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The Sir Quinton Hazell Molecular Medicine Research Center, Department of Biological Sciences, University of Warwick, Coventry, United Kingdom CV4 7AL
Address all correspondence and requests for reprints to: Dr. D. Grammatopoulos, The Sir Quinton Hazell Molecular Medicine Research Center, Department of Biological Sciences, University of Warwick, Gibbet Hill Road, Coventry, United Kingdom CV4 7AL. E-mail: chdg{at}dna.bio.warwick.ac.uk
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Abstract |
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CRH receptor binding affinity for CRH and activation of adenylate cyclase were reduced in the presence of OT in pregnant (at term, but not preterm) human myometrium. OT action was mediated via pertussis toxin-sensitive G proteins, which directly inhibit adenylate cyclase and, via activation of protein kinase C, phosphorylate the CRH receptor, leading to desensitization. Activation of protein kinase C by OT could be partially inhibited in human pregnant myometrial cells by OT antagonists (F327 and CAP476; 1 µM) or phospholipase C inhibitors (U73122; 10 µM).
These results suggest that in term myometrium, CRH receptor function is modulated by OT, leading to reduced biological activity, lower cAMP levels, and a subsequent shift in favor of contractility rather than relaxation.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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toward
term and labor (4). As the uterus becomes prepared for labor, it is likely that uterine activation occurs in response to paracrine/autocrine action of a variety of uterotonins. Oxytocin (OT) is the most potent known endogenous uterotonic agent (5), and the major factor influencing its bioactivity is an increase in the number of myometrial OT receptors at term (6). Local OT production by uterine tissues is also increased (7). In mice, however, OT may not play a critical role, because the induction of labor and parturition can proceed normally in mice in which the OT gene has been deleted (8). In the human myometrium, the effects of OT are mediated through a seven-transmembrane domain receptor (9) linked to both Gi and Gq proteins (10), which activate phospholipase C (PLC) with subsequent production of inositol 1,4,5-triphosphate. This has two important consequences: 1) the rapid release of calcium ions from intracellular stores in the sarcoplasmic reticulum, and 2) the accumulation of diacylglycerol (DAG), which remains membrane bound. Elevated levels of intracellular calcium activate some types of cytosolic protein kinase C (PKC) and induce its translocation to the cellular membrane, where it is further activated by DAG (11). PKC is a serine/threonine kinase and acts as a key mediator in signal transduction events (12). The family of PKC has several members, which have been divided into a Ca2+-dependent or conventional PKC group and a Ca2+-independent or novel PKC group (12). One of the most important functions of PKC in signal transduction is that it mediates cross-talk between different signaling pathways.
In the human myometrium one of the most important signaling pathways is
the adenylate cyclase-cAMP system (13), which can be activated by the
CRH receptor (2). Molecular characterization has revealed that this
receptor belongs to the calcitonin/vasoactive intestinal
polypeptide/GRF/PTH subfamily of G protein-coupled receptors. There are
two groups of CRH receptors (CRH-R1 and CRH-R2) arising from separate
genes (14, 15), both of which have several variants arising from
alternative splicing. The R1 gene encodes four known subtypes: R1,
R1ß (with a 29-amino acid insertion in the first intracellular loop)
(14), R1c (with a 40-amino acid exon deletion from the N-terminal
domain) (16), and R1d (with a 12-amino acid exon deletion from the
seventh transmembrane domain; our unpublished observations). The
CRH-R2 also has three spliced variants, R2, R2ß, and R2
, each
the product of differential 5'-exon splicing and each with distinct
tissue distributions (17, 18, 19). All of these subtypes of the CRH
receptor, with the exception of R2, exist in the human pregnant
myometrium and have been identified using PCR, sequencing of the
amplified DNA fragments, and fluorescent in situhybridization (20). Interestingly, the complementary DNA
sequences of the CRH-R1 and -R2 receptors encode four potential PKC
phosphorylation sites, identical in both CRH receptors, which are
located in the first and second intracellular loops as well as in the
C-terminus.
These PKC phosphorylation sites may be involved in the modulation of CRH receptor signal transduction. Their precise role, however, has not yet been characterized. In view of this, we searched for potential mechanisms of cross-talk between OT and the CRH system that might be involved in the regulation of human myometrial contractility during human pregnancy.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For myocyte isolation and culture, fresh tissue was placed in 20 ml ice-cold DMEM culture medium containing 200 IU penicillin/ml and 200 mg streptomycin/ml. Myocytes were prepared by enzymatic dispersion (see below).
