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Endocrinology Vol. 140, No. 10 4601-4608
Copyright © 1999 by The Endocrine Society

ARTICLES

Molecular Identification of Adenylyl Cyclase 3 in Bovine Corpus Luteum and Its Regulation by Prostaglandin F2{alpha}-Induced Signaling Pathways1

Roni Mamluk, Nicole Defer, Jacques Hanoune and Rina Meidan

Department of Animal Sciences (R.Ma., R.Me.), Faculty of Agricultural, Food and Environmental Quality Sciences, Hebrew University, Rehovot 76100 Israel and INSERM U99 Hopital Henri Mondor (N.D., J.H.), 94010 Creteil, France

Address all correspondence and requests for reprints to: Rina Meidan, Department of Animal Sciences, Faculty of Agricultural, Food and Environmental Quality Sciences, Hebrew University, Rehovot 76100, Israel. E-mail: rina{at}agri.huji.ac.il


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The involvement of cAMP in various aspects of ovarian steroidogenic cells functions has been extensively studied. However, the adenylyl cyclase (AC) types expressed in ovarian cells, of any species, are not yet determined. The present study was undertaken to identify AC types present in bovine luteal cells and their regulation by various stimuli. AC isoforms 2, 3, 5, 6, 7, 8, and 9 were detected in the bovine brain by Northern blotting analysis, whereas the bovine corpus luteum (CL) only expressed AC3 and 6 mRNAs, with AC3 being more abundant than AC6. The use of AC3-specific primers in RT-PCR reaction verified the presence of AC3 mRNA in both bovine and rat CL tissue as well as in bovine steroidogenic luteal cells. Because these two AC isoforms, AC3 and 6, exhibit distinct regulatory patterns we have next examined the effects of various signaling pathways on AC activity in luteal cells. These studies have shown that: 1) prostaglandin (PG) F2{alpha} and phorbol 12-myristate 13-acetate markedly elevated agonist-stimulated cAMP synthesis (these effects were inhibited by addition of highly specific PKC inhibitor, bisindolylmaleimide); 2) depletion of Ca2+ from the incubation medium inhibited AC activity; 3) physiological concentrations of Ca2+ ions (up to 5 mM) significantly stimulated cAMP production in luteal cells; and 4) the effects of Ca2+ on cAMP synthesis were evident only in the presence of forskolin. These regulatory characteristics of AC activity are consistent with the molecular identification of ACs indicating the presence of AC3 in luteal cells.

The reported data may delineate the cross-talk between physiological activators of AC in the CL (such as LH, PGE2, and PGI2) and other ligands (such as PGF2{alpha} and endothelin-1), which indirectly modulate AC activity. Therefore, the identification of AC isoforms present in luteal cells is an important step toward understanding the mode of action of a wide array of hormones regulating ovarian cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LH SURGE TRIGGERS a cascade of events leading to the development of the corpus luteum (CL). These effects are exerted mainly by activation of adenylyl cyclase (AC) (1). Our previous studies have shown that LH binding and LH receptors mRNA were present mainly in the theca-derived small luteal cells (2, 3). This probably explains why, in ruminants, LH-induced AC activity is only observed in the small luteal cells (2, 4). Nevertheless, the granulosa-derived large luteal cells, which produce the major part of luteal progesterone, have a functional AC and respond to physiological concentrations of prostaglandin (PG) I2 and PGE2 with elevated cAMP and progesterone production (5, 6).

Nine different mammalian AC isoforms were identified and cloned so far. There is approximately 50% amino acid sequence similarity among these forms, limited mainly to the two cytoplasmic domains. Within each domain, two regions are highly conserved and have up to 93% sequence identity (7). All AC isoforms are activated by the {alpha} subunit of the stimulatory G protein-coupled(G{alpha}s) and the diterpene forskolin. However, they differ in their regulation by other G-proteins (non G{alpha}s) subunits, Ca2+ ions and protein kinase C (PKC) (8, 9). Therefore, ligands that are not coupled to G{alpha}s still have the potential to modulate AC activity, allowing a dynamic cross-talk among different signaling pathways. The diversity in regulation is particularly intriguing in light of the distinct, cell-specific expression of AC isoforms. Whereas all forms are expressed in brain, peripheral tissues have been shown to express various AC isoforms in a cell- and development-specific patterns (10, 11, 12, 13).

