0013-7227/90/1271-0272$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society

Vol. 127, No. 1 Printed in U.S.A.

Characterization and Distribution of Binding Sites for the Hypothalamic Peptide, Pituitary Adenylate CyclaseActivating Polypeptide* PAUL E. GOTTSCHALL, ICHIRO TATSUNO, ATSURO MIYATAf AND AKIRA ARIMURA U.S.-Japan Biomedical Research Laboratories, Tulane University, Hebert Center, Belle Chasse, Louisiana 70037; the Departments of Medicine (P.E.G., I.T., A.M, A.A.), Anatomy (A.A.), and Physiology (A.A.), Tulane University School of Medicine, New Orleans, Louisiana 70112

sites. A variable distribution of binding sites was observed between PACAP27 and VIP when binding to different tissue membranes was measured with 125I-labeled peptides. Very high specific binding of both PACAP27 and VIP was observed in lung membranes. An almost identical relative magnitude of binding was observed between PACAP27 and VIP in lung, liver, duodenum, ovary, and thymus. However, whereas PACAP27 binding to hypothalamic and pituitary membranes was great, VIP binding to these tissues was almost absent. To determine if VIP and PACAP might share a binding site in peripheral tissues, displacement curves were generated using [125I]PACAP27 binding to lung membranes and VIP, PACAP27, and PACAP38 as unlabeled ligands. VIP was highly potent in displacing [125I] PACAP27 binding in lung membrane, and the IC50 values for all three of these peptides were between 1-10 nM. These results suggest that 1) a saturable, high affinity binding site for PACAP is present on anterior pituitary membranes; 2) PACAP27 and PACAP38, but not VIP, share this binding site in the anterior pituitary and possibly the hypothalamus; and 3) PACAP27, PACAP38, and VIP share a similar or identical binding site on lung membranes and possibly other peripheral tissues. (Endocrinology 127: 272-277, 1990)

ABSTRACT. A novel bioactive peptide was recently isolated from ovine hypothalamus and was named PACAP (pituitary adenylate cyclase-activating polypeptide). PACAP was present in two bioactive, amidated forms, PACAP27 and PACAP38 (27 and 38 amino acids, respectively), and showed a 68% sequence homology with vasoactive intestinal peptide (VIP) in the Nterminal 28 residues. PACAP38 was at least 1000 times more potent than VIP in stimulating adenylate cyclase in pituitary cells, but both peptides exhibited comparable vasodepressor activity. Thus, we sought to determine whether PACAP acts on specific binding sites in the anterior pituitary or other tissues and whether these binding sites are different from those of VIP. Binding of [125I] PACAP27 to freshly prepared rat anterior pituitary membranes in the presence and absence of 212 nM unlabeled PACAP27 was specific, saturable, and more rapid at 22 C than at 4 C. Scatchard analysis of this binding site using increasing doses of unlabeled PACAP27 revealed a single high affinity site with a Kd of 446 ± 141 pM and a maximum number of sites of 1312 ± 182 fmol/mg protein. These results do not exclude the possibilty of a second pituitary binding site with significantly lower affinity. Unlabeled PACAP38 and PACAP38OH exhibited significantly higher affinity binding (3to 5-fold) than PACAP27 with a similar number of pituitary

A

NOVEL bioactive peptide was recently isolated from ovine hypothalmus in an effort to discern any unidentified hypothalamic-hypophysiotropic releasing hormones that regulate anterior pituitary hormone secretion (1). This peptide was named PACAP (pituitary adenylate cyclase-activating polypeptide) because it stimulates the accumulation of intracellular and extracellular cAMP in monolayer cultures of rat anterior

pituitary cells. Elucidation of the primary structure of PACAP revealed it to be a C-terminus-amidated peptide, 38 amino acid residues in length (PACAP38), and a member of the vasoactive intestinal polypeptide (VIP) family of peptides. PACAP-(l-28) showed a 68% amino acid sequence homology with porcine VIP; however, the sequence of the C-terminal region (amino acids 29-38) was determined to be unique. PACAP contained a GlyLys-Arg sequence at positions 28-30, suggesting the possibility of the presence of a shorter amidated peptide with 27 residues. In fact, our laboratory has isolated the 27-amino acid form of PACAP (PACAP27) from ovine hypothalamus (2). The cloning of a single, hybridizing cDNA from an ovine hypothalamic cDNA library (3) suggests that both of these forms are derived from a single 176-amino acid precursor.

