Distribution of PACAP (pituitary adenylate cyclase-activating polypeptide)-like and helospectin-like peptides in the teleost gut

Pituitary adenylate cyclase-activating polypeptide (PACAP) and helospectin are two vasoactive intestinal polypeptide (VIP)-related neuropeptides that have recently been demonstrated in the mammalian gut; the aim of this study was to reveal their occurrence and localisation in the gastrointestinal tract, swimbladder, urinary bladder and the vagal innervation of the gut of teleosts, using immunohistochemical methods on whole-mounts and sections of these tissues from the Atlantic cod, Gadus morhua and the rainbow trout, Oncorhynchus mykiss. Both PACAP-like and helospectin-like peptides were present in the gut wall of the two species. Immunoreactive nerve fibres were found in all layers but were most frequent in the myenteric plexus and along the circular muscle fibres. Immunoreactivity was also demonstrated in nerves innervating the swimbladder wall, the urinary bladder and blood vessels to the gut. Immunoreactive nerve cell bodies were found in the myenteric plexus of the gut and in the muscularis mucosae of the swimbladder. In the vagus nerve, non-immunoreactive nerve cells were surrounded by PACAP-immunoreactive fibres. Double staining revealed the coexistence of PACAP-like and helospectin-like peptides with VIP in all visualized nerve fibres and in some endocrine cells. It is concluded that PACAP-like and helospectin-like peptides coexist with VIP in nerves innervating the gut of two teleost species. The distribution suggests that both PACAP and helospectin, like VIP, are involved in the control of gut motility and secretion.     

Characterization of the oligosaccharide moiety of VIP receptor from the human pancreatic cell line BxPC-3

The human pancreatic cell line BxPC-3 displays two classes of binding sites with high and low affinity for VIP. The order of potency of VIP-related peptides in inhibiting either [¹²⁵I]VIP or [¹²⁵I]N-AcPACAP27 binding and in stimulating cAMP production was typical of the human VIP receptor. By combining affinity labeling with glycosidase treatments, we have characterized the VIP receptor as a M_r = 68,200 glycoprotein, consisting of a M_r = 39,300 polypeptide core with at least three N-linked oligosaccharide chains. In addition, our results revealed the presence of a low amount of sialic acid residues in the carbohydrate moiety of receptor.     

Contrasting effects of PACAP and carbachol on [Ca]i and inositol phosphates in human neuroblastoma NB-OK-1 cells

The effects of PACAPs on [Ca²⁺]_i were compared to those of carbachol in human neuroblastoma NB-OK-1 cells. PACAP(1–27) and PACAP(1–38) increased [Ca²⁺]_i in a biphasic manner: a transient rise and a secondary plateau. The transient phase reflected the mobilization of [Ca²⁺]_i pool(s) via the inositol phosphate pathway. The modest sustained plateau required extracellular Ca²⁺. Carbachol also increased [Ca²⁺]_i in a biphasic manner, but it mobilized intracellular Ca²⁺ pool(s) with a higher efficacy than PACAPs, then greatly increased Ca²⁺ entry, this being accompanied by a more marked and prolonged elevation of IP₃ and IP₄ than with PACAPs. It is likely that cAMP-mediated phosphorylations due to PACAPs facilitated desensitization at the PACAP receptor-phospholipase C level, so that there was less Ca²⁺ handling through PACAP receptors than with muscarinic M₁ receptors.     

Expression patterns of pituitary adenylate cyclase activating polypeptide and its type I receptor mRNAs in the rat placenta

Pituitary adenylate cyclase activating polypeptide (PACAP) was first isolated from ovine hypothalamus and is known to act as a tropic factor in various cells. Recent report revealed the expression of PACAP and the PACAP type I (PAC(1)) receptor in human and rat placentas at term. Placenta is a critical organ that synthesizes several growth and angiogenic factors for its own growth as well as fetal development. However, there is little information regarding the expression pattern and cellular localization of PACAP and PAC(1) during pregnancy. The aim of this study was to define the expression and distribution of PACAP and PAC(1) receptor mRNAs in the rat placenta during pregnancy. PACAP and PAC(1) receptor mRNAs were expressed in decidual cells, chorionic vessels, and stromal cells of the chorionic villi. Interestingly, the expression of these genes varied with the day of gestation. For example, PACAP and PAC(1) receptor mRNAs expressed in decidual cells on day 13.5 and 15.5, their expression was strong in chorionic vessels and stromal cells of the chorionic villi within the labyrinth zone on day 17.5, 19.5, and 21.5. In fact, as gestation advanced, the expression of PACAP and PAC(1) receptor mRNAs in the decidua cells disappeared, as their high expression became evident in the chorionic vessels and stromal cells of the chorionic villi. Our finding that PACAP and the PAC(1) receptor are co-localized and their genes seemingly co-regulated within specific placental areas, strongly suggest that this peptide may play an important role, as an autoregulator or pararegulator via its PAC(1) receptor, in physiological functioning of the placenta for gestational maintenance.     

