Distribution, characterization, and growth hormone-releasing activity of pituitary adenylate cyclase-activating polypeptide in the European eel, Anguilla anguilla

The complementary DNA encoding pituitary adenylate cyclase-activating polypeptide (PACAP) has been cloned from two species of teleost fishes, the Sockeye salmon and the Thai catfish, and the amino acid sequence of PACAP has been determined in another teleost, the stargazer. However, to date, the detailed distribution of PACAP immunoreactivity has never been investigated in the fish brain. In the present study, we have determined the localization of PACAP-immunoreactive neurons in the central nervous system of a primitive teleost fish, the European eel Anguilla anguilla, using an antiserum raised against PACAP27. PACAP-positive perikarya were exclusively observed in the diencephalon, i.e. in the preoptic nucleus of the hypothalamus and in the dorsal and ventral nuclei of the thalamus. PACAP-immunoreactive fibers were detected in various areas of the brain, notably in the ventral telencephalon, the diencephalon, the mesencephalon, the cerebellar valvula, and the medulla oblongata. In addition, a dense accumulation of PACAP-containing nerve terminals was found in the pars distalis of the pituitary. The PACAP-like immunoreactivity contained in the eel brain was characterized by HPLC analysis combined with RIA quantification. The major form of PACAP-immunoreactive material coeluted with mammalian PACAP38. Molecular cloning of the PACAP precursor has previously shown that in fish, PACAP and GH-releasing hormone (GHRH) originate from the same precursor. We have thus investigated the effects of PACAP and GHRH on GH secretion from eel pituitary cells in primary culture. Dose-response experiments revealed that PACAP27 and PACAP38 possessed the same efficacy, but PACAP38 was 12 times more potent than PACAP27 in stimulating GH release (ED50 = 4.3 x 10(-10) and 3.5 x 10(-9) M, respectively). In contrast, GHRH, even at a high concentration (10(-6) M), had no effect on GH release. Taken together, these data indicate that in the eel, PACAP may play a significant role in the regulation of somatotrope cells: 1) PACAP-immunoreactive neurons are exclusively located in the diencephalon and send numerous projections in the pars distalis; and 2) PACAP, but not GHRH, dose dependently stimulates GH secretion from cultured eel pituitary cells.     

Purification and primary structure of pituitary adenylate cyclase activating polypeptide (PACAP) from the brain of an elasmobranch, stingray, Dasyatis akajei

Pituitary adenylate cyclase activating polypeptide (PACAP) was isolated from ovine hypothalami and found to exist as two amidated forms with 38 (PACAP 38) and 27 (PACAP 27) residues. The amino acid sequences of PACAPs isolated from the vertebrates, such as a bird, a frog and teleost fish, appear to be well conserved. In the present study, we attempted to isolate PACAP from the brain of an elasmobranch fish, Dasyatis akajei (stingray), which belongs to the Chondrichthyes (cartilaginous fish), by extraction of the acetone-dried powder with acetic acid, followed by successive high-performance liquid chromatography (HPLC) on a gel-filtration, a cation-exchange and two reverse-phase columns. Purification was monitored by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and Western blotting analysis using an anti-PACAP 27 serum. The PACAP thus obtained consisted of 44 residues. The amino acid sequence of the comparable portion of its N-terminal 38 residues showed 92%, 89%, 89%, and 82% identity with those of mammalian, chicken, frog and teleost PACAPs with 38 residues, respectively. The extra six C-terminal residues of the stingray resembled those of tetrapod and teleost PACAP precursors which were deduced from the respective cDNAs. These results indicate that PACAP, which has an amino acid sequence showing high similarity with those of tetrapod and teleost PACAPs, is present in the elasmobranch brain.     

