Intracellular mechanisms involved in copper-gonadotropin-releasing hormone (Cu-GnRH) complex-induced cAMP/PKA signaling in female rat anterior
pituitary cells in vitro

The copper-gonadotropin-releasing hormone molecule (Cu-GnRH) is a GnRH analog, which preserves its amino acid sequence, but which contains a Cu2+ ion stably bound to the nitrogen atoms including that of the imidazole ring of Histidine2 . A previous report indicated that Cu-GnRH was able to activate cAMP/PKA signaling in anterior pituitary cells in vitro, but raised the question of which intracellular mechanism(s) mediated the Cu-GnRH-induced cAMP synthesis in gonadotropes.

To investigate this mechanism, in the present study, female rat anterior pituitary cells in vitro were pretreated with 0.1 µM antide, a GnRH antagonist; 0.1 µM cetrorelix, a GnRH receptor antagonist; 0.1 µM PACAP6–38, a PAC-1 receptor antagonist; 2 µM GF109203X, a protein kinase C inhibitor; 50 mM PMA, a protein kinase C activator; the protein kinase A inhibitors H89 (30 µM) and KT5720 (60 nM); factors affecting intracellular calcium activity: 2.5 mM EGTA; 2 µM thapsigargin; 5 µM A23187, a Ca2+ ionophore; or 10 µg/ml cycloheximide, a protein synthesis inhibitor. After one of the above pretreatments, cells were incubated in the presence of 0.1 µM Cu-GnRH for 0.5, 1, and 3 h. Radioimmunoassay analysis of cAMP confirmed the functional link between Cu-GnRH stimulation and cAMP/PKA signal transduction in rat anterior pituitary cells, demonstrating increased intracellular cAMP, which was reduced in the presence of specific PKA inhibitors. The stimulatory effect of Cu-GnRH on cAMP production was partly dependent on GnRH receptor activation. In addition, an indirect and Ca2+-dependent mechanism might be involved in intracellular adenylate cyclase stimulation. Neither activation of protein kinase C nor new protein synthesis was involved in the Cu-GnRH-induced increase of cAMP in the rat anterior pituitary primary cultures.

Presented data indicate that conformational changes of GnRH molecule resulting from cooper ion coordination affect specific pharmacological properties of Cu-GnRH molecule including specific pattern of intracellular activity induced by complex in anterior pituitary cells in vitro.

1. Introduction

The secretory action of pituitary gonadotropes, which synthe- size and release luteinizing hormone (LH) and follicle-stimulating hormone (FSH), depends on a complex neuroendocrine con- trol network that includes hypothalamic, gonadal, and locally produced hormones. Of these, the neuropetide gonadoliberin (GnRH) plays a central role in mammalian reproductive func- tions (Naor and Huhtaniemi, 2013). GnRH acts in the ante- rior pituitary gland gonadotropin-releasing hormone receptors (GnRH-R), G-protein-coupled cell surface receptors with seven- transmembrane-spanning domains connected by extracellular and intracellular loops. GnRH-Rs are mainly coupled to phospholipase Cβ via Gαq/11 protein, and their activation leads to a rapid increase in diacylglycerol (DAG) and inositol triphosphate (IP3) second mes- sengers, which propagate signaling cascades that account for the specific physiological effects of GnRH. IP3 mobilizes Ca2+ from intra- cellular stores that, together with GnRH-stimulated Ca2+ influx, regulates acute gonadotropin release. In addition, GnRH-induced DAG activates protein kinase C (PKC) isoforms that mediate MAP kinase (MAPK) activation by GnRH, and regulation of gonadotropin subunit gene expression (Schang et al., 2012). In contrast to the well-established IP3/PKC pathway that activates the complex sig- naling network responsible for the biological effects of GnRH, the contribution of the cAMP/PKA pathway to the action of GnRH is much less understood. However, the importance of cAMP/PKA sig- naling in gonadotrope function is underscored by the fact that pituitary adenylate-cyclase activating peptide (PACAP) is the most potent hypothalamic activator of cAMP production in pituitary cells, and is recognized as a key player in reproduction through its autocrine/paracrine action (Winters and Moore, 2011). An array of intracellular effects exerted by cAMP pathway are attributed not only to well recognized PKA activation but also to Epac 1 and Epac 2 proteins (Bos, 2003). In the pituitary, cAMP cascade plays a crucial role both in normal and tumor-induced cell functions (Peverelli et al., 2014; Vitali et al., 2014). At the gene level, cAMP effects depend on cyclic-AMP response element binding protein (CREB) activation as observed in anterior pituitary (Monteserin- Garcia et al., 2013), and neuronal cells (Alzoubi and Alkadhi, 2014; Kida and Serita, 2015).