For all biopsies ethical approval was obtained from the local ethical committee, and each patient gave informed consent to the study.
Chemicals
Ovine (o) CRH, Tyr-oCRH, and all other chemicals were purchased
from Sigma Chemical Co. (Poole, UK). Waters Sep-Pak
C18 columns were obtained from Millipore Corp.
(UK) Ltd. (Watford, UK). OT antagonists, CAP476
(1-deamino-2-D-Tyr-(OEt)-4-Thr-8-Orn-OT) and F327
(des-Gly9-[D-Tyr(Et)2,Thr4,Orn8]dC6-OT),
were gifts from Ferring Pharmaceuticals Ltd. (Malmo,
Sweden).
125I-Labeled Nle21,Tyr32-oCRH (SA, 922 Ci/mmol), human/rat (h/r) CRH, and OT were obtained from Peninsula Laboratories (Merseyside, UK).
Phorbol myristate acetate (PMA), H-7 and H-8 (PKC inhibitors), and U73122 were obtained from Calbiochem (La Jolla, CA). The myristoylated PKC peptide (Myr-PKC) inhibitor was purchased from Promega Corp. (Madison, WI). The cAMP assay kits were obtained from DuPont-New England Nuclear (Hertfordshire, UK). Protein A-Sepharose beads (CL-4B) were purchased from Pharmacia Biotech (Uppsala, Sweden).
A specific CRH receptor antibody was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). This is a goat polyclonal antibody raised against a peptide corresponding to amino acids 425–444 mapping at the C-terminus of the human CRH-R1 precursor. The antibody reacts with both human CRH-R1 and -R2 receptors, and this has been verified in preliminary studies by Western blotting and immunohistochemistry.
Myocyte isolation and culture: preparation of myometrial membranes
from biopsies
Pieces of myometrium were transferred into DMEM containing
collagenase (300 U/ml), deoxyribonuclease (30 U/ml), penicillin (200
U/ml), and streptomycin (200 mg/ml) and incubated at 37 C for 30 min.
After filtration and centrifugation, cells were suspended in DMEM
containing 10% FCS, penicillin (100 U/ml), streptomycin (100 mg/ml),
and fungizone (2.5 µg/ml). The cells were kept at 37 C in a
humidified atmosphere of 95% air and 5% CO2 until
confluent (2–4 weeks). The purity of myometrial muscle cells was
assessed by immunocytochemical staining. Mouse antihuman smooth muscle
actin-specific monoclonal antibody and peroxidase-conjugated rabbit
antimouse antibody were used. Human fibroblast cells and omission of
the primary antibody were used as negative controls; frozen myometrial
tissue was used as a positive control.
Myometrial membranes were prepared by homogenization and differential centrifugation as described previously (1). Protein concentrations were measured using the bicinchoninic acid method (21). The final pellet was resuspended in 3 ml extraction buffer and after homogenization was aliquoted in 1.5 ml Eppendorf tubes (50 µg protein/tube) and stored at -70 C until use.
Cultured human myometrial cells were used only for those experiments on the OT-induced activation and subsequent translocation of PKC from the cytosol to the cell membrane. The rest of the experimental studies used membrane homogenates prepared from myometrial biopsies at term or preterm.
Binding studies on myometrial membranes: Scatchard analysis
Scatchard analysis was performed as previously described (1).
All experiments were performed at 22 C for 2 h. For the control
Scatchard plot, myometrial membrane suspensions (100–150 µg protein)
were incubated with 50 µl of different concentrations of
125I-labeled Nle21,Tyr32-oCRH
(10,000–700,000 cpm) and unlabeled oCRH (1,000-fold molar excess) in
50 µl incubation buffer A [50 mM Tris-HCl, 2
mM EGTA, 10 mM MgCl2, 0.1% BSA
(wt/vol), and 0.15 mM bacitracin, pH 7.2]. In an
additional series of polypropylene tubes, 10 different concentrations
of 125I-labeled Nle21,Tyr32-CRH
(10,000–400,000 cpm) were incubated in the presence of varying
concentrations of OT (1–500 nM) and unlabeled CRH
(1,000-fold molar excess). Binding data were analyzed by computer
analysis with EBDA (22) and LIGAND (23).
