Current Pharmaceutical Design, 2004, 10, 2499-2517 2499

Regulation of Myometrial Smooth Muscle Functions

F. Hertelendy1,* and T. Zakar2

1
Department of Obstetrics, Gynecology and Women’s Health, Saint Louis University School of Medicine, St. Louis,
Missouri, USA and 2Mothers and Babies Research Centre and Department of Obstetrics and Gynaecology, University of
Newcastle, Newcastle, Australia

Abstract: Regulation of myometrial functions during gestation, labor and birth are in the forefront of research in
reproductive sciences. The complexity of the problem is reflected by our scant understanding of the intimate cellular and
molecular events underlying these phenomena, despite extensive efforts spanning several decades. Unlike other smooth
muscles, the myometrium is, to a large extent, under hormonal control. Of these, the steroid hormones, progesterone and
estrogen, play dominant roles in terms of uterine growth, the maintenance of quiescence during gestation and the
preparation of the uterus for labor and delivery. In addition to steroid hormones, there are a number of factors that
modulate myometrial contractility (oxytocin, prostaglandins, endothelin, platelet activating factor) and relaxation
(corticotropin releasing hormone, prostacyclin, nitric oxide). Although notable advances have been made towards
understanding some of the key steps in receptor signaling that define the actions of these factors, a good deal of new
information is needed to fully understand this fundamental life process. Pharmaceutical agents have been used extensively
to induce labor or to prolong pregnancy in the case of preterm labor that represents the major cause of perinatal morbidity
and mortality. Because preterm labor is a syndrome of multiple etiologies, pharmacologic agents will have to be targeted
accordingly. This review attempts to present a critical overview of these topics.

Key Words: myometrium, parturition, preterm labor, progesterone, oxytocin, prostaglandins, cytokines, endothelin, nitric
oxide, CRH, cell signaling, mechanical stretch, tocolysis.

INTRODUCTION leads to increased tension, resulting from the activation of

The myometrium, forming the muscular wall of the
uterus, has two principal functions. By growth and stretch, it
provides the protective environment for the developing fetus
and during labor, by powerful contractions, it lends much of
the mechanical force to expel the products of conception.
Although the regulation of neither of these myometrial func-
tions is fully understood, there is good evidence that endo-
crine, paracrine, as well as mechanical factors, are involved.
The aim of this review is to provide the reader with an
overview of our current understanding of potential mechan-
isms that maintain the myometrium in a relatively quiescent
state, refractory to various uterotonic substances during
much of pregnancy, as well as the cellular and molecular
events that may account for the conversion to an active,
contractile organ at the onset of labor, responsive to various
uterotonins.
Although a number of factors are believed to be involved
in the maintenance of quiescence during gestation, there is
general consensus that progesterone, the principal gestational
hormone, plays a dominant role in this process and there is
growing evidence that a functional withdrawal of proges-
terone precedes the evolution of myometrial activation
leading to labor. The onset of labor is heralded by phasic
myometrial contractions that are driven by action potentials.
The ensuing influx of calcium via voltage-gated channels
*Address Correspondence to that Author at the Department of Obstetrics,
Gynecology and Women’s Health, Saint Louis University School of
Medicine, St. Louis, Missouri, USA; E-mail: hertelf@slu.edu
myosin light chain kinase (MCLK) by the calcium-calmo-
dulin complex. MLCK, as in other smooth muscle cells,
phosphorylates the light chain of myosin, allowing it to
interact with actin. The actomyosin complex, in turn, can
convert the chemical energy of ATP to the mechanical
energy necessary for myometrial contraction. Calcium may
also enter the intracellular compartment by way of receptor
regulated mechanisms. A ligand activated receptor can
increase [Ca2+]i by enhancing calcium entry, as well as
promoting calcium release from intracellular stores. Such a
mechanism has been amply demonstrated to operate in
myometrial cells. Conversely, G-protein-coupled receptor
(GPRC) activation may transmit the signal via Gαs,
activating the appropriate isoforms of adenylate cyclase
(AC), causing a rise in cAMP levels.
Even though the relaxing effect of cAMP on myome-
trium is well established (tocolysis using β2-adrenergic
receptor agonists that activate the AC/cAMP system is based
on this principle), the molecular mechanism underpinning
this phenomenon is still uncertain. It is known, however, that
cAMP-induced activation of protein kinase A (PKA I and
PKA II), in association with a family of A-kinase anchor
proteins [1,2], is involved in the phosphorylation of various
target proteins (calcium channels, MLCK, IP3 receptors in
the sarcoplasmic reticulum, etc.) leading to myometrial
smooth muscle relaxation. Elevated levels of cAMP found in
the pregnant myometrium may also be amplified by proges-
terone-inhibited phosphodiesterase activity, suppressing
cAMP breakdown.

1381-6128/04 $45.00+.00 © 2004 Bentham Science Publishers Ltd.

2500 Current Pharmaceutical Design, 2004, Vol. 10, No. 20
The intimate mechanism responsible for the switch from
a quiescent state to an active, contractile state at the end of
pregnancy, i.e. the mechanism underlying the onset of labor
in the human, remains largely obscure. However, because the
steroid hormones, progesterone and estradiol, directly or
indirectly regulate the biosynthesis of key proteins involved
in uterine physiology, we shall first address these issues in
some detail, followed by the reviewing of a number of
factors that modulate myometrial smooth muscle functions
during gestation and parturition. Several recent reviews,
which the reader may wish to peruse [3-5], have addressed
the regulation of parturition at term and preterm.
PROGESTERONE
Almost half a century ago Arpad Csapo [6] recognized
that uterine quiescence during pregnancy results from the
blocking effect of progesterone on myometrial contractility.
The progesterone “block” is lifted at term, and the
developing uterine activity soon culminates in the delivery of
the fetus. Progesterone has also been implicated in the
maintenance of the closed cervix, and the removal of the
progesterone block is considered critical for the opening of
the birth canal. Consistent with this model, progesterone
levels in the maternal plasma are high during pregnancy and
fall shortly before labor in many mammalian species. Early
withdrawal of progesterone, for example by removing the
ovaries of pregnant rats or mice, causes preterm delivery,
while progesterone administration prolongs gestation beyond
term. In other species including the human, however, labor
and delivery occur without a fall of circulating progesterone
levels. Attempts to identify alternative mechanisms, such as
increased target tissue metabolism [7] or the presence of a
local progesterone binding substance in the fetal membranes
[8], failed to show a drop in tissue progesterone levels before
the onset of labor. Therefore, progesterone withdrawal
before labor in women is believed to be “functional”,
involving a mechanism that affects the myometrium in a way
analogous to a drop in circulating or tissue progesterone
levels. The existence of functional progesterone withdrawal
is supported by the observations that administration of the
progesterone antagonist mifepristone (RU486) increases
myometrial contractility in early pregnancy [9, 10] and
promotes cervical dilatation and delivery at term [11, 12].
Thus, progesterone is required for the maintenance of
pregnancy and the myometrium is released from the relaxing
influence of the hormone before labor and delivery occur.
In order to understand the way progesterone suppresses
myometrial contractile activity during most of pregnancy
and influences the timing of labor in women, two distinct,
but related questions need to be addressed. The first one
relates to the mechanisms by which progesterone suppresses
uterine activity during pregnancy, and the second one
addresses the mechanisms that cause functional progesterone
withdrawal before labor. There are no satisfactory answers to
these questions yet, but some intriguing possibilities are
emerging in view of recent research.
Progesterone Receptor-Mediated Mechanisms
Progesterone, being a steroid hormone, exerts its effects
by influencing the activity of progesterone responsive genes.
Hertelendy and Zakar
Cells that are targets of progesterone express receptor
proteins (PRs) that bind the hormone with high specificity.
The progesterone-receptor complex then adopts a confor-
mation that enables it to function as a transcription factor. In
this process, the active PR-progesterone complex forms
dimers and binds to palindromic recognition sequences,
called PRE (AGAACANNNTGTTCT is the ideal consensus
sequence), present in the promoter regions of progesterone
responsive genes. This interaction stimulates the transcrip-
tion of the target genes. In the absence of progesterone, the
receptor is bound to chaperone proteins, predominantly
HSP90, and is inactive as a transcription factor.
The above description, however, is a much simplified
overview of the molecular mechanisms of progesterone
action. First, two forms of PRs exist, PR-A and PR-B. PR-A
is an N-terminally truncated variant of PR-B, and the two
receptor isoforms can associate to homo- and hetero-dimers.
The transcriptional activity of the three receptor dimers is
different and varies in a target gene- and target cell-specific
fashion [13]. Second, PRs undergo multiple phosphorylation
after ligand binding which may modulate their transcrip-
tional activity [14]. Third, PRs bind to promoters as part of a
multiprotein complex. This complex includes the basic
transcription factors (TBP, polymerase-II, TAFs), coactiva-
tors such as CBP/p300, and the steroid receptor coactivator
proteins and also corepressor proteins (reviewed in ref. [15]).
The expression of the cofactors varies in different tissues and
physiological states, and their contribution to progesterone-
dependent regulation may vary according to target genes.
The activity of some cofactors may also be modulated by
protein phosphorylation. Fourth, the PR may interact with
other transcription factors without binding to the PRE
sequence, repressing their activity. This transrepression is
mutual with RelA(p65) [16], while PR represses the activity
of the glucocorticoid and estrogen receptors [13, 17, 18].
Furthermore, PR activity on a PRE-controlled gene may be
inhibited by the AP1 component, c-fos, in a cell line-specific
fashion [19]. These regulatory interactions have been
identified in cultured cells after co-transfection with reporter
constructs and vectors overexpressing the transcription
factors under study. They indicate a great variety of possible
PR modulations at the functional level. Work to translate
these basic findings to physiological and pathophysiological
situations is still in the initial phase. In the next sections, we
will review the data relevant to the progesterone dependent
regulation of human myometrial function during pregnancy
and labor and will discuss the possible mechanisms of func-
tional progesterone withdrawal, in view of the information
available on the molecular mechanisms of PR regulation.
Connexin43
The PR is expressed in the pregnant human uterus [20-
22], therefore progesterone may directly control myometrial
function by regulating the expression of progesterone
responsive genes. Among these, much attention has been
devoted to the gene encoding connexin43 (Cx43). The Cx43
protein is a major component of myometrial connexons,
which are pore structures between myometrial cells allowing
cell-to-cell communication via diffusible intracellular com-
ponents. Clusters of connexons form gap junctions, which
provide the structural basis of intercellular communication
Regulation of Myometrial Smooth Muscle Functions
necessary for the coordinated, phasic contractions of labor.
In support of this assumption, the density of myometrial gap
junctions has been shown to increase in labor [23-25], and
Cx43 mRNA [26] and protein expression [27] have been
found to increase in the uterine muscle in late gestation and
during labor. Negative regulation of Cx43 gene activity by
progesterone during pregnancy, but not during labor, would
be consistent with functional progesterone withdrawal at the
gene expression level.
