Synthetic Pituitary Adenylate Cyclase-activating Polypeptide Research Articles

Pituitary Adenylate Cyclase-Activating Polypeptide Alleviates Intestinal, Extra-Intestinal and Systemic Inflammatory Responses during Acute Campylobacter jejuni-induced Enterocolitis in Mice.

Human Campylobacter jejuni infections are emerging, and constitute a significant health burden worldwide. The ubiquitously expressed pituitary adenylate cyclase-activating polypeptide (PACAP) is well-known for its cell-protective and immunomodulatory effects. In our actual intervention study, we used an acute campylobacteriosis model and assessed the potential disease-alleviating effects of exogenous PACAP. Therefore, secondary abiotic IL-10−/− mice were perorally infected with C. jejuni and treated with synthetic PACAP38 intraperitoneally from day 2 until day 5 post-infection. Whereas PACAP did not interfere with the gastrointestinal colonization of the pathogen, mice from the PACAP group exhibited less severe clinical signs of C. jejuni-induced disease, as compared to mock controls, which were paralleled by alleviated apoptotic, but enhanced cell proliferative responses in colonic epithelia on day 6 post-infection. Furthermore, PACAP dampened the accumulation of macrophages and monocytes, but enhanced regulatory T cell responses in the colon, which were accompanied by less IFN-γ secretion in intestinal compartments in PACAP versus mock-treated mice. Remarkably, the inflammation-dampening properties of PACAP could also be observed in extra-intestinal organs, and strikingly, even the systemic circulation on day 6 post-infection. For the first time, we provide evidence that synthetic PACAP might be a promising candidate to combat acute campylobacteriosis and post-infectious sequelae.

Protective Effects of Pituitary Adenylate Cyclase-Activating Polypeptide and Vasoactive Intestinal Peptide Against Cognitive Decline in Neurodegenerative Diseases.

Cognitive impairment is one of the major symptoms in most neurodegenerative disorders such as Alzheimer’s (AD), Parkinson (PD), and Huntington diseases (HD), affecting millions of people worldwide. Unfortunately, there is no treatment to cure or prevent the progression of those diseases. Cognitive impairment has been related to neuronal cell death and/or synaptic plasticity alteration in important brain regions, such as the cerebral cortex, substantia nigra, striatum, and hippocampus. Therefore, compounds that can act to protect the neuronal loss and/or to reestablish the synaptic activity are needed to prevent cognitive decline in neurodegenerative diseases. Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are two highly related multifunctional neuropeptides widely distributed in the central nervous system (CNS). PACAP and VIP exert their action through two common receptors, VPAC1 and VPAC2, while PACAP has an additional specific receptor, PAC1. In this review article, we first presented evidence showing the therapeutic potential of PACAP and VIP to fight the cognitive decline observed in models of AD, PD, and HD. We also reviewed the main transduction pathways activated by PACAP and VIP receptors to reduce cognitive dysfunction. Furthermore, we identified the therapeutic targets of PACAP and VIP, and finally, we evaluated different novel synthetic PACAP and VIP analogs as promising pharmacological tools.

Pituitary Adenylate Cyclase-Activating Polypeptide Modulates Hippocampal Synaptic Transmission and Plasticity: New Therapeutic Suggestions for Fragile X Syndrome.

Pituitary adenylate cyclase-activating polypeptide (PACAP) modulates glutamatergic synaptic transmission and plasticity in the hippocampus, a brain area with a key role in learning and memory. In agreement, several studies have demonstrated that PACAP modulates learning in physiological conditions. Recent publications show reduced PACAP levels and/or alterations in PACAP receptor expression in different conditions associated with cognitive disability. It is noteworthy that PACAP administration rescued impaired synaptic plasticity and learning in animal models of aging, Alzheimer’s disease, Parkinson’s disease, and Huntington’s chorea. In this context, results from our laboratory demonstrate that PACAP rescued metabotropic glutamate receptor-mediated synaptic plasticity in the hippocampus of a mouse model of fragile X syndrome (FXS), a genetic form of intellectual disability. PACAP is actively transported through the blood–brain barrier and reaches the brain following intranasal or intravenous administration. Besides, new studies have identified synthetic PACAP analog peptides with improved selectivity and pharmacokinetic properties with respect to the native peptide. Our review supports the shared idea that pharmacological activation of PACAP receptors might be beneficial for brain pathologies with cognitive disability. In addition, we suggest that the effects of PACAP treatment might be further studied as a possible therapy in FXS.

