 2000, 2001). Moreover, TAC1
expression was found to be absent from the placenta (Page et al.,
2001, 2003). However, we now know that the placenta is an abundant
source of the recently discovered SP-like endokinins, leading to the
proposal that the endokinins are the peripheral SP-like endocrine/
paracrine agonists where SP is not expressed (Page et al., 2003; Page,
2004). Moreover, pre-eclampsia has been associated with increased
plasma levels of NKB (Page et al., 2000; D’Anna et al., 2004), where
in the placenta it is expressed by the outer syncytiotrophoblast in an
ideal position to be secreted into the maternal bloodstream (Page
et al., 2000). Concentrations of NKB in the middle and late pregnancy
have been found to be significantly higher than non-pregnant concen-
trations and decrease rapidly after delivery. This indicates that NKB
secretion into both the fetal and the maternal circulation is derived
mainly from the placenta (Page et al., 2000; D’Anna et al., 2002;
Sakamoto et al., 2003; Schlembach et al., 2003; Tjoa et al., 2004).
A role for the tachykinins in the placenta has remained as yet unde-
fined. Nevertheless, recent and consistent evidence suggests that they
may play a role in utero-placental haemodynamic adaptation by inducing
uterine and placental vasodilatation, thereby increasing placental blood
flow (Page et al., 2000, 2001; Brownbill et al., 2003; D’Anna et al.,
2004; Laliberte et al., 2004). In contrast, in the peripheral mammalian
vasculature, NKB has been shown to have hypertensive effects at its pre-
ferred receptor, NK3 (Mastrangelo et al., 1987; D’Orleans-Juste et al.,
1991; Thompson et al., 1998). Indeed, the placenta has been shown to
play a major role in the pathogenesis of pre-eclampsia (reviewed in
Scott, 1958; Shembrey and Noble, 1995; Palma Gamiz, 1998), where
after poor placental perfusion (Pijnenborg et al., 1980, 1981), a process
is initiated leading to placental hypoxia, ischaemia, ensuing oxidative
stress and the symptoms of pre-eclampsia (reviewed in Page, 2002). To
date, no study has yet analysed the molecular regulation of TAC3 in the
placenta. In an attempt to clarify this issue, we have identified the pla-
centa as the predominant site of TAC3 gene expression and determined
the changes in tachykinin and tachykinin receptor expression between
normal and pre-eclamptic placentae at term. We have also studied the
effects of proposed pathophysiological triggers of pre-eclampsia, such as
hypoxia and oxidative stress, on TAC3 and NK3 receptor expression.
Materials and methods
Animals and human subjects
Adult female time-mated Crl : WI rats (Charles River, Margate, UK) were kept
in standard laboratory conditions with a 12 h light and a 12 h dark cycle. All
Mol. Hum. Reprod. Advance Access published May 18, 2006
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N.M.Page, J.Dakour and D.W.Morrish
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procedures for the collection of tissues were followed according to the
accepted standards of animal care. Human tissues were collected with approval
from the local Research Ethics Committee (The University of Alberta) and in
compliance with their guidelines. Term placentae from both normal (n = 12)
and pre-eclamptic (n = 12) pregnancies were obtained. Samples for extraction
were chosen randomly from cotyledons in areas without obvious infarcts or
other pathology. A large portion of each placenta (∼100–200 g) was used. All
tissues were collected fresh and immediately frozen by immersing in isopen-
tane with dry ice and transferred to –80°C for storage. Pre-eclampsia was
defined according to the Report of the National High Blood Pressure Educa-
tion Program Working Group criteria as hypertension starting during preg-
nancy after 20 weeks’ duration with blood pressure greater than 140 mm Hg
systolic and greater than 90 mm Hg diastolic with proteinuria of at least 0.3 g
protein in a 24 h urinary specimen or >1+ (National Institutes of Health, 2000).
Clinical characteristics of the normal controls and patients with pre-eclampsia
whose placentae were used are summarized in Table I. Of the pre-eclamptic
pregnancies, none had intrauterine growth restriction (IUGR) as previously
defined (Mayhew et al., 2003), and none were smokers. One had only
pregnancy-induced hypertension without proteinuria. Only one control was a
smoker, and none had proteinuria or hypertension.