Although the involvement of cAMP in various aspects of steroidogenic cells functions has been extensively studied, the AC types expressed in ovarian cells have not been determined. The aim of the present study was to identify AC types present in bovine CL and their regulation by PGF2{alpha}, Ca2+ ions and PKC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
DMEM and Ham’s F-12 1:1 (vol/vol) nutrient mixture (DMEM-F12) and Moloney murine leukemia virus SuperScript II H- Reverse Transcriptase were from Life Technologies, Inc. (Gaithersburg, MD); penicillin, streptomycin, neomycin, and FCS were from Biological Industries, (Beit HaEmek, Israel); forskolin, insulin, phorbol 12-myristate 13-acetate (TPA), bisindolylmaleimide, isobutylmethylxanthine (IBMX), ionomycin and PGF2{alpha} were from Sigma (St. Louis, MO); deoxynucleotide triphosphate, random hexamers oligodeoxynucleotides were from Promega Corp., (Madison, WI); Taq DNA polymerase was from Farmentas (Vilnius, Lithuania); 125I-labeled tyrosine methyl ester-cAMP was from Amersham Pharmacia Biotech (Buckinghamshire, UK).

CL collection
Stage of bovine luteal phase was determined according to Fields and Fields (14). Ovaries were collected at the abattoir and CL were frozen immediately in liquid N2. Rat CL were obtained as previously described (15). Briefly, PMSG treated immature rats were mated following hCG injection and killed 20 h later.

Cell cultures
Granulosa and theca cells were isolated from healthy bovine preovulatory follicles, as previously described (2, 16). Cells from individual follicles were cultured in 24-well plates (8 x 104 granulosa and 3 x 104 theca cells per well) for 8 days in DMEM-F12 media (basal media) containing 1% FCS, insulin (2 µg/ml) and forskolin (10 µM)—conditions previously shown to induce cell luteinization (2, 16). Our previous studies have established that these cells have similar characteristics to cells enriched from the bovine CL (3, 16). Luteinized theca and granulosa cells are referred to as LTC and LGC, respectively.

Determination of cAMP production by luteal cells
On the eighth day of culture, cells were washed and incubated for 24 h in basal media containing 1% FCS to reduce forskolin-stimulated AC activity.

Effects of PGF2{alpha} and TPA on forskolin-stimulated cAMP production were determined as follows: cells were preincubated for 10 min in the presence of the phosphodiesterase (PDE) inhibitor, isobutylmethylxanthine (IBMX; 200 µM), which was also present during the entire incubation period. Incubation was carried out for 40 min with basal media containing forskolin (10 µM) alone, forskolin + PGF2{alpha} (1 µg/ml), or forskolin + TPA (200 nM) in the presence or absence of a highly specific PKC inhibitor, bisindolylmaleimide (100 nM). At the end of incubation period, media were collected, boiled for 4 min, and frozen until assayed for cAMP by RIA. A similar experimental design was used to examine the effects of PGF2{alpha} on LH-stimulated cAMP production.

The effects of Ca2+ ions on cAMP production were examined as follows: cells were preincubated for 10 min at 37 C with either Krebs buffer or a nominally Ca2+-free Krebs buffer containing IBMX (200 µM), which was also present during the incubation period. The Krebs buffer consisted of 120 mM NaCl, 4.75 mM KCl, 1 mM KH2PO4, 5 mM NaHCO3, 1.44 mM MgSO4, 1.1 mM CaCl2, 0.1 mM EGTA, 11 mM glucose, 25 mM HEPES, and 0.1% BSA adjusted to pH 7.4 with 2 M Tris base. The nominally Ca2+-free Krebs buffer contained 120 mM NaCl, 4.75 mM KCl, 1 mM KH2PO4, 5 mM NaHCO3, 1.44 mM MgSO4, 11 mM glucose, 25 mM HEPES, and 0.1% BSA adjusted to pH 7.4 with 2 M Tris base. The use of Ca2+-free Krebs buffer in experiments denotes the addition of 0.1 mM EGTA to the nominally Ca2+-free Krebs. Incubation was carried out at 37 C for 5 min. Reaction was terminated by aspiration of the buffers and addition of 200 µl of 0.2 M HCl. The content of each well was collected, neutralized with NaOH and frozen until assayed for cAMP by RIA.

