Molecular Human Reproduction, Vol. 7, No. 4, 397-402,
April 2001
© 2001 European Society of Human Reproduction and Embryology
EP4 receptors mediate prostaglandin E2-stimulated glycosaminoglycan synthesis in human cervical fibroblasts in culture
1 INSERM U 361, Paris and 2 Maternité Port-Royal, Hopital Cochin, AP-HP, Université René Descartes, Paris V, France
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
The aim of this study was to determine the prostaglandin E (EP) receptors and second messengers implicated in glycosaminoglycan (GAG) synthesis by human cervical fibroblasts in culture. Human cervical fibroblasts were obtained from cervical biopsies in pre-menopausal, cycling women. Cultured cells were incubated with prostaglandin E2 (PGE2) and an array of agonists and antagonists. Glycosaminoglycan synthesis was assayed after extraction by measuring the [3H]glucosamine and [35S]sulphate incorporated into GAG and cAMP production was determined by radioimmunoassay. PGE2 significantly stimulated GAG synthesis. Neither 17-phenyl-trinor-PGE2, the EP1 selective agonist, nor sulprostone, an EP3 agonist, had any effect on GAG production. Butaprost, the EP2 selective agonist, also failed to increase GAG synthesis. AH6809, an EP2 antagonist, had no effect on PGE2-stimulated GAG production. AH23848, an EP4 antagonist, inhibited the GAG synthesis provoked by PGE2. PGE2 and butaprost significantly increased cAMP production. Both AH6809 and AH23848 inhibited the PGE2-stimulated cAMP production. H89, a cAMP-dependent protein kinase (PKA) inhibitor, did not inhibit PGE2-stimulated GAG synthesis and Sp-cAMPS, a selective PKA activator, failed to increase GAG production. In conclusion, both EP4 and EP2 receptors are present and functional in human cervical fibroblasts. Only EP4 receptors mediate PGE2 stimulated GAG synthesis in a PKA-independent pathway.
cervical ripening/EP receptors/glycosaminoglycans/human parturition/prostaglandin E2
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
The uterine cervix plays an essential role during pregnancy; it stays tough and closed during gestation to allow intrauterine growth of the conceptus, but becomes a soft and compliant tissue capable of dilating to accommodate the passage of the fetus during labour. Understanding the mechanisms underlying cervical ripening in human pregnancy could be of great importance in clinical practice allowing, on the one hand, the induction of labour when a fetal or maternal pathology implies the termination of pregnancy, or, on the other hand, the postponement of delivery in threatened preterm labour. Cervical ripening is a complex combination of biochemical and structural processes resulting in alterations of the cervical connective tissue and leading to an extensible organ. Softening of the cervix is characterized by an oedema, a dispersion of the collagenic network and a glycosaminoglycan (GAG) redistribution (Uldbjerg et al., 1983
) which reflects metabolic changes in proteoglycans. Changes in GAG mainly consist of an increased cervical concentration of hyaluronic acid (Uldbjerg et al., 1983; Osmers et al., 1993), a hydrophylic hydrogenated GAG that plays a major role in oedema constitution, and a relative augmentation in sulphated GAG, with a decrease in dermatan sulphate (Osmers et al., 1993), implicated in the fibrillar organization of collagen (Uldbjerg et al., 1983).
Local application of prostaglandin E2 (PGE2) has been used clinically for several years to induce softening of the cervix at term pregnancy (Shepherd et al., 1979; Ekman et al., 1983). The changes in the composition of the cervical connective tissue after PGE2-induced cervical ripening are similar to those occurring in spontaneous cervical ripening. Indeed, PGE2 reproduces in vitro (Carbonne et al., 1996) and in vivo (Norström et al., 1982; Cabrol et al., 1987; Osmers et al., 1991; Norman et al., 1993) most of the biochemical features of physiological ripening of cervical tissue. PGE2 acts through seven-transmembrane domain, G protein-coupled receptors, called EP receptors (Narumiya et al., 1999). These receptors are present in human myometrium and cervix (Adelantado et al., 1988; Senior et al., 1991, 1993). The EP receptor family has been further classified into four subtypes: EP1, EP2, EP3 and EP4 (Coleman et al., 1984, 1994a). The coupling of each receptor subtype to different G proteins, activating different second messenger pathways, further increases the diversity of PGE2 cellular effects. In myometrium, mainly EP3 and EP1 are involved in contraction (Asboth et al., 1996), via the Ca2+/phospholipase C pathway, whereas EP2 provokes relaxation (Asboth et al., 1997–98; Hillock and Crankshaw, 1999), via the cAMP/adenylate cyclase pathway and the subsequent activation of protein kinase A (PKA). There is as yet no evidence for the presence of a functional EP4 receptor in the myometrium (Hillock and Crankshaw, 1999) and almost nothing is known about the presence, distribution and functionality of each EP receptor subtype in human cervix. However, we have already demonstrated that PGE2 stimulates GAG synthesis in cultured human cervical cells in a cAMP-dependent pathway (Carbonne et al., 1996).
