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European Institute for Peptide Research (IFRMP 23), Laboratory of Cellular and Molecular Neuroendocrinology, Institut National de la Santé et de la Recherche Médicale U413, UA CNRS, University of Rouen (L.Y., V.C., G.B., C.D., H.V.), 76821 Mont-Saint-Aignan, France; Service of Endocrinology (L.B., N.G.-P.), Department of Medicine, Department of Biochemistry (J.-G.L.), Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4; and INRS Santé, University of Quebec (A.F.), Pointe-Claire, Quebec, Canada H9R 1G6
Address all correspondence and requests for reprints to: Dr. Hubert Vaudry, European Institute for Peptide Research (IFRMP 23), Laboratory of Cellular and Molecular Neuroendocrinology, Institut National de la Santé et de la Recherche Médicale U413, UA CNRS, University of Rouen, 76821 Mont-Saint-Aignan, France. E-mail: hubert.vaudry{at}univ-rouen.fr
Abstract
The distribution and pharmacological properties of pituitary adenylate cyclase-activating polypeptide (PACAP) receptors were studied in the fetal human adrenal gland during the second trimester of gestation. Autoradiographic studies, using [125I]PACAP27 as a radioligand, revealed that PACAP-binding sites are exclusively located on chromaffin cells of adrenals from fetuses 14–20 weeks old. Biochemical characterization of binding revealed the occurrence of a single class of PACAP-binding sites with a dissociation constant value of 0.32–0.74 nmol/L and a binding capacity of 0.30–0.81 pmol/mg wet tissue. PACAP27 and PACAP38 were equipotent in competing for [125I]PACAP27 binding (IC50 = 0.28–0.64 nmol/L and 0.15–0.81 nmol/L, respectively), and the Hill coefficients were close to 1. In contrast, vasoactive intestinal polypeptide was much less efficient in displacing the tracer (IC50 = 4–362 nmol/L), and the Hill coefficients were less than 0.6. PACAP38 induced a dose-dependent increase in cAMP production in fetal human adrenal cell suspension (ED50 = 0.07 ± 0.02 nmol/L), as well as in cells maintained in culture for 5 days (5.4 ± 1.8 nmol/L). In constrast, PACAP38 induced a modest increase in inositol phosphate formation. These data indicate that type I PACAP receptors are present in the early stages of the human medulla organization during the process of migration of chromaffin cells from the periphery to the central part of the gland. The present results suggest that PACAP could be involved in the regulation of the human adrenochromaffin cells during ontogenesis.
PITUITARY adenylate cyclase-activating
polypeptide (PACAP) is a 38-amino acid 
-amidated neuropeptide
initially isolated from the ovine hypothalamus because of its ability
to stimulate adenylyl cyclase activity in rat anterior pituitary cells
(1). PACAP38 possesses an internal cleavage-amidation consensus
sequence (Gly28-Lys29-Arg30) and
can thus generate a 27-residue -amidated peptide (PACAP27) that
exhibits the same biological activity as PACAP38 (2, 3). The
NH2-terminal region of PACAP shows 68% sequence similarity
with vasoactive intestinal polypeptide (VIP), identifying PACAP as a
member of the VIP/secretin/glucagon superfamily. The primary structure
of PACAP has been remarkably conserved during evolution (4), indicating
that the peptide plays important biological functions.
Two types of PACAP-binding sites have been characterized so far
(see Ref. 5 for review). Type I binding sites have a high affinity
(dissociation constant (Kd) 
0.2 nmol/L) for PACAP38 and
PACAP27 and a much lower affinity (Kd 1 µmol/L) for
VIP (5, 6), whereas type II binding sites have similar affinity for
PACAP38, PACAP27, and VIP (7). Both receptor subtypes belong to the
seven-transmembrane domain G protein-coupled receptor superfamily
(8, 9, 10) and are differentially coupled to adenylyl cyclase and
phospholipase C (10). PACAP and PACAP-binding sites are widely
distributed in the central nervous system and in peripheral organs (7, 11). In particular, the occurrence of PACAP and its recognition sites
has been demonstrated in various endocrine glands, including the
pituitary, pancreas, testis, ovary, and adrenal (3, 5, 7, 12, 13).
