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Endocrinology Vol. 143, No. 6 2366-2375
Copyright © 2002 by The Endocrine Society

NEUROENDOCRINOLOGY

Pituitary Adenylate Cyclase-Activating Polypeptide Acts as a Paracrine Regulator of Melatonin-Responsive Cells of the Ovine Pars Tuberalis

Perry Barrett, Sophie Messager, Carole Schuster, Kim M. Moar, Julian G. Mercer and Peter J. Morgan

Rowett Research Institute and Aberdeen Center for Energy Regulation and Obesity, Bucksburn, Aberdeen, United Kingdom AB21 9SB

Address all correspondence and requests for reprints to: Dr. Perry Barrett, Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen, United Kingdom AB21 9SB. E-mail: . pb{at}rri.sari.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The pars tuberalis (PT) region of the anterior pituitary plays a physiological role in seasonal animals. The primary signal transduction mechanism of the melatonin receptor in this tissue is an inhibition of cAMP signaling. However, nothing is known about the endocrine signals that activate cAMP synthesis in the cells of the PT, as previous studies relied on the pharmacological tool, forskolin, to stimulate cAMP synthesis. Here we show that pituitary adenylate cyclase-activating polypeptide (PACAP) activates cAMP synthesis in the cells of the PT. The pharmacology of cAMP activation by PACAP peptides suggests that cAMP activation is mediated by the type I PACAP receptor. PACAP treatment of PT cells results in cellular responses that are consistent with cAMP activation in these cells, including activation of MAPK and elevation of melatonin receptor mt1 mRNA expression. These responses can be inhibited by melatonin, demonstrating that activation of cAMP occurs within the melatonin-responsive cells. However, although PACAP activates cAMP in the cells of the PT, the effect of PACAP may not be direct, as colocalization in situ hybridization studies demonstrates that the type I PACAP receptor and the melatonin mt1 receptor do not colocalize on the cells of the PT.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE PARS TUBERALIS (PT) region of the anterior pituitary gland expresses high levels of melatonin receptors in almost all mammalian species (1). Photoperiod regulates melatonin secretion from the pineal gland to provide an endocrine signal, which regulates circadian and seasonal activities of the PT (2, 3).

The principle effect of melatonin on primary culture of ovine PT cells is an inhibition of the adenylate cyclase enzyme to reduce the intracellular levels of cAMP and inhibit cellular processes regulated by this second messenger. These include the secretion of proteins, phosphorylation of cAMP response element-binding protein (CREB), and transcriptional events dependent on cAMP activation (4, 5, 6, 7, 8). The availability of this tissue has provided the biological material on which much of the pioneering work on the mechanism of action of melatonin has been elucidated. Nevertheless, the natural ligand that stimulates cAMP synthesis in PT cells has been elusive. Although a number of peptides and neurotransmitters have previously been analyzed for a stimulatory effect on cAMP synthesis in PT cells, none has been found to be effective (1). To date, the studies performed on PT cells have used forskolin as the agent to stimulate cAMP synthesis.

Melatonin receptors are also found in the suprachiasmatic nucleus (SCN) of the hypothalamus (9, 10). Although details of the mode and mechanism of action of melatonin are not as complete as for the PT, much more is known about the neuropeptides and transmitters that act upon the SCN. The SCN acts as the primary endogenous pacemaker and receives photic inputs from the retina via the retino-hypothalamic tract, and entrainment of the endogenous clock is regulated by the release of neurotransmitters through this input. PACAP released from the retino-hypothalamic tract was suggested as a putative transmitter in conveying environmental stimuli to the SCN. This peptide has subsequently been shown to elevate cAMP levels in cells of the SCN with consequential phosphorylation of CREB (11). Depending upon the time of application, PACAP phase-shifts the circadian clock, and this effect can be blocked by melatonin (12). Thus, one action of melatonin in the SCN may be to fine-tune the action of PACAP and its consequences on components of the circadian clock. More recently, melatonin has been shown to have a relationship with PACAP on the cells of the neonatal rat pituitary (13), where melatonin was shown to inhibit PACAP-induced cAMP synthesis and cAMP-induced GnRH release.

