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European Institute for Peptide Research, Laboratory of Cellular and Molecular Neuroendocrinology, INSERM U-413, Unité Affiliée au Centre National de la Recherche Scientifique, University of Rouen (M.M., L.Y., H.V.), 76821 Mont-Saint-Aignan; and the Laboratory of General and Comparative Physiology, Unité de Recherche Associée au Centre National de la Recherche Scientifique, National Museum of Natural History (K.R., S.D.), 75005 Paris, France; U.S.-Japan Biochemical Research Laboratories, Tulane University, Hebert Center (A.A.), Belle Chasse, Louisiana 70037; and Institut National de la Recherche Scientifique-Santé, University of Quebec (A.F.), Pointe-Claire, Quebec, Canada H9R1G6
Address all correspondence and requests for reprints to: Dr. Hubert Vaudry, European Institute for Peptide Research (IFRMP 23), Laboratory of Cellular and Molecular Neuroendocrinology, INSERM U-413, Unité Affiliée au Centre National de la Recherche Scientifique, University of Rouen, 76821 Mont-Saint-Aignan, France. E-mail: hubert.vaudry{at}univ-rouen.fr
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In mammalian vertebrates, PACAP and GHRH originate from two distinct precursors (18, 19). In contrast, the recent cloning of the complementary DNA encoding the PACAP precursor in salmon (5), catfish (6), chicken (7), and tunicate (8) has revealed that in all of these species, PACAP and GHRH originate from a single common precursor. Although the functional significance of the cosynthesis of PACAP and GHRH is still a matter of speculation, it has been reported that PACAP actually stimulates GH secretion by salmon (20), frog (21), rat (22), and cow (23) somatotrophs as well as by human tumor cells (24).
In teleost fish, GH plays an important role not only in the stimulation of growth (25, 26), but also in the regulation of hydromineral homeostasis (27, 28, 29) and reproductive functions (30, 31, 32, 33). In the eel, it has been recently shown that animals submitted to starvation exhibit a differential pattern of GH regulation depending on their physiological stage (34), suggesting that GH may be involved in the metabolic adaptation during the long period of fasting associated with the reproductive migration of this animal.
In the present study, we have determined the anatomical distribution of PACAP-containing neurons in the brain and pituitary of the eel. The molecular forms of PACAP were characterized by combining HPLC analysis with RIA quantification. Finally, the effect of PACAP on GH secretion by cultured pituitary cells was investigated.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Reagents and test substances
Medium 199 (Earle’s salts with sodium bicarbonate), penicillin,
streptomycin, and fungizone were purchased from Life Technologies
(Cergy-Pontoise, France). BSA was obtained from Boehringer Mannheim
(Mannheim, Germany), and Na125I was obtained from Amersham
(Les Ulis, France). Mammalian PACAP38 was synthesized by the solid
phase methodology as previously described (13), and its identity was
confirmed by amino acid analysis and mass spectrometry. PACAP27 was
purchased from Bachem (Voisins-le-Bretonneux, France). Chicken VIP
(cVIP), human GHRH (hGHRH), 3-aminobenzoic acid ethyl ester, gelatin,
trifluoroacetic acid (TFA), polyethylene glycol, and polylysine were
supplied by Sigma (St. Quentin Fallavier, France). Acetonitrile was
obtained from Carlo Erba (Milan, Italy).