Developmental studies have shown that, in the brain, the expression of PACAP and PACAP-binding sites undergoes important variations during ontogenesis. For instance, a high concentration of PACAP has been detected in the rat cerebellum between postnatal days 4 and 20 (14, 15). Similarly, intense expression of PACAP receptors has been observed in the proliferative zone of the cerebellar cortex during the postnatal period (16, 17), suggesting that PACAP exerts neurotrophic activities during development. In support of this hypothesis, it has been shown that PACAP promotes cell survival and neurite outgrowth in cultured cerebellar granule cells (18) and stimulates proliferation of folliculo-stellate cells (19).
The occurrence of PACAP and PACAP receptors in the adult adrenal gland (13, 20, 21, 22) and the observation that PACAP modulates proliferation and differentiation of adrenal chromaffin cells (23, 24, 25) suggest that PACAP may play a role in the ontogeny of the adrenal gland. The fetal human adrenal gland is composed of morphologically and functionally distinct steroid-secreting tissues: a thin neocortex that produces cortisol and a large fetal zone that produces massive amounts of dehydroepiandrosterone sulfate (26, 27). During fetal life, cells derived from the ectoderm migrate inside the gland to form the medulla (28, 29). After birth, the fetal zone involutes, and the cortex develops and differentiates into the three characteristic zones present in the adult adrenal gland. These profound changes in the organization of the gland suggest that trophic factors, such as PACAP, may control the modeling of the tissue during ontogenesis. However, the distribution and function of PACAP receptors have not yet been studied in the adrenal during development.
In the present study, we have localized PACAP-binding sites in the fetal human adrenal gland by autoradiography, and we have investigated the effect of PACAP on second-messenger systems in suspended and cultured fetal adrenal cells.
Materials and Methods
Reagents and peptides
Myo-[3H]inositol (10–20 Ci/mmol) and [3H]adenine (24 Ci/mmol) were purchased from Amersham (Oakville, Ontario, Canada). Na125I (2000 Ci/mmol) was obtained from Amersham (Les Ulis, France). ATP, cAMP, and deoxyribonuclease were supplied by Sigma (St. Louis, MO). Eagle’s MEM, collagenase, and Opti-MEM medium were obtained from Gibco (Burlington, Ontario, Canada). PACAP27 was from Bachem (Marina Delphen, CA). BSA was from Boehringer (Mannheim, Germany). PACAP38 and VIP were synthesized by the solid-phase methodology previously described (30).
Monoiodinated PACAP27 was used as a tracer. Iodination of synthetic PACAP27 was performed by means of the lactoperoxidase technique, and the radioligand was purified by reverse-phase high-performance liquid chromatography, as previously described (31, 32). The specific radioactivity of the radioligand was approximately 800 Ci/mmol.
Tissue preparation
Fetal human adrenal glands were obtained from fetuses between 14 and 20 weeks old (post conception) at the time of therapeutic abortion. Fetal ages were estimated by foot length and time after the last menstruation, according to Streeter (33). After removal, glands were kept on ice in McCoy’s medium and transported within 4 h to the laboratory. The project was approved by the human subject review committees at our institutions. Immediately after collection, glands were cleansed of fat and were either frozen at -80 C or processed for cell dispersion.