The purpose of this study, performed in cultured human cervical fibroblasts, was to use an array of EP agonists and antagonists to determine the EP receptor subtypes implicated in PGE2-stimulated GAG synthesis which is usually considered to reflect PGE2-induced cervical ripening, and to explore the potential role for the suspected cAMP-dependent PKA transduction pathway in this process.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Culture of human cervical fibroblasts
Cervical biopsies were obtained after hysterectomy on hormonally active woman. Special care was taken in removing exoand endocervical epithelia. Cell cultures were performed as previously described (Carbonne et al., 1996
, 2000; Cavaillé et al., 1996). Briefly, explants were minced and plated out in 60 mm diameter plastic dishes in culture medium [Dulbecco's modified Eagle's medium (DMEM) containing 20% inactived fetal calf serum (FCS), 2 mmol/l glutamine and penicillin-streptomycin (100 UI/ml-100 µg/ml)]. Dishes were placed in a 5% CO2/95% air humidified incubator at 37°C. After 7 days, cells had started growing out from the explants and the medium was then replaced by DMEM containing only 10% FCS. Cells became confluent about 3 weeks after tissue collection and were then subcultured every 7 days in DMEM containing 10% FCS by trypsination. All experiments were performed between passages 3 and 6, on cells from five different explants. There was no noticeable morphological difference observed with cells from different passages, or with cells obtained from different uteri. The viability of the cells was checked by Trypan Blue exclusion.
GAG synthesis assay
Cervical cells were subcultured onto 24-well plastic culture plates at a density of 5x104 cells per well and allowed to grow to confluence. Cells were rinsed with PBS and incubated in serum-free DMEM without Phenol Red in the presence of PGE2 or an agonist (17-phenyl-trinor-PGE2, butaprost, sulprostone, Sp-cAMPS) or vehicle. Cells were preincubated with inhibitors (AH6809, AH23848, rolipram or H89) 30 min before adding the agonists. The radiolabelled GAG precursors, 2.5 µCi/ml [3H]glucosamine (precursor of hydrogenated GAG) and 2.5 µCi/ml [35S]sulphate (precursor of sulphated GAG), were then added for 24 h.
GAG extraction was conducted as previously described (Wasteson et al., 1973) and modified (Redini et al., 1991). Briefly, at the end of the labelling period, monolayer cells were washed with phosphate-buffered saline and digested with pronase (1 mg/ml) in 100 mmol/l Tris-HCl (pH 7.5)/5 mmol/l CaCl2. Proteolysis continued for 24 h at 56°C. GAG were precipitated at 37°C with 1% w/v cetylpyridinium chloride (CPC), in the presence of carriers (hyaluronic acid 1 mg/ml, chondroitin-4-sulphate and chondroitin-6-sulphate 0.5 mg/ml). The GAG-CPC complex was recovered by centrifugation and dissolved in 2 mol/l MgCl2. GAG were then precipitated overnight at 4°C with cold ethanol. The final pellet was dissolved in 75 mmol/l NaCl. The radioactivity incorporated into GAG was measured by liquid scintillation (Beckman LS 6000IC). For each experiment, assays were performed in quadruplicate. The intra-assay and inter-assay coefficents of variance were 6.5 and 25% for [3H]glucosamine and 9 and 38% for [35S]sulphate incorporation into GAG.
cAMP production assay
Human cervical cells were plated out on 35 mm diameter plastic culture dishes at a density of 8x104 cells per dish and allowed to grow to confluence as described above. Confluent cultures were washed and preincubated for 30 min with or without AH6809 or AH23848 in serum-free DMEM without Phenol Red. Reactions were initiated by adding PGE2 or butaprost in the presence 0.5 mmol/l IBMX. After 15 min incubation at 37°C, reactions were stopped by adding 10% trichloroacetic acid (TCA) solution. cAMP cell content was determined following a published method (Negishi et al., 1989) using a commercially available radioimmunoassay kit after TCA extraction with diethylether. For each experiment, assays were performed in duplicate.