Isoelectric focusing
Myometrial membrane suspensions (300–350 µg protein) were
incubated with 50 µl [125I]oCRH (200,000–300,000 cpm)
in the presence or absence of OT (100 nM) diluted in buffer
A. Nonspecific binding was determined in the presence of unlabeled CRH
(1 µM). The reaction was carried out at 22 C for 2
h. After the binding assay, pellets were resuspended in 500 µl
incubation buffer A containing 1% Triton X-100, incubated for 1–2 h
at 0 C, and centrifuged at 12,000 rpm for 10 min. The solubilized
membrane suspensions were collected, concentrated using Centricon-30
microconcentrators, and fractionated by isoelectric focusing as
previously described (24). Gels were stained and dried, and
autoradiography was performed for 5–10 days at -70 C using Fuji Photo Film Co. Ltd. x-ray film (Tokyo, Japan) and intensifying
screens. The relative distribution of the human myometrial CRH receptor
isoforms was measured by optical density scanning using the Image
analysis program (Wayne Rasband, NIH, Bethesda, MD).
In preliminary experiments (24), we confirmed that the two isoforms at pI 4.6 and 4.9 represent free radioligand preparation and not receptor-ligand complex; thus, they were excluded from further analysis. Furthermore, no differences in the isoform profile were found when CRH was covalently cross-linked to its binding sites using disuccinimidyl suberate (final concentration, 1.5 mM; for 10 min at room temperature); therefore, disuccinimidyl suberate was not used in subsequent experiments.
cAMP studies
Human pregnant (at term or preterm) myometrial membrane
preparations (50 µg protein) were preincubated with different
concentrations of h/rCRH (0.1–1000 nM) or OT (100
nM) in 50 µl extraction buffer in the presence or absence
of the Myr-PKC inhibitor (final concentration, 100 µM)
for 30 min at 22 C before the addition of 100 µl 50 mM
Tris-HCl containing 10 mM MgCl2, 1
mM EGTA, 0.1% BSA, 1 mM ATP, ATP regeneration
system (7.4 mg/ml creatine phosphate and 1 mg/ml creatine
phosphokinase), 100 µM isobutyrmethylxanthine
(phosphodiesterase inhibitor), and 0.15 mM bacitracin, pH
7.4 (cAMP assay buffer), at 37 C. The reaction was terminated after 10
min by the addition of 1 ml 0.1 M imidazole buffer, pH 7,
followed by heating the tubes in boiling water for 5 min.
For the pertussis toxin (PT) pretreatment of the myometrial membrane preparations, PT was preactivated with 50 mM dithiothreitol. Human pregnant myometrial membranes at term were incubated for 50 min at 30 C with 500 µl 50 mM Tris-HCl containing 1 mM ATP, 1 mM thymidine, 0.1 mM GTP, 10 mM MgCl2, 1 µg/ml digitonin, 1 mM NAD, 7.4 mg/ml creatine phosphate, and 1 mg/ml creatine phosphokinase, pH 7.4, in the presence or absence of PT (final concentration, 25 µg/ml) for 45 min at 30 C. The reaction was stopped by the addition of 1 ml ice-cold extraction buffer, followed by centrifugation at 10,000 rpm in a Beckman Coulter, Inc., J20 centrifuge (Palo Alto, CA) for 15 min. The pellet was then washed with extraction buffer and centrifuged at 10,000 rpm for 15 min (three times). The final pellet was resuspended in extraction buffer and homogenized. Pretreated human myometrial membranes were preincubated with different concentrations of OT (1–500 nM) for 30 min at 22 C before the addition of 50 µl cAMP assay buffer and initiation of the cAMP release reaction at 37 C.
The amount of cAMP in the incubate was estimated in the supernatants by RIA using commercial cAMP RIA kits. Standard cAMP concentrations, covering the range 100–0.5 pmol/ml, were used for determination of the standard curve of the RIA. The interassay coefficient of variation was 5%.