Autoradiographic studies
Frozen adrenal glands were sliced into 10-µm sections on a cryostat (Frigocut, Reicher-Jung, Germany). Sections were thaw-mounted on gelatin-coated slides, placed in a desiccator overnight at 4 C, and processed for autoradiography, as previously described (16). Tissue slices were preincubated for 30 min at 24 C in 50 mmol/L Tris buffer (pH 7.4) containing 1% BSA, 5 mmol/L MgCl2, 32 mmol/L sucrose, and 0.5 µg/mL bacitracin. The sections were incubated with [125I]PACAP27 (50 pmol/L) for 2 h at 24 C in the same buffer, supplemented with 2% BSA and 100 KIU/mL aprotinin (Hoechst Laboratories, Puteaux, France). Nonspecific binding was determined by adding 1 µmol/L PACAP27. For competition studies, the sections were incubated with 50 pmol/L tracer in the presence of various concentrations of unlabeled PACAP27, PACAP38 (10-10 to 10-6 mol/L) or VIP (10-9 to 10-6 mol/L). The slices were washed three times for 5-min periods at 4 C in 50 mmol/L Tris buffer containing 0.1% BSA, 5 mmol/L MgCl2, and 0.5 µg/mL bacitracin. Finally, the sections were dried under a cold-air stream and apposed onto Amersham Hyperfilm-3H for 10 days.
The gray levels of the autoradiograms were measured by means of a computer-assisted image analysis station BIO 500 (Biocom, Les Ulis, France), as previously described (34). The concentration of ligand which causes 50% inhibition of the binding values (IC50) and Hill coefficients were determined using the Sigma Plot program (Jandel Scientific, Corte Madera, CA).
Histochemical and immunological procedures
After exposure for autoradiography, adrenal sections were fixed
with vapors of paraformaldehyde (24 h, 60 C). Tissue slices were either
stained with hematoxylin-eosin or immunostained with antibodies against
human chromogranin A (CgA; Dako, Glostrup, Denmark), a marker of
chromaffin cells (29). Briefly, tissue sections were incubated
overnight at 4 C with the CgA antiserum, diluted 1:100 in phosphate
buffer (PB; 0.1 mol/L, pH 7.4) containing 0.3% Triton X-100 and 1%
BSA. After several rinses in PB, the sections were incubated for 90 min
at room temperature with fluorescein isothyocyanate-conjugated goat
antirabbit 
-globulins (GAR/FITC; Caltag Laboratories, San Francisco,
CA) diluted 1:100. Finally, the sections were rinsed in PB and mounted
in PB/glycerol (1:1). The preparations were examined on a Leitz
Orthoplan (Heidelberg, Germany) microscope equipped with a photographic
system.
Dissociation of adrenal cells
The adrenal glands from 18- to 20-week-old fetuses were finely chopped into 1- to 2-mm pieces. The tissue fragments were subjected to enzymatic dissociation in Eagle’s MEM containing collagenase (2 mg/mL) and deoxyribonuclease (25 µg/mL), as previously described (35). The tissue was mechanically dissociated by gentle aspiration, and dispersed cells were filtered and centrifuged for 10 min at 100 x g. The cell pellet was resuspended in Opti-MEM containing 2% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin. Cells were plated in 35-mm Petri dishes at a density of about 2.5 x 105 cells per dish and were grown in a humidified atmosphere of 95% air-5% CO2 at 37 C. The culture medium was changed 24 h after seeding, and cells were used after 5 days of culture. For studies using suspended cells, cells were incubated in culture medium for a resting period of 12 h. After centrifugation (10 min; 100 x g), cells were suspended in Hank’s buffered saline (HBS)-glucose (HBS: NaCl, 130 mmol/L; KCl, 3.5 mmol/L; CaCl2, 1.8 mmol/L; MgCl2, 0.5 mmol/L; NaHCO3, 2.5 mmol/L; HEPES, 5 mmol/L) at a density of 5 x 105 cells/tube.