A few early studies indicated that GnRH stimulates cAMP accu- mulation in rat hemipituitaries (Naor et al., 1975; Borgeat et al., 1972). Since then, however, several studies on dispersed rat pitu- itary cell cultures, and later on the αT3-1 gonadotrope cell line, were unable to substantiate the former early findings (Theoleyre et al., 1976; Conn et al., 1979; Horn et al., 1991). More recently, studies using LβT2 cells concluded that cAMP analogs were able to mimic most of the effects of GnRH, including increased luteinizing hormone (LH) release, increased expression of LHβ and α sub- units, and increased expression of GnRH-R and NOS I mRNAs and proteins (Garrel et al., 2002; Horton and Halvorson 2004). CREB protein was also shown to affect FSHβ promoter activity in this line (Thompson et al., 2013). In fact, several genes including follis- tatin (Winters et al., 2007; Mutiara et al., 2009), Nur77 (Hamid et al., 2008), Adcyap1 (Grafer et al., 2009), and Nos1 (Garrel et al., 2010) are known to be regulated by cAMP/PKA-mediated GnRH signal- ing, providing further support for the importance of this pathway in gonadotrope function.

Although metal ions can directly affect cellular function (Mlyniec et al., 2015), their coordination by biological compounds is needed to maintain conformational structure and biological activity in around one-third of all proteins (Shi et al., 2005; Shi and Chance, 2008, 2011). This can also result in the mod- ification of biological activity of non-peptide metal complexes (Mazur et al., 2012; Alomar et al., 2013) as well as metallopeptide analogs (Matusiak et al., 2014; Kuczer et al., 2015). Interest- ingly, gonadotropin-releasing hormone analogs containing PtCl2 or trans-bis(salicylaldoximato) copper(II), coordinated in a D- Lys6-GnRH molecule, exhibited profoundly enhanced affinity to GnRH membrane receptors of rat pituitary and human breast cancer cells (Bajusz et al., 1989), as well as increased potency in LH release. Modified biological activity was also attributed to the Cu-GnRH molecule, a peptide with the native decapep- tide amino acid sequence of GnRH, but with a Cu2+ ion stably bound to the nitrogen atom of the imidazole ring of His2 (Gaggelli et al., 2005). Relative to GnRH, the copper complexed variant stimulated LH release more potently in female rats (Kochman et al., 1992), in porcine primary pituitary cell culture (Michaluk et al., 2006) and bound GnRH receptors with greater affinity (Kochman et al., 1997). Furthermore, Cu-GnRH and Co-GnRH analogs increased cAMP accumulation in porcine gonadotrope pri- mary cultures, whereas the non-complexed GnRH failed to do so (Kochman et al., 2005; Blitek et al., 2005). The surprisingly dif- ferent sensitivity of cAMP/PKA signaling when comparing GnRH and Cu-GnRH stimulation raised questions regarding the possible intracellular mechanism(s) responsible for the complex-induced up-regulatory effect exerted on this signal transduction system in anterior pituitary cells. To identify this process, this study tested several hypothetical mechanisms, including regulatory engage- ment of GnRH and/or PACAP specific receptors, protein kinase A and C activity, intracellular calcium homeostasis, and a potential requirement for new protein synthesis. We utilized an in vitro model based on female rat anterior pituitary primary cell cul- tures, with the use of specific pharmacological approaches directed toward the above mentioned intracellular regulatory and signaling systems.