RNA extraction and RT-PCR
Total RNA was extracted from cells by the guanidinium thiocyanate method (17). RT-PCR was conducted as previously described (3, 18). AC3 specific primers: sense 5'-cctgcctgtgcctgcctcgct-3'; antisense 5'-gtagtaggacatgatgcccac-3' were synthesized according to the rat and human AC 3 sequences (GenBank accession numbers M55075 and AF033861, respectively). The primers were designed to amplify a region of 581 bp, conserved in the AC3 sequences of both species. To avoid amplification of other ACs, we have chosen a region outside the boundaries of the two cytoplasmic domains. Computer searches, primer designs and sequence alignments were performed by means of software from Genetics Computer Group Inc. (Madison, WI). The PCR product amplified from bovine CL and luteal cells were subjected to direct sequence analysis using both sense and antisense AC3 oligonucleotides.

Northern blot analysis
The cDNA probes used for Northern blotting (ranging from 300 to 800 bp) were described in previous reports (12). Total RNA was extracted by the guanidinium thiocyanate method (17) and Poly(A)+ RNA was obtained by oligo(dT) cellulose chromatography. Five micrograms of Poly(A)+ RNA (extracted from bovine tissues) or 20 µg of total RNA (from rat brain) were electrophoresed on 1% agarose gel containing 2.2 M formaldehyde and transferred overnight onto a Hybond-N+ membrane by capillary blotting. Hybridization with specific cDNA probes was carried out using Rapid-hyb buffer (Amersham Pharmacia Biotech) according to the manufacturer’s instructions. The membranes were washed once in 2 x SSC (1 x SSC = 0.15 M NaCl and 15 mM Na3C6H5O7, pH 7.0), 0.1% SDS for 15 min at room temperature, and twice in 0.5 x SSC, 0.1% SDS for 15 min at 65 C, and exposed to film with intensifying screens at -80 C for 6–14 days.

RIA for cAMP
cAMP was determined after acetylation, using 125I-labeled tyrosine methyl ester-cAMP as tracer (19). The sensitivity limit of this assay was 1.2 fmol/tube.

Statistical analysis
Cells for each experiment were derived from individual follicle (one per cow). Experiments were repeated three times and each treatment was examined in triplicates. Data are presented as means ± SEM. One-way ANOVA was used to determine statistical difference between treatments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Molecular identyfication of adenylyl cyclase isoforms in bovine corpus luteum and luteal cells
To identify AC isoforms expressed in the bovine CL, we have employed Northern blot analysis using rodent cDNA probes. Their suitability for the bovine system was determined by hybridization to brain tissue, which is expected to express all AC isoforms. Isoforms 2, 3, 5, 6, 7, 8, and 9 were detected in the bovine brain (Fig. 1Go, A and B), whereas the bovine CL only expressed AC3 and 6 mRNAs, with AC3 being much more abundant than AC6 (Fig. 1BGo). AC1 was not examined as it is confined to the brain; AC4 did not hybridize to bovine brain; therefore, it is unknown whether it is expressed in the bovine CL. Another verification for the Northern blot analysis in the bovine species were the findings that AC6 mRNA, but not that of AC3, was detected in bovine kidney (Fig. 1BGo), in accordance with data previously reported in the rat (10, 11). Although Northern blot analysis revealed that AC3 was the most abundant type in CL, it was important to find out whether the steroidogenic luteal cells express the AC3 gene. This was achieved by means of RT-PCR using AC3 specific primers (please refer to Materials and Methods section). In an RT-PCR reaction, these primers amplified a product of an expected size in rat and bovine brain tissues. On the other hand, the heart and kidney, which do not express AC3, gave no product (Fig. 2AGo). The amplified fragment from the bovine CL was sequenced (Fig. 3Go); this cDNA sequence was highly homologous to AC3 of human (88%) and rat (86%). In good agreement with results presented in Fig. 1Go, AC3 mRNA was readily detected (by RT-PCR) in bovine and rat CL tissues (Fig. 2Go, B and C), as well as in the two steroidogenic luteal-like cells.