Term human cytotrophoblasts were prepared as previously described using
trypsin–DNase I digestion which produces a cell preparation over 95% pure
for cytotrophoblast with fewer than 5 vimentin-positive cells per 105 cells
(Morrish et al., 1987; Guilbert et al., 2002). Cells were plated at 6–8 × 106
cells/dish in 100 mm Petri dishes (Corning, Corning, NY, USA) for mRNA
studies and cultured in 10%-fetal bovine serum (FBS) Dulbecco’s modified
Eagle’s medium (DMEM)–penicillin–streptomycin, as previously described
(Morrish et al., 1997). Cells were attached for 2 h, then the medium changed to
serum-free DMEM. These cells rapidly differentiate spontaneously over 24 h
towards a syncytial phenotype including the up-regulation of most syncytial
gene products and the formation of some morphological syncytium (Guilbert
et al., 2002). Two sets of experiments were performed. In the first set (n = 6
separate placental preparations), immediately after attachment and changing to
serum-free medium, cells were exposed to either hypoxia (2% O2) using an
hypoxic incubator (BioSpherix, Redfield, NY, USA), or 18 μM peroxynitrite,
or 100 μM xanthine/5 μU/ml of xanthine oxidase, or control for 24 h, then
scraped gently from the plate with a plastic policeman and frozen at –70oC. In
the second set of experiments (n = 3 placental preparations), half the cells were
immediately exposed to experimental conditions as described above (n = 6 pla-
cental preparations for these experiments), and the other half of the cells (n = 3
preparations) were cultured for 48 h in 10% FBS plus 10 ng/ml of epidermal
growth factor (EGF) to induce syncytialization, then changed to serum-free
medium and exposed to the same experimental conditions (control 20% oxy-
gen, 2% hypoxia, peroxynitrite, xanthine/xanthine oxidase) for a further 24 h.
Cells were then harvested for mRNA. Cultured cells immediately after attach-
ment (T = 0) were stained with cytokeratin, as previously described (Morrish
et al., 1987, 1997). At 72 h, after cytotrophoblast cells had been partially dif-
ferentiated into syncytium as described above, control cells were stained with
desmoplakin to define syncytial unit formation, as previously described
(Morrish et al., 1997).
Total RNA was isolated from rat placenta at days 16, 19 and 21 of gestation
and from human placental tissues at term using Tri Reagent (Sigma-Aldrich,
Poole, UK). Contaminating genomic DNA was removed by DNase I treatment
and the RNA then re-purified. Total human RNA from each of 24 tissues
(brain, heart, kidney, liver, lung, colon, bone marrow, small intestine, spleen,
stomach, thymus, prostate, skeletal muscle, testis, uterus, fetal brain, fetal
liver, thyroid, placenta, adrenal gland, pancreas, salivary gland, trachea and
mammary gland) was also obtained from a human RNA master panel (BD Bio-
sciences, Oxford, UK). First-strand cDNA synthesis was performed using 1.5
μg of each RNA using the Powerscript™ reverse transcriptase kit (BD Bio-
sciences) in the presence of 300 ng of random hexameric primers (Invitrogen,
Paisley, UK) by following the manufacturer’s instructions. The resulting
cDNA was diluted by adding 300 μl of nuclease-free H2O. One microlitre of
this cDNA was used in each quantitative PCR. Specific Taqman® probes and
primer sets were designed using Primer Express™ 1.5 to span where possible
an exon–exon junction (Applied Biosystems, Foster City, CA, USA). The
sequences of the primers and probes used are listed in Table II. All probes and
primers were synthesized by Sigma-Genosys (Pampisford, UK). Reactions
were set up in triplicate in 25 μl using ABsolute™ Quantitative PCR ROX
master mix (ABgene, Epsom, UK) following the manufacturer’s instructions.
PCR cycling was performed in an ABI PRISM® 7700 sequence detector under
the following conditions: initial denaturation/activation of the Thermo-Start®
DNA polymerase at 95°C for 15 min and then 40 cycles of 95°C for 15 s and
60°C for 1 min. The optimal concentrations of the primers (50–900 nM) and
probe (25–225 nM) used to amplify each target gene were calculated using
those combinations that gave the lowest threshold cycle (CT) and highest nor-
malized reporter (Rn) values. The derived optimal concentrations are summa-
rized in Table II. The standard curve method for relative quantitation was used
with normalization to the endogenous control, 18S rRNA. Separate standard
curves were generated using a 10-fold serial dilution of template cDNA for
each respective target gene and the endogenous control gene. The target
amount was divided by the endogenous control reference to obtain a normal-
ized target value to generate the relative expression levels; these levels were
arbitrarily presented as percentages. The level of 18S rRNA in each tissue/
sample was assessed using the Taqman® rRNA control reagents following the
manufacturer’s instructions (Applied Biosystems). Controls containing no
reverse transcriptase, no template and no probe were included.
