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Endocrinology Vol. 141, No. 8 2795-2804
Copyright © 2000 by The Endocrine Society

ARTICLES

Characterization of Neuropeptide Y-Mediated Corticotropin-Releasing Factor Synthesis and Release from Human Placental Trophoblasts1

Jacques Robidoux2, Lucie Simoneau, Serge St-Pierre, André Masse and Julie Lafond

Programme des Sciences Biomédicales and Gynecology-Obstetrics Department (J.R., A.M., J.L.), Faculté de Médecine, Université de Montréal; Départements des Sciences Biologiques (J.R., L.S., J.L.)and Chimie (S.St-P.), Université du Québec à Montréal, Canada, H3C 3P8

Address all correspondence and requests for reprints to: Prof. Julie Lafond, Université du Québec à Montréal, Département des Sciences Biologiques, C.P. 8888, Succursale "Centre-Ville," Montréal, Québec, Canada H3C 3P8. E-mail: lafond.julie{at}uquam.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Neuropeptide Y (NPY) is a CRF secretagogue for human placental cells in culture. We have studied the involvement of intracellular calcium and calcium-dependent signaling in the NPY-induced CRF release in trophoblastic cells. The incubation of trophoblasts with NPY for 3 and 8 h led to a dose-dependent increase in CRF secretion. Also, NPY stimulated synthesis of this peptide hormone upon an 8-h incubation period. BIBP3226, a selective Y1 receptor antagonist, and pertussis toxin (PTX) eliminated these effects. NPY-stimulated CRF secretion was mostly prevented by loading cells with BAPTA-AM, suggesting that elevation of intracellular calcium is responsible for the increase of CRF secretion. However, this calcium chelator had no effect on CRF synthesis. Furthermore, U-73122, a phospholipase C-ßs (PLC) inhibitor or xestospongin C, an inositol triphosphate receptor (InsP3-R) blocker, have partially prevented the effect of NPY on CRF synthesis and secretion. Therefore, the increase in CRF synthesis and secretion rely in part on the release of calcium from intracellular store. Interestingly, SKF 96365, an inhibitor of store operated calcium (SOC) influx, also partially blocked the NPY stimulatory effect on CRF release but not its synthesis, suggesting that calcium influx is also involved in this stimulation. In the syncytiotrophoblast, known to possess a NPY-activated protein kinase C (PKCs) activity, NPY also stimulated calcium calmodulin kinase II (CaMKII) and extracellular regulated kinase (ERK1/2) activities. In the present study, we observed that bisindolylmaleimide (BIM), a nonspecific PKCs inhibitor partially prevented the NPY-induced CRF release. On the other hand, autocamtide-2 related inhibitory peptide (AIP), a CaMKII inhibitor, prevented most of the stimulatory effect of NPY on both CRF synthesis and release. Gö6976, an inhibitor of the conventional and µ PKCs and PD 098059, an inhibitor of the ERK cascade, had no effect on neither CRF synthesis nor release. Altogether, these results support a Y1 receptor-mediated PTX-sensitive induction on CRF synthesis and release by NPY from human placental trophoblasts. The stimulation of CRF synthesis by NPY seems to depend mainly on a PLC-ß to InsP3-R axis and on CaMKII activity. Also, the release of CRF depends on the PLC-ß to InsP3-R axis and CaMKII activity but also entails the participation of a calcium-independent PKCs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE FUNCTIONAL placental unit, which includes fetal membranes (amnion and chorion), plays an important role in the maintenance of pregnancy, in the growth of the fetus, and in birth’s timing. These pivotal roles of the placental unit are mainly accountable to its ability to produce a large variety of hormones and to its intimate relationship with both fetal and maternal compartments (1). The ultimate outcome of pregnancy, parturition, is still partially elucidated. Interestingly, the plethora of hormones, growth factors, and cytokines produced by the placental unit during spontaneous labor and delivery indicates a well-organized process (2).

During the last few years, significant interest has been given to placental CRF. This great interest arises from the apparent link between the production of this peptide and the duration of pregnancy (3). In that regard, several studies indicated that maternal concentration of CRF is elevated during pregnancy, reaching its highest level at the onset of labor (4, 5), particularly during an abnormal pregnancy complicated by preterm labor (6).

The syncytiotrophoblast layer appears to be, at least in the third trimester, the principal cellular source of CRF (7) and therefore, most studies dealing with the control of this peptide’s synthesis and release were conducted on trophoblast cells isolated from human term placenta. Isolated trophoblast cells from human term placenta undergo spontaneous syncytiotrophoblastic-like morphological and biochemical differentiation in vitro (8) and would reflect in vivo syncytiotrophoblast. Several studies (for a review , see Ref. 9) pointed glucocorticoids and progesterone as the major regulators of placental CRF synthesis. However, many other putative CRF secretagogues have been identified, such as acetylcholine, angiotensin II, arginine vasopressin, catecholamines, interleukin-1, NPY, oxytocin, and prostaglandins (10, 11, 12). Nitric oxide has been reported as a negative regulator (13).

