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Original Studies |
The Sir Quinton Hazel Research Centre for Molecular Medicine, Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom
Address correspondence and requests for reprints to: Professor E. W. Hillhouse, Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom. E-mail: eh{at}dna.bio.warwick.ac.uk
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Using the nonhydrolysable photoreactive analog [
-32P]
GTP-azidoanilide and peptide antisera raised against G protein
-subunits, we studied coupling of CRH receptors to G proteins in
both placental and fetal membranes. Treatment of placental membranes
with human CRH (100 nM) increased the labeling of Gq, Go,
and Gz but not Gi and Gs. Treatment of fetal membranes with human CRH
(100 nM) increased the labeling of Go and Gq but not Gi,
Gs, and Gz. These results were supported by experiments that showed
that CRH failed to activate adenylate cyclase in these tissues, but
induced an increase in inositol phosphates instead. These findings
provide new insights into the components of the signal transduction
machinery in both fetal and placental membranes and suggest that CRH
Type-1 receptors can couple to different G proteins in different
tissues. The physiological significance of these observations remains
to be elucidated.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The human placenta and fetal membranes synthesize and secrete large amounts of CRH (7, 8, 9), the circulating concentrations of which rise exponentially in the third trimester of pregnancy. In late pregnancy, there is a concomitant fall in concentrations of CRH-binding protein, which increases CRH bioactivity (10, 11). The timing of those events coincides with the onset of parturition, suggesting that CRH may act directly as a trigger for parturition in humans (11).
The addition of CRH and a CRH-like peptide, Urocortin, to primary
trophoblast cell cultures stimulates ACTH secretion in a dose-dependent
manner (12, 13). In addition, CRH stimulates prostaglandin production
in human fetal membranes and placenta (14) and induces vasodilation in
the human fetal-placental circulation via a nitric oxide-cGMP-mediated
pathway (15). These effects are mediated by specific CRH receptors,
localized in the feto-placental tissues (16, 17, 18, 19). We have recently
demonstrated the presence of CRH-R1 and CRH-Rc receptor subtypes,
but not the Type-2 in placenta and fetal membranes (20).
In most tissues, such as pituitary, myometrium, heart, and brain, CRH exerts its actions by stimulating adenylate cyclase and cAMP production (21, 22, 23). In cultured myometrial cells expressing functional CRH receptors, CRH was able to stimulate cAMP production in a dose-dependent manner (24). Other agents, such as the vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypetide, also caused a dose-dependent increase in cAMP in myometrial cells (25). This has also been shown in stably transfected cells that the type 1 CRH receptor can couple to adenylate cyclase and weakly to phospholipase C (26, 27). The CRH-Rc receptor variant has an exon deletion (40 amino acids) from the amino-terminal domain that impairs its ability to bind to CRH (28).
Because guanine nucleotide-binding proteins provide a regulatory link between action at a receptor site and second messengers, the identification and functionality of the G proteins involved in the feto-placental activity are essential in understanding the influence exerted by extracellular ligands. In view of all these observations, we sought to investigate the functional characteristics of the CRH receptors in human fetal and placental tissues and their potential implication in the onset of labor and parturition in humans.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fetal and placental membranes were obtained from women at term undergoing normal uncomplicated vaginal deliveries (n = 8). Immediately after delivery, the maternal and fetal surfaces of the placenta were dissected off, and fetal membranes were peeled away from the placenta. Samples were washed in phosphate-buffered saline (PBS) and immediately snap-frozen in liquid nitrogen. Informed consent was obtained from each woman, and ethical approval was granted.
Chemicals
Human CRH, isoproterenol, cholera and pertussis toxins,
forskolin, GTP, GppNHp and GTP
s, and all other chemicals were
purchased from Sigma (Poole, UK). The antisera against Gz
-subunit was purchased from Calbiochem (Nottingham,
UK). Anti-G protein antibodies GC/2, AS/7, RM/1, and QL, directed
against the -subunits, were obtained from New England Nuclear-DuPont
(Boston, MA). All primary antibodies were raised in rabbits. AS/7,
RM/1, and QL were polyclonal, whereas GC/2 and Gz antibodies were
monoclonal.