Statistical analysis was performed with one-way ANOVA used in conjunction
with the post hoc Fisher’s protected least significant difference test for multi-
ple comparisons and Student’s t-tests to compare the means of two groups.
These analyses were undertaken using StatView (version 5.01) and plotted
using GraphPad PRISM (version 3.0). P < 0.05 was considered to be signific-
ant. All values are expressed as mean ± SEM, where n represented the number
in the group used.
Quantitative PCR demonstrated the major site of expression of TAC3 to
be the placenta (Figure 1) with levels 2.6-fold higher than those in the
whole brain. Significant, but lower levels of TAC3 mRNA transcripts
were distributed, in the fetal brain, with significant expression also in
the endocrine/reproductive organs of the testis and mammary gland.
The expression pattern of the tachykinin genes (TAC1, TAC3 and
TAC4) and tachykinin receptor genes (TACR1 [NK1], TACR2 [NK2]
and TACR3 [NK3]) was investigated between control and pre-eclamptic
Table I. Characteristics of normal controls and patients with pre-eclampsia
CX, caesarean section; IV, induced vaginal delivery; ND, not determined;
PIH, pregnancy-induced hypertension; SV, spontanoeus vaginal delivery.
Control patients had no other disease; five were on ampicillin and one smoked.
Pre-eclampsia patients: epilepsy (n = 1); smokers (n = 2), dexamethasone
(n = 1), HELLP (hemolysis, elevated liver function tests, low platelet count)
syndrome (n = 1). One patient had PIH without proteinuria.
Number of patients
Systolic (mm Hg ± SEM)
Diastolic (mm Hg ± SEM)
Gestation at delivery (weeks ± SEM)
Fetal birth weight (g ± SEM)
Placental weight (g ± SEM)
123 ± 5
72 ± 2
39.4 ± 0.4
3497 ± 119
721 ± 46
SV (n = 10),
CX (n = 2)
None (n = 12)
171 ± 5
101 ± 3
37.0 ± 1.1
2748 ± 264
862 ± 289
CX (n = 8),
IV (n = 4)
1+ (n = 7),
3+ (n = 2)
4+ (n = 1),
ND (n = 1)
PIH (n = 1)
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Neurokinin B gene expression in pregnancy
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placentae at term using real-time quantitative PCR. TAC1 mRNA
expression levels in both control and pre-eclamptic placentae at term
were found to be either absent (9 of 24 placenta) or present at
extremely low levels, except for one pre-eclamptic placenta that was
found to express much higher levels of TAC1 (Figure 2). A signifi-
cantly higher level of TAC3 mRNA expression of 1.7-fold (P < 0.05,
unpaired t-test, n = 12) was found in the pre-eclamptic placentae when
compared with the control placentae (n = 12) (Figure 2). No significant
alterations in TAC4 mRNA expression levels were observed between
the pre-eclamptic and control groups (Figure 2). No significant alterations
in any of the mRNA expression levels of the tachykinin receptors
(TACR1, TACR2 or TACR3) were observed between the pre-eclamptic
and control groups (Figure 2).
To determine whether proposed pathophysiological triggers of pre-
eclampsia were responsible for the elevated TAC3 gene expression in
the pre-eclamptic group, we analysed the effects of hypoxia and oxi-
dative stress in term human placental cytotrophoblast and syncytiotro-
phoblast cultures. Significant decreases in TAC3 mRNA expression
levels in differentiating placental cytotrophoblasts grown in hypoxic
conditions (2% O2) were found when compared with those grown in
Table II. The sequences of the forward (F) and reverse (R) primers and Taqman® probes (P) used for quantitative PCR
Taqman® probes have the fluorescent reporter dye FAM™ covalently linked to the 5′-end and the quencher TAMRA™ dye
located at the 3′-end. The final optimized concentrations of the primers and Taqman® probes are shown. ‘r’ and ‘h’ before the
gene name denote rat and human, respectively.
Gene Primers/probes Sense Concentration (nM)
Figure 1. Real-time quantitative PCR analysis of the tissue distribution of the TAC3 gene in 24 human tissues relative to the expression of 18S rRNA. The intensi-
ties are not a guide to expression between different genes, as the values are relative and not absolute.