Distribution and effects of pituitary adenylate cyclase-activating peptide in the rabbit eye

Pituitary adenylate cyclase-activating peptide (PACAP)-like immunoreactivity was demonstrated by immunocytochemistry together with calcitonin gene-related peptide (CGRP)-like immunoreactivity in small to medium-sized neurons in the trigeminal ganglion and in nerve fibers in the iris, ciliary body, cornea, choroid and sclera of the rabbit eye. The regional distribution of PACAP-27- and PACAP-38-like immunoreactivity in the eye was studied by radioimmunoassay: the highest concentrations were found in the iris sphincter and ciliary body. The distribution pattern resembled that of CGRP-like immunoreactivity, which is a well-known constituent of sensory C-fibre neurons. Intravitreal injection of PACAP-27 or PACAP-38 induced conjunctival hyperemia, swelling of the anterior segment of the eye, miosis and breakdown of the blood-aqueous barrier, manifested as a marked aqueous flare response. Tetrodotoxin pretreatment inhibited the conjunctival hyperemia, the swelling of the anterior segment of the eye, and the miosis but not the aqueous flare response. The concentration of PACAP-like immunoreactivity in the aqueous humor was increased greatly following infrared irradiation of the iris, topical application of formaldehyde to the cornea, or intravitreal injection of endotoxin or bovine serum albumin. Also the concentration of CGRP-like immunoreactivity in the aqueous humor was increased greatly. Both in vivo and in vitro studies showed that capsaicin caused a parallel release of PACAP-like immunoreactivity and CGRP-like immunoreactivity from the uvea. Injection of PACAP-27 and PACAP-38 resulted in the release of CGRP-like immunoreactivity (and PACAP-like immunoreactivity) into the aqueous humor and PACAP-27 and PACAP-38 were also found to evoke tachykinin-mediated contractions of the isolated iris sphincter muscle, indicating that PACAP induces positive feedback on C-fibres. Thus, PACAP is a sensory neuropeptide in the eye. Since the PACAP-induced ocular responses mimicked the symptoms of inflammation, and since the PACAP-like immunoreactivity concentration in the aqueous humor was greatly increased following noxious stimulation, we suggest that it takes part in the inflammatory responses of the rabbit eye.     

PACAP (pituitary adenylate cyclase-activating peptide)-like immunoreactivity in the gill arch of the goldfish, Carassius auratus: distribution and comparison with VIP

Pituitary adenylate cyclase-activating peptide (PACAP) is a novel vasoactive intestinal peptide (VIP)-like peptide isolated from ovine hypothalamus. It is present in neuronal elements of a number of peripheral organs. We have examined whether PACAP occurs in the gill arch of Carassius auratus L. in which our recent studies have shown the presence of VIP-like peptide. Immunohistochemistry has revealed PACAP-like immunoreactivity in the anterior branches of the post-trematic glossopharyngeal and vagus nerves. PACAP-immunoreactive nerve cell bodies and fibers are present in connective tissue on the oral side of the gill arch. Colocalization studies carried out by the application of double immunofluorescence show that a PACAP-like peptide coexists with VIP in the same nerve cell bodies and fibers. The localization pattern of PACAP in the gill arch of goldfish suggests its possible involvement in the regulation of secretory activities.     

Immunohistochemical demonstration of the intracellular localization of pituitary adenylate cyclase activating polypeptide-like immunoreactivity in the rat testis using the stamp preparation

Radioimmunoassay has revealed an abundance of PACAP in the rat testis. In the present study, a novel stamp preparation together with light microscopic immunohistochemistry were used to investigate in detail the intracellular localization of PACAP-like immunoreactivity (PACAP-LI) in rat germ cells. Samples were obtained by pressing the freshly cut surfaces of the testes against glass slides. PACAP-LI was clearly identified in developing acrosomes using five antisera which recognize PACAP 38, or both PACAP 27 and PACAP 38. Immunoreactivity with the antisera specific to PACAP38 was strictly localized in the developing acrosomes of the spermatids but vanished in the mature spermatids. Using an antiserum which detects PACAP 27 specifically, little staining was observed. Based on the specificities of antisera used, it was suggested that the PACAP-LI in the acrosomes represents mainly PACAP 38-LI. In addition, the present results supported the usefulness of the stamp preparation for immunohistochemical study of testicular tissues.     