Pituitary Adenylate Cyclase-Activating Polypeptide-A Neuropeptide as Novel Treatment Option for Subacute Ileitis in Mice Harboring a Human Gut Microbiota.

The neuropeptide Pituitary adenylate cyclase-activating polypeptide (PACAP) is well-known for its important functions in immunity and inflammation. Data regarding anti-inflammatory properties of PACAP in the intestinal tract are limited, however. In our present preclinical intervention study we addressed whether PACAP treatment could alleviate experimental subacute ileitis mimicking human gut microbiota conditions. Therefore, secondary abioitic mice were subjected to human fecal microbiota transplantation (FMT) and perorally infected with low-dose Toxoplasma gondii to induce subacute ileitis on day 0. From day 3 until day 8 post-infection, mice were either treated with synthetic PACAP38 or placebo. At day 9 post-infection, placebo, but not PACAP treated mice exhibited overt macroscopic sequelae of intestinal immunopathology. PACAP treatment further resulted in less distinct apoptotic responses in ileal and colonic epithelia that were accompanied by lower T cell numbers in the mucosa and lamina propria and less secretion of pro-inflammatory cytokines in intestinal ex vivo biopsies. Notably, ileitis-associated gut microbiota shifts were less distinct in PACAP as compared to placebo treated mice. Inflammation-ameliorating effects of PACAP were not restricted to the intestines, but could also be observed in extra-intestinal including systemic compartments as indicated by lower apoptotic cell counts and less pro-inflammatory cytokine secretion in liver and lungs taken from PACAP treated as compared to placebo control mice, which also held true for markedly lower serum TNF and IL-6 concentrations in the former as compared to the latter. Our preclinical intervention study provides strong evidence that synthetic PACAP alleviates subacute ileitis and extra-intestinal including systemic sequelae of T cell-driven immunopathology. These findings further support PACAP as a novel treatment option for intestinal inflammation including inflammatory bowel diseases (IBD).

Characterizations of a synthetic pituitary adenylate cyclase-activating polypeptide analog displaying potent neuroprotective activity and reduced in vivo cardiovascular side effects in a Parkinson's disease model

Parkinson's disease (PD) is characterized by a steady loss of dopamine neurons through apoptotic, inflammatory and oxidative stress processes. In that line of view, the pituitary adenylate cyclase-activating polypeptide (PACAP), with its ability to cross the blood–brain barrier and its anti-apoptotic, anti-inflammatory and anti-oxidative properties, has proven to offer potent neuroprotection in various PD models. Nonetheless, its peripheral actions, paired with low metabolic stability, hampered its clinical use. We have developed Ac-[Phe(pI)6, Nle17]PACAP(1-27) as an improved PACAP-derived neuroprotective compound. In vitro, this analog stimulated cAMP production, maintained mitochondrial potential and protected SH-SY5Y neuroblastoma cells from 1-methyl-4-phenylpyridinium (MPP+) toxicity, as potently as PACAP. Furthermore, contrasting with PACAP, it is stable in human plasma and against dipeptidyl peptidase IV activity. When injected intravenously to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice, PACAP and Ac-[Phe(pI)6, Nle17]PACAP(1-27) restored tyrosine hydoxylase expression into the substantia nigra and modulated the inflammatory response. Albeit falls of mean arterial pressure (MAP) were observed with both PACAP- and Ac-[Phe(pI)6, Nle17]PACAP(1-27)-treated mice, the intensity of the decrease as well as its duration were significantly less marked after iv injections of the analog than after those of the native polypeptide. Moreover, no significant changes in heart rate were measured with the animals for both compounds. Thus, Ac-[Phe(pI)6, Nle17]PACAP(1-27) appears as a promising lead molecule for the development of PACAP-derived drugs potentially useful for the treatment of PD or other neurodegenerative diseases.