The effect of progesterone on myometrial cell Cx43
expression has been studied in cell culture experiments.
Andersen et al. [28] and Zhao et al. [29] showed that the
synthetic progestin medroxyprogesterone acetate repressed
Cx43 gene transcription (measured by nuclear run-on
analysis), mRNA abundance and protein abundance
(measured by Northern and immunoblot analyses, respec-
tively) in confluent primary myocyte cultures from non-
pregnant uteri and uterine leiomyomas. At 100 nM progestin
concentration, repression of all three parameters was partial.
Repression by progesterone of Cx43 protein levels and
Cx43-gap junction density has also been reported in primary
myocyte cultures from term pregnant women [30]. These
data strongly suggest that progesterone exerts an inhibitory
effect on Cx43 gene expression in the human myometrium.
The promoter of the human Cx43 gene has been cloned,
sequenced [31] and analyzed functionally by transfection
studies with myometrial cells [29, 31, 32]. The 3.0 kilobase
upstream sequence of the Cx43 promoter contains a TATA-
box sequence, several AP-1, AP-2 and SP-1 regulatory
elements, 6 half palindromic estrogen regulatory elements
(EREs) and half palindromic PREs. A reporter construct
containing a thymidine kinase promoter and a PRE upstream
of the chloramphenicol acetyltransferase (CAT) gene has
been transfected into primary myometrial cells [29]. The
construct exhibited progestin-stimulated CAT expression
indicating that the cells contained functional PRs. In these
cells, however, progestin suppressed Cx43 transcription,
which argues against the involvement of the half PRE sites
in the regulation. Interaction of the PR with transcription
factors controlling Cx43 expression via the AP-1 or SP-1
sites is a possibility to be explored, because these sites and
factors are involved in the control of the human myometrial
Cx43 gene [31-33]. The nature and changes of these
interactions may be informative regarding the molecular
mechanisms that maintain the progesterone-repression of the
Cx43 gene during pregnancy and withdraw progesterone
action functionally at labor.
The Oxytocin Receptor (OTR)
Oxytocin is a potent and specific stimulant of uterine
contractions (see below). Oxytocin receptor expression in the
myometrium, measured as specific binding of radioactive
oxytocin to crude membrane fractions, increases about 12-
fold from early pregnancy (13-17 weeks) to term, and a
further increase occurs during labor [34-36]. A similar surge
of OTR protein and mRNA abundance accompanies the
increase of hormone binding activity [37]. The up-regulation
of myometrial OTR expression results in enhanced
sensitivity of the uterus to oxytocin at term [38], which,
according to Mitchell and Schmid [39], argues for a vital
Current Pharmaceutical Design, 2004, Vol. 10, No. 20 2501
role of the OTR/oxytocin system in the mechanism of labor
onset and maintenance.
The time course of OTR expression in the uterus
corresponds to the pattern of progesterone withdrawal in late
pregnancy [40]. This is best documented in mice [41], where
failure of parturition in a prostaglandin FP-receptor deficient
strain is due to the lack of luteolysis at the end of the normal
gestational period. In these animals, high progesterone levels
are maintained and the OTR is not induced in the uterus at
term. Ovariectomy results in a drop of circulating proges-
terone concentrations, induction of OTR and delivery of the
pups. The oxytocin receptor gene promoter, however, does
not contain a PRE in mice or any other species studied so
far, including the human [40, 42]. This suggests that the
progesterone dependent suppression is either indirect or
mediated by a hitherto unrecognized progesterone-sensitive
regulatory sequence of the OTR gene. In a study where the
effects of sex steroids were examined in human uterine cell
cultures, Adachi and Oku have indeed found that proges-
terone suppressed the stimulatory effect of estrogen on OTR
levels measured in a binding assay [43]. On the other hand,
administration of mifepristone, a progesterone receptor
antagonist, in early human pregnancy has been reported to
increase spontaneous uterine contractions without enhancing
the sensitivity to oxytocin [9]. In this study, uterine contrac-
tions were recorded using a pressure transducer inserted into
the uterine cavity. Similar results were reported by Webster
et al. [44], who monitored intrauterine pressure through a
balloon catheter inserted into the uterus of early pregnant
women. The women were treated with epostane, a 3-beta
hyroxysteroid dehydrogenase inhibitor, in a dosage that
reduced maternal serum progesterone concentrations by 77%
and estrogen concentrations by 45% without affecting
cortisol levels. Epostane increased uterine activity, but
subsequent oxytocin injections showed no increase in
oxytocin sensitivity. Selinger et al. conducted a similar study
on mid-trimester pregnancies, also showing no effect of
epostane treatment on myometrial oxytocin sensitivity [45].
In these women, epostane reduced the levels of progesterone,
but not of estrogen or cortisol. Thus, there is no consistent
clinical or experimental evidence implicating progesterone in
the control of OTR expression or function in the pregnant
human uterus. More data are available on the non-genomic
actions of progesterone and its metabolites on OTR function,
but this evidence, as discussed below, is also controversial.
Prostaglandins (PGs)
Prostaglandins are important paracrine regulators of
uterine function in normal and pathological pregnancies and
are used clinically to induce abortion and stimulate parturi-
tion. Their involvement in myometrial regulation is discus-
sed later; here we summarize those aspects that link intrau-
terine PGs to the action of progesterone. Pharmacological
progesterone withdrawal by mifepristone [9] or epostane in
early or mid-gestation [45, 46] increases the sensitivity of the
uterus to prostaglandins. This synergism may explain the
efficiency of combined mifepristone/PG-analog treatments
to terminate early pregnancies [47]. The way mifepristone
increases PG-sensitivity, however, is unclear. For example,
Norman et al. [10] found that the mifepristone-enhanced
2502 Current Pharmaceutical Design, 2004, Vol. 10, No. 20
myometrial contractions, monitored by intrauterine pressure
recordings, were not attenuated by indomethacin in a dose
that reduced decidual PGF2α production. The obvious
explanation that progesterone controls myometrial PG-
sensitivity by influencing FP-receptor or other PG-receptor
expression is supported by evidence in rats and mice [48-51].
Data on pregnant human myometrium, on the other hand, is
very limited [52]. Cell and tissue culture studies may
establish unequivocally whether progesterone controls PG-
receptor expression in the pregnant human uterus.
Substantial evidence suggests that progesterone up-
regulates the expression of 15-hydroxyprostaglandin dehyd-
rogenase (PGDH) in uterine smooth muscle, placenta and the
chorion membrane. The PGDH enzyme catalyzes the key
step of prostaglandin catabolism and has a major influence
on the tissue level of uterotonic prostaglandins such as PGE2
and PGF 2α. Since PGs can diffuse between adjacent tissues,
PGDH activity in the myometrium, as well as in the
juxtaposed chorion laeve and in the nearby placenta, can
affect the myometrium. Greenland et al . studied thoroughly
the steroid regulation of the human PGDH promoter in
primary myometrial and endometrial (stromal) cell cultures
[53]. They have transfected the cells with a series of PGDH
promoter-reporter constructs and detected a marked stimu-
lation of reporter expression by progesterone. To achieve
stimulation, however, it was necessary to overexpress the
progesterone receptors by co-transfecting a PR-expression
vector. Overexpressed PR-A and PR-B were both active in
the assays, and a cAMP-analog synergized with progesterone
in the presence of PR-B but not of PR-A. Notably, the
endogenous PR, present in the endometrial cells, was unable
to mediate the stimulatory effect of progesterone on PGDH
promoter-driven reporter expression. Furthermore, no
consensus PRE was identified in the 2.3 kilobase 5’ flanking
sequence of the human PGDH gene [53]. Thus, the
physiological significance of these PGDH promoter-trans-
fection experiments is unclear. In vivo, myometrial PGDH
activity and protein abundance are unchanged during human
pregnancy, but decrease at term and preterm labor [54].
Patel et al. characterized the steroid regulation of PGDH
activity in primary cultures of chorionic and placental
trophoblasts [55-57]. Using a variety of natural and synthetic
progestins, glucocorticoids and hormone antagonists, they
have concluded that progesterone up-regulates and cortisol
down-regulates the activity of PGDH in these cells. The
regulation appears to be mediated by the progesterone and
glucocorticoid receptors and involves changes in PGDH
mRNA levels. In the absence of identified consensus or cell-
specific PRE and GRE sequences in the PGDH promoter,
these steroid effects are likely indirect and require further
characterization to assess their physiological roles.
Calcitonin Gene-Related Peptide (CGRP) and its
Receptors
The 37-amino acid neuropeptide, CGRP, is a potent
vasodilator present in the maternal plasma in increasing
concentrations as pregnancy advances [58, 59]. Dong et al.
[60] have shown that CGRP is also an effective inhibitor of
the spontaneous contractions of myometrial strips isolated
from non-laboring women, but it is much less effective in
Hertelendy and Zakar
relaxing myometrial strips from women in labor. The
importance of the decreased sensitivity of the myometrium
to CGRP at labor is highlighted by the observations of Florio
et al. [61], who reported that maternal CGRP levels do not
change in labor. Accordingly, Dong et al. [60] detected a
marked decrease of immunoreactive (60 kDa) CGRP-
receptor protein levels in laboring myometrium and a
concomitant decrease of CGRP (Type-1)-receptor mRNA
measured by polymerase chain reaction using primers that
hybridize to a cognate receptor-cDNA sequence [62]. The
group has since characterized the steroid responsiveness of
the myometrial CGRP-receptor[63]. Using a monoclonal
antibody that appears to recognize a new variant of the
CGRP-receptor, called CGRP-B receptor [64], they have
concluded that progesterone stimulates CGRP receptor
protein-B expression dose dependently almost six-fold in
non-laboring myometrial explants. Estradiol alone is
inhibitory, but together with progesterone stimulates further
the level of the receptor protein. It will be important to
compare the effects of steroids on tissues obtained before vs.
during labor and to monitor specific mRNA levels once the
CGRP-B receptor mRNA is characterized. Based on the
available data, the myometrial CGRP-B receptor is a
promising candidate to study functional progesterone
withdrawal at the gene expression level.
Progesterone Receptors
Changing PR abundance in the myometrium can
potentially influence the progesterone responsiveness of the
tissue and underpin, at the molecular level, the concept of
functional progesterone withdrawal. Early studies using
ligand-binding assays and an enzyme-linked immunoassay
(EIA) found that PR levels were markedly lower in the
pregnant than in the non-pregnant myometrium [20, 65, 66].
At the same time, a characteristically large proportion of the
binding sites was associated with the nuclear fraction in
pregnant myometrium, which was concordant with the
persistent high tissue progesterone concentrations during
pregnancy. Subsequently, several groups have confirmed the
predominant nuclear localization of PR in gravid
myometrium using immunohistochemistry [21, 33, 67].