Among these secretagogues, NPY, a peptide produced by cytotrophoblastic cells, amnion, chorion, and decidua (11, 14) is abundant in both plasma and amniotic fluids of woman throughout pregnancy (15). NPY binds to a mixed population of Y1 and Y3 receptors on the brush border membranes of human syncytiotrophoblast (16). These receptors are linked to multiple signal transduction cascades. Until now, no study intended to elucidate the mechanisms implicated in the modulation of placental CRF release. For that reason, we decided to investigate the mechanisms implicated in the NPY effects. The purpose of this study was to investigate the role of calcium signaling on the NPY induced CRF synthesis and release in cultured human trophoblast cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Placental trophoblast isolation and culture
Normal term placentas from either vaginal or caesarian delivery were obtained in accordance with the established guidelines of the ethical committee of the Centre Hospitalier de l’Université de Montréal. After delivery, the placentas were kept in 500 ml of cold (4 C) DMEM)(Life Technologies, Inc., Burlington) containing: penicillin (200 U/ml), streptomycin (200 µg/ml), emphotericin B (5 µg/ml), and gentamycin (50 µg/ml). The trophoblast cells were isolated as previously described by Thiede (17), following the modifications reported by Stromberg et al. (18) and Winkel et al. (19). Briefly, the villous tissue (~75 g) was grossly minced, rinsed in HBSS containing the additives mentioned above, and cut again into smaller pieces, before being rinsed in 100 ml HBSS without calcium and magnesium on a rotary shaking incubator (50 rpm) for 10 min at 32 C. The rinsed tissue was after subjected to two consecutive digestions of 10 min in 120 ml of a similar medium containing 0.125% trypsin (ICN Biochemicals, Inc., Montréal, Québec, Canada) and 0.02% deoxyribonuclease type I (Roche Molecular Biochemicals, Laval, Canada). Both supernatants were filtered over a succession of 200 µm and 60 µm sieves to remove aggregates. The digestion was stopped by the addition of 4% (vol/vol) of FCS (HyClone Laboratories, Inc. Logan, UT). The cell suspension was centrifuged at 400 g for 10 min. The red blood cells were lysed in 140-mM ammonium chloride-Tris-HCl buffer at 100 rpm for 15 min. The cells were than centrifuged at 350 g for 5 min, resuspended in HBSS, layered over a 10% percoll (Amersham Pharmacia Biotech, Baie d’Urfé, Canada) and centrifuged at 450 g for 10 min. The resulting pellet was washed twice in HBSS (Life Technologies, Inc.) and once in culture medium by centrifugation at 350 g for 5 min. The isolated trophoblast cells were suspended in DMEM containing FCS (10%), fatty acid-free BSA (0.5%) (ICN Biochemicals, Inc.), penicillin (100 U/ml), streptomycin (100 µg/ml), Amphotericin (2.5 µg/ml) and gentamycin (50 µg/ml). After cells counting and evaluation of viability by trypan blue exclusion (viability usually greater than 90%), the cells were plated at a density of 1.7 x 106 per well in 24 well plates (Sarstedt, Montréal, Québec, Canada). The cells were incubated in a humidified atmosphere (95% air and 5% CO2 at 37 C). The culture medium was changed after the first 3 h and changed every day thereafter. All experiments were done at the fourth day of culture. On that day, cells count was around 106 per well. This variation is inherent to the methods and the model, but intrapreparation variability as judged by protein determination of cellular lysate was not more than 15%.

Evaluation of trophoblast phenotype
Using the above isolation procedure, we usually obtained cultures consisting of more than 90% trophoblast cells as judged by morphological and immunocytochemical determination of pan-cytokeratin-positive (epithelial cell marker; Sigma, St. Louis, MO) and vimentin-positive cells (mesenchymal cell marker; Sigma) (20). Briefly, cells were growth for 12 h on plastic coverslips and were fixed for 10 min in acetone at -20 C. After evaporation, endogenous peroxidase activity was quenched by a 10 min incubation at 4 C with 3% H2O2 in methanol. Nonspecific binding was blocked by a 3 h incubation at room temperature with 5% skimmed milk, 3% FCS and 1% BSA. Primary antibody was diluted (1:200 for vimentin, 1:400 for cytokeratin) in TBS containing 0.1% Tween and incubated for 1 h. The biotinylated second antibody included in histochemical ABC kit (Oncogene Research, Cambridge, MA) was diluted in the same buffer and incubated for 1 h. An irrelevant antibody [trp E (Ab-1)], included in the kit, was used as first antibody for the control. All the following steps were done in accordance with the Oncogene’s technical protocol, except that we also used the DAB enhancer from Sigma.

Measurements of CRF release by cultured trophoblasts
The levels of CRF release in the medium and in the cells were determined by RIA kit (Phoenix Pharmaceuticals, Inc., Mountain View, CA), following the Phoenix’s technical protocol. The sensitivity of the CRF RIA kit was 23.9 ± 2.5 pg/tube. The interassay and within-assay variation coefficients at 16 pg/tube were 8.4% and 3.8%, respectively. On the fourth day of culture, the content of CRF was determined in the absence (basal) and the presence of NPY for 3 h and 8 h. For all experiments, the treated cells were compared with cells exposed to the appropriate vehicle(s). Before each experiment, cells were serum starved for 1 h in DMEM containing 0.1% BSA. Afterward, the medium was replaced by 200 µl of medium containing a 2x concentration of the agent used as treatment. Ten minutes later, NPY (200 µl of a 2 x 107 M stock) was added and incubated for the appropriate time. The medium present in each well was collected, centrifuged at 350 x g for 5 min, evaporated to dryness before being stored at -20 C until assayed. For the intracellular CRF content, cells were lysed by the addition of 200 µl of HBSS containing 0.1% Nonidet P-40, followed by a 5 sec sonication. The resulting medium was treated as described above.

Evaluation of the promotion of the autonomous form of CaMKII
The method used was essentially described by Abraham et al. (21) with minor modifications. Briefly, cells were serum starved for 1 h in DMEM containing 0.1% BSA, than the medium was changed for 200 µl of fresh medium and preincubated for 10 min. Afterward, NPY (200 µl of a 2 x 10-7 M stock) was added and the cells were incubated for different interval of time. At the end of the incubation, the medium was aspired and replaced by 75 µl of an ice-cold lysis buffer constituted of an 1:1 mixture of calcium/magnesium-free HBSS and 50 mM MOPS buffer containing 1% Nonidet P-40, 20 mM sodium pyrophosphate, 4 mM dithiothreitol, 2 mM NaF, 2 mM EGTA, 1 mM ammonium molybdate, 200 µM PMSF, 20 µM leupeptine, and 1 µg/ml aprotinin. Following a 5-sec sonication, the protein content of the lysates (75.9 ± 11.4 µg/well) was quantified by the BCA protein assay (Pierce Chemical Co., Rockford, IL). The autonomous CaMKII activity of the lysate was assayed by incubating 5 µg of the lysate in a total 25 µl mixture containing 50 µM autocamtide-2 and 10 µM [{gamma}-32P]ATP in 10 mM MOPS (pH 7.4) containing 10 mM magnesium chloride and 1 mM EGTA, at room temperature for 5 min. The reaction was stopped by the addition of 150 µl of ice cold 10% trichloroacetic acid. Samples were incubated for 30 min at 4 C and centrifuged at 10,000 x g for 3 min. An aliquot (150 µl) of the supernatant was added to an equal volume of 30% trichloroacetic acid and the mixture was adsorbed on a MultiScreen phosphocellulose plates (Millipore Corp., Nepean, Canada). Following three washes, the filters were punched into vials and counted using a liquid scintillation ß counter after addition of 5 ml of Ready protein+ liquid scintillation cocktail (Beckman Coulter, Inc., Missisauga, Canada). The specific incorporation into the synthetic peptide was determined as the differentiation in its presence or absence.

