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41 Placental expression of neurohormones and other neuroactive molecules



Expression and localization
With respect to gestational tissues, NPY is produced by human placenta, mater-
nal decidua and fetal membranes. Acidic extracts of human placental tissue
collected at term pregnancy contained high immunoreactive NPY (ir-NPY)
concentrations. The extracted ir-NPY eluted from high-pressure liquid chromato-
graphy (HPLC) with the same retention time as synthetic NPY. Its presence
in placental cells was con¬rmed by immunohistochemical ¬ndings showing
an intense NPY in the cytoplasm of the epithelial amnion cells and of the
cytotrophoblast cells, and intermediate trophoblast of the chorion. To further
support the local production of NPY, primary cultures of human placental cells
released ir-NPY into the culture medium and the addition of high K concen-
trations increased the release of the peptide (Petraglia et al., 1989a; 1993b).


Receptors
Binding sites for NPY are present in all peripheral cells of placental terminal villi
(Petraglia et al., 1989a; Robidoux et al., 1998). All NPY receptors mediate their
responses through pertussis toxin sensitive G-proteins of the Gi/0 family, result-
ing in inhibition of adenylate cyclase activity, but they are also able to increase
intracellular Ca2 levels (Balasubramaniam, 2003). A variety of receptor sub-
types for NPY exists, that is Y1, Y2, Y3, Y4, Y5, Y6 receptor, and NPGPR
(Balasubramaniam, 2003). The Y1 (Wharton et al., 1993) and Y3 receptor
(Robidoux et al., 1998) and NPGPR (Cikos et al., 1999) have been identi¬ed also
within placenta. The NPY1R and NPY3R are located on brush-border membranes
of syncytiotrophoblastic cells of placental villi (Robidoux et al., 1998).


Biological ¬‚uids
During pregnancy, NPY is secreted from human placental tissues in maternal
and fetal circulation and, in amniotic ¬‚uid, NPY levels are higher than in non-
pregnant women, without signi¬cant changes throughout gestation. Maternal
plasma levels increased threefold during labor, thus suggesting that the peptide
may play a role in the stress response of parturition. In fact, during labor mater-
nal plasma NPY levels progressively increased, matching the highest levels at the
most advanced stages of cervical dilation and at the time of VD (Petraglia et al.,
1989b) (Figure 1.5). Moreover, plasma NPY values fall immediately after deliv-
ery, supporting the placental origin of the circulating NPY during pregnancy.
The NPY is also measurable in amniotic ¬‚uid and umbilical cord serum, and
levels are comparable to those found in maternal circulation, being highest at
term and mainly during the early or late stages of labor (Petraglia et al., 1989b).
42 F. Petraglia et al.



A recent study by the use of radioimmunoassay showed that maternal plasma
NPY levels in pregnant women with eclampsia and preeclampsia are signi¬-
cantly elevated with respect to that in normotensive pregnant women (Table
1.2). At 6 days after delivery the concentration of plasma NPY was signi¬cantly
decreased in women with eclampsia and preeclampsia, and in women with nor-
motension, compared with the value measured on admission. Probably, ele-
vated plasma NPY levels may play a key role in the development of eclampsia
and preeclampsia (Khatun et al., 2000).




Box 1.6 Calcitonin-gene-related peptide

The CGRP is a 37 amino acid neuropeptide produced by tissue-speci¬c alter-
native splicing of the primary transcript of the calcitonin gene (Poyner et al.,
2002). A second gene encoding a similar peptide ( -CGRP) has also been iden-
ti¬ed in rat and human (Poyner et al., 2002), and various tissues, including the
CNS, the heart and kidney, are able to express the peptide.
The distribution of CGRP-producing cells and pathways in the brain and
other tissues suggests functions for CGRP in nociception, ingestive behavior,
and modulation of the autonomic and endocrine systems. Moreover, CGRP
also shows potent vasodilator actions and probably is an important regulator of
vascular tone and blood ¬‚ow (Poyner et al., 2002).