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  		Figure 1. Northern blot analysis of AC mRNA expression. RNA was extracted, electrophoresed, and transferred to nylon membrane as described in Materials and Methods. Blots were hybridized with [{alpha}-32P]dCTP-labeled rat cDNA and exposed to film with intensifying screens at -80 C for 6–14 days. A, Hybridization with AC cDNA types 2, 5, 7, 8, and 9; B, AC types 3 and 6. The following preparations of RNA were loaded: lane 1, rat brain (20 µg total RNA); lane 2, bovine CL; lane 3, bovine kidney; lane 5, bovine brain (each 5 µg poly A+ RNA).

 


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  		Figure 2. Detection of AC3 in rat and bovine tissues by RT-PCR. Total RNA from rat and bovine tissues were reversed transcribed and amplified using the following primers: sense 5'-cctgcctgtgcctgcctcgct-3'; antisense 5'-gtagtaggacatgatgcccac-3'. Products were electrophoresed on agarose gels. The inverse images of the ethidium bromide stained gels (A–C) are presented.

 


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  		Figure 3. The cDNA sequence of bovine AC3 fragment amplified by RT-PCR and its alignment with human and rat AC 3 sequences. Boldface letters indicate mismatching between the bovine and either human or rat sequences.

 
Functional characteristics of adenylyl cyclase activity in bovine luteal cells
To elucidate the physiological relevance of AC isoforms present in the CL, the regulation of AC activity by different signaling pathways was studied. These experiments were conducted in the presence of a PDE inhibitor, IBMX, and therefore data of the following experiments reflect cAMP synthesis, rather than inhibition of its degradation.

Effect of PGF2{alpha}
Forskolin stimulated cAMP production in both luteal cell types, however, the effect was much higher in LTC than in LGC: 250 and 22-fold increase over basal levels, respectively (from basal values of 0.06.2 ± 0.02 and 0.05 ± 0.01 to forskolin-stimulated response of 14.9 ± 2.8 and 1.09 ± 0.15 pmol/well for LTC and LGC, respectively). PGF2{alpha} alone had no effect on cAMP production by luteal cells (Figs. 4Go and 5Go). However, in the presence of forskolin, PGF2{alpha} synergistically increased cAMP production in a dose-dependent manner, and cAMP production was increased by up to 6- and 10-fold in LGC and LTC, respectively (Fig. 4Go). The increments induced by forskolin and PGF2{alpha} were generated rapidly; significant elevations were evident already after 10 min of incubation (data not shown). Similarly to its effects on AC activated by forskolin, the stimulation by PGF2{alpha} was also evident in cells challenged with LH, acting via G{alpha}s (Table 1Go). As expected, LH elevated cAMP levels in LTC in a dose-dependent manner. PGF2{alpha} synergistically facilitated LH response, the maximal stimulation in the presence of PGF2{alpha} was 10 fold higher than the effect of LH alone (Table 1Go).



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  		Figure 4. Effects of PGF2{alpha} on forskolin-stimulated cAMP (pmol/well) production by luteal cells. Luteinized theca and granulosa cells (LTC and LGC, respectively) were washed thoroughly and incubated for 24 h in basal media containing 1% FCS followed by 40 min incubation in basal media containing IBMX (200 µM) and PGF2{alpha} (0–1000 ng/ml) with or without forskolin (10 µM). Data (mean ± SEM) are from three separate experiments. *, Statistically significant difference (P < 0.01) compared with forskolin alone.

