Pituitary adenylate cyclase activating polypeptide (PACAP), a member of the secretin/glucagon/vasoactive intestinal polypeptide family of peptides, consists of a 38-residue (PACAP 38) and a truncated 27-residue (PACAP 27) form that play several roles in mammals. Recently, we isolated a PACAP-like peptide with a sequence highly homologous with that of tetrapod PACAP from the brain of a teleost, the blue spotted-stargazer (Gnathagnus elongatus). In this study, a PACAP-like peptide obtained from the brain of the stargazer, Uranoscopus japonicus, was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting analysis using an anti-PACAP 27 serum. The stargazer PACAP-like peptide and synthetic human PACAPs were reacted with this antiserum. The distribution of PACAP-like immunoreactivity in the stargazer brain was also studied immunohistochemically using the peroxidase-antiperoxidase method. PACAP-like immunoreactive (LI) cells were found in the nucleus preopticus, pars parvicellularis of the hypothalamus. PACAP-LI nerve fibers and terminals were observed in the anterior neurohypophysis and in the rostral pars distalis, and many PACAP-LI nerve fibers were seen in the medulla oblongata. These results suggest that PACAP-like peptide may be involved in pituitary regulation and/or other neural functions in the stargazer brain.
INTRODUCTION
Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide that was first isolated from the ovine hypothalamus (Miyata et al., 1989) and two molecular forms of PACAP have been identified: a 38-residue peptide and a shorter 27-residue form (Miyata et al., 1990). Structurally, PACAP is a member of the secretin/glucagon/vasoactive intestinal polypeptide family of peptides and ovine, rat and human PACAPs are identical. In addition to stimulating adenylate cyclase activity in the pituitary, PACAP has been reported to have various actions in mammals (see Arimura, 1992; Arimura and Shioda, 1995; Arimura et al., 1994). In non-mammalian species, PACAP comprising 38 amino acid residues with only one substitution compared with mammalian PACAP 38 that stimulated adenylate cyclase activity in the pituitary gland was isolated from the brain of the frog, Rana ridibunda (Chartrel et al., 1991, 1995). Nerve perikarya and fibers showing PACAP-like immunoreactivity were found to be distributed widely in the hypothalamo-pituitary complex as well as the central nervous system of R. ridibunda (Yon et al., 1992, 1993).
Recently, we isolated a PACAP-like peptide with a highly homologous sequence with those of mammalian and frog PACAPs from the brain of a teleost, the blue spotted-stargazer (Gnathagnus elongatus) (Matsuda et al., 1996). In this study, a PACAP-like peptide obtained from the brain of another teleost, the stargazer (Uranoscopus japonicus) was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting analysis using an anti-PACAP 27 serum and the distribution of PACAP-like immunoreactivity in the stargazer brain was investigated immunohistochemically.
MATERIALS AND METHODS
Animals
Adult stargazers, Uranoscopus japonicus (Order Perciformes), were obtained from the fishermen in Yokata fishery harbor of Toyama City, Japan. All animal experiments were conducted in accordance with Toyama University's guidelines for the care and use of animals.
Antiserum
The primary anti-PACAP 27 serum (No. 92112-4) was raised in a rabbit, and the details of its production and characterization are described elsewhere (Köves et al., 1990).
Electrophoresis and Western blotting analysis
An extract was obtained from 50 stargazer brains with 0.5 M acetic acid. A purified stargazer PACAP-like peptide was prepared from this extract by chromatography on a Sep-Pak C18 cartridge (Waters Associates, Milford, MA, USA) and successive high-performance liquid chromatography (Model 510, Waters Associates, Milford, MA, USA) on gel-filtration (Superose-12 HR 10/30, Pharmacia Biotech, Uppsala, Sweden), cation-exchange (CM-TOYOPEARL 650S, TOSOH, Tokyo, Japan) and two reverse-phase (Puresil C18, Waters Associates, Milford, MA, USA; Inertsil Phenyl, Gas-Liquid Science, Tokyo, Japan) columns. The details of the purification procedure are described elsewhere (Matsuda et al., 1997).