Expression and localization
The CGRP mRNA is expressed by human placenta, but mainly by decidual cells
(Graf et al., 1996; Knerr et al., 2002; Tsatsaris et al., 2002; Yallampalli et al., 2002).
The mRNAs levels measured in the human placenta by RT-PCR in normal and
preeclamptic women were signi¬cantly reduced in PE compared with controls,
in chorionic plate but not in villi specimens. In general, CGRP gene expression
indicated by mRNA amounts was slightly higher in chorionic plate tissue than
in placental villi (Knerr et al., 2002). Moreover, in placentae of preeclamptic and
HELLP syndrome women, a reduction of CGRP mRNAs has been shown in con-
trast to unchanged mRNA levels of their receptors (Knerr et al., 2002).
With respect to decidual cells, they are an important source of a CGRP-like
substance within the placenta that may regulate vasodilation and in¬‚uence pla-
cental hormone secretion (Graf et al., 1996). Moreover, decidual cells express
both CGRP mRNA and protein, that is secreted by decidual cells in vitro
(Tsatsaris et al., 2002).
43 Placental expression of neurohormones and other neuroactive molecules



Receptors
Two classes of CGRP receptors exist: one is sensitive to hCGRP(8“37) C-terminal
fragment, while the other is insensitive to this fragment. The CGRP acts at the
cellular level by binding to a seven-transmembrane domain GPCR, and recep-
tors are linked to the activation of adenilate cyclase in several systems and in
intracellular calcium level modulation (Born et al., 2002). In human placentae
there are speci¬c binding sites for CGRP, able to bind - and -CGRP in a dose-
dependent and saturable manner consistent with a single binding site of high
af¬nity, with a low af¬nity for calcitotin (Foord and Craig, 1987).
The CGRP receptors are localized on human syncytiotrophoblast brush-
border membrane (facing the mother) and in basal plasma membrane (facing the
fetus), and are able to bind CGRP in a speci¬c, rapid, time dependent and of high-
af¬nity manner (Lafond et al., 1997). The expression of CGRP receptors has been
also detected by Southern blot hybridization and RT-PCR in decidual cells and
extravillous trophoblast cells (Tsatsaris et al., 2002). In addition to placental and
decidual sites, CGRP receptors are also expressed by human myometrium (Casey
et al., 1997; Dong et al., 1999) and, the myometrial expression is increased during
pregnancy and signi¬cantly downregulated after labor (Dong et al., 1999). Indeed,
CGRP receptors are abundant in myometrial cells of pregnant women who are
not in labor and, are minimal in uterine specimens from women in labor and in
the non-pregnant state (Dong et al., 1999). Finally, the sensitivity of myometrial
tissues to CGRP signi¬cantly decreases at term labor (Chan et al., 1997).
Levels in biological ¬‚uids
The CGRP is secreted in maternal and fetal circulation in increasing amounts from
early to term gestation (Yallampalli et al., 2002). Pregnant women at term have
higher plasma CGRP levels than non-pregnant women and spontaneous labor
does not alter maternal CGRP levels, as levels do not differ between VD and ECS
section, and do not correlate with cervical ripening throughout labor (Florio et al.,
2001b).
There is a controversial report about maternal plasma CGRP concentrations
in PE. In fact, no differences were found between severe PE and normal preg-
nancy, as levels were similar to those in non-pregnant women (Schiff et al.,
1995). Also fetal plasma CGRP do not change and levels in the supernatants of
placental extracts do not differ between preeclamptic and normal pregnancies
(Schiff et al., 1995). On the contrary, recently maternal circulating CGRP con-
centrations were reported signi¬cantly lower in women with PE, thus contribu-
ting to the development and maintenance of hypertension during pregnancy
(Halhali et al., 2001) (Table 1.2).
44 F. Petraglia et al.


relaxant of a variety of smooth muscle tissues (Brain et al., 1985). In fact, CGRP can
induce dose-dependent relaxation in spontaneously contracting pregnant
myometrium, via activation of adenylyl cyclase (Casey et al., 1997), and this relax-
ing effect of CGRP is lower in myometrium obtained from women after labor and
in non-pregnant women (Chan et al., 1997; Dong et al., 1999). The inhibitory
action of CGRP on myometrial contractions may be also dependent on nitric
oxide (NO) formation (Shew et al., 1993), but also involves the hyper polarization of
cell membrane potentials via activation of membrane potassium channels (Chan
et al., 1997). The CGRP relaxation induced in uterus collected after spontaneous or
OT-induced labor was 60 times less effective than in tissues from pregnant women
not in labor (Chan et al., 1997).