All electrophoretic reagents were of the highest grade available and
obtained from Bio-Rad Laboratories, Inc. (Richmond, CA).
cAMP RIA kit was purchased from New England Nuclear-DuPont, and
myo-[2-3H] inositol and
[-32P] GTP (1000 Ci/mmol) were purchased
from Amersham Pharmacia Biotech (Little Chalfont, UK).
4-azidoaniline hydrochloride and
1-(3-dimethylaminopropyl)-3-ethylenecarbodiimide were from
Aldrich Chemical Co., Inc. (Milwaukee, WI).
Preparation of fetal and placental membranes
Tissues were weighed and homogenized in 6 mL Dulbecco’s PBS containing 10 mM MgCl2, 2 mM EGTA, 0.15% BSA (wt/vol), 0.15 mM bacitracin, and 1 mM phenylmethylsulfonyl fluoride (pH 7.2; extraction buffer) at 22 C for 40 sec. The homogenate was centrifuged at 3000 rpm for 30 min at 4 C. The resultant pellet was washed, resuspended in extraction buffer, and spun at 19,000 rpm for an additional 60 min at 4 C. The final pellet was resuspended in extraction buffer using homogenizer for 20 sec.
Binding studies
Fetal and placental membrane suspensions (100 µg) were added to polypropylene tubes with 50 µL 125I-oCRH (50,000 cpm) and 50 µL extraction buffer or unlabeled peptide (diluted in buffer). The tubes were incubated at 22 C for 2 h, after which the reaction was terminated by the addition of 1 mL ice-cold polyethylene glycol (20% wt/vol). The tubes were spun at 3000 rpm for 30 min at 4 C. The supernatant was discarded, and the membrane-bound radioactivity present in the pellet was measured in a gamma-counter.
cAMP studies
Fetal and placental membrane suspensions (100 µg) were incubated with increasing concentrations of CRH and other agonists for 30 min at 22 C in 50 µL incubation buffer, containing 50 mM Tris-HCl, 10 mM MgCl2, 1 mM EGTA, 0,15% BSA (wt/vol), 0.15 mM bacitracin, and 500 mM isobutyl methyl xanthine (pH 7.4). Fetal and placental membranes were incubated with 5.5 µL/tube for 10 min at 37 C of an ATP generation system composed of ATP, creatine phosphate, and phosphocreatine kinase. The reaction was terminated by the addition of 1 mL 0.1 M Imidazole buffer, followed by heating the tubes in boiling water for 10 min. The amount of cAMP in the incubate was determined by RIA. Standard cAMP concentrations, covering the range 0.138–100 pmol/mL, were used for determination of the standard curve of the RIA. The interassay coefficient of variation was 8%. cAMP assay buffer (without any membrane preparations) was used as the negative control.
Effect of CRH on inositol phosphate accumulation
Human fetal membrane and placental explants (n = 6) were teased into small pieces, carefully removing any obvious blood vessels or clots. The tissues were re-washed several times with PBS and placed afterward in sterile centrifuge tubes with 1.5 mL inositol-free DMEM: Ham’s F12 nutrient mixture (1:1) containing 1% BSA, 50 U/mL penicillin, 50 µg/mL streptomycin, and 10 µCi/mL of myo-[2-3H] inositol for 6–8 h at 37 C in a 5% CO2 incubator. After incubation, explants were washed once with DMEM/Ham’s F12 containing 1% BSA, 50 U/mL penicillin, and 50 µg/mL streptomycin. Inositol-free DMEM/Ham’s F12 medium containing 0.1% BSA and 30 mM LiCl was then added for further incubation for 30 min at 37 C in a 5% CO2 incubator. The addition of LiCl is necessary because in many mammalian tissues LiCl has been shown to increase greatly the accumulation of inositol phosphates. Phosphoinositide turnover was stimulated with human/rat (h/r) CRH (100 nM) in the presence of 30 mM LiCl, and the reactions were stopped by the addition of chloroform/methanol/hydrochloric acid (50:100:1) at specified time intervals. The contents transferred to borosilicate glass tubes were centrifuged for 10 min at 4000 rpm. The transparent, supernatant phase was then applied into prefilled Poly-Prep columns (Dowex 1-X8 100–200 mesh, chloride form; Bio-Rad Laboratories, Inc., York, UK) and elute by 14 mL of: 1) water for free inositol; 2) 5 mM sodium tetraborate/150 mM ammonium formate for inositol phosphate (IP); 3) 100 mM formic acid/400 mM ammonium formate for inositol biphosphate (IP2); and 4) 100 mM formic acid/0.8 M ammonium formate for inositol triphosphate (IP3). To determine the radioactivity of each fraction by liquid scintillation counting, each sample of the column eluates was mixed with 3.5 mL scintillation fluid (Wallac, Inc. Turku, Finland).