Central administration of PACAP stimulates gastric secretion mediated through the vagal pathway in anesthetized rats

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide that was originally isolated from ovine hypothalamic tissue. The peptide has two amidated forms, PACAP38 and PACAP27. In this study, we examined the effects of centrally administered PACAP38 and PACAP27 on gastric secretion in anesthetized rats. Centrally administered PACAP stimulated gastric acid and pepsin secretion in a dose-dependent manner. PACAP38 was 1.5-2 times more potent than PACAP27 on gastric secretion. By contrast, intravenously administered PACAP38 had no effect on basal or pentagastrin-stimulated gastric secretion. PACAP6-38, a PACAP antagonist, by itself at high doses also stimulated gastric and pepsin secretion, but at lower doses had no effect. Centrally administered PACAP6-38 at a dose that had no effect on gastric secretion, atropine pretreatment, or vagotomy pretreatment, suppressed the stimulatory effect of PACAP38. It is concluded that centrally administered PACAP may have a regulatory effect on gastric secretion through PACAP receptors and the vagal pathway.     

Gene expression of pituitary adenylate cyclase activating polypeptide (PACAP) in the rat hypothalamus

Pituitary adenylate cyclase activating polypeptide (PACAP) isolated from ovine hypothalamus is considered to be a member of the vasoactive intestinal peptide/glucagon/secretin/growth hormone-releasing hormone family of peptides. Two forms of PACAP, PACAP38 and PACAP27, have been demonstrated in the rat hypothalamus. The PACAP precursor contains another peptide called PACAP-related peptide (PRP), but so far no information on this peptide in tissue exists. We have developed three radioimmunoassays specific for PACAP38, PACAP27 and PRP and demonstrate that all three preproPACAP peptides are expressed in the rat hypothalamus, the PACAP38/PACAP27 ratio being around 60 and the PACAP38/PRP ratio being around 10. HPLC analysis of hypothalamic extract showed that PACAP38 and PACAP27 are found in only one form corresponding to the respective synthetic peptides, whereas PRP eluted in two peaks, the predominant form corresponding to synthetic PRP1-29. The cellular distribution of PACAP38, PACAP27, and PRP and corresponding mRNA in the hypothalamus was determined with immunohistochemistry and in situ hybridization histochemistry. PACAP- and PRP-immunoreactive neuronal perikarya were observed in the medial parvocellular part of the paraventricular nucleus (PVN) in colchicine pretreated rats. Some cell bodies of magnocellular variety were found in the PVN. PACAP mRNA containing cells were observed in moderate numbers in the same parts of the paraventricular nucleus. PACAP- and PRP immunoreactive fibres and varicosities were distributed in the PVN and in the periventricular nucleus. These data show that PACAP38, PACAP27 and PRP are expressed in the parvocellular part of the PVN, implying roles as hypothalamic regulatory peptides.     

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP-27, but not PACAP-38) degradation by the neutral endopeptidase EC 3.4.24.11

VIP (vasoactive intestinal polypeptide) and PACAP (pituitary adenylate cyclase-activating polypeptide), which are potent relaxing agents in the airways, were submitted to in vitro degradation by the neutral endopeptidase EC 3.4.24.11 (NEP), one of the most active peptidase in the lung, to test their relative resistance to proteolysis. Both VIP and PACAP(1-27) were cleaved by NEP, but PACAP(1-38) was not. The main fragments produced were VIP(1-22) and VIP(1-25), and PACAP(1-22) and PACAP(1-25), respectively. The degradation of VIP(1-27), PACAP(6-27), and PACAP(13-27) was also hindered by extending their C-terminal ends with the (28-38) sequence of PACAP(1-38). The sensitivity to enzyme degradation was gradually reduced when the C-terminal extension was increased from PACAP(1-27) to PACAP(1-29), PACAP(1-32) and PACAP(1-38). The biological activities of the degradation products were evaluated on the three classes of PACAP/VIP receptors, with VIP(1-25) and PACAP(1-25) retaining an important part of their activities on the VIP1 receptor. Thus, the degradation of VIP and PACAP(1-27) by the neutral endopeptidase 24.11 might produce a VIP1 receptor-selective active metabolite, provided that very high VIP or PACAP(1-27) concentrations are achieved in the receptor vicinity.     