Immunohistochemical features of paragangliomas

Immunohistochemistry is part of the routine diagnosis of the neuroendocrine tumors. In our study, we included 52 paragangliomas with various localizations by routine histology and immunohistochemistry. In order to increase the diagnostic specificity, a complex immunohistochemistry panel has been performed consisting of Bcl-2, Ki-67, Bax and Pituitary Adenylate Cyclase-Activating Peptide (PACAP), somatostatin, VIP and Calcitonin Gene Related Peptide (CGRP). After heat induced antigen retrieval, the immunostaining was performed by StreptABC using DAB as a chromogen. We were the first to demonstrate the presence of Bax and PACAP in paragangliomas. Some of the used markers are of prognostic value. The relationship between Bcl-2 and Bax is decisive in generating the final response to the input apoptotic signals. The Ki-67 antigen staining has gained wide acceptance in prognostic evaluation of other tumor types. We noted a small number of Ki-67 positive cases, which signifies a low mitotic activity of these tumors and a relatively high number of Bax positivities (32.9%) and the much lower number of Bcl-2 positivities (11.39%), and could explain the benign behaviour of paragangliomas.     

Effects of pretreatment with PACAP on the infarct size and functional outcome in rat permanent focal cerebral ischemia

PACAP exerts neuroprotective effects under various neurotoxic conditions in vitro. In vivo, it reduces brain damage after global and transient focal ischemia. The present study investigated whether PACAP has neuroprotective effects when applied before the onset of permanent ischemia. Rats were given bolus injections of PACAP38 intracerebroventricularly, and then underwent permanent middle cerebral artery occlusion. The results show that 2 μg of PACAP significantly reduced the infarct size measured 12 and 24 h after the onset of ischemia. No further reduction was obtained by a 7-day pretreatment. PACAP also ameliorated certain sensorimotor deficits. Our present study provides further evidence for the neuroprotective effects of PACAP, and implies that it might be a promising preventive therapeutic agent in ameliorating ischemic brain damage.     

Expression of mRNA for PACAP and its receptors in intra- and extra-adrenal human pheochromocytomas and their relationship to catecholamine synthesis

Purpose: Pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the secretin/glucagons/vasoactive intestinal peptide family, induces the expression of catecholamine-synthesizing enzymes in adrenal medullary cells. In addition, PACAP and its receptor have been detected in human pheochromocytoma tissues, though it is not yet known whether PACAP enhances the expression of genes encoding catecholamine-synthesizing enzymes. To address this question, we analyzed PACAP, PACAP receptor, and tyrosine hydroxylase (TH) and phenylethanolamine-N-methyltransferase (PNMT) mRNAs in pheochromocytomas. Methods: The levels of the mRNA for PACAP and vasoactive intestinal peptide (VIP), and their receptors, and for TH and PNMT were measured by RT-PCR or real-time PCR analysis, and the concentrations of catecholamines were measured by HPLC in 24 intra-adrenal and six extra-adrenal pheochromocytomas. Results: mRNA expression of PACAP and its receptor VPAC1R were detected in many pheochromocytomas (24/30 and 29/30, respectively), but mRNA expression of the PAC1R and VPAC2R receptor subtypes were detected in only one of six extra-adrenal pheochromocytomas. PACAP mRNA expression correlated with TH (p=0.0018) and PNMT (p=0.05) mRNA expression, as well as epinephrine (p=0.0342) levels in 16 intra-adrenal pheochromocytomas. Conclusion: Our findings support a possible role for PACAP in the regulation of expression of genes encoding catecholamine-synthesizing enzymes in intra-adrenal pheochromocytomas.