Reagents
DMEM, FCS, penicillin, streptomycin, trypsin EDTA and phosphate-buffered saline were from Gibco BRL Life Technologies (Cergy Pontoise, France). [3H]Glucosamine, [35S]sulphate and the 125I-cAMP assay kit (Biotrak) were obtained from Amersham (Les Ulis, France). Pronase (from Streptomyces grisens) was purchased from Boehringer Mannheim (Meylan, France). 17-Phenyl-trinor-PGE2 and sulprostone were from Cayman chemicals (Ann Arbor, MI, USA). Butaprost was a gift from Bayer-AG (Leverkussen, Germany). AH6809 and AH23848 were given by GlaxoWellcome (Stevenage, Herts, UK). Sp-cAMPS was purchased from Biolog (Bremen, Germany). H89 was obtained from TEBU (Le Perray-en-Yvelines, France) and rolipram from Schering (Burgess Hill, Sussex, UK). PGE2 and all other reagents were from Sigma (St Louis, MO, USA).
Statistical analysis
The non-parametric Wilcoxon-Mann-Whitney test for paired samples was applied to compare the GAG synthesis or the cAMP production in cervical cells in culture. Results are expressed as mean ± SEM. The difference was considered significant when P < 0.05.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Effects of PGE2 and EP agonists and antagonists on GAG production
As previously demonstrated (Carbonne et al., 1996
), we confirmed that PGE2 produced a significant increase in the incorporation of both [3H]glucosamine and [35S]sulphate by human cervical fibroblasts in culture (Figure 1A, B). The stimulatory effect at 10–6 mol/l PGE2 was prominent on [3H]glucosamine uptake since the maximum incorporation into GAG was more than twice the control value for [3H] glucosamine and only 1.45 times for [35S]sulphate. PGE210–4 mol/l further increased the synthesis of hydrogenated GAG (Figure 1A).
|
Neither 17-phenyl-trinor-PGE2 10–6 mol/l, an EP1 selective agonist (Kiriyama et al., 1997
), nor sulprostone 10–6 mol/l, an EP3 >> EP1 agonist (Abramovitz et al., 2000) increased [3H]glucosamine and [35S]sulphate uptake into GAG (Figure 1A, B
). Butaprost 10–6 and 10–5 mol/l, the EP2 selective agonist (Gardiner, 1986), caused a slight augmentation in both [3H]glucosamine and [35S]sulphate incorporation into GAG but did not reach significance (Figure 1A, B). AH6809 10–4 mol/l, an EP2 antagonist (Woodward et al., 1995) and AH23848 10–4 mol/l, an EP4 selective antagonist (Coleman et al., 1994b) had no effect alone on GAG synthesis (Figure 2). AH6809 10–4 mol/l was ineffective in reducing PGE2-induced stimulation (Figure 2A, B). Conversely, AH23848 10–4 mol/l, the EP4 selective antagonist, dramatically decreased PGE2-induced GAG production (Figure 2A,B), whatever the PGE2 concentration used.
|
Effects of PGE2 and EP agonist and antagonists on cAMP production
When cervical fibroblasts were treated with PGE2, cAMP production significantly increased in a dose-dependent manner (Figure 3
), describing a curve with a slope rupture at 10–5 mol/l PGE2. AH6809 10–4 mol/l, an EP2 antagonist, significantly decreased the cAMP production provoked by PGE2 at all concentrations used. AH23848 10–4 mol/l, the EP4 selective antagonist, had no effect before 10–5 mol/l PGE2-stimulated cAMP production but significantly inhibited the cAMP production stimulated by 10–4 mol/l PGE2. Butaprost, the EP2 selective agonist, induced a significant augmentation in cAMP production only at 10–4 mol/l. AH6809 completely inhibited the butaprost-stimulated cAMP production.