Immunohistochemical procedure
Eels were anesthetized by immersion in a solution of 0.1% of
3-aminobenzoic acid ethyl ester in water. The animals were perfused via
the aortic bulb with a 0.9% NaCl solution. The perfusion was continued
with 150 ml of a freshly prepared fixative solution consisting of 4%
paraformaldehyde and 0.2% picric acid in 0.1 M phosphate
buffer (pH 7.4). The brains with the attached pituitaries were
carefully removed and postfixed overnight at 4 C in the same fixative
solution. The tissues were cryoprotected by immersion in 15% sucrose
in phosphate buffer overnight (4 C) and then transferred into 30%
sucrose. Brains were embedded in Tissue-Tek (Jung, Nussloch, Germany)
and frozen on dry ice. Frontal or parasagittal sections (10 µm thick)
were cut in a cryostat (Frigocut 2800E, Reichert-Jung, Nussloch,
Germany) and mounted on glass slides coated with 0.5% gelatin, 5%
chrom-alum, and 50 µg/ml polylysine. Slices were processed for the
indirect immunofluorescence technique, as previously described (13),
with an antiserum raised against PACAP27 (11). Briefly, tissue sections
were incubated overnight at 4 C with the PACAP antiserum diluted 1:100
in 0.1 M Coons buffer (sodium diethylmalonylurea; pH 7.4)
containing 0.3% Triton X-100 and 1% BSA. The sections were rinsed in
Coons buffer for 30 min and incubated for 1 h at room temperature
with fluorescein isothiocyanate-conjugated goat antirabbit
-globulins (Caltag Laboratories, San Francisco, CA) diluted 1:100.
Finally, the slices were rinsed in Coons buffer, mounted with
buffer/glycerol (1:1), coverslipped, and observed on a Leitz Orthoplan
microscope (Leitz, Heidelberg, Germany) equipped with a
Vario-Orthomat photographic system. Selected slices were also
analyzed using a confocal laser scanning microscope (Leica, Heidelberg,
Germany) equipped with a Diaplan optical system and an
argon/krypton ion laser (excitation wavelengths, 488/568/610 nm).
Several types of controls were performed to verify the specificity of the immunostaining: 1) substitution of the primary antiserum with Coons buffer or 2) nonimmune rabbit serum, and 3) preincubation of the PACAP antibodies (diluted 1:100) with synthetic PACAP27, mammalian PACAP38 (mPACAP38), or cVIP (10-6 M each).
The nomenclature of eel brain areas was based on the atlas of Peter and Gill (35) and reports by Fremberg et al. (36), Kah et al. (37), and Medina et al. (38, 39).
Tissue extraction
Eels were decapitated, and the brains were dissected in three
regions: olfactory bulbs and telencephalon, diencephalon and
mesencephalon, and metencephalon and myelencephalon, as previously
described (40). Each brain region was frozen and kept at -20 C until
extraction. The tissues were immersed for 10 min in a cooled solution
of ethanol-hydrochloric acid-water (75:18:7, vol/vol/vol) and
sonicated. The homogenates were centrifuged (13,000 x
g, 4 C) for 30 min. The supernatants were collected, dried
by vacuum centrifugation (Speed-Vac concentrator, AES 2000, Savant,
Hicksville, NY) and kept in a dry atmosphere until direct RIA or
chromatographic analysis. The protein concentration in the pellet was
determined by the Lowry method (41).
Characterization of PACAP in eel brain extracts
Dried samples were reconstituted in 2 ml of a
water-TFA-acetonitrile solution (89.9:0.1:10, vol/vol/vol) and loaded
onto three Sep-Pak C18 cartridges (Alltech Europe, Eke,
Belgium) connected in series. The cartridges were rinsed with 20 ml of
the same solution, and the bound material was eluted with 10 ml of a
water-TFA-acetonitrile solution (43.9:0.1:56, vol/vol/vol). The solvent
was evaporated in a Speed-Vac concentrator. The samples were
reconstituted in 500 µl of a water-TFA-acetonitrile solution
(89.9:0.1:10, vol/vol/vol) and centrifuged (13,000 x
g, 4 C). The supernatant was injected onto a 0.46 x
25-cm Vydac C18 column (Hesperia, CA) equilibrated with a
solution of 20% acetonitrile and 0.1% TFA at a flow rate of 1.5
ml/min. The concentration of acetonitrile in the eluting solvent was
raised to 40% over 20 min using a linear gradient. HPLC standards
consisted of synthetic PACAP27 and mPACAP38 (1 µg each). Fractions of
0.75 ml were collected, evaporated, and kept dry until RIA.