Although the acute effect of melatonin on the cells of the PT is to inhibit the cAMP second messenger system, melatonin can also have an a stimulatory effect on the adenylate cyclase-generating system through prolonged exposure of PT cells to melatonin. Thus, exposure of PT cells to melatonin for 8–16 h, followed by removal of melatonin and stimulation of PT cells with forskolin, result in much enhanced cAMP levels (14, 15). This ability of melatonin to sensitize PT cells is a time-dependent phenomenon and is a possible mechanism by which PT cells decode the seasonal change in the length of the melatonin signal. We hypothesized, therefore, that a stimulatory ligand for the cAMP signal transduction pathway may require sensitization of PT cells before a stimulatory ligand becomes physiologically relevant or that the action of the ligand could be seen in standard signal transduction assay measurements. Although VIP, a member of the PACAP family of peptides, has no effect on cAMP synthesis in PT cells (1), the interaction of melatonin with PACAP in two other tissues (12, 13) suggests the potential of melatonin to interact with PACAP on the cells of the PT after sensitization of the adenylate cyclase enzyme. We have examined this possibility.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of PT cells
PTs were collected from sheep of mixed breed and sex killed at a local abattoir. Cell preparations were made throughout the year. The preparation of PT cells was performed as described previously (8).

Pretreatment of primary cell cultures
After dispersal of cells from the PT tissue and overnight culture, the cells were harvested and counted. Cells (0.5 x 106) were aliquoted to each well of a 24-well plate treated with poly-D-lysine. Overnight treatments contained, as appropriate, one of the following: vehicle, 10 nM melatonin, 1 µM herbimycin, or 10 nM melatonin plus 1 µM herbimycin. The following day, the medium was removed, the cells were washed three times, and fresh medium containing the appropriate treatments was added to the culture dishes.

Measurement of intracellular cAMP levels
For these experiments 0.5 x 106 cells were plated into 24-well plates. After pretreatments, cells were washed three times with ice-cold serum-supplemented medium. Drug treatments made up in serum-supplemented medium containing 0.1 mM isobutylmethylxanthine and chilled on ice were added to wells. The reactions were initiated by placing the plate in a 37 C water bath and were allowed to proceed for 30 min. The medium was then removed, and the cells were washed three times with ice-cold PBS [137 mm NaCl, 2.7 mm KCl, 4.3 mM Na2HPO4, and 1.47 mM KH2PO4 (pH 7.1)]. The cells were lysed by the addition of 200 µl 5% trichloroacetic acid and were left at room temperature for 30 min.

Melatonin receptor mRNA measurement
For these experiments 107 cells were aliquoted into 60-mm petri dishes and pretreated for 16 h. Cells were washed three times with prewarmed serum-supplemented medium before the addition of medium containing the drug treatments. Cells were returned to the incubator for a further 4 h before harvesting the cells.

Measurement of the melatonin mt1 receptor mRNA was made using the ribonuclease protection assay (RPA) as described previously (8, 16).

MAPK activation
For these experiments PT cells were harvested on the day after preparation and were washed three times in serum-free medium. Cells (2 x 106) were aliquoted into 35-mm petri dishes. The cells were simultaneously serum-starved and pretreated for 16 h. Cells were washed three times with prewarmed serum-free medium before the addition of serum-free medium containing the drug treatments. Cells were returned to the incubator for an additional 5 min before harvesting the cells. After the 5-min incubation period, cells were washed twice with ice-cold buffer [10 mM HEPES (pH 7.8) and 150 mM NaCl] and scraped up in 100 µl of this buffer. An equal volume of 2x SDS gel loading buffer [125 mM Tris-HCl (pH 6.8), 20% glycerol, 4% SDS, and 10% ß-mercaptoethanol] was added to the cells and vortexed. Cells were left on ice until all stimulations had been completed. The cells were then heated to 95–100 C for 5 min, followed by sonication for 5 sec at a 5-µm setting (MSE Soniprep 150, Sanyo Gallenkamp, UK). Twenty microliters of sample were loaded onto duplicate 7 x 8-cm 10% SDS-polyacrylamide gels and electrophoresed at 150 V for 2–3 h. Proteins were transferred to polyvinylidene difluoride membrane (Millipore Corp., Watford, UK) by the method of Towbin et al. (17) using a transfer apparatus (Bio-Rad Laboratories, Inc., Hercules, CA). One membrane was immunoblotted with antibodies raised to phosphorylated activated forms of ERK42/44, whereas the duplicate blot was immunoblotted with antibodies raised to the nonphosphorylated proteins (to verify protein loading) using the p42/44 MAPK assay kit (NEB Biolabs, Hitchin, UK) according to the manufacturer’s instructions.

PCR amplification, cloning, and Northern blot analysis
Poly(A)+ RNA was isolated directly from frozen tissues using the message maker kit (R&D Systems, Abingdon, UK). For Northern analysis 20 µg poly(A)+ RNA were electrophoresed on a 1% denaturing formaldehyde gel and transferred to nylon. The nylon filter was probed with a 32P-labeled DNA fragment of the PACAP type I receptor. Hybridization was performed at 65 C for 1 h in Quickhyb solution (Stratagene, Amsterdam, The Netherlands), and washes were performed twice in 2x SSC/0.1% SDS for 15 min/wash at room temperature, then in 0.2x SSC/0.1% SDS for 30 min at 60 C.