(E) Role of PTHrP
Recent evidence from sheep suggest that parathyroid hormone-related peptide
(PTHrP; Box 1.7) may be an important modulator of placental calcium transport.




Box 1.7 Parathyroid hormone-related peptide

The PTHrP is a 141 amino acids protein involved in endochondral bone devel-
opment and epithelial“mesenchymal interactions during the formation of the
mammary glands and teeth (Strewler, 2000). Eight of the ¬rst 13 amino acids
in the mature PTHrP peptide are identical to those of PTH but the sequence
diverges completely after amino acid 13, and the subsequent region accounts for
the distinctive biological actions of the two peptides (Strewler, 2000). The PTHrP
regulates local tissue functions, in contrast to the systemic hormonal function of
PTH. However, PTHrP functions as a poly-hormone that gives rise to several
biologically active peptides, each of which presumably has it own receptor
(Strewler, 2000). PTHrP is produced by many tissues, binds to the same receptor
as PTH and has major effects on development (Fiaschi-Taesch and Stewart, 2003).

Expression and localization
The placenta and the mammary glands are the main sources of PTHrP (Ardawi
et al., 1997). In fact, its mRNA has been identi¬ed in placenta, myometrium,
decidua and fetal membranes (Ferguson et al., 1992; Bowden et al., 1994; Emly
et al., 1994; Curtis et al., 1997) and the peptide is localized in both syncytio-
trophoblast and cytotrophoblast cells (Clemens et al., 2001). With respect to
mRNA levels, the expression is higher in placental amnion than in re¬‚ected
amnion (Ferguson et al., 1992). By using immunohistochemistry, a differential
45 Placental expression of neurohormones and other neuroactive molecules



localization of immunoreactive PTHrP (ir-PTHrP)(1“34) and ir-PTHrP(67“86)
in the human placenta and fetal membranes was found (Ramirez et al., 1995),
with PTHrP(1“34) localized strongly to the syncytiotrophoblast of the placenta,
while PTHrP(67“86) was present predominantly in the endothelial cells of cap-
illaries in the placental villi. Moreover, the staining for ir-PTHrP(1“34) was less
in placenta and membranes obtained from women at the time of labor than at
ECS section in the absence of labor, whereas ir-PTHrP(67“86) staining did not
differ signi¬cantly (Ramirez et al., 1995).
Receptors
The PTH/PTHrP receptor is a seven-transmembrane domain, G-protein-linked
receptor which signals via both adenilate cyclase and phospholipase C (Strewler,
2000). Using real-time protein-coupled receptor (RT-PCR), PTH/PTHrP receptor
mRNA was expressed in the myometrium and in preterm and term samples of pla-
centa, amnion over placenta, re¬‚ected amnion and choriodecidua (Curtis et al.,
1998). In details, PTHrP receptor has been found in human trophoblast in prox-
imity to sites of PTHrP expression (Ferguson et al., 1998), thus suggesting possible
autocrine and paracrine functions of PTHrP in all preterm and term tissues,
including amnion, chorodecidua, placenta and myometrium (Curtis et al., 1998;
Ferguson et al., 1998).
Levels in biological ¬‚uids
Plasma levels of PTHrP increase throughout pregnancy with higher levels at
term (Hirota et al., 1997) and PTHrP produced in either the feto-placental unit
or the breast, or both, can reach the circulation of pregnant women in the third
trimester and at 1 month postpartum in women with breast- and mixed-feeding
(Hirota et al., 1997).
The PTHrP is detectable in fetal blood and concentrations are lower in
maternal blood (Bucht et al., 1995; Papantoniou et al., 1996). Moreover, PTHrP
levels were higher in fetal than maternal circulation (Bucht et al., 1995) and,
concentrations in the umbilical artery are higher than in the vein, thus suggest-
ing that the fetus is the main source of PTHrP in the cord blood circulation
(Papantoniou et al., 1996). The concentrations in umbilical cord plasma were
increased in intrauterine growth restriction (IUGR), but unaltered in diabetes
(Strid et al., 2003) (Table 1.2). In preeclamptic women, the PTHrP expression in
placenta and amnion was not increased in association with maternal hyperten-
sion, placental insuf¬ciency and vasoconstriction. The PTHrP mRNA expres-
sion was decreased in choriodecidua in association with term but not preterm
PE, thus suggesting that PTHrP is not involved in the placental pathophysiology
of PE in late gestation (Curtis et al., 1998; Clemens et al., 2001).
46 F. Petraglia et al.