PACAP RIA
The concentrations of PACAP-like material in the eel brain were
measured by RIA. Iodination of synthetic PACAP27 was performed by means
of the lactoperoxidase technique as previously described (42). The
radioligand was purified by reverse phase HPLC on a 0.46 x 25-cm
Vydac C18 column using a mobile phase of acetonitrile in
0.1% TFA. Monoiodinated PACAP27 eluted at 36% acetonitrile. The RIA
was performed in veronal buffer (0.02 M; pH 9.1) containing
0.1% Triton X-100 and 3% BSA, using an antiserum against PACAP38 at a
dilution of 1:20,000 (43). After a 2-day incubation at 4 C, the
antibody-bound fraction was immunoprecipitated by addition of 200 µl
goat antirabbit -globulins (1:30), 200 µl normal rabbit serum
(1:150), and 500 µl 5% polyethylene glycol 8000. After a 2-h
incubation at room temperature, the mixture was centrifuged, and the
pellet containing the bound fraction was counted on a -counter
(LKB-Wallac, Rockville, MD). The standard curves were set up with
synthetic PACAP27 and mPACAP38 at concentrations ranging from 0.1–333
ng/tube.
Primary culture of eel pituitary cells
Dispersion of eel pituitary cells was performed using an
enzymatic and mechanical procedure as previously described (44). Forty
to 100 animals were used for each experiment. Cells were cultured at a
density of 62,500 cells/250 µl in each well on
poly-L-lysine-precoated 96-well plates (Costar, Brumath,
France) in serum-free culture medium (medium 199 containing Earle’s
salts with 1.25 g/liter sodium bicarbonate, 100 U/ml penicillin, 100
µg/ml streptomycin, and 250 ng/ml fungizone) (45). Cells were
incubated at 18 C in a humidified incubator (Napco, Tualotin,
OR) with an atmosphere of CO2-air (3:97). After 1
day of culture, the medium was renewed, and the cells were cultured for
2 more days. The effect of PACAP-related peptides on GH secretion was
then studied during a 24-h incubation.
Stock solutions of synthetic PACAP27, mPACAP38, cVIP, and hGHRH were diluted in distilled water at a concentration of 10-4 M. Final dilutions were prepared in culture medium just before addition to the wells. Six-well replicates were used for each peptide treatment. After a 24-h incubation, the culture medium was collected and kept frozen until RIA.
GH RIA
The concentration of GH secreted in the culture medium by
cultured pituitary cells was measured in duplicate using a homologous
RIA for eel GH, as previously described (34). Briefly, the antiserum
generated against eel GH was used at a dilution of 1:20,000. Purified
eel GH was radioiodinated by the chloramine-T method and purified on a
Sephadex G-50 column (Pharmacia, Uppsala, Sweden). Results are
expressed as nanograms of GH per 62,500 cells.
Statistical analysis
The effect of each neuropeptide on GH secretion was tested in
six wells, and the results were expressed as the mean ±
SEM. The experiments were repeated three times
independently. The significance of the differences between control and
treated cells was assessed by ANOVA (GraphPad Software, Inc., San
Diego, CA) followed by a post-hoc Tukey’s test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and 2.
Table 2 summarizes the location and
relative density of immunoreactive cell bodies and fibers in the eel
brain.
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, B–E). Parasagittal sections at the level of the
hypothalamus revealed the presence of clusters of immunoreactive cell
bodies along the rostrocaudal axis of the preoptic nucleus (Figs. 1 and 3A). Groups of immunoreactive perikarya
were also found in the thalamus within the ventral and dorsal thalamic
nuclei (Fig. 2, E and F). The olfactory bulbs, telencephalon,
mesencephalon, metencephalon, and myelencephalon were devoid of
PACAP-positive cell bodies.