Evaluation of the ERKs activation
The phosphorylation status of ERKs is a quantitative indicator of ERKs activation. Therefore, the evaluation of ERKs activation was done by Western blot using an anti-phosphoERKs antibody purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Briefly, cells were serum-starved for 3 h, preincubated for 10 min in 200 µl fresh serum-free medium and incubated in the presence of NPY 10-7 M, for an additional 0, 2.5, 5, 7.5, 10, 15, 30, and 180 min. At the end of the incubation, the medium was aspired and replaced by 75 µl of SDS loading buffer containing 80 µM sodium vanadate, 80 µM sodium pyrophosphate, 10 mM EDTA, 0.8 mg/ml benzamidine, 80 µM PMSF, 0.8 µg/ml pepstatin, and 0.4 µg/ml aprotinin. The protein content of the lysates (80.2 ± 12.0 µg/well) was quantified by a modification of the Bradford method (22). Proteins (10 µg) were loaded on an alkaline tricine-SDS-PAGE system consisting of a 4% stacking gel and a 12% acrylamide-glycerol separating gel (23), using a Mini-Protean II system (Bio-Rad Laboratories, Inc. Hercules, CA). After migration, proteins were transferred to PVDF membrane (Roche Molecular Biochemicals) using a semidry system (Millipore Corp.). The membrane was blocked overnight at 4 C in Tris-buffered saline containing 0.05% Tween (TBS-T), 5% skimmed milk, 3% FCS, and 1% BSA. These membranes were blotted for 1 h at room temperature with the anti-phosphoERKs antibody (1:1000 in TBS-T), washed three times with TBS-T, and incubated for 1 h in TBS-T containing 0.5% skimmed milk, 0.3% FBS, and 0.1% BSA and a horseradish peroxidase-coupled antirabbit IgG antibody (1:1250) (Roche Molecular Biochemicals). Finally, the immunoreactive bands were developed by chemiluminescent (Roche Molecular Biochemicals). The specific bands were quantified by densitometry scanning of the x-ray film using the Personal Densitometer (Molecular Dynamics, Inc., Sunnyvale, CA) and analyzed with ImageQuant software (Sunnyvale, CA).

Data analysis
For each cells preparation (n = 3–5), experiments were done in duplicate or triplicate, as described, at a given condition or time point. The mean of such replicates was used as a single datum point for analysis. When time-course or dose-response protocols were analyzed, ANOVA on repeated measures followed by Dunnett’s posthoc test were used. Otherwise, Newman-Keuls test was used to compare multiple condition experiments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Modulation of CRF synthesis and release by NPY
Addition of NPY to trophoblastic cells stimulated the release of CRF in a concentration-dependent manner after 3 h and 8 h (P < 0.001; Fig. 1Go, a and b). For the 3 h incubation (Fig. 1aGo), only the CRF release was significantly increased, the NPY effect was perceived at concentration ranging from 10-8 M (p < 0.05) to 10-6 M (p < 0.01). After a 8 h incubation with NPY, long enough to observe CRF synthesis, the peptide increased both CRF synthesis (total CRF content) and release at concentrations ranging from 10-8 to 10-6 M (Fig. 1bGo; P < 0.01). Therefore, from now on, when we will mention NPY-induced CRF synthesis we will always refer to the 8 h experiment because no modification of total CRF content was observed during any of the 3 h experiments (Figs. 1aGo, 2bGo, 3bGo, 4bGo, and 7bGo). The NPY (10-7 M) increased CRF release by 2- and 3-fold at 3 h and 8 h, respectively. The NPY potency to induce CRF release was slightly different between the 3 h and 8 h incubations (with PEC50 of 9.59± 0.61 and 8.93 ± 0.38 respectively; P < 0.05). However, the NPY potency to induce CRF release at the longer incubation time was identical to its potency to induce CRF synthesis (PEC50 8.93 ± 0.62).



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  		Figure 1. Effects of NPY on total CRF content and CRF release from human placental trophoblasts. The cells in their fourth day of culture were serum starved for 1 h and incubated for 3 or 8 h (a and b, respectively) in presence of increasing concentration of NPY. CRF content was determined in the medium and cells lysate by RIA. Data represent the mean ± SE from three different placental cell preparations. (*P < 0.05, ** P < 0.01 vs. basal.)

 


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  		Figure 2. Effect of BIBP3226 (Y1 antagonist) and PTX on NPY-induced CRF synthesis and release. PTX treatments (500 ng/ml) were done overnight, then the cells were serum starved for 1 h. The BIBP3226 (5 µM) was added 10 min before NPY (10-7 M) and incubated for 3 (a, b) or 8 h (c, d). CRF release (a, c) and total CRF content (b, d) were determined by RIA. Data represent the mean ± SE from three different placental cell preparations. (*P < 0.05, *** P < 0.001 vs. basal and +P < 0.05,++P < 0.01 vs. control.)

 


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  		Figure 3. Effect of BAPTA/AM and nitrendipine on NPY-induced CRF synthesis and release. Cells were loaded with BAPTA/AM (20 µM) for 30 min and serum starved for 1 h. Nitrendipine (2 µM) was added 10 min before NPY (10-7 M) and incubated for 3 (a, b) or 8 h (c, d). CRF release (a, c) and total CRF content (b, d) were determined by RIA. Data represent the mean ± SE from four different placental cell preparations. (*P < 0.05, ** P < 0.01 *** P < 0.001 vs. basal and +P < 0.05,+++P < 0.001 vs. control.)

 


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  		Figure 4. Effect of U-73122, xestospongin C, and SKF 96365 on NPY-induced CRF synthesis and release. Cells were serum starved 1 h. U-73122 (5 µM), xestospongin C (1 µM) or SKF 96365 (50 µM) were added 10 min before NPY (10-7 M) and incubated for 3 (a, b) or 8 h (c, d). CRF release (a, c) and total CRF content (b, d) were determined by RIA. Data represent the mean ± SE from three different placental cell preparations. (*P < 0.05, ** P < 0.01 *** P < 0.001 vs. basal and +P < 0.05, 2+P < 0.01 +++P < 0.001 vs. control.)

 


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  		Figure 7. Effect of BIM, Gö6976, AIP and PD 098056 on NPY-induced CRF synthesis and release. Cells were serum starved for 1 h. BIM (50 nM), Gö6976 (1 µM), AIP (1 µM), or PD 098056 (40 µM) were added 10 min before NPY (10-7 M) and incubated for 3 (a, b) or 8 h (c, d). CRF release (a, c) and total CRF content (b, d) were determined by RIA. Data represent the mean ± SE from five different placental cell preparations. (*P < 0.05, ** P < 0.01 *** P < 0.001 vs. basal and +P < 0.05 vs. control.)