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N.M.Page, J.Dakour and D.W.Morrish
Page 4 of 7
normoxic conditions (21% O2). These were represented by a 1.8-fold
(P < 0.05, paired t-test, n = 5) decrease in TAC3 expression levels as
shown by quantitative PCR (Figure 3). There was a slight tendency
for the increased expression of the NK3 receptor; however, this was
not significant (Figure 3). In contrast, quantitative PCR of TAC3
mRNA expression levels in differentiated placental syncytiotrophob-
lasts (generated by 48 h exposure to EGF and 10% FBS) exposed to
the conditions of oxidative stress (induced by the presence of perox-
ynitrite or xanthine/xanthine oxidase for 24 h) or of hypoxia (2% O2)
for 24 h demonstrated no significant changes in the expression of
TAC3 (Figure 3). Neither were significant changes in the expression
of TAC3R found under the same conditions of oxidative stress and
hypoxia (data not shown). Cultured mononuclear cytotrophoblast
cells before experimental treatment are shown in Figure 4A. Cytoker-
atin staining confirms their trophoblast identity. Partly syncytialized
cells at the end of the experimental period (72 h) are shown in Figure 4B.
Approximately 50% of cytotrophoblasts have fused to form multinu-
clear syncytial units that are outlined by desmoplakin staining.
As we were unable for ethical reasons to obtain longitudinal placen-
tal samples throughout the course of the third trimester of human
pregnancy, we investigated the expression pattern of TAC3 and
TACR3 in the rat placenta at days 16, 19 and 21 of gestation using
quantitative PCR. TAC3 mRNA levels were found to decline through-
out the duration of late pregnancy by 2.0-fold at day 19 and 2.9-fold at
day 21 when compared with day 16, respectively. This decline was
found to be significant between days 16 and 21 (P < 0.05, ANOVA,
n = 4) (Figure 5). There was a significant decrease in TACR3 mRNA
expression levels between days 16 and 19 of 90.6-fold (P < 0.05,
ANOVA, n = 4) and of 58.6-fold between days 16 and 21 (P < 0.05,
ANOVA, n = 4). The TACR3 gene appeared to be virtually switched
off by day 19 (Figure 5).
The mammalian tachykinins are a family of peptides that have been
traditionally classified as neurotransmitters; however, we have found
the placenta, a tissue devoid of nerves, to be the most abundant site of
TAC3 mRNA expression. Furthermore, there is growing evidence to
suggest that the tachykinins may play a significant role in the regula-
tion of many different reproductive functions (Page et al., 2000, 2001;
Pintado et al., 2003; Loffler et al., 2004). One such proposed repro-
ductive function has come from the discovery of elevated levels of
NKB detected in the plasma of third-trimester pre-eclamptic pregnan-
cies (Page et al., 2000; D’Anna et al., 2004). It has been proposed that
in response to defective trophoblast invasion, which is not rectified
after the first trimester of pregnancy, the placenta will start secreting
ever-increasing amounts of NKB into the maternal circulation (Page
et al., 2001). We have found TAC3 mRNA expression levels to be sig-
nificantly higher in pre-eclamptic placenta at term, a result consistent
with the theory that TAC3 expression could be up-regulated in
Figure 2. Real-time quantitative PCR analysis of the expression of the tachykinin genes (TAC1, TAC3 and TAC4) and the tachykinin receptor genes (TACR1,
TACR2 and TACR3) in normal control (N) and pre-eclamptic (PE) placentae. Their expression is compared with that of the human β-actin gene and normalized to
that of 18S rRNA. Each bar represents the mean of 12 different normal and 12 different pre-eclamptic placental samples, with SEM shown by vertical lines.
aP < 0.05, significant difference versus mRNA levels from pre-eclamptic placentae.
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Neurokinin B gene expression in pregnancy
Page 5 of 7
response to poor placental perfusion after defective trophoblast inva-
sion. We have believed, like many others (reviewed in Roberts, 1998;
Taylor et al., 1998; VanWijk et al., 2000), that a poorly diffused and
ischaemic placenta releases an excess of such factor(s) including NKB.
