0013-7227/91/1295-2787$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society

Vol. 129, No. 5 Printed in U.S.A.

Tissue Distribution of PACAP as Determined by RIA: Highly Abundant in the Rat Brain and Testes Akira Arimura*, Aniko Somogyvaxi-Vigh, Atsuro Miyata, Keiko Mizuno, David H. Coy and Chieko Kitada U.S.-Japan Biomedical Research Laboratories, Tulane University Hebert Center, Belle Chasse, LA 70037 (AA, ASV, AM, KM); Departments of Medicine (AA, ASV, AM, KM, DHC), Physiology (AA) and Anatomy (AA), Tulane University School of Medicine, New Orleans, LA 70112; Tsukuba Research Laboratories, Takeda Chemical Industries, Ltd., Tsukuba, Japan (CK) Abstract: A heterologous RIA method for pituitary adenylate cyclase activating polypeptide with 38 residues (PACAP38) and a homologous RIA method for a shorter form of PACAP with 27 residues (PACAP27) were established to determine PACAP content in central and peripheral tissues in rats. The highest concentration of radioimmunoassayable PACAP38 was found in the hypothalamus, but other brain regions also contained considerable amounts of PACAP38. PACAP38 concentration in the posterior pituitary was comparable with that in the extrahypothalamic brain, but its concentration in the anterior pituitary was very low. Unexpectedly, the testis contained a high abundance of PACAP38, and the total amount of PACAP in both testes exceeded its content in the whole brain. Reverse phase HPLC suggested that the major testicular PACAP38 immunoreactivity represents PACAP38. Among peripheral tissues, adrenal gland contained the second highest concentration of PACAP. Smaller amounts of PACAP were widely distributed in the digestive tract and other peripheral tissues. The concentration of PACAP in stomach, duodenum and jejunum appeared to be greater than in other portions of the gut. In all tissues, PACAP27 represented only a minor portion of total PACAP immunoreactivity. Pituitary adenylate cyclase activating polypeptide (PACAP) is a new member of the secretin/glucagon/vasoactive intestinal peptide (VIP) family, and was isolated from ovine hypothalamic tissues using adenylate cyclase stimulating activity in rat pituitary cell cultures as a response parameter for screening (1). Accumulation of cAMP in cultured rat pituitary cells can be used for a standard bioassay for PACAP and its analogs. Two forms with 38 (PACAP38) and 27 residues (PACAP27) (2) are derived from the 175 amino acids precursor (3). The N-terminal 28 residues of PACAP has 68 % homology with VIP, but its adenylate cyclase stimulating activity in rat pituitary cell cultures are 1,000 - 10,000 times greater than VIP (1). The presence of at least two types of binding site for PACAP, one specific for PACAP and another shared with VIP, was demonstrated in various tissues (4, 5). Although a variety of biological actions of PACAP in addition to activation of pituitary adenylate cyclase were demonstrated, its physiological roles remain unclear. For physiological studies, it is, therefore, necessary to establish a sensitive and specific method for determining PACAPs in the tissues and biological fluids. The present paper describes the RIA methods for PACAP38 and PACAP27 with highest tissue content found in brain and testis.