PKC assay in human myometrial cell cultures and myometrial
membranes
Human pregnant myometrial cells, at a density of around
106 cells/flask, were preincubated for 15 min at 37 C with
5 ml PBS containing OT (1–100 nM) in the presence or
absence of 10 µM U73122 (PLC inhibitor) or OT inhibitors
(1 µM). After the incubation period, cells were washed
with 5 ml PBS (twice) and were removed from the bottom of the dishes
and resuspended in 0.5 ml chilled extraction buffer [Dulbecco’s PBS
containing 10 mM MgCl2, 2 mM EGTA,
1.5 µg/liter BSA (wt/vol), 10 mM ß-mercaptoethanol,
0.15 mM bacitracin, and 1 mM
phenylmethylsulfonylfluoride, pH 7.2]. The suspensions were
homogenized for a few seconds and centrifuged for 1 h at
50,000 x g. Supernatants were stored (at 0 C), and the
membrane fraction was resuspended in 0.5 ml extraction buffer
containing 1% Triton X-100 and solubilized for 1 h at room
temperature. After centrifugation and collection of the resulting
supernatants, all samples (supernatants) were loaded onto 1-ml
diethylaminoethyl (DEAE)-cellulose columns, and the PKC-containing
fraction was eluted using 1 ml extraction buffer containing 200
mM NaCl. Samples were frozen at -70 C until assayed for
PKC activity.
Human myometrial membranes (250 µg/tube) were resuspended in 50 µl buffer containing 20 mM Tris-HCl and 10 mM MgCl2, pH 7.5. Membrane suspensions were incubated in the presence or absence of PKC activators (PMA, 200 nM; OT, 1–100 nM) or inhibitors (H-7, 100 µM; H-8, 100 µM; myristoylated PKC peptide inhibitor, 100 µM) for 30 min at room temperature. Membranes were solubilized, and PKC was extracted as described using DEAE-cellulose columns.
The activity of PKC recovered from either human myometrial cells or
membrane preparations was assayed in a 10 mM Tris-HCl
buffer (pH 7.5) containing 10 mM MgCl2, 0.2
mM CaCl2, 0.5 mM EGTA, and 0.1
mg/ml BSA, using a commercial PKC assay system in which the
incorporation of 32P into a specific PKC biotinylated
peptide substrate (neurogranin-(28–43)-AAKIQASFRGHMAR KK) (25) using
[-32P]ATP (3000 Ci/mmol) as a 32P donor
was measured by the binding of the substrate to streptavidin disks and
monitoring the radioactivity using a scintillation counter.
In vitro phosphorylation of the myometrial CRH receptor
Human pregnant (at term or preterm) myometrial membrane
suspensions (500 µg/tube) were incubated in the presence or absence
of OT (100 nM) with or without myristoylated PKC peptide
inhibitor (100 µM) for 10 min at 30 C.
[-32P]ATP (3000 Ci/mmol) was added as
32P- donor (4 µCi/tube). The reaction was stopped by
centrifugation (15,000 rpm in a microfuge), and membranes were
solubilized for 2 h on ice in 500 µl solubilization buffer
containing 10 mM Tris-HCl, 10 mM EDTA, 500
mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 1
mM phenylmethylsulfonylfluoride, and 1 mM
bacitracin, pH 7.4. After centrifugation, immunoprecipitation of the
CRH-receptor complex was performed by adding 25 µl CRH receptor
antiserum to the supernatant and incubating overnight at 4 C. Isolation
of the immune complexes was carried out using protein A-Sepharose beads
(100 µl), rotation for 20 min at 4 C, centrifugation, washing of the
resulting pellet with 1 ml Tris-EDTA buffer (three times), and
resuspension in SDS buffer. Proteins were resolved on 8% SDS-PAGE, and
gels were dried and subjected to autoradiography (-70 C, 10–14 days)
using intensifying screens.
Statistical analysis
Data are shown as the mean ± SEM of each
measurement. Comparison between group means was performed using the
nonparametric statistical method of Kruskal-Wallis. P
< 0.05 was considered significant.
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Results |
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Saturation analysis of 125I-labeled
Nle21,Tyr32-oCRH binding to human pregnant
myometrial membranes demonstrated that binding was saturable and of
high affinity (Fig. 1a
). When the
myometrial membranes were incubated with radiolabeled oCRH in the
presence of OT for 2 h, the receptor required a greater
concentration of radioligand to reach saturation. Scatchard analysis of
these data (Fig. 1b) showed that the OT effect was due to a reduction
in the affinity of the CRH receptor. The dissociation constant
(Kd) was increased from 84 ± 4 to 165 ± 12
pM, whereas the maximum receptor concentration was
unchanged (11 ± 2 fmol/mg membrane protein). This effect was OT
concentration dependent, was apparent at OT concentrations greater than
1 nM, and was maximal at a concentration around 100
nM (Table 1). This OT
concentration was used in all subsequent experiments.