The chloride form of Dowex resin was converted to the formate form before use by passage of 3 M ammonium formate through the resin in a 40 mm x 450-mm column until acidified silver nitrate gave no reaction, followed by deionized water to neutrality.
Immunoblotting
Fetal and placental membranes (100 µg) were centrifuged at 13,000 rpm for 15 min at 4 C. The supernatant was then discarded, and the resultant pellets were solubilized with Laemmli buffer [5 M urea, 0.17 M SDS, 0.4 M dithiothreitol, and 50 mM Tris-HCl (pH 8.0)], mixed and placed in a boiling-water bath for 5 min, and allowed to cool at room temperature.
Samples were separated on a SDS-10% polyacrylamide gel, and the
proteins were electrophoretically transferred to a nitrocellulose
filter at 250 mA for 16–18 h in a transfer buffer containing 20
mM Tris, 150 mM glycine, and 20% methanol. The
filter was then blocked in PBS containing 0.1% Tween 20 and 5% dried
milk powder (wt/vol), for 2 h at room temperature. After three
washes with PBS-0.1% Tween, the nitrocellulose filters were incubated
with primary antibodies against all different G protein -subunits.
All primary antisera were used at a 1:1000 dilution in PBS-0.1% Tween
for 1 h at room temperature. The filters were washed thoroughly
for 30 min with PBS-0.1% Tween, before incubation with the secondary
antirabbit horseradish peroxidase-conjugated immunoglobulin (1:2000)
for 1 h at room temperature and further washing for 30 min with
PBS-0.1% Tween.
To detect the antibody complexes, solution A containing 100 mM Tris (pH 8.0) and 30% H2O2 was mixed with solution B containing 100 mM Tris (pH 8.0), 90 mM coumaric acid, and 250 mM Luminol, and applied to immunoblots for 2 min at room temperature. Immunoblottings were visualized using Kodak (Rochester, NY) Biomax MR x-ray film, and were replicated at least twice on each batch of tissue.
Treatment of membranes with pertussis and cholera toxins
Both pertussis (50 µg/mL) and cholera (150 µg/mL) toxin were preactivated in 0.05 M Tris buffer (pH 7.5) containing 20 mM dithiothreitol (DTT) and 50 mM glycine for 45 min at 37 C in a final volume of 50 µL and cooled on ice for 20 min. Fetal and placental membranes (100 µg) were incubated in 20 mM Tris (pH 7.5) containing 1 mM EDTA, 1 mM DTT, 1 mM ATP, 1 mM GTP, 5 mM MgCl2 10 mM thymidine, 10 µM NAD, and 5 µCi [32P]NAD, together with the preactivated toxins. All reactions were carried out at 37 C for 30 min, and the incubations were terminated with 0.7 mL ice-cold 20 mM Tris buffer (pH 7.5) containing 1 mM EDTA. Control samples were prepared by incubating membranes in the same medium, but in the absence of any toxin. After termination, samples were centrifuged at 13,000 rpm for 20 min, and the pellets were washed and respun three times. The resultant pellets were resuspended in 100 µL 2% SDS and 320 µL buffer D [1% (v/v) Triton X-100, 1% deoxycholate, 0.5% (w/v) SDS, 150 mM NaCl, 10 mM Tris-HCl (pH 7.4), 1 mM DTT, 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluuoride, and 10 µg/mL aprotinin]. Resuspended samples were centrifuged at 11,000 rpm for 10 min at room temperature, and the resulted supernatants were equally aliquoted (200 µL). Into each of these aliquots 10 µL of Gi, Go, and Gs antisera were added and left for continuous agitation for 2 h, followed by the addition of 60 µL protein A-Sepharose per tube and further agitation overnight at 4 C. The samples were then centrifuged at 12,000 rpm for 10 min, and the pellets were solubilized with Laemmli buffer and mixed and placed in a boiling-water bath for 5 min before cooling to room temperature. Each sample was loaded on an SDS-10% polyacrylamide gel, and after electrophoresis, the gels were dried and autoradiographed using Kodak x-ray film, to assess the extent of ADP-ribosylation.