Radioimmunoassay (RIA): PACAP27 and PACAP24-38 were radiolabelled with Na l25 I using the lactoperoxidase method and purified by reverse phase HPLC with Vydac C18 column (Rainin Instruments, Woburn, MA) as described previously (4). The diluent used in RIA was 0.05 M phosphate buffer/ 0.15 M NaCl/ 0.025 M disodium EDTA 0.1 % Triton X-100 0.4 % Bmercaptoethanol/1 % BSA/0.02 % sodium azide, pH 7.5. The standards, samples, antiserum and tracer were diluted in the above diluent containing 0.1 % normal rabbit serum. PACAP27 or PACAP38 as the standard or the sample was dissolved in 100 |j.l diluent, placed in a 12 x 75 mm polystyrene culture tube, and 300 (il appropriately diluted antiserum was added. After incubation for 24 hr at 4 C, 100 \i\ of tracer was added and incubated at 4 C overnight. Then 100 \ll sheep anti-rabbit IgG diluted to 1 : 100 and 500 ji.1 of 10 % polyethylene glycol (mol wt 8,000) were added and mixed. After one hr incubation at room temperature, the tubes were centrifuged at 3000 x g at 4 C for 30 min, the supernatant was decanted and the pellet counted by an auto-gamma counter (Micromedic 4/600) with an on-line data processor. Extraction of tissues : Whole or a portion of various tissues were collected from CD strain rats of either sex, 300 400 g body weight (Charles River Breeding Labs, Wilmington, MA) immediately after decapitation under ether anesthesia. Each of the tissues was weighed, placed in a 16 x 100 mm test tube, 10 vol of chilled water were added and then kept in ice until collection of tissues, was completed. These tubes were then placed in heated oil at 110 C for 10 min, cooled in ice, and acetic acid was added to a final concentration of 0.5 M. The tissue was then homogenized by the Ultra-Turrax homogenizer (Janke & Kunkel, Germany), and the extract was diluted with the same volume of distilled water and stored at 4 C overnight. After centrifugation at 12000 x g at 4 C for 30 min, the supernantant was decanted on a C18 Sep-Pak cartridge (Waters Chromatography Division Milford, MA), washed with 10 ml water and then eluted with 3 ml 60 % CH3CN/O.I % TFA. The eluent was evaporated by a Speed-Vac Concentrator (Savant Instruments, Inc. Hicksville, N.Y.) and lyophilized. Immediately before the assay, the residue was dissolved in the diluent.

Materials and Methods Peptides and reagents: PACAP38, PACAP27, Cys23PACAP24-38OH, PACAP24-38 and other PACAP related peptides were synthesized by the solid phase method. PACAP27 was conjugated with bovine thyroglobulin (BTG) using l-ethyl-3-(3-dimethylarninopropyl)-carbodiirnide-HCl (ECDI). Cys23PACAP24-38OH was also conjugated with BTG using m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), so that the peptide is coupled with BTG through the N-terminal cysteine. The conjugate was emulsified with complete Freund's adjuvant and injected into mixed-bred female rabbits sc in the nuchal area and ip at one month intervals until a high-titer antibody was generated. MBS was purchased from Pierce, Rockford, IL; Freund's adjuvant from Difco Labs, Detroit, Michigan; sheep anti-rabbit IgG from The Arnel Products Co., New York, NY; sodium phosphate and EDTA from Mallinckrodt, Paris, KY; Na 125 I from Amersham, Arlington Heights, IL; sodium azide from Kodak, Rochester, NY; and all other reagents from Sigma, St. Louis, MO.

HPLC : Testicular extracts were purified on reverse phase HPLC using a gradient of CH3CN/O.I % TFA as described in the legend for Fig. 3, and eluents were monitored for UV absorbance at 210 nm and assayed for PACAP38 or PACAP27 by RIA.

ELJSA : The antibody titer in the rabbit serum was monitored by ELISA as described previously (6). One of the rabbits immunized with PACAP27/BTG conjugate showed high titer antibody against PACAP27 and, to a lesser extent, against PACAP38 at the third bleeding. The antiserum was named 88123-3. Another rabbit immunized with Cys23PACAP24-38OH showed a high titer for PACAP38 at the third bleeding, and the antiserum named 88111-3. These two antisera were used in RIA for PACAP27 and PACAP38, respectively.

Data analysis : Tissue content of PACAP immunoreactivity was expressed by ng per g wet tissue. The mean of five determinations for each tissue was subjected to analysis, using a one-way analysis of variance followed by Duncan's multiple range test when appropriate. P < 0.05 was considered significant.

Received in Iowa City on June 12, 1991.

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Endo • 1991 Vol 129 • No 5

RAPID COMMUNICATIONS

2788 RIA for PACAP27 with antiserum 8 8 1 2 3 - 3

RIA for PACAP38 with antiserum 8 8 1 1 1 - 3

10000

Fig. 1. Standard curve of PACAP27 RIA. O : PACAP27. • : peptides corresponding to PACAP38 and PACAP fragments 113, 1-20, 1-23, 1-24, 1-38, 2-23, 2-27, 3-27, 4-27, 31-38, and hypothalamic and pituitary peptides, including VIP, PHI, neuropeptide Y, CCK, rat CRF, rat GRF, human GRF, TRH, somatostatin, LH-RH, substance P, arginine vasopressin, oxytocin, secretin, glucagon, ACTH1-24, B-endorphin, Metenkephalin, Leu-cnkephalin.