It has been suggested that the key physiological activators responsible
for the production of these factors are hypoxia (Kingdom and
Kaufmann, 1997) and oxidative stress (Roberts and Hubel, 1999). Nev-
ertheless, the evaluation of these triggers demonstrated no evidence that
they were responsible for increasing TAC3 mRNA expression. Paradox-
ically, the hypoxic conditions proposed to occur in pre-eclamptic
placentae were found to significantly down-regulate TAC3 mRNA
expression in cytotrophoblast cultures. These data do not support a
role for placental hypoxia as the underlying cause of elevated NKB
levels in pre-eclampsia, either in cytotrophoblast differentiating into
syncytium or in mature syncytium in vitro (Figure 3). Regardless, this
outcome is similar to that found for activin A in cytotrophoblasts,
whose gene is likewise down-regulated by hypoxia, but associated
with higher circulating levels of protein during pre-eclampsia
(Blumenstein et al., 2002). Indeed, the concept of placental hypoxia in
pre-eclampsia has been previously challenged, with some theories
believing the converse that hyperoxic conditions develop during pre-
eclampsia (Kingdom and Kaufmann, 1997). It is unlikely that serum
deprivation accounted for either the decrease in TAC3 with hypoxia or
prevented its increase, as the effect of hypoxia was selective for TAC3
and not seen for TAC4 or TACR1, TACR2 and TACR3 (data not
shown). Similarly, short-term serum deprivation in past studies using
this culture system has not been found to impair hormonal secretion
from cyto- or syncytiotrophoblast (Morrish et al., 1987, 1997).
As we have previously found using semi-quantitative PCR, human
placentae were either devoid of TAC1 expression (Page et al., 2001,
2003), or as in this study, some were found to express extremely low
levels of TAC1. The latter observation perhaps arises owing to the more
sensitive nature of the real-time quantitative PCR used in this study.
Such low levels of TAC1 may originate from invading immune cells, as
these have been previously shown to be a source of TAC1 expression
(Weinstock et al., 1988; O’Connor et al., 2004). It is plausible that the
much higher level of TAC1 expression seen in one of the pre-eclamptic
placenta represents such an increased immune cell invasion. In regard to
TAC4 expression, there was no overall difference in expression between
the normal and pre-eclamptic groups, making TAC3 the only tachykinin
gene significantly elevated during pre-eclampsia. Both Page et al.
(2000) and D’Anna et al. (2004) have reported higher differences in
circulating NKB peptide levels in pre-eclampsia (9.6- and 2.1-fold,
respectively) than we report here in placental mRNA expression levels
(1.7-fold). It is likely that circulating peptide levels will be determined
by many other key factors including the manner of processing, turnover,
storage, secretion and degradation, which are not reflective of mRNA
levels. For example, in the case of corticotrophin releasing hormone
(CRH), it has been demonstrated that most CRH in the human normo-
tensive placenta exists as largely unprocessed/partially processed pro-
CRH, with very little in the form of cleaved CRH except in the case of
pre-eclampsia (Ahmed et al., 2000).
Figure 3. Effects of hypoxia on the expression of TAC3. Term cytotrophob-
lasts were cultured in serum-free conditions in either normoxic (21% O2) (col-
umn 1) or hypoxic (2% O2) (column 2) culture conditions for a period of 24 h.
aP < 0.05 showed a significant difference versus mRNA levels under normoxic
conditions using paired Student’s t-test. Expression was also assessed in term
cytotrophoblasts that had been differentiated into syncytiotrophoblasts using
EGF and 10% fetal bovine serum for 48 h under normoxic (21% O2) condi-
tions. Cells were then exposed for 24 h to normoxia (21% O2) (column 3),
hypoxia (2% O2) (column 4), oxidative stress with 100 μM xanthine/5 μU/ml
of xanthine oxidase (column 6) or 18 μM peroxynitrite (column 8) both under
normoxic (21% O2) conditions. Control syncytiotrophoblasts exposed to nor-
moxia are shown in columns 5 and 7. All expressions were assessed using
quantitative PCR and normalized to those of 18S rRNA. Each bar represents
the mean of six individual experiments for cytotrophoblasts and three for syn-
cytiotrophoblasts, with SEM shown by vertical lines.
Figure 4. Trophoblast culture. (A) Cytotrophoblast after attachment at T = 0. Dark mononulcear cytotrophoblast cells are stained with cytokeratin. (B) Syncytial-
ized cells at 72 h culture (end of experimental period) stained with desmoplakin, outlining cell groups. Many cells have fused to form syncytial units (S) with some
mononuclear cytotrophoblasts (C) remaining. Magnification, ×250.