Received January 29, 1990. Address all correspondence and requests for reprints to: Paul E. Gottschall, Ph.D., U.S.-Japan Biomedical Research Laboratories, Tulane University Hebert Center #30, 3705 Main Street, Belle Chasse, Louisiana 70037. * This work was supported in part by grants from Takeda Chemical Industries Ltd. and NIH Grant AM-09094 (to A.A.). t Present address: Center of National Cardiovascular Research Institute, Department of Pharmacology, 7-1 Fujishiro-dai, 5-chome, Suita, Osaka 565, Japan.

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PACAP-BINDING SITE PACAP activated adenylate cyclase in a rapid, potent, and dose-dependent fashion, as evidenced by increased cAMP levels in cultured anterior pituitary cells and the media from those cells (1). Despite this action, PACAP was unable to alter the secretory rate of any known anterior pituitary hormone when assayed in a static pituitary monolayer culture, nor did PACAP modify the release of anterior pituitary hormones in response to a "cocktail" of the known hypothalamic releasing factors. Preliminary results also suggested that injection of PACAP in vivo to male rats also did not stimulate or inhibit the release of any anterior pituitary hormone (4). However, PACAP stimulated the release of GH, PRL, and ACTH from superfused rat pituitary cells, albeit with bell-shaped dose-response curves (1). Therefore, although PACAP stimulated an intracellular messenger, i.e. cAMP, in cultured, anterior pituitary cells, the specific physiological end point of PACAP action on the anterior pituitary as well as the target cell type for PACAP remain in question. Nevertheless, PACAP was at least 1000 times more potent than VIP in stimulating adenylate cyclase in pituitary cells (and its potency was similar to that of CRF), although both PACAP and VIP exhibited comparable vasodepressor activity when injected into pentobarbital-anesthetized rats (4). Thus, it was of interest to determine if PACAP acts on specific binding sites in the anterior pituitary or other tissues and whether these sites are different from those for VIP. The objective of these experiments was to characterize the binding sites for PACAP in the anterior pituitary, describe the distribution of PACAP-binding sites in other selected tissues, and determine if the PACAPbinding sites are different from or the same as those for VIP. Materials and Methods Chemicals and materials PACAP27 was synthesized by Dr. Chieko Kitada, Takeda Industries Ltd., Tsukuba Research Laboratories (Tsukuba, Japan). PACAP38 was synthesized by the American Peptide Co. (Santa Clara, CA), and PACAP38OH and the C-terminal fragment (PACAPCys23-(24-38)] were synthesized by Dr. David Coy (Department of Medicine, Tulane University School of Medicine, New Orleans, LA). All peptides were synthesized using the solid phase method. The purity and sequence of these peptides were determined by amino acid analysis using the Pico Tag System (Waters Associates, Milford, MA) and sequence analysis using model 477A/120A (Applied Biosystems, Foster City, CA) and reverse phase HPLC in our laboratory. Porcine VIP, secretin, glucagon, peptide histidine isoleucine amide-(l27) (porcine), and LHRH were purchased from Peninsula Laboratories, Inc. (Belmont, CA). Epidermal growth factor was obtained from Calbiochem (La Jolla, CA), and human recom-