|
Involvement of cellular cAMP and PKA-dependent mechanisms in the effects of PGE2 on GAG production
We next examined whether the cAMP/PKA transduction pathway was implicated in the effects of PGE2 on GAG synthesis by human cervical fibroblasts in culture. Since phosphodiesterase-4 (PDE-4) is the predominant family responsible for cAMP hydrolysis in cervix cells, i.e. 70% (data not shown), rolipram, a selective cAMP-specific PDE-4 inhibitor, was used. Rolipram caused a 1.44-fold increase in [3H]glucosamine uptake into GAG. Simultaneous addition of PGE2 and rolipram resulted in a further enhancement of [3H]glucosamine incorporation into GAG (2.88-fold) as compared to that elicited by PGE2 alone (2.18-fold) (Figure 4
).
|
Secondly, with the aim to determine whether PKA was also implicated in the PGE2-stimulated GAG production, we investigated the effects of Sp-cAMPS, a membrane-permeable cAMP analogue that strongly activates PKA (Rothermel et al., 1988
), and H89, a selective PKA inhibitor (Chijiwa et al., 1990), on [3H]glucosamine uptake into GAG. Sp-cAMPS at 10–4 mol/l failed to increase GAG production while 10–4 mol/l H89 did not inhibit the GAG synthesis stimulated by 10–6 mol/l PGE2 (Figure 5).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
In order to clarify the EP receptor subtypes which could be involved in PGE2-stimulated GAG synthesis, we examined the effects of PGE2 and an array of EP agonists and antagonists on GAG production by human cervical fibroblasts in culture. PGE2 10–6 mol/l provoked a 2-fold increase in the incorporation of [3H]glucosamine and [35S]sulphate into GAG, while 10–6 mol/l 17-phenyl-trinor-PGE2 (EP1 selective agonist) and 10–6 mol/l sulprostone (EP3 >> EP1 agonist) produced no significant stimulation, ruling out the involvement of EP1 and EP3 receptor subtypes in PGE2-stimulated GAG synthesis. Similarly, both of the concentrations of butaprost (EP2 selective agonist) failed to stimulate GAG synthesis. The fact that the EP2 receptor subtype should not be implicated in PGE2-stimulated GAG production was confirmed by the lack of inhibition of the EP2 antagonist, AH6809, on PGE2-stimulated GAG synthesis. Taken together, these results suggested that the effects of PGE2 on GAG production were mediated through the remaining functional EP receptor subtype, the EP4 receptor. This hypothesis was confirmed by the observation that an EP4-selective antagonist, AH23848, inhibited the PGE2-induced synthesis of hydrogenated and sulphated GAG. AH23848 inhibited both untreated and PGE2-stimulated sulphated GAG synthesis, suggesting that the EP4 receptor could also be implicated in the basal production of sulphated GAG.
Both EP2 and EP4 receptor subtypes stimulate adenylate cyclase (Bastien et al., 1994; Coleman et al., 1994b; Regan et al., 1994). Because we have already demonstrated that cAMP is involved, at least in part, in PGE2-stimulated GAG synthesis (Carbonne et al., 1996) and because EP4 receptors appear to be the only EP receptor subtype implicated in this process, we next examined whether PGE2 modulates cAMP production via the functional EP2 and EP4 receptor subtypes in the human cervix. Butaprost (EP2 selective agonist) enhanced cAMP production, indicating that the EP2 receptor subtype is present in cervical cells and is implicated in PGE2-induced cAMP augmentation. To investigate the relative implication of the EP2 and EP4 receptor subtypes in PGE2-stimulated cAMP accumulation, cells were pretreated with AH6809 (EP2 antagonist) or AH23848 (EP4 selective antagonist). AH6809 strongly inhibited PGE2and butaprost-stimulated cAMP production, at each concentration of these compounds, further establishing the involvement of the EP2 receptor subtype in cAMP production. The complete inhibition of the EP2 agonist effects clearly confirmed that AH6809, first described as an EP1 antagonist (Senior et al., 1991), was also an EP2 receptor antagonist in our system, as in other systems (Woodward et al., 1995; Abramovitz et al., 2000). Conversely, AH23848 inhibited PGE2-induced cAMP production only at 10–4 mol/l PGE2, confirming the implication of the EP4 receptor subtype in PGE2-induced cAMP production. This last result, in association with the shape of the PGE2-stimulated cAMP curve with a slope rupture at 10–5 mol/l PGE2 and the strict parallelism between the PGE2-stimulated cAMP curve in presence of AH23848 and the butaprost-stimulated cAMP curve, suggest that PGE2-induced cAMP augmentation is mainly mediated by EP2 receptor subtype below 10–5 mol/l PGE2, and by both EP2 and EP4 receptor subtypes above 10–5 mol/l PGE2. Such differences in the involvement of each receptor subtype in PGE2-induced cAMP production may be due to different levels of coupling of EP2 and EP4 receptors with adenylate cyclase. Actually, EP4 receptor is reported to be more sensitive than the EP2 receptor to uncoupling reagents like GTP
S (Abramovitz et al., 2000).