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, A and B). These processes probably originated
from perikaria located in the preoptic region of the diencephalon. In
this latter area, a very dense network of PACAP-positive fibers was
observed (Fig. 2, C and D), and some of these immunoreactive elements
established contacts with the cerebrospinal fluid. Bundles of labeled
fibers were seen ascending dorsally within the thalamus along the
ependyma of the third ventricle (Fig. 2, E and F). Another bundle of
nerve processes was observed in the ventral part of the diencephalon,
oriented toward the pituitary (Fig. 2, E and F). At the level of the
pituitary stalk, a dense tract of PACAP-positive fibers entered the
neurohypophysial digitations (Figs. 2F and 3B). Parasagittal sections
revealed that these PACAP-immunoreactive fibers were located in the
digitations invading the anterior part of the pituitary (Fig. 3C),
whereas no immunoreactivity was detected in the posterior part (Fig. 1). In the mesencephalon, ascending PACAP-containing fibers innervated
the torus semicircularis and the stratum album centrale of the optic
tectum (Fig. 2, G and H). The locus coeruleus and the lateral parts of
the mesencephalon exhibited a moderate density of immunoreactive
processes (Fig. 2, H and I). Scattered PACAP-positive fibers were also
found in the cerebellar valvula and in the granular eminence of the
cerebellum (Fig. 2I). In the ventral zone of the medulla oblongata,
PACAP-immunoreactive fibers were seen running
ventrally to the central canal (Figs. 1 and 3D). No labeling was observed when the PACAP antiserum was replaced with Coons buffer or nonimmune rabbit serum. Similarly, preincubation of the primary antiserum with synthetic PACAP27 or mPACAP38 totally abolished immunostaining. In contrast, preincubation of the antiserum with VIP did not modify the distribution or the intensity of the immunostaining.
Characterization of PACAP-like material in eel brain
extracts
Synthetic PACAP27 and mPACAP38 displaced antibody-bound
[125I]PACAP27 with similar efficacies (Fig. 4). In contrast, cVIP did not displace
binding of the tracer. Serial dilutions of extracts from the three
different regions of the eel brain generated displacement curves that
were parallel to those obtained with synthetic PACAP27 or mPACAP38
(Fig. 4). The apparent concentration of PACAP-immunoreactive material
contained in the tissue homogenates was 2272 pg/mg protein in the
olfactory bulb-telencephalon region, 4054 pg/mg protein in the
diencephalon-mesencephalon region, and 1640 pg/mg protein in the
metencephalon-myel-encephalon region. The elution profiles of
synthetic standards and eel diencephalon-mesencephalon extract are
shown in Fig. 5. The retention times of
mPACAP38 and PACAP27 were 12 and 14.6 min, respectively, as determined
by their optical absorbance at 215 nm (Fig. 5A). HPLC analysis of an
eel brain extract resolved a major peak of
PACAP-immunoreactive material that coeluted with
synthetic mPACAP38 (Fig. 5B).
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. Both
peptides induced a dose-dependent stimulation of GH release. PACAP38
appeared to be more potent than PACAP27 (ED50 = 4.3 ±
0.23 x 10-10 and 3.5 ± 0.8 x
10-9 M, respectively), although the two
peptides exhibited similar efficacies (2.7- and 3.2-fold over control
values, respectively). In contrast, cVIP only induced a significant
stimulation of GH release at a concentration of 10-6
M (data not shown).
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). In addition, concomitant
administration of PACAP27 and hGHRH did not modify the stimulatory
effect of PACAP27 on GH release (Fig. 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In agreement with the immunohistochemical distribution of PACAP-positive elements, quantification of PACAP-like immunoreactivity in crude brain extracts has shown that the diencephalon-mesencephalon region contains the highest concentration of peptide. HPLC analysis combined with RIA detection has revealed that the major immunoreactive component coeluted with mPACAP38. Consistent with this observation, it has been previously shown that PACAP38 is the predominant molecular form in the brain of mammals (47).