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N.M.Page, J.Dakour and D.W.Morrish
Page 6 of 7
NKB plasma levels equivalent to those found during pre-eclampsia
are shown to be vasodilatory in the normal placental vasculature
(Brownbill et al., 2003; Laliberte et al., 2004), where they cause their
effects by solely activating the NK1 receptor subtype (Brownbill
et al., 2003). Consequently, as there are no differences in the expres-
sion of the three tachykinin receptors during pre-eclampsia, NKB is
also expected to induce placental vasodilatation in the pre-eclamptic
placenta and would therefore not contribute to enhancing any placen-
tal vasoconstrictor response. In addition, evidence has been presented
that the vasoconstrictor NK3 receptor is either absent or expressed at
extremely low levels in the human placenta at term compared with
those of NK1 and NK2 receptors (Brownbill et al., 2003). This would
certainly advocate a mechanism in the human placenta, whereby high
NKB levels induce placental vasodilatation predominantly via the
NK1 receptor. The rat model consolidates such a mechanism where
during late pregnancy there is a significant decrease in placental
TACR3 mRNA expression as term approaches without any significant
change in NK1 receptor expression (data not shown). Similar declines
in TAC3 and TACR3 gene expression have also been observed in the
rat uterus, where there is a significant reduction in their expression
throughout gestation (Candenas et al., 2001; Patak et al., 2005). Fur-
thermore, we have also observed a significant decline in TAC3 expres-
sion during late pregnancy in the rat placenta. In essence, we conclude
that normal late pregnancy, at least in the rat, is associated with a
down-regulation of the NKB/NK3 ligand–receptor pair in both the
placenta and the uterus.
Estrogen is a prime candidate responsible for the down-regulation
of the NKB/NK3 ligand–receptor pair in late pregnancy. The placenta
is the major site of estrogen production during pregnancy where its
synthesis occurs exponentially during gestation with the highest
serum levels obtained in late pregnancy (Candenas et al., 2001). There
is consistent evidence to show that TAC3 and TACR3 expressions are
strongly down-regulated by estrogen in many different biological sys-
tems (Rance and Young, 1991; Pinto et al., 1999; Candenas et al.,
2001; Cintado et al., 2001; Pillon et al., 2003). Cintado et al. (2001)
have tentatively hypothesized that the NKB/NK3 ligand–receptor pair
could be involved or, at least, be an indicator of estrogen-related
pathophysiologies. In this context, low circulating levels of estrogens
have been associated with pre-eclampsia (Innes and Byers, 1999). In
the case of pre-eclampsia, possible mechanisms of high NKB expres-
sion include disruption/failure to down-regulate the NKB/NK3 system
during the third trimester as observed in the rat, or an unknown stimu-
lus inducing NKB secretion. Current data in humans, which lack early
pregnancy expression patterns in the third trimester, do not allow dif-
ferentiation of these possibilities for ethical reasons.
Our data support previous findings that argue for a role for NKB
and the NK3 receptor in the placenta and during pregnancy. Indeed,
female rats treated with the NK3 receptor antagonist SR142801 before
mating exhibited a tendency towards decreased fertility with a signi-
ficant reduction in their litter sizes (Pintado et al., 2003). In addition,
the highest levels of uterine NK3 receptor expression in the pregnant
rat (peaking at day 3) were detected before and at the time of implan-
tation (Candenas et al., 2001). NKB could play a key role in early
pregnancy at the time of implantation. In humans, elevated plasma
levels of NKB have also been associated with IUGR that likewise
occurs with impaired placental implantation (D’Anna et al., 2004). In
our studies, none of the placentae used came from patients with
IUGR. Nonetheless, such increases in NKB production may represent
sustained synthesis because of the early failure of trophoblast invasion
into the uterus although the precise mechanism remains unknown.
In summary, the main site of TAC3 expression was found to be the
placenta. TAC3 expression was significantly elevated in pre-eclamptic
human placenta at term, but hypoxia and oxidative stress, presumed
etiologic factors in pre-eclampsia, did not induce TAC3 expression
in vitro, with hypoxia in fact reducing TAC3 expression. In contrast,
in the rat, there is a down-regulation of TAC3 in the third trimester,
suggesting either a failure of a similar down-regulation in human pre-
eclamptic pregnancies or the presence of an as-yet-unknown stimulus
to induce TAC3 expression.
The Medical Research Council (UK) and Canadian Institutes of Health
Research (Canada) supported this work.
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Blumenstein M, Mitchell MD, Groome NP and Keelan JA (2002) Hypoxia
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TACR3 during late rat pregnancy. Expression was normalized to that of 18S
rRNA. Each bar represents the mean tissue expression from four different ani-
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aP < 0.05, significant difference versus mRNA levels from day 16; bP < 0.05,
significant difference versus mRNA levels from day 16; cP < 0.05, significant
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Submitted on January 16, 2006; resubmitted on February 1, 2006; accepted on
February 6, 2006
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