It is well established that the EP4 receptor subtype is positively coupled to adenylate cyclase via a stimulatory guanine nucleotide binding protein Gs (Bastien et al., 1994, Narumiya et al., 1999). Through this receptor activation, PGE2 enhances production of cAMP and PKA activity (Takahashi et al., 1999). Consistent with this postulate, we investigated whether cAMP and PKA were involved in the stimulation of GAG synthesis by PGE2 via the EP4 receptor. We previously demonstrated (Carbonne et al., 1996) that 8-Br-cAMP, a cAMP analogue resistant to PDE, significantly increases [3H]glucosamine incorporation into GAG, suggesting that PGE2-induced GAG synthesis was mediated, at least in part, by cAMP. We have now shown that rolipram, a selective PDE-4 inhibitor, increases GAG synthesis and further enhances PGE2-stimulated GAG production, confirming the involvement of cAMP, as a second messenger implicated in GAG synthesis. The facts that PGE2 enhances GAG synthesis via the EP4 receptor subtype, whatever the PGE2 concentration used, and that the cellular cAMP concentration is increased by 10–6 M PGE2 mainly via the EP2 receptor subtype, suggest that a minor EP4-induced cAMP augmentation is sufficient to provoke GAG production. The ability of a minor EP4-induced cAMP increase to provoke GAG synthesis could result from a compartmentalization of the cAMP signal in our model, as has been already described (Houslay et al., 1997).
Because PKA is the major target of cAMP, we examined whether PKA was involved in the signal transduction pathway underlying the PGE2 stimulation of GAG synthesis. Surprisingly, the PKA inhibitor, H89, failed to decrease PGE2-stimulated GAG synthesis and the PKA activator, Sp-cAMPS, did not increase basal GAG production. It has been recently reported that cAMP can act independently of PKA activation and can activate other protein kinases. In vascular smooth muscle cells, cAMP activates cGMP-dependent protein kinase (PKG) (Jiang et al., 1992; Kawada et al., 1997; Han et al., 1999), whereas in chondrocytes, PGE2-stimulated cAMP production enhances protein kinase C (PKC) activity (Schwartz et al., 1998). Furthermore, cAMP has been identified as an activator of small GTPase proteins, such as Ras and Rap-1 in thyroid and brain (de Rooij et al., 1998; Pham et al., 2000; Tsygankova et al., 2000). These findings cast doubt on the putative involvement of PKA in mediating PGE2 stimulation of GAG synthesis, but since the examination of the mechanims involving PKG, PKC or small GTPase proteins was beyond the scope of this study, we have not yet investigated these transduction pathways. Studies evaluating the implication of each EP receptor subtype on PGE2-stimulated collagen degradation, the other fundamental aspect of cervical ripening, are also now in progress. They may clarify the role of the EP2-induced cAMP production.
In conclusion, PGE2-stimulated GAG synthesis, usually considered to reflect PGE2-induced cervical ripening, is mediated through the EP4 receptor subtype in a PKA-independent pathway in human cultured cervical fibroblasts. Knowing that studies with human pregnant cervical tissue are very difficult to perform for ethical reasons, cultured cells provide a convenient model where the hormonal environment can be modulated. It has allowed us to work with high concentrations of PGE2 as the goal of this study was more to determine the pharmacological mechanisms by which PGE2 induces cervical ripening, than the physiological role of this prostaglandin in this process. The identification of an EP receptor subtype implicated in the biochemical features of PGE2-induced cervical ripening, distinct from those involved in myometrial contraction, i.e. EP1 and EP3, is of major clinical importance. The control of cervical ripening through the stimulation or inhibition of the EP4 receptor subtype, using selective agonists or antagonists, could be very helpful in daily clinical practice. It would allow the preparation of an unfavourable cervix before inducing labour or the inhibition of preterm cervical ripening in threatened preterm labour.