For PCR amplification of PACAP type I receptor sequences, 1 µg poly(A)+ RNA was primed with random primer and reverse transcribed using 200 U SuperScript II at 42 C for 60 min in a reaction volume of 20 µl. The amplification reaction contained approximately 100 ng cDNA, 100 pmol primers, 2.5 U Taq DNA polymerase (Promega Corp., Southampton, UK), 10 mM Tris-HCl (pH 9 at 25 C), 1.5 mM MgCl2, 50 mM KCl, 1% Triton X-100, and 250 µM each dNTP. The primers used for PCR amplification were 5'-CCGRATCTTCAACCCRGACCAAG-3' and 5'-AGCTGCTCTTGCTCAGGATGGA-3' corresponding to position 791–813 and 1971–1992 of the bovine PACAP receptor sequence (GenBank accession no. D17290) Amplification conditions were preamplification at 96 C for 1 min and at 50 C for 30 sec, then cycled 35 times at 96 C for 30 sec, 50 C for 1 min, and 72 C for 90 sec. A band of approximately 1.2 kb was cloned into pGEM-T (Promega Corp.) and sequenced for verification of the PACAP receptor.

[125I]PACAP 27 binding assays on PT membranes
PT tissue was homogenized in cold binding [20 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 2 mM EGTA, 1% BSA, and 5 mg/ml bacitracin]. Homogenized tissue was centrifuged in microfuge tubes for 2 min at 10,000 x g. The supernatant was discarded, and the pellet was washed twice with the same buffer. The membrane pellet was resuspended in binding buffer at 1 PT equivalent/400 µl buffer. For each reaction, 0.16 PT equivalent of membrane was incubated with 12–400 pM [125I]PACAP 27 (NEN Life Science Products) with or without cold competitor ligands, PACAP 38, PACAP 27, and VIP at a final concentration of 0.1 µM in a final volume of 500 µl. Membranes were incubated for 2 h at room temperature, then centrifuged for 2 min at 4 C at 10,000 x g. The supernatant was discarded, and the pellet was resuspended in 500 µl binding buffer and repelleted. The bound counts were determined by counting the pelleted membranes in a {gamma}-counter. All experiments were performed with duplicate or triplicate reactions, and the data were averaged. Kd and binding capacity (Bmax) values were determined by Grafit software (Sigma, Poole, UK).

In vitro autoradiography
Blocks of ovine brain tissue with the PT and PD intact were dissected at the abattoir and immediately frozen on dry ice. Sections of tissue (20 µM) were thaw-mounted onto poly-L-lysine-coated slides. The slides were dried at room temperature for 15 min before preincubation with blocking buffer [50 mM Tris-HCl (pH 7.4), 32 mM sucrose, 1% BSA, 5 mM MgCl2, and 0.5 µg/µl bacitracin] for 30 min. The sections were then covered with 300 µl buffer (as described above but with 3% BSA) containing 50 pM [125I]PACAP 27 with or without cold competitor ligand at a final concentration of 1 µM. The sections were incubated at room temperature for 1 h, then washed three times in buffer equilibrated to 4 C containing 50 mM Tris-HCl (pH 7.4), 0.1% BSA, 5 mM MgCl2, and 0.5 µg/µl bacitracin. The slides were briefly dipped in distilled water and air-dried before apposing them to autoradiographic film (Hyperfilm ß-Max, Amersham Pharmacia Biotech, Little Chalfont, UK) for 6 d at 4 C. Localization of melatonin receptors using [125I]melatonin was carried out as previously described (18).

In situ hybridization
The plasmid containing the PACAP type I receptor sequence was linearized with appropriate restriction enzymes for the generation of T7 or SP6 transcripts for use as sense and antisense transcripts. Similarly, a plasmid (pBKCMV Mel131) containing the ovine melatonin receptor was linearized for the generation of T7 or T3 transcripts (antisense and sense transcripts). PACAP receptor riboprobes were labeled by in vitro transcription containing [35S]UTP, and melatonin receptor probes were labeled with (digoxygenin)-11-UTP. The labeling reactions and dual in situ hybridization protocol were described previously (19).

Protein estimations
Protein estimations were made using a detergent-compatible protein estimation kit (Bio-Rad Laboratories, Inc.) with BSA as a standard.

