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Oxytocin receptor system and human repro-duction
Human parturition is a complex sequence of events that concludes in the coordinated processes of cervical dilation and
uterine contraction. For most of human pregnancy the myometrium remains relatively quiescent, allowing development of
the fetus. Towards the end of pregnancy, there is a relatively gradual change from myometrial quiescence to a more
contractile state. The transformation of the uterine muscle from quiescent to contractile was termed "activation" and is
thought to involve increased synthesis of contraction-associated
proteins[1,2]. Activation prepares the uterus for labor and
alters it from a relatively insensitive organ to a sensitive, pro-contractile organ. Once the uterus has become activated, a
process termed "stimulation" initiates the powerful coordinated contractions of labor. It is accepted that nonapeptide
hormone oxytocin has a fundamental role in the stimulation process, and there is growing evidence that this hormone might
also contribute to the process of activation as well.
Oxytocin was first extracted from the human posterior pituitary in1906 by Sir Henry
Dale[3]. The chemical structure of oxytocin was determined in 1953 by Du Vigneaud
et al[4]. Soon after the discovery of its uterotonic activity, the pituitary
extract was used for treatment of postpartum
hemorrhage[5]. Half a century later, obstetricians started using oxytocin for
induction of labor[6]. In recent years, it has become apparent that oxytocin is involved in a much greater number of
reproduction-related processes. Apart from modulating the uterine contractions during parturition, oxytocin is essential for milk
ejection during lactation[7]. Furthermore, the widespread distribution of oxytocin receptors in the brain and the specific
behavioral effects of centrally applied oxytocin point towards its role as a central neurotransmitter mediating
reproduction-related processes such as maternal behavior, sexual receptivity and partnership
bonding[8]. For in-depth discussion on
different aspects of oxytocin biology the reader is referred to several detailed reviews published in the last few
years[9_13]. In the present paper, we will reflect on our own work and the work of other groups on oxytocin-induced modulation of uterine
contractility. Because oxytocin is one of the most potent uterotonic agents known, its effect on uterine contractility is of
major pharmacological importance. Thus, synthetic analogs of oxytocin are widely used to induce labor and to treat
postpartum hemorrhage. Premature activation of the oxytocin system might be a leading cause of preterm labor, therefore the
antagonists of oxytocin receptors have been extensively tested as means of inhibiting premature uterine contraction. The
effectiveness of atosiban, one of the most potent oxytocin receptor antagonists, has been tested in clinical
trials[14,15]. Based on these studies, atosiban is currently approved for clinical use in 29 countries. Despite the extensive clinical use of oxytocin
and oxytocin receptor inhibitors, the molecular and cellular mechanisms underlying oxytocin-induced modulation of uterine
contractility are not completely understood. The oxytocin receptor belongs to the G-protein coupled receptor family. After
binding oxytocin, the receptor activates
phospholipase-Cb by coupling to
Gq/G11 GTP-binding
proteins[16]. This leads to the generation of the 2 second messengers: inositol 1,4,5-trisphosphate
(IP3); and diacylglycerol (DAG). Both second
messengers are believed to be involved in the generation of the physiological response to
oxytocin[17,18], although their relative
contributions to the overall effect are unknown. There is substantial evidence in published reports that cytoplasmic
Ca2+ concentration
([Ca2+]i) determines myometrial contraction and is
involved in stimulatory action of
oxytocin[19]. However impor-tant, the
IP3-mediated
[Ca2+]i release does not appear to be the
whole story. Disabling the sarcoendoplasmic reticulum (SR) using a specific SR
Ca2+ ATP-ase inhibitor, thapsigargin, does
not abolish the oxytocin-induced increase in the amplitude of myometrial contractions (Figure 1). An example of changes in
spontaneous contractions of an isolated strip of human myometrium in response to oxytocin application is given in Figure
1A. At least three distinct components can be discerned in the effect of oxytocin on human uterine smooth muscle: (1)
increase in frequency of contractions; (2) initial transient increase in the base tone (incomplete relaxation); and (3)
long-lasting increase in the amplitude and duration of phasic contractions. Even prolonged treatment (more than 1 h) of the
myometrium with thapsigargin to disable the SR abolishes only the first 2 components of the oxytocin effect and does not
change the third. That is, the oxytocin-induced increase in frequency of contractions and the transient rise in base tone are
eliminated, but not the oxytocin-induced potentiation of force (Figure 1B). These data indicate that, in addition to the
IP3-mediated Ca2+ release, other processes might be involved in oxytocin-induced modulation of myometrial contractility. We
will discuss these after reviewing the oxytocin-induced
[Ca2+]i signaling.