 
Syncytiotrophoblast presents a mixed population of binding sites for NPY, namely Y1 and Y3 (16). To further characterize the NPY receptor subtype(s) involved in the NPY effects, we preincubated the cells for 10 min with 5 µM BIBP 3226, a specific antagonist of the Y1 subtype (24). The results showed that this subtype is fully accountable for the effect of NPY 10-7 M on both CRF synthesis an release (Fig. 2Go, a, c, and d) because BIBP 3226 prevented both effects. In most cell types, the Y1 receptors are linked to PTX-sensitive G proteins. Because no studies have reported this kind of coupling in trophoblastic cells, we preincubated the cells overnight with 500 ng/ml of PTX. As expected, the toxin eliminated the effect of NPY on both CRF synthesis and release (Fig. 2Go, a, c, and d).

Implication of calcium in NPY-induced CRF synthesis and release
To examine the involvement of intracellular calcium in NPY-induced CRF synthesis and release in trophoblastic cells, we loaded the cells (30 min) with 20 µM BAPTA/AM, an intracellular calcium chelator. As shown in Fig. 3Go, a and c, BAPTA prevents 50% of the NPY-induced CRF release at 3 h (P < 0.05) and 75% at 8 h (P < 0.01). Nonetheless, no effect of the calcium chelation could be discerned on CRF synthesis (Fig. 3dGo). The calcium implicated in the NPY-induced CRF-release could originate from either extracellular calcium influx or intracellular store release. Therefore, we investigated both possibilities. Since depolarization-induced CRF release has been reported (13), we investigated the involvement of L-type calcium channels in the NPY effects. The result showed that the L-type calcium channels specific blocker, nitrendipine (2 µM) did not prevent the effects of NPY on either CRF synthesis or release (Fig. 3Go, a, c, and d). In syncytiotrophoblasts, NPY is known to activate PLC-ß (16). Two strategies have been elaborated to examine the contribution of the NPY to InsP3-R pathway: 1) cells incubation with U-73122 (5 µM), a inhibitor of G protein-coupled receptors activation of PLC-ß (25), and 2) cells incubation with xestospongin C (1 µM), an InsP3-R blocker (26). As shown in Fig. 4Go, a and c, both agents reduced CRF release after 3 h and 8 h (P < 0.01). Surprisingly, U-73122 and xestospongin C treatments eliminated (P < 0.001) the NPY effects on CRF synthesis (Fig. 4dGo). Because prolonged stimulation of calcium release will empty the stores without refilling them, we incubated the cells with SKF 96365 (50 µM), a SOC influx blocker (27). Figure 4Go, a and c, showed that SKF 96365 reduces the NPY-induced CRF release by 65% after 3 h (P < 0.05) and 50% after 8 h (P < 0.05). Such an effect of the blocker on NPY-induced CRF synthesis (Fig. 4dGo) was less obvious since NPY-stimulated CRF release was still important (P < 0.01).

Activation of CaMKII and ERKs by NPY
Because of the importance of calcium in the NPY-induced CRF release, we postulated that calcium dependent kinases might be involved in this process. Because we have evidences of PKCs (16) and calcium-dependent ERKs activation (data not shown) by NPY, we investigated the activation of both CaMKII and ERK by NPY to ascertain their involvement in the signal transduction pathways connecting NPY to CRF synthesis and release.

The CaMKII is characterized by an activation-dependent autophosphorylation leading to the promotion of a calcium/calmodulin independent activity (28). Therefore, a time-course experiment was conducted to evaluate the induction of the autonomous form of the kinase in presence of NPY (10-7 M). As shown in Fig. 5Go, NPY produced a rapid promotion of the autonomous form of CaMKII. This activation was already apparent after 15 sec (P < 0.05) and the activity was maintained for an additional 30 sec, before gradually returning to the basal level.



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  		Figure 5. Time-course of CaMKII activation by NPY. Cells were serum starved 1 h. The medium was replaced by 200 µl of fresh medium 10 min before the addition of NPY (10-7 M). At different interval of time, the medium was retrieved and ice-cold lysis buffer (75 µl) was added. The autonomous CaMKII activity was than measured as described in Materials and methods. Data represent the mean ± SE from three different placental cell preparations. (*P < 0.05 vs. basal.)

 
ERKs are catalytically activated by phosphorylation (29), which can be monitored by Western blot using a phosphoERKs antibody. A time-course experiment was then conducted to evaluate the induction of the phosphorylated form of the kinases in presence of NPY (10-7 M). As shown in Fig. 6Go, the incubation of cells with NPY leads to a sustained ERKs activation.



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  		Figure 6. Time-course of ERKs activation by NPY. Cells were serum starved 1 h. The medium was replaced by 200 µl of fresh medium 10 min before the addition of NPY (10-7 M). At different interval of time, the medium was retrieved and ice-cold lysis buffer (75 µl) was added. The phosphoERKs immunoreactivity was than measured as described in Materials and methods. Data represent the mean ± SE from three different placental cell preparations. (*P < 0.05, **P < 0.01 vs. basal.)

 
Effect of the protein kinase inhibitors on NPY induced synthesis and release of CRF
To evaluate the involvement of the activation of PKCs, CaMKII, and ERKs by NPY on the stimulation of CRF synthesis and release, the cells were incubated in presence of specific inhibitors of the three-kinase families. As shown in Fig. 7Go, a and c, cell incubation with BIM (50 nM), a nonisotype specific inhibitor of PKCs (30), inhibited NPY-induced CRF release (P < 0.05) but had no effect on CRF synthesis (Fig. 7dGo). Interestingly, the use of Gö6976 (1 µM), a specific inhibitor of the conventional (calcium-dependent) and µ PKCs (31, 32), had no effect on both NPY-induced release and synthesis (Fig. 7Go, a and c, and d). On the other hand, CaMKII, another calcium-dependent kinase, appears to be linked to both synthesis and release because the presence of AIP (1 µM), a specific inhibitor of this kinase (33), reduced NPY-induced CRF release by 45% after 3 h and totally prevented NPY-induced CRF synthesis and release after 8 h (Fig. 7Go, c and d). Finally, the cells incubation with PD 098056 (40 µM), an ERK kinase inhibitor (34), had no significant effect on NPY-induced CRF synthesis or release (Fig. 7Go, a and c and d).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The syncytiotrophoblast layer of human placenta has been suggested to be the main source of immunoreactive and biologically active CRF during the third trimester of human pregnancy (7, 35). In this regard, cytotrophoblasts freshly isolated from human term placenta have an undetectable level of CRF (36). However, in parallel with their spontaneous differentiation to syncytiotrophoblast-like structure in vitro, they acquire the ability to produce and release CRF (10, 36). NPY is also produced by the placental unit (11, 14), and could be implicated in the stimulation of CRF release by the syncytiotrophoblast (11).