Cyclic AMP determination
Cyclic AMP production was determined by measuring the conversion of [3H]ATP into [3H]cAMP, as previously described (36). Briefly, cultured cells were incubated at 37 C for 1 h in Opti-MEM culture medium containing 2 µCi/mL [3H]adenine. The cultures were washed with HBS buffer and incubated in the same buffer, containing 1 mmol/L isobutyl methylxanthine, for 15 min at 37 C. Cultured cells were incubated with PACAP38 for an additional 15 min at 37 C. The reaction was stopped by aspiration of the medium and addition of 1 mL 5% trichloroacetic acid. Cells were scraped with a rubber policeman, and 100 µL of cold solution of ATP and cAMP (5 mmol/L each) was added to the mixture. Cell membranes were pelleted at 5,000 x g for 15 min, and the supernatants were sequentially chromatographed on Dowex and alumina columns, as previously described (37), allowing the separation of [3H]ATP nucleotide from [3H]cAMP. cAMP formation was expressed as: % conversion = ([3H]cAMP/([3H]cAMP + [3H]ATP) x 100 per 15 min.
Measurement of inositol phosphate (InsP) formation
The effect of PACAP on polyphosphoinositide metabolism was investigated as described previously (38). Briefly, cells were grown for 2 days in Opti-MEM culture medium containing 2 µCi/mL myo-[3H]inositol. The radioactive medium was then discarded, and the cells were incubated in isotope-free and serum-free culture medium. After 30 min, cells were washed, incubated for 15 min in HBS-glucose/LiCl (10 mmol/L) medium, and incubated for another 15 min at 37 C with PACAP38 in the same medium. The incubation was stopped by aspiration of the medium and addition of 1 mL of 5% (vol/vol) perchloric acid and 200 µL BSA (20 mg/mL). Total InsPs were separated by ion exchange chromatography on (1 x 8 cm) Dowex columns. The radioactivity found in the InsP fractions was determined by scintillation counting in gel phase in a Beckman ß counter.
Data analysis
The data are presented as means ± SE. Statistical analysis was performed using the one-way ANOVA test. Homogeneity of variance was assessed by Bartlett’s test, and P values were obtained from Dunnett’s tables.
Results
Localization of PACAP-binding sites
The autoradiographic localization of
[125I]PACAP27-binding sites was performed in the adrenal
of fetuses at 14–20 weeks of gestation. The distribution of the
binding sites in a 16-week-old fetus is illustrated in Fig. 1A
. Labeling of consecutive sections with
antibodies against human CgA demonstrated that the autoradiographic
labeling was restricted to the chromaffin tissue (Fig. 1, A and B).
Histochemical staining of tissue sections with hematoxylin-eosin showed
that chromaffin cells formed radial clusters, penetrating the
eosinophilic cells of the fetal zone (Fig. 1C). At all developmental
stages studied, PACAP-binding sites were found only in the fetal
medullary zone.
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Figure 2 illustrates the
displacement of [125I]PACAP27 binding (Fig. 2A) by
increasing concentrations of synthetic PACAP27 (Fig. 2, B–D), PACAP38
(Fig. 2, E–G), or VIP (Fig. 2, H–J) on consecutive sections of a
fetal adrenal gland at 15 weeks of gestation. Incubation of tissue
slices with 10-8 mol/L PACAP27 (Fig. 2C) or PACAP38 (Fig. 2F) completely abolished the autoradiographic labeling. In contrast, at
the same dose, VIP induced only a slight displacement of
[125I]PACAP27 binding (Fig. 2I).
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, A–F). Scatchard plot analysis of
PACAP27 inhibition curves (Fig. 3B inset) revealed that the
affinity of PACAP-binding sites and the density of sites were
relatively constant throughout the gestation period studied (Table 1). PACAP27 and PACAP38 were equally
potent in displacing [125I]PACAP27, whereas VIP was 20 to
500 times less potent than PACAP27 in competing with the tracer during
the whole period studied (Table 2). The
values of the Hill coefficient were close to 1 for PACAP27 and slightly
lower for PACAP38, but systematically inferior to 0.6 for VIP (Table 2).
