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Endocrinology Vol. 142, No. 4 1616-1625
Copyright © 2001 by The Endocrine Society

ARTICLES

Early Expression of Pituitary Adenylate Cyclase-Activating Polypeptide and Activation of its Receptor in Chick Neuroblasts1

Nola M. Erhardt, Erica A. Fradinger, Laura A. Cervini, Jean E. Rivier and Nancy M. Sherwood

Department of Biology, University of Victoria, (N.M.E., E.A.F., N.M.S.),Victoria, British Columbia, Canada, V8W 2Y2; and The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute (L.A.C., J.E.R.), La Jolla, California 92037

Address all correspondence and requests for reprints to: Nancy M. Sherwood, Ph.D., University of Victoria, Department of Biology, P.O. Box 1700, Victoria, British Columbia V8W 2Y2, Canada. E-mail: nsherwoo{at}uvic.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To investigate the involvement of pituitary adenylate cyclase- activating polypeptide (PACAP) and GH-releasing factor (GRF) during early chick brain development, we established neuroblast- enriched primary cell cultures derived from embryonic day 3.5 chick brain. We measured increases in cAMP generated by several species-specific forms of the peptides. Dose-dependent increases up to 5-fold of control values were measured in response to physiological concentrations of human/salmon, chicken, and tunicate PACAP27. Responses to PACAP38 were more variable, ranging from 5-fold for human PACAP38 to 4-fold for chicken PACAP38, to no significant response for salmon PACAP38, compared with control values. The responses to PACAP38 may reflect a greater difference in peptide structure compared with PACAP27 among species. Increases in cAMP generated by human, chicken, and salmon/carp GRF were not statistically significant, whereas increases in response to lower-range doses of tunicate GRF27-like peptide were significant, but small. We also used immunocytochemistry and Western blot to show synthesis of the PACAP38 peptide. RT-PCR was used to demonstrate that messenger RNAs for PACAP and GRF and a PACAP-specific receptor were present in the cells. This is a first report suggesting an autocrine/paracrine system for PACAP in early chick brain development, based on the presence of the ligand, messages for the ligand and receptor, and activation of the receptor in neuroblast-enriched cultures.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PITUITARY ADENYLATE CYCLASE-ACTIVATING polypeptide (PACAP) has been isolated and sequenced for sixteen vertebrates and one invertebrate and is the most highly conserved member of the glucagon superfamily of hormones (1). A remarkably high maintenance of sequence identity, across species and through time, suggests an important role for this peptide. PACAP is produced in the central nervous system, throughout the peripheral nervous system, and in nonneural tissues (1). The peptide is known to be active in neural system function, smooth and cardiac muscle function, bone metabolism, immune system function, and paracrine, endocrine, and exocrine secretions. Although the major function of this hormone is yet to be elucidated, PACAP appears to play an important role in regulation of cell cycle, by enhancing cellular survival, and by enhancing or inhibiting cellular proliferation and differentiation. The particular effect depends on concentration, interaction with other factors, and utilization of receptor variants (1). In rodents, a response to physiological doses of PACAP has been recorded for primordial germ cells, splenocytes, thymocytes, and astrocytes (2, 3, 4, 5). In the nervous system of rats, effects on survival, proliferation, and differentiation have been reported for chromaffin cells, and cells in the superior cervical ganglion, cerebral cortex, and septum (6, 7, 8, 9, 10, 11, 12). An important aspect of this function is regulation of cell cycle during nervous system development. In the embryonic rat, PACAP increased cAMP, and affected survival, proliferation, and differentiation of neuroblasts in the superior cervical ganglion, dorsal root ganglion, hippocampus, cerebral cortex, and cerebellar granule layer (9, 10, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24). PACAP messenger RNA (mRNA) has been detected in the brain as early as embryonic day 9.5 (E9.5) in mouse and E14 in rat (1).

The receptors for PACAP belong to a subset of the seven-transmembrane receptor family and are linked through a G protein to adenylyl cyclase. Two major types of PACAP receptor have been described, based on binding affinities (1). One type (VPAC1-R and VPAC2-R) binds PACAP with equal affinity to vasoactive intestinal polypeptide (VIP), and the other (PAC1R) binds PACAP with 100-1000 times greater affinity than VIP. Inclusion or exclusion of three cassettes (hip, hop1, hop2), singly or in combination, in the third intracellular loop of PAC1R creates six isoforms of the receptor. In addition, a 21-amino acid deletion in the extracellular domain creates another variation (very short). An eighth form, PAC1R-TM4, does not activate adenylyl cyclase but instead acts through a calcium channel. PAC1R has been isolated from human, cow, rat, chicken, frog, and goldfish and is the predominant PACAP receptor expressed in the brain during development (1). PACAP receptor mRNA and binding sites for PACAP have been reported by E14 in rat brain (25).