273

binant interleukin-1/3 was a gift from Otsuka Pharmaceutical Co. (Tokushima, Japan). Iodination of PACAP27 and VIP Synthetic PACAP27 and VIP were labeled with 125I (Amersham Corp., Arlington Heights, IL) by the lactoperoxidase method. The products of the iodination were separated on a Vydac C18 reverse phase HPLC column (Rainin Instruments, Woburn, MA), with a 60-min linear gradient of 10-60% acetonitrile in 0.1% trifluoro-acetic acid (TFA) and the monoiodinated peptide was separated from multiiodinated peptides and free iodide. The peak of monoiodinated peptide was collected and used for the binding assays. The range of specific activities of both peptides was 484-502 nd/ng (for five iodinations), as assessed by the self-displacement method in immunoassay. Iodinated peptide was stored at —20 C in 60% acetonitrile in 0.1% TFA containing 2 mg/ml bacitracin (Sigma Chemical Co., St. Louis, MO) and demonstrated consistent bindability for 68 weeks. Membrane preparation Adult female CD rats (Charles River, Wilmington, MA), weighing 160-180 g, were donors for the tissues used for all of the experiments. The animals were killed, and tissues were collected and briefly rinsed in saline to remove excess blood. All procedures were performed at 4 C unless otherwise indicated and were modified from the protocol of Robberecht et al. (5) for VIP binding to human lung membranes. The tissue was placed in at least a 10-fold volume of membrane isolation buffer (MIB): 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 0.5 mg/ml bacitracin, 2 Mg/ml phenylmethylsulfonylfluoride (Sigma), and 200 U/ml Trasylol (FBA Pharmaceuticals, New York, NY). The tissue was homogenized by hand using 15 strokes/tissue in a Teflon-glass homogenizer and centrifuged at 250 X g for 10 min. The supernatant was collected and centrifuged at 50,000 x g for 30 min. The pellet was resuspended in a similar volume of MIB by passing the suspension several times through a 21-gauge needle and then through a 27-gauge needle. The suspension was centrifuged again at 50,000 X g for 30 min. The crude membrane pellet was diluted so as to provide the appropriate protein concentration per 100 /xl MIB. An aliquot was taken for measurement of protein by using a Bio-Rad protein assay kit (Bio-Rad, Richmond, CA). Receptor binding assay The buffer used for dilution of the labeled peptide or unlabeled peptides was MIB containing 1% BSA. Appropriate concentrations of the labeled and unlabeled peptides were diluted in 100-jitl aliquots and pipeted into 12 X 75-mm tubes. The membrane preparation was the final 100-^1 aliquot to be added to the tubes. The tubes were incubated at 4 or 22 C for various time periods. Unless otherwise indicated, incubations were performed at 22 C for 90 min. At the end of the incubation, 4 ml washing buffer [50 mM Tris-HCl (pH 7.4), 0.5 mM EDTA, and 0.2% BSA] were added to each assay tube. Bound hormone was then separated from free hormone by passing the membrane suspension through Whatman GF/B membranes (Bran-

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PACAP-BINDING SITE

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del, Gaithersburg, MD) presoaked overnight in 0.5% polyethylenimine (Sigma) using a 48-well cell harvester (Brandel). The filters were washed twice with 4 ml washing buffer, the filter paper circles were punched out into glass tubes, and the tubes were counted in a 7-counter. Because PACAP is a very basic peptide, the iodinated peptide (and presumably the unlabeled peptide) was sticky. Therefore, nonspecific binding was moderately greater than that in most RRAs, usually 5-10% of the total counts. Nonspecific binding was even larger than this when anterior pituitary membrane was used. 60

90

120

150

TIME (min)

Data analysis and statistics The displacement curves for [125I]PACAP27 binding to anterior pituitary membrane preparations were analyzed using Ligand (Peter Munson, NICHHD, NIH). The IC50 values and the slopes for the displacement curves of the lung membrane assays were determined by ALLFIT (David Rodbard, NICHHD, NIH). Determination of the differences between experimental groups were performed by analysis of variance, and pairwise comparisons were made using Tukey's Honestly Significant Difference (HSD) test. A rejection level of P < 0.05 was considered significant. Unless otherwise indicated, data are expressed as the mean ± SEM. Assays were performed multiple times, with duplicate tubes per assay point.

30000

0

20

40

60

80

100

MEMBRANE PROTEIN fog/tube)

Results Characterization of PACAP27-binding site (5) Specific binding of [125I]PACAP27 to crude anterior pituitary membranes in the absence and presence of 212 nM unlabeled PACAP27 was more rapid at 22 C than at 4 C (Fig. 1, top panel). Binding at 22 C reached equilibrium after 60 min of incubation. The binding of [125I] PACAP27 increased linearly with increasing amounts of anterior pituitary membrane included in the assay (Fig. 1, bottom panel). Binding was saturable when anterior pituitary membranes were incubated with increasing concentrations of [125I]PACAP27 and was linear when plotted by the method of Scatchard. Saturating concentrations of [125I]PACAP27 were determined to be between 100-150 pM (Fig. 2). Specificity, Kd, and binding capacity (Bn binding site

of PACAP-

To determine the specificity of the PACAP-binding site, anterior pituitary membranes were incubated with [125I]PACAP27 in the presence of increasing concentrations of four PACAP-related peptides: the two naturally occurring forms, PACAP27 and PACAP38; the free acid of the 38-amino acid form, PACAP38OH; and PACAPCys23-(24-38). Four VIP-related peptides were tested (VIP, secretin, glucagon, and peptide histidine isoleucine) in addition to three structurally unrelated peptides or proteins (LHRH, interleukin-1/?, and epider-