Synthesis and purification of
[-32P]GTP-azidoanilide (GTP-AA)
[-32P]GTP (1 mCi) was evaporated to
dryness under vacuum. The residue was dissolved in 60 µL of a
solution of 1-(3-dimethylaminopropyl)-3-ethylenecarbodiimide (30 mg/mL)
in 0.1 M MES 2-[N-morpholino]ethanesulfonic acid, (pH 5.6
adjusted with NaOH 10 M), plus 40 µL of a suspension of
azidoaniline-HCl (40 mg/mL) in 1,4-dioxane. The reaction mixture was
incubated overnight at room temperature, in the dark with constant
agitation. The GTP-AA was purified by hydrophobic interaction
chromatography using a C-18 Sep Pak cartridge. The cartridge was
prewetted with 5 mL methanol and equilibrated with 97% buffer A and
3% buffer B. Buffer A (100 mM triethylamine in water) and
buffer B (100 mM triethylamine in ethanol) were gassed with
neat CO2 to obtain a pH of 7.0 at room
temperature. The sample (reaction mixture) was dissolved in 1 mL
equilibrating buffer (97% buffer A and 3% buffer B) and applied to
the cartridge. The cartridge was washed with 10 mL equilibrating
buffer, and the solution was collected in 1-mL aliquots (tubes 1–10).
The GTP-AA was eluted from the cartridge with 5 mL 10% buffer A and
90% buffer B and collected in 0.5-mL fractions. Aliquots (1/10,000)
were added to vials containing scintillation liquid, and
32P was quantified by scintillation
spectrophotometry. Fractions containing GTP-AA were combined,
evaporated to dryness under vacuum, and resuspended in water to yield a
concentration of 1 µCi/µL The overall yield of GTP-AA varied from
45–60%. All procedures were performed in a darkened room.
Photoaffinity labeling of -subunits
Plasma membranes (150–200 µg) were incubated for 3 min at 30 C with the agonist (CRH, 100 nM) in buffer C (50 mM HEPES, 30 mM KCl, 10 mM MgCl2, 1 mM benzamidine, and 0.1 mM EDTA), followed by the addition of 5 µM GDP and 6 µCi GTP-AA. After incubation for 3 min at 30 C in a darkened room, membranes were placed on ice and collected by centrifugation at 13,000 rpm for 15 min at 4 C. The supernatant was carefully removed, and the membrane pellet was resuspended in 120 µL modified buffer C (1.6 mg DTT in 5 mL buffer C). Samples were vortexed and irradiated for 5–10 min at 4 C with an ultraviolet light (254 nm, 0.16 A, 115V) from a distance of 5 cm, to cross-link the GTP-AA to the G proteins.