Allogeneic bone marrow transplantation for chronic myelomonocytic leukemia in childhood: a report from the European Working Group on Myelodysplastic Syndrome in Childhood

The purpose of this study was to investigate the mechanisms of action of pituitary adenylate cyclase-activating polypeptide (PACAP) in stimulating aldosterone production in two different models: bovine adrenal zona glomerulosa (ZG) cells in primary culture and the human adrenocortical carcinoma cell line H295R. PACAP binds to two major groups of receptors: type I, which prefers PACAP38 and PACAP27 over vasoactive intestinal peptide (VIP); and type II, which has approximately equal affinity for PACAP38, PACAP27, and VIP. The type I subclass comprises multiple splice variants that can be distinguished by their specificity to PACAP38 and PACAP27 in their activation of adenylate cyclase and phospholipase C. Type II PACAP/ VIP receptors couple only to AC. In bovine ZG cells, PACAP38 and PACAP27 stimulated aldosterone production in a dose-dependent manner, whereas VIP was ineffective. In H295R cells, PACAP38, PACAP27, and VIP dose-dependently stimulated aldosterone production with roughly the same ED50. In bovine ZG cells, PACAP38 and PACAP27 stimulated cAMP production with similar efficacy, whereas VIP had no effect. In H295R cells, all three peptides stimulated cAMP accumulation. PACAP38 and PACAP27 also activated PLC in bovine ZG cells as they induced an increase in Ins(1,4,5)Ps production. In H295R cells, neither of these peptides was able to stimulate IP turnover. These results indicate that PACAP stimulation of aldosterone production is mediated by the PVR1s or the PVR1hop splice variants of the type I PACAP-specific receptor subtype in bovine ZG cells, whereas only type II PACAP/VIP receptors seemed to occur in the human H295R cell line. In addition, PACAP-stimulated aldosterone production was inhibited by atrial natriuretic peptide in bovine and human adrenocortical cells, however not by the same mechanism. This further supports species-specific and/or cell type-specific signaling pathways for PACAP in the regulation of aldosterone production.     

Pituitary adenylate cyclase-activating polypeptide, helospectin, and vasoactive intestinal polypeptide in human corpus cavernosum

1. The distribution and effects of pituitary adenylate cyclase-activating polypeptide (PACAP-27 and -38), helospectin (Hel-1 and Hel-2), and vasoactive intestinal polypeptide (VIP), were investigated in isolated preparations of human corpus cavernosum (CC). 2. Immunohistochemistry revealed coinciding profiles of nerve structures that showed immunoreactivities for VIP and PACAP, and VIP and Hel. Confocal microscopy showed the co-existence of VIP- and PACAP-immunoreactivities, and VIP- and Hel-immunoreactivities in most (90%) varicose nerve structures. 3. As determined by radioimmunoassay, the amounts of VIP, PACAP-27, and PACAP-38 in the preparations were 61.7 +/- 11.6, 0.1 +/- 0.05, and 3.7 +/- 0.5 pmol g-1 wet weight of tissue (pmol g-1 wet wt.), respectively. In tissue from patients with diabetes, the content of VIP was lower (13.7 +/- 0.5 pmol g-1 wet wt.), whereas that of PACAP (-27 and -38) was unchanged. 4. Cyclic nucleotide levels were determined in preparations exposed to PACAP-27, PACAP-38, Hel-1, Hel-2, and VIP. All the peptides, but Hel-2, significantly increased the concentrations of cyclic AMP, whereas the levels of cyclic GMP were unchanged. 5. The peptides concentration-dependently relaxed noradrenaline-contracted preparations. The order of potency was VIP > PACAP 27 > Hel-1 > Hel-2 > PACAP-38. 6. Hel-1, VIP and PACAP-27 effectively counteracted electrically induced contractions. At 10(-6) M, the highest peptide concentration used, the inhibitory effects obtained reached 96 +/- 3%, 87 +/- 6%, and 80 +/- 3%, respectively. 7. The results suggest that PACAP and Hel-1 are co-localized with VIP in nerve structures within the human cavernous tissue, and that the peptides are effective relaxants of CC preparations in vitro. The role of the investigated peptides for penile erection remains to be established.     