The occurrence of a dense network of PACAP-immunoreactive terminals in the pars distalis indicated that in the eel, PACAP could act as a hypophysiotropic neurohormone. In fact, cloning of the complementary DNA encoding the PACAP precursor has shown that in all submammalian vertebrate species studied to date, GHRH and PACAP originate from the same precursor (5, 6, 7). The observation that in the eel, PACAP-immunoreactive perikarya are located in the parvo- and magnocellular portions of the preoptic nucleus where GHRH-positive cell bodies have been previously detected (48) supports the view that in A. anguilla, the two peptides may also originate from a common precursor. These data prompted us to investigate the effects of PACAP and GHRH on GH secretion. The present study has demonstrated that PACAP27 and PACAP38 both induce a dose-dependent stimulation of GH release from cultured eel pituitary cells. These findings are consistent with previous reports showing that PACAP stimulates GH secretion from bovine (23), sheep (49), porcine (50), rat (51), chicken (52), frog (21), and salmon (20) pituitary cells. PACAP was also found to increase cytosolic calcium concentrations in GH cells from the rat (53, 54) and frog (17) pituitary. In the eel, PACAP38 was approximately 12 times more potent than PACAP27, but the two peptides exhibited the same efficacy to stimulate GH secretion. In contrast, VIP had no effect at doses ranging from 10-10-10-7 M and stimulated GH secretion only at a concentration of 10-6 M, indicating that the action of PACAP on eel somatotrope cells was mediated through a receptor exhibiting the pharmacological profile of type I mPACAP receptor (55). In agreement with this finding, it has been recently shown that GH-enriched rat pituitary cells express exclusively type I PACAP receptors (56).
It has been previously reported that GHRH stimulates GH secretion in various species of teleosts, including the Sockeye salmon (20), the rainbow trout (57), and the goldfish (58). In contrast, the present data have shown that in the eel, hGHRH has no effect, even at a high concentration, on GH release. Moreover, GHRH did not modify the response of eel somatotrope cells to PACAP27. These observations indicate that in the primitive teleost fish, PACAP rather than GHRH plays a role in the regulation of GH secretion. Consistent with this finding, it has been reported that in the salmon, PACAP is far more efficient than native GHRH in stimulating GH release (20). Alternatively, the lack of effect of hGHRH on GH secretion could be ascribed to species specificity, inasmuch as the structure of GHRH has been less conserved than that of other hypophysiotropic neuropeptides. In particular, catfish GHRH (6) has only 58% identity to salmon GHRH (5) and 60% identity to carp GHRH (58). Molecular cloning of the eel PACAP precursor is clearly required to determine whether it possesses a GHRH-like sequence and, if so, whether this peptide has any GH-releasing activity.
In conclusion, the present study has provided the first detailed mapping of PACAP-immunoreactive neurons in a fish. The presence of a population of PACAP-containing perikarya in the preoptic area, the dense accumulation of PACAP-immunoreactive fibers in the distal lobe of the pituitary, and the stimulatory effect of PACAP on GH secretion in vitro indicate that PACAP may act as a physiological GH-releasing factor in the eel. The lack of effect of GHRH on GH secretion suggests that in this primitive teleost fish, PACAP may play a crucial role in the control of somatotrope cell activity.
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Acknowledgments |
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Footnotes |
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2 Recipient of a fellowship from the Ministère de
l’Enseignement Supérieur et de la Recherche.
3 Affiliated Professor at the INRS-Santé (Montreal,
Canada).
Received March 10, 1998.
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), mPACAP38 (•), cVIP (
), and serial
dilutions of eel brain extracts. Region 1 corresponds to the olfactory
bulbs and telencephalon (
), region 2 corresponds to the diencephalon
and mesencephalon (
), and region 3 corresponds to the metencephalon
and myelencephalon (
). Each tissue extract contained the equivalent
of 16 brain regions.