Synthetic peptides: human PACAPs 27 and 38, porcine vasoactive intestinal polypeptide, porcine secretin and human glucagon were purchased from Peptide Institute Inc., Osaka, Japan. Aliquots of the extract (10 μg protein), purified peptide (60 ng) and synthetic peptides (1.5-2.5 μg) were subjected to SDS-PAGE with 15% polyacrylamide gels containing 8 M urea and 0.1% SDS at pH 8.9 on a pair of gels at 50 V, one of which was stained with 0,25% coomassie brilliant blue R-250 (MERCK, Darmstadt, Germany) and the other was used for blotting analysis. The proteins and peptides were transferred electrophoretically to a nitrocellulose membrane (Hybond-C Super, Amersham Int. plc, Buckinghamshire, UK). After the membrane was incubated with 3% bovine serum albumin (BSA; Sigma Chemical Co., St. Louis, MO, USA) in 0.01 M PBS for 1 hr, it was immunostained by the peroxidase-antiperoxidase (PAP) method using an anti-PACAP 27 serum (diluted 1:2,000) for 24 hr. Then, the membrane was treated with swine anti-rabbit immunoglobulin (diluted 1:100, DAKO A/S, Denmark) for 1 hr, incubated with the rabbit peroxidase-antiperoxidase (PAP) complex (diluted 1:100, DAKO A/S, Denmark) for 1.5 hr and treated with 20 mg 3, 3′-diaminobenzidine-4 HCI (Dojin, Tokyo, Japan) and 0.005% H2O2 in 100 ml 0.1 M Tris-HCI buffer (pH 7.6). The specificity of the immunostaining was checked by incubating a blotted membrane with normal rabbit serum (diluted 1:50, DAKO A/S, Denmark) instead of primary antiserum and with 1 ml primary antiserum (diluted 1:2,000) preincubated with excess (60 μg, 20 μM) synthetic human PACAP 27. Marker proteins (Rainbow™ colored protein molecular weight markers, Amersham Int. plc, Buckinghamshire, UK) were used to estimate the molecular weight of each band.
Immunohistochemical staining
Six fishes of both sexes were anesthetized with MS-222 (Sankyo Co., Tokyo, Japan), blood was flushed from the blood vessels by infusing 80 ml physiological saline for teleosts containing heparin through the aorta and then the animals were perfused with 80 ml 4% paraformaldehyde and 0.3% picric acid in 0.1 M PBS for 1 hr. The whole brains were removed immediately after perfusion, postfixed with the latter solution at 4°C for 24 hr, dehydrated, embedded in paraffin and serial frontal and sagittal sections (8 μm thick) were cut. Several sections were selected for normal staining with 0.1% cresyl violet acetate (EM Science, Gibbstown, NJ, USA). Neighboring sections were treated with the normal swine serum (diluted 1:50, DAKO A/S, Denmark), then stained with the anti-PACAP 27 serum (diluted 1:4,000) at room temperature for 24 hr, treated with swine anti-rabbit immunoglobulin (diluted 1:100, DAKO A/S, Denmark) for 1 hr, incubated with the rabbit PAP complex (diluted 1:100, DAKO A/S, Denmark) for 1.5 hr, and finally treated with 20 mg 3, 3′-diaminobenzidine-4 HCI (Dojin, Tokyo, Japan) and 0.005% H2O2 in 100 ml 0.05 M Tris-HCI buffer (pH 7.6). After immunostaining, all the sections were counterstained with Mayer's hematoxylin. The specificity of the immunostaining was checked by incubating sections with normal rabbit serum (diluted 1:50, DAKO A/S, Denmark) instead of the primary antiserum and with 1 ml primary antiserum (diluted 1:4,000) preincubated with excess (30 μg, 10 μM) synthetic human PACAP 27. The nomenclature of stargazer brain nuclei was based on the papers of Tuge et al. (1968) on the blue spotted-stargazer and Anglade et al. (1993) on the goldfish.