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). Analysis of membrane
preparations incubated with a 1000-fold excess of unlabeled h/rCRH
showed an equal displacement of bound [125I]oCRH from
each of the five receptor isoforms.
Effect of OT on basal and CRH-stimulated cAMP production
This OT effect was analyzed in more detail at a functional level.
In membranes prepared from human pregnant term myometrium, OT (at a
concentration range of 0.1–500 nM) was able to reduce the
basal cAMP production (maximum inhibition, 37–42 ± 6% at an OT
concentration of 100 nM; Fig. 2a). One possible explanation for the
effect of OT on cAMP release is the involvement of an inhibitory
guanyl-nucleotide binding protein (Gi) in the
regulation of adenylate cyclase activity. To test for this possibility,
myometrial membranes were pretreated with PT, which catalyses the
ADP-ribosylation and inactivates the Gi
subunit of the guanine nucleotide regulatory component, thus preventing
inhibition of adenylate cyclase. The OT effect was reversed when
myometrial membranes were pretreated with PTX (25 µg/ml; Fig. 2a).
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, open squares). To test whether the inhibitory effect of OT
on adenylate cyclase was mediated via PT-sensitive G proteins (probably
a Gi), we used PT pretreatment of the myometrial membranes.
Such pretreatment completely abolished the OT-induced inhibition of
basal adenylate cyclase activity and partially reversed the OT-induced
inhibition of CRH-stimulated cAMP production. Furthermore, it reduced
both the sensitivity and the magnitude of the CRH effect (Fig. 2b, closed triangles). These data suggest the involvement of
another component in this phenomenon that is PTX insensitive, and that
OT might target the activity of the CRH receptor itself.
Studies on PKC activity of human myometrium
Using a specific PKC assay system, we measured the PKC
activity in human pregnant (term and preterm) myometrial membranes.
Significant PKC activity was present in the pregnant (term) myometrial
membranes (Fig. 3); furthermore, PMA (200
nM) and OT (1–100 nM) were able to stimulate
PKC activity. PKC activity was reduced in the presence of PKC
inhibitors (H-7, 100 µM; myristoylated PKC peptide
inhibitor, 100 µM), with the myristoylated PKC peptide
inhibitor being the most potent inhibitor. H-8 at an equimolar
concentration (100 µM) had no effect on PKC activity. In
contrast, no measurable PKC activity was detected in pregnant (preterm)
membranes, and agents such as PMA and OT could not exert any
stimulatory action (data not shown).
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).
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). In
contrast, in preterm myometrium there was no effect of OT or the PKC
inhibitor on cAMP levels. CRH was able to activate adenylate cyclase at
lower concentrations, consistent with previous findings (4). In both
membrane preparations, adenylate cyclase activities were similar and
were unaffected by inhibition of PKC, as forskolin was able to increase
cAMP levels to a similar level (10- to 12-fold increase in basal
values) in both membrane preparations in the presence or absence of PKC
inhibitor (data not shown).
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, inset). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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At term, the ability of the myometrial CRH receptors to activate adenylate cyclase is impaired (3), and our results implicate OT in this phenomenon. The decidua has been shown to express high concentrations of messenger RNA for OT (7), and the translated peptide may reach high concentrations locally and exert a paracrine effect. Furthermore, the number of OT receptors can increase as much as 200-fold (6). We now report that OT is able to reduce CRH binding to its myometrial receptor by reducing the affinity of the CRH receptor without affecting the number of receptors. This effect is mediated by at least four CRH receptor isoforms, as identified by isoelectric focusing. Previously, we have shown that the human myometrium expresses five isoforms of the CRH receptor, which can be separated by charge (24). However, a paradox appears to exist, as Scatchard analysis suggested a single population of high affinity CRH receptors. It is possible that the different isoforms separated with isoelectric focusing might represent receptor heterogeneity due to different glycosylation forms of the same receptor during posttranslational modification that display the same binding affinity. It is well known that there are several potential N-glycosylation sites in both R1 and R2 CRH receptor amino acid sequences (14, 15). In support of this hypothesis was the observation that the isoform profile was identical in both pregnant and nonpregnant myometrium (24), which suggests that the difference in CRH binding affinity and function in these two tissues is not due to differences in the receptor isoform population.