It has been demonstrated that partially puri¬ed fetal parathyroid extracts of
PTHrP increased placental calcium transport (Rodda et al., 1988; Care et al., 1990),
and that parathyroid hormones (PTH) and PTHrP(1“34) regulate the calcium
transport across the fetal facing, but not the maternal facing, of the syncytiotro-
phoblast (Farrugia et al., 2000). Moreover, acting through the PTH/PTHrP recep-
tor, the two molecules may contribute to the overall maintenance of calcium
transfer across placenta (Rodda et al., 1988; Care et al., 1990).
Calcium is a factor that is also related to the physiology of the myometrium and
calcium channel blockers effectively inhibit undesired uterine activity (Challis
et al., 2000). The PTH/PTHrP regulates calcium homeostasis in various target tis-
sue, and hyperparathyroidism complicating pregnancy involves an increased inci-
dence of premature birth but no statistically signi¬cant differences were observed
in the levels of calcium and other minerals salts, between preterm labor, preterm
non-labor, term labor and term non-labor (Lurie et al., 1997). However, in rats
PTH/PTHrP(1“34) acts on myometrial smooth muscle to cause relaxation (Shew
et al., 1984; Williams et al., 1994), and inhibits OT-induced rat uterine contractions
in vitro (Dalle et al., 1992). Moreover, PTHrP may facilitate the myometrial quies-
cence characteristic of the ¬rst 95% of normal pregnancy (Figure 1.5).


Peptide signaling and the control of fetal“placental blood ¬‚ow

The control of fetal“placental blood ¬‚ow is very important throughout pregnancy,
as the nutrients and oxygen to the fetus come from the mother and have to pass the
placental barrier. In fact, the fetal vessels of the human placenta are not inner-
vated, so that the control of blood ¬‚ow in this vascular bed is partly dependant on
locally produced and circulating vasoactive factors (Boura et al., 1994). The tight
regulation of the blood ¬‚ow through the uterine arteries (from the mother to the
placenta) and the umbilical cord (from the placenta to the fetus) is critical for the
growth and differentiation of the embryo/fetal tissues (Reis et al., 2002). In healthy
pregnant women an increase in the uterine blood ¬‚ow and a decrease in uterine
vascular resistance are typical features. The mechanism causing this decreased vas-
cular resistance is poorly understood. It is probably the result of multiple factors,
including a loss of smooth muscle in myometrial resistance vessels (spiral arteries
and terminations of radial arteries), an increased angiogenesis, as well as an
augmented local uterine artery vasodilation probably related to an increased role
of endogenous vasodilators (Kuo et al., 1990; Poston et al., 1995).
Pregnancy is associated with various cardiovascular changes such as increased
blood volume and cardiac output, and decreased blood pressure and peripheral
vascular resistance. The decrease in peripheral vascular resistance occurring in
pregnancy (Poston et al., 1995) has been attributed to increased production of
47 Placental expression of neurohormones and other neuroactive molecules


vasorelaxant, which acts on the vascular endothelium to cause the release of several
relaxant factors (Moncada and Vane, 1979; Furchgott, 1993), as well as directly on
vascular smooth muscle causing relaxation (Brayden and Nelson, 1992). CRH, NPY,
CGRP and PTHrP play a major role in regulating locally the tone of blood vessels.

(A) Effects of CRH and NPY

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