2. Material and methods

2.1. Animals

Ninety random-cycling 4–5 month old female Wistar rats (230–250 g) were maintained for three weeks under controlled lab- oratory conditions on a 12/12 h light/dark cycle (lights on at 07.00 am) and under constant temperature (22 ◦C), with access to pel- leted food (Murigran, Poland) and tap water ad libitum. All animal procedures were performed in accordance with the Guiding Prin- ciple for the Care and Use of Research Animals, and were approved by the local Ethics Committee at the University of Agriculture in Warsaw.

2.2. Cu-GnRH complex synthesis

GnRH was purchased from Sigma–Aldrich (St. Louis, MO). Cu-GnRH complex was synthesized according to the method previ- ously described (Kozlowski et al., 1990). Briefly, GnRH and copper acetate (Cu(CH3COO)2·2H2O) were mixed in an equimolar ratio, then stirred and simultaneously heated up to 40 ◦C for 1 h, and finally ice-cooled. After the water was evaporated, the stoichiom- etry and purity of the obtained complex were determined by elementary analyses and electrospray ionization–mass spectrom- etry (ESI–MS).

2.3. Primary culture of anterior pituitary cells

Anterior pituitary glands were obtained from all 90 rats. Asepti- cally dispersed pituitary cells underwent a sequential 0.25% trypsin dissociation procedure according to methods described previously (Wasilewska-Dziubin´ ska et al., 2011). Briefly, after dispersion the pituitary cells were centrifuged at 800 × g for 10 min, then washed with M199 medium (Sigma–Aldrich). They were then resuspended and counted on a hemocytometer. Cell viability (96–98%) was determined by trypan blue dye exclusion. Finally, dispersed cells were plated into a 24-well plate at a density of 5 × 105 cells/ml, in M199 containing 10% fetal calf serum (FCS), penicillin (100 U/ml), and streptomycin (100 µg/ml), and then incubated for 72 h at 37 ◦C in a humidified atmosphere with 5% CO2. On the day of the experi- ments, cells were washed twice with fresh M199 medium without serum, and then incubated in 1 ml of bacitracin (2 × 10−5 M) containing serum-free M199 medium. In this study, distinct conditions concerning culture incuba- tion patterns have been applied: pretreatment refers to sequential exposure of cells to the experimental drug/hormone, treatment refers to a single experimental drug/hormone being added to the incubation media and co-treatment refers to the simultaneous addition of the experimental factors to the media.

2.4. cAMP determination

To prevent cAMP degradation, cultured pituitary cells were first pre-incubated for 30 min with 100 µM of the phosphodiesterase inhibitor IBMX (Sigma–Aldrich). Parallel incubations with 10 µM forskolin, an adenylate cyclase activator, served as positive con- trols. To determine extracellular cAMP, 750 µl of medium was boiled for 5 min, and stored at −80 ◦C. To determine intracellular cAMP, cells were washed with PBS, scraped off in 80% ethanol for 5 min, and centrifuged (800 × g for 15 min at 4 ◦C). Supernatants were dried, resuspended with 250 µl of PBS, and stored at −80 ◦C until use in the assay. In each well, intra- and extracellular cAMP levels were analyzed in duplicate via RIA, using an Immunotech cAMP kit (Immunotech, Czech Republic) according to the manufac- turer’s protocol. The standard curve ranged from 50 to 5000 fmol cAMP/0.1 ml, and intra-assay coefficient of variation was 2.1%.

2.5. LH radioimmunoassay

For LH radio-iodination, 125INa from Hartmann Analytic (Munich, Germany) was purchased. LH concentration in the medium was measured by RIA using antibodies and LH prepa- rations generously supplied by Dr. A.F. Parlow and the National Hormone & Peptide Program (NHPP) (Torrance, USA). Values were expressed in terms of rat LH (RP-3) preparations. Intra-and inter- assay coefficients of variations were 5.9% and 7.8%, respectively.