GH-releasing factor (GRF) is also a member of the glucagon superfamily. The gene has been isolated and sequenced for six vertebrates and one invertebrate (1). Although it is best known for release of GH from the pituitary, GRF has varied functions (1). It is produced in the central nervous system in the hypothalamus, throughout the peripheral nervous system, and in nonneural tissues. GRF is involved in paracrine and endocrine secretions, fetal growth, and in the functions of the immune, reproductive, and digestive systems. GRF also affects the cell cycle by enhancing proliferation and differentiation of pituitary cells (26, 27, 28). Few effects have been described during nervous system development, but the peptide has been observed to influence survival and proliferation of developing spinal cord cells in chick (29). However, because it is found on the same gene as PACAP in most vertebrate groups and protochordates, it is possible GRF and PACAP have coordinated functions. In mammals, where GRF is found on a separate gene from PACAP, the former peptide has been detected at E16.5 in mouse brain and E18 in rat brain (1). The GRF receptor, which is structurally related to PAC1R, is synthesized in both a long and short form (1). GRF receptors have been isolated from human, pig, rat, mouse, and goldfish but have yet to be examined during development (1).

Isolation of PACAP/GRF-like genes in the tunicate with high sequence identity to vertebrate forms reinforced the importance of these peptides. The tunicates are considered to be an ancient group that may have given rise to the vertebrates (30).

To investigate the effects of PACAP and GRF on developing chick brain, we used primary cell culture to record increases in cAMP, a measure of adenylyl cyclase activation. This paper determines whether a dose-dependent increase in cAMP production occurs in neuroblast-enriched cultures derived from E3.5 chick brain in response to physiological concentrations of PACAP27, including the human/salmon (h/s; the peptides are identical), chicken (c) and tunicate (t) forms, and in response to physiological concentrations of hPACAP38, cPACAP38, and sPACAP38. Also, changes in cAMP were measured in response to hGRF29, cGRF29, carp GRF28 (identical to sGRF28 except for one conservative amino acid substitution at position 25), tGRF27-like peptide, hGRF44, cGRF46, and sGRF45. We used immunocytochemistry and Western blot to examine synthesis of PACAP by the cells, and RT-PCR and sequencing to determine whether PACAP, GRF, and PAC1 receptor transcripts were expressed in the cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
White leghorn chicken eggs were incubated in a humidified, forced air incubator that automatically rocked the eggs. Brains were removed from the embryos at Stage 21–22 (31) after 3.5 days of incubation (Fig. 1Go). Brains were washed 3 times for 10 min in PBS containing 500 µg/ml streptomycin and 500 U/ml penicillin. The tissue was briefly minced with a sterile razor blade, placed in culture medium, and slowly triturated 30 times through a sterile 20 gauge needle. The mixture was allowed to settle, and individual cells in the supernatant were used for experiments. Cells were counted using a Neubauer hemacytometer and diluted to 0.9–1.1 x 106 cells/ml, then plated 0.5 ml/well in 24-well, flat-bottom, tissue culture-coated plates (Corning, Inc., Acton, MA). Cells were kept on ice until plated. Cells were cultured in Neurobasal Medium (Life Technologies, Inc., Burlington, Ontario, Canada), with manufacturer’s recommended supplements, in a humidified atmosphere of 5% CO2 at 37 C. Peptides were dissolved in PBS and added to the cultures immediately upon plating. Because of the presence of methionine residues in the peptides, ascorbic acid (0.5 mM) was added with the peptides to prevent oxidation, following tests to confirm that this concentration had no effect on basal cAMP production. 1-Isobutyl-3-methylxanthine (0.01%) was added at the same time to preserve the cAMP, following similar tests.



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  		Figure 1. Chick embryo after 3.5 days of 21-day gestation period. Development is to Hamburger and Hamilton Stage 21–22 (31 ). Heads were used for experiments. Figure is adapted from Belairs and Osmond (55 ). Bar represents 1 mm.

 
Peptides
Six species-specific forms of PACAP were tested, including h/sPACAP27, cPACAP27, tPACAP27, hPACAP38, cPACAP38, and sPACAP38. Seven forms of GRF were tested, including hGRF29, cGRF29, carp GRF28 (used in place of sGRF28), tGRF27-like peptide, hGRF44, cGRF46, and sGRF45. Human PACAP27 and PACAP38 were purchased from Peninsula Laboratories, Inc. (Belmont, CA). All other peptides were synthesized at the Salk Institute for Biological Studies (La Jolla, CA) by two of us (L.A.C., J.E.R.).