 


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  		Figure 5. Effects of forskolin, PGF2{alpha}, and TPA on cAMP (pmol/well) production by luteal cells. Luteinized theca cells (LTC) were washed thoroughly and preincubated for 24 h in basal media containing 1% FCS followed by 40 min incubation with basal media containing IBMX (200 µM) and PGF2{alpha} (1 µg/ml) or TPA (200 nM) with (+) or without (-) forskolin (10 µM). Bisindolylmaleimide (100 nM), a highly specific PKC inhibitor, was maintained in the appropriate treatments during the entire incubation period. Data (mean ± SEM) are from three separate experiments. *, Statistically significant difference (P < 0.01) from forskolin alone. {dagger}, Statistically (P < 0.05) significant inhibition by bisindolmaleimide.

 

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  		Table 1. Effects of PGF2{alpha} on LH-stimulated cAMP production by luteinized theca cells

 
Effects of PKC
Treatment with a PKC activator, TPA alone, had no effect on cAMP production in luteal cells. However, when incubated with forskolin, it synergistically stimulated cAMP production by 5-fold. Although TPA and PGF2{alpha} were used in saturating concentrations (200 nM and 1 µg/ml, respectively), PGF2{alpha} induced higher cAMP levels than TPA; cAMP levels were 120 and 51 pmol/well in the presence of PGF2{alpha} and TPA, respectively (Fig. 5Go).

To further examine the role of PKC in AC activation, we employed a highly specific PKC inhibitor-bisindolylmaleimide. This agent significantly (P < 0.01) inhibited the stimulation cAMP production by PGF2{alpha} and TPA (Fig. 5Go). The specificity of this inhibitor was confirmed by the lack of inhibition in cAMP increase induced by forskolin alone. cAMP elevation by TPA was completely abolished in the presence of PKC inhibitor, and cAMP production was decreased to levels obtained in the presence of forskolin alone (Fig. 5Go). In contrast to TPA, AC activity in the presence of PGF2{alpha} was only partially inhibited (by ~40%) suggesting that effects of PGF2{alpha} on cAMP may not involve only PKC activation.

Effects of Ca2+ ions
Depletion of Ca2+ from the culture media by the divalent cation chelator, EGTA, significantly (P < 0.01) reduced forskolin-stimulated cAMP synthesis by LTC (Fig. 6AGo). A stimulatory effect of Ca2+ on cAMP production was also demonstrated in Fig. 5BGo: Ca2+ (3 mM) significantly (P < 0.05), elevated cAMP levels by 60% compared with cells incubated in nominally Ca2+-free Krebs buffer. Addition of ionomycin, a Ca2+ ionophore, to the media did not affect cAMP production (Fig. 6Go, A and B).



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  		Figure 6. Effects of extracellular Ca2+ on forskolin-stimulated cAMP production by luteal cells. Luteinized theca cells (LTC) were washed thoroughly and preincubated for 24 h in basal media containing 1% FCS. A, Ten minutes before the beginning of the experiment, cells were incubated at 37 C in Krebs buffer containing IBMX (200 µM). Incubation was carried out for 5 min in Krebs buffer containing forskolin (10 µM) alone, control (C), or with EGTA (2 mM), in the presence or absence of ionomycin (400 nM; IM). *, Statistically significant difference (P < 0.05) from controls. B, Cells were incubated for 40 min in Krebs buffer followed by 10 min incubation with nominally Ca2+-free Krebs containing IBMX (200 µM). Cells were incubated for 4 min in Ca2+-free Krebs, treatments were added and incubation was continued for additional 5 min. + or -, Presence or absence of 3 mM extracellular Ca2+, the presence of ionomycin (400 nM) is also indicated (+IM). Forskolin (10 µM) was present in all treatments. Data (mean ± SEM) are from three separate experiments. *, Statistically significant difference (P < 0.05) from forskolin alone (without Ca2+or ionomycin)

 
The interactions between forskolin and Ca2+ on cAMP production are shown in Figs. 6Go and 7Go. In the absence of forskolin, Ca2+ had no significant effect on cAMP production. However in its presence (forskolin 10 µM), Ca2+ (0–5 mM) elevated cAMP production in a dose-dependent manner with a maximal increase being 3-fold over levels induced by forskolin alone (Fig. 7AGo). The effects of Ca2+ on the dose-response curve of forskolin (0–100 µM,) are demonstrated in Fig. 7BGo. In the presence of Ca2+ (2 mM) the dose-response curve of forskolin was shifted to the left, again suggesting a facilitatory role of Ca2+ on cAMP production. Similar findings were obtained using LGC (data not shown).