RESULTS
Figure 1 shows the SDS-PAGE (A) and Western blotting (B) patterns of each material. The acid extract of the whole brains, purified PACAP-like peptide and synthetic human PACAPs were reacted with an anti-PACAP 27 serum. The lanes containing the acid extract and purified peptide each showed a single band with a mobility similar to that of synthetic human PACAP 38 that was stained by the antiserum, whereas the other peptides demonstrated no immunoreactivity (Fig. 1B). The normal rabbit serum and the antiserum preadsorbed with PACAP 27 stained no bands (data not shown), Figure 2 shows the immunohistochemical results for the stargazer brain. A population of PACAP-like immunoreactive (LI) neuronal cell bodies was found in the nucleus preopticus, pars parvicellularis (Fig. 2A), PACAP-LI nerve fibers and terminals were located on the anterior neurohypophysis (Fig. 2B), and scattered PACAP-LI nerve terminals were seen in the rostral pars distalis (Fig. 2C). The dorsolateral zone of the medulla oblongata also exhibited a dense plexus of intensely immunoreactive fibers (Fig. 2D). No immunoreactivity was detected when the sections were treated with normal rabbit serum and antiserum preadsorbed with synthetic PACAP 27 (data not shown). Diagrams of sections showing the distributions of PACAP-LI cells and fibers throughout the brain of the stargazer are shown in Fig. 3.
Fig. 1
Detection of PACAP-like immunoreactivities (PACAP-LI) in each material by Western blotting analysis. (A) SDS-PAGE patterns stained with coomassie brilliant blue. (B) Western blotting patterns stained with an anti-PACAP 27 serum. M, molecular weight markers. Lanes 1, Acid extract of stargazer brain; 2, purified stargazer PACAP-like peptide; 3, synthetic PACAP 27; 4, synthetic PACAP 38; 5, synthetic vasoactive intestinal polypeptide; 6, synthetic secretin; 7, synthetic glucagon.
Fig. 2
(A) PACAP-LI neuronal cell bodies in the nucleus preopticus, pars parvicellularis. (B) PACAP-LI nerve fibers and terminals on the anterior neurohypophysis. (C) PACAP-LI nerve terminals in the rostral pars distalis. (D) Dense bundle of PACAP-LI nerve fibers on the dorsolateral zone of the medulla oblongata. The sections were counterstained with Mayer's hematoxylin. PACAP-LI neurons and nerve fibers are shown by arrowheads. Scale bar: 20 μm.
Fig. 3
Diagrams showing the distribution of PACAP-LI neuronal cell bodies (asterisks) and nerve fibers and terminals (dots) throughout the brain. Abbreviations: CC, corpus cerebelli; Dd, dorsal zone of area dorsalis telencephali; Dl, lateral zone of area dorsalis telencephali; Dm, medial zone of area of dorsalis telencephali; FL, facial lobe; MLF, medial longitudinal fascicle; MO, medulla oblongata; NPOpc, nucleus preopticus, pars parvicellularis; ON, optic nerve; OTe, optic tectum; PT, pituitary gland; RP, recessus posterior; SV, saccus vasculosus; VC, valvula cerebelli; VL, vagal lobe. (A) Schematic parasagittal section. (B) and (C) Schematic frontal sections at levels a and b shown in (A).
DISCUSSION
Immunohistochemical examinations have revealed PACAP-like immunoreactivity in some peripheral organs, such as the chromaffin tissues (Reid et al., 1995) and gastrointestinal tracts (Olsson and Holmgren, 1994) of fishes, but the present study is the first to show the distribution and localization of PACAP-like immunoreactivity in the brain of a teleost.