Peptide synthesis
Peptides were made by the solid-phase approach (32), either manually or on a Beckman Coulter, Inc. (Fullerton, CA) 990 Peptide Synthesizer, using p-methylbenzydrylamine or chloromethyl resins. Couplings on 1–2 g resin per peptide were mediated for 2 h by diisopropylcarbodiimide in CH2Cl2, dimethylformamide (DMF) or N-methylpyrrolidinone (NMP) solvents for Asn, Gln, Ile, Leu, and Arg(Tos) and monitored by the qualitative ninhydrin test (33). Difficult couplings were mediated with benzotriazolylyoxy-Tris (dimethylamino) phosphonium hexafluorophosphate, O-(benzotriazol-1-yl)N,N,N',N'-tetramethyluronium tetrafluoroborate or O-(benzotriazol-1-yl)N,N,N',N'-tetramethyluronium hexafluorophosphate in DMF or NMP and adjusted to pH 9 with diisopropylethylamine. Boc-Asn and Boc-Gln were coupled in the presence of 1.5 eq 1-hydroxybenzotriazole. A 2.5 eq excess of amino acid based on the original substitution of the resin was used in most cases. Coupling steps were followed by acetylation [10% (CH3CO)2O in CH2Cl2] for 10–15 min as necessary. The Boc-protecting group was removed during a 20-min reaction with 50% trifluoroacetic acid (TFA) in CH2Cl2, containing 1–2% ethanedithiol (EDT) or m-cresol as cation scavenger. An isopropyl alcohol (1% EDT or m-cresol) wash followed TFA treatment and then successive washes with triethylamine (TEA) solution (10% in CH2Cl2), methanol, TEA solution, methanol, and CH2CL2 completed the neutralization sequence. The completed peptides were cleaved from the resin by reacting with anhydrous hydrofluoric acid (HF), containing 10% anisole and 2–5% dimethysulfoxide as scavengers, for 1.5 h at 0 C. After HF distillation, the crude peptides were precipitated with diethyl ether, filtered, and dissolved in 10% aqueous acetic acid or 25% aqueous acetonitrile. The products were then shell-frozen and lyophilized.

Peptide purification
Peptides were purified (34) via reversed phase HPLC on a 5 x 30 cm cartridge packed in the laboratory with reversed-phase 300 Å Vydac C18 silica (15–20 µm particle size). The crude, lyophilized peptides (1–3 g) were dissolved in a minimum amount (300 ml) of 0.25 N triethylammoniumphosphate (TEAP) pH 2.25 and acetonitrile and loaded onto the HPLC. The peptides eluted with a flow rate of 100 ml/min using a linear gradient of 1% B per 3 min increase from the baseline %B. (Eluent A = 0.25 N TEAP, pH 2.25; eluent B = 60% CH3CN, 40% A). Generally, purifications in TEAP pH 2.25 followed by TEAP, pH 6.5, were necessary to achieve the desired purity level. As a final step, the TEAP salt of the peptide was exchanged for the TFA salt using a gradient of 1% B/min where A = 0.1% TFA.

Peptide characterization
Peptide purity was determined from two systems, by analytical HPLC in TEAP pH 2.5 buffer and capillary zone electrophoresis (CZE). Analytical HPLC analysis employed a Vydac C18 column (0.46 x 25 cm, 5 µm particle size, 300 Å pore size). CZE analysis employed a field strength of 10–20 kV at 30 C with a buffer of 100 mM sodium phosphate, pH 2.5, on either a Beckman Coulter, Inc. eCAP or a Supelco P175 fused silica capillary (363 µm od x 75 µm id x 50 cm length). Purity was determined to be >95% for all peptides but sPACAP38 and tPACAP27, which had purities >80%. No single impurity was greater than 10% of the desired product. Liquid secondary ion mass spectra were measured with a JEOL JMS-HX110 double-focusing mass spectrometer fitted with a Cs+ gun. An accelerating voltage of 10 kV and Cs+ gun voltage between 25 and 30 kV were employed. The samples were added directly to a glycerol and 3-nitrobenzyl alcohol (1, 1) matrix. The mass of each analog was measured and the observed monoisotopic (M + H)+ values were within 100 ppm of the calculated (M + H)+ values.