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  		Figure 7. Interactions between forskolin and Ca2+ on cAMP production. Luteinized theca cells (LTC) were washed thoroughly and preincubated for 24 h in basal media containing 1% FCS. Cells were than incubated for 40 min in Krebs buffer followed by 10 min incubation with nominally Ca2+-free Krebs containing IBMX (200 µM). Incubation was carried out for 4 min in Ca2+-free Krebs into which treatments were added for additional 5 min stimulation. A, Varying concentrations of extracellular Ca2+, with or without forskolin (10 µM). B, Varying concentrations of forskolin, with or without Ca2+ (2 mM). Data are of one representative out of two separate experiments.

 
In the range of Ca2+ ions concentration examined (0–5 mM), there was no inhibition of cAMP production in luteal cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The current paper, using a series of molecular and functional studies, demonstrates that the CL expresses the adenylyl cyclase 3 isoform.

AC3 mRNA was detected by two independent methods, Northern blotting and RT-PCR. The functional studies have shown that: 1) TPA markedly elevated agonist-stimulated cAMP synthesis; 2) depletion of Ca2+ from the incubation medium inhibited AC activity; 3) physiological concentrations of Ca2+ ions (up to 5 mM) significantly stimulated cAMP production in luteal cells; and 4) the effects of Ca2+ on cAMP synthesis were evident only in the presence of forskolin. These regulatory characteristics of AC activity are consistent with those of AC3 (20, 21) and therefore support the molecular identification of this enzyme in luteal cells. Nevertheless, it is difficult to extrapolate mRNA data to actual protein, and the positive identification of AC3, at the protein level, awaits further research. The presence of AC3 was not only demonstrated in whole CL tissue (of both bovine and rat) but also in the two luteal steroidogenic cell types. This observation is physiologically significant since the CL is a heterogeneous tissue and is comprised of steroidogenic and non steroidogenic cell types (22).

The mammalian ACs are divided into four subfamilies according to different sequence elements and their regulatory functions (9, 20, 21). AC1, AC3, and AC8 comprise a subfamily whose activity is stimulated by Ca2+/calmodulin (CaM). Although AC3 is a member of the Ca2+-stimulable subfamily, its structural and regulatory features are distinct. AC1 and AC8 expression is mostly confined to the brain and Ca2+/CaM stimulated their basal activity. AC3, on the other hand, is expressed in discrete brain areas and in various peripheral tissues (13). In addition, unlike types 1 and 8, the activation of AC3 by Ca2+/CaM requires the presence of forskolin or an active G{alpha}s.

In addition to AC3 we have also observed AC6 expression in the CL, the latter isoform together with another member, AC5, comprise a subfamily whose activity is inhibited by low concentration of Ca2+ (in the µM range). (23). However, under no conditions examined could we observe a Ca2+-induced inhibition, suggesting that AC6 was present at lower levels than AC3, as indeed suggested by the Northern blotting.

Our data demonstrating stimulation of AC activity by Ca2+ are in accord with other reports using nontransformed cells in which AC3 naturally predominates (24, 25). In smooth muscle cells, activation of the phospholipase C (PLC) pathway by arginine vasopressin (acting via V1 receptors) or angiotensin II (acting via angiotensin type 1 receptors), elevated agonist-stimulated cAMP production. This elevation was blocked by an inhibitor of CaM (24). Similarly, Burnay et al. (25) had shown that in bovine adrenal glomerulosa cells, angiotensin II potentiated ACTH-stimulated AC activity through mobilization of Ca2+. Conflicting data has been reported regarding the effects of Ca2+ on AC3 activity in human embryonic kidney (HEK) 293 cells. In these cells, transfected with rat AC3 (26, 27, 28, 29), CA2+/CaM inhibited cAMP production (27) due to activation of CaM kinase II (28). Inadequate compartmentalization of AC and/or availability of endogenous CaM (or CaM kinase) may possibly underline differences between transfected cells vs. cells naturally expressing the enzyme.