Anorexigenic action of pituitary adenylate cyclase-activating polypeptide (PACAP) in the goldfish: Feeding-induced changes in the expression of mRNAs for PACAP and its receptors in the brain, and locomotor response to central injection

Our recent research has indicated that intracerebroventricular (ICV) administration of pituitary adenylate cyclase-activating polypeptide (PACAP) suppresses food intake in the goldfish. We therefore examined feeding- and fasting-induced changes in the expression of mRNAs for PACAP and its receptors in the goldfish brain, and the effect of ICV administration of synthetic PACAP on locomotor activity in the goldfish. Semiquantitative analysis revealed that the expression of mRNAs for PACAP and the PAC 1 receptor was significantly increased by excessive feeding (daily food supply at ≥5% of the body weight [BW]) for 7 days. ICV administration of PACAP at 20 pmol/g BW induced a significant decrease in locomotor activity during the 60-min post-treatment observation period. These results suggest that PACAP may have an anorexigenic action via hypomotility in goldfish.

Inhibitory effects of pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) on food intake in the goldfish, Carassius auratus

Pituitary adenylate cyclase-activating polypeptide (PACAP) has a similar structure to that of vasoactive intestinal peptide (VIP) and both the polypeptides belong to the same molecular group, the secretin–glucagon superfamily. PACAP and VIP have possible potency as hypothalamic factors mediating the release of pituitary hormones in the fish pituitary. However, the roles of PACAP and VIP in the central nervous systems of fish have not yet been made clear. Recently, it was reported that PACAP and/or VIP are involved in the feeding behavior of the mouse and chick. Therefore, we investigated the effects of intracerebroventricular (ICV) and intraperitoneal (IP) administration of synthetic PACAP and VIP on food intake in the goldfish, Carassius auratus. Cumulative food intake was significantly decreased by ICV injection of PACAP (11 or 22 pmol/g body weight) or VIP (11 or 22 pmol/g) during a 60-min observation period after treatment. IP administration of PACAP (44 or 88 pmol/g) or VIP (22 or 44 pmol/g) induced a significant decrease in food intake during a 60-min observation period after treatment. These results suggest that PACAP and VIP may be involved as feeding regulators in goldfish.

Pituitary adenylate cyclase-activating polypeptide stimulates both c-fos gene expression and cell survival in rat cerebellar granule neurons through activation of the protein kinase A pathway

A high density of pituitary adenylate cyclase-activating polypeptide (PACAP) receptors coupled to both adenylyl cyclase and phospholipase C is found in the external granule cell layer of the rat cerebellum during postnatal development. It has recently been reported that synthetic PACAP promotes cell survival and neurite outgrowth in immature granule cells. In the present study, we have investigated the transduction pathways that mediate the neurotrophic activity of PACAP in cultured granule cells from eight-day-old rat cerebellum. The effect of PACAP on cell survival was mimicked by dibutyryladenosine 3′, 5′-cyclic-monophosphate but not phorbol 12-myristate 13-acetate suggesting that only the adenylyl cyclase pathway is involved in the neurotrophic activity of PACAP. PACAP also induced a transient increase in c-fos messenger RNA level. The ability of PACAP to stimulate c-fos gene expression was mimicked by dibutyryladenosine 3′, 5′-cyclic-monophosphate but not phorbol 12-myristate 13-acetate. Similar effects of PACAP on granule cell survival were observed whether the cells were continuously incubated with PACAP for 48 h or only exposed to PACAP during 1 h. The protein kinase A inhibitor H89 significantly reduced the effect of PACAP on c-fos messenger RNA level whereas the specific protein kinase C inhibitor chelerythrine did not modify c-fos gene expression. These data indicate that the action of PACAP on cerebellar granule cell survival and c-fos gene expression are both mediated through the adenylyl cyclase/protein kinase A pathway. The observation that a short-term stimulation by PACAP can be converted into a long-lasting response indicates that the effect of the peptide on cell survival must involve immediate-early gene activation. The fact that a brief exposure to PACAP causes both c-fos gene expression and promotes cell survival strongly suggests that c-fos is involved in the trophic effect of PACAP on immature cerebellar granule cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an islet substance serving as an intra-islet amplifier of glucose-induced insulin secretion in rats.