Statistics
The effect of treatments on cAMP responses or mt1 mRNA levels in the RPA assay were analyzed by one-way ANOVA using the SigmaStat package (Jandel, San Ramon, CA). For analysis of the cAMP responses, to avoid the assumption in ANOVA of equal variability in all groups, the forskolin value was omitted for the analysis. Differences were assessed using Tukey’s test for multiple comparisons.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PACAP activates adenylate cyclase in ovine PT cells
Primary cultures of PT cells that had or had not received a pretreatment protocol to sensitize the adenylate cyclase response were treated with forskolin, PACAP with or without melatonin, and melatonin alone. As shown in Fig. 1AGo for the control, nonsensitized PT cells, a robust increase in cAMP was achieved with the pharmacological adenylate cyclase activator, forskolin (Fsk), a response that was inhibited by melatonin. Likewise, PACAP stimulated an increase in cAMP in PT cells, albeit of smaller magnitude than that produced by forskolin (1.5- to 7-fold). Importantly, this PACAP-induced increase in cAMP was inhibited by melatonin.



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  		Figure 1. Stimulation of adenylate cyclase by PACAP in sensitized and nonsensitized PT cells. Primary cell cultures of ovine PT cells were pretreated with or without a sensitization protocol before attempted stimulation of adenylate cyclase. A, Control cells, no sensitization. B, Sensitized with herbimycin. C, Sensitized with melatonin. D, Sensitized with herbimycin plus melatonin. After removal and washout of the overnight treatment medium, the cells received a 30-min exposure to the treatments indicated: Con, medium only; Fsk, 1 µM forskolin; Mel, 10 nM melatonin; F/M, 1 µM forskolin plus 10 nM melatonin; PACAP, 100 nM PACAP 38; P/M, 100 nM PACAP 38 plus 10 nM melatonin. Shown are the data from one representative experiment repeated in triplicate. *, Significant increase in cAMP synthesis relative to control (P < 0.05).

 
In previous experiments we have demonstrated that pretreatment of PT cells with melatonin for 16 h sensitizes the adenylate cyclase enzyme, resulting in a greater production of cAMP after treatment with forskolin. This sensitized response can be induced by treatment with the tyrosine kinase inhibitor, herbimycin (15). An even greater sensitized response can be achieved when both melatonin and herbimycin are added during the pretreatment period (15). We reasoned that sensitization of PT cells may be required for the ability of endocrine signals to fully activate the adenylate cyclase enzyme in these cells.

Although the data in Fig. 1AGo indicate that sensitization is not a prerequisite for cAMP activation in PT cells, sensitization does have a profound effect on the amount of cAMP produced even in response to PACAP. In sensitized PT cells the amplitude of cAMP increases in response to PACAP and is of greater magnitude than in control cells (4- to 6-fold for herbimycin treatment, 3- to 12-fold for melatonin treatment, and 2- to 3-fold for herbimycin plus melatonin treatment; Fig. 1Go, A–D). In addition to the increased amplitude of the response relative to basal levels, there was also an increased amount of cAMP produced relative to that in nonsensitized PT cells, with the herbimycin plus melatonin treatment producing the largest amounts of cAMP after stimulation.

We next sought to determine which PACAP receptor was involved in transducing the PACAP signal. Figure 2Go shows that using a combined herbimycin and melatonin pretreatment to maximize the cAMP response obtainable, PACAP 38 and PACAP 27 stimulated cAMP synthesis equally well at 1 nM or 1 µM. VIP, on the other hand, stimulated cAMP, but even at 1 µM was not as effective as either PACAP 27 or PACAP 38. This observation is consistent with the activation of type I PACAP receptors, which are fully activated by PACAP 38 and PACAP 27, but are only partially activated by VIP.



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  		Figure 2. Effects of PACAP and VIP on cAMP synthesis in PT cells. Primary cell cultures of ovine PT cells were pretreated overnight with herbimycin and melatonin to cause sensitization of the adenylate cyclase system, then treated with for 30 min with the following: Fsk, 1 µM forskolin; PAC38, 1 µM or 1 nM PACAP 38; PAC27, 1 µM or 1 nM PACAP 27; VIP, 1 µM or 1 nM VIP. All treatments produced an increase in cAMP relative to control (P < 0.05). *, 1 nM VIP treatment significantly different from 1 nM Pac38 and Pac 27 treatments (P < 0.05).

 
Receptor saturation binding assays and Scatchard analysis using membranes prepared from whole PT tissue reveal a high affinity site for PACAP 38 (Kd, 79 ± 20 pM) and a low receptor density (Bmax, 1.31 ± 0.18 fmol/mg protein). A similar analysis performed on membranes prepared from primary cell cultures could not detect any binding. The reason for this is probably due to the localization of PACAP receptors (see below). Activation of adenylate cyclase by PACAP in primary cell cultures of ovine PT cells was dose dependent, with a pEC50 (negative value for the log of the EC50 value) of 10.88 ± 0.15 pM.