Results The PACAP27 antiserum 88123-3 diluted to 1:1,000,000 bound to about 31% of total 12 5l-PACAP27 (specific activity : 482-502 |iCi/(ig in five iodinations) with a low non-specific binding (5.8 %). The binding was displaced in a dose-dependent manner by unlabeled PACAP27 in a range between 4.2 and 1000 pg (Fig. 1). The minimum detectable level was 4.2 pg PACAP27 per tube. Coefficients of intra- and interassay variation were 4.5 % and 6.2 %, respectively. The binding was not altered by addition of PACAP38, PACAP fragment peptides, and other hypothalamic and pituitary peptides as described in the legend of Fig. 1 in a dose up to 100 ng per tube. Likewise, antiserum 88111-3 with 125I-PACAP24-38 as the tracer (specific activity : 500 - 545 (iCi/ng) showed a linear displacement curve for unlabeled PACAP38 in a range between 4 and 800 pg (Fig. 2). The minimum detectable amount was 4 pg PACAP38/tube. This RIA did not recognize PACAP27. Coefficients of intra- and inter-assay variation were 4.2 % and 6.8 %, respectively. The binding was not altered by PACAP27 and other hypothalamic and pituitary peptides in a dose up to 100 ng per tube. However the binding was displaced by two PACAP fragment peptides, PACAP16-38 and PACAP24-38. The extraction method eliminated substances which interfered with the RIA for both PACAPs and yielded a satisfactory recovery (91 ± 4.2 % of added PACAP38). Table 1 shows tissue distribution of radioimmunoassayable PACAP38 and PACAP27. As expected, the hypothalamus contained the highest concentration of either PACAP38 and PACAP27, but the amount of PACAP38 was nearly 15 times greater than PACAP27. Other brain areas also contained significant amounts of the two PACAPs. The posterior pituitary PACAP38 concentration was comparable to extrahypothalamic areas of the brain, but the anterior pituitary contained only a small amount of the peptide. Unexpectedly, the testis contained the highest concentration of PACAP38 in the peripheral tissues, comparable to the concentration in the extrahypothalamic brain. PACAP27 concentration in the testis was about 1/600 of PACAP38. The serial dilution of the testicular extracts yielded a displacement curve parallel to that for synthetic PACAP38 (Fig. 2) and PACAP27 in respective RIA (data not shown). Reverse phase HPLC of the testicular extracts showed that the major PACAP38 immunoreactivity was co-eluted with synthetic PACAP38 (Fig. 3). The total content of PACAP38 in both testes were about 160 ng, corresponding to about two times greater than the total amount of PACAP38 in the whole brain. The adrenal gland also contained a significant amount of PACAP38, greater than in other peripheral tissues. The gastrointestinal tract also contained a moderate amount of PACAP38, but the concentration of the peptide in the stomach, duodenum and jejunum appeared to be slightly higher than other areas of the digestive tract. In all tissues, PACAP27 represented

Fig. 2. Standard curve of PACAP38 RIA. • : PACAP38, A : PACAP16-38, X :PACAP24-38, D : rat testicular extracts, A : PACAP27 and peptides corresponding to PACAP fragments 2-27, 3-27, 4-27, 14-38, and hypothalamic and pituitary peptides shown in the legend for Fig. 1. TABLE 1. Tissue concentration of immunoreactive PACAP38 and PACAP27 Tissue/Brain region hypothalamus cortex hippocampus anterior pituitary posterior pituitary lung atrium

liver spleen

pancreas stomach duodenum jejunum ileum colon

kidney adrenal gland testis epididymis ovary

PACAP38 540.00 24.20 36.54 3.32 40.23 1.72 0.81 0.35 0.74 0.62 6.62 8.76 6.08 3.84 2.64 0.86 14.42 54.56 1.72 2.00

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

19.90 3.89 7.84 0.72 4.16 0.74 0.32 0.10 0.16 0.15 0.30 1.28 1.31 0.31 0.10 0.20 0.69 3.95 0.15 0.22

PACAP27 34.90 2.70 1.08 0.99 2.81 0.32 0.32 0.10 0.22 0.07 0.66 0.82 0.30 0.28 0.78 0.22 0.22 0.09 0.17 0.50

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.91 0.12 0.42 0.11 0.33 0.07 0.12 0.04 0.03 0.03 0.04 0.05 0.12 0.04 0.23 0.06 0.04 0.02 0.01 0.05