1. We examined whether pituitary adenylate cyclase-activating polypeptide with 38 or 27 residues (PACAP-38 or PACAP-27) serves as an intra-islet regulator of glucose-induced insulin secretion in rats. PACAP antiserum specific for PACAP-38 and PACAP-27 was used to neutralize the effect of endogenous PACAP in islets. PACAP release from islets was bioassayed using the response of cytosolic Ca2+ concentration ([Ca2+]i) in single beta-cells, monitored by dual-wavelength fura-2 microfluorometry. Expression of PACAP mRNA was studied by reverse transcription-polymerase chain reaction (RT-PCR), while expression of PACAP was studied by metabolic labelling and immunoblotting. Localization of PACAP receptors was studied immunohistochemically. 2. High glucose-stimulated insulin release from isolated islets was attenuated by PACAP antiserum but not by non-immune sera. 3. The islet incubation medium with high glucose (Med) possessed a capacity, which was neutralized by PACAP antiserum, to increase [Ca2+]i in beta-cells. PACAP antiserum also neutralized the [Ca2+]i-increasing action of synthetic PACAP-38 and PACAP-27, but not that of vasoactive intestinal polypeptide (VIP) and glucagon. 4. Both Med and synthetic PACAP increased [Ca2+]i in beta-cells only in the presence of stimulatory, but not basal, glucose concentrations. In contrast, ATP, a substance that is known to be released from beta-cells, increased [Ca2+]i in beta-cells at both and stimulatory glucose concentrations. 5. Expression of PACAP mRNA and biosynthesis of PACAP-38 were detected in islets and a beta-cell line, MIN6. 6. Immunoreactivity for PACAP-selective type-I receptor was observed in islets. 7. [Ca2+]i measurements combined with immunocytochemistry with insulin antiserum revealed a substantial population of glucose-unresponsive beta-cells, many of which were recruited by PACAP-38 into [Ca2+]i responses. 8. These results indicate that PACAP-38 is a novel islet substance that is synthesized and released by islet cells and then, in an autocrine and/or paracrine manner, potentiates and arouses beta-cell responses to glucose, thereby amplifying glucose-induced insulin secretion in islets.

Identification of Endogenous Sympathetic Neuron Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP): Depolarization Regulates Production and Secretion through Induction of Multiple Propeptide Transcripts

The vasoactive intestinal peptide/pituitary adenylate cyclase-activating polypeptide (PACAP)/secretin/glucagon family of peptides displays numerous physiological roles in autonomic nervous system development and function. The regulated endogenous production and release of PACAP peptides in sympathetic neurons of the superior cervical ganglion (SCG) was investigated. The two posttranslationally processed forms of PACAP, PACAP27 and PACAP38, were identified in rat adult, neonatal, and cultured SCG neurons. PACAP38 levels were approximately 5-10 fmol/adult SCG and approximately 2 fmol/neonatal SCG; PACAP27 levels were comparable. The authenticity of peptide immunoreactivity in these tissues was verified by coelution with synthetic PACAP in reverse-phase HPLC analysis. Reverse transcription-PCR and sequence-specific hybridization revealed PACAP mRNA in adult, neonatal, and cultured SCG neurons; in situ hybridization histochemistry and immunocytochemistry localized the PACAP peptide and proPACAP mRNA to a subset of the SCG neuronal population. Basal and stimulated release of endogenous PACAP38 from cultured sympathetic neurons was established, suggesting that these peptides may function as signaling molecules at target tissues. Chronic depolarization with 40 mM potassium stimulated the PACAP secretory rate 10- to 20-fold, with concomitant increases in cellular PACAP peptide and mRNA levels. When examined using Northern analysis, depolarizing conditions not only stimulated the 2.2 kb form of PACAP mRNA, but also induced the expression of a shortened, 0.9 kb, transcript. Further reverse-transcription PCR analysis demonstrated that this smaller transcript was not identical to the unique testicular message. These studies identify PACAP38 and PACAP27 as regulated endogenous releasable peptides contributing to the functional diversity and phenotypic plasticity of the sympathetic nervous system.

Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) and Its Receptors: Neuroendocrine and Endocrine Interaction

The recent progress of research on the functions of pituitary adenylate cyclase activating polypeptide (PACAP), especially endocrine and neuroendocrine interactions, is described. Studies of the genes encoding the PACAP precursor and the type I PACAP receptor provide information on the control of PACAP gene expression and on the relationship between the structure of the receptor subtypes and the activation of various signal transduction pathways. The availability of specific antisera against PACAP and the type I PACAP receptor made it possible to examine their distributions in the brain and other tissues. Immunohistochemical studies and physiological studies with synthetic PACAP indicate that PACAP is a new type of hypophysiotropic hormone and also functions as a neurotransmitter, neuromodulator, and neurotrophic factor in the central nervous system. The abundance of PACAP and its type I receptors in the adrenal medulla and the results of studies with synthetic PACAP suggest that PACAP is a potent noncholinergic secretogue for catecholamines. PACAP and its receptors are also present in the pancreas and appear to play a regulatory role in insulin secretion at extremely low concentrations in a glucose-dependent manner. Immunohistochemical demonstration of PACAP and its receptors in the testicular spermatids at early stages suggests an important role of testicular PACAP in spermiogenesis. Together with its actions on pituitary gonadotropes, this suggests that it plays a key role in reproduction.