Immunoprecipitation
GTP-AA-labeled membranes were collected by centrifugation at 13,000 rpm, for 15 min at 4 C, and solubilized by repeated pipetting in 120 µL 2% SDS, and 360 µL buffer D were added on ice for 30 mins. Solubilized membranes were divided into 120-µL aliquots. Each aliquot was incubated with 10 µL undiluted G protein antisera at 4 C, with continuous agitation. After 2 h, 60 µL of a suspension of protein A-Sepharose was added, and the incubation was continued overnight at 4 C, with continuous agitation. The protein A-Sepharose beads were collected by centrifugation at 13,000 rpm, for 10 min at 4 C, and washed twice with 30 µL of a solution containing 300 mM NaCl, 100 mM Tris-HCl (pH 7.4), and 10 mM EDTA, followed by a further centrifugation at 13,000 rpm for 15 min at 4 C. The supernatant was discarded, and the immune complexes were dissociated from protein A-Sepharose by reconstitution in Laemmli buffer (20–40 µL) and boiling for 5 min. Proteins were resolved by electrophoresis through 10% SDS-polyacrylamide gels. The gels were dried under vacuum for 90 min and exposed to an autorad Kodak Biomax MR film for 3–7 days at -70 C.
Statistical analysis
Data are shown as the mean ± SEM of each measurement. In each case, results were evaluated between groups by using a two-tailed Student’s t test, with significance determined at the level of P < 0.05. Statistical ANOVA was also performed measuring the intensity of immunoreactive staining using a scanning densitometer (Scion Image, Frederick, MD).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The presence of functional CRH receptors in both tissues was
confirmed by binding displacement studies. Human/rat CRH was able to
displace radiolabeled ovine CRH (oCRH) from its binding sites in
a concentration-dependent manner in both tissues (Fig. 1
). The specificity of the receptor was
assessed by incubating with the unrelated peptide arginine vasopressin
(at concentrations up to 100 nM), which was unable to
displace radiolabeled oCRH binding from either group.
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Next, we determined the effect of incubation with CRH on cAMP
production. When either fetal and placental membranes were incubated
with CRH (10 pM-100 nM) for 30 min at room
temperature, there was no significant increase in cAMP production. As a
positive control, we used the ß2 adrenergic
agonist isoproterenol (10 µM), which was able to
stimulate cAMP production in both tissues (Table 2) (P
< 0.05).
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-helical
CRH (9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41), at a concentration of 100 µM/L. This
produced a 47% and 54% inhibition of the CRH inositol phosphate
production for the fetal and placental membranes, respectively. There
was no effect on IP3, IP2,
or IP production with isoproterenol (10 µM) treatment
(Table 2
). Effect of pertussis toxin pretreatment
One possible explanation for the inability of CRH to stimulate
cAMP production may be the involvement of inhibitory G proteins. To
test this, both membranes were pretreated with pertussis toxin, which
catalyzes the transfer of the ADP-ribose of NAD+
to a cysteine residue close to the carboxy terminus of the -subunit
of the G inhibitory proteins. The ADP-ribosylated form of Gi cannot
sustain the inhibition of adenylate cyclase. Both fetal and placental
membranes were pretreated with three different concentrations of
pertussis toxin (25, 50, and 100 µg/mL) and were further incubated
with h/r CRH 100 nM for 30 min at room temperature.
However, none of the above concentrations altered significantly the
effect of h/r CRH on cAMP production (data not shown).
Effect of forskolin and guanine nucleotides
The functional capacity of adenylate cyclase in both tissues was
tested with the use of the diterpene forskolin, which acts directly on
the catalytic subunit of the adenylate cyclase. We found that forskolin
stimulated adenylate cyclase activity in a dose-dependent manner in
both tissues, thus confirming the functional integrity of adenylate
cyclase in our preparations. Interestingly, higher doses of forskolin
(10-4–10-6
M), produced a significantly higher stimulation of
adenylate cyclase activity in fetal than placental membranes
(P < 0.001) (Fig. 2A).
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s > GppNHP > GTP. The lower stimulation of
cAMP with GTP is consistent with the fact that the proteins possess an
endogenous GTPase activity. In fetal membranes, however, GTPs
induced a significantly (P < 0.001) higher increase in
adenylate cyclase activity than in placental membranes (Fig. 2B). Immunoblotting
To confirm the presence of the relevant G protein subunits in both
tissues, immunoblotting was performed using antibodies to the specific
G protein subunits. The antibodies used in these studies are listed in
Table 1.
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i1 and i2. This
antibody detected a band of 41 kDa in both fetal and placental
membranes (Fig. 3), suggesting the
presence of Gi1/2 subunit.