Oxytocin-mediated Ca2+ release and excitation-contraction coupling in myometrium
Myometrium is a phasic smooth muscle with an intrinsic ability to generate spontaneous contractions. It is generally
accepted that myometrial contractions are myogenic, that is, neural or hormonal stimuli are not required for the contractions
to occur, but do modulate them. Slow depolarization of the myometrium preceding the discharge of action potentials is
attributed to the activity of certain cells, considered
pacemaker cells. In some types of visceral smooth muscle, for example,
gastrointestinal[20],
urogenital[21] and
vascular[22], a specialized type of cell called interstitial cells of Cajal (ICC) had been found.
In some, but not all tissues, these cells constitute a pacemaking mechanism. Our recent study has identified the ICC-like cells
in human and rat myometrium[23]. It is therefore a plausible scenario that the multiple components in the effect of oxytocin are
due to its action on different cell types within the myometrium. The ICC-like cells might mediate changes in frequency,
whereas smooth muscle cells are responsible for the increase in amplitude of contrac-tions. More research into this area is
needed to make informed conclusions regarding the role of ICC-like cells in the regulation of myometrial contractility.
Several research groups have investigated oxytocin-induced
[Ca2+]i signaling using
Ca2+ sensitive dyes in primary cultures of
rat[24], human myometrial
cells[25] and in a cell line derived from pregnant human
myometrium[26,27]. In all types of cells
studied, these transients persisted in
Ca2+-free solution and were abolished by thapsigargin, indicating their SR origin. In the
absence of extracellular Ca2+, even a brief application of oxytocin to primary human myometrial cells caused a full-sized
[Ca2+]i transient, but subsequent applications of agonist were ineffective. Desensitization of the oxytocin receptors can be ruled
out because readmission of extracellular
Ca2+ led to restoration of the oxytocin-induced
[Ca2+]i transients with 2
min[25]. These data might suggest that oxytocin completely emptied the store during the first application and there was nothing left for the
subsequent applications. Alternatively, the ineffectiveness of multiple applications of oxytocin might reflect a steep
dependence of the IP3-induced
Ca2+ release on intraluminal
Ca2+ concentration
([Ca2+]L), so that even a moderate decrease in
[Ca2+]L will render the
IP3 receptors insensitive to
IP3[28]. The extent to which a single application of oxytocin depletes the SR is
important because store depletion can trigger capacitative
Ca2+ entry through store-operated
Ca2+ channels on the surface membrane. Several isoforms of TrpC have been identified in the
myometrium[29]. Whether or not these channels contribute
to the excitation-contraction coupling should depend on the amount of
Ca2+ displaced from the SR by oxytocin via
IP3-mediated Ca2+ release. It is difficult to estimate the extent of the SR depletion without actually measuring intraluminal
Ca2+. In recent years, new powerful experimental approaches to direct measurement of
[Ca2+]L have been developed. These include
Ca2+-sensitive fluorescent and luminescent proteins targeted to the SR and low-affinity
Ca2+-sensitive dyes selectively loaded into the SR. A detailed discussion of these methods, their advantages and pitfalls has been
published[30]. In our study, we combined the low-affinity
Ca2+ indicator Mag-Fluo-4 with a high-affinity dye Fura-2 to measure
[Ca2+]L and
[Ca2+]i simultaneously in freshly isolated uterine
myocytes[31]. The data obtained in our experiments on rat myometrial cells suggest
a very steep dependence between the SR filling state and the effectiveness of the
IP3-mediated Ca2+ release in intact
cells[32]. That is, even a moderate depletion of the