Localization of pituitary adenylate cyclase-activating polypeptide (PACAP) in the hypothalamus-pituitary system in rats: light and electron microscopic immunocytochemical studies.

The localization of pituitary adenylate cyclase-activating polypeptide (PACAP) in the hypothalamus-pituitary system in rats was examined in light and electron microscopic immunocytochemistry using a specific antiserum to synthetic PACAP 1-38 (R0831). In light microscopic study, intensely PACAP-immunostained perikarya were observed in the supraoptic and paraventricular magnocellular nucleus in the hypothalamus. In the median eminence, many immunoreactive nerve fibers were observed in the internal layer, but a few immunoreactive terminals were noticed in the external layer. In the pituitary gland, numerous immunoreactive nerve fibers were observed in the posterior lobe. In the intermediate lobe, moderately immunostained cells were observed, but in the anterior lobe no immunostained cells were noticed. In electron microscopic study, PACAP-immunoreactivity was examined by avidin-biotin peroxidase complex method. In the perikarya of the supraoptic and paraventricular magnocellular nucleus, DAB-reaction products were distributed diffusely in the cytoplasmic matrix, frequently attaching to the rough-surfaced endoplasmic reticulum. In the nerve terminals of the posterior lobe, reaction products were observed among the secretory granules, but sometimes upon them. In the cells of the intermediate lobe, reaction products were also distributed in the cytoplasmic matrix.

Pituitary Adenylate Cyclase-Activating Polypeptide Dilates Cerebral Arterioles of Newborn Pigs

The actions of the 38- and 27-amino acid forms of synthetic pituitary adenylate cyclase-activating polypeptide (PACAP-38 and PACAP-27) on cerebral arterioles were tested in anesthetized newborn pigs equipped with closed cranial windows. The diameter changes of pial arterioles to topical PACAP were measured and cortical periarachnoid cerebrospinal fluid samples were collected for measurement of cAMP. The 38- and 27-amino acid forms of PACAP produced similar dose-dependent vasodilations. The increases of pial arteriolar diameter produced by PACAP-38 were 6 +/- 1%, 15 +/- 2%, 23 +/- 3%, and 38 +/- 3%, and those produced by PACAP-27 were 6 +/- 1%, 15 +/- 2%, 27 +/- 5%, and 38 +/- 8% at 10((-9), 10(-8), 10(-7), and 10(-6) M, respectively. Arteriolar diameter began to increase 1-2 min and reached a maximum 6-8 min after topical application of PACAP. Vasoactive concentrations of PACAP-38 and PACAP-27 increased cerebrospinal fluid cAMP levels dose dependently. Thus, PACAP-38 and PACAP-27 appear to stimulate cerebral adenylate cyclase and are potent dilators of newborn pigs pial arterioles.

Pituitary adenylate cyclase activating polypeptide (PACAP)-like immunoreactivity in pheochromocytomas

Pituitary adenylate cyclase activating polypeptide (PACAP) is a novel hypothalamic peptide consisting of 38 amino acids [PACAP(1–38)] with a potent stimulatory action on adenylate cyclase in rat pituitary. The presence of immunoreactive (IR-) PACAP in the tumor tissue of pheochromocytomas was studied by radioimmunoassay and immunocytochemistry. The antibody to PACAP was raised in a rabbit injected with a peptide containing amino acids 28–38 of PACAP. This antibody showed no significant cross-reactivity with either PACAP(1–27) or vasoactive intestinal polypeptide. The tumor tissue concentrations of IR-PACAP(1–38) were 0.5–57.5 pmol/g wet weight ( n = 13) ( 24.5 ± 22.4 pmol/g wet weight, mean ± SD), while those in the normal adrenal glands were 3.58 ± 2.02 pmol/g wet weight ( n = 7) and those in the adrenal cortical tumors were 5.58 ± 2.02 pmol/g wet weight ( n = 6). The IR-PACAP(1–38) concentrations in 7 out of 13 pheochromocytomas exceeded 18 pmol/g wet weight. Sephadex G-50 column chromatography revealed that the IR-PACAP(1–38) in extracts of pheochromocytomas eluted in both the positions of PACAP(1–38) and a larger molecular weight. Reverse-phase high performance liquid chromatography of the tumor extracts revealed a peak in the position of PACAP(1–38) and at least four other peaks. Immunocytochemistry of pheochromocytomas showed numerous immunoreactive cells. The immunostaining was abolished by absorption of the antiserum with synthetic PACAP(1–38). These findings indicate that multiple forms of IR-PACAP(1–38) are present in pheochromocytomas.

