ABSTRACT
Endogenous levels of pituitary adenylate cyclase-activating polypeptide (PACAP) and its binding site densities were measured in eight brain regions in rats of different ages (2-240 days) by sandwich-enzyme immunoassay and autoradiography. PACAP levels were quite low at day 2, peaked in 30-60 days, and then remained constant in most regions. Such ontogenetic changes are similar to those of vasoactive intestinal polypeptide (VIP) and classical neurotransmitters. PACAP-binding sites were already dense at day 2 and varied only slightly up to day 240. These results suggest that PACAP may have modulatory effects on brain development.
Subject(s)
Brain Chemistry/physiology , Brain/growth & development , Neuropeptides/biosynthesis , Neurotransmitter Agents/biosynthesis , Receptors, Pituitary Hormone/biosynthesis , Animals , Autoradiography , Binding Sites/physiology , Brain/anatomy & histology , Immunoenzyme Techniques , Male , Neuropeptides/immunology , Neurotransmitter Agents/immunology , Pituitary Adenylate Cyclase-Activating Polypeptide , Rats , Rats, Wistar , Receptors, Pituitary Adenylate Cyclase-Activating PolypeptideABSTRACT
Vasoactive intestinal polypeptide (VIP) has been suggested to have a presynaptic effect on cholinergic terminals in the rat hippocampus, which results in an activation of acetylcholine (ACh) synthesis. Recently, a VIP-related novel peptide, pituitary adenylate cyclase activating polypeptide (PACAP) was isolated from the ovine hypothalamus, and we previously demonstrated in the rat that PACAP binding site densities were high in the hippocampus. In the present study, we investigated the effects of VIP and PACAP on the release of ACh from the rat hippocampus. We succeeded in detecting the spontaneous release of ACh from the dorsal hippocampus in the conscious rat using microdialysis and HPLC-ECD. VIP, PACAP38 and PACAP27 were applied through a microinjection cannula placed in a region adjacent to the tip of a microdialysis tube. Injections of VIP, PACAP38 and PACAP27 (12, 120 pmol) resulted in dose-related increases in ACh release. The ability to enhance ACh release was VIP > PACAP38 > PACAP27. The increased release of ACh caused by these peptides was highly calcium-dependent. Tetrodotoxin (10(-6) M) added to the perfusion medium significantly reduced both the release of ACh enhanced by these peptides and the basal release. The present results suggest that VIP, PACAP38 and PACAP27 presynaptically stimulate cholinergic activity in the hippocampus, which may be reflected by an increase in ACh synthesis to maintain releasable terminal stores of ACh.
Subject(s)
Acetylcholine/metabolism , Brain/metabolism , Hippocampus/metabolism , Neuropeptides/pharmacology , Vasoactive Intestinal Peptide/pharmacology , Animals , Brain/drug effects , Calcium/pharmacology , Dialysis/methods , Hippocampus/drug effects , Kinetics , Male , Microchemistry , Pituitary Adenylate Cyclase-Activating Polypeptide , Rats , Rats, Wistar , Tetrodotoxin/pharmacology , Time FactorsABSTRACT
We investigated endogenous levels of a novel peptide, pituitary adenylate cyclase activating polypeptide (PACAP), in the rat central nervous system. The amount of PACAP was measured by means of highly specific and sensitive sandwich-enzyme immunoassay. This assay system following HPLC analysis revealed that PACAP38 was a major portion of the total PACAP immunoreactivity and PACAP27 levels were negligibly low in the brain. Therefore, we measured the amount of PACAP38 in 62 regions punched out from frozen tissue sections. High amounts of PACAP38 were found in the lateral septal nucleus (intermediate part), diagonal band, central amygdaloid nucleus, several parts of the hypothalamus (suprachiasmatic, supraoptic, periventricular and arcuate nuclei), central gray, interpeduncular nucleus and dorsal raphe. The suprachiasmatic, paraventricular and periventricular hypothalamic nuclei showed the highest levels. A moderate amount of the peptide was observed in the lateral septal nucleus (dorsal part), medial septal nucleus, medial amygdaloid nucleus, thalamus (paraventricular, paratenial, central medial, ventromedial, reuniens and rhomboid nuclei), hypothalamus (lateral hypothalamic area and mammillary body), ventral tegmental area, interfascicular nucleus and in the locus coeruleus. Such a distribution of endogenous PACAP38 did not parallel the localization of PACAP binding sites which we had demonstrated recently. Moreover, the topographical distribution of PACAP38 observed in the present study differed from that of VIP which had been previously reported. The present results suggest that PACAP38 may have a neurotransmitter/neuromodulator role which is different from that of VIP in the central nervous system.
Subject(s)
Brain Chemistry/physiology , Neuropeptides/metabolism , Neurotransmitter Agents/metabolism , Pituitary Gland/chemistry , Animals , Chromatography, High Pressure Liquid , Immunoenzyme Techniques , Male , Pituitary Adenylate Cyclase-Activating Polypeptide , Rats , Rats, Wistar , Sensitivity and SpecificityABSTRACT
Production of immunoreactive (ir-) endothelin-2 (ET-2) in renal adenocarcinoma cells, ACHN, was reduced by transforming growth factor-beta, basic fibroblast growth factor, transforming growth factor-alpha and, most strikingly, by epidermal growth factor (EGF). These growth factors did not show such inhibitory effects on the secretion of ir-ET-1 in ET-1-producing cells, indicating that the production of ET-2 and ET-1 is regulated differently by the growth factors. EGF specifically reduced the secretion of not only ir-ET-2 but also ir-big ET-2 with only a small decrease in total protein synthesis. Northern blot analysis indicated that EGF controls the ET-2-production at the transcription levels of ET-2 gene.