PCR amplification
Using primers based on a conserved sequence of the bovine, human, and rat type I PACAP receptors, a 1.4-kb DNA fragment was amplified from ovine PT cDNA and cloned. Sequence analysis verified that this fragment contained a portion of the type I PACAP receptor. This fragment was used to probe a Northern blot containing RNA prepared from ovine hippocampus, cerebellum, cortex, pars distalis (PD), and PT. As expected, a 6.5-kb RNA species was observed in the cerebellum and hippocampus, consistent with the known expression of this receptor in these areas of the brain (20). This 6.5-kb RNA species was also found in the PT, but not in the PD or cortex (Fig. 3Go).



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  		Figure 3. Detection of the type I PACAP receptor in PT tissue. Northern blot analysis was performed on 20 µg poly(A)+ RNA isolated from the tissues indicated and probed with a 32P-labeled ovine type I PACAP receptor DNA fragment. The expected size of the type I PACAP receptor is 6.5 kb.

 
Functional responses to PACAP
Activation of the adenylate cyclase pathway in PT cells results in an up-regulation of cAMP-regulated cellular processes. These include activation of PKA, phosphorylation of ERK42/44 MAPKs and CREB, and an increase in the transcription of cAMP-responsive genes (6, 7, 8, 21, 22). Therefore, the effect of PACAP on two of these cAMP-regulated processes was investigated. Firstly, the effect of PACAP was assessed on the ability to increase the transcription of the melatonin mt1 receptor mRNA in PT primary cell cultures. Activation of the adenylate cyclase pathway, but not the diacylglycerol-Ca2+-PKC pathway, increased mRNA expression of the mt1 receptor gene (16). Figure 4Go shows that in both melatonin-sensitized and nonsensitized PT cells, PACAP increased mt1 receptor mRNA.



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  		Figure 4. Effect of PACAP on melatonin receptor mRNA mt1 expression. Ten micrograms of total RNA prepared from treated ovine PT cells were used in an RPA. The PT cells were treated as follows: Con, vehicle only (lanes 1 and 4); Fsk, 1 µM forskolin (lanes 2 and 5); PAC, 100 nM PACAP 38 (lanes 3 and 6). A, A representative RPA autoradiogram; lanes 1–3, samples from nonsensitized PT cell; lanes 4–6, samples from melatonin-sensitized PT cells. B, Data averaged from three experiments. The protected band for the melatonin receptor mRNA (mt1) or glyceraldehyde-3-phosphate dehydrogenase (G3PDH; used as control for normalization) is shown. **, Significant increase relative to the nonsensitized control sample; ++, significant increase relative to the sensitized control (P < 0.05).

 
MAPK (p42 and p44) can be activated by both cAMP and the diacylglycerol-Ca2+-PKC pathway in PT cells (21). Stimulation of nonsensitized primary cell cultures of ovine PT cells with PACAP 38 robustly activated MAPK (Fig. 5Go). Melatonin, however, was unable to fully inhibit this activation, suggesting that activation of MAPK may have occurred by both the Gs- and Gq-coupled PACAP receptor activation. Activation of MAPK by PACAP also occurs in sensitized PT cells. However, in these cells melatonin effectively inhibits PACAP-activated MAPK (Fig. 5Go). PACAP 27 and VIP were also tested in this assay and were found to activate MAPK (data not shown).



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  		Figure 5. Effect of PACAP on MAPK activation. Primary cultures of ovine PT cells were incubated overnight in the presence or absence of melatonin and herbimycin. After an extensive washout of the overnight treatment medium, cells were treated as indicated: Con, medium only; Fsk-5 and Fsk-6, forskolin at 10 µM and 1 µM, respectively; F/M, forskolin plus melatonin at 1 µM and 10 nM, respectively; PAC38, 100 nM PACAP 38; P/M, PACAP 38 plus melatonin at 100 nM and 10 nM, respectively; Mel-8, melatonin at 10 nM. Stimulation was for 5 min before termination. Activated MAPK (p42 and p44) was detected using an antiphospho-MAPK antibody by Western analysis and detection using chemiluminescence. Con (H/M), Control sample from the sensitized PT cells. The anti-Pan ERK antibody was used to check for equal loading of the lanes (data not shown). Shown is a representative experiment.