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s
antibody RM1 (C terminus), detected four s species of apparent
molecular weight of 45, 47, 54, and 67 kDa, respectively, in placental
membranes. In fetal membranes, however, only the 45-, 47-, and 67-kDa
bands were detected (Fig. 3). The additional band present in the
placental samples may be due to alternative splicing of the single
precursor of Gs messenger RNA.
Detection of Gq.Probing with QL, a specific antibody for
q, 11 (C terminus), detected one band at 42.5 kDa in both fetal
and placental membranes (Fig. 3).
Detection of Go.Immunoblotting with a specific o antibody
(GC/2) (N terminus) detected two bands of 40.5 kDa in fetal and
placental membranes (Fig. 3).
Detection of Gz.Probing with this specific antibody (N
terminus), which does not cross-react with any of the other inhibitory
G proteins, we were able to detect z as a 40-kDa protein in both
tissues (Fig. 3).
ADP ribosylation
Cholera toxin treatment.Incubating membranes with cholera
toxin resulted in the incorporation of [32P] ADP ribose
into three bands of 45, 47, and 67 kDa for the placental membranes and
into two bands of 47 and 67 kDa for the fetal membranes. Incorporation
of label was similar for both tissues (Fig. 4a).
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i1 and
Go, in both fetal and placental membranes (Fig. 4b). Incorporation
of label into the 40.5-kDa band was the same for both tissues, whereas
more label was incorporated into the Gi of the fetal membranes. No
incorporation of label was seen in either fetal or placental tissues in
the absence of the pertussis toxin. Photoaffinity labeling with GTP-AA
To determine which G proteins are coupled to the CRH receptors in
fetal and placental membranes, we used GTP-AA to label G protein
-subunits activated by CRH (100 nM). Both membranes were
labeled with GTP-AA in the presence or absence of CRH, and the
-subunits of various G proteins (i.e. Gs, Gq, Gi, Go, and
Gz) were immunoprecipitated. A significant amount of GTP-AA was
incorporated into -subunits even in the absence of agonist.
Treatment of placental membranes with CRH increased the labeling of Gq,
Go, and Gz but not Gi and Gs (Fig. 5).
Treatment of fetal membranes with CRH increased the labeling of Go and
Gq but not Gi, Gs, and Gz (Fig. 5). Quantification of the amount of
radioactivity bound by immunoprecipitated -subunits is shown in Fig. 6.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and CRH-Rc receptors, but not CRH-R2 receptors, are
present in placenta and fetal membranes (20). Although CRH exerts an
effect on prostaglandin production in the feto-placental unit (14),
little is known about how CRH mediates its effects. Using
125I-oCRH, we confirmed the presence of specific,
high-affinity, saturable binding sites in the human placental and fetal
membranes (17).
CRH receptors in most tissues are coupled preferentially to adenylate
cyclase, except in the rat Leydig cells (29), where they couple to
phospholipase C. Our results demonstrate for first time that the CRH
receptors in human placental and fetal membranes do not couple to Gs
and adenylate cyclase, although significant (P < 0.05)
increase in total inositol phosphate (IP3,
IP2, IP) accumulation was detected in response to
CRH (100 nM) (Table 2). There was a 7-fold increase in total
IP production in placental membranes and a 5-fold increase in fetal
membranes. This increase was blocked when the CRH anatagonist
-helical was used, suggesting that CRH exerts its effects via
specific CRH receptors that are coupled to Gq -subunits.
Gs, however, and the main classes of G proteins are expressed in
human placenta (30, 31) and fetal membranes. Immunoblotting analysis
for Gs -subunits revealed the presence of four splice variants in
the human placenta with molecular weights at 44, 47, 54, and 67 kDa,
whereas in the fetal membranes the 54 kDa was not present. The
functional significance of this different pattern is unknown, but is
currently under investigation.