Attempts to assess the tissue abundance of PR by scoring the
intensity of the immunohistochemical staining provided
conflicting results. How et al. [21] reported a decrease of
staining with advancing gestation and with term and preterm
labor, while Roh et al. [67] detected no change of PR
abundance with term labor and decreased abundance in post-
term pregnancies. Patient numbers were rather low in both
studies (between 3 and 10 per group) lowering the power of
the semi-quantitative scoring even further. Using the
quantitative EIA methodology, Rezapour et al. [22]
examined myometrial PR levels in a larger cohort of women
(n=18-19 per group). Significantly, these investigators took
biopsies not only from the lower uterine segment, where the
incision for cesarean delivery and the sampling of
myometrium are usually performed, but also from the fundus
of the uterus. The double sampling enabled them to detect
regional differences in myometrial PR expression. They have
found that PR abundance increases in the fundus but not in
the lower segment myometrium at term labor. Thus,
according to this set of data, labor is associated with
Regulation of Myometrial Smooth Muscle Functions
increased PR expression in the upper segment but not in the
lower segment of the uterus. In oxytocin-resistant labor,
however, PR levels did not increase in the fundus and
decreased in the lower segment as compared to the term not-
in-labor group. Winkler et al. [68] used EIA to measure PR
levels in the lower segment myometrium during the course
of labor. This study detected a transient decrease of PR
abundance during active labor, reaching a nadir at 2-3.9 cm
cervical dilation. The temporary and regional differences in
PR levels may indicate a dynamic spatio-temporal regulation
of uterine PR levels including, for example, the changing
cellular composition of the uterine tissue [69] at term.
However, differences in investigative techniques may also
contribute to the variability.
To assess the functional consequences of changing PR
levels, it is necessary to consider the isoform distribution of
PRs in the various parts of the uterus and during the various
stages of pregnancy. The antibodies used in the EIA and
immunohistochemistry measurements discussed above did
not differentiate between PR-A and PR-B, although both PR
isoforms are expressed in the pregnant human myometrium
[21]. As pointed out earlier, the transcriptional activity of the
two PRs differs in a promoter- and cell-specific fashion;
however, the human PR-A, in general, has a dominant
negative effect over other steroid receptors including PR-B
[13, 18]. The inhibitory action of PR-A over PR-B has also
been confirmed in transfection studies using myometrial cell
cultures from term pregnant women [70]. Mesiano et al.
have recently examined the abundance of PR-A and PR-B
mRNAs in myometrial biopsies obtained before and during
term labor [71]. They have found that both PR-A and PR-B
mRNA levels increase in labor, but the increase of PR-A
mRNA abundance is more marked resulting in an elevated
PR-A/PR-B mRNA ratio. Interestingly, the PR-A/PR-B
mRNA ratio correlated in the same laboring tissues with the
level of HOXA 10 mRNA, which is an mRNA species
down-regulated by progesterone in non-pregnant myometrial
cell cultures [72]. This observation suggests that the PR-
A/PR-B mRNA ratio may be relevant functionally. Further
data on PR-A/PR-B protein levels and expression studies on
the PR-A/PR-B dependent regulation of progesterone
responsive genes are needed to corroborate the possibility
that preferential PR-A expression is a molecular mechanism
of functional progesterone withdrawal in laboring human
myometrium.
As discussed in the preceding sections, a major difficulty
impeding the study of progesterone action during pregnancy
is that no myometrially expressed human genes were found
that are well-characterized structurally and functionally, have
critical roles in pregnancy, and respond to progesterone
robustly in vivo and in vitro. There are candidate progesterone
responsive genes in addition to those discussed, such as
HOXA 10 [72], TGFβ1, TGFβ receptor type 1 [73, 74], or
hemoxygenase-1 and 2 [75], although the involvement of the
hemoxygenases in maintaining uterine quiescence has been
questioned [76]. Large-scale functional genomic studies,
which screen hundreds or thousands of genes for differential
expression, may uncover new candidates for progesterone-
controlled genes in the myometrium. From three recent
studies using functional genomic approaches, an overall
pattern of labor-related myometrial gene expression is
Current Pharmaceutical Design, 2004, Vol. 10, No. 20 2503
emerging [77-79]. Of the three, the team of Charpigny et al.
examined the highest number of genes, 2352, using two
macroarrays. They found that only 4% of the screened genes
exhibited altered expression with advancing gestation and
labor at term. Remarkably, many developmental, prolife-
ration-related and cell-adhesion-associated genes were
massively down-regulated during late gestation, and a lower
number of pro-inflammatory, contraction- and apoptosis-
related genes were up-regulated relatively modestly during
late pregnancy or labor. Results from the other two studies
were in general agreement with these findings. Clearly,
functional progesterone withdrawal in the period leading up
to term labor is associated with the repression of many genes
involved in pregnancy maintenance possibly giving way to
pro-inflammatory processes at labor. Traditional approaches
will be needed to establish which of the regulated genes are
controlled directly by progesterone.
New findings by Condon et al. [15] suggest a plausible
mechanism by which a number of progesterone-controlled
genes can be functionally down-regulated in term myomet-
rium. These investigators have reported that the levels of
three PR coactivators, SRC-2, -3 and CBP decrease in the
human (fundal) myometrium in labor. The decrease of both
mRNA and protein levels was detected. Since the coacti-
vators participate in the actions of a variety of transcription
factors in addition to PR [80], reduced levels may suppress
the expression of many genes depending on the specificity of
transcription factor interactions. Moreover, these coactiva-
tors have intrinsic protein acetyltransferase activity, which is
involved in general chromatin remodeling, for example, by
histone acetylation. In agreement with this, Condon et al.
found decreased levels of acetylated histone H3 in laboring
myometria, indicating a relatively closed chromatin struc-
ture. Treatment of pregnant mice with trichostatin A (TSA),
a histone deacetylase inhibitor, increased histone H3
acetylation and significantly prolonged pregnancy. In a cell
culture study, however, histone hyperacetylation by TSA
blocked the PR-dependent transcription of a mouse
mammary tumor virus (MMTV) promoter-driven reporter
[81]. Collectively, these data indicate that protein or histone
acetylation remodels chromatin and modulates progesterone
action in a promoter-specific manner.
None-Genomic Actions of Progesterone
Strips of pregnant myometrium, when suspended
vertically in an organ bath and perifused with oxygenated
and temperature-controlled nutrient buffer, start to contract
spontaneously within 50-90 minutes. Usually one or two
contractions occur per 10 minutes, which may continue for
several hours. Connected to an isometric recording device,
this arrangement is often used as an model of phasic myo-
metrial activity characteristic of labor. Progesterone added to
the perifusion medium diminishes the spontaneous contrac-
tions within minutes [82, 83]. The inhibition is reversible
and stops within minutes after progesterone is washed out.
Detailed analysis of the contraction parameters showed that
progesterone decreased the amplitude and the work area of
the contractions and increased the contraction frequency and
the tone of the muscle [84, 85]. The effects of the steroid
were also dose dependent, and importantly, were observed
only at supraphysiological progesterone concentrations
2504 Current Pharmaceutical Design, 2004, Vol. 10, No. 20
(IC50=225µM). (Progesterone concentration is 0.2-0.3µM in
uterine tissue [86] and approximately 2µM in venous uterine
blood [87]). The rapidity of action and the requirement of
non-physiological hormone concentrations suggested that
progesterone inhibited the myometrial contractions by a non-
genomic, receptor-independent mechanism. Further support
for this suggestion was provided by steroid specificity
studies, which showed that the progesterone receptor
antagonist mifepristone (RU 486) did not block the action of
progesterone. Moreover, progesterone metabolites, especially
the 3α-hydroxy-5α-reduced progesterone derivative and 5β-
dihydro-progesterone, were even more effective than
progesterone (IC50 values: 35µM and 81µM, respectively)
[83, 88]. According to work by Fu et al. [89], 15µM and
150µM progesterone elevated the cAMP content of the
perifused myometrial strips and increased the release of
cAMP into the medium, suggesting that the anti-contractile
effect was mediated at least in part by cAMP. Further,
Kofinas et al. [86] have shown that pharmacological concen-
trations of progesterone inhibited the activity of cAMP-
phosphodiesterase in a cell-free assay with myometrial
extracts. Thus, progesterone may cause cAMP accumulation
by directly inhibiting the breakdown of the cyclic nucleotide.
Following up on early observations [90], Fu et al.
examined the influence of progesterone on oxytocin-
stimulated myometrial contractions [91]. Addition of 16µM
progesterone together with oxytocin (10 or 100 mU/ml)
decreased the amplitude and the activity area of the
contractions, increased the frequency of the contractions and
elevated the tonus of the muscle. These effects were similar
to those seen on the spontaneously contracting myometrium.
At the same time, progesterone prevented the tachyphylaxis
of the oxytocin response normally seen after longer exposure
to the peptide. The physiological significance of the anti-
tachyphylactic effect is not clear; however, it indicates that
progesterone interfered with OTR-dependent signalling
under the experimental conditions employed. Grazzini and
co-workers reported a direct interaction between the human
OTR and the progesterone metabolite 5β-dihydro-proges-
terone (5β-DHP) [92]. The interaction was demonstrated in
CHO (Chinese hamster ovary) cells stably transfected with
the human OTR. The binding of oxytocin to membranes
from the transfected cells was inhibited by 5β-DHP with a Ki
of 32 nM. Moreover, 5β-DHP inhibited the oxytocin-induced
inositol phosphate production in the CHO cells with a very
low Ki (0.25nM). Progesterone was ineffective in this
system, but had analogous inhibitory actions on the rat OTR.
The data by Grazzini et al. indicated that 5β-DHP was an
effective oxytocin antagonist in addition to its inhibitory
effect on the spontaneous myometrial contractions discussed
above. Subsequently, Thornton et al. [88] tested the influ-
ence of 5β-DHP on oxytocin-augmented myometrial contrac-
tions. They found that 100µM 5β-DHP in the medium
decreased the oxytocin sensitivity of the myometrium,
measured as activity area or peak contraction force, by about
10-fold. This suggested that 5β-DHP might be a tocolytic
oxytocin antagonist. The encouraging results prompted
further studies to explore the interaction 5β-DHP with the
OTR. Burger et al. [93] transfected several G-protein-
coupled receptors, including the human OTR, into various
cell lines including CHO cells. They have measured the
Hertelendy and Zakar
effects of progesterone, 5β-DHP and other steroids on ligand
binding and calcium signal generation. Nearly all tested
steroids inhibited ligand binding and calcium signaling by all
receptors to a degree dependent on the particular receptor
and cell line. The effective concentration range was 10-
500µM. The steroid effects were clearly non-genomic and
were related to membrane composition and/or properties
[94]. Overall, the results did not support the possibility of
direct interaction of the steroids with the receptor proteins.
Finally, Astle et al. re-examined the effect of 5β-DHP on
oxytocin binding and (human) OTR-dependent signaling
[95]. The data in this report demonstrated that 5β-DHP (up
to 100µM) had no effect on the binding of oxytocin to the
OTR either in membranes from pregnant human myomet-
rium or in CHO cells expressing OTR. Furthermore, 5β-
DHP (1µM) had no influence on the inward calcium current
in a whole-cell patch-clamp arrangement with primary
human myometrial cells.