Immunocytochemistry
To detect neurons and glia, cells were plated 4.5 x 105 cells/chamber in 8-chamber, tissue culture-treated Falcon CultureSlides (Becton Dickinson and Co., Franklin Lakes, NJ). Cells were cultured for 6 days, to allow for attachment and differentiation. Medium was gently aspirated and chambers allowed to air-dry 1.5 h in a sterile environment. Cells were fixed by addition of ice-cold 100% acetone for 1 min, air dried, and rehydrated in ice-cold PBS (Life Technologies, Inc.). Nonspecific binding was blocked by addition of 5% sheep serum in PBS for 40 min at room temperature. To detect neurons, undiluted rabbit antiserum raised against neuron-specific enolase (INCSTAR Corp., Stillwater, MN) was added. To detect glia at the same time as neurons, a 1:100 dilution of monoclonal mouse anti-glial fibrillary acidic protein conjugated to Cy3 (Sigma, Oakville, Ontario, Canada), diluted in PBS was added. Both primary antisera contained 5% sheep serum. A control lacking both primary antisera was included. Each primary antibody with its conjugate was also tested separately on the cells. The cells were incubated in a humidity chamber for 48 h at 4 C, then washed 3 times for 5 min in cold PBS. Incubation for 2 h at room temperature followed, in goat antiserum raised against rabbit IgG and conjugated to FITC (Sigma), diluted 1:60 in PBS. The cells were washed again, then mounted using a SlowFade Light Antifade kit (Molecular Probes, Inc., Eugene, OR). Slides were examined and photographed under a Leitz Aristoplan epifluorescent microscope.

To detect PACAP, cells were plated 1 x 106 cells/chamber in identical Supercells, cultured for 2 days, and processed as above with the following changes: cells were fixed in 4% paraformaldehyde and incubated in primary antiserum for 24 h. The primary antiserum was undiluted HB7, a rabbit antiserum raised in our laboratory against human PACAP38 conjugated to bovine thyroglobulin.

Western blot
Protein was extracted from E3.5 chick brain using 400 µl lysis buffer (4% wt/vol SDS, 5% vol/vol 2-mercaptoethanol, 5% wt/vol sucrose). The protein extract (6 µl) was separated on a 16% Tris-Tricine gel (Bio-Rad Laboratories, Inc. Hercules, CA) at 100 V until the dye front reached the bottom of the gel. The protein was transferred to a polyvinylidene dilfluoride membrane (NEN Life Science Products, Boston, MA) at 100 V for 30 min in transfer buffer (12.5 mM Tris-HCl, pH 8.2, 200 mM glycine, 10% methanol). Immunolocalization was performed using a Vectastain Elite ABC kit (Vector Laboratories, Inc. Burlingame, CA), according to the manufacturer’s instructions. The membrane was blocked with 10 ml of Tris buffered saline with 0.05% Tween-20 (TBST) containing three drops of goat serum, for 1 h at room temperature. Rabbit antisera against human PACAP27 and human PACAP38 (Peninsula Laboratories, Inc.), were diluted 1:2000 in 10 ml of TBST with one drop of goat serum and added to the membrane for overnight incubation at 4 C. The membrane was washed with TBST three times for 5 min, then incubated with one drop of goat antiserum raised against rabbit IgG, diluted in 10 ml of TBST with three drops of goat serum, for 45 min at room temperature. The membrane was washed again, incubated in ABC reagent for 30 min at room temperature, and washed again. The DAB substrate with NiCl was added and the color allowed to develop for 3–5 min. The reaction was stopped by rinsing the membrane two times for 5 min in distilled water, and the membrane was allowed to air dry.

mRNA isolation and complementary DNA (cDNA) synthesis
Freshly dissected E3.5 chick brain cells were harvested and flash frozen on dry ice. The cells were ground to a fine powder using a micropestle (Diamed, Missisauga, Ontario, Canada) in 1.5 ml tubes chilled with liquid nitrogen. Duplicate samples of mRNA were isolated using the Poly (A) Pure Kit (Ambion, Inc., Austin, TX), as outlined by the manufacturer. Single-stranded cDNA was synthesized using 2 mM of oligo (dT20) in 1x First Strand Buffer, 2 mM dNTPs, 10 mM DTT, 5 U Ribonuclease Inhibitor (Life Technologies, Inc.) and 200 U Superscript II (Life Technologies, Inc.) to a final volume of 50 µl. The mRNA was combined with the oligo (dT) primer and the mixture was heated to 70 C for 7 min, then placed on ice. The remaining reagents were added, the reaction was incubated at 42 C for 90 min, then Superscript II was denatured at 95 C for 10 min. The quality of the cDNA was verified by PCR amplification of tubulin using the primers T10 (5'-CAGGTGTCCACGGCTGTGGTG-3') and T11 (3'-AGGGCTCCATCGAAACGCAG-5').