 
Localization of PACAP receptors
PACAP receptors were localized in sections of sheep brain and pituitary using the techniques of in vitro autoradiography with [125I]PACAP 27 and in situ hybridization with 35S-labeled antisense riboprobes for the type I PACAP receptor mRNA. To compare the localization of PACAP receptors with that of melatonin receptor, [125I]iodomelatonin binding was performed on adjacent sections of the brain and pituitary taken from the same animals.

Using [125I]PACAP 27, binding was observed to the brain, PD, and an area corresponding to the median eminence. Competition binding with cold ligand showed that all binding was displaceable and specific, except for that observed in the PD. Figure 6Go shows that [125I]PACAP 27 was displaceable with cold PACAP 38 and PACAP 27, but not with cold VIP at 1 µM, consistent with the pharmacology of the type I PACAP receptor. Figure 6EGo shows the specific binding of [125I]iodo-melatonin to the melatonin receptors of the PT. Apposition of the autoradiographs for [125I]melatonin binding and [125I]PACAP 27 binding showed little or no overlap, suggesting that these two receptors do not colocalize. An absence of colocalization of the melatonin and PACAP receptors was confirmed by performing dual in situ hybridization experiments using 35S-labeled type I PACAP receptor antisense riboprobe and a digoxygenin-labeled ovine mt1 receptor antisense riboprobe. Figure 7Go, A and B, shows only a uniform distribution of silver grains over the cells of the PT, demonstrating the absence or very low levels of PACAP receptor expression in this tissue where digoxygenin labeling due to the presence of the melatonin receptor is apparent. However, silver grains are easily observed in the cells of the median eminence where melatonin receptors are not located.



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  		Figure 6. Analysis of the competitive displacement of [125I]PACAP 27 by in vitro autoradiography. Adjacent sagittal sections of the ovine pituitary/PT and brain incubated with [125I]PACAP 27 in the presence of no competitor (A) 1 µM cold PACAP 38 (B), 1 µM PACAP 27 (C), and 1 µM VIP (D). E, Localization of melatonin receptors to the PT by [125I]iodomelatonin. The arrows in A and E indicate the position of the PT.

 


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  		Figure 7. Localization of melatonin and PACAP type I receptors by in situ hybridization. A, An antisense digoxigenin-labeled mt1 melatonin receptor probe (purple coloration, white arrow) and an antisense 35S-labeled PACAP type I receptor probe (silver grains, black arrow) were hybridized to coronal sections through the PT/median eminence. The digoxigenin signal is confined to the PT, whereas the 35S hybridization signal is confined to the median eminence (ME). B, Close-up of the region where the median eminence interfaces with the PT. Note silver grains over cells in the ME, but no silver grains are observed over the digoxigenin-labeled cells of the PT.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study provides the first data to show cAMP stimulation in melatonin-responsive cells of the PT in response to a receptor-mediated ligand. However, the data also suggest that the relationship between PACAP and melatonin may not be a simple one, but may involve an intermediate endocrine-signaling molecule communicating between cells of the PT and cells in the external zone of the median eminence, as PACAP receptors and melatonin receptors do not appear to be coexpressed in the same cell type.

Several peptides and biogenic amine molecules have previously been tested for their potential to stimulate cAMP in PT cells (1). These included LH-releasing hormone, NPY, VIP, dopamine, serotonin, and others. However, either these ligands showed no cAMP stimulation, or when stimulation of cAMP did occur, there was no inhibitory effect of melatonin, indicating that cAMP stimulation occurred in cells that were not responsive to melatonin.

Because there is an established physiological relationship between PACAP and melatonin in the SCN and rat neonatal pituitary cells (12, 13), we investigated the possibility that PACAP may provide a stimulatory input to the cells of the PT.

When PACAP 38 was applied to primary cell cultures of PT cells presensitized with herbimycin and/or melatonin, an increase in intracellular cAMP was obtained. These data therefore demonstrate that PACAP can induce cAMP stimulation in PT cells. The response to PACAP 38 was dose dependent, with a pEC50 value of 10.88 ± 0.15 consistent with the dose required to obtain half-maximal cAMP stimulation in a heterologous cell system (23).

The PACAP/VIP receptor family consists of three subtypes of receptor. Type I has a higher affinity for PACAP peptides and activates both cAMP and the diacylglycerol-Ca2+ pathway. Type II and III receptors have high affinity for PACAP and VIP and activate the cAMP pathway only, but equally well (24). In PT cells the pharmacology of the cAMP response to the three peptides were PACAP 38 = PACAP 27 > VIP. This is consistent with effect of PACAP mediated through the type I PACAP receptor.