Despite the controversy surrounding the actions of
progesterone and 5β-DHP on OTR-signalling, clinical data
show that progesterone is effective in preventing preterm
birth in high-risk pregnancies. Recently, the results of a
multicenter clinical trial have been published, where 463
high risk women were enrolled in a double-blind placebo-
controlled design [96]. The treatment group (310 partici-
pants) received weekly injections containing 250 mg of 17α-
hydroxyprogesterone caproate, which was an effective dose
in previous pilot studies (reviewed in ref. [96]). High risk
was determined as spontaneous preterm delivery in a
previous pregnancy. The treatments started at 16-20 weeks
of pregnancy and resulted in a significant reduction of the
incidence of preterm deliveries before the 37th (term), 35th
and 32nd weeks of gestation. In an other clinical study
involving 142 women with high risk pregnancy [97], vaginal
suppositories containing 100 mg progesterone were
administered weekly from 25-27 weeks of gestation. The
design was also randomized, double blind and placebo-
controlled (treatment group: n=72). Preterm birth rate and
delivery rate before 34 gestational weeks were significantly
lower in the treatment group. Uterine activity, monitored by
an external tocodynamometer, has been also reduced in the
progesterone-treated group. These clinical studies strongly
suggest that the prophylactic use of progesterone may
decrease the probability of preterm birth in a high risk group
of pregnant women, and importantly, may improve birth
outcomes [96]. The mode of action of progesterone is
unknown, but it is tempting to speculate that the non-
genomic pharmacological action(s) of progesterone play a
role opposing the consequences of any functional progeste-
rone withdrawal that may occur pathologically before term.
ESTROGEN
In the sheep [98] or the rat [99], the fall of progesterone
levels is followed by a sharp rise in estrogen concentrations
in the maternal plasma at term. Thus, progesterone
withdrawal is accompanied by estrogen activation in these
animal species, and the hormonal changes are believed to
bring about labor and delivery by acting together. In the
human, progesterone and estradiol concentrations increase
gradually in the maternal plasma during the second and third
trimesters and show no change in this tendency at term
Regulation of Myometrial Smooth Muscle Functions
before labor onset [100]. The ratio of estrogen to progeste-
rone concentrations in the maternal saliva also remains
unchanged during the last twenty weeks of pregnancy,
reflecting the unchanged ratio of unconjugated (free)
hormone levels in the plasma [101]. Furthermore, estradiol
levels do not change within human myometrium in labor
[86]. Collectively, these data suggest that estrogen activa-
tion, like progesterone withdrawal, must be functional in
human pregnancy at term. The symptoms of the recently
described congenital aromatase deficiency syndrome [102]
are in agreement with this assumption. The estrogens of
human pregnancy (including estradiol and estriol) are
produced by placental aromatase, which converts fetal
adrenal androgens to estrogens. Aromatase deficiency,
caused by the recessive autosomal mutations of the CYP19
gene, results in low estrogen levels in pregnancy, virilization
of the female fetus and the mother, but no reported disorder
of the parturition process. Maternal estrogens, of course, are
present in these pregnancies, but their concentrations are
grossly abnormal. It is reasonable to conjecture that in these
patients, the maternal estrogen is sufficient to activate the
estrogen receptors in the uterus, and any change in estrogen
action at term is the result of regulation at the receptor or
post-receptor levels.
Estrogen Receptor-Dependent Regulation
The pregnant human myometrium is an estrogen target
tissue expressing estrogen receptors (ERs) [20]. The ERs
bind the hormone with high affinity and specificity, then
undergo a conformational change and gain ability to function
as a transcription factor. Estrogen responsive genes contain a
nucleotide sequence within the promoter (the ideal
palindrome is AGGTCANNNTGACCT), which binds the
active ER dimer resulting in transcriptional activation. These
steps are akin to those described for progesterone and the
PRs. Likewise, the outline of the ER-dependent gene
regulation is very simplified. There are two ER isoforms,
ERα and ERβ, produced by separate genes. In vitro gene
transfer experiments suggested that the two ERs have
distinct modes of action on the same promoters and have
different ligand activation properties [103]. Moreover, ERβ,
in general, is a transrepressor of ERα [103]. These
differences also exhibited cell line-dependent variations.
Further, the two receptor isoforms showed opposing actions
on a promoter containing an AP1 site [104]. Growth factors,
such as EGF, and cAMP activate ERα independently of the
steroid ligand [105], which represents an additional level of
regulation involving the phosphorylation of ERα by the
MAP-kinase system [106], and possibly by PKA. ERα
interacts with a large number of cofactors when it performs
its gene-regulatory function (reviewed by McDonnel and
Norris [107]). The cofactors can be either activators or
repressors and are involved in chromatin remodeling by
histone acetylation, and also in establishing a link between
the receptor and the basic transcription factors. Finally, ER is
subject to transrepression by other transcription factors, such
as PR-A (discussed above), and NF-κB p65 [108].
The bulk of data summarized above on the molecular
regulation of ER function has been obtained in gene
transfection experiments. In these experiments, estrogen-
responsive promoter-reporter constructs and various ER-
Current Pharmaceutical Design, 2004, Vol. 10, No. 20 2505
overexpressing vectors are co-transfected into cell lines in
order to perform functional analyses. The regulatory
interactions discovered in these models indicate several
mechanistic possibilities that are in line with the concept of
functional estrogen activation. For example, the change of
ER-isoform and cofactor levels, increased growth factor
signaling, altered chromatin structure and changing trans-
cription factor transrepression profile may all potentially
increase the estrogen sensitivity of a target cell. Further work
is needed to determine which of the possible regulatory
mechanisms are involved in the physiological and patho-
physiological regulation of the myometrium by estrogens.
One critical issue is to identify functionally important estro-
gen responsive genes in the pregnant human myometrium.
Information about some of these genes is summarized below.
Connexin 43
Estrogen treatment of cultured human myometrial cells
increases the level of Cx43 protein [28, 30, 33, 109], sugges-
ting that the Cx43 gene is estrogen responsive. The human
Cx43 gene promoter contains 6 half-ERE elements [31];
however, no evidence suggests so far that these sequences
are involved in the up-regulation of Cx43 gene activity in
uterine myocytes. Nuclear run-on, DNAse I footprinting, gel
mobility shift and transfection assays indicated that the AP1
and SP1 sites within the proximal Cx43 promoter were
important positive regulatory elements, but these sites and
the corresponding transcription factors did not mediate the
estrogen stimulation, even when the ER was overexpressed
in primary myometrial cells [32, 33]. Thus, the mechanism
of the estrogen stimulation of human Cx43 expression
remains unknown. An interesting possibility has been raised
by new work from Oltra et al. [110], who isolated a small
zinc finger protein from an estrogen induced rat myometrium
expression library. In cotransfection assays, this protein,
called Ini, enhanced the estrogen response of the Cx43 gene
in a cell specific fashion. The reporter construct used in the
experiments contained the proximal 145bp region of the
human Cx43 gene promoter, which does not possess half
palindromic EREs. Ini is a widely expressed and highly
conserved transcriptional cofactor also found in humans. The
potential involvement of Ini in regulating the estrogen
sensitivity of the Cx43 or other genes in the pregnant human
myometrium deserves further study.
The Oxytocin/OTR-System
Estrogen stimulates myometrial OTR gene expression in
ovariectomized rats [111]. Primary cultures of myometrial
cells from non-pregnant women also respond to estrogen
with increased OTR expression, as determined by a ligand-
binding assay [43]. The 1.8 kb upstream promoter region of
the human OTR gene contains 3 half-palindromic EREs
[42], but these elements do not mediate estrogen stimulation
in transfection assays even when the cells are cotransfected
with ER-expressing vectors [112]. Thus, the mechanism by
which estrogens regulate the OTR gene is unclear. Estrogen
may stimulate the oxytocin responsiveness of human
myometrial cells at a post-receptor step. This was suggested
by Phaneuf et al. [113], who measured the activation of
phospholipase C (PLC) by oxytocin, fluoroaluminate (a G-
protein activator) and PGF2α in primary (non-pregnant)
2506 Current Pharmaceutical Design, 2004, Vol. 10, No. 20
myometrial cell cultures. Estrogen pretreatment increased the
efficiency of all three PLC activators, and tamoxifen par-
tially reversed the estrogen effect suggesting an involvement
of the ERs. The immediate target of estrogen regulation in
the signal transduction system has not been identified.
The oxytocin gene is expressed in the human decidua and
the fetal membranes [114]. Oxytocin mRNA levels increase
in chorio-decidua explants incubated with 1 and 10 nM
estradiol, and the stimulation is partially abolished by co-
incubation with tamoxifen [114, 115]. Estrogens, therefore,
may potentially stimulate myometrial contractions indirectly
by increasing the production of oxytocin in the decidua,
which is the tissue lining the internal surface of the pregnant
uterus. The human oxytocin gene contains several imperfect
half-palindromic EREs, and one of these, located at approxi-
mately 160 bases upstream of the transcriptional start site,
confers estrogen responsiveness to a reporter-construct in
neuroblastoma cells overexpressing ER [116]. This regula-
tory element is highly conserved and appears to mediate
regulation by several transcription factors including orphan
receptors, like SF-1 and COUP-TF [40]. Interestingly, ER
was reported to bind this element with very low affinity [40],
which raises the possibility that the stimulation of the
oxytocin gene by estrogens is not direct, but mediated by
other estrogen-controlled transcription factors or by proteins
that anchor the ER to the regulatory DNA sequence. No such
factors or mechanisms have been demonstrated so far.
Similarly, no information is available about the time course
of decidual oxytocin expression during pregnancy or with
labor.
It is likely that estrogen regulates more genes in the
pregnant human myometrium than just Cx43 and OTR. For
example, CGRP-B receptor protein levels increase in
estrogen-treated myometrial explants from term pregnant
women [63], and TGFβ1 secretion and TGFβ receptor type-1
abundance increases and decreases, respectively, in (non-
pregnant) human myometrial cell cultures [74] treated with
estradiol. Given the high number of differentially expressed
genes in the human myometrium in late pregnancy [79],
other estrogen responsive genes may be identified in the
future.
ER in the Pregnant Human Myometrium
The level and the activity of ERs may influence the
estrogen responsiveness of the myometrium. This important
possibility has prompted several groups of investigators to
measure ER expression in the pregnant myometrium.
Hormone-exchange assays indicated the presence of ERs in
the nuclear and cytoplasmic fractions of term pregnant
myometria [20, 117, 118], and immmunohistochemistry
demonstrated the presence of ERα protein in myometrial cell
nuclei [21]. Others detected faint and diffuse ER-staining by
immunohistochemistry [67] and low but measurable levels
of ERα protein by EIA [22]. The majority of studies found
no difference in ER localization and abundance with normal
term or preterm labor; however, Winkler et al. [68] reported
that ERα protein levels transiently decreased in the
myometrium during the early phase of active labor at term.
Overall, available data do not support the possibility that
functional estrogen activation occurs by increased ER
Hertelendy and Zakar
expression at the time of labor. Nevertheless, there is still no
information about myometrial ER expression during
gestation, prior to labor onset.