Amplification and sequencing of cDNA
PCR amplification of the PACAP receptor was performed using primers 5'-GCGTTGTACACAGTTGGATA-3' and 5'-TTGAATTGGGACTGGGATCT-3' designed against transmembrane regions 1 and 7 of the chicken PAC1 receptor (35). Amplification of ligands was performed using primers 5'-CAAAGCCTACAGGAAACTCCTGGGCC-3' and 5'-CGCTATTTGTAGGATGAGCAACCGCC-3' designed against the 5' region of GRF and the 3' UTR of the chicken PACAP gene (36). A 2 µl volume of cDNA was added to a 50 µl volume containing 200 µM dNTPs, 2 mM MgCl2, 0.4 µM of each primer and 2.5 U of Taq DNA polymerase (Life Technologies, Inc.). The reaction was heated to 94 C for 2 min, then cycled 30 times at 94 C for 30 sec, 52 C for 45 sec, and 72 C for 1 min. A 10 µl aliquot of PCR product was separated on a 1.5% agarose gel. The PCR product was ligated into pGEM-T vector as specified by the manufacturer (Promega Corp., Madison, WI) and cloned. Two recombinant plasmids from each of the duplicate samples were sequenced using an ABI Prism 377 DNA Sequencer.

cAMP RIA
Cells were lysed 0.5, 1, 2, and 24 h after plating, and cAMP was assayed from the combined cells and medium. Cells were lysed by addition of ice-cold 100% ethanol to the medium, to a final concentration of 65% ethanol. The mixture was allowed to settle and the supernatant collected. The settled material was washed with 200 µl of ice-cold 100% ethanol, and the wash combined with the supernatant before centrifugation at 5000 rpm for 15 min at 4 C. The supernatant was collected and the ethanol was evaporated by vacuum centrifugation at 4 C. Samples were covered and stored at 4 C until assayed. RIA (125I) kits were supplied by PerSeptive Biosystems (Framingham, MA) for all assays except those which measured a response to tPACAP and tGRF-like peptide. When the PerSeptive kit was no longer available, a kit supplied by NEN Life Science Products was used to assay the tunicate peptides, and h/sPACAP27 was reassayed using this kit for comparison purposes. A minimum of three independent values, obtained in two separate experiments, were averaged to obtain each data point. The data were analyzed by ANOVA, followed by Dunnett’s test. Dunnett’s test was chosen because it is considered a stringent test for comparing treatment means with a control mean, and allows unequal sample sizes. Scheffé’s method was used to compare two treatment means (100 nM and 1000 nM hPACAP38) when it appeared that the lower concentration might be generating a greater response and thus altering a generally observed dose-dependence response curve.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of cells
At the time of plating, primary cultures from E3.5 chick brain contained proliferating cells, and within 12 h some cells developed processes and began to aggregate (Fig. 2Go). By several days in culture, most cells were aggregated and fasciculated axons connected the aggregates. Cells surrounding the aggregates were elongating and forming tracts or sheets of flattened cells (Fig. 2Go). To show that the cultures consisted primarily of neuroblasts, cells were cultured for 6 days and stained with antibodies that recognize neurons and glial cells. Although evidence was difficult to obtain because of the tendency of the cells to detach during the staining procedure, photographs of a typical culture reveal an abundance of nerve cells and no glial cells (Fig. 2Go). Cultures stained with single antibodies confirmed the results of the double labeling procedure. Antiserum against neuron-specific enolase stained cells in 6-day cultures, but did not stain cells before 4 days in culture (data not shown). This evidence shows that the E3.5 cells were undifferentiated, because neuron-specific enolase is found only in differentiated cells.



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  		Figure 2. Chick brain cells, cultured after 3.5 days of 21-day gestation period. Cultures after one day (top left) contained many smoothly rounded cells, which were proliferating, and cells with more varied morphology, which were differentiating and beginning to aggregate. After 3 days in culture (top right) most cells were aggregated, and fasciculated axons connected the aggregates. Cells surrounding the aggregates were elongating and forming tracts or sheets of flattened cells. After 6 days in culture, cells were stained for neurons (middle left) and glial cells (middle right). Photographs show the same frame. Neurons were visualized with antiserum against neuron-specific enolase conjugated to FITC. The middle left photograph shows a dense aggregate of neuronal cells with indistinct cell membranes in the upper left corner, and a smaller aggregate of flattened and elongated cells, only a few layers thick, in the lower right corner. Glial cells were visualized with antiserum against glial fibrillary acidic protein conjugated to Cy3. Lack of staining in the middle right photograph suggests that the cultures were virtually free of glial cells. To show that the cells were producing the PACAP peptide, cells were stained after 2 days in culture using antiserum against PACAP38 conjugated to FITC (bottom left). Bright fluorescence in the treated wells compared with minimal color in the control wells (bottom right), confirmed the presence of the peptide. Magnifications are: 700x (top left), 1000x (top right), 625x (middle), 1000x (bottom). Bars represent 8 µm.

 
Synthesis of PACAP and expression of PACAP and its receptor
Photographs of E3.5 chick brain cells cultured for 2 days and stained to reveal the presence of PACAP38 showed that most or all the cells contained the protein (Fig. 2Go). Western blot analysis of protein isolated from freshly dissected cells revealed a band of approximately 4.5 kDa. This corresponded to the PACAP38 standard, and confirmed the presence of PACAP38, but not PACAP27, in the cells (Fig. 3Go).



