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Pituitary adenylate-cyclase-activating polypeptide (PACAP), a novel brain-gut hormone, was isolated from ovine hypothalami and represents the latest mammalian member of the secretinglucagon 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 '251-labelled PACAP(1-27) to plasma membranes from rat brain. We identified a single high-affinity binding site (Kd, 340 pM and B,,,, 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), and PACAP(3 -25), nor vasoactive intestinal peptide (VIP), secretin and growth-hormone-releasing factor (GRF) revealed significant binding at concentrations up to 1 FM. 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-l -propane sulfonate (Chapso) or Triton X-100 and was characterized as a high-molecular-mass receptor complex (400 kDa) by nonreducing 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. Pituitaryadenylate-cyclase-activating polypeptide (PACAP) has been recently isolated from ovine hypothalamic extracts [l]. This C-terminally amidated peptide comprises 38 or 27 amino acid residues and reveals a high potency for adenylate-cyclase activation on rat pituitary cells [2]. PACAP(1-27) possesses full biological activity and shows 68% similarity to vasoactive intestinal peptide (VIP). Therefore, PACAP is regarded as a novel member of the secretin- Dedication. This publication is dedicated to Prof. Dr Norbert Hilschmann, Max-Planck-Institute for Experimental Medicine, Gottingen on the occasion of his 60th birthday. glucagon family, a group of structurally related regulatory peptides.cDNA cloning of the PACAP precursor from ovine, rat and human sources revealed that the primary structures of human [3] and rat [4] PACAP molecules are identical to the ovine peptide, suggesting a highly conserved and crucial role as a neuropeptide and/or as a gastrointestinal hormone. In superfused rat pituitary cells, PACAP stimulates the release of adrenocorticotropin, growth hormone, prolactin and luteinizing hormone [2]. However, the regional distribution of PACA...

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The present study was performed to characterize a direct influence of cyclosporine A (CsA) on exocrine pancreatic enzyme secretion. CsA inhibited dose-dependently amylase release from isolated rat pancreatic acini in response to carbachol or cholecystokinin octapeptide (CCK-8). A significant reduction in amylase release by 17% was observed at 0.2 μmol/l CsA (p < 0.001) when compared to controls. At 0.2 mmol/l, CsA reduced amylase release in response to both secretagogues by maximally 45%, whereas basal secretion was not affected. CsA had no influence on CCK-8-stimulated increase in intracellular Ca²⁺ concentrations or amylase release in response to the Ca²⁺ ionophore A 23187. In contrast, the dose-response curve for amylase secretion induced by the phorbol ester phorbolmyristate-13-acetate was shifted to the right without a reduction of the maximal secretory response. Unexpectedly, vasoactive-intestinal-polypeptide- and secretin-stimulated acinar secretion was not diminished by CsA. In isolated pancreatic lobules exposed to 0.1 mmol/l CsA, amylase release stimulated by cerulein or veratridine was reduced by 26.7 ± 2 or 28.3 ± 4%, respectively. CsA had no influence on the displacement of ¹²⁵I-Bolton-Hunter-labeled CCK-8 from acinar CCK receptors. The ultrastructure of cellular organelles in isolated lobules was not altered after incubation with 0.1 mmol/l CsA for 60 min. Our data suggest that CsA interferes with proteinkinase-C-mediated signal transduction in isolated rat pancreatic acini, without affecting cAMP-dependent signalling.

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