Functional responses downstream of cAMP stimulation were also observed after the application of PACAP to PT cells. This includes the transcription of the ovine melatonin mt1 receptor and activation of MAPK (ERK1/2) in PT cells, both of which are responsive to cAMP (8, 16, 20). MAPK activation was obtained by application of PACAP 38, PACAP 27, and VIP. Melatonin, however, was not effective at inhibiting PACAP-activated MAPK phosphorylation in nonsensitized PT cells. This suggests that MAPK phosphorylation occurs largely through the diacylaglycerol-Ca2+-activated pathway in these cells. However, in cells pretreated with melatonin and herbimycin, melatonin did inhibit PACAP-activated MAPK phosphorylation, suggesting this pretreatment has eliminated the diacylglycerol-Ca2+-activated pathway. In this respect, this pretreatment reduces MAPK phosphorylation, as demonstrated by comparing the basal phosphorylation status in the control treatments of the pretreated and nonpretreated cells.

Receptor binding assays performed using membrane preparations from whole PT tissues confirmed the presence of a high affinity PACAP receptor. The estimated Bmax value is low at approximately 1 fmol/mg protein. However, this estimate is unlikely to represent the true receptor density in cells expressing the PACAP receptor, because the tissue from which the membranes were prepared is a heterogeneous population of cells containing PT cells and cells or membranes present in the median eminence region of the pituitary stalk. Attempts to estimate binding affinity and Bmax values on primary cell cultures were unsuccessful, probably due to loss of the majority of cells or membranes bearing PACAP receptors during the cell dispersion procedure.

At this point suspicion falls on PACAP as the principal mediator of cAMP stimulation in cells of the PT through activation of the type I PACAP receptor. However, both in vitro autoradiography with [125I]PACAP 27 and in situ hybridization for the colocalization of PACAP and melatonin receptors provide a most unexpected finding, that PACAP receptors were localized to cells in the median eminence region of the pituitary stalk and are apparently not present in cells of the PT. Thus, to account for the observed responses to PACAP, there are three possible explanations. The first is that melatonin-responsive PT cells do contain a very small number of PACAP receptors that cannot be detected by these conventional means. The second possible explanation is that in the primary cell cultures, PACAP activates receptors in cells derived from the median eminence region cause the release of another molecule to activate adenylate cyclase in cells of the PT. This could be either through release into the medium, with the released bioactive compound available to act on all cells or through direct action on cells in the vicinity of PACAP-stimulated cells. At present there is no direct evidence for either of these two explanations. A third possibility is that PT cells contain an unidentified PACAP receptor subtype with insufficient sequence homology with the type I PACAP receptor to allow cross-hybridization and detection in the PT. However, the second explanation is favored over the other reasons. 1) The PACAP receptor mRNA and protein are observable in cells of the median eminence. 2) The size of the cAMP response in sensitized PT cells is significant in the absence of detectable PACAP receptors in primary cultured cell membranes. 3) PACAP receptors present on the cells of the ovine PD (a tissue with the same ontogenic origin) reportedly desensitize within 5 min of agonist exposure (25). In the experiments conducted here PACAP application to PT cells was performed for 30 min before terminating the reaction. Furthermore, it is not surprising that the PT primary cell cultures would contain cells originating from the median eminence, as it is impossible to remove the material that traverses the pituitary stalk during or after dissection of the tissue. Glial/astrocyte type cells of the nervous system are more readily cultured than neuronal cells and may therefore survive the dissociating procedure involved in the preparation of the primary cell cultures. Attempts to use an antibody to the PACAP receptor (26) to identify PACAP-responsive cells were not successful. This may be due to the low density of the receptors or insufficient sensitivity of the antibody. Attempts to identify glial/astrocyte-type cells with an anti-GFAP antibody were also unsuccessful due to nonspecific binding of the antibody to cells of the PT (data not shown).