FIG. 1. Time course for specific binding of [125I]PACAP27 to anterior pituitary membranes at 4 and 22 C (top panel). Anterior pituitary membranes (38 fig) were incubated with 124 pM [125I]PACAP27 (100,000 cpm) in the absence or presence of 212 nM unlabeled PACAP27 for various time periods at two different temperatures. Specific binding was calculated as the difference between counts per min bound in the absence and presence of unlabeled PACAP27. Results were expressed as counts per min specifically bound. The specific binding of [125I]PACAP27 to increasing concentrations of anterior pituitary membrane is shown {bottom panel). Various concentrations of membrane were incubated with 124 pM [125I]PACAP27 in the absence or presence of 212 nM unlabeled PACAP27 at 22 C for 90 min. Results were calculated and expressed as described in the top panel.

mal growth factor). As is evident by a representative experiment shown in Fig. 3, high affinity competition curves were obtained with increasing concentrations of PACAP27, PACAP38, and PACAP38OH. However, PACAPCys23-(24-38), any of the VIP-related peptides, or any of the unrelated peptides did not displace [125I] PACAP27 binding. Therefore, the competition curves for PACAP27, PACAP38, and PACAP38OH were repeated several times to determine if parallel curves were obtained. These curves were analyzed by Scatchard analysis, and a Kd and Bmax were calculated for each curve using the curve-fitting program Ligand. These values are shown in Table 1. Both PACAP38 and PACAP38OH exhibited similar Kd values; however, the Kd for PACAP27 was about 3- to 5-fold greater (P < 0.05). The mean Bmax values were not different among the three peptides (P > 0.05).

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TABLE 1. Kj and Bmax values from Scatchard analysis of peptide displacement curves of [125I]PACAP27 binding in anterior pituitary membranes PACAP27 (n = 6)

ID

o

PACAP38 (n = 4)

PACAP38OH (n = 4)

Kd (pM) Bma]t (fmol/mg protein)

CD

O O

LU Q_

en 0

100

200

300

400

500

TOTAL [125I]-PACAP27 (pM)

446 ± 141 138 ± 29° 93 ± 26° 1312 ± 182 1181 ± 244 1326 ± 200 Data were analyzed using the Ligand curve-fitting program. Results are expressed as the mean ± SEM. D Significantly less compared to PACAP27 (P < 0.05). 1200

LU 111 DC «= Q

o CO

0

1

2

3

AP HYPO ADR KID

4

SPECIFIC BOUND (fmol)

FIG. 2. Equilibrium binding of [125I]PACAP27 to anterior pituitary membranes (top panel). Anterior pituitary membranes (24 fig) were incubated with increasing concentrations of [125I]PACAP27 in the absence or presence of 212 nM unlabeled PACAP27 at 22 C for 90 min. Specific binding was calculated as the difference between femtomoles bound in the absence and presence of unlabeled PACAP27. Scatchard analysis of the saturation curve is shown (bottom panel).

• a o

.1

1

10

100

PACAP27 PACAP38OH PACAP38 PACAPCys23(24-38 VIP secretin glucagon PHI LHRH IL-1 EGF

1000

DOSE OF PEPTIDE (nM)

FIG. 3. Representative displacement curves of [125I]PACAP27 binding by unlabeled PACAP27, PACAP38, and PACAP38OH in anterior pituitary membranes. The figure also demonstrates the specificity of [12BI]PACAP27 binding with VIP-related peptides and unrelated peptides. Anterior pituitary membranes (41 /ug) were incubated with 124 pM [125I]PACAP27 with increasing concentrations of unlabeled hormones at 22 C for 90 min. Results are expressed as a percentage of binding in the absence of unlabeled hormone. PHI, Peptide histidine isoleucine; IL-1, interleukin-1; EGF, epidermal growth factor.