2.6. Statistical analysis

Given values are the mean ± SEM of six to eight separate incu- bations for each experimental group. Statistical evaluations were carried out using the nonparametric Mann–Whitney U-test pro- vided by Statistica 6.0 PL (StatSoft, Inc., Tulsa, USA). Differences with p ≤ 0.05 were considered statistically significant.

3. Results

3.1. Cu-GnRH activated the cAMP pathway and stimulated LH release in female-derived pituitary cells

At first, coupling of the Cu-GnRH complex to the AC/cAMP pathway was examined by incubating anterior pituitary cells for increasing periods of time (0.5 h, 1 h, or 3 h) with Cu-GnRH in the presence of 100 µM IBMX, and measuring cAMP intracellular accumulation (Fig. 1A). When compared to the respective non- stimulated controls, intracellular cAMP increased by 114% at 1 h, and by 150% at 3 h. Parallel incubations with 10−7 M GnRH were completely ineffective in promoting intracellular and extracellular cAMP accumulation. At three tested time points, cAMP levels were sustained at the same level as their non-treated respective controls (figure not shown). As expected, PACAP1–38 significantly increased intracellular cAMP accumulation at all time-points tested. Simi- larly, costimulation of cells with both peptides was effective during all periods of incubation. Moreover, it led to an additional signifi- cant enhancement of intracellular cAMP levels of 70% and 43% at 1 h and 3 h, respectively, as compared to the PACAP1–38-stimulated groups. In the medium, a ligand-dependent pattern of extracellu- lar cAMP accumulation was induced, but neither PACAP1–38 nor Cu-GnRH was effective prior to the 1-h long incubation (Fig. 1B). Although both peptides significantly increased LH release at all time points tested, Cu-GnRH was more potent that PACAP1–38. In co-treated cells, an additive effect on LH release was observed only after 0.5 h incubation, with a 45% increase in comparison to the respective Cu-GnRH cells, and a 114% increase vs the PACAP- stimulated group (Fig. 2A).

3.2. New protein synthesis was not essential for Cu-GnRH increases in cAMP accumulation

To examine whether the observed latency of cAMP stimulation by Cu-GnRH may be a consequence of intermediate protein syn- thesis, cells were pretreated with cycloheximide for 1 h (10 µg/ml medium), and then incubated in the presence of 10−7 M Cu-GnRH for 1 and 3 h. This treatment, however, did not affect intracellular cAMP increase in response to either 1 h (207 ± 11 vs 183 ± 8% over basal with and without cycloheximide, respectively) or 3 h long Cu- GnRH stimulation (202 ± 10 vs 196 ± 7% over basal with or without cycloheximide, respectively). Thus, new protein synthesis was not required for this process (figure not shown).

3.3. Ca2+ is required for Cu-GnRH-induced cAMP production

As GnRH-R activation leads to an immediate increase in cytoso- lic calcium concentration, an indirect, Ca2+-dependent mechanism of AC activation might be responsible for the observed lag in the cAMP response. To study this, anterior pituitary cells were incu- bated for 15 min in the presence of the Ca2+-selective ionophore A23187 (5 µM), or in the presence of the calcium chelator EGTA (to deplete calcium) with and without thapsigargin (2 µM) for 30 min, before basal and Cu-GnRH-stimulated cAMP (1 h) was measured. Increasing Ca2+ influx failed to increase cAMP synthesis (Fig. 3A), whereas pharmacological depletion of intracellular and extracel- lular calcium with 2.5 mM EGTA and EGTA/thapsigargin (which inhibits refilling of the sarcoplasmic reticulum Ca2+ pool), abolished by 44% and 65%, respectively, Cu-GnRH-induced cAMP intracellular accumulation (Fig. 3B). Thus, the calcium dependence of Cu-GnRH- induced activation of some AC isoforms cannot be excluded.