In human pregnant myometrium at term, OT was able to reduce both basal and CRH-stimulated cAMP levels, in agreement with previous data (26). The OT effect on basal adenylate cyclase activity appears to be mediated by a PTX-sensitive G protein, most likely Gi protein, which is consistent with previous observations (10). Such PTX-sensitive as well as PTX-insensitive G proteins have been shown to be involved in OT stimulation of phosphoinositol hydrolysis in human myometrial cells (10). Inhibition of Gi activity by PTX, however, did not completely reverse the OT effect on CRH-stimulated cAMP, suggesting the presence of an additional mechanism. Subsequently, we showed that OT as well as phorbol esters (PMA) were able to stimulate PKC in human pregnant (at term but not preterm) myometrial membranes, an effect that could be reversed in the presence of PKC inhibitors. A possible explanation for the lack of PKC activity in preterm myometrium is the immaturity of the myometrium, which lacks sufficient OT receptors as well as sensitizing stimuli capable of inducing myometrial PKC activity in the cell membrane.
Most importantly, we showed that in human pregnant myometrial cells, OT
was able to induce PKC translocation from the cytosol to the cell
membrane. This phenomenon has been suggested previously, and
interactions between PKC activation/inhibition and the OT signaling
pathway have been described in human myometrium (27) and amnion cells
(28), although no direct PKC stimulation has been shown. Several
isoforms of PLC (ß1, ß2, ß3, 1, and ß2) have been described
in human myometrial cells (26), and this pathway was investigated in
more detail using specific OT antagonists and the PLC inhibitor U73122
(29); partial inhibition of the OT-induced translocation of PKC
activity was achieved. Only one dose of OT antagonists was used, so as
to demonstrate the effect of the antagonists rather than investigate
dose-response characteristics. The actions of OT antagonists used have
been well described; they inhibit OT-induced calcium influx (30) and
inositol triphosphate generation in myometrial cells (31).
These results in association with the presence of four potential PKC phosphorylation sites in each of the three intracellular loops as well as in the C-terminus of the complementary DNA sequence of the CRH-R1 and -R2 receptors led us to investigate whether the inhibitory action of OT was mediated by PKC-induced phosphorylation and desensitization of the CRH receptor. There are several examples of 7TMD receptors where PKC-induced phosphorylation and desensitization are accompanied by a reduction in the receptor affinity for the agonist, presumably by impairment of receptor-G protein coupling (32, 33, 34). Furthermore, interactions between the CRH signaling pathway and PKC have been described previously; in the pituitary, PKC activation potentiates CRH-stimulated cAMP production (35). In term pregnant myometrium, the inhibitory action of OT on CRH-stimulated cAMP production was reversed in the presence of the Myr-PKC inhibitor, and the sensitivity of the CRH receptor was increased. In preterm myometrium there was no effect of either OT or the PKC inhibitor on CRH-stimulated cAMP production. Although we cannot exclude other actions of OT on the CRH receptor resulting in increased sensitivity, we obtained further evidence for a direct phosphorylation effect of PKC (induced by OT) on the CRH receptor only in term myometrium with a direct phosphorylation assay.
At present, it is not clear how PKC can potentiate CRH action in the
pituitary while inhibiting it in the myometrium at term. It is possible
that different PKC isoenzymes are involved with tissue-specific action
or that the PKC actions target different CRH-R subtypes. Recently, in
pregnant myometrium we have identified several CRH-R messenger RNA
transcripts (20). These correspond to four spliced variants of the R1
receptor (1, 1ß, 1C, and a novel form named 1d) and the
R2 receptor. At the moment it is uncertain which of these subtypes
mediates the OT effect, and studies using recombinant receptor subtypes
are necessary to delineate these events.