[Ca2+]L (approximately 20% of its normal content) abolishes the agonist-induced
cytoplasmic Ca2+ transients. Thapsigargin treatment causes
slow decrease in the luminal Ca2+ due to unbalanced passive
Ca2+ leak and abolishes the agonist-induced
[Ca2+]i
transients[32]. An example of simultaneous recording of
[Ca2+]L and
[Ca2+]i is given in Figure 2. A measurable decrease in
[Ca2+]L and a transient rise in
[Ca2+]i is seen after application of oxytocin in the absence
of extracellular Ca2+ (Figure 2A). After transient increase, the cytoplasmic
Ca2+ returns to its resting level in the presence of
oxytocin. The intraluminal Ca2+ decreases at the beginning of the oxytocin application and remains at a decreased level for
as long as the cell is exposed to Ca2+-free extracellular solution, even though the oxytocin has been washed out. After
readmission of extracellular Ca2+, the
[Ca2+]L returns to its initial level within 2_3 min. However, under physiological
conditions (ie, in the presence of extracellular
Ca2+), a completely different result is obtained: the cytoplasmic
Ca2+ rises to a somewhat higher level and, more importantly, remains at this high level for long period of time (Figure 2B). The dynamics of
luminal Ca2+ is also different under these conditions. Thus, only a transient fall in the
[Ca2+]L level occurs at the beginning of
oxytocin application, immediately followed by the increase above resting level, suggesting uptake of
Ca2+ into the SR. These data indicate that, under physiological conditions, oxytocin triggers a complex
[Ca2+]i response consisting of initial
Ca2+ release from the SR followed by
Ca2+ entry from outside. This oxytocin-induced
Ca2+ entry leads to sustained elevation in
[Ca2+]i that underlies increased uptake of
Ca2+ into the SR. The process is unlikely to be triggered by store depletion, as there
is no persistent decrease in
[Ca2+]L. However, the possibility of the TrpC involvement in the oxytocin-induced
[Ca2+]i signaling still exists. In a recent study, a direct activation of extracellular
Ca2+ entry through TrpC by
1-oleoyl-2acetyl-sn-glycerol, a membrane-permeant analog of DAG, was found in both primary uterine myocytes and immortalized myometrial cell
line derived from pregnant uterus[18].
It has to be borne in mind that, under physiological conditions, the excitation-contraction cycle occurs spontaneously
and the SR Ca2+ release and entry induced by continuous oxytocin application will be imposed on the action potential
triggered [Ca2+]i transients. Therefore, the phasic effect of the elevated
IP3 level has to be viewed through the prism of the
excitation_contraction coupling process. A generally accepted mechanism of the myometrial excitation-contraction coupling
is that the action potential-induced rise in
[Ca2+]i triggers acto-myosin interaction by
Ca2+-calmodulin-mediated phosphorylation of the regulatory myosin light
chains[33]. In the presence of oxytocin, or indeed any
IP3 producing agent, the amplitude of the action potential-induced
[Ca2+]i transients can, in principle, be potentiated by sustained elevation of
IP3. The possibility of such potentiation stems from the bell-shaped
Ca2+ dependence of the IP3 receptor sensitivity to
IP3[34]. That is, at sub-maximal levels of
IP3, the rise in
[Ca2+]i produced by the inward
Ca2+ current will augment the
IP3-mediated SR Ca2+ release at
200_300 nmol/L [Ca2+]i and diminish it when
[Ca2+]i exceeds 500_600 nmol/L. The mechanism of oxytocin-induced changes in
the frequency of contractions has not been elucidated yet. As the frequency effect of oxytocin is abolished by thapsigargin, it must
be related to the IP3-mediated
Ca2+ release from the SR and concomitant activation of
Ca2+ activated
channels[35,36]. Clearly, the mechanism(s) of oxytocin-induced increase in frequency of uterine contractions can only be addressed once we understand the
mechanism(s) of myometrial autorhythmicity.