Experiments arereportedin ng/g wet tissue derivedfrom5 replicates (mean ± SE).

only a small portion of the total PACAP immunoreactivity, but the ratio PACAP27 to PACAP38 varies between tissues, suggesting different post-translational processing between tissues. Discussion A heterologous RIA for PACAP38 and a homologous RIA method for PACAP27 were established and found to be specific and sufficiently sensitive for determining tissue contents of these peptides. The homologous RIA method for PACAP38 was problematical because 125I-PACAP38 was found practically unusable due to an extremely high non-specific binding. When PACAP38 was first isolated at our laboratories, the C-terminus appeared to be free. Therefore, we first synthesized PACAP38 with a free carboxyl terminus. During the synthesis, a half of the resin was separated, and Cys 23 PACAP24-38OH was synthesized to conjugate with BTG through Cys residue using MBS, so that the antibody generated against such conjugate would recognize the C-terminal portion of PACAP38 (7) that is absent in PACAP27. As expected, the antiserum 88111-3 bound to the peptides corresponding to the C-terminal portion of PACAP38 and did not recognize PACAP27 in the RIA method. The antiserum effectively bound to 125I-PACAP24-38 with an amidated C-terminus, and its binding was displaced by unlabeled PACAP38 in a dose-dependent manner, enabling us to establish a heterologous RIA method for PACAP38. It was interesting that

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RAPID COMMUNICATIONS PACAP27 antiserum 88123-3 was extremely specific, not recognizing even PACAP 1-24 and PACAP2-27 (Fig. 1). It is possible that the recognition site of the antibody was formed against the N- and C-termini which were closely opposed in the conformation of the conjugated PACAP27. Therefore, deletion of either N- or C-terminal amino acids may result in a loss of bindability to the antibody. A similar antibody which requires both N- and C-terminal amino acids for binding was generated against LHRH previously (8). Although the highest concentration of PACAP38 was found in the hypothalamus, other brain areas also contained considerable amounts of the peptide, in agreement with our immunohistochemical findings in the sheep (9), human, primate (10) and rat brain (11). These findings suggest that PACAP, in addition to its role as a hypophysiotropic hormone, may play as a role as a neurotransmitter/neuromodulator. A high concentration of PACAP38 in the posterior pituitary determined by RIA is also in agreement with our immunohistochemical finding in sheep (9) and rats (11). A dense network of PACAP fibers in the rat posterior pituitary appeared to be similar to that of oxytocin (11). Its physiological role of the posterior pituitary PACAP, however, remains to be investigated. The abundance of PACAP38 in the testis was unexpected. Its concentration was comparable to or even greater than that in the extrahypothalamic areas of brain, and the total content in the two testes was approximately two times greater than total brain PACAP. To our knowledge, the content of PACAP far exceeded the amount of any other neuropeptides present in the rat testis. The major testicular PACAP38 immunoreactivity was coeluted with synthetic PACAP38 in a reverse phase HPLC. Since the retention time of synthetic PACAP16-38 was very close to that of PACAP38, the presence of the PACAP 16-38 in the testicular extracts cannot be ruled out. However, the regression line for PACAP 16-38 in RIA was shallower than that of the testicular extracts which were parallel to that of PACAP38 (Fig. 2). We recently isolated rat testicular PACAP immunoreactivity in a pure form and the amino acid sequential analysis of the purified material indicated the presence of the same sequence as PACAP38 (in preparation). Therefore, the testicular immunoreactivity may mainly represent PACAP38. However, the possibility of the presence of other forms of PACAP in the testis cannot be excluded. Based on our preliminary finding of abundant radioimmunoassayable PACAP in the rat testis, a cDNA of the PACAP precursor protein was successfully cloned from a human testicular cDNA library (3). Although the radioreceptor assay of the rat testicular membrane preparations failed to reveal a significant amount of specific high affinity binding site for PACAP, our recent study of autoradiography using 125I-PACAP27 showed specific binding sites for PACAP on germ cells (5). These findings suggest that PACAP is expressed in the testicular cells and may play an important role in testicular function, in particular in maturation and/or function of germ cells. It is intriguing that the ovary did not contain a significant amount of PACAP. Although the concentration of PACAP in other peripheral tissues is not as high as in the brain and testes, these findings do not preclude a role in these peripheral tissues, including the gut, lung, and adrenal glands. These tissues contain high affinity binding sites for PACAP (4, 5). Like VIP, PACAP may play multifunctional physiological roles in different tissues. Acknowledgement We thank Dr. Brenda Shivers for her critical reading of the manuscript and editorial help, Mrs. Linda Bustoz for preparing the manuscript, and Dr. Ding Chang, Peninsula Laboratories for PACAP16-38. This study was supported in part by NIH grant DK09094 and a grant from Takeda Chemical Industries, Ltd.