|
Acknowledgements |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
T.S. was supported by the French National Academy of Medecine. We are grateful to Dr Lister for kindly providing AH6809 and AH23848. We also thank Dr Stünkel for generously donating butaprost. We are indebted to Dr C.Méhats for helpful discussions and to Dr O.Parkes for reviewing the English text.
|
Notes |
|---|
3 To whom correspondence should be addressed at: INSERM U 361, Pavillon Baudelocque, 123, Bd de Port-Royal, F-75014 Paris, France. E-mail: tsn{at}club-internet.fr
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Abramovitz, M., Adam, M., Boie, Y. et al. (2000) The utilization of recombinant prostanoid receptors to determine the affinities and selectivities of prostaglandins and related analogs. Biochim. Biophys. Acta, 1483, 285–293.[Medline]
Adelantado, J.M., Lopez Bernal, A. and Turnbull, A.C. (1988) Topographical distribution of prostaglandin E receptors in human myometrium. Br. J. Obstet. Gynaecol., 95, 348–353.[Web of Science][Medline]
Asboth, G., Phaneuf, S., Europe-Finner, G.N. et al. (1996) Prostaglandin E2 activates phospholipase C and elevates intracellular calcium in cultured myometrial cells: involvement of EP1 and EP3 receptor subtypes. Endocrinology, 137, 2572–2579.[Abstract]
Asboth, G., Phaneuf, S. and Lopez Bernal, A.L. (1997–98) Prostaglandin E receptors in myometrial cells. Acta Physiol. Hung., 85, 39–50.[Medline]
Bastien, L., Sawyer, N., Grygorczyk, R. et al. (1994) Cloning, functional expression and characterization of the human prostaglandin E2 receptor EP2 subtype. J. Biol. Chem., 269, 11873–11877.
Cabrol, D., Dubois, P., Sedbon, E. et al. (1987) Prostaglandin E2-induced changes in the distribution of glycosaminoglycans in the isolated rat uterine cervix. Eur. J. Obstet. Gynecol. Reprod. Biol., 26, 359–365.[Web of Science][Medline]
Carbonne, B., Jannet, D., Dallot, E. et al. (1996) Synthesis of glycosaminoglycans by human cervical fibroblasts in culture: effects of prostaglandin E2 and cyclic AMP. Eur. J. Obstet. Gynecol. Reprod. Biol., 70, 101–105.[Web of Science][Medline]
Carbonne, B., Dallot, E., Haddad, B. et al. (2000) Effects of progesterone on prostaglandin E2-induced changes in glycosaminoglycan synthesis by human cervical fibroblasts in culture. Mol. Hum. Reprod., 6, 661–664.
Cavaillé, F., Cabrol, D. and Ferré, F. (1996) Human myometrial smooth muscle cells and cervical fibroblasts in culture. In Jones, G.E. (ed.), Methods in Molecular Medicine: Human Cell Culture Protocols. Humana Press, Totowa, NJ, pp. 335–344.
Chijiwa, T., Mishima, A., Hagiwara, M. et al. (1990) Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide (H89), of PC12D pheochromocytoma cells. J. Biol. Chem., 265, 5267–5272.
Coleman, R., Humphrey, P., Kennedy, I. et al. (1984) Prostanoid receptors | the development of a working classification. Trends Pharmacol. Sci., 5, 303–306.