Another important regulator of AC activity is PKC, as with Ca2+ ions, it exerts isoform-specific actions (21, 30, 31). It has been demonstrated that activation of PKC by TPA enhanced the forskolin responsiveness of AC3 (30, 31), and had no significant effect on AC6 (31). In bovine (this study) and porcine (32) luteal cells, TPA markedly elevated agonist-stimulated cAMP synthesis. These data further imply that the regulation of AC activity in luteal cells exhibit the characteristics of AC3 rather than those of AC6.

Because both Ca2+ and PKC enhanced forskolin-stimulated cAMP synthesis in bovine luteal cells, it is reasonable to predict that factors acting via Gq-coupled receptors will elevate cAMP production in the CL when present simultaneously with a cAMP activator. One of key hormones regulating luteal function via activation of the PLC pathways is PGF2{alpha}. Its receptors are coupled to Gq protein leading to diacylglycerol activation of PKC and inositol triphosphate-induced elevation of intracellular Ca2+ (33, 34, 35). Indeed, the current study demonstrated that PGF2{alpha} markedly elevated agonist-stimulated cAMP synthesis by luteal cells. Moreover, the stimulatory effects of PGF2{alpha} were higher than those of TPA and were only partially blocked by PKC inhibitor, supporting the notion that PGF2{alpha} exerted its effects via both Ca2+ and PKC activation.

Luteal PGF2{alpha} and its receptors are induced by the ovulatory surge of LH (36, 37). The developing CL produces also two other prostaglandins, PGE2 and PGI2, shown to be highly luteotrophic (5, 6, 38). Their receptors are coupled to G{alpha}s and similarly to LH they elevate cAMP in luteal cells. In addition, LH, together with its activation of G{alpha}s, can elevate intracellular Ca2+ ion concentrations (39, 40). It appears, therefore, that the presence of AC3 in luteal cells is instrumental in achieving high cAMP levels in response to LH and the luteal prostaglandins. High cAMP levels would inevitably increase steroidogenic activity in the CL because it acts as a positive regulator of steroidogenic acute regulatory protein (at the transcriptional and posttranslational levels (18, 41, 42). Based on the above it is not surprising that PGF2{alpha} acutely augmented LH-induced progesterone production by luteal cells in vitro (2, 4).

In contrast to its luteotrophic effects under in vitro conditions, uterine PGF2{alpha} has long been recognized as the physiological trigger of luteolysis (43). Data we had published in recent years suggested that the acute luteolytic effects of PGF2{alpha} are mediated by endothelin-1 (44, 45). The involvement of endothelin -1, produced by endothelial cells in response to exogenous PGF2{alpha}, may provide an explanation for different modes of PGF2{alpha} action (endocrine vs. paracrine). This contention is supported by the fact that luteal and uterine PGF2{alpha} appear at different stages of the estrous cycle; at early (36) and late luteal phases, respectively (43).

The reported data may delineate the cross-talk between physiological activators of AC in the CL (such as LH, PGE2 and PGI2) and other ligands (such as PGF2{alpha} and endothelin-1), which indirectly modulate AC activity. Therefore, the identification of AC isoforms present in luteal cells is an important step toward the understanding of molecular mechanisms underlying hormonal regulation of luteal cell functions.


   Acknowledgments
 
We are particularly grateful to Drs. Ruth Braw-Tal of the ARO Volcani Center and Nava Dekel of the Weizmann Institute for critical review of the manuscript and Drs. Michal Kobo and Nava Dekel of the Weizmann Institute for kindly providing us with the rat CL. We also wish to thank Nitzan Levy for his assistance.


   Footnotes
 
1 We thank the Federation of European Biochemical Societies (FEBS) for their financial support. Back

Received May 5, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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