Mesiano et al. [71] have recently examined myometrial
ER expression using the sensitive and highly specific real-
time quantitative RT-PCR technique. These investigators
have found a significant increase of ERα mRNA abundance
in myometrial biopsies with term labor. The levels of ERβ
mRNA, which encodes the dominant negative ERβ isoform,
were much lower and constant in the same cohort of
myometrial biopsies. Interestingly, OTR mRNA levels also
increased with labor, and ERα mRNA abundance showed
significant positive correlation with OTR mRNA abundance
in non-laboring tissues. These results, if confirmed by
protein measurements, may support the possibility of ERα-
mediated estrogen activation and OTR induction before or
during labor.
Functional Progesterone Withdrawal and Estrogen
Activation: Concepts in Need of Further Testing
The hypothesis of functional progesterone withdrawal
and estrogen activation was formulated as an extension to
humans of the reproductive endocrinology of animal species,
where circulating progesterone levels drop and estrogen
levels rise before labor onset. Its central postulates are that
the responsiveness of the human myometrium to progeste-
rone decreases and to estrogen increases in term pregnancies.
Despite several decades of investigations, however, no
conclusive evidence for, or against, the existence of such
mechanisms has been found. The slow progress is due to
major obstacles to research in this area of medical science.
Tissue availability is limited to small biopsy samples excised
from the myometrium during elective or emergency cesarean
section. Myometrial cells in primary culture show instable
phenotype, and immortalized pregnant myometrial cell lines
[119] are still not generally available and characterized.
There are only a few genes known to be under progesterone
and/or estrogen control in pregnant myometrial cells, but
remarkably, reporter construct assays and transfection
studies were not successful in determining the molecular
mechanisms of the steroid regulation of these genes. Without
this information, gestational age-dependent changes of
steroid responsiveness cannot be examined at the single gene
level. Cross-talk between the PR and ER pathways has also
been proposed, especially the inhibition of ER-signaling by
PRs [17], by low PR-A to PR-B ratio [71], or possibly by
PR-A-transrepression, as discussed. The involvement of
these mechanisms in the physiological actions of progeste-
rone or estrogen in pregnancy remains to be tested. If the
progesterone and/or estrogen receptor pathways play a
central role in the regulation of myometrial gene expression
during pregnancy, molecular studies of hormone action will
likely lead to the discovery of new drug targets and drugs.
Hopefully, a new generation of drugs targeting key steps in
the maturation of the myometrium to a contractile phenotype
will effectively prevent or treat various forms of
dysregulated myometrial function such as preterm labor,
dystocia, or post-term pregnancy.
Having reviewed the regulatory functions of female sex
steroids we shall now turn to what we prefer to term as
Regulation of Myometrial Smooth Muscle Functions
factors modulating myometrial smooth muscle functions
during pregnancy and labor.
Prostaglandins
Prostaglandins have long been considered to play impor-
tant roles in uterine physiology and pathology, based on their
ability to induce abortion as well as parturition at term, to
treat postpartum hemorrhage and to affect myometrial
contractility. In addition, the progressive increase in amnio-
tic fluid PG levels prior to labor and the ability of PG
synthesis inhibitors to prolong gestation have further
underscored this belief. Due to the ubiquity of arachidonic
acid (AA) as a component of membrane phospholipids, PGs
are readily synthesized, depending on the presence and
activity of the cyclooxygenase enzymes (COX-1&2)
catalyzing the rate-limiting step in the metabolism of AA,
and the specific synthases that convert PGH2, the common
precursor, to the bioactive end products. Of the two cyclo-
oxygenase isoenzymes, COX-1 is expressed constitutively
and apparently does not change during gestation and labor
[120]. The expression of COX-2 mRNA in association with
labor is less clear cut. Some investigators found a decrease,
while others reported no change or an increase in COX-2
message and protein. It would appear that such inconsistent
findings maybe due in part to the site of myometrial tissue
excision. Specimens collected during labor at cesarian
section from the lower segment of the uterus yielded up to
15-fold higher levels of COX-2 mRNA than similar tissue
samples collected from non-laboring patients [121]. It seems,
therefore, that the major PGs, prostacyclin (PGI2) and PGE 2,
produced by the myometrium are directed towards cervical
ripening rather than enhancing myometrial contractions. This
may also explain in part the relaxant action of PGI2 on
myometrial smooth muscle. Once again, the choice of
experimental approach and the qualitative or quantitative
nature of employed methodology may contribute to the
variability of reported observations. It is also important to
note that measuring the message and protein levels may only
reflect the transcriptional and translational status of the cell.
Prostaglandin levels will depend on the activity of the
biosynthetic enzymes, as well as the presence and activity of
PGDH, the principal PG-metabolizing enzyme. Thus,
dynamic changes in bioactive PG levels can occur if the
balance is upset from either direction, e.g. in the case of
intrauterine infection attended by a robust induction of COX-
2 (see under cytokines). Moreover, it should be emphasized
that the myometrium is exposed to PGs produced by
neighboring intrauterine tissues such as the decidua, the
chorion laeve and the amnion. PG biosynthesis and
degradation in these tissues have been studied extensively
during the past decades, predominantly because of the
possible effects of the generated PGs on myometrial
functions. The results have been summarized often and the
interested reader is referred to some of the more recent
reviews for additional information [122-126].
How do PGs act to modulate myometrial functions? The
first step involves the recognition by specific receptors.
Crude particulate preparations from myometrial homo-
genates contain high affinity PGE and PGF2α binding sites
indicating the presence of membrane receptors [127-129].
The number and affinity of these binding sites were not
Current Pharmaceutical Design, 2004, Vol. 10, No. 20 2507
different in the upper and lower segments and remained
unchanged during pregnancy and labor. Furthermore,
receptor expression was generally lower in the pregnant than
in the non-pregnant myometrium. The various types of PG-
receptors have been identified and characterized originally in
functional studies, using smooth muscle preparations for
bioassays and a series of PGs, PG analogs and PG
antagonists as effectors [130, 131]. Such a pharmacological
approach has been applied subsequently to the pregnant
myometrium by Senior et al. [132]. The results suggest that
in the muscle bath experiments, the inhibitory PGE2 response
was mediated by the EP 2 receptor, while the excitatory PGE2
response was triggered by the EP3 receptor, since the other
excitatory PGE2 receptor, EP1, was not detectable using a
specific EP 1 antagonist. Stimulation by PGF2α was mediated
by the excitatory FP receptor. PGI2 relaxed the myometrium
acting through the IP receptor, while the mechanism of PGI 2-
evoked contractions was unclear. PGD2 also exhibited a
biphasic effect on myometrial contractility, like PGE2 and
PGI2. The relaxing action of PGD2 was effected by the DP
receptor, and the excitation was possibly mediated by the
cross-stimulation of FP receptors by the DP-agonists, as
suggested by Fernandes and Crankshaw [133]. Thromboxane
A2 analog was excitatory, acting through the TP receptor.
Thus, the pharmacological characterization revealed that
multiple PG-receptors participate in the contractile and
relaxing effects of prostaglandins on the myometrium.
The pharmacological classification of PG receptors has
been confirmed by the molecular cloning of the PG receptor
cDNAs and genes and also by studies of the coupled
signalling pathways. The reader is referred to excellent
comprehensive and specialized review articles for this body
of information [134, 135]. In the human myometrium,
Matsumoto et al. [136] detected EP3 and FP mRNAs by
northern blotting of polyA+ RNA. The abundance of both
mRNAs was lower in pregnancy than in the non-pregnant
state. Using semi-quantitative RT-PCR, Brodt-Eppley and
Myatt measured FP and EP2 mRNA abundance in the
myometrium at different times of gestation [52]. Overall,
they detected a gestational age-dependent decrease in the
(relative) abundance of both mRNAs. In addition, FP mRNA
levels increased with term labor, but preterm labor had no
effect on the myometrial levels of EP2 or FP mRNAs. The
processing of the primary EP3 gene transcript is especially
complex, giving rise to alternatively spliced C-terminal
receptor variants. Six variants have been identified so far,
and the EP3 subtypes are coupled to various signaling
systems that can increase or decrease intracellular cAMP
levels, stimulate phosphatidyl-inositol turnover and activate
the MAP-kinase pathway [137, 138]. These variants,
therefore, are capable of transmitting both smooth muscle
contracting and relaxing signals. The expression of EP3-II,
EP3-III, EP 3-V, and EP3-VI mRNAs has already been confirmed
in the human myometrium [137, 139]. In pregnancy,
myometrial EP3-II mRNA levels decreased and EP3-VI mRNA
levels increased as compared to the non-pregnant state [139].
The significance of the EP3 variant distribution and changes
in the modulation of myometrial contractility by PGs still
remains to be established.
The molecular characterization of the myometrial PG
receptors and the coupled signaling pathways is an area of
2508 Current Pharmaceutical Design, 2004, Vol. 10, No. 20
intensive research. The work is expected to identify
prospective drug targets and new drugs for the control of
uterine activity in pathological conditions such as preterm
labor. Examples are the FP receptor and the new FP receptor
antagonist THG113, which effectively delayed delivery in an
animal model of preterm birth [140]. The development of
various selective PG-receptor type- and subtype-specific
agonists and antagonists currently in progress is likely to
offer new hope for the better treatment of preterm labor,
early cervical maturation or recurrent preterm birth.
The postreceptor actions of PGs are still uncertain. It is
generally believed that the heptahelical G-protein coupled
receptor of the uterotonic PGF2α transmits the signal via the
phosphoinositide cycle, generating the two intracellular
signaling molecules, IP3 and DAG. IP3, in turn, mobilizes
calcium from intracellular stores, while DAG is an
endogenous activator of PKC. The latter is involved inter
alia in AA release by way of activating PLA2, thus
contributing to the provision of ample precursor pool.
However, it is not known if such a mechanism is of
physiological significance. Comparing the effects of PGF2α
with oxytocin in primary cultures of human myometrial
cells, Molnár and Hertelendy [141] observed that both
agonists were equipotent in raising [Ca2+]i in the presence of
1mM extracellular calcium. However, in calcium free
medium only oxytocin could increase [Ca2+]i, albeit to a
considerably lesser extent, confirming a previous report by
Anwer and Sanborn using rat myometrial cells [142].
Further, at concentrations which raised [Ca2+]i, PGF2α failed
to activate the phosphoinositide cycle, responding only at
micromolar concentrations unlikely to be encountered in vivo.
This observation, combined with the finding that the calcium
channel blocker verapamil inhibited PGF2α-promoted rise in
[Ca2+]i, led to the conclusion that the primary effect of PGF2α
in myometrial cells is to promote calcium entry. Prostacyc-
lin, on the other hand, activates the AC/cAMP system which,
as pointed out elsewhere, is involved in myometrial
relaxation and is down regulated at the onset of labor [143].