3.4. Cu-GnRH increases anterior pituitary cell sensitivity to forskolin

We next tested whether the pre-exposure of anterior pituitary cells to Cu-GnRH may potentiate the cAMP response to forskolin. As presented in Fig. 4, 3 h long Cu-GnRH pretreatment enhanced forskolin-induced cAMP accumulation by 35%, as compared to not pre-treated forskolin group, suggesting that previous exposure to Cu-GnRH modifies intrinsic AC activity.

3.5. GnRH-R activation, but not PKC activity, is required for Cu-GnRH-induced cAMP production

We next performed preincubations with GnRH or GnRH-R antagonists (antide and cetrorelix, respectively) to determine whether GnRH-R was involved in Cu-GnRH-evoked cAMP synthe- sis. Antide and cetrorelix were both applied to take advantage of their relatively different inhibitory effects on GnRH-R activity, so we could estimate the receptor downregulating effect result- ing from different pharmacological approaches. Pharmacological blockade of GnRH-R activity markedly diminished Cu-GnRH- evoked increases in cAMP accumulation. In the presence of antide, Cu-GnRH effectiveness was reduced by 37% (1 h) and 62% (3 h), and in the presence of cetrorelix, effectiveness was diminished by 35% (1 h) and 64% (3 h) (Fig. 5). It is well-established that GnRH-R stimulation, in addition to a transient cytosolic Ca2+ increase, also rapidly induces PKC activity. Therefore, the possible involvement of PKC in Cu-GnRH-mediated cAMP synthesis was evaluated. Prein- cubation of anterior pituitary cells with the phorbol ester PMA, known to directly activate most PKC isoforms, did not affect basal cAMP levels. Similarly, costimulation of cells with PMA and Cu- GnRH did not significantly increase cAMP versus Cu-GnRH alone (data not shown). In addition, the broad-spectrum PKC inhibitor bisindolylmaleimide I (GF109203X, 2 µM) induced no changes in Cu-GnRH-evoked intracellular cAMP accumulation at either 1 or 3 h treatment (Fig. 6).

3.6. Cu-GnRH-induced cAMP production requires PAC-1 receptor and PKA activity

The potential dependence of Cu-GnRH activity on Gsα/AC/PKA- coupled PAC-1 receptor activity was also evaluated. Pre-incubation of cells with 10−7 M µM PACAP6–38, a specific PAC-1 receptor antagonist, diminished cAMP accumulation by 33% (1 h Cu-GnRH treatment) and 30% (3 h treatment) (Fig. 7A). As a positive con- trol, the effect of PACAP6–38 on PACAP-induced cAMP production was also analyzed. With the PAC-1 antagonist, PACAP1–38-induced cAMP levels were reduced by 30% (1 h) and 52% (3 h) relative to PACAP1–38-only treated groups (Fig. 7B).

Pretreatment of cells with pharmacological agents known to inhibit PKA activity revealed its importance for maintaining the stimulatory effects of Cu-GnRH on cAMP production. In the presence of 30 µM broad-spectrum PKA inhibitor H89, Cu-GnRH- evoked cAMP production was reduced by 41% (1 h) and 42% (3 h), whereas at the same time points PKA-specific KT5720 (6 × 10−8 M) treatment reduced the effectiveness of Cu-GnRH by 44% (1 h) and 35% (3 h) when compared to their respective Cu-GnRH-only treated groups (Fig. 7A). The specificity of endogenous PKA inhibition in the primary cell cultures was validated by parallel 1 h and 3 h incubations with PACAP1–38. As shown in Fig. 7B, both PKA inhibitors abolished PACAP-induced intracellular cAMP accumulation.