LUN DUO UTR OVR THY

FIG. 4. Distribution of [125I]PACAP27 and [126I]VIP to various tissue membranes. About 120 pM [125I]PACAP27 (n = 4) or [126I]VIP (n = 3) was incubated with different tissue membranes in the absence or presence of 200 nM of the appropriate unlabeled peptide, PACAP27 or VIP, at 22 C for 90 min. Results are expressed as the mean ± SEM, calculated by the difference between femtomoles bound in the absence of unlabeled peptide and femtomoles bound in the presence of unlabeled peptide. The mean protein levels for seven assays are: anterior pituitary (AP), 19 fig; hypothalamus (HYPO), 28 ug; adrenal (ADR), 27 fig; kidney (KID), 25 ug; liver (LIV), 49 fig; lung (LUN), 15 fig; duodenum (DUO), 40 fig; uterus (UTR), 9 fig; ovary (OVR), 30 Mg; thymus (THY), 49 Mg.

Distribution of PACAP-binding sites among various tissues Because PACAP was isolated from ovine hypothalamus, like other hypothalamic peptides, the presence of peptide and the peptide-binding sites may be distributed among diverse tissue and cell types. Thus, it was of interest to determine the tissue distribution of PACAPbinding sites and compare it to that of the PACAP related peptide, VIP. Crude membranes from various tissues were incubated with 124 pM [125I]PACAP27 or [125I]VIP either with or without 212 nM unlabeled PACAP27 or 200 nM VIP. The results of specific binding from each tissue are shown in Fig. 4. Since the affinity of the PACAP-binding site is in the same range as the known binding affinity for VIP sites (Kd = 80-1000 pM) (5-9), single point determinations of binding reveal information about the relative distribution of binding sites. The relative magnitude of binding between PACAP and VIP was similar in certain peripheral tissues, i.e. lung, liver, duodenum, ovary, and thymus. Specific binding of

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[125I]PACAP or [125I]VIP was small in uterus, adrenal, and kidney. Whereas large specific binding was observed in anterior pituitary and hypothalamus to [125I] PACAP27, [125I]VIP binding to these tissues was almost absent. Thus, these data suggested that the peripheral binding site for PACAP27 might be the same or similar to that for VIP, but the hypothalamic and anterior pituitary sites are different. To address this question, crude pituitary and hypothalamic membranes were incubated with [125I]PACAP27 with and without 212 nM VIP. As shown in Fig. 5, VIP did not displace [125I] PACAP27 binding in either the pituitary or hypothalamus. Competition curves with crude lung membrane Previous data suggested that the pituitary and hypothalamic binding sites for [125I]PACAP27 were different from those for VIP. Since the tissue distribution in peripheral tissues was similar for PACAP27 and VIP, it was of interest to determine if these peptides share a receptor in lung. Therefore, lung tissue was incubated with [125I]PACAP27 and increasing amounts of unlabeled PACAP27, PACAP38, or VIP. All three competition curves were parallel (Fig. 6). These curves were repeated, and IC50 values and slopes were calculated; these are shown in Table 2. There was no difference in the slopes of these curves, although PACAP27 and PACAP38 were only slightly more potent in displacing [125I]PACAP27 compared to VIP.

Discussion The isolation and characterization of PACAP were based on the peptide's ability to stimulate adenylate

?

20

Q Z Z)

O CO C\J CL

O CL i

CM •-

u"1

" AP HYPO FlG. 5. Displacement of [126I]PACAP27 binding to anterior pituitary (AP) and hypothalamic (HYPO) membranes in the absence and presence of 200 nM VIP. Membranes were incubated with 124 pM [125I] PACAP27 with and without unlabeled VIP at 22 C for 90 min. Results are expressed as the mean ± SEM (n = 3) of the total femtomoles bound.

Q Z

o CO

i

Endo• 1990 Vol 127 • No 1

uuPACAP38 VIP PACAP27

o



80\

\

60"

\

v

^o

\

Vs.