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The capacity of PACAP38 to stimulate cAMP production was
measured both in cell suspensions (used after a 12-h resting period)
and in cells cultured for 5 days. In suspended cells, PACAP38 induced a
dose-dependent increase in the conversion of ATP into cAMP (Fig. 4A). The ED50 value was
0.07 ± 0.02 nmol/L, and the maximum effect was reached at a dose
of 10 nmol/L, with a stimulation ratio of 2.55 ± 0.25 (n =
7, P < 0.01) over basal value. In cultured cells, the
basal conversion of ATP into cAMP was significantly lower than in
suspended cells. The ED50 value was 5.4 ± 1.8 nmol/L,
and the maximal effect was reached at a dose of 100 nmol/L, with a
stimulation ratio of 3.4 ± 0.40 (n = 9, P <
0.001) over basal value. In contrast, PACAP38 did not affect InsP
accumulation in suspended adrenal cells from 20-week-old fetuses but
only induced a modest increase of InsP content (1.30-fold over control)
in cells from 15- to 16-week-old fetuses (data not shown).
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The presence of PACAP in the adrenal gland and the effect of the neuropeptide on catecholamine and steroid secretion have been documented in various mammalian species. In contrast, little is known concerning the possible role of PACAP in the human adrenal gland. The present study provides the first evidence for the occurrence of functional PACAP receptors in fetal chromaffin cells.
Localization and characterization of PACAP-binding sites
Autoradiographic labeling, using [125I]PACAP27 as a radioligand, revealed the presence of a high density of binding sites in the central zone of the gland. Immunohistochemical staining of adrenal slices with antibodies against human CgA, a selective marker of neuroendocrine cells (29), demonstrated that PACAP-binding sites are exclusively borne by chromaffin cells. The strong autoradiographic signal observed at the earlier stage studied (14-week-old fetuses) is consistent with previous reports indicating that, in the developing human adrenal gland, chromaffin cells establish their phenotype as early as 6 weeks of gestation, as evidenced by the expression of CgA and tyrosine hydroxylase (29, 39). The fetal human adrenal cortex is composed of a thin outer zone (neocortex) and a large fetal zone that represents over 80% of the gland (26, 27). The present study showed that both of these cortical zones are virtually devoid of PACAP-binding sites. In agreement with this observation, autoradiographic labeling, membrane binding assay, and in situ hybridization immunohistochemistry studies indicate that, in the adult rat adrenal gland, PACAP-binding sites are located only on chromaffin cells (20, 21, 22).
Scatchard plot analysis of PACAP-binding in the fetal human chromaffin tissue showed the existence of high-affinity binding sites with Kd values ranging from 0.32–0.74 nmol/L. Displacement experiments demonstrated that PACAP27 and PACAP38 were equally potent in competing with the radioligand at all developmental stages studied, whereas VIP was a much weaker competitor. These data revealed that the binding sites evidenced with [125I]PACAP27 correspond predominantly to type I PACAP receptors. The fact that the Hill coefficients measured with PACAP27 were close to 1 suggested that the peptide interacts with a single class of receptors. However, the lower Hill coefficient value calculated with VIP as a competitor would indicate the existence of a heterogeneous population of PACAP/VIP receptors, as previously reported in rat (20, 40, 41) and human adult adrenal medulla (42).