In summary, these data suggest that in human pregnant (at term)
myometrium there is an inhibitory action of OT on CRH biological
activity that is mediated via activation of PKC and subsequent
phosphorylation and desensitization of the CRH receptor. CRH-OT
interactions may play a primary, and important, role in the fine
balance of myometrial contractility. Furthermore, these interactions
appear to be dependent on both OT receptor expression (36) and
up-regulation of PKC activity at term. Currently, the role of CRH
during pregnancy is unknown, and the presence of multiple CRH receptor
subtypes in the human pregnant (at term) myometrium suggests distinct
functional roles for each receptor during pregnancy and raises the
possibility of multiple roles for CRH and/or related peptides. It is
attractive to speculate that the OT-sensitive CRH receptors are
responsible mainly for the generation of cAMP and the stimulation of
myometrial relaxation; inhibition of their biological activity by OT
may enable CRH to play a different role in the control of uterine
contractility and the mechanism of labor. We speculate that the major
CRH receptor subtype influenced by OT is R1, as in transfection
studies we have shown that this subtype is most efficiently linked to
adenylate cyclase (unpublished observations). The interactions of the
CRH and OT signal transduction pathways are shown diagrammatically in
Fig. 6. Further studies are under way to
investigate in-depth the role of phosphorylation on the function of the
CRH receptor by using mutant receptor technology where the potential
PKC phosphorylation sites have been mutated.
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Footnotes |
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2 Holds the WPH Charitable Trust Chair of Medicine.
Received July 22, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and CRF2ß receptor mRNAs are
differentially distributed between the rat central nervous system and
peripheral tissues. Endocrinology 136:4139–4142[Abstract]
:
a novel CRH2 receptor isoform found in the human brain. Soc Neurosci
Abstr 23:1765 (Abstract)
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R. L. Hauger, J. A. Olivares-Reyes, S. Braun, K. J. Catt, and F. M. Dautzenberg Mediation of Corticotropin Releasing Factor Type 1 Receptor Phosphorylation and Desensitization by Protein Kinase C: A Possible Role in Stress Adaptation J. Pharmacol. Exp. Ther., August 1, 2003; 306(2): 794 - 803. [Abstract] [Full Text] [PDF]
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 E. Aggelidou, E. W. Hillhouse, and D. K. Grammatopoulos Up-regulation of nitric oxide synthase and modulation of the guanylate cyclase activity by corticotropin-releasing hormone but not urocortin II or urocortin III in cultured human pregnant myometrial cells PNAS, February 14, 2002; (2002) 52296399. [Abstract] [Full Text] [PDF]
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 G. Gimpl and F. Fahrenholz The Oxytocin Receptor System: Structure, Function, and Regulation Physiol Rev, April 1, 2001; 81(2): 629 - 683. [Abstract] [Full Text] [PDF]
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J. R.G. Challis, S. G. Matthews, W. Gibb, and S. J. Lye Endocrine and Paracrine Regulation of Birth at Term and Preterm Endocr. Rev., October 1, 2000; 21(5): 514 - 550. [Abstract] [Full Text]
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D. K. Grammatopoulos, Y. Dai, H. S. Randeva, M. A. Levine, E. Karteris, A. J. Easton, and E. W. Hillhouse A Novel Spliced Variant of the Type 1 Corticotropin-Releasing Hormone Receptor with a Deletion in the Seventh Transmembrane Domain Present in the Human Pregnant Term Myometrium and Fetal Membranes Mol. Endocrinol., December 1, 1999; 13(12): 2189 - 2202. [Abstract] [Full Text]
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 D. K. Grammatopoulos and E. W. Hillhouse Basal and Interleukin-1{beta}-Stimulated Prostaglandin Production from Cultured Human Myometrial Cells: Differential Regulation by Corticotropin- Releasing Hormone J. Clin. Endocrinol. Metab., June 1, 1999; 84(6): 2204 - 2211. [Abstract] [Full Text]
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E. Aggelidou, E. W. Hillhouse, and D. K. Grammatopoulos Up-regulation of nitric oxide synthase and modulation of the guanylate cyclase activity by corticotropin-releasing hormone but not urocortin II or urocortin III in cultured human pregnant myometrial cells PNAS, March 5, 2002; 99(5): 3300 - 3305. [Abstract] [Full Text] [PDF]
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 E. S. Park, C. O. Echetebu, S. Soloff, and M. S. Soloff Oxytocin stimulation of RGS2 mRNA expression in cultured human myometrial cells Am J Physiol Endocrinol Metab, March 1, 2002; 282(3): E580 - E584. [Abstract] [Full Text] [PDF]
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