All studies took place on the fourth day of culture because preliminary experiments have showed that this period coincides with the peak of secretory competence for both CRF and hCG (data not shown). This is in accordance with the syncytiotrophoblastic origin of these peptides. The syncytiotrophoblast model used, even though costly, time-consuming, and variable from preparation to preparation, has the advantage to rely on nontransformed cells containing the endogenous CRF gene. The present study shows that addition of NPY to trophoblastic cells, on their fourth day in culture, results in an increase in CRF synthesis and release. The stimulation of CRF release by NPY from these cells during the 3-h incubation period is similar to previous result reported by Petraglia et al. (11). Moreover, we showed for the first time that NPY modulates CRF synthesis in trophoblastic cells. An up-regulation of CRF synthesis has been reported in Y2 receptor bearing SK-N-BE2 cells transfected with the human CRF promoter (37). Taking into account the up-regulated synthesis, the NPY effect on CRF release between 3 h and 8 h appears mainly derived from this novel synthesis. After consideration, it could mean that during a short incubation period (3 h), the NPY induces the release of preformed vesicles containing CRF, while longer incubation period (8 h) allows the production and the processing of new CRF by the trophoblastic cells.

The syncytiotrophoblast displays on its maternal surface two types of NPY binding sites, the Y1 and Y3 subtypes (16). Because NPY could stimulate CRF synthesis and release by interacting with either one of them, we explored this possibility with the highly specific Y1 antagonist, BIBP3226 (24). This molecule completely abolished the NPY-induced CRF synthesis and release, thus strongly supporting the involvement of Y1 receptor subtype in the neuropeptide effects. Our results also showed that a pretreatment of trophoblastic cells with PTX, known to inactivate Gi and Go proteins by ADP ribosylation (38), caused a total inhibition of the NPY-induced CRF synthesis and release, suggesting that some PTX-sensitive G-proteins are involved in this syncytiotrophoblast response to NPY. This result is not surprising because, in most cell types studied, the Y1 receptor is PTX-sensitive (39).

Calcium-dependent CRF secretion has already been suggested in placental explants and cells (13, 36, 40). In these studies, KCl induced a marked increase in CRF release, which could be attributable to a depolarization-induced calcium influx in secreting cells. In that respect, a study done on microdialyzed median eminence has shown that basal and KCl-induced CRF releases are mediated by L-type calcium channel activation (41). Because syncytiotrophoblast has been shown to possess these channels (42), as well as L-type calcium channels dependent secretion process (43, 44), we used BAPTA/AM and nitrendipine to investigate the possible involvement of calcium and these channels in the NPY effects. The reduction of NPY-induced CRF release in presence of BAPTA/AM strongly suggest the involvement of calcium in CRF release, whereas the involvement of L-type calcium channels is unlikely according to the nitrendipine ineffectiveness. The fact that this blocker has no effect on NPY-induced CRF release is not surprising because Y1 receptors, at the exception of those found in vascular smooth muscles, are generally linked to L-type calcium channels inhibition (39) and therefore cannot account for the stimulation.

Our previous study (16) has demonstrated that Y1 receptors of human syncytiotrophoblasts are linked to PLC-ß activation. This activation leads to the cleavage of PtdInsP2, which generates 1,2 diacylglycerol (an activator of cPKCs and nPKCs) and Ins (1, 4, 5)P3 (which has a permissive effect on IP3-R) (45). The inhibition of NPY-induced CRF release and synthesis by U-73122 indicates that PLC-ß activation is partially responsible for the NPY effect, and almost totally accountable for the NPY-induced CRF synthesis. However, a report showing a decrease of PYY binding to Y1 receptor following U-73122 incubation (46) prompted us to further investigate the involvement of InsP3-releasable intracellular stores, by using the InsP3-R blocker, xestospongin C. The results obtained with xestospongin C reflect those obtained using the aminosteroid inhibitor of PLC-ß, indicating that a decrease of NPY binding, while not excluded, cannot explain the inhibition seen with the use U-73122. Therefore, we propose that in trophoblastic cells, NPY binds to its receptors which in turn activates PLC-ß. The generated InsP3 binds its receptor, induces calcium release from the intracellular store, which is responsible for the NPY-induced CRF synthesis and release.

The use of SKF 96365 [an inhibitor of the receptor-mediated calcium influx, the voltage-operated calcium channels (47) and SOC influx (27)] was initially planned to investigate the influence of SOC calcium influx on the NPY-induced CRF synthesis and release. The lack of specificity of this wide-range tool is not a problem because many aspects of the calcium entry in syncytiotrophoblasts remain to be elucidated. Also, it is likely to involve multiple calcium influx pathways. In the first time, our results showed that SOC influx is implicated only in NPY-induced CRF release. In the presence of NPY, either SKF 96365 or BAPTA/AM should induce the emptying of the calcium stores, while the presence of U-73122 or xestospongin C should not. These results suggest that the emptying of the store mediated by NPY could induced the activation of a pathway implicated in CRF synthesis.

Our results obtained in presence of BAPTA lead also to another interrogation: Why the calcium influx, which should be induced by the presence of this chelator alone (48), is not enough by itself to induce the release of CRF? A possible explanation is that calcium influx following the emptying of intracellular stores, even if SKF 96365 seems to be as efficient as U-73122 or xestospongin C in reducing NPY-induced CRF release, is not the sole factor to its release, but rather a cofactor.

In the placenta, where PKCs are activated by NPY (16), nothing has been published on either CaMKII or ERKs activities. To our knowledge, our results report a novel relationship between phosphoERKs immunoreactivity or activity and G protein-coupled receptor in trophoblastic cells. Our results clearly show that NPY promotes the autonomous form of CaMKII. The kinetic of the stimulation is rapid and transient, meaning that either the NPY effect on CaMKII could be rapidly desensitized or that the receptor could be rapidly internalized. In this regard, ERKs activation has been reported to follow G protein-coupled receptor endocytosis (49).