It has also been suggested that PGs may function as
ionophores [144], or by altering membrane fluidity [145],
leading ultimately to increased calcium entry. More recent
studies indicate that PGs may activate the peroxisome-
proliferation-activated receptor signaling pathway affecting
gene transcription [146]. Gene transcription may also be
modulated by PGs in an autocrine fashion via intracellular
receptors identified in the perinuclear envelope [147].
Collectively, PGs, in conjunction with their numerous
receptors that have been characterized, activate multiple
signaling pathways to bring about their multifaceted actions
in uterine smooth muscle cells. Clearly, more work is needed
to elucidate the intimate mechanisms of action of the
principal PGs and other bioactive eicosanoids in association
with the pregnant and parturient uterus.
Cytokines
The inflammatory cytokines, chiefly IL-1, TNF and IL-6,
have long been linked to intrauterine infection-driven
preterm labor [148]. There is accumulating evidence,
however, that pro-inflammatory cytokines are also involved
in normal term labor [149]. In fact, it has been proposed that
Hertelendy and Zakar
human parturition represents an inflammatory process. This
assumption is supported by the observation that leukocytes
infiltrate gestational tissues and may also be the major
source of cytokines in the uterus [150]. Further, increased
expression of the cytokines IL-1, IL-6, IL-8 and TNF has
been documented in the myometrium and cervix in
association with parturition [69,151]. Here we shall focus on
IL-1, the prototypical proinflammatory cytokine, in terms of
its action on the myometrium..
The labor-promoting action of IL-1 has been attributed at
least in part to the induction of cPLA2 and COX-2 and the
ensuing rise in PG production by uterine tissues, including
the myometrium [121,152-155]. IL-1 signaling in various
cell types involves multiple pathways leading to the
expression of more than a hundred different genes. In intact
human myometrial cells, IL-1β binds with high affinity
(Kd=2 x 10-10M) [156]. Although two IL-1 receptors, each
having a single membrane-spanning domain, have been
identified, Scatchard analysis indicated the presence of a
single class of binding sites in human myometrial cells. Post
receptor steps in the signaling pathways leading to COX-2
expression involve the activation of the MAP kinase family
of enzymes, of which p38 MAP kinase is functionally linked
to COX-2 mRNA and protein expression, because SB
203580, a selective inhibitor of p38 action blocked the effect
of IL-1 in primary cultures of human myocytes [157,158].
However, the role of jun-terminal kinase (JNK), which
responds robustly to IL-1 stimulation, is uncertain and a
crosstalk between MAP kinases is not uncommon in various
other systems. Another signaling pathway that leads to
activation and nuclear translocation of NF-κB has also been
described in an immortalized human myometrial cell line
[159]. The nuclear transcription factor NF-κB in its inactive
form is localized in the cytosol bound to the inhibitory
protein IκB. Upon stimulation with IL-1, IκB is rapidly
degraded allowing the translocation of NF-κB to the nucleus
where it can interact with one or both of the NF-κB binding
motifs present in the promoter of human COX-2 gene.
Stimulation of myometrial cells caused a rapid degradation
of IκB (as assessed by immunoblotting) and nuclear binding
of NF-κB (as demonstrated by electrophoretic mobility shift
assay) [159]. Inhibition of IκB degradation prevented IL-1
induced COX-2 mRNA and protein expression, as well as
PGI2 synthesis. Combined stimulation of myocyte cultures
with TNF greatly potentiated PGE2 and PGI 2 production, but
another cytokine, interferon-γ, significantly suppressed IL-
induced COX-2 expression and PG production [160].
Interestingly, IL-1-induced activation of MAPK, COX-2
expression and PG synthesis was also partially inhibited by
pertussis toxin, a widely used tool to probe for the
involvement of certain G-proteins in signal transduction
[161]. Thus, IL-1 receptor that does not belong to the G-
protein coupled receptors with seven membrane spanning
domains, interacts in some fashion with the latter. In a
fascinating recent study, Tribe et al. [162] investigated the
effects of IL-1β on the expression of sarcoplasmic reticulum
calcium ATPase (SERCA) 2b and Ca2+ fluxes in primary
cultures of myometrial cells isolated from tissue specimens
obtained at cesarian section from women not in labor. They
found a significant increase in SERCA 2b protein expression
and calcium mobilization after 24 h treatment with 10ng/ml
Regulation of Myometrial Smooth Muscle Functions
IL-1β. The rise in [Ca2+]i was apparently due to increased
release, as well as entry of Ca2+; the latter independent of
altered activity of voltage-gated calcium channels.
In conclusion, proinflammatory cytokines, especially IL-
1β, are powerful inducers of COX-2 and PG production in
human myometrium. Interleukin’s multifunctional character
is reflected by the activation of multiple signaling pathways,
which may include mechanisms that influence intracellular
calcium levels and, hence, myometrial contractility.
Oxytocin
Even though oxytocin has been widely used in clinical
practice for the pharmacologic induction or augmentation of
labor [163], its physiologic role in the initiation of parturition
has been disputed, owing to a lack of its demonstrable
increase in plasma levels prior to the onset of labor. More
recently, however, an increase in oxytocin mRNA and
peptide has been demonstrated in human and rat uterine
tissues [114, 164]. This finding, together with the striking
increase in oxytocin receptors (OTR) at term and,
particularly, during labor, lends support to a paracrine role
for oxytocin in the regulation of myometrial function
associated with the birth process. Since oxytocin sensitivity
of the myometrium parallels the progressive rise in OTRs
during gestation, it suggests that it is the OTR that is of
primary importance in promoting myometrial contractility
[165]. This notion is supported by the efficacy of various
OTR antagonists to suppress uterine contractions and their
clinical applications as tocolytic agents (see below). It
should be noted, however, that OTR antagonists can only
delay, but not block parturition, indicative of the operation of
additional pathways responsible for the initiation and
maintenance of parturition.
OTR belongs to the superfamily of the heptahelical, G-
protein-coupled receptors. Ligand binding activates multiple
signaling pathways. Of these, the activation of phospholipase
Cβ (PLCβ), which breaks down PIP2 to IP3 and DAG, has
been amply demonstrated in human [166-168] and animal
[169, 170] myometrial tissues and cells. The ensuing rise in
IP3 releases Ca 2+ from the sarcoplasmic reticulum, causing a
sudden rise in intracellular Ca2+ ([Ca2+]i), whereas DAG
activates protein kinase C (PKC).
Another important function of oxytocin is the promotion
of PG production by stimulation of arachidonic acid (AA)
release by the sequential hydrolysis of DAG and by G-
protein-coupled activation of PLA2, that may also be
activated by oxytocin-induced increase in [Ca2+]i. In
addition, oxytocin-induced activation of the MAP kinase
pathway [171], coupled to the activation of cytosolic PLA2
(cPLA2), should also enhance the release of AA, the
common precursor of prostaglandins and other eicosanoids.
Prolonged exposure of human myometrial cells to oxytocin
has been shown to promote COX-2 gene transcription and
sustained PG production [172]. Such a signaling pathway for
oxytocin has also been demonstrated in bovine uterine cells
[173, 174]. Finally, oxytocin can also enhance myometrial
contraction by a calcium-independent mechanism involving
the RhoA/Rho-kinase cascade [175]. Studies using Y-27632,
a specific inhibitor of Rho-kinase, have demonstrated an
inhibition of oxytocin-induced myometrial contraction
Current Pharmaceutical Design, 2004, Vol. 10, No. 20 2509
without affecting a rise in [Ca2+]i. However, oxytocin-
induced phosphorylation of MLCK was attenuated, as was
oxytocin-induced phosphorylation of MLC phospatase,
which inhibits the activity of the enzyme. There is also
evidence that the expression of Rho-kinase is increased in
the myometrium during pregnancy [175, 176].
In summary, the oxytocin/OTR system plays an impor-
tant, though perhaps not a decisive role in the regulation of
myometrial function in the context of labor and delivery.
Endothelin
The vasoconstrictor, endothelin (ET-1), is a potent
stimulant of uterine smooth muscle contraction. The action
of ET-1 is mediated by the A-type receptors (ETA) coupled
to the PLC-phosphoinositide-Ca2+-signaling pathway, with
the ensuing phosphorylation of MLC and force generation
[177, 178]. ETA-receptors in the myometrium increase as
pregnancy progresses towards labor [178, 179], supporting a
physiologic role for ET-1 in the regulation of parturition.
ET-1 has also been found to stimulate the expression of
COX-2 and PG production in human myometrial cells [180].
Recent studies provide experimental evidence for the
operation of an additional signaling pathway activated by
ET-1 [181]. Stimulation of human myometrial cells activated
an atypical protein kinase C isoform (PKCξ) that is not
regulated by DAG or calcium, but by phospholipidic
mediators, products of phosphatidylinositol 3-kinase (PI3-
kinase) [182]. Inhibition of PI3-kinase by LY-294002 or by
an antisense against PKCξ significantly suppressed ET-1-
induced myometrial contractions [181]. Activated PKCζ
associates with the actin component of the cytoskeleton of
human myometrial cells isolated near term and may regulate
the phosphorylation or binding properties of actin. Interest-
ingly, ET-1-activated PKC may also interact with the
signaling pathway of inflammatory cytokines (IL-1β, TNFα)
which regulate the multifunctional nuclear transcription
factor NF- κB [183, 184]. Finally, ET-1 stimulates myomet-
rial cell proliferation [185,186], perhaps contributing as a
paracrine regulator to uterine growth during pregnancy.
Platelet Activating Factor (PAF)
Activation of PLA2, e.g. by inflammatory cytokines,
leads not only to the release of AA and the subsequent
generation of prostanoids modulating myometrial functions,
but can also give rise to PAF, by way of the acetyl-CoA
acetyltransferase catalyzed acetylation of phosphatidyl-
choline. (In addition to this “remodeling pathway” in the
central nervous system and some other tissue, a “de novo”
pathway can also generate PAF via the synthesis of 1-O-
alkyl-2-acetylglycerol, which is then converted to PAF by a
specific dithiothreitol-insensitive CDP-choline-1-alkyl-2-
acetyl-sn-glycerocholine-phosphotrans-ferase [187]).
PAF produced in endothelial cells may act in a paracrine
fashion on myometrial cells. Stimulation of myometrial
contractions by PAF has been observed both in vivo and in
vitro [188]. Its putative role, such as premature rupture of the
membranes and preterm labor, particularly in complicated
pregnancies, is supported by the detection of PAF in amnio-
tic fluid [189]. PAF receptor antagonists have been shown to
delay parturition in rats [190]. In human myometrial cells, as
2510 Current Pharmaceutical Design, 2004, Vol. 10, No. 20
in endothelial cells, PAF activates the phosphoinositide cycle
after binding to specific G-protein-coupled receptors,
causing an increase in [Ca2]i due, in part, to the release of
bound intracellular calcium [191]. This response to PAF was
inhibited by L-659,989, a PAF-receptor antagonist. Stimula-
tion of eicosanoid production may also contribute to the
uterotonic action of PAF [192]. Thus, there is a close analogy
between the action of ET-1 and PAF on myometrium. This is
further underscored by the possible involvement of PAF in
PKC-mediated cytoskeletal rearrangement as demonstrated
in endothelial cells [193].