Time-resolved fluoroimmunoassay for pituitary adenylate cyclase activating polypeptide 27 (PACAP27) using europium (III) ion chelate labeled streptavidin-biotin complex

Pituitary adenylate cyclase activating polypeptide (PACAP) is a novel peptide hormone and has a variety of biological action. In studies of the physiological behaviour of endogenous PACAP, the determination of PACAP levels in biological materials require a highly sensitive and specific method. Therefore, we developed a sensitive time-resolved fluoroimmunoassay (TR-FIA) for PACAP27 which is the biologically important fragment of PACAP. Accordingly, we developed TR-FIA using a biotinylated PACAP27 (b-PACAP27) as a tracer and europium (III) chelate labeled streptavidin-biotinylated bovine serum albumin complex as a detection of biotin on solid phase. A measurable range of PACAP27 was 7.8-1000 pg m1-1 by the proposed TR-FIA. For measurement of biological samples, the samples were purified to eliminate substances which interfered with the TR-FIA. The mean recovery of PACAP27 using commercially reversed phase column was 74.7% (n = 12). The various tissues, extracts and plasma concentrations of rat could be measured by the proposed TR-FIA.     

PACAP stimulates pancreatic exocrine secretion via the vagal cholinergic nerves in sheep

The present study evaluates the possible role of the vagus nerves in mediating the stimulatory effect of PACAP-27, PACAP-38 and VIP on the exocrine pancreas, especially on enzyme secretion which is atropine sensitive in sheep. The animals were equipped with two cannulae into the common bile duct, a duodenal cannula, and a ruminal cannula under anesthesia. The bilateral cervical vagus nerves were coiled with a cooling device. In conscious animals, the peptides were infused intravenously for 10 min at 10 pmol kg(-1)min(-1) in phase II of the duodenal migrating motor complexes and the same peptide infusion was repeated in the reversible cooling blockade of the vagus nerves. Increment in fluid secretion was not significantly altered by the vagal blockade in all the peptide infusions, while increment in bicarbonate ion by only PACAP-27 was inhibited by the vagal blockade. Increments in protein and amylase output decreased significantly to 32.0+/-5.0 and 23.2+/-2.6% in PACAP27, and to 26.1+/-7.7 and 20.8+/-6.4% in PACAP-38 in the vagal blockade, but the increments by VIP did not decrease. These results demonstrate that circulating PACAP stimulates pancreatic enzyme secretion via the vagal cholinergic preganglionic neurons in sheep, suggesting the central action of PACAP.     

Pituitary adenylate cyclase-activating polypeptide (PACAP) 38 and PACAP27 differentially stimulate growth hormone release and mRNA accumulation in porcine somatotropes

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been suggested to regulate growth hormone (GH) secretion in several species. Here, we analyzed the in vitro effects of PACAP38 and PACAP27 on the secretory activity of porcine somatotropes. Cultures of porcine pituitary cells were treated with PACAP38 and PACAP27, and GH release, intracellular GH content, and GH mRNA levels were evaluated. Also, the time course of changes in the somatotrope content of GH and its mRNA in response to PACAPs were measured. Both PACAPs stimulated GH release from porcine somatotropes in a broad range of doses (10(-10)-10(-6) M), yet only PACAP27 elicited a dose-dependent response. GH cell content remained essentially unchanged after PACAP treatment. In contrast, both PACAPs induced significant and sustained increases in GH mRNA cell content, although the response to PACAP27 appeared faster (8 h) than to PACAP38 (16 h). These results demonstrate that PACAP stimulates GH production in porcine somatotropes. Furthermore, the differential responses induced by PACAP38 and PACAP27 suggest that distinct mechanisms mediate their effects on this cell type.     