Oxytocin-mediated Ca2+sensitization
Almost half a century of experimental research on many types of smooth muscle provided convincing evidence that
contractility of smooth muscle is regulated not only by electromechanical coupling but also by membrane
potential-independent, pharmacomechanical
coupling[37]. Many agonists induce sensitization of contractile apparatus to
Ca2+ by inhibiting myosin phosphatase and decreasing the rate of myosin regulatory chain dephosphorylation.
This prolongs RhoA
myosin light chain phosphorylation, leading to enhanced tension without influencing
[Ca2+]i[38,39]. The contractile
apparatus can therefore produce more force at given
[Ca2+]i. This also implies that the rate of relaxation will be decreased in the
presence of agonist. Indeed, oxytocin decreases the rate of relaxation in human
myometrium[40]. Figure 3A shows
superimposed traces of force (upper traces) and its first derivative (lower traces) in control (blue traces) and in the presence of
oxytocin (red traces). It is clear that oxytocin not only increases the amplitude and duration of contraction but also decreases
the peak rate of relaxation. Similar results were obtained after thapsigargin treatment (Figure 3B) indicating SR
Ca2+ release is not involved in this process. Simultaneous measurements of
[Ca2+]i and force from human myometrium revealed that oxytocin
applied to strips depolarized by KCl could increase force without affecting
[Ca2+]i[41]. In our experiments, oxytocin increased
force without affecting peak amplitude of
[Ca2+]i
transients[40]. Molecular mechanisms involved in
Ca2+ sensitization are currently being investigated in several laboratories. In many tissue types, RhoA, a monomeric G-protein, can mediate
Ca2+ sensitisa-tion[42]. Activation occurs on stimulation of receptors coupled to
Ga12,13; Gaq or
Gai[43], which convert inactive
RhoA·GDP to active RhoA·GTP through the action of guanine nucleotide exchange factors. RhoA·GTP acts through a
serine/threonine kinase (Rho kinase) that phosphorylates the regulatory subunit of MLCP, thereby inhibiting phosphatase
activity. It has been shown that upregulation of
and Rho kinase occurs in human myometrium during
pregnancy[44]. It has also been demonstrated that inhibition of Rho kinase
activity by Y-27632 reduces tension but not
[Ca2+]i during spontaneous and oxytocin-induced contractions in human
myometrium[41]. Thus, Rho kinase mediates (at least in part) oxytocin-induced increase in myometrial contractility. This
mechanism is summarized in Figure 4. It remains to be determined whether Rho A is the activator of Rho kinase in myometrium.
The oxytocin receptor has not been shown to couple to
Ga12,13 although activation of Rho A could occur through
Gaq. The situation becomes even more complicated
because arachidonic acid can also activate Rho kinase (Figure 4) and might
mediate Rho kinase-dependent Ca2+
sensitization[45].
In addition to Rho kinase, protein kinase C modulates myosin light chain phosphatase activity either by direct
phosphorylation or a smooth-muscle specific inhibitor called CPI-17.
Activation (phosphorylation) of CPI-17 inhibits the
catalytic subunit of MLCP leading to increased light chain phosphorylation and enhanced contraction at a given
[Ca2+]i[46]. As Rho
kinase[47] and arachidonic
acid[45] also activate CPI-17, this suggests that these pathways could converge on CPI-17 to
increase the [Ca2+]i sensitivity of contractile apparatus.
It appears that Ca2+ sensitization is the longest lasting effect of oxytocin on human myometrium. In fact, contractions of
potentiated amplitude can be recorded for many hours, whereas increase in the base tone (incomplete relaxa-tion) takes place
only at the beginning of oxytocin application and lasts only a short time. Clearly, tonic contraction of the uterus during labor
is undesirable as it would lead to foetal distress through decreased blood supply to the foetus. An increase in the amplitude
and frequency of phasic contractions will aid successful labor. The data discussed in this review suggest that the tonic
contraction seen at the beginning of oxytocin application (see Figure 1) and changes in frequency of contractions are
mediated by the SR Ca2+, and potentiation of contraction amplitude is achieved by sensitization of contractile machinery to
Ca2+. This process appears to be the most relevant physiologically and should be concentrated on in the future research.
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