Distribution, molecular characterization of pituitary adenylate cyclase-activating polypeptide and its precursor encoding messenger RNA in human and rat tissues.

The distribution of a novel neuropeptide, pituitary adenylate cyclase-activating polypeptide (PACAP), was studied in the brain of the rat and man and a variety of other rat tissues using Northern blot hybridization and two radioimmunoassays for PACAP 1-38 and PACAP 1-27. The assay, using PACAP 1-38 as standard and an antibody to PACAP 21-38 and radiolabelled tracer, revealed immunoreactive PACAP in all brain regions examined, with the highest concentrations in the rat being in the hypothalamus, nucleus accumbens and substantia nigra (380 +/- 34, 310 +/- 37 and 346 +/- 30 pmol/g wet tissue, means +/- S.E.M., n = 5 respectively), whilst in man the highest concentrations were found in the pituitary gland (15.8 +/- 4.7 pmol/g). Immunoreactive PACAP 1-38 was also detected in the rat gastrointestinal tract, adrenal gland and testis. The assay using PACAP 1-27 as standard and label and an antibody to PACAP 1-27 detected immunoreactive PACAP only in the rat hypothalamus (12.6 +/- 1.8 pmol/g wet tissue, n = 5). PACAP mRNA of approximately 2.7 kb in size was detectable in all brain regions of both rat and man, and its distribution paralleled that of the immunoreactive peptide. Gel permeation chromatography of different regions of human and rat hypothalamus, and also rat spinal cord and small intestine, showed a broad immunoreactive peak corresponding to PACAP 1-38. Fast protein liquid chromatography (FPLC) resolved this peak into two immunoreactive peaks, the majority eluting in the position of synthetic PACAP 1-38.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of a guanosine-nucleotide-binding-protein-coupled receptor for pituitary adenylate-cyclase-activating polypeptide on plasma membranes from rat brain.

Pituitary adenylate-cyclase-activating polypeptide (PACAP), a novel brain-gut hormone, was isolated from ovine hypothalami and represents the latest mammalian member of the secretin-glucagon peptide family. PACAP exists in two C-terminally amidated molecular forms, PACAP(1-27) and PACAP(1-38), comprising 27 or 38 amino acid residues, respectively. In order to identify a specific receptor for PACAP, we studied binding of 125I-labelled PACAP(1-27) to plasma membranes from rat brain. We identified a single high-affinity binding site (Kd, 340 pM and Bmax, 3.34 pmol/mg), specific for synthetic PACAP(1-38) and PACAP(1-27). Hormone binding was reversible and time, protein and temperature dependent. In contrast, neither the analogues PACAP(1-23), PACAP(18-38) and PACAP(3-25), nor vasoactive intestinal peptide (VIP), secretin and growth-hormone-releasing factor (GRF) revealed significant binding at concentrations up to 1 microM. A specific receptor protein, with an apparent molecular mass of 60 kDa, was identified by means of affinity cross-linking with disuccinimidyl suberate (DSS) and ethylene glycol disuccinimidyl suberate (EGS). PACAP receptors are associated with a GTP-binding protein as determined by the influence of different nucleotides on PACAP binding. PACAP-binding activity was solubilized with the detergents 3-[(3-cholamidopropyl)dimethylammonio]2-hydroxy-1-propane sulfonate (Chapso) or Triton X-100 and was characterized as a high-molecular-mass receptor complex (400 kDa) by non-reducing size-exclusion chromatography on Sepharose CL-6B. These data imply the following: high-affinity PACAP receptors are expressed abundantly on rat-brain plasma membranes; PACAP receptors are specific for PACAP and show no affinity for VIP, secretin and GRF; the PACAP receptor molecule has an apparent molecular mass of 60 kDa; the PACAP receptor complex is associated with a GTP-binding protein.