ERKs are known as downstream effectors of Y1 receptors (50, 51). We have measured the ERKs activation by their phosphorylation status (52), in presence of NPY. Our results showed a biphasic activation of the ERKs by NPY, a first activation that is rapid, followed by a sustained phase. Usually, the studies on ERKs activity induced by G protein-coupled receptors look at short period because a peak of ERKs activity is normally found between 2 and 10 min. The significance of the sustained ERK activation is intriguing, but not uncommon for receptor linked to PTX-sensitive G proteins (53). Phosphatidylinositol 3-kinase, Ras- and PKC-dependent processes have been proposed for the Y1 receptor-mediated ERKs activation (50, 51). Nevertheless, a considerable amount of work has to be done to ascertain which of these or other pathways are implicated in trophoblastic cells.

Many studies, mainly done on hypothalamic cells, have involved protein kinase A (PKA) and PKCs as inducers of CRF synthesis and release (54, 55). However, in syncytiotrophoblast, there is only evidence of the involvement of PKC in the inhibition of CRF release (36). Our results indicate that CaMKII, ERKs, and calcium-independent PKCs are regulators of CRF release and/or synthesis. The use of the nonisotype specific PKC inhibitor, BIM, inhibited NPY-induced CRF release but not its synthesis, while the conventional and µ PKCs inhibitor, Gö6976, did not. This means that novel or atypical subtypes of PKCs are implicated in the NPY-induced CRF secretion and could represent the cofactors associated with SOC influx responsible for CRF release. However, the PKC isotypes activated following NPY receptors activation are not known in syncytiotrophoblast. Previous studies have shown the presence of the three subfamilies of PKCs in syncytiotrophoblast (56, 57). Because Y1 receptors are linked to PLC and to phosphatidylinositol 3-kinase (16), both novel and atypical PKCs could be involved. The nonimplication of PKCs in the CRF synthesis induced by NPY is probably tissue specific because PKCs have been shown to be implicated in CRF gene expression in transiently transfected chicken macrophages (58).

CaMKII is a ubiquitous enzyme that has been implicated in peptide hormone secretion (59, 60). Our results with AIP clearly showed the involvement of this kinase in NPY-induced CRF synthesis and release. The exact nature of the substrates involved in the CaMKII action on CRF release is still unclear. In mouse pancreatic B-cells, this kinase is involved in the early steps of the exocytotic machinery leading to insulin release (60). The CaMKII has been implicated in the increasing of the size of the readily releasable vesicle pool and in the decreasing time for the refilling of the vesicles. Then, the implication of CaMKII in the NPY-stimulated CRF synthesis is in accordance with previous studies (37, 61). In these reports, NPY has been shown to induce CaMKII activity, which phosphorylates the cAMP response element binding protein (CREB). The phosphorylated CREB interacts with the cAMP response element (CRE) within several genes and leads to their transcription (37). Because CREs are found in the CRF promoter (58, 62), we could hypothesize that a similar mechanism could be involved in trophoblastic cells. Nevertheless, there is still a considerable amount of work that has to be done to ascertain the role of CREB in the NPY-induced CRF synthesis.

In conclusion, our study indicated that Y1 receptor links NPY to CRF synthesis and release in trophoblastic cells. Furthermore, our results indicate that these NPY effects are involved the PLC-InsP3-R axis and the CaMKII. They also showed that calcium independent PKCs are implicated in the stimulatory effects of NPY on CRF release. Our results suggest the existence of a diversity of biochemical pathways connecting the Y1 receptor to the CRF synthesis and release. Nonetheless, further studies are necessary to elucidate the sequence of their involvement. Our results also suggest that we can add PTX-sensitive G protein-coupled receptors, like the NPY-Y1 receptor, to the growing list of modulators of CRF synthesis and release.


   Acknowledgments
 
We express our gratitude to Mrs. Christiane Paré (Chief of Nursing) and the staff of the Department of Obstetrics and Gynecology of Pavillon St-Luc of the Centre Hospitalier Universitaire de Montréal (CHUM) for the donation of placentas. We are also grateful to Dr. Robert Moreau for his critical comments and suggestions and to Mélanie Laramée for editorial assistance.