We have described the purification and characterization of a PACAP-like peptide with an amino acid sequence that shows high homology with those of mammalian and frog PACAPs, but contains several amino acid residue substitutions, from the blue spotted-stargazer brain on the basis of its immunoreactivity with an anti-PACAP 27 serum (Matsuda et al., 1996). In this study, the results of SDS-PAGE and Western blotting analysis of the stargazer brain indicated that this anti-PACAP 27 serum recognizes the substance with a molecular weight of approximately 5,000. This substance is a PACAP-like peptide that is distinct from secretin-, glucagon-and VIP-like peptides. Recently, we actually characterized and sequenced this stargazer PACAP-like peptide, which has a molecular mass of 4623, comprises 38 amino acid residues and its amino acid sequence shows 89 and 87% homology with those of mammalian and frog PACAPs, respectively (Matsuda et al., 1997).
We showed that PACAP-LI neuronal cell bodies and fibers were located in the diencephalon, hypophysis and medulla oblongata. Neuronal cell bodies with PACAP-like immunoreactivity were observed in the nucleus preopticus, pars parvicellularis. PACAP-LI nerve fibers were observed in the anterior neurohypophysis. In teleosts, the neurohypophysis penetrates deeply into adenohypophysis and the neuronal cell bodies in the preoptic area which contain neuropeptides, such as galanin (Batten et al., 1990; Holmqvist and Ekström, 1991), corticotropin releasing factor (Olivereau and Olivereau, 1988) and growth hormone releasing hormone (Olivereau et al., 1990) may project their fibers to the neurohypophysis. Indeed, using a retrograde transport technique, Anglade et al. (1993) demonstrated that hypophysiotrophic areas are essentially restricted to the preoptic area of the goldfish brain. The distribution of PACAP-LI neurons is similar to those of neurons as mentioned above. Therefore, the preoptic area appears to be the major source of the PACAP-LI nerve fibers in the anterior neurohypophysis of the stargazer. We also observed scattered PACAP-LI nerve terminals in the rostral pars distalis suggesting that some of PACAP-LI fibers through the anterior neurohypophysis terminate in it. The dorsolateral zone of the medulla oblongata contained a dense bundle of PACAP-LI fibers. Any PACAP-LI cell bodies were not observed in the midbrain and the hindbrain.
PACAP-containing neurons in the central nervous systems of tetrapods have been well demonstrated in detail. In the mammalian hypothalami, PACAP-LI cells were located in the supraoptic and paraventricular nuclei and a dense network of PACAP-LI fibers was distributed throughout the hypothalami (Kivipelto et al., 1992; Köves et al., 1990, 1991; Vigh et al., 1991). PACAP has been considered to be a pleiotropic neuropeptide with several functions, such as regulation of anterior and postenor pituitary hormone and neurotransmitter release, and to act as a neurotrophic factor (see Arimura, 1992; Arimura and Shioda, 1995; Arimura et al., 1994; Murase et al., 1993; Shioda et al., 1996; Uchida et al., 1996). PACAP-Ll cells and fibers were also found to be distributed widely throughout the frog brain and PACAP induced concentration-dependent stimulation of cAMP production in frog isolated pituitary fragments (Yon et al., 1992, 1993) and stimulated calcium mobilization in frog dispersed distal pituitary cells (Gracia-Navarro et al., 1992). These findings indicated that frog PACAP may also act as a hypophysiotropic hormone.
In our preliminary experiments, synthetic human PACAP 27, which has a one-amino acid residue substitution relative to the comparative portion of stargazer PACAP-like peptide, at higher concentration, stimulated cAMP production in the stargazer pituitary in vitro (Matsuda et al., unpublished data). Our results suggest that human PACAP 27 activates adenylate cyclase in the stargazer pituitary, but the homologous peptide may be essential for full expression of this function. Although further studies are necessary to elucidate the physiological functions of stargazer PACAP-like peptide, it seems likely that it may be involved in the regulation of the pituitary hormone secretion and synthesis and in other functions as a neurotransmitter, neuromodulator or neurotrophic factor in the brain of stargazer.
Acknowledgments
We express our appreciation to the members of our laboratory of Toyama University for their help collecting the fishes. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan to K.M. and by a research grant from the Tamura Foundation for Promotion of Science and Technology to M.U.