40-

\ LU O CC LU CL

20-

n.01

.1

1

10

100

1000

DOSE OF PEPTIDE (nM) FIG. 6. Representative displacement curves of [125I]PACAP27 binding by unlabeled PACAP27, PACAP38, and VIP in lung membranes. Lung membranes (17 Mg) were incubated with 124 pM [126I]PACAP27 with increasing concentrations of unlabeled hormones at 22 C for 90 min. Results are expressed as a percentage of binding in the absence of unlabeled hormone. TABLE 2. Analysis of peptide displacement curves (IC50 and slope) of [125I]PACAP27 binding in lung membranes

IC50 (nM) Slope

PACAP27 (n = 4)

PACAP38 (n = 3)

VIP (n = 4)

1.56 ± 0.13 0.72 ± 0.02

3.13 ± 1.04° 0.65 ± 0.08

9.29 ± 1.23° 0.64 ± 0.04

Data were analyzed using the Allfit curve-fitting program. Results are expressed as the mean ± SEM. " Significantly greater compared to PACAP27 (P < 0.05).

cyclase activity in cultures of anterior pituitary cells. After the primary structure was elucidated, it was determined that PACAP showed the greatest amino acid sequence homology with VIP and a number of related peptides. The present data have validated and demonstrated the existence of saturable, high affinity binding sites for PACAP27 in anterior pituitary membrane preparations, which were clearly distinct from VIP-binding sites. In addition to the pituitary, PACAP-binding sites were shown to be present, and of greater density, in the hypothalamus and lung. The most surprising finding, however, was that although VIP was unable to compete with PACAP27 for high affinity sites in the anterior pituitary, VIP was highly potent (IC50 = 9.3 nM) in displacing PACAP27 binding on lung membranes. This suggests a molecular heterogeneity between the binding sites for PACAP located in the pituitary and lung. Clearly, an answer to this speculation awaits experiments designed to analyze the molecular characteristics of the PACAP-binding sites in different tissues. The affinity of PACAP27 for high affinity binding sites in the anterior pituitary (Kd = 446 pM) was similar

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PACAP-BINDING SITE to the affinity of some of the hypothalamic hypophysiotropic hormones for their respective sites in the pituitary, i.e. the Kd for LHRH agonist analogs in the anterior pituitary is about 200 pM (10), and that for CRF is 600 pM (11). Although the data from our study revealed only a single class of high affinity binding sites in the anterior pituitary for PACAP27, they do not eliminate the possibility of the existence of a lower affinity site (Kd > 200 nM) for PACAP. Displacement of [125I]PACAP27 binding by PACAP38 or the free acid of PACAP38 occurred at lower concentrations than displacement by unlabeled PACAP27, and therefore, the affinity of these C-terminus elongated peptides was greater than that of PACAP27. The reason for this is unclear, especially since the dose-response curves for PACAP27 and PACAP38 in activating adenylate cyclase do not appear to be different (1); however, this difference is very reproducible from assay to assay. It is possible that the different affinities of PACAP27 and PACAP38 for the pituitary binding sites would allow for a discrete cellular regulation of adenylate cyclase activity. Recently, it has been shown that heterologous receptors for the homologous peptide hormones a-atrial naturetic peptide and brain naturetic peptide may be differentially and preferentially stimulated by one or the other of the hormones (12). For example, the atrial naturetic peptide-B receptor, which is found exclusively in brain, is preferentially stimulated by brain naturetic peptide. It is possible that a parallel phenomenon may eventually turn out to exist for PACAP. To test this hypothesis, it would be necessary to label PACAP38, but we have yet to determine adequate assay conditions for using the very basic peptide [125I]PACAP38 as the labeled ligand because the peptide is sticky, and nonspecific binding is high. The relative magnitude of binding between PACAP27 and VIP in selected tissues was strikingly similar, except in the anterior pituitary and hypothalamus, where PACAP27 binding greatly exceeded that of VIP. In fact, the Bmax in the anterior pituitary reported here for PACAP27 was substantially higher than that reported for the hypophysiotropic hormones. The number of PACAP27 sites (1312 fmol/mg protein) was 5-fold greater than the number of sites reported for LHRH (10) and 53-fold greater than that reported for CRF (11). Several criteria have been satisfied to establish PACAP as a hypothalamic hypophysiotropic hormone. Immunoreactive PACAP has been observed in the external zone of the median eminence (13); specific, high affinity binding sites for PACAP were demonstrated in anterior pituitary membranes; and PACAP stimulated an intracellular messenger (adenylate cyclase) in cultured anterior pituitary cells (1). However, evidence to satisfy two