   Footnotes
 
1 This study was supported by grants from Université du Québec à Montréal (to J.L.). Back

2 Recipient of a FRSQ-FCAR santé doctoral studentship. Back

Received October 6, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Evain-Brion D 1999 Maternal endocrine adaptations to placental hormones in humans. Acta Paediatr Suppl 428:12–16
  2. Petraglia F, Florio P, Nappi C, Genazzani AR 1996 Peptide signaling in human placenta and membranes: autocrine, paracrine, and endocrine mechanisms. Endocr Rev 17:156–186[CrossRef][Medline]
  3. McLean M, Bisit A, Davies J, Woods R, Lowry PJ, Smith R 1995 A placental clock controlling the length of human pregnancy. Nat Med 1:460–463[CrossRef][Medline]
  4. Goland RS, Waedlaw SL, Stark RI, Brown LS, Frantz AG 1986 High levels of corticotropin-releasing hormone immunoreactivity in maternal and fetal plasma during pregnancy. J Clin Endocrinol Metab 63:1199–1204[Abstract]
  5. Campbell EA, Linton EA, Wolfe CDA, Scraggs PR, Jones MT, Lowry PJ 1987 Corticotropin-releasing hormone concentrations during pregnancy and parturition. J Clin Endocrinol Metab 64:1054–1059[Abstract]
  6. Warren WB, Patrick SL, Goland RS 1992 Elevated maternal plasma corticotropin-releasing hormone levels in pregnancies complicated by preterm labor. Am J Obstet Gynecol 166:1198–1202[Medline]
  7. Riley SC, Walton JC, Herlick JM, Challis JRG 1991 The localization and distribution of corticotropin-releasing hormone in the human placenta and fetal membranes throughout gestation. J Clin Endocrinol Metab 72:1001–1007[Abstract]
  8. Kliman HJ, Nestler JE, Sermasi E, Sanger JM, Strauss JF 1986 Purification, characterization, and in vitro differentiation of cytotrophoblasts from human term placenta. Endocrinology 118:1567–1582[Abstract]
  9. Karalis K, Goodwin G, Majzoub JA 1996 Cortisol blockade of progesterone: a possible molecular mechanism involved in the initiation of human labor. Nature Med 2:556–560[CrossRef][Medline]
  10. Petraglia F, Sutton AN, Vale W 1989a Neurotransmitters and peptides modulate the release of immunoreactive corticotropin-releasing factor from cultured human placental cells. Am J Obstet Gynecol 160:247–251
  11. Petraglia F, Calzà L, Giardino L, Sutton S, Marrama P, Rivier AR, Genazzani AR, Vale W 1989b Identification of immunoreactive neuropeptide-{chi} in human placenta: localization, secretion, and binding sites. Endocrinology 124:2016–2022
  12. Petraglia F, Garuti GC, De Ramundo B, Angioni S, Genazzani AR, Bilezikjan LM 1990 Mechanisms of action of interleukin 1ß in increasing corticotrophin-releasing factor and adrenocorticotrophin hormone released from cultured human placental cells. Am J Obstet Gynecol 163:307–312
  13. Sun K, Smith R, Robinson PJ 1994 Basal and KCl-stimulated corticotropin-releasing hormone release from placental syncytiotrophoblasts is inhibited by sodium nitroprusside. J Clin Endocrinol Metab 79:519–524[Abstract]
  14. Petraglia F, Calzà L, Giardino L, Zanni M, Florio P, Ferrari AR, Nappi C, Ganazzani AR 1993 Maternal decidua and fetal membranes contain immunoreactive neuropeptide Y. J Endocrinol Invest 16:201–205[Medline]
  15. Petraglia F, Coukos G, Battaglia C, Bartolotti A, Volpe A, Segre A, Genazzani AR 1989c Plasma and amniotic fluid immunoreactive neuropeptide Y level changes during pregnancy, labor and at parturition. J Clin Endocrinol Metab 69:324–328
  16. Robidoux J, Simoneau L, St-Pierre S, Ech-Chadli H, Lafond J 1998 Human syncytiotrophoblasts NPY receptors are located on BBM and activate PLC-to-PKC axis. Am J Physiol 274:E502–E509
  17. Thiede HA 1960 Studies of the human trophoblast in tissue culture. I. Cultural methods and histochemical staining. Am J Obstet Gynecol 79:636–647
  18. Stromberg K, Azizkhan JC, Speeg KV 1978 Isolation of functional human trophoblast cells and their partial characterization in primary culture. In Vitro 14:631–638[Medline]
  19. Winkel CA, Smyder JM, Macdonald PC, Simpson ER 1980 Regulation of cholesterol and progesterone synthesis in human placental cells in culture by serum lipoproteins. Endocrinology 106:1054–1060[Medline]
  20. Contractor SF, Routledge A, Sooranna SR 1984 Identification and estimation of cell types in mixed primary cell cultures of early and term human placenta. Placenta 3:41–54
  21. Abraham ST, Benscoter H, Schworer CM, Singer HA 1996 In situ Ca2+ dependence for activation of Ca2+/calmodulin-dependent protein kinase II in vascular smooth muscle cells. J Biol Chem 271:2506–2513[Abstract/Free Full Text]
  22. Pande SV, Murthy MSR 1994 A modified micro-Bradford procedure for elimination of interference from sodium dodecyl sulfate, other detergents, and lipids. Anal Biochem 220:424–426[CrossRef][Medline]
  23. Schägger H, von Jagow G 1987 Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166:368–379[CrossRef][Medline]
  24. Rudolf K, Eberlein W, Engel W, Wieland HA, Willim KD, Entzeroth M, Wienen W, Beck-Sickinger AG, Doods HN 1994 The first highly potent and selective non-peptide neuropeptide Y Y1 receptor antagonist: BIBP3226. Eur J Pharmacol 271:R11–R13
  25. Thompson AK, Mostafapour SP, Denlinger LC, Bleasdale JE, Fisher SK 1991 The aminosteroid U-73122 inhibits muscarinic receptor sequestration, and phosphoinositide hydrolysis in SK-N-SH neuroblastoma cells. A role for Gp in receptor compartimentation. J Biol Chem 266:23856–23862[Abstract/Free Full Text]
  26. Gafni J, Munsch JA, Lam TH, Catlin MC, Costa LG, Molinski TF, Pessah IN 1997 Xestospongins: potent membrane permeable blockers of the inositol 1,4,5-triphosphate receptor. Neuron 19:723–733[CrossRef][Medline]
  27. Mason MJ, Mahaut-Smith MP, Grinstein S 1991 The role of intracellular Ca2+ in the regulation of plasma membrane Ca2+ permeability of unstimulated rat lymphocytes. J Biol Chem 266:10872–10879[Abstract/Free Full Text]
  28. Hanson PI, Schulman H 1992 Neuronal Ca2+/calmodulin-dependent protein kinases. Annu Rev Biochem 61:559–601[CrossRef][Medline]
  29. Cobb HM, Goldsmith EJ 1995 How MAP kinases are regulated. J Biol Chem 270:14843–14846[Free Full Text]
  30. Toullec D, Pianetti P, Coste H, Bellevergue P, Grand-Perret T, Ajakane M, Baudet V, Boissin P, Boursier E, Loriolle F, Duhamel L, Charon D, Kirilovsky J 1991 The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. J Biol Chem 266:15771–15781[Abstract/Free Full Text]
  31. Martiny-Baron G, Kazanietz MG, Mischak H, Blumberg PM, Kochs G, Hug H, Marmé D, Schächtele C 1993 Selective inhibition of protein kinase C isozymes by the indocarbazole Gö 6976. J Biol Chem 268:9194–9197[Abstract/Free Full Text]