Second-messenger coupling of PACAP receptors
There is now clear evidence that type I PACAP receptors can be coupled to adenylyl cyclase and phospholipase C (6, 7, 10). We have thus investigated the transduction mechanisms associated with the PACAP binding sites in fetal human adrenomedullary cells. We found that PACAP38 stimulated, in a dose-dependent manner, cAMP production from both isolated and cultured adrenal cells. The concentration of ligand which causes 50% inhibition of the binding (IC50) for PACAP27 binding and the ED50 for cAMP formation were in the same range (0.3 and 0.07 nmol/L, respectively), showing the existence of a good correlation between binding and coupling potency. These data indicate that the binding sites visualized by autoradiography actually correspond to functional receptors positively coupled to adenylyl cyclase. However, although PACAP38 stimulates both adenylyl cyclase and phospholipase C activity in porcine adrenochromaffin cells (43), we found that PACAP causes only a slight increase in InsP formation. The modest effect of PACAP on phospholipase C activity suggests that fetal human adrenal cells express either the SV1 or the SV3 splice variants of the PACAP receptor, which exhibit a low efficacy for phospholipase C activation (44). Alternatively, the Gq protein, which is required for receptor coupling to phospholipase C, may be expressed at a very low level at this stage of fetal development in human adrenochromaffin cells. In support of this latter hypothesis, we found that fluoroaluminate, a nonspecific activator of all heterotrimeric G proteins, induces a 2-fold increase in InsP production in fetal adrenal cells, compared with a 15-fold increase in adult cells (data not shown).
Functional implications
The role of PACAP in the human adrenal during development is currently unknown. In vivo and in vitro studies on animal models have shown that PACAP stimulates tyrosine hydroxylase activity and catecholamine secretion from chromaffin cells (21, 43, 45). It has recently been reported that PACAP also enhances catecholamine release from adult human adrenal explants (46). These data suggest that PACAP may also exert a stimulatory effect on catecholamine secretion by fetal adrenochromaffin cells. In support of this hypothesis, immunohistochemical studies have shown the presence of both tyrosine hydroxylase and dopamine ß-hydroxylase in human chromaffin cells at early stages of development (28, 39).
In the adult human adrenal gland, Neri et al. (46) have shown that PACAP stimulates aldosterone and 18-OH-corticosterone secretion, probably via an indirect mechanism involving the release of catecholamines by chromaffin cells. This paracrine effect of PACAP in the adult adrenal tissue is facilitated by the existence of islets and medullary rays within the adrenal cortex (47, 48). In the fetal human adrenal, a paracrine mode of action is even more plausible, inasmuch as, during ontogenesis, chromaffin cells migrate centripetally through the cortex towards their central destination (29, 39).
PACAP is known to act as a growth factor, modulating proliferation, survival, and differentiation of various types of neuronal cells (18, 49, 50). In particular, PACAP has been shown to induce neurite outgrowth and tyrosine hydroxylase gene expression in cultured rat chromaffin cells (24, 25) and PC12 cells (51). It is thus conceivable that PACAP also may affect the migration and/or differentiation of immature human chromaffin cells.
In conclusion, the present study has shown that type I PACAP receptors are expressed in human chromaffin cells in 14- to 20-week-old fetuses and that these receptors are functionally coupled to adenylyl cyclase. These data suggest that PACAP may play a role in the differentiation and/or secretory activity of chromaffin cells in the fetal human adrenal gland.
Acknowledgments
The authors would like to thank Ms. Huguette Lemonnier (INSERM U413) and Lucie Chouinard (Service of Endocrinology, Sherbrooke) for skillful technical assistance. We appreciate the advice from Drs. Magali Basille and Bruno Gonzalez (INSERM U413) for autoradiography.
Footnotes
1 This work was supported by the Medical Research Council of Canada
(MRC MT13679, to N.G.-P. and J.-G.L.), the Institut National de la
Santé et de la Recherche Médicale (INSERM U413), a
France-Québec exchange programme (Coopération Scientifique
et Technologique Franco-Québécoise (PVP-73–9, to N.G.-P.
and H.V.), and the Conseil Régional de Haute-Normandie. 
2 Recipient of studentship from the Fonds de la Recherche en
Santé du Québec et Fonds pour la Formation de Chercheurs et
l’Aide à la Recherche.
3 Recipient of a studentship from the Conseil Régional de
Haute-Normandie.
4 Affiliated Professor at the Institut National de la Recherche
Scientifique-Santé, Montréal.
Received September 9, 1997.
Revised December 22, 1997.
Accepted December 31, 1997.
References
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