4. Discussion

To identify the intracellular mechanism(s) mediating Cu-GnRH stimulation of cAMP/PKA signaling in rat anterior pituitary cells, we pharmacologically targeted GnRH-R, PAC-1, protein kinases A and C, and intracellular calcium activity. Coupling of the Cu-GnRH complex to the cAMP/PKA pathway was evident by the increased cAMP accumulation found in cells incubated solely with Cu-GnRH, as well as in the relative loss of effectiveness upon pretreatment with specific PKA inhibitors. The Cu-GnRH-evoked cAMP accumu- lation in our rat anterior pituitary cell cultures is in accordance with previous studies on metal-GnRH complexes in pig pituitary primary cultures (Blitek et al., 2005; Kochman et al., 2005). This finding sup- ports the hypothesis that metal-induced changes in GnRH molecule conformation (D’Amelio et al., 2003; Nakamura et al., 2005) may contribute to modification of the intracellular signaling pattern.

Our results also revealed that GnRH-R played a role in mediating Cu-GnRH activity, given the reduced effectiveness of the complex in cells pretreated with the GnRH antagonist antide or GnRH-R antagonist cetrorelix. Although early studies (Borgeat et al., 1972; Naor et al., 1975) indicated cAMP accumulation after long (3 h) exposures to GnRH in hemipituitaries, there was debate as to whether the phenomenon occurred in cultured pituitary cells (Theoleyre et al., 1976; Conn et al., 1979). Nevertheless, the link between GnRH-R and the cAMP pathway was reported in several heterologous cultures (Lin et al., 1998; Arora et al., 1998; Oh et al., 2005). In a gonadotrope cell line (LβT2), sustained GnRH-R activa- tion led to coupling to adenylyl cyclase via Gαs (Liu et al., 2002). In addition, high-frequency pulses of GnRH did not desensitize the same cell line to cAMP activation (Tsutsumi et al., 2010). A functional link between cAMP pathway activation and high GnRH pulsatility was also identified in the proestrus-specific elevation of pituitary cAMP levels typical of the rat ovarian cycle (Kimura et al., 1980; Garrell et al., 2010). On the other hand, the unique struc- tural feature of mammalian GnRH-R, lack of a carboxy-terminal tail, is now recognized to be responsible for low level constitu- tive receptor internalization, as well as for its inability to undergo rapid agonist-induced internalization (Pawson et al., 2008). Thus, GnRH-R can maintain cAMP pathway activation under stimulation conditions that would induce desensitization in most G-protein coupled receptors, GPCRs (Millar et al., 2004; Cohen-Tannoudji et al., 2012).

Recently, the endogenously expressed proto-oncogene SET, a GPCR-interacting protein, was shown to bind to GnRH-R intracel- lular domains and switch the receptor to cAMP signaling in the gonadotrope αT3-1 cell line (Avet et al., 2013). In our study, how- ever, the complex was not applied in a pulsatile manner, but in a single bolus. This pattern of Cu-GnRH stimulation was still effective in promoting cAMP synthesis. Existing data suggest that specific properties of the Cu-GnRH molecule might act to prolong GnRH- R stimulation and thus contribute to enhanced cAMP synthesis. Indeed, Cu-GnRH interacts with GnRH receptors with greater affin- ity than GnRH (D’Amelio et al., 2003), but is also less susceptible to proteolytic degradation (Herman et al., 2012). Cu-GnRH-induced cAMP levels were observed in the presence of IBMX, a potent phos- phodiesterase inhibitor, indicative of activation of AC rather than inhibition of cAMP degradation. The possibility that AC intrinsic activity can be modified by previous exposure to Cu-GnRH was sup- ported by the observation that the complex potentiated the cAMP response to forskolin. Given the long time needed for Cu-GnRH stimulation to elevate intracellular cAMP compared to PACAP, it is possible that an indirect, Gαs-independent mechanism could be involved in Cu-GnRH coupling to the cAMP pathway. GnRH- R induces a rapid increase of cytosolic Ca2+ and PKC activity in gonadotropes, which can increase activity of several AC isoforms (Ferguson and Storm, 2004).