Pituitary adenylate cyclase activating polypeptide (PACAP) is localized in human dermal neurons and causes histamine release from skin mast cells

OBJECTIVE AND DESIGN: Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide homologous with vasoactive intestinal polypeptide (VIP) which is known to induce histamine release in human skin mast cells. PACAP has not been detected in human skin. The purposes of the study were to investigate the occurrence of PACAP in human skin and to evaluate the histamine releasing activity of the two common pro-PACAP products, PACAP-27 and PACAP-38. MATERIAL: Fourteen human surgical skin samples were obtained. PACAP and VIP were visualized by immunohistochemistry. A microdialysis technique was used to measure histamine release in intact skin samples following intradermal injections of the peptides. RESULTS: PACAP and VIP were localized in dermal nerves in connection with sweat glands. Intradermal injection of 3 or 10 microm PACAP significantly released histamine. Kinetics of histamine release showed peak release 2-4 min after skin challenge. Ten microm of PACAP-27, VIP and somatostatin caused histamine release with similar efficacy, whereas PACAP-38 was less effective. Substance P was twice as efficient as PACAP-27, whereas calcitonin gene-related peptide did not release histamine. CONCLUSIONS: PACAP is found in human skin and is capable of releasing histamine from skin mast cells.     

Dimensionality of depression in acute schizophrenia: a methodological study using the Bech-Rafaelsen Melancholia Scale (BRMES)

Ca2+ plays an essential role in pituitary adenylate cyclase-activating polypeptide (PACAP)-stimulated growth hormone (GH) secretion from porcine somatotropes. Here, Indo-1 microfluorimetry was used to investigate the dynamics of free cytosolic Ca2+ concentration ([Ca2+]i) in single porcine somatotropes in response to PACAP38 and PACAP27. We also evaluated the relative contributions of extra- and intracellular Ca2+ sources and of cAMP-dependent protein kinase (PKA) and phospholipase C (PLC). A high proportion of somatotropes responded to PACAP38 (79.4%) and PACAP27 (68.4%) with [Ca2+]i rises that could be followed by a refractory plateau (type 1 response), or by a decrease in [Ca2+]i during which somatotropes were responsive to a subsequent PACAP pulse (type II response). Although Ca2+ profiles in response to both peptides were similar, PACAP38-induced [Ca2+]i rises were higher. Somatotrope response to PACAP38 or PACAP27 was markedly reduced by removing extracellular Ca2+, blocking Ca2+ entry through L-type voltage sensitive Ca2+ channels (VSCC), or inhibiting PKA. Conversely, Ca2+ depletion from intracellular stores or PLC inactivation did not affect the response to PACAP27 but considerably reduced maximal [Ca2+]i induced by PACAP38. We conclude that both peptides stimulate extracellular Ca2+ influx through L-type VSCC by a PKA-dependent mechanism. However, PACAP38 also triggers a PLC-mediated Ca2+ mobilization from intracellular stores, thereby indicating that the two molecular forms of PACAP activate common and distinct second messenger pathways within porcine somatotropes.     

Lipoprotein(a) in patients with carotid atherosclerosis and ischemic cerebrovascular disorders

Two novel peptides, named PACAP (pituitary adenylate cyclase activating polypeptide) containing 38 (PACAP38) and 27 residues (PACAP27) were recently isolated from ovine hypothalami. In order to investigate the pituitary cell type(s) that bear a receptor for PACAP, PACAP38 was biotinylated and used for cytochemical examination of binding. The cells were also identified by immunocytochemical methods using the antisera against each of the rat anterior pituitary hormones or an antiserum against S-100 protein, a marker for pituitary folliculo-stellate (FS) cells. Biotinylated PACAP38 (biot-PACAP) exhibited adenylate cyclase stimulating activity (ACSA) comparable to PACAP38 in rat pituitary cell cultures, and displaced the bound 125I-PACAP27 to the rat pituitary membrane preparation to the same extent as PACAP38. After 2-4 days of culture, dispersed rat pituitary cells were incubated with varying concentrations of biot-PACAP at room temperature or 4 degrees C. The bound biot-PACAP38 was visualized by avidin-biotin-peroxidase complex (ABC) method with nickel intensification. Biot-PACAP-positive and pituitary hormone or S-100-positive cells were counted. More than 90% of S-100-positive cells bound biot-PACAP38. A considerable number of GH and PRL cells and a lesser number of ACTH cells also bound biot-PACAP38, whereas only a few identified LH, FSH, or TSH cells bound biot-PACAP38. These results suggest that FS cells are a major target cell type for PACAP. A recent study from our laboratory demonstrated that PACAP stimulated the release of interleukin (IL)-6 in rat pituitary cell cultures. FS cells are known to produce IL-6.