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  		Figure 3. Western blot analysis of protein isolated from E3.5 chick brain cells, using antisera against human PACAP38 and human PACAP27. Because initial staining of the standards for PACAP38 and PACAP27 was very heavy (lanes 4 and 5), standards were repeated on a separate gel to enhance band clarity (lanes 6 and 7). Chick brain extract, 6 µl (lane 1), SDS-PAGE low-range standards (lane 2), chick brain extract, 3 µl (lane 3), 100 ng human PACAP38 (lane 4), 100 ng human PACAP27 (lane 5), 4 pg human PACAP38 (lane 6), 4 pg human PACAP27 (lane 7). The molecular masses of the PACAP38 peptide (4.5 kDa) and the PACAP27 peptide (3.1 kDa) are indicated.

 
RT-PCR on mRNA isolated from E3.5 chick brain cells using exact match primers for PACAP receptor and its ligand yielded 708 bp and 276 bp bands, respectively (Fig. 4Go). Sequence analysis confirmed that the 708 bp band was the PAC1-R (short) transcript (Fig. 5Go). Our sequence has 1 nucleotide change at position 555 (Fig. 5Go) when compared with the previously isolated cDNA (35). This change does not cause an amino acid change. The transcript did not contain the TM-4 variation. The 276 bp band was confirmed to be the chicken GRF-PACAP transcript (Fig. 6Go). Our sequence has 3 nucleotide changes at positions 43, 46, and 124 (Fig. 6Go) when compared with the previously isolated GRF-PACAP gene (36). The nucleotide change at position 46 causes an amino acid change from Asp to Lys.



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  		Figure 4. RT-PCR amplification of mRNA for the PACAP receptor and GRF-PACAP ligand isolated from E3.5 chick brain cells. A, 123-bp ladder (lane 1), PAC1 receptor (lane 2), tubulin control (lane 3), and negative control (lane 4). B, 123-bp ladder (lane 1), GRF-PACAP ligand (lane 2), tubulin control (lane 3), and negative control (lane 4).

 


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  		Figure 5. Partial nucleotide sequence from cDNA isolated from E3.5 chick brain cells encoding the chicken PAC1 receptor (short) isoform. Transmembrane domains are underlined and primer sequences are in bold italics (first 20 and last 21 nucleotides). The arrow at nucleotide 555 indicates the site of a nucleotide change from a previously reported sequence (35 ).

 


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  		Figure 6. Partial nucleotide sequence from cDNA isolated from E3.5 chick brain cells encoding GRF and PACAP. The sequence corresponding to the GRF peptide is double underlined, and the sequence corresponding to the PACAP peptide is single underlined. Primer sequences are in bold italics (first and last 26 nucleotides), and arrows indicate sites of nucleotide changes from a previously reported sequence (36 ).

 
Activation of cAMP pathway
A general dose-dependent increase in cAMP production in neuroblast-enriched cultures was observed in response to physiological concentrations of h/sPACAP27, cPACAP27, hPACAP38, and cPACAP38 (Fig. 7Go). In many cases, a statistically significant response compared with control was evident for the lowest concentration administered, 1 nM. Administration of 10 nM enhanced production further, and 100 nM concentrations resulted in peak production levels. Statistically, values did not rise further with administration of 1000 nM hormone, indicating that a plateau had been reached. An apparent drop in production above 100 nM in response to hPACAP38 was not borne out statistically. Responses to h/sPACAP27 obtained with the NEN Life Science Products kit were lower than with the PerSeptive Biosystems kit, with values reaching 50–75% of those initially recorded (Fig. 8Go). However, the typical trend in cAMP production is evident in response to tPACAP27 when measured with this manufacturer’s kit, and the magnitude of the response is similar to that elicited by h/sPACAP27 using the same kit (Fig. 8Go). Statistical increases were not recorded in response to sPACAP38 for any concentration tested, at any point in time up to 24 h (Fig. 7Go).



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  		Figure 7. Response by E3.5 chick brain cells to nM concentrations of human/salmon and chicken PACAP27, and human, chicken, and salmon PACAP38. Each data point is the mean of at least three independent determinations. Arrow indicates cAMP production at the time the hormone was administered. Clear asterisks indicate a significant difference from control at P < 0.05; dark asterisks indicate significance at P < 0.01.

 


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  		Figure 8. Response by E3.5 chick brain cells to nM concentrations of human and tunicate PACAP27, using a different manufacturer’s RIA kit than was used to collect the data in Fig. 7Go. Each data point is the mean of at least three independent determinations. Arrow indicates cAMP production at the time the hormone was administered. Clear asterisks indicate a significant difference from control at P < 0.05; dark asterisks indicate significance at P < 0.01.