Reduced Cu-GnRH-induced cAMP accumulation observed after depletion of intracellular and extracellular calcium stores indicated that calcium signaling could be involved in Cu-GnRH coupling to the cAMP/PKA pathway. In fact, calcium ions were believed to play a crucial role in GnRH-induced cAMP accumulation (Grosse et al., 2000). However, neither basal nor GnRH-stimulated cAMP production were affected by pharmacologically induced changes in intracellular Ca2 concentration when tested in gonadotrope LβT2 cells (Lariviere et al., 2007). Nevertheless, the cAMP-dependence on calcium homeostasis observed in our study might reflect Ca2+/calmodulin-mediated activation of AC isoforms (Sunahara et al., 1996). Indeed, the AC-1 enzyme was shown to be highly expressed in the pituitary gland (Antoni et al., 1995).

GnRH-R activation leads both to transient enhancement of cytosolic Ca2+ and rapid activation of PKC in gonadotrope cells (Counis et al., 2009). Interestingly, studies on the LβT2 line revealed that GnRH-mediated cAMP accumulation was markedly reduced by the PKC inhibitor bisindolylmaleimide, whereas long-term GnRH pre-treatment revealed that novel δ and ‹ PKC isoforms medi- ated GnRH-induced cAMP accumulation in these cells (Lariviere et al., 2007). In the same study, PKC-sensitive AC isoforms (AC5 and/or AC7) were also suggested to act as potential targets for GnRH stimulation. In the present study, however, a PKC inhibitor did not prevent Cu-GnRH-induced cAMP accumulation. Furthermore, in cells costimulated with Cu-GnRH and the phorbol ester PMA, neither altered the effect exerted by Cu-GnRH alone. The existing discrepancy concerning PKC involvement in cAMP activation might result, at least in part, from differences in the in vitro models i.e., a gonadotrope LβT2 line vs an anterior pituitary primary cell cul- ture. Furthermore, time-dependency cannot be excluded, since a preventive effect of novel PKC isoforms on the downregulation of cAMP production was observed in LβT2 cells after 24 h of GnRH treatment.

The additive effect of Cu-GnRH and PACAP1-38 on cAMP accumu- lation further suggests GnRH receptor involvement in mediating the effects of the complex. However, the increased cAMP accu- mulation found after our combined Cu-GnRH and PACAP1–38 treatment is in contrast to the effects exerted by GnRH in LβT2 cells, where GnRH agonist markedly inhibited the PACAP-induced cAMP increase (Lariviere et al., 2006, 2008). The ability of GnRH to counteract PACAP-stimulated cAMP accumulation has been also reported in αT3-1 cells (McArdle et al., 1994). Our results, how- ever, further underscore the ability of Cu-GnRH to synergize with PACAP1–38 to promote cAMP synthesis.

Although GnRH-R was believed to be the main target for intra- cellular Cu-GnRH signaling, the complex may also interact with the PACAP receptor. Activation of membrane receptors by pep- tide ligands correlates with the conformation of the ligand in the membrane-bound state (Wakamatsu et al., 1987). In the case of PACAP, conformational changes associated with the specific inter- actions of the receptor are limited to N-terminal residues 1–7,
including His1, where the characteristic β-coil structure is induced upon receptor binding (Inooka et al., 2001). In the Cu-GnRH com- plex, however, a square planar configuration between the Cu2+ ion and Glu-His-Trp-NHCH3 is favored (Nakamura et al., 2005). Thus, further detailed studies are required to elucidate the conforma- tional aspects of a possible Cu-GnRH-PAC-1 receptor interaction.

In conclusion, a functional link between Cu-GnRH stimulation and cAMP/PKA signal transduction in rat anterior pituitary cells was identified, such that the complexed peptide enhanced intra- cellular cAMP levels in a manner sensitive to PKA inhibitors. The effect required GnRH-R activation, as well as some dependence on intracellular calcium activity. However, neither activation of pro- tein kinase C nor de novo protein synthesis was necessary. Taking into account the present results, future research using gonadotrope LβT2 cells should clarify whether specific PKC isoforms are involved in Cu-GnRH coupling to 3r, 5r-cyclic adenosine-5r monophosphate pathways, and should also focus on the role of Ca2+/calmodulin- mediated AC isoforms could have in mediating the action of Cu-GnRH.