Coleman, R., Smith, W. and Narumiya, S. (1994a) VIIIth International Union of Pharmacology: classification of prostanoid receptors: properties, distribution and structure of the receptors and their subtypes. Pharmacol. Rev., 46, 205–229.[Web of Science][Medline]
Coleman, R., Grix, S., Head, S. et al. (1994b) A novel inhibitory prostanoid receptor in piglet saphenous vein. Prostaglandins, 47, 151–168.[Web of Science][Medline]
de Rooij, J., Zwartkruis, F.J., Verheijen, M.H. et al. (1998) Epac is a Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP. Nature, 396, 474–477.[Medline]
Ekman, G., Forman, A., Marsal, K. et al. (1983) Intravaginal versus intracervical application of prostaglandin E2 in viscous gel for cervical priming and induction of labor at term in patients with unfavorable cervical state. Am. J. Obstet. Gynecol., 147, 657–661.[Web of Science][Medline]
Gardiner, P.J. (1986) Characterization of prostanoid relaxant/inhibitory receptors (
) using a highly selective agonist, TR4979. Br. J. Pharmacol., 87, 45–56.[Web of Science][Medline]
Han, G., Kryman, J.P., McMillin, P. et al. (1999) A novel transduction mechanism mediating dopamine-induced vascular relaxation: opening BKCa channels by cyclic AMP-induced stimulation of cyclic GMP-dependent protein kinase. J. Cardiovasc. Pharmacol., 34, 619–627.[Web of Science][Medline]
Hillock, C.J. and Crankshaw, D.J. (1999) Inhibitory prostanoid EP receptors in human non pregnant myometrium. Eur. J. Pharmacol., 378, 99–108.[Web of Science][Medline]
Houslay, M.H. and Milligan, G. (1997) Tailoring cAMP-signalling responses through isoform multiplicity. Trends Biochem. Sci., 22, 217–224.[Web of Science][Medline]
Jiang, H., Colbran, J.L., Francis, S.H. et al. (1992) Direct evidence for cross-activation of cGMP-dependent protein kinase by cAMP in pig coronary arteries. J. Biol. Chem., 267, 1015–1019.
Kawada, T., Toyosato, A., Islam, O. et al. (1997) cGMP-Kinase mediates cGMPand cAMP-induced Ca2+ desensitization of skinned rat artery. Eur. J. Pharmacol., 323, 75–82.[Web of Science][Medline]
Kiriyama, M., Ushikubi, F., Kobayashi, T. et al. (1997) Ligand binding specificities of the eight types of mouse prostanoid receptors expressed in Chinese hamster ovary cells. Br. J. Pharmacol., 122, 217–224.[Web of Science][Medline]
Narumiya, S., Sugimoto, Y. and Ushikubi, F. (1999) Prostanoid receptors: structures, properties and functions. Physiol. Rev., 79, 1193–1226.
Negishi, M., Ito, S. and Hayaishi, O. (1989) Prostaglandin E receptors in bovine adrenal medulla are coupled to adenylate cyclase via Gi and to phosphoinositide metabolism in a pertussis toxin-insensitive manner. J. Biol. Chem., 264, 3916–3923.
Norman, M., Ekman, G. and Malmström, A. (1993) Prostaglandin E2-induced cervical ripening of the human cervix involves changes in proteoglycan metabolism. Obstet. Gynecol., 82, 1013–1020.[Web of Science][Medline]
Norström, A. (1982) Influence of prostaglandin E2 on the biosynthesis of connective tissue constituents in the pregnant human cervix. Prostaglandins, 23, 361–367.[Web of Science][Medline]
Osmers, R., Rath, W., Adelmann-Grill, B.C. et al. (1991) Collagenase activity in the human cervix uteri after prostaglandin E2 application during the first trimester. Eur. J. Obstet. Gynecol. Reprod. Biol., 42, 29–32.[Web of Science][Medline]
Osmers, R., Rath, W., Pflantz, M.A. et al. (1993) Glycosaminoglycans in cervical connective tissue during pregnancy and parturition. Obstet. Gynecol., 81, 88–92.[Web of Science][Medline]
Pham, N., Cheglakov, I., Koch, C.A. et al. (2000) The guanine nucleotide exchange factor CNrasGEF activates ras in response to cAMP and cGMP. Curr. Biol., 10, 555–558.[Web of Science][Medline]
Redini, F., Daireaux, M., Mauviel, A. et al. (1991) Characterization of proteoglycans synthesized by rabbit articular chondrocytes in response to transforming growth factor-ß (TGF-ß). Biochim. Biophys. Acta, 1093, 196–206.[Medline]
Regan, J.W., Bailey, T., Pepperl, D. et al. (1994) Cloning a novel human prostaglandin receptor with characteristics of pharmacologically defined EP2 subtype. Mol. Pharmacol., 46, 213–220.[Abstract]
Rothermel, J.D. and Parker Botelho, L.H. (1988) A mechanistic and kinetic analysis of the interactions of the diastereoisomers of adenosine 3', 5'-(cyclic) phosphorothioate with purified cyclic AMP-dependent protein kinase. Biochem. J., 251, 757–762.[Web of Science][Medline]
Schwartz, Z., Gilley, R.M., Sylvia, V.L. et al. (1998) The effect of prostaglandin E2 on costochondral chondrocyte differentiation is mediated by cyclic adenosine 3', 5'-monophosphate and protein kinase C. Endocrinology, 139, 1825–1834.