Arachidonic Acid (AA)
AA is distributed throughout the body as a polyunsatu-
rated fatty acid component of membrane phospholipids. Its
physiological role in the myometrium and various other
tissues has been attributed to its acting as the principal
precursor of prostaglandins and other biologically active
eicosanoids. There is good evidence, however, that AA itself
may serve as a cell signaling molecule [194]. AA, when
added to various cell types, including human myometrial
cells [195], has been shown to stimulate the release of
intracellular calcium, even in the presence of inhibitors that
block the conversion to eicosanoids.
Stimulation of [3H]-AA-labeled human myometrial cells
with PGF2a caused a rapid release of AA, chiefly from
phosphatidylethanolamine, indicative of PLA2 activation,
whereas oxytocin liberated AA mainly from phosphoino-
sitides, suggestive of G-protein-coupled PLC activation. AA
may also participate in intracellular signaling by activating a
specific PKC that is distinct from the “classic” DAG-
sensitive enzyme [196]. During labor, AA levels in amniotic
fluid increase several-fold and intramniotic administration of
AA was shown to induce abortion during the second
trimester [197].
Thrombin
Intrauterine bleeding, which is estimated to occur in 15-
20% of pregnancies, has been recognized by clinicians as a
risk factor for placental abruption, spontaneous abortion and
preterm birth. These pathological conditions are associated
with increased uterine contractions. Indeed, addition of
whole blood to rat myometrial strips promoted contractility
that was attributed to thrombin, a known stimulant of smooth
muscle contractions [198, 199]. Subsequent studies by the
same group have demonstrated the presence of prothrombin
in rat myometrium and its expression in endometrium and
placenta, in the absence of bleeding [200]. Importantly,
thrombin-induced tension in rat myometrium was much
enhanced in pregnant myometrium compared to tissue from
non-pregnant animals. This could be attributed to a
corresponding rise in [Ca2+]i, and to a 10-fold increase in
thrombin receptor mRNA [201]. These observations, and the
well-established contractile effect of thrombin in a variety of
other smooth muscle preparations, support the notion of a
functional role for thrombin in the regulation of myometrial
contractions.
The physiologic function of thrombin has long been
attributed to its role as a key intermediate in the coagulation
cascade. More recently, it has become evident that thrombin
Hertelendy and Zakar
is a multifunctional serine protease, affecting a wide variety
of cellular functions [202]. It is generated by catalytic
cleavage from its precursor, prothrombin, by the activated
factor Xa. Most of its actions are mediated by protease
activated receptors (PARs), belonging to the superfamily of
heptahelical G-protein-coupled receptors. Activation of PAR
involves the enzymatic cleavage of the amino-terminal
exodomain of the G-protein-coupled PAR, revealing a new
amino-terminal that acts as a tethered ligand, binding intra-
molecularly to the body of the receptor initiating transmem-
brane signaling. Synthetic peptides coinciding with the
amino acid sequence of the new amino-terminal (SFLLRN),
referred to as TRAP (thrombin receptor activating peptide),
can activate PAR independently of thrombin without the
splitting off of the N-terminal segment of the receptor.
Unlike other G-protein-coupled receptors, PAR activation is
irreversible and the receptor is rapidly internalized and
degraded. Replenishment is from preformed intracellular
stores. Like oxytocin and other uterotonins, thrombin-
activated PAR is coupled to the phosphoinositide signaling
cascade, generating IP 3 and DAG as intracellular messengers
with the subsequent rise in [Ca2+]i and activation of PKC. In
addition, however, thrombin activates a number of other
signaling pathways involving, e.g., the MAP kinase family
of enzymes, PI3 kinase, JAK-2/STAT-1, etc. (reviewed in
[203]).
A novel aspect of thrombin action in human myometrial
cells has been described recently [204]. Stimulation of
primary cultures of human myocytes with thrombin upregu-
lated COX-2 expression and PGE2 production in a time- and
dose-dependent manner. These responses were completely
abolished by actinomycin D, indicative of transcriptional
regulation of COX-2 expression, but were not affected by
antagonists to PLC, PKC and ERK. However, SB203580, a
selective inhibitor of p38 MAP kinase, abrogated thrombin
action. Thus, the action of thrombin on human myometrial
cells is analogous to that of oxytocin in terms of induction of
COX-2, activation of the phosphoinositide cycle and
promotion of contractility. Intracellular signaling, however,
may follow diverging pathways.
Thrombin in the plasma is rapidly bound to antithrombin
(AT) in a 1:1 ratio and the formed TAT complex can be
accurately measured, giving quantitative values for thrombin
generation. Using this analytical approach, Uszynski [205]
reported a 10-fold increase in TAT levels at the end of
pregnancy and a further transient 15-fold increase after
separation and expulsion of the placenta. Increased TAT
levels have also been reported in preterm labor and preterm
premature rupture of membranes (PPROM), as well as in
patients with small-for-gestational-age fetuses [206, 207]. It
has been suggested that the plasma level of TAT may serve
to predict PPROM [208].
Stretch
Mechanoreception is the most broadly evolved signal
recognition in living organisms, from bacteria (turgor
pressure induces the expression of osmoregulatory genes), to
plants (geotropism) and the animal kingdom [209, 210]. It
has been known for a long time that mechanical stretch
imposed on skeletal, cardiac or smooth muscle, leads to
Regulation of Myometrial Smooth Muscle Functions
hypertrophy [211]. In 1965, Csapo et al. placed closed
liquid-filled rubber balloons into single horns of the
bicornuate uteri of ovariectomized rabbits and observed that
the stretched horns increased in weight compared to the
contralateral unstretched uterine horns. Based on RNA/DNA
ratio and myometrial contractility they concluded that the
weight increase was largely due to de novo protein synthesis
[212, 213]. Similar observations were reported using
continuous distension of non-pregnant rat uteri [214].
Interestingly, mechanical stretch of uterine muscle not only
induces growth, but also prevents postpartum involution
undergoing at the same time by the unstretched contralateral
horn [215]. Using the rat model, the Toronto group extended
these observations in a series of elegant experiments
(reviewed in [216]), and demonstrated the expression of a
cassette of proteins involved in myometrial activation at
term, including OTR, Cx43, and PGF2α receptor (FP), that
could be reproduced by mechanical stretch of the non-gravid
horn, provided that progesterone withdrawal had occurred.
Importantly, progesterone administration prevented both the
expression of these proteins and the onset of parturition.
They concluded that the regulation of these key proteins is
under both hormonal and mechanical factors. In subsequent
experiments, Lye and his colleagues have demonstrated that
the transcription factor AP-1 is upregulated in rat myomet-
rium shortly before the onset of labor [217]. They also
observed the activation of the MAP kinase family of
enzymes (ERK, JNK, p38) in stretched rat myometrial
smooth muscle cells, leading to the expression of the early
gene c-fos [218]. Using selective inhibitors they demons-
trated that the activity of all three MAP kinases are required
for the upregulation of c-fos mRNA and that the
mechanotransduction is linked upstream to tyrosine kinase
activation. Less is known about the molecular aspects of
mechanotransduction in human myometrium. Nonetheless,
several lines of evidence support the concept of stretch-
induced activation of the uterus. The incidence of preterm
labor is markedly higher in multifetal pregnancies, polyhydr-
amnios or somatomegaly in singleton pregnancies [219,
220]. Yet in a recent study, Lyall et al. [221] found no
difference between the expression of Gαs, gap junction
proteins or PGE2 receptors in mechanically stretched human
myometrial cells isolated from singleton and multiple
pregnant uteri. Other investigators reported increased Cx43
and c-jun and c-fos expression in term-pregnant human
myometrium [33]. Upregulation of COX-2 and prostaglandin
production appear to be the most consistent responses to
myometrial stretch in both animal and human studies [222,
223].
In summary, mechanogenetic regulation of protein
synthesis in the myometrium during gestation and at term
may play important functional roles in both pregnancy
maintenance (by promoting uterine growth) and parturition
(by inducing the synthesis of key proteins).
Corticotropin Releasing Hormone (CRH)
CRH, a hypothalamic peptide regulating pituitary ACTH
secretion, is also expressed locally in a variety of tissues,
including the human placenta and myometrium (reviewed in
[224, 225]. Its production and release into the circulation
Current Pharmaceutical Design, 2004, Vol. 10, No. 20 2511
increases exponentially towards the end of pregnancy, but its
physiological role in pregnancy maintenance and parturition
remains an enigma. CRH binds to G-protein-coupled recep-
tors, of which two subtypes, R1 and R1, have been identified
and cloned. In addition, a number of isoforms of both
receptors, resulting from alternative gene splicing, are known.
Importantly, R1 receptors are significantly upregulated in
human myometrium at the time of labor, lending support to
the notion that the CRH/R1 system is involved in myo-
metrial activity. Because CRH-activated receptors are
coupled to Gαs and the generation of the myometrial relaxant,
cAMP, a role in the maintenance of uterine quiescence,
particularly during the rapid expansion of uterine volume,
has been suggested. CRH may also promote uterine
relaxation by activating the NO/cGMP signaling pathway
[226]. The inhibition of basal IL-1β- and oxytocin-
stimulated PGE2 production, as demonstrated in human
myometrial cell cultures [227], could potentiate the relaxant
effect. The paradoxical rise in CRH/R1 levels at labor may
promote the birth process by relaxing the lower uterine
segment, rather than potentiating uterotonin-driven contrac-
tions in the fundus [228]. The myometrial relaxant effect
may also be obtunded by the downregulation of Gαs at the
onset of labor [229]. In addition, the effect of CRH may be
reduced due to phosphorylation of CRH receptor by
oxytocin-activated PKC [230]. All in all, further evidence is
required in support of a physiological role for CRH in the
regulation of myometrial functions.
Nitric Oxide (NO)
NO (originally thought to act only as a potent vasodilator
released from the endothelium) is a multifunctional signaling
molecule widely distributed in the body, affecting a variety
of cellular functions, including myometrial smooth muscle
contractility (for review, see [231]). NO is generated from
the amino acid, L-arginine, during its conversion to L-
citrulline by NO synthase (NOS). There are three NOS
isozymes, the Ca-dependent constitutively expressed eNOS
and nNOS and the cytokine-inducible iNOS that does not
require a rise in Ca2+ for activity. In most systems, NO
activates the guanylate cyclase/cGMP signaling pathway,
leading to relaxation. Several lines of evidence, using
various NO donors, indicate that in human non-pregnant,
laboring and non-laboring myometrium, NO-induced relaxa-
tion is independent from cGMP [232-234]. Interestingly,
human myometrial strips are much less sensitive to the
relaxing effect of cGMP than precontracted bovine trachea
smooth muscle [235]. Further, the inconsistent reports failed
to provide concrete evidence to support the role of NO in
maintaining uterine quiescence. In one study, Norman et al.