Our results show that the integrity of the membrane-bound
components of the signal transduction system is intact. In the present
study, we have used GTP-AA, a nonhydrolysable, photoreactive analog, to
label G protein -subunits that are coupled to the CRH receptor. The
main reason why CRH fails to activate Gs is that the CRH receptors
do not couple to this subunit, as shown by the photo-affinity labeling
experiments. However, the CRH receptors in the placenta membranes can
activate the Gq, Go, and Gz in placental membranes. More importantly,
coupling of the receptor with the PTX-insensitive Gz inhibitory protein
(32), would also be responsible for low levels of cAMP production,
because Gz can replace Gi in mediating inhibition of cAMP accumulation,
but not in the stimulation of phospholipace C (33, 34). This is the
first time that CRH receptors have been shown to activate this
ubiquitous G inhibitory protein. In addition, a small but significant
increase in labeling of Go and Gq but not Gs, Gi, and Gz was observed
in fetal membranes. The fact that CRH receptors do not couple to Gz may
be due to extremely low protein levels of this protein in these
tissues.
It has been demonstrated, using photo-affinity labeling, that CRH
and urocortin can couple to and activate different G proteins in human
pregnant myometrium (35), and CRH can activate a number of different G
proteins in HEK 293-cells transfected with the CRH-1 receptor (36).
The findings of the present study provide a better understanding of the
functionality of the CRH receptors in the feto-placental unit and their
potential to couple to multiple G proteins that may subserve
tissue-specific functions. Moreover, it has been also demonstrated (37)
that Gs regulates the trans-Golgi apical surface transport pathway in
an epithelial cell line. Therefore, it has been suggested (38) that Gs
of the human syncytiotrophoblasts might play a role in intracellular
transport in the fetoplacental unit, rather than having a signaling
function.
In a number of different tissues, CRH exerts its actions in a cAMP-independent manner. In rat Leydig cells, CRH receptors interact with a PTX-insensitive G protein, leading to the rapid translocation of protein kinase C (PKC) (26, 39) and inhibition of stimulation of cAMP. Moreover, CRH increases calcium influx via an activation of CRH receptors in a cAMP-independent mechanism (40) in cultured rat astrocytes, suggesting that the increase in calcium is due to direct coupling with receptor-linked channels. CRH has also provoked significant increases in inositol trisphosphate (IP3) in human fetal adrenal cortical cells that was dose and time dependent, whereas cAMP levels remained unaltered (41).
The functional coupling of the CRH receptors to Gq and increase in phosphoinositol turnover would account for the induction of prostaglandin biosynthesis by CRH in both tissues (14). A number of studies have shown that activation of PKC has been shown to stimulate the release of arachidonic acid and prostaglandins in human amnion cells (42, 43). In addition, PKC activation is a fundamental requirement for oxytocin-induced prostaglandin production in human amnion cells (44) and may also regulate the release of arachidonic acid through inhibition of acyltransferase, thereby increasing free available arachidonate and stimulating prostaglandin synthesis in human platelets (45). Moreover, diacylglycerol usually contains arachidonic acid and can directly provide this precursor for prostaglandin synthesis within the cell (46).
Studies have shown that protein kinase A activators, such as forskolin or cAMP analogs, inhibit agonist-induced prostaglandin production in human fetal membranes (47). Evidence also suggests that CRH induces the transcription of the type-2 cyclo-oxygenase gene in human fetal membranes. Once again, the involvement of a Gq/PKC pathway, rather the Gs/cAMP pathway, seems to be more important because PKC activation has been shown to promote type-2 cyclooxygenase messenger RNA expression in human fetal membranes (48, 49). These data, therefore, would provide a physiological explanation why CRH cannot activate the adenylate cyclase/cAMP system in human placenta and fetal membranes.
These findings illustrate the diversity of CRH receptor signaling pathways in human placental and fetal membranes and further support an autocrine role for placental CRH during pregnancy.
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Footnotes |
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2 Wellcome Career Development Fellow.
3 WPH Charitable Trust Chair of Medicine.
Received August 4, 1999.
Revised November 10, 1999.