Senior, J., Marshall, K., Sangha, R. et al. (1991) In vitro characterization of prostanoid EP-receptors in non pregnant human myometrium. Br. J. Pharmacol., 102, 747–753.[Web of Science][Medline]
Senior, J., Marshall, K., Sangha, R. et al. (1993) In vitro characterization of prostanoid receptors on human myometrium at term pregnancy. Br. J. Pharmacol., 108, 501–506.[Web of Science][Medline]
Shepherd, J., Pearce, J.M. and Sims, C.D. (1979) Induction of labor using prostaglandin E2 pessaries. Br. Med. J., 2, 108–110.
Takahashi, S., Takeuchi, K. and Okabe, S. (1999) EP4 receptor mediation of prostaglandin E2-stimulated mucus secretion by rabbit gastric epithelial cells. Biochem. Pharmacol., 58, 1997–2002.[Web of Science][Medline]
Tsygankova, O.M., Kupperman, E., Wen, W. et al. (2000) Cyclic AMP activates Ras. Oncogene, 19, 3609–3615.[Web of Science][Medline]
Uldbjerg, N., Ekman, G., Malmström, A. et al. (1983) Ripening of human uterine cervix related to changes in collagen, glycosaminoglycans and collagenolytic activity. Am. J. Obstet. Gynecol., 147, 662–666.[Web of Science][Medline]
Wasteson, A., Uthne, K. and Westermark, B. (1973) A novel assay for the biosynthesis of sulphated polysaccharides and its application to studies on the effects of somatomedin on cultured cells. Biochem. J., 136, 1069–1074.[Web of Science][Medline]
Woodward, D.F., Pepperl, D.J., Burkey, T.H. et al. (1995) 6-Isopropoxy-9-oxoxanthene-2-carboxylic acid (AH6809) a human EP2 receptor antagonist. Biochem. Pharmacol., 50, 1731–1733.[Web of Science][Medline]
Submitted on September 29, 2000; accepted on February 2, 2001.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  K. S. Song, Y. H. Choi, J.-M. Kim, H. Lee, T.-J. Lee, and J.-H. Yoon Suppression of prostaglandin E2-induced MUC5AC overproduction by RGS4 in the airway Am J Physiol Lung Cell Mol Physiol, April 1, 2009; 296(4): L684 - L692. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Schmitz, B. A Levine, and P. W Nathanielsz Localization and steroid regulation of prostaglandin E2 receptor protein expression in ovine cervix. Reproduction, April 1, 2006; 131(4): 743 - 750. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Anderson, N. Brown, M. S. Mahendroo, and J. Reese Utilization of Different Aquaporin Water Channels in the Mouse Cervix during Pregnancy and Parturition and in Models of Preterm and Delayed Cervical Ripening Endocrinology, January 1, 2006; 147(1): 130 - 140. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Schmitz, M.J. Leroy, E. Dallot, M. Breuiller-Fouche, F. Ferre, and D. Cabrol Interleukin-1{beta} induces glycosaminoglycan synthesis via the prostaglandin E2 pathway in cultured human cervical fibroblasts Mol. Hum. Reprod., January 1, 2003; 9(1): 1 - 8. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Tintut, F. Parhami, A. Tsingotjidou, S. Tetradis, M. Territo, and L. L. Demer 8-Isoprostaglandin E2 Enhances Receptor-activated NFkappa B Ligand (RANKL)-dependent Osteoclastic Potential of Marrow Hematopoietic Precursors via the cAMP Pathway J. Biol. Chem., April 12, 2002; 277(16): 14221 - 14226. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Fujimoto, R. C. Savani, M. Watari, A. J. Day, and J. F. Strauss III Induction of the Hyaluronic Acid-Binding Protein, Tumor Necrosis Factor-Stimulated Gene-6, in Cervical Smooth Muscle Cells by Tumor Necrosis Factor-{alpha} and Prostaglandin E2 Am. J. Pathol., April 1, 2002; 160(4): 1495 - 1502. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||