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Fig. 3. Reverse phase HPLC of rat testicular extracts. Column : TSK ODS-120T, 5 u.m, 4.6 x 250 mm. Solvent system: Linear gradient elution from (A) to (B) for 40 min; (A) H2O : CH3CN : 10% TFA = 90 : 10 : 1 (v/v) (B) H2O : CH3CN : 10%TFA = 40 : 60 : 1 (v/v). The black bar shows PACAP38 immunoreactivity. The major immunoreactivity was coeluted with synthetic PACAP38 at 30 - 30.5 min. Solid line shows UV absorbance at 210 nm and dotted line indicates concentration of CH3CN. A: PACAP24-38, B: PACAP16-38.

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References 1. Miyata A, Arimura A, Dahl RR, Minamino N, Uehara A, Jiang L, Culler MD, Coy DH 1989 Isolation of a novel 38 residue-hypothalamic polypeptide which stimulates adenylate cyclase in pituitary cells. Biochem Biophys Res Commun 164:567-574

"A 1 " 1

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Miyata A, Jiang L, Dahl RD, Kitada C, Kubo K, Fujino M, Minamino N, Arimura A 1990 Isolation of a neuropeptide corresponding to the N-terminal 27 residues of the pituitary adenylate cyclase activating polypeptide with 38 residues (PACAP38). Biochem Biophys Res Commun 170:643648 Kimura C, Ohkubo S, Ogi K, Hosoya M, Itoh Y, Onda H, Miyata A, Jiang L, Dahl RR, Stibbs HH, Arimura A, Fujino M 1990 A novel peptide which stimulates adenylate cyclase: molecular cloning and characterization of the ovine and human cDNAs. Biochem Biophys Res Commun 166: 81-89 Gottschall PE, Tatsuno I, Miyata A,Arimura A 1990 Characterization and distribution of binding sites for the hypothalamic peptide, pituitary adenylate cyclase activating polypeptide. Endocrinology 127:272-277 Shivers BD, Gores TJ, Gottschall PE, Arimura A 1991 Two high-affinity binding sites for pituitary adenylate cyclase activating polypeptide have different tissue distributions. Endocrinology 128:3055-3065 Engvall E, Perlmann P 1971 Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunohistochemistry 8:871-874 Arimura A, Sato H, Coy DH, Worobec RB, Schally AV, Yanaihara N, Hashimoto T, Yanaihara C, Sukura N 1975 The antigenic determinant of the LH-releasing hormone for three different antiserums. Acta Endocrin 79:222-231 Millar, RP, Tobler C, King JA, Arimura, A 1984 Regionspecific antisera in molecular biology of neuropeptides: Application in quantitation, structural characterization, and metabolism of luteinizing hormone-releasing hormone. In: Soreg H (ed) Molecular Biology Approach to the Neurosciences. John Wiley & Sons, New York, 221-230 Koves K, Arimura A, Somogyvaxi-Vigh A, Vigh S, Miller J 1990 Immunohistochemical demonstration of a novel hypothalamic peptide, pituitary adenylate cyclase activating polypeptide, in the ovine hypothalamus. Endocrinology 127:264-271 Vigh S, Arimura A, Koves K, Somogyvari-Vigh A, Sitton J, FerminCD 1991 Immunohistochemical localization of the neuropeptide, pituitary adenylate cyclase activating polypeptide (PACAP), in human and primate hypothalamus. Peptides 12: 313-318 Koves K., Arimura A, Gores TG, Somogyvari-Vigh A 1991 Comparative distribution of immunoreactive pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) in rat forebrain. Neuroendocrinology 54:159-169

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Tissue distribution of PACAP as determined by RIA: highly abundant in the rat brain and testes.

A heterologous RIA method for pituitary adenylate cyclase activating polypeptide with 38 residues (PACAP38) and a homologous RIA method for a shorter ...
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