Accepted January 18, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and the CRH-C variant receptor. J Clin Endocrinol Metab. 83:1376–1379.-subunit of
Gz couples neurotransmitter receptors to ion channels in sympathetic
neurons. Neuron. 21:1201–1212.[CrossRef][Medline]
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T. Teli, D. Markovic, M. A. Levine, E. W. Hillhouse, and D. K. Grammatopoulos Regulation of Corticotropin-Releasing Hormone Receptor Type 1{alpha} Signaling: Structural Determinants for G Protein-Coupled Receptor Kinase-Mediated Phosphorylation and Agonist-Mediated Desensitization Mol. Endocrinol., February 1, 2005; 19(2): 474 - 490. [Abstract] [Full Text] [PDF]
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N. Papadopoulou, J. Chen, H. S. Randeva, M. A. Levine, E. W. Hillhouse, and D. K. Grammatopoulos Protein Kinase A-Induced Negative Regulation of the Corticotropin-Releasing Hormone R1{alpha} Receptor-Extracellularly Regulated Kinase Signal Transduction Pathway: The Critical Role of Ser301 for Signaling Switch and Selectivity Mol. Endocrinol., March 1, 2004; 18(3): 624 - 639. [Abstract] [Full Text] [PDF]
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 E. Karteris, E. W. Hillhouse, and D. Grammatopoulos Urocortin II Is Expressed in Human Pregnant Myometrial Cells and Regulates Myosin Light Chain Phosphorylation: Potential Role of the Type-2 Corticotropin-Releasing Hormone Receptor in the Control of Myometrial Contractility Endocrinology, February 1, 2004; 145(2): 890 - 900. [Abstract] [Full Text] [PDF]
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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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 K. J. McKeown and J. R. G. Challis Regulation of 15-Hydroxy Prostaglandin Dehydrogenase by Corticotrophin-Releasing Hormone through a Calcium-Dependent Pathway in Human Chorion Trophoblast Cells J. Clin. Endocrinol. Metab., April 1, 2003; 88(4): 1737 - 1741. [Abstract] [Full Text] [PDF]
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E. Karteris, A. Goumenou, E. Koumantakis, E. W. Hillhouse, and D. K. Grammatopoulos Reduced Expression of Corticotropin-Releasing Hormone Receptor Type-1{alpha} in Human Preeclamptic and Growth-Restricted Placentas J. Clin. Endocrinol. Metab., January 1, 2003; 88(1): 363 - 370. [Abstract] [Full Text] [PDF]
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 S. Zervou, E. Karteris, E.W. Hillhouse, and R.W. Old Steroids mediate the expression of cytoplasmic and membrane-linked components in human myometrial cells Mol. Hum. Reprod., July 1, 2002; 8(7): 597 - 605. [Abstract] [Full Text] [PDF]
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 E. Dermitzaki, C. Tsatsanis, A. Gravanis, and A. N. Margioris Corticotropin-releasing Hormone Induces Fas Ligand Production and Apoptosis in PC12 Cells via Activation of p38 Mitogen-activated Protein Kinase J. Biol. Chem., March 29, 2002; 277(14): 12280 - 12287. [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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H. S. Randeva, E. Karteris, D. Grammatopoulos, and E. W. Hillhouse Expression of Orexin-A and Functional Orexin Type 2 Receptors in the Human Adult Adrenals: Implications for Adrenal Function and Energy Homeostasis J. Clin. Endocrinol. Metab., October 1, 2001; 86(10): 4808 - 4813. [Abstract] [Full Text] [PDF]
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E. Karteris, H. S. Randeva, D. K. Grammatopoulos, R. B. Jaffe, and E. W. Hillhouse Expression and Coupling Characteristics of the CRH and Orexin Type 2 Receptors in Human Fetal Adrenals J. Clin. Endocrinol. Metab., September 1, 2001; 86(9): 4512 - 4519. [Abstract] [Full Text] [PDF]
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S. R. Smith, L. de Jonge, M. Pellymounter, T. Nguyen, R. Harris, D. York, S. Redmann, J. Rood, and G. A. Bray Peripheral Administration of Human Corticotropin-Releasing Hormone: A Novel Method to Increase Energy Expenditure and Fat Oxidation in Man J. Clin. Endocrinol. Metab., May 1, 2001; 86(5): 1991 - 1998. [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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