[236] found an increase in the constitutive NOS expression
in the human myometrium during pregnancy, while a
subsequent report by the same group failed to observe any
change in NOS activity in the uterus and placenta during
parturition [237], contrary to the findings of Bansal et al.
[238], who reported a decline in myometrial NOS expression
associated with labor and delivery. Dennes et al. [239], on
the other hand, reported that myometrial NOS mRNA
expression did not change during gestation or with the onset
of labor, nor was iNOS induced by cytokines in cultured
cells [240].
2512 Current Pharmaceutical Design, 2004, Vol. 10, No. 20
In a more recent study, Batra et al. [241], using identical
methodologies, compared the cellular distribution, activity
(formation of 14C-citrulline from 14C-arginine) and
expression (Western blot) of NOS in human myometria and
vagina with those of the rat. They were able to detect only
very low NOS activity in human myometrium compared to
that measured in the corresponding tissue of the rat.
Surprisingly, vaginal NOS activity was 4-5-fold higher than
that found in myometrium of either species. In view of these
observations, the authors suggested that, in human preg-
nancy, the paracrine action of NO derived from the fetal
membranes and placenta may contribute to the maintenance
of uterine quiescence. It may be pertinent to note here that,
compared to mouse macrophages, the iNOS gene of human
macrophages is hyporesponsive to induction by lipopoly-
saccharide (LPS) + interferon-γ + IL-1β cocktail [242].
However, the same regimen effectively induces iNOS gene
in human hepatocytes [243]. Attempts to induce the expres-
sion of iNOS in human myometrial cells with cytokines and
LPS have failed (F. Hertelendy, unpublished). Hence, it
appears that the regulation and activity of NOS enzymes in
both species are cell specific.
Tocolysis
Early diagnosis of preterm labor, a complex syndrome of
multiple etiologies, is critical for the implementation of
appropriate clinical management in an attempt to delay or, if
possible, prevent premature birth, the leading cause of
neonatal morbidity and mortality in developed countries.
There are a variety of drugs available for pharmacologic
intervention to suppress myometrial contractions and the
literature is replete with reports and reviews of clinical trials
evaluating the efficacy, as well as shortcomings, of such
therapies. In general, first line (acute) tocolytic therapy may
prolong pregnancy for 48h or longer, allowing the
administration of corticosteroids to enhance lung maturation
and, if available, for transfer to a tertiary medical facility for
high risk pregnancy. Prolonged tocolytic therapy has little or
no beneficial effect and may be harmful for both mother and
fetus.
Beta Mimetics
Beta Mimetics (Isosuprine, salbutamol, terbutaline, feno-
terol, hexoprenaline, nylidrin, ritodrin). These drugs activate
β1 and β2 adrenergic receptors, raising cAMP levels resulting
in reduced intracellular Ca2+ concentration and, ultimately,
myometrial contractions. Although beta mimetics have been
used extensively, there is substantial evidence linking such
therapy with adverse effects for both mother and fetus
without clinically significant improvement in perinatal or
neonatal outcomes [244,245].
Magnesium Sulfate
Magnesium sulfate acts at high concentrations by
displacing calcium from the endoplasmic reticulum. It is a
popular tocolytic and anticonvulsant agent in the USA, even
though there is no adequate supporting evidence for its use
for the prevention of preterm labor following acute tocolysis
[246]. In one trial (n=156) there was no difference between
magnesium sulfate therapy and placebo in terms of preterm
delivery [247]. Comparing i.v. magnesium with the calcium
Hertelendy and Zakar
channel blocker, nicardipine, for short term tocolysis,
Larman et al. [248] found no differences with regard to
prolongation of pregnancy.
Calcium Channel Blockers
Calcium channel blockers (nicardipine, nifedipine) act by
preventing calcium entry into myocytes, as well as suppres-
sing intracellular calcium release. In a comprehensive meta-
analysis, Tsatsaris et al. [249] compared nifedipine with β-
mimetic therapy. The results indicated that the calcium
antagonist was more effective in prolonging pregnancy
beyond 48h than β-adrenergic agonists, with fewer side
effects, but without affecting neonatal mortality rate.
Non-Steroidal anti-Inflammatory Drugs (NSAID)
Indomethacin, an inhibitor of both COX-1 and 2 activity,
has been the most extensively evaluated NSAID for tocolysis.
Although indomethacin, or a related drug, sulindac, have
been shown in placebo controlled trials to be effective in
prolonging pregnancy for 48h or longer, there are undesir-
able side effects that include, in addition to the well
documented constriction of ductus arteriosus, intraventricu-
lar hemorrhage, olygohydramnios, pulmonary dysplasia and
necrotizing enterocolitis [250, 251]. In a randomized
controlled prospective trial, indomethacin and nylhidrin were
found to be equipotent in delaying delivery [252]. Since
labor has been associated with upregulation of COX-2,
selective inhibitors targeted at this enzyme may eventually
prove to be more effective tocolytics with fewer side effects.
Indeed, a small recent trial (n=24) found celecoxib to be as
effective as indomethacin in prolonging pregnancy, but
without constricting the ductus arteriosus [253]. Evidently,
larger, well designed multi center trials with clearly defined
end points are required to evaluate the possible benefits of
selective COX-2 inhibitors vs. traditional tocolytics.
Oxytocin Receptor Antagonists
It was pointed out elsewhere in this review that OTR
density in the myometrium increases strikingly as pregnancy
progresses to term. Hence, the idea that an OTR blocker
could prolong pregnancy and delay delivery in uncomplicated
preterm labor appeared a promising avenue to pursue. Atosi-
ban, a drug that binds to OTRs (though even more avidly to
arginine vasopressin receptors) offered such a promise.
However, after large scale multi center, multinational trials
comparing it to placebo or the beta-agonist terbutalin, it was
concluded that atosiban was not more efficacious in terms of
clinical significance than placebo or terbutalin, although its
safety profile was superior to that of the beta agonist [254-
257].
Nitric Oxide
The rationale to use NO donors stems from animal
experiments, which showed that such compounds relaxed,
and NOS inhibitors promoted, uterine activity. In human,
however, the putative role of NO in myometrial relaxation is
controversial (see above under Nitric Oxide). Duckitt and
Thornton [258] have recently reviewed the results of several
trials, comparing the effects of transdermally or intrave-
nously administered nitroglycerine, with placebo or no
treatment vs. beta receptor agonists (ritodrine, albuterol) and
magnesium sulfate. They found no differences between NO
Regulation of Myometrial Smooth Muscle Functions Current Pharmaceutical Design, 2004, Vol. 10, No. 20 2513

donors and placebo or no treament, concluding: “ The nitric CBP = CREB (cAMP-response element-binding
oxide donors appear to have a more favorable side effect protein)-binding protein
profile than some other tocolytic agents but in the absence of
cGMP = Guanosine-3’-5’-cyclic monophosphate
beneficial effects on perinatal morbidity and mortality, their
routine administration cannot be advocated”. Larger, better- COUP-TFs = Chicken ovalbumin upstream promoter-
controlled trials may uncover additional information that associated transcription factors
may modify this view and include NO-donors among the DAG = 1,2-diacyl-glycerol
therapeutic modalities for the treatment of preterm labor.
DHP = Dihydro-progesterone
Taken together, there are no satisfactory tocolytic drugs
that have influenced the outcome of preterm labor during the EGF = Epidermal growth factor
past three decades in the USA. Because preterm labor is EP1, -2, -3, -4
multifactorial, combination therapies may offer some receptors = PGE2 receptor type-1, -2, -3, -4
advantages over single tocolytic therapies. But, ultimately,
elucidating the etiologies of preterm labor may offer more ERK = Extracellular signal-regulated protein
promising avenues for the design of new classes of tocolytic kinase
drugs. FP receptor = PGF2α receptor

CONCLUSION Gαs = G-protein alpha-s

Pregnancy maintenance and parturition are regulated by HOXA-10 = Gene 10 of the homeotic selector
complex interactions of endocrine, paracrine and mechano- complex A
genetic factors. The unraveling of these mechanisms has HSP90 = Heat-shock protein 90
posed a monumental task for reproductive biologists. Much
IL-1, IL-1β = Interleukin-1 (beta)
of our knowledge relies on data from animal experiments,
the results of which cannot always be extrapolated to the IL-6 = Interleukin 6
human. Because of ethical considerations, studies on human IL-8 = Interleukin 8
subjects are limited and information is gathered mainly from
experiments using tissue explants or cultured cells. Whereas IP receptor = Prostacyclin receptor
the molecular mechanism of myometrial smooth muscle IP3 = Myo-inositol 1,4,5-trisphosphate
activity has been quite well mapped out, the intimate
mechanism responsible for converting the quiescent pregnant JAK-2 = Janus tyrosine kinase-2
uterus to the parturient organ remains largely elusive. What LPS = Lipopolysaccharide
has emerged over the last decade or so are the multiple levels
of regulation involving key steps in receptor signaling MAP-kinase,
leading to gene regulation. Compounding the complexity of MAPK = Mitogen-activated protein kinase
normal parturition at term are the multiple etiologies of MLC = Myosine light chain
preterm labor, such as intrauterine infection, psychosocial
stress, multifetal pregnancy, drug abuse, poor prenatal care, NO = Nitric oxide
malnutrition, placental abruption, etc. Future work will have PGD2 = Prostaglandin D2
to focus on early recognition of these risk factors, coupled
PGE2 = Prostaglandin E2
with the development of reliable diagnostic tests. This
should allow early intervention aimed at intercepting key PGF2α = Prostaglandin F2α
upstream steps that would prevent the evolution of labor by, PGH2 = Prostaglandin endoperoxide H2
for example, blocking the synthesis of key proteins involved
in the activation of the myometrium. Additional difficulty in PIP2 = Phosphatidyl inositol-4, 5-bisphopsphate
designing new drugs targeted at key steps in cell signaling SF-1 = Steroidogenic factor-1
pathways may lie in the intrinsic promiscuity of key
enzymes and their multiple isoforms (e.g., PKC, tyrosine SRC-2, -3 = Steroid receptor coactivator-2, -3
kinase, IκB/NF-κB system, MAPK, etc.). A concerted effort TBP = TATA box-binding protein
by pharmacologists, biologists, chemists and clinicians may
bring us closer to understanding the intricate regulation of RT-PCR = Reverse transcriptase-coupled
labor and birth, that may allow the design of new approaches polymerase chain reaction
for the diagnosis and prevention of preterm birth and other STAT-1 = Signal transducer and activator of
pathologies of this reproductive process. transcription-1
TAFs = TBP(TATA-binding protein)-associated
ABBREVIATIONS
factors
AP1, AP2 = Activator protein complex -1, -2 TGF-β1 = Transforming growth factor beta-1
ATP = Adenosine-5’-triphosphate
TNF, TNFα = Tumor necrosis factor (alpha)
cAMP = Adenosine-3’,5’-cyclic monophosphate
TP receptor = Thromboxane A2 receptor
2514 Current Pharmaceutical Design, 2004, Vol. 10, No. 20 Hertelendy and Zakar

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