  32. Gschwendt M, Dieterich S, Rennecke J, Kittstein W, Mueller HJ, Johannes FJ 1996 Inhibition of protein kinase C µ by various inhibitors. Differentiation from protein kinase c isoenzymes. FEBS Lett 392:77–80[CrossRef][Medline]
  33. Ishida A, Kameshita I, Okuno S, Kitani T, Fujisawa H 1995 A novel highly specific and potent inhibitor of the calcium-dependent protein kinase II. Biochem Biophys Res Commun 212:806–812[CrossRef][Medline]
  34. Alessi DR, Cuenda A, Cohen P, Dudley DT, Saltiel AR 1995 PD 098056 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. J Biol Chem 270:27489–27494[Abstract/Free Full Text]
  35. Riley SC, Challis JRG 1991 Corticotrophin-releasing hormone production by the placenta and fetal membranes. Placenta 12:105–119[Medline]
  36. Ni X, Chan EG, Fitter JT, Smith R 1997 Nitric oxide inhibits corticotropin-releasing hormone exocytosis but not synthesis by cultured human trophoblasts. J Clin Endocrinol Metab 82:4171–4175[Abstract/Free Full Text]
  37. Sheriff S, Dayal R, Kasckow, J, Regmi A, Chance W, Fischer J, Balasubramaniam A 1998 NPY upregulates genes containing cyclic AMP response element in human neuroblastoma cell lines bearing Y1 and Y2 receptors: involvement of CREB. Regul Pept 75–76:309–318
  38. Spiegel AM 1987 Signal transduction by guanine nucleotide binding proteins. Mol Cell Endocrinol 49:1–16[CrossRef][Medline]
  39. Michel MC, Beck-Sinkinger A, Cox H, Doods HN, Herzog H, Larhammar D, Quirion R, Schwartz T, Westfall T 1998 XVI. International union of pharmacology recommendations for the nomenclature of neuropeptide Y, peptide YY, and pancreatic polypeptide receptors. Pharmacol Rev 50:143–150[Abstract/Free Full Text]
  40. Thomson M, Chan EC, Falconer J, Madsen G, Smith R 1988 Secretion of corticotropin-releasing hormone by superfused human placental fragments. Gynecol Endocrinology 2:87–100[Medline]
  41. Gabr RW, Gladfelter WE, Birkle DL, Azzaro AJ 1994 In vivo microdialysis of corticotropin-releasing factor (CRF): calcium dependence of depolarization-induced neurosecretion of CRF. Neurosci Lett 169:63–67[CrossRef][Medline]
  42. Cimerikic B, Zamah R, Ahmed MS 1998 Identification of L-type calcium channels associated with kappa opiod receptors in human placenta. J Mol Neurosci 10:261–272[Medline]
  43. Petraglia F, Lim ATW, Vale W 1987 Adenosine 3',5'-monophosphate, prostaglandins, and epinephrine stimulate the secretion of immunoreactive gonadotropin-releasing hormone from cultured human placental cells. J Clin Endocrinol Metab 65:1020–1025[Abstract]
  44. Petit A, Gallo-Payet N, Vaillancourt C, Bellabarba D, Lehoux JG, Belisle S 1993 A role for extracellular calcium in the regulation of placental lactogen release by angiotensin-II and dopamine in human term trophoblastic cells. J Clin Endocrinol Metab 77:670–676[Abstract]
  45. Berridge MJ, Irvine RF 1989 Inositol phosphates and cell signalling. Nature 341:197–205[CrossRef][Medline]
  46. Parker SL, Parker MS, Sweatman T, Crowley WR 1998 Characterization of G protein and phospholipase C-coupled agonist binding to the Y1 neuropeptide Y receptor in rat brain: sensitivity to G protein activators and inhibitors and to inhibitors of phospholipase C. J Pharmacol Exp Ther 286:382–391[Abstract/Free Full Text]
  47. Merritt JE, Armstrong WP, Benham CD, Hallam TJ, Jacob R, Jaxa-Chamiec A, Leigh BK, McCarthy SA, Moores KE, Rink TJ 1990 SK&F 96365, a novel inhibitor of receptor-mediated calcium entry. Biochem J 271:515–522[Medline]
  48. Parekh AB, Penner R 1997 Store depletion and calcium influx. Physiol Rev 77:901–930[Abstract/Free Full Text]
  49. Daaka Y, Luttrell, LM, Ahn S, Della Rocca GJ, Fergusson SSG, Caron MG, Lefkowitz RJ 1998 Essential role for G protein-coupled receptor endocytosis in the activation of mitogen-activated protein kinase. J Biol Chem 272:685–688
  50. Nakamura M, Sakanaka C, Aoki Y, Ogasawara H, Tsuji T, Kodama H, Matsumoto T, Shimizu T, Noma M 1995 Identification of two isoforms of mouse neuropeptide Y-Y1 receptor generated by alternative splicing. J Biol Chem 270:30102–30110[Abstract/Free Full Text]
  51. Mannon PJ, Raymond JR 1998 The neuropeptide Y/peptide YY Y1 receptor is coupled to MAP kinase via PKC and Ras in CHO cells. Biochem Biophys Res Commun 246:91–94[CrossRef][Medline]
  52. Gorenne I, Su X, Moreland RS 1998 Inhibition of p42 and p44 MAP kinase does not alter smooth muscle contraction in swine carotid artery. Am J Physiol 275:H131–H138
  53. Meloche S, Seuwen K, Pagès G, Pouysségur J 1992 Biphasic and synergistic activation of p44mapk (ERK1) by growth factors: correlation between late phase activation and mitogenicity. Mol Endocrinol 6:845–854[Abstract]
  54. Widmaier EP, Lim AT, Vale W 1989 Secretion of corticotropin-releasing factor from cultured rat hypothalamic cells: effects of catecholamines. Endocrinology 124:583–590[Abstract]
  55. Emanuel RL, Girard DM, Thull DL, Majzoub JA 1990 Second messengers involved in the regulation of corticotropin-releasing hormone mRNA and peptide in cultured rat fetal hypothalamic primary cultures. Endocrinology 126:3016–3021[Abstract]
  56. Amane A, Simoneau L, Savard R, Lafond J 1995 Identification and chromatographic separation of protein kinase C isotypes in syncytiotrophoblast brush border and basal plasma membranes of human placenta. Int J Biol Chromatogr 1:237–246
  57. Karl PI, Divald A 1996 Protein kinase C in cultured human placental trophoblasts: identification of isoforms and role in cAMP signalling. Biochem J 320:831–836
  58. Van LP 1993 Phorbolester stimulates the activity of human corticotropin-releasing hormone gene promoter via 3',5'-cyclic adenosine monophosphate response element in transiently transfected chicken macrophages. Endocrinology 132:30–34[Abstract]
  59. Waters WW, Chen PL, McArthur NH, Moreno PA, Harms PG 1998 Calcium/calmodulin-dependent protein kinase II involvement in release of gonadotropin-releasing hormones. Neuroendocrinology 67:145–152[CrossRef][Medline]
  60. Gromada J, Hoy M, Renstrom E, Bokvist K, Eliasson L, Gopel S, Rorsman P 1999 CaM kinase II-dependent mobilization of secretory granules underlies acetylcholine-induced stimulation of exocytosis in mouse pancreatic B-cells. J Physiol 518:745–759[Abstract/Free Full Text]
  61. Sheriff S, Chance W, Fischer J, Balasubramaniam A 1997 Neuropeptide Y treatment and food deprivation increase cyclic AMP response element-binding in rat hypothalamus. Mol Pharmacol 51:597–604[Abstract/Free Full Text]
  62. Seasholtz AF, Thompson RC, Douglass JO 1988 Identification of a cyclic adenosine monophosphate-responsive element in the rat corticotropin-releasing hormone gene. Mol Endocrinol 2:1311–1319[Abstract]



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