The presence of PACAP receptors in the external zone of the ovine median eminence is consistent with the presence of neuronal fibers containing the PACAP peptide in this region (27). It was anticipated that from the observation of PACAP in the external zone of the median eminence, PACAP would be measurable in the hypophyseal portal blood system into which it would be released for subsequent action on the cells of the PD. However, Clarke et al. (28) demonstrated that many of the neuropeptides found in the neuronal fibers of the ovine median eminence, including NPY and VIP (PACAP was not measured in this study), are not released into the hypophyseal portal blood supply in sheep. This observation is consistent with in vitro studies demonstrating that neither VIP nor PACAP stimulates PRL release in cultured sheep PD cells and can only stimulate GH release at high concentrations of the peptide (29). This contrasts with the finding made in rats, where several peptides present in the median eminence are found in high concentrations in hypophyseal portal blood supply (30, 31, 32). In this species this correlates well with the ability of both VIP and PACAP to cause PRL secretion from rat PD cell cultures (33, 34). Therefore, although PACAP does not cause direct release of PRL from cells of the ovine PD, the ability of PACAP to stimulate PRL release may not be lost, but may be mediated through an indirect mechanism. A precedent for this indirect route of PACAP action is seen in the action of PACAP to stimulate a paracrine factor from folliculo-stellate cells of the pituitary to release follistatin. The released follistatin causes an inhibition of activin A-induced secretion of gonadotropins from PD cells (35). Therefore, one can speculate that an indirect mechanism, employing the intermediate cells of the PT may be an adaptation to impose a level of seasonal regulation on PRL secretion. In this respect the effect of PACAP on PRL release has been measured mainly in nonseasonal animals, humans, rats, and chickens. Sheep are the only seasonal animal in which the effect of PACAP on PRL secretion was determined. In this scenario, PACAP releases a substance from PACAP-responsive cells of the median eminence to stimulate cAMP in PT cells, in turn releasing tuberalin (5) from PT cells to increase PRL secretion from the PD. A diagrammatic representation of a possible pathway of communication between cells that produce PACAP and the melatonin-responsive cells is shown in Fig. 8Go. Seasonal regulation could be imposed by melatonin acting on the cAMP signal transduction pathway in the PT. However, a primary role for PACAP in seasonal PRL regulation from the PD seems unlikely, because in hypophyseal-pituitary-disconnected rams in which the connection between the brain and pituitary is interrupted and the internal and external zones of the median eminence are removed (36), seasonal regulation of PRL secretion is still demonstrated. Furthermore, PRL secretion is highest at night when melatonin production is also elevated (37). At this time melatonin would be expected to inactivate any PACAPinduced cAMP synthesis. Nevertheless, the possibility still exists for PACAP to play a secondary role in PRL release in sheep.



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  		Figure 8. Diagrammatic summary of the possible relationship between cells synthesizing and releasing PACAP and the effect on cells of the PT. PACAPsynthesizing neurons in the hypothalamus produce and release PACAP at nerve terminals in the median eminence. An intermediate factor of unknown identity is released by PACAP-responsive cells of the median eminence and acts to stimulate cAMP in melatonin-responsive cells of the PT. cAMP has been shown to activate PKA and MAPK in PT cells (21 22 ). One or both of these kinase activation events results in the increase in cAMP-responsive gene expression, including the synthesis of mRNA for the mt1 melatonin receptor, c-fos, and other genes as shown above (3 5 8 39 ). cAMP also results in the synthesis and release of an unidentified hormone named tuberalin. This, in turn, acts on lactotroph cells of the pituitary gland to release PRL (5 ). Also summarized in this diagram is the possible interaction between a tyrosine kinase and adenylate cyclase (15 ) and the interaction of the melatonin receptor with the G protein Gq (40 ) in addition to the inhibitory G proteins Gi and Gz (15 ).

 
The other issue this study raises is the role of sensitization resulting from prolonged melatonin exposure. It is tempting to hypothesize that sensitization is part of the mechanism required to decode the melatonin signal into a seasonal signal. In the case of PACAP, sensitization has allowed us to reveal the action of this peptide in the primary cell cultures of the PT cells through a consistent and robustly measurable increase in cAMP levels. Sensitization may not be a requirement for PACAP to activate PT cells, as an effect can be seen on mt1 mRNA transcription and MAPK activation in the absence of sensitization; however, sensitization may affect more profoundly and be more physiologically relevant to the response of the PT to other peptides. Alternatively, sensitization of the cAMP system may be a prelude to desensitizing the action of stimulatory inputs to the cells of the PT, such as seen by prolonged activation of the cAMP system in pituitary cells (38).


   Acknowledgments
 
We thank Catriona Webster, Dana Wilson, Shaun Conway, and Ted Howell. We are grateful to Keith Pennie for his assistance with collecting the PT material at the abattoir. The anti-PACAP receptor antibody was supplied by Dr. Arimura (Tulane University, New Orleans, LA).


   Footnotes
 
This work was supported by the Scottish Executive Environmental Rural Affairs Department and a fellowship (to C.S.) from the Fondation pour la Recherche Médicale (Paris, France).

Abbreviations: Bmax, Binding capacity; CREB, cAMP response element-binding protein; PACAP, pituitary adenylate cyclase-activating polypeptide; PD, pars distalis; PT, pars tuberalis; RPA, ribonuclease protection assay; SCN, suprachiasmatic nucleus.

Received December 18, 2001.

Accepted for publication February 13, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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