277

of the most important criteria must be established before PACAP may be considered a hypophysiotropic hormone, i.e. 1) the presence of PACAP in the hypothalamic hypophyseal portal blood in levels sufficient to activate its physiological action on the anterior pituitary, and most significantly, 2) the ability of PACAP to regulate (stimulate or inhibit) the synthesis and secretion of a specific (or several) anterior pituitary hormone or factor. Acknowledgments The authors would like to thank Peter Munson and David Rodbard for providing the software for Ligand and Allfit. We thank Drs. David H. Coy, Tulane University; Chieko Kitada, Takeda Chemical Industries Ltd.; and Janos Varga, American Peptide Co., for the synthesis of the peptides.

References 1. Miyata A, Arimura A, Dahl RR, Minamino N, Uehara A, Jiang L, Culler M, Coy DH 1989 Isolation of a novel 38 residue-hypothalamic polypeptide which stimulates adenylate cyclase in pituitary cells. Biochem Biophys Res Commun 164:567-74 2. Miyata A, Katsuura G, Gottschall PE, Dahl RR, Coy DH, Fujino M, Arimura A, Isolation and characterization of a novel ovine hypothalamic polypeptide which stimulates adenylate cyclase in rat pituitary cell cultures. 19th Annual Meeting of the Society for Neuroscience, Phoenix AZ, 1989, p 1277 (Abstract) 3. 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 166:81-9 4. Arimura A, Katsuura G, Gottschall PE, Dahl RR, Biological actions of a novel hypothalamic polypeptide with 38 residues. 71st Annual Meeting of The Endocrine Society, Seattle WA, 1989, p 263 (Abstract) 5. Robberecht P, Waelbroeck M, deNeef P, Camus JC, Coy DH, Christophe J 1988 Pharmacological characterization of VIP receptors in human lung membranes. Peptides 9:339-45 6. Taylor DP, Pert CB 1979 Vasoactive intestinal polypeptide: specific binding to rat brain membranes. Proc Natl Acad Sci USA 76:660-4 7. Ogawa N, Mizuno S, Mori A, Nukina I, Yanaihara N 1985 Properties and distribution of vasoactive intestinal polypeptide receptors in the rat brain. Peptides 6:103-9 8. Besson J 1989 Distribution and pharmacology of vasoactive intestinal peptide receptors in the brain and pituitary. In: Said S, Mutt V (eds) Vasoactive Intestinal Peptide and Related Peptides. New York Academy of Sciences, New York, vol 527:204-19 9. Patthi S, Simerson S, Velicelebi G 1988 Solubilization of rat lung vasoactive intestinal peptide receptors in the active state. J Biol Chem 263:19363-9 10. Clayton RN, Catt KJ 1980 Receptor-binding affinity of gonadotropin-releasing hormone analogs: analysis by radioligand-receptor assay. Endocrinology 106:1154-9 11. Grino M, Guillaume V, Castanas E, Boudouresque F, Conte-Devolx B, Oliver C 1987 Effect of passive immunization against corticotropin-releasing factor (CRF) on the postadrenalectomy changes of CRF binding sites in the rat pituitary gland. Neuroendocrinology 45:492-7 12. Chang M, Lowe DG, Lewia M, Hellmiss R, Chen E, Goeddel DV 1989 Differential activation by atrial and brain natriuretic peptides by two different receptor guanylate cyclases. Nature (Lond) 341:68-72 13. Koves K, Arimura A, Somogyvari-Vigh A, Vigh S, Miller J 1990 Immunocytochemical demonstration of a novel hypothalamic peptide, pituitary adenylate cyclase activating polypeptide, in the ovine hypothalamus. Endocrinology 127:264-71

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Characterization and distribution of binding sites for the hypothalamic peptide, pituitary adenylate cyclase-activating polypeptide.

A novel bioactive peptide was recently isolated from ovine hypothalamus and was named PACAP (pituitary adenylate cyclase-activating polypeptide). PACA...
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