 
Peptide purity was determined to be >95% for all analogs but sPACAP38 and tPACAP27, which had purities >80%. Because N-terminally deleted fragments of PACAP have been shown to have antagonistic activity at PACAP-specific receptors (37), a question arises as to whether the synthetic peptide mixtures could have included truncated forms that affected our results. However, the two most potent N- terminally deleted forms of PACAP include one that is missing the first amino acid and another that is missing the first five amino acids (37). Of these, only the latter acts as an antagonist. In addition, PACAP (6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27) has only 1/200th and PACAP (6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38) has only 1/10th the affinity of the parent compound for the receptor (37). Given that no single impurity exceeded 10% of the desired product, and given the low potency of antagonists that may have been present, it is not plausible that antagonists affected the results.

Although a trend was not evident, a statistical increase in cAMP production over control was measured in response to the tGRF-like peptide. Increases were generated in response to a 1 nM concentration 0.5 h after plating, and a 10 nM concentration 1 h after plating (Fig. 9Go). The response to the tGRF-like peptide was measured using the RIA kit that produced lower values overall, but it is also evident that the magnitude of increase is not as great as the increases recorded in response to hPACAP and cPACAP (Figs. 7Go and 9Go). Although P values between 0.04 and 0.12 were obtained for responses to cGRF29, significance was not confirmed by Dunnett’s test. No statistically significant increases were recorded in response to hGRF29, carp GRF28, hGRF44, cGRF46, or sGRF45 (data not shown). P values were between 0.08 and 0.96.



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  		Figure 9. Response by E3.5 chick brain cells to nM concentrations of tunicate GRF27-like peptide. (Note different scale than in Figs. 7Go and 8Go.) Each data point is the mean of at least three independent determinations. Arrow indicates cAMP production at the time the hormone was administered. Clear asterisks indicate a significant difference from control at P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Hormones and growth factors during chick nervous system development
It is clear that E3.5 chick brain cells produce mRNA for PACAP and that this transcript is translated to yield the mature PACAP38 peptide. Although the PACAP27 peptide was not detected, it is possible that it is present at levels not detectable by Western blot. In the adult rat, PACAP27 accounted for less than 10% of total PACAP in the brain (38). E3.5 chick brain cells also produce mRNA for a PACAP-specific receptor, and physiological doses of PACAP strongly activated the cAMP pathway. This suggests that an autocrine/paracrine system may be present. GRF, which is encoded on the same gene as PACAP in chickens, was also shown to be present as mRNA at this stage. However, only one of seven forms of GRF tested produced a statistical increase in cAMP production. Only the tunicate GRF-like hormone initiated a response, and then only in response to a 1 nM concentration 0.5 h after plating, and a 10 nM concentration 1 h after plating. A critical question is whether the GRF receptor is expressed in E3.5 chick brain cells. To date, cDNA for the chicken GRF receptor has not been reported, although the receptor has been cloned in mammals and goldfish (1). It is also possible that GRF has an effect at this early stage, but that the effect is not mediated through the cAMP pathway.

We argue that the cell cultures in these experiments were neuroblast-enriched based on three lines of evidence. Firstly, the cells were cultured in medium designed to virtually eliminate glial cell growth (39), and this effect was confirmed by immunocytochemical results. Secondly, gliogenesis in chick brain does not begin until E8 in vivo. Thirdly, concentrations of antiserum against neuron-specific enolase that identified neurons in 6-day cultures did not recognize cells in 3-day cultures. Because neuron-specific enolase does not recognize undifferentiated nerve cells, this supports the conclusion that the labeled cells in these experiments, cultured only up to 24 h, were neuronal precursors or neuroblasts.

There are few reports on the involvement of PACAP in chick brain development. Between E3.5 and E9, cell numbers increased in the dorsal root ganglion and lumbar motor column when treated with PACAP in ovo (40). These increases were ascribed to a decrease in programmed cell death because massive proliferation has stopped in the spinal cord at this stage. In postnatal chicken hypothalamus and cerebral cortex, which are still developing, PACAP increased cAMP (41). Reports on GRF involvement are also limited. When administered at E1–3 in ovo or in culture, hGRF influenced the expression of neurotransmitters later in development (42, 43). In E10 spinal cord cultures, GRF increased survival, proliferation and differentiation of cells, and influenced the expression of neurotransmitters (29). Therefore, the present study is the first one to show an immediate activation of chick brain cells by PACAP.

Also, little is known about the role of other hormones and growth factors at this stage of chick brain development. There is evidence to suggest that fibroblast growth factor is required in utero for acquisition of neural cell fate (44), and plays a role in organization of the midbrain by E2 (45). Two soluble factors secreted by specialized cells of the developing neural tube, bone morphogenetic protein and sonic hedgehog, are active during this time, inducing differentiation of neural cell types in the brain (46, 47). There are reports of early involvement by nerve growth factor (NGF), brain- derived neurotrophic factor (BDNF), and insulin-like growth factor I (IGF-I). NGF mRNA transcripts have been located in the chick brain at E3.5 (48), and receptors are present by at least E5 (49). Increases in survival have been recorded in E3.5 cell populations in response to NGF (50). BDNF was reported to be active in chicken embryos at E4 (51). Receptors that bind these neurotrophins are present at this stage (52). A peak in the production of IGF-I peptide and receptors was reported between E3 and E6 in chick brain (53, 54).

Receptor activation in chick neuroblast-enriched cultures
Our results suggest that the physiological actions of PACAP in neuroblast-enriched cultures from E3.5 chick brain are mediated through one of the PACAP-specific receptors, PAC1-R (short isoform). The PAC1-R (short) and the PAC1-R hop1 isoforms were expressed in the brain of the adult chicken (35). However, we found only the PAC1-R (short) isoform in the developing brain of E3.5 chick. This could indicate that the PAC1-R (short) is the only form, or the predominant isoform of this receptor in neuronal precursors. The PAC1-R (short) is strongly coupled to the cAMP pathway (1), indicating that the observed increase in cAMP in these cultures in response to hormone addition is likely achieved by activation of this receptor. However, the possibility exists that these effects are due to activation of a VPAC receptor because we have not yet shown the presence of the receptor protein.

Possible downstream effects of increased cAMP production
The current study does not elucidate the nature of downstream effects resulting from increases in cAMP production. Although the protocol was not designed to rule out simple trophic effects, it can be expected that this alone would not account for the magnitude of response generated during the first 2 h of culture, a time when the cells should be healthy. It is possible that the increase in second messenger leads to enhancement of proliferation, or differentiation, or both. Both proliferating and differentiating cells were evident during the first 24 h, and increases in cAMP can enhance both these processes. Proof that these cells were differentiating is shown by the presence of neuron-specific enolase after 4 days in culture. Studies on chick embryogenesis show that by E3.5 the neural tube has closed, and cranial nerves, sensory organs, Rathke’s pouch and the infundibulum (which will become the pituitary), are forming (55). Generally, cells are still in a proliferative state, but some are differentiating in the hindbrain, olfactory epithelium and optic tectum (56).

Evolution and function of PACAP and GRF
This study suggests that the single amino acid differences between the human/salmon, chicken, and tunicate forms of PACAP27 (Table 1Go) do not cause an ascertainable difference in production of cAMP in neuroblast-enriched chick brain cell cultures. However, substitutions in the C-terminal region of PACAP38 appear to have a marked effect on the function of the peptide. There are three amino acid substitutions in the C-terminal region (amino acids 28–38) of sPACAP38, compared with hPACAP38 and cPACAP38 (Table 1Go). It is possible that the C-terminal changes lessen the effectiveness of the peptide in other species, probably by altering its binding capabilities to receptors. In support of this, it has been reported that hPACAP29–38 extension analogs of hVIP enhanced binding to PAC1, suggesting a stabilizing effect of the C-terminus of PACAP38 on optimal peptide conformation (57).


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  		Table 1. PACAP and GRF amino acid sequences

 
The only form of GRF that prompted a statistical increase in cAMP was the tunicate GRF-like peptide. Examining the structure of this peptide compared with the other GRF hormones tested provides a possible explanation. Within the first 27 amino acids, tGRF-like peptide is more similar in sequence to hPACAP27 than are hGRF, cGRF, sGRF, or carp GRF. The latter four peptides have nine amino acids in common with hPACAP27, but the tGRF-like peptide has 12 that match the human peptide (Table 1Go). As well, within the glucagon superfamily of hormones, four amino acids at the N terminus of the ligands have been identified as crucial for binding to the PAC1 receptor, and tGRF-like peptide is the only GRF tested here that includes all four of these amino acids. These crucial amino acids include histidine in position 1, serine in position 2, phenylalanine in position 6, and threonine in position 7 (57). Two of four of these amino acids are changed in each of hGRF, cGRF, sGRF, and carp GRF (Table 1Go). Because it is not known whether cells at this stage express the GRF receptor, and because the GRF and PACAP receptors are related, it is possible that the tGRF-like peptide is binding to the PACAP receptor.


   Acknowledgments
 
We thank Carol Warby and Dr. Tom Mommsen for advice on cAMP assays, Diana Wang for advice on cell culture and immunocytochemical techniques, and Ron Kaiser, Dean Kirby, Charleen Miller, and Anthony G. Craig for assistance with peptide synthesis, purification and characterization.


   Footnotes
 
1 This work was supported by Grants from the Natural Sciences and Engineering Research Council and the Canadian Medical Research Council (to N.M.S.) and Grant DK-26741 from the N.I.H. (to